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TABLE OF CONTENTS 2081782961

I I Pnj,-jo Mo49A -1) 1 ~ International Symposium ~ on Lifestyle Factors and Human Lung Cancer I I I I I I I 1 I I I December 12-16, 1994 N Guangzhou, People's Republic of China ° -4 w N tC , W O

PAPERS 2081782971

2081782972

8. The Relationship Between Histologic Types of Lung Cancer and Cigarette Smoking Zhou Bao-sen, He An-guang and Wang Tian jue China Medical University, Shenyang, China 9. Progressive Changes in the Relative Distribution of Different Histological Types of Lung Cancer in Guangzhou, China Li Lie, Huang Shu-wei, Lu Zhen-jie and Wan Guang-ai Department of Pathology, Guangzhou Medical College, Guangzhou, China 10. Induction of DNA-Protein Crosslink in Rat Lung and Blood by the Carcinogen Nickel Lei Yi-idong*, Zhang Qiao** and Zhang Zhi-xiong* * Department of Hygiene, Guangzhou Medical College, Guangzhou, China ** Research Unit of Genotoxicology, Sun Yat-sen University of Medical Sciences, Guangzhou, China 11. Molecular Epidemiologic Study of Coal Smoke-Generated Envionmental Carcinogens and Lung Cancer in Humans Me Huei-jiang, Chen Xiao jia, Wang Su-min and Zheng Su-hua Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China 12. A Study on the Relationship Between P53 Mutation and Smoking in Human Non-Small Cell Cancer Li Jin-han, Bi X.J. and Peng Z.H. Department of Oncology, Nan Fang Hospital, First Military Medical University, Guangzhou, China I I I I I I I I I I I I I I

I 8. A Study of Association of Female Squamous Cell Carcinoma and Adenocarcinoma in the Lung and History of Menstruation Liao Mei-lin, Wang Jian-hwa, Wang Hwei-min, Ou Ai-qin, Wang Xiao jun and Long Wan-qing Shanghai Chest Hospital, Shanghai, China 9. Combined Analysis of Case-Control Studies of Smoking and Lung Cancer in China Yu Shun-zhane and Zhao Ning Shanghai Medical University, Shanghai, China 10. A Case-Control Study of Childhood and Adolescent Household Passive Smoking (PS) and the Risk of Female Lung Cancer Wang Fu-line*, Edgar J. Love** and Dai Xu-dong* * Heilongjiang Institute for Cancer Research, Harbin, China ** University of Calgary, Calgary, Canada 11. A Comparative Study of the Risk Factors for Lung Cancer in Guangdong, China Wang Shen-yong*, Hu Yi-ling*, Wu Yi-long**, Li Xin*, Chi Gui-bo*, Chen Ying *** and Dai Wen-Sha* * Medical College of Jinan University, Guangzhou, China ** Tumor Hospital of Sun Yat-sen Medical University, Guangzhou, China *** People's Hospital of the Yue-xiu District of Guangzhou, China 12. Analyses and Estimates of Attributable Risk Factors for Lung Cancer in Nanjing, China Shen Xiao-bing, Wang Guo-xiong*, Huang Yuan-zhu**, Xiang Long-sheng* and Wang Xing-he* *Nanjing Railway Medical College, Nanjing, China **Nanjing Medical University, Nanjing, China 13. Diet as a Confounder of the Association Between Air Pollution and Female Lung Cancer: Hong Kong Studies on Exposures to Environmental Tobacco Smoke, Incense and Cooking Fumes as Examples Linda C. Koo and John H-C Ho Cancer Research Laboratory, Hongkong Anticancer Society, Nam Long Hospital, Hong Kong 14. Indoor Burning Coal Air Pollution and Lung Cancer - A Case-Control Study in Fuzhou, China Luo Ren-Jda*, Wu Bin**, Yi Ying-nan** and Lin Ru-tao** * Fuzhou Senior Medical School, Fuzhou, China ** Fujian Medical College, Fuzhou, China I I I ' I I I I I I I I I I I I I I

I I I I . TABLE OF CONTENTS PAPERS An Epidemiological Investigation of Risk Factors for Lung Cancer in Guangzhou, China Du Ying-xiu*, Cha Qing*, Chen Xiao-wei* Chen Yong-Zhong* Huan Lan-fan ** I I . , , g g , Feng Zhen-zhi** and Wu Xia-fen** * Department of Hygiene, Guangzhou Medical College, Guangzhou, China **The Municipal Health & Antiepidemic Station of Guangzhou, Guangzhou, China Aspects of the Epidemiology of Lung Cancer in Smokers and Nonsmokers in the I United States Geoffrey C. Kabat I 3. Albert Einstein College of Medicine, Bronx, New York, USA Risk Factors for Lung Cancer Among Nonsmokers With Emphasis on Lifestyle Factors Gao Yu-tan¢ I I 4. Shanghai Cancer Institute, Shanghai, China Attributable Risk of Lung Cancer in Nonsmoking Women Michael C.R. Alavania*, Ross C. Brownson**, Jacques Benichou* Christine I I . , Swanson* and John D. Boice, Jr. * * Epidemiology and Biostatistics Program, National Cancer Institute, Bethesda, Maryland, USA ** Department of Community Health, Saint Louis University School of Public Health, St. Louis, Missouri, USA The Etiology of Lung Cancer in Nonsmoking Females in Harbin, China I Dai Xu-don¢, Lin Chun-yan, Sun Xi-wei, Shi Yu-bo and Lin Ying ji I 6. Cancer Research Institute, Harbin Medical University, Harbin, China Lung Cancer in Nonsmoking Chinese Women: A Case-Control Study Wang Tian-iue, Zhou Bao-sen and Shi Jin-pu I 7. China Medical University, Shenyang, China Lung Cancer, Smoking and Diet Among Swedish Men Ragnar Rylander*, Gosta Axelsson*, Lars Andersson**, Tomi Liljequist* and I Bengt Bergman*** * Department of Environmental Medicine, University of Gothenburg, Gothenburg, N O 00 I Sweden ** The Pulmonary Clinic, North Alvsborg General Hospital, Trollhattan, Sweden i 00 I I *** Institute of Lung and Heart Diseases, Sahlgren's Hospital, Gothenburg, Sweden N co w N

I 22. Lifestyle, Environmental Pollution and Lung Cancer in Cities of Liaoning in Northeastern China Xu Zhao-vi*, Linda Brown**, Pan Guo-wei*, Li Guang* and Feng Yi-ping* * Liaoning Public Health and Antiepidemic Station, Shenyang, China ** National Cancer Institute, Biostatistics Branch, Rockville, Maryland, USA 23. Determination of Personal Exposure of Nonsmokers to Environmental Tobacco Smoke in the United States Roeer A. Jenkins, M.A. Palausky, R.W. Counts, M.R. Guerin, A.B. Dindal and C.K. Bayne Oak Ridge National Laboratories, Oak Ridge, Tennessee, USA 24. Bayesian Meta-Analysis with Application to Studies of ETS and Lung Cancer Richard L. Tweedie, D.J. Scott, B.J. Biggerstaff and K.L. Mengersen Department of Statistics, Colorado State University, Fort Collins, Colorado, USA 25. The Relationship Between Smoking and Lung Cancer in Humans Geng_,Guan-vi, Liang Zhong-hua, Xu Rui-heng, Liu Jing-ying and Shi Pei-ying Tianjin Medical University, Tianjin, China 26. Some Lifestyle Factors and Human Lung Cancer: A Case-Control Study Among 792 Lung Cancer Cases Lei Yi-xion¢, Chen Yong-zhong and Du Ying-xiu Department of Hygiene, Guangzhou Medical College, Guangzhou, China 27. Health Impacts by Lifestyle and Behavioral Factors in Guangdong, China Zhou Jiona-Iiang, Liang Hao-cai, Wang Zhi jin and Liu Oing Institute of Preventive Medicine, Sun Yat-sen University of Medical Sciences, Guangzhou, China 28. Low Risk Epidemiology and Good Epidemiology Practice RaPnar Rylander Department of Environmental Medicine, University of Gothenburg, Gothenburg, Sweden I I I I I I I I I I I I I I I I

 ' I I 1. SUPPLEMENTS Recent Deveopments in the Epidemiology of Lung Cancer, Seminars in Surgical Oncology 9:73-79 (1993) (Reproduced with permission of the publisher) Geoffrey C. Kabat I 2. Albert Einstein College of Medicine, Bronx, New York, USA Recent Progress in the Epidemiology of Lung Cancer in Humans ' Du Ying-xiu I 3. Guangzhou Medical College, Guangzhou, China Exposure to Environmental Tobacco Smoke and the Incidence of Lung Cancer -- A Review Du Ying-xiu* and Joseph M. Wu** I I . * Guangzhou Medical College, Guangzhou, China ** Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, USA Etiology of Lung Cancer in Women Du Ying-xiu I 5. Guangzhou Research Center for Lung Cancer, Guangzhou, China Indoor and Outdoor Air Pollution and Lung Cancer I Du Ying=xiu*, Huang Lan-fang**, Feng Zhen-zhi** and Feng Jian-wei* I . * Department of Hygiene, Guangzhou Medical College, Guangzhou, China ** The Municipal Health & Antiepidemic Station of Guangzhou, Guangzhou, China Study of the Relation Between Smoking as a Lifestyle Factor and Lung Cancer in I Beijing Area of China Fan Ruo-Ian*, Zheng Su-hua*, Wu Zhao-su**, Wu Zhao-ru*, Zhang Rui-song**, I I . Cao Li-hua* and Li Yu-zhen* * Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China ** Beijing, Heart, Lung and Blood Vessel Medical Center, Beijing, China Analyses of Sex Differentials in Risk Factors for Primary Lung Adenocarcinoma Shen Xiao-bine*, Wang Guo-xiong*, Xiang Long-sheng* and Huang Yuan-zhu** I * Nanjing Railway Medical College, Nanjing, China ** Nanjing Medical University, Nanjing, China 00 ~ cNo C) 1 °'

 ' 15. The Effect of Beta-Carotene on Lung Cancer I Wang Hui and Lai Bai-tan¢ I 16. Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China A Matched Case-Control Study on the Relationship Between the Beta-Carotene Intake and Lung Cancer Tan Ai-Jun, He Shang-pu, Huang Ming-xi, Li Guo-Guang and Shi Lu-yuan I 17. Department of Epidemiology, Tongji Medical University, Wuhan, China Modulation of Molecular Mechanisms by Dietary Restriction in Rats I Beverly D. Lyn-Cook, Jin Bo and Ronald W. Hart I 8. Nutritional Modulators of Toxicity Program, National Center for Toxicological Research, Jefferson, Arkansas, USA Transformation of Tracheal Epithelial Cells and the Role of Transforming Growth 1 Factors (TGF) and P53 in the Lung Cancer Progression Wang Hong, Cheng Shu jun, Lin Li-min, Chen Lei, Guo Shu-pin, Fen Ji-nong, Han I 9. Nai jun and Sun Han-xiao Cancer Institute, CAMS and PUMC, Beijing, China Bioassays of Benzo(a)pyrene and Lung Cancer I Wu Zhong-Gang*, Chen Jia-kun*, Zhan De jin*, Jin Bo*, He Ling* Du Ying-xiu* I I 0. , and Joseph M Wu** * Guangzhou Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China ** Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, USA The Study of Correlation Between GSTµ Gene Deletion and Susceptibility to Lung I Cancer Sun Gui-fan*, Pi Jing-bo*, Zheng Quan-mei** and Zheng Mei-zhen*** I ' 1. * Laboratory of Occupational Medicine, Department of Preventive Medicine, China Medical University, Shenyang, China ** Cancer Prevention Center of China Medical University, Shenyang, China *** Liaoning Cancer Hospital, Shenyang, China A Retrospective Lung Cancer Mortality Study of People Exposed to Insoluble Arsenic Salts and Radon Liu Yu-tang and Chui Jin , Institute of Occupational Medicine, Chinese Academy of Preventive Medicine, Beijing, China n~ ao ~ V DD t0 , I O> A

! I 18. Point Mutations of Ha-ras and Ki-ras Oncogenes in Sputum Specimens from Lung I I 9. Cancer Patients He Lin¢, Chen Jia-kun, Yi Fei, Wu Zhong-liang and Du Ying-xiu Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China Effect of Dietary Restriction on Benzo(a)pyrene (B(a)P) Metabolic Activation and I Pulmonary B(a)P-DNA Adduct Formation in Mice Chen Wen* and Chou Ming W** I I 0. * Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China ** National Center for Toxicological Research, Jefferson, Arkansas, USA Natural Killer (NK) Cell Activity Assessment and NK Cell Activation by rhIL-2 in Patients With Lung Cancer Zhang Oiu-wang, Yang Ying, Cheng Xiao-yang, Fang Xiang, Mo Shu-xia and Liang I I 1. Xi-ruo Department of Microbiology and Immunology, Guangzhou Medical College, Guangzhou, China A Retrospective Cohort Study of Proportional Cancer Mortality Among Chinese Tar I Felt Workers Gong De-tian, Feng Ke-yu, Liu Chuen-hwa and Cui Xiu juen I 2. Construction and Waterproof Material Manufacturing Plant, Workers' Hospital, Qiqihar, Heilongjiang, China Environmental Risk Factors for Lung Cancer Among Swedish Men I Cecilia Modieh, GSsta Axelsson and Ragnar Rylander I I I I I Department of Environmental Medicine, University of Gothenburg, Gothenburg, Sweden N O eo i ~ I I N cc V O

 , I 1. ABSTRACTS Analysis of Lung Cancer Risk Factors in Guangzhou, China I Chen Xiao-wei, Lei Yi-xiong and Du Ying-xiu I 2. Department of Hygiene, Guangzhou Medical College, Guangzhou, China Passive Smoking and Lung Cancer Among Nonsmoking Women in Harbin China , Sun Xi-wei, Dai Xu-dong, Lin Chun-yan, Shi Yu-bo, Ma Yu-yan and Li Wei I 3. Heilongjiang Cancer Research Institute, Harbin, China Analysis of the Relationship Between Smoking and Lung Cancer Xie Cai-liang, Yang Wei-hua, Lian Ruan-shen, Huan Xia and Yang Xiao jian I I 4. Shanxi Tumor Hospital, China The Trend of Lung Cancer Death Rates in Guangdong Province, China Li Te-vou, Zhai Shao jiang and He Chai-gang I . The Cancer Research and Control Office, Public Health Bureau of Guangdong Province, Guangzhou, China Mortality Trend From Lung Cancer From 1976 to 1992 in Guangzhou, China I Zhou Xiu-zhen, Yang Zhi-cong, Liu Jin-cheng and Liu Shu-guo I 6. The Municipal Health & Antiepidemic Station of Guangzhou, Guangzhou, China Analysis of the Correlation Between Atmospheric Pollution and Lung Cancer in Guangzhou, China Huang Lan-fane*, Feng Zhen-zhi*, Wu Xia-feng*, Yan Li-ying** and Du Ying- I I . xiu*** * The Municipal Health & Antiepidemic Station of Guangzhou, Guangzhou, China ** Guangzhou Environmental Monitoring Center, Guangzhou, China *** Guangzhou Research Center for Lung Cancer, Guangzhou, China Relationship Between Lifestyle Factors and Lung Cancer in Humans Based on Trend I Analysis of Lung Cancer Incidence in Xuanwei, China Xu Chong-wan¢ I 8. Tumor Prevention and Treatment Research Center of Xuanwei County, Yunnan, China Psychological Factors and Lung Cancer ~ Yu Zheng-feng, Li Kang, Lu Bo, Hu Tian-ming and Liu Shu-qing N 0 Harbin Medical University, Harbin, China eo ~ , ~l 00 9. Environmental Factors and Lung Cancer N Yu Zheng-feng, Li Kang, Lu Bo, Hu Tian-ming and Fu Ti-sheng co rn ' Harbin Medical University, Harbin, China eo 

I I I I I S 1 I I I I The effect of smoking on lung cancer cell-type was also investigated by correlating the smoking status and the smoking index. 5. Case-Control Studies. (a) Effects of Smoking and Coal Fume Exposure. In 1985, there were 849 lung cancer deaths (571 males and 298 females). They were matched for sex, age ( t 2 years), and residence with nonlung cancer-related deaths. They were analyzed according to Mantel-Haenszel and by the stratification method, from which the relative risks associated with smoking status and contact with smoke from burning coal were obtained. (b) Effects of Other Risk Factors: Study Involving Nonsmokers. In the 849 lung cancer death cases, 120 cases (28 males and 92 females) were never-smokers. To investigate the effects of risk factors other than smoking, a separate case-control study, matched 1:2 for sex, age, and residence, was performed using two groups of controls. The first group consisted of 120 cases of never smokers who died from nonrespiratory illnesses. The second group consisted of never smokers whose deaths were caused by tumors outside of the respiratory system. Items investigated include: X1-history of respiratory disease; X2-consumption of fresh vegetables; X3-history of contact with toxic substances prior to death; X4-ETS exposure; X5-indoor air pollution; X6-size of living quarters; X7-size of kitchen; X8-cooking fuel; X9-participation in cooking; X10-family history of cancer. The contribution of each of these items was analyzed using conditional logistic regression. 6. Factors Affecting the Distribution of Lung Cancer Cell Tvnes In this study, we examined the influence of active and passive smoking, air pollution (indoor and outdoor), and occupational exposures on lung cancer cell types in both males and females. Eight cell- types of lung cancer were identified. These include squamous cell, small cell, adeno, large cell, epi- adeno, carcinoid, bronchial gland, and others. Over 80% of the total cases can be classified into squamous cell carcinoma and adenocarcinoma. The occurrence of these cell types shows a significant difference between males and females. Results 1. Deaths Attributed to Five Leading Cancers in Guangzhou From 1976 to 1989 Table 1 shows the results of the regression analysis of deaths due to the five leading tumors. In the case of lung cancer, a significant increase was observed in both males and females (p<0.01). Little change was found in liver and stomach cancers (p>0.05). Deaths due to nasopharynx and esophageal cancers show a decline (p<0.05) in the same period. O ~ ~ v 00 ~ v N I

I I I I I ~ I I I I I I I I investigated may be causal as well as consequential, due consideration must be given to confounding in connection with attempting to elucidate the effects of a single agent (or event) on the development of lung cancer. For example, the real impact of indoor air pollution on female lung cancer should be addressed in the context of confounding due to occupational exposure and cigarette smoking. Because the cell-types in male and female lung cancer are distinctly different, with squamous cell carcinoma being most predominant in males and adenocarcinoma prevailing in females, and because numerous studies have already concluded that unique lung cancer cell-types are caused by different carcinogenic factors, it is of interest to determine the cell-type in lung cancer cases that are attributed to indoor air pollutants. In this report, we have systematically analyzed the risk factors for female lung cancer deaths and have compared the associated risk factors between males and females. Materials and Methods Because of the long latency of lung cancer, and since the three factors (air pollution, occupational exposure, and cigarette smoking) being investigated in relation to lung cancer deaths may have complex interactive effects, a broad database, collected over a long period of time, is required for reaching meaningful conclusions. 1. Case History. In Guangzhou (population 2,000,000), there are four districts with 63 local police stations. Each station has a complete registry, containing information on age, sex, occupation, residence, and time/cause of death. In 1976, we began a detailed analysis of the registry, concentrating on cases in which death was caused by cancers of the lung, liver, nasopharynx, stomach, and esophagus. The annual crude and age- adjusted death rate, as well as the wotld age-adjusted death rate, was calculated yearly in order to ascertain the trend of deaths attributed to the five cancers. Beginning in 1980, every case of death from lung cancer was further analyzed using a standardized questionnaire containing 31 questions. Information was obtained retrospectively from relatives and verified by comparison with hospital records. The questionnaires were administered by trained medical personnel, and the data were entered into a computer. Because all deaths in China, including time and cause, must be reported to the local police station, and the report must agree with information provided to cremation centers, the data generated were considered to be highly reliable and accurate. 2. Analysis of Outdoor and Indoor Air Pollutants. ~ The city of Guangzhou, with an area of 55 square kilometers, can be divided into the four districts of Liwan, Yuexiu, Dongshan, and Haizhu. Atmospheric pollution status was systematically ' monitored by the Guangzhou Health and Antiepidemic Station(8), by the Guangzhou Environmental Monitoring Center(9), and by International Atmospheric Pollution Centers(8) established and managed by the World Health Organization. The information collected over the past two decades was used to ~ calculate the Air Pollution Index according to the following equations(10): p W i V ~ 00 3- N cc V N I

a I I I I t I I I I I I 1 W I 1 AN EPIDEMIOLOGICAL INVESTIGATION OF RISK FACTORS FOR LUNG CANCER IN GUANGZHOU, CHINA Du Yine-xiu*, Cha Qing*, Chen Xiao-wei*, Chen Yong-zhong* Huang Lan-fang**, Feng Zhen-zhi** and Wu Xia-fen** * Department of Hygiene, Guangzhou Medical College, Guangzhou, China ** The Municipal Health & Antiepidemic Station of Guangzhou, Guangzhou, China Abstract Lung cancer is one of the five leading tumors in the city of Guangzhou and has been increasing steadily in both males and females since the 1970s. In this report, more than 6,000 cases of lung cancer deaths, accumulated over the past nine years, were analyzed. Significant differences were found between males and females with respect to lung cancer risk factors. In a case-control study, 849 cases (571 males and 278 females) and a conditional logistic regression analysis of 120 nonsmokers (28 males, 92 females) were studied on the relative contributions of smoking, occupational exposure and indoor air pollution as risk factors for the rising incidence of lung cancer. The conclusions were as follows: In females, indoor air pollution, derived primarily from burning coal, is a highly significant risk factor for lung cancer. In males, however, cigarette smoking and occupational exposure play a more important role. Diet, especially vegetable intake, afforded positive protection for lung cancer. Estrogen changes are suggested to be significantly involved in the increased incidence of female adenocarcinoma. Introduction Statistics published by the National Bureau of Public Health in China show that the overall population death rate in the sixteen largest cities from 1982 to 1988 remained relatively constant at 565/100,000 (regression coefficient b=0.001, p> 0.05), while the mortality rate attributable to all forms of cancer has steadily increased from 100/100,000 in 1982 to 125/100,000 in 1988 (b=0.0117, p<0.05). Of particular note is the change in the lung cancer death rate, having increased from 25/100,000 in 1982 to 32/100,000 in 1988 (b=0.0151, p<0.001), which accounts for 25-26% of all cancer-related deaths. Lung cancer deaths in the city of Guangzhou rank as the third highest in the nation, behind the cities of Chungking and Shanghai. Since the early 1970s, lung cancer has been the foremost cause of death among the leading tumor-induced deaths. In 1989, the world standardized mortality rate (per 100,000) for the five leading tumors ranked, respectively, as follows: lung cancer (39.79), liver cancer (24.12), stomach cancer (9.67), nasopharynx cancer (6.07) and esophageal cancer (5.00), with lung cancer deaths amounting to almost the total of those originating from the liver, the stomach, and the nasopharynx. ' Cigarette smoking, occupational exposure, and air pollution (indoor and outdoor) are generally believed to be the three major risk factors for lung cancer. The relative importance of each of these N I factors, however, is known to vary with sex as well as with region and location. For example, in the city pC)p of Guangzhou, the ratio of the incidence of lung cancer in males and females is approximately 2.1:1; ~ however, cigarette smoking is much more prevalent among males (43 % of males, age 15 and above, are OVo ` smokers) compared to females (only 4%).(1) Forty percent (40%) of female lung cancer deaths were ~ ! V W I

1 n Ci API = E where: Ci -- Measured concentration of pollutants Si -- Permissible level of pollutants To assess the effects of indoor air pollution, two investigations were conducted in 1984 and 1985 as follows: Five families were randomly selected in each of the four districts. Samples were obtained over a 5-day period at each of the four seasons during the year. Daily samples were collected as follows: SO2 and NOx samples were obtained every 2 hours from 7 a.m. to 7 p.m. for a total of 7 samples per day; TSP and B(a)P were determined daily. To compare the levels of indoor and outdoor pollution, samples of SO2, NOx, TSP and B(a)P were also collected in the immediate outdoor vicinity of the selected families.(11) The levels of indoor and outdoor radioactivity in Guangzhou were measured by Wu(11) as follows: ten families were randomly selected in each of the four districts in July of 1984 and in February of 1985. Radon, thoron, and their daughters were measured. To determine whether the measurements may be affected by the construction material and by the type of cooking fuel used, houses constructed with different materials and homes using either coal or liquified petroleum for cooking were used. The major source of indoor pollution came from cooking. This is especially evident when burning coal was used. Thus, a comparative study was conducted in 1986-1987 in which total suspended particulate (TSP), TSP-B(a)P, sedimentary dust (SD), SD-B(a)P and B(a)P in the urine of housewives were determined. To avoid contamination from industrial sources, only families located far from factories and highways were used. The age of housewives ranged from 40-70. They were nonsmokers and had been working at home for at least 1 year. 3. Occupation Analysis. The majority of the participants were males with steady, well-defined jobs. To be eligible, a person must have worked in the same job for a minimum of 10 years. To determine the true risk potential of the different occupations, Hench's method was used to calculate the Standard Mortality Rate (SMR) and the Population Attributable Risk (PAR). The relationship between lung cancer deaths and occupation was ranked as "None," "Probable." and "Obvious." Such a ranking system permits a systematic comparison of the relative contribution of occupation on the incidence of lung cancer deaths. 4. Active Smoking. Smoking history includes age at which smoking began, number of cigarettes smoked per day, number of years smoked, and the type of tobacco consumed. A smoking index was obtained by multiplying the number of cigarettes smoked per day by the number of years smoked. In addition to case-control studies, we further analyzed whether smoking is a confounding factor for the effects of occupational exposure. Since it was not possible to obtain the rate of smoking for every case in each occupation, the effects of smoking on the standard mortality rate (generated from occupational exposure) were determined by rank correlation. 4 N 0 ~ ~ V Co N tD V rn I 11 I I t M l I I I I I I I I

' 1 10. Analyses of the Relationship Between Smoking, Passive Smoking and Lung Cancer Cell Type Cha in , Chen Yong-zhong and Du Ying-xiu I 11. Department of Hygiene, Guangzhou Medical College, Guangzhou, China Amplification and Point Mutation of Ha-ras Oncogene in Lung Cancer I Chen Jia-kun, He Ling, Wu Zhong-liang and Du Ying-xiu 12. Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China Amplification of C-myc, C-Ha-ras and C-sis Oncogenes in Human Lung Cancer I I He Ling, Zhan De jin, Chen Jia-kun, Wu Zhong-liang and Du Ying-xiu 3. Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China Expression of P53 and C-myc in Mouse Lung Cancer Induced by Coal Burning I Lin Chun-van, Dai Xu-dong, Song Lan-ying, Shi Yu-bo and Sun Xi-wen I 14. Heilongjiang Cancer Research Institute, Harbin, China Point Mutation at Codon 11 and 12 of H-ras and K-ras Oncogenes in Human Fetal I Epithelial Cells Treated with Benzo(a)pyrene trans-7,8-diol-anti-9,10-epoxide Zhan De-iin, Chen Jia-kun, Jin Bo, Yi Fei and Wu Zhong-liang 15. Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China Analysis of P53 and K-ras Mutational Patterns in Lung Cancer I I Gao Hong=g.uang*, Chen Jia-kun**, Wu Zhong-liang**, Whong Wen-zong* and Ong 6. Tong-man* * Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA ** Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou, China Methylation Profile and Amplification of Proto-Oncogenes in Caloric Restriction BKF I I I Rat Pancreas Jin B* and Beverly D. Lyn-Cook** Guangzhou Medical College * Institute for Chemical Carcinogenesis N  , , o !!! China Guangzhou 00 , ~ ** National Center for Toxicological Research, Jefferson, Arkansas, USA ~ 17. An Analysis of Seven Metal Elements in Lung Cancer Tissues in Guangzhou, China Population N ~ rn 10 Zhong Sai-xian, Chai Cheng-keng, Zhao Zhen-xin and Chen Cheng-zhang ' Department of Environmental and Health, Sun Yat-sen University of Medical Sciences, Guangzhou, China I I

I found to be never smokers, suggesting that, in females, factors other than smoking must exist and contribute significantly to lung cancer deaths. In an occupational analysis involving 5546 cases of lung cancer deaths, it was found that the percentage attributable to occupational exposure is small and can account for no more than 15 % of the cases in both males and females.(2) In an earlier investigation, Du et al.(3) concluded that in the various city districts of Guangzhou in which significantly different lung cancer death rates exist (ranging from a low of 20/100,000 to a high of 48/100,000), lung cancer incidence was correlated with the severity of atmospheric pollution. These findings show that even within the same city, the incidence of lung cancer can be influenced by the complex interaction of numerous known and unknown factors. Our previous studies have also revealed an association between indoor air pollution and the incidence of female lung cancer deaths.(4) Similar results have been obtained in other cities in China. In a case-control study on the prevalence of female lung cancer in the city of Shanghai, Gao, etal.(5) suggested that indoor air pollution and the related use of rapeseed oil in cooking may significantly contribute to the recent pronounced increase in lung cancer cases. Similarly, in a case-control study in Harbin, Dai eta l.(6) observed that the risk for adenocarcinoma was correlated with lower I3-carotene consumption and the use of coal for heating. Risk was also related to the generation of smoky conditions during heating. In the same study, it was reported that the frequency of squamous cell carcinoma was significantly associated with smoking, a history of bronchitis, and a prevailing smoky environment during heating. In Xuanwei county, Yunnan Province (documented to have the highest national female lung cancer mortality of 121/100,000 based on statistics published in 1973-1975), He et al.(7) reported in a case-control study that extremely high lung cancer mortality was caused by indoor air pollution and by "smoky" coal combustion. Substantial differences exist regarding the nature of indoor air pollution between a still developing country such as China and developed countries. Since cooking in industrialized nations primarily uses electricity or gas and seldom involves deep or stir frying over high heat, insignificant levels of respirable particulate matter and B(a)P are generated during cooking. Consequently, cooking is not considered a significant source of indoor air pollution. On the other hand, there are substantial particulates liberated from carpets, walls, ceilings and other types of indoor decorations, which would constitute the bulk of indoor air pollutants in families of developed countries and which are rarely encountered in China. In China, families in rural villages use wood and straw for cooking and heating, while in cities, coal is the primary fuel source for cooking. Cities in northern China are additionally dependent on burning coal for heating. These social habits, coupled with China's population density and, hence, small-size living area, have compounded the severity of indoor air pollution. Although many of the published case-control epidemiologic studies investigating the relationship between indoor air pollution and the incidence of female lung cancer have suggested a causal relationship, it is important to emphasize that such information is merely a clue and must be complemented by laboratory investigations in order to definitively demonstrate the biological plausibility of a causal relationship. For example, the notion that indoor coal burning is a major factor for female lung cancer would require studies showing that: 1) coal consumption indoors can generate significant levels of potentially carcinogenic substances and 2) such materials are also found to be present in exposed subjects at concentrations sufficiently high for cancer to be induced. In addition, because the development of lung cancer is likely to involve multiple factors and has a relatively long latency, and since factors being 2 I I I 1 I I• I I I i I I I I

2. Analysis of Lung Cancer Death Records. Between 1980 and 1989, 6,812 cases of lung cancer deaths, with 4,615 males and 2,197 females (sex ratio of 2.1:1), were recorded. For the present study, the following death cases were excluded: 1) those not involving primary lung cancer and 2) those residing for less than 10 years in Guangzhou. A total of 5,546 cases (3,760 males and 1,786 females) were included in the analysis (sex ratio 2.1:1). The youngest subject was 25 years old and the oldest was 98 years of age. The average death age was 64 in males and 65 in females. There were more cases of death due to cancer of the right lung, and the majority of these cases had central rather than peripheral foci. Metastasis was observed in 60% of the cases. 3. Atmospheric Pollution Between 1972 and 1990, 3 large-scale samplings of atmospheric pollutants were conducted. The results were transformed into an Atmospheric Pollution Index (API), which measured the concentrations of S02 and TSP. The TSP were further checked for their mutagenic activity using the Ames test (TA98, S9-) as described by Li et al.(12) Results in Table 2 show that atmospheric pollution was most severe in Liwan and was also correlated with the greatest incidence of lung cancer deaths. Likewise, TSP in Liwan demonstrated the most pronounced mutagenic activity (Table 2. 4. Indoor Air Pollution An investigation into indoor air pollution over a two year period shows that the level of pollutants is higher indoors than outdoors (Table 3, due to the fact that most of the factories are located in the outskirts of the city and because of the infrequent automobile use in Guangzhou in the year 1984-1985. Three daily peaks (7:00 a.m., 11:00 a.m., and 7:00 p.m.) in the levels of SO2 and NOx were observed. The levels were higher in winter and spring (when the windows were usually closed) than in summer and autumn (when the windows were open), suggesting that the major source of indoor air pollution came from cooking (Table 4. In Guangzhou, the traditional use of wood for cooking in the 1950s was replaced by coal in the 1960s, Beginning in 1980, some of the families began using liquefied petroleum. In 1992, about 50% of the families used gas for cooking. Table 5 compares the levels of indoor air pollutants and B(a)P in the urine of housewives among families using burning coal and those using gas. The levels are significantly higher in coal-buming families. The presence of B(a)P in the urine suggests that the amounts generated during cooking are readily taken up by the body. Table 6 illustrates the levels of radon and thoron (and their daughters) inside and outside of the home. The levels are correlated with the type of construction materials Table 7 and cooking fuel used (Table 8. Specifically, houses constructed with green and red bricks emit more radon and thoron than houses constructed with concrete. Moreover, thoron and radon levels were further elevated by the presence of burning coal, although never exceeding the National Standards (GB 4792-84). -6- i 1 11 I I R I I I I I I I ^ 1

a I I I I I I I I I I 5. The Effects of Occupational Exposure In the 5,546 cases of lung cancer, 6,167 personal occupation files were found. Approximately 600 people spent 10 years in 2 or more different professions. Occupation distribution is illustrated in Table 9 and Table 10. About 15 % of the cases may have exposure to job-related pollutants. To correlate occupational exposure and incidence of lung cancer deaths, a Standardized Mortality Ratio (SMR) was calculated for 434 cases of lung cancer deaths in the year, 1982 (Table 11). The Population Attributable Risks (PAR) for the 12 occupations are shown in Table 12. Chemists have the highest SMR in males, whereas homemakers have the highest SMR in females. Homemakers also have the highest PAR. 6. Active Smoking About 93% and 59% of the 3,755 and 1,784 cases of male and female lung cancer deaths, respectively, had a smoking history record. 95.3%, 2.8%, 1.4% and 0.5% of the smokers smoked cigarettes, water pipes, pipes, and cigars, respectively. The smoking history in the 5,539 cases of male/female lung cancer deaths is illustrated in Table 13. About 70% of the smokers began smoking before age 20. Cigarette consumption is higher in males (25 per day) than in females (20 per day). The smoking index is also higher in males (670) compared to females (630). The longer duration of smoking in females is probably due to their longer life expectancy. Using a rank correlation method, the contribution of smoking as a confounding factor on "Occupation-SMR" was studied and is illustrated in Table 14. In subjects with identical occupation, female "Occupation-SMR" was not affected by smoking, whereas in males, smoking did significantly influence "Occupation-SMR". Cigarette sales (packs/person/year) in Guangzhou between the years 1961-1974 were correlated with lung cancer death rates between the years 1976-1989. The coefficient of correlation (r) was 0.86 (p<0.01) in males and 0.71 (p>0.05) in females, showing that cigarette consumption is more closely associated with male lung cancer deaths. 7. Case-Control Studies The 849 (571 males and 278 females) lung cancer deaths in 1985 were further analyzed in two case-control studies. In the first study, in addition to evaluating the relative risks associated with cigarette smoke and contact with burning coal, the estimated annual death rate, the attributable death rate, and attributable risk were also evaluated and are listed in Table 15. The effect of cigarette smoke on lung cancer is much less important in females than males. By ] contrast, indoor air pollution is a highly significant risk factor for female lung cancer deaths but had no ' effect in males. The estimated death rate in males attributable to cigarette smoking is 31 % higher than the standardized death rate (68.5 compared to 52.2), while that attributable to contact with burning coal N ~ is much lower (38%, 19.9/52.2). In the case of females, the corresponding percentages are 49% p~p _L ti co 7_ y ~ M tc 1

i (12.3/24.7) and 77.7% (19.2/24.7), further showing that smoking is a more important risk factor for males, while contact with burning coal is a highly significant risk factor for females. ~ i In the second study, conditional logistic regression analysis was performed on the 120 cases of nonsmokers (28 males and 92 females) matched 1:2 with controls. ~ When lung cancer cases were matched with nonrespiratory cancer, we obtained results that are described by the following equations: ~ Males: logit Pi= ai + 0.045X3 11 Females: logit Pi= ai - 0.663X2 + 0.129X5 - 0.217X7 When lung cancers were matched with deaths not involving the respiratory system, the observed results were shown to fit the following equations: Males: logit Pi= ai - 1.330X2 + 0.0481X3 Females: Logit Pi = ai - 0.796X2 + 0.032X3 + 0.216X5 - 0.548X7 These results suggest that the consumption of fresh vegetables (X2) was a protective factor for lung cancer, whereas contact with toxic substances (X3) increases the risk for lung cancer. It is worth noting that in nonsmoking females, indoor air pollution (X5) and size of kitchen (X7) are risk factors for lung cancer, whereas ETS exposure (X4), respiratory disease history (Xl), familial history of cancer (X10), living conditions (X6), use of cooking fuel (X8), and participation in cooking (X9) had no effect whatsoever on female lung cancer deaths. The exclusion of X8 and X9 in the regression equations suggest that cooking fuel use and the degree of cooking participation may have been quite similar between the lung cancer cases and the matched controls. In the case of nonsmoking males, the major risk factors were contact with toxic substances and occupational exposure. 8. Factors Affecting the Distribution of Lung Cancer Cell Types (a) Effects of Gender, 1,093 of the 5,546 lung cancer death cases (804 males and 289 females) contain information on lung cancer cell type (19.7%). In males, the most common cell type is squamous cell carcinoma (58%) followed by adenocarcinoma (24%). In females, the converse was observed, with 48% adenocarcinoma and 29% squamous cell carcinoma (Table 16 . (b) Contribution of Cigarette Smoking. These results are shown in Table 16. In addition, the relationship between smoking index and lung cancer cell types was also studied (Table 17 . A significant difference between the ratio of the various lung cancer cell types was shown to exist between smokers and nonsmokers (males, X2=15.74, p<0.01; females, X2=8.55, p<0.05). In males, the smoking index is proportional to the percentage of squamous cell carcinoma (p <0.01) and inversely proportional to the percentage of adenocarcinoma (p<0.0001). The smoking index did not affect the lung cancer cell type in females (p>0.05). In both males and females, the incidence of squamous cell carcinoma is higher in smokers than in nonsmokers, whereas the frequency of adenocarcinoma is lower in smokers than in -8- I I I I

t I I I I nonsmokers. These results suggest that smoking may be correlated with an increased incidence of squamous cell carcinoma. (c) Occupational Exposure and Lung Cancer Cell TjMe. These results are shown in Table 18. In males, occupations requiring significant exposure to toxic substances showed a greater proportion of squamous cell carcinoma lung cancer than adenocarcinoma. No difference was observed between the two cell types in occupations lacking such exposures. Such effects were not observed in females. (d) The Relationship between Lung Cancer Cell Type and Indoor (coal fume exposure) or Outdoor (houses surrounded with pollution sources) Air Pollution. These results are shown in Table 19. No difference in cell types was observed between the "exposed" and "nonexposed" groups in both males and females, except that an increasing trend of squamous cell carcinoma was noted in females exposed to coal fumes (p<0.05). Table 20 provides a comparison and a summary of the lung cancer associated risk factors in males and females. Discussion I 1 I I Our studies clearly show that major differences exist between males and females with respect to risk factors for lung cancer. In males, cigarette smoking and occupational exposure are important considerations, whereas in females, indoor air pollution appears to be most significant. A similar conclusion has been reached in numerous epidemiological investigations carried out in China. Although these studies have provided important leads with respect to factors that could play a significant role in the etiology of lung cancer, it is necessary to emphasize that the epidemiological clues must be supplemented with laboratory investigations in order to unequivocally confirm their biological plausibility and to further show that they are at least mechanistically compatible with the pathogenesis of lung cancer. 1. Cigarette Smoking Cigarette smoking is widely accepted as a major risk factor for lung cancer in males. In our studies, 93% of the males have a history of smoking. The calculated relative risk (RR) of smoking for male lung cancer was 3.54(95% CI=2.44-5.11) (p<0.001). In females, the RR was 1.93 (95% CI =1.30-2.87)(p < 0.01). Numerous studies have focussed on the chemical composition of mainstream and sidestream tobacco smoke.(13) Among the 108 chemicals that have been identified, 2-naphthylamine and 4- aminobiphenyl are considered by IARC (International Agency for Research on Cancer) to be human carcinogens.(14) Benzo(a)pyrene, N-nitrosodimethylamine, formaldehyde, and acetamide are suggested to be probable carcinogens, while 1,3-butadiene, nitrosonornicotine, N-nitrosopyrrolidine and indino(1,2,3-cd)pyrene are listed as possible carcinogens. Benzo(a)pyrene has been studied most extensively. A number of studies have shown the concentrations of benzo(a)pyrene to be elevated in the urine of smokers. N O 00 ~ V 9- 00 N CO 00 s I

3. While epidemiological studies have provided some clues to the etiology of lung cancer, they must be complemented with laboratory studies in order to conclusively demonstrate the mechanisms underlying the pathogenesis of lung cancer. I 4. Currently available data do not provide an adequate explanation on the recent global rise in lung cancer incidence. Further studies must be conducted to further elucidate the mechanisms underlying the etiology and pathogenesis of lung cancer. V tb -13- cNp I

As a pro-carcinogen, benzo(a)pyrene is actively metabolized to phenols, quinones, and dihydrodiols by microsotttal enzymes. While no carcinogenic effects have been associated with phenols and quinones, some of the dihydrodiols have been demonstrated to be highly reactive with DNA. We have treated human fetal tracheal epithelial cells (HFTE) with four different dihydrodiols (i.e., anti- BPDE, syn-BPDE, 3-hydroxy-BP, and 9-hydro-BP) and found that cells exposed to anti-BPDE are accompanied by an increase in micronuclei content, an induction of unscheduled DNA synthesis, and point mutations in codon 12 of the H-ras gene.(15, 16) These results are similar to the findings of Kapitulnik et al.,(17) showing that anti-BPDE was capable of inducing lung tumors in mice. Mutations in codon 12 of the H-ras gene have been similarly established in squamous cell carcinoma and adenocarcinoma of the lung. Squamous cell carcinoma and adenocarcinoma constitute 58 % and 23 % of lung cancer in males, whereas in females, the most commonly observed lung cancer is adenocarcinoma (48 %), with squamous cell carcinoma constituting only 29 %. Insofar as the incidence of squamous cell carcinoma is concerned, the 59% and 36% observed in male and female smokers are significantly higher than that observed in nonsmokers (40% in males and 22% in females, p<0.01), suggesting that cigarette smoking is mainly associated with an increased incidence of squamous cell carcinoma. In addition, we observed that the ratio of squamous cell carcinoma to adenocarcinoma in male smokers and nonsmokers is 2.57:1 and 1.14:1, respectively. In females, the ratio of squamous cell carcinoma to adenocarcinoma is 0.89:1 in smokers and 0.39:1 in nonsmokers. Thus, no significant difference can be observed in the incidence of squamous cell carcinoma to adenocarcinoma in nonsmokers (p>0.05), further supporting the role of cigarette smoking in inducing an increase in the incidence of squamous cell carcinoma. In female nonsmokers, 56% of the lung cancer is of the adenocarcinoma type. The incidence of squamous cell carcinoma in smoking females (36%) is slightly higher than the average 29% observed in the general female population, but is still lower than the observed 40% adenocarcinoma in nonsmoking females, suggesting that other factors must account for the high incidence of adenocarcinoma and that the same factor(s) must also somehow counteract the effects of cigarette smoking in inducing squamous cell carcinoma. Analysis of cell types in 1,048 cases of lung cancer deaths show that in males, squamous cell carcinoma is the most prevalent, followed by adenocarcinoma. In females, adenocarcinoma is more prevalent than squamous cell carcinoma. 2. Air Pollution Unlike other tumors, lung cancer is mainly caused by inhalation of carcinogenic substances. In our studies, areas with the highest air pollution index have the most cases of lung cancer deaths, clearly pointing to atmospheric pollution being associated with lung cancer deaths. A similar conclusion was reached by Yu et al.(18) in their studies on mutagenicity of size-fractionated air particles. It is necessary to point out that, although the magnitude of air pollution in an urban setting can be influenced by factors such as population density; the degree of industrialization and development; the source of energy; the quality and quantity of traffic; the geographical location of city and its design; etc., the major source of atmospheric pollution in Chinese cities comes from coal burning in connection with home heating and cooking. Because females stay indoors longer than males, indoor air pollution has been established as a major risk factor for female lung cancer deaths. Such a conclusion has been reached by numerous studies carried out in different parts of China. -10- a I I I I I

I R i I Our studies show that cooking fuel and household coal consumption is a significant risk for female lung cancers (RR= 2.21, 95% CI=1.16-4.21, p<0.01) but not male lung cancers (RR= 0.90, P>0.05). This is further supported by the fact that housewives have the highest SMR. Conditional regression analysis of case-control studies involving nonsmokers show that, whereas the major risk factors for lung cancer in males are occupational exposure to chemicals and a deficiency in fresh vegetable intake, indoor air pollution and the small size of the kitchen are important considerations in females. Previously the lung cancer death rate in 23 major cities in China has been correlated with the coal consumption and the city latitudes. Moreover, a positive correlation was shown to exist in females (r= 0.41-0.49, p< 0.05) and not in males (r =0. 16-0.08, p> 0.05). Since cities located at higher latitudes tend to have longer winters requiring proportionally more home heating, it is further suggested that contact with indoor air pollutants, generated from household coal consumption, is associated with female lung cancer. Many studies in China have analyzed and compared chemicals derived from burning coal with those generated from burning gas and have generally concluded that the concentrations of SO2, C02, CO, NOx, TSP, SD, radon, thoron and B(a)P are much higher in households using coal. Furthermore, the mutagenicity associated with TSP and RSP has been established by laboratory studies. Li et al. (19) have studied organic extracts prepared from inhalable particles derived from coal burning and have compared these to similar extracts derived from wood burning, using both the Ames test and the two-stage skin carcinogenesis test. The results show that inhalable particles generated from burning coal are more carcinogenic and mutagenic. Similar studies and conclusions were also reached by Liang et al.(20) and Guan et al.(21) Our studies show that TSP and SD in kitchens burning coal have concentrations of B(a)P that are significantly higher than those burning gas. Moreover, the concentrations of B(a)P in the urine of housewives are also elevated in coal-burning households, providing direct evidence that B(a)P present in indoor air can be taken up by humans present in such an indoor environment. In addition to burning coal, the concentration of B(a)P is also affected by the type of cooking methods. Investigations carried out in the same kitchens show that, whereas the base values of TSP and B(a)P in indoor air are 107 µg/m3 and 0.41 µg/100 m3, they are elevated to 219 µg/m3 and 0.65 µg/100 m3 when soup is being prepared and greatly increased to 521 µg/m3 and 2.64 µg/100 m3 as meat is stir- fried. These studies show clearly that B(a)P is significantly generated by the method as well as the type of ingredients used during food preparation. Wang et al.(22) in a matched case-control study concluded that cooking oil fumes are a risk factor for lung cancer. 3. Occupational Exnosure Occupational exposure to arsenic, chromium, nickel carbonyl, bis(chloromethyl)ether and chloromethyl methyl ether are known to induce lung cancer. Likewise, the presence of 2-naphthylamine, beryllium, isopropyl oils, mustard gas, and asbestos have been shown to increase the incidence of lung cancer. Underground haematite mining and iron and steel founding are also significant risk factors for human lung cancer. In Guangzhou, the majority of occupational exposure involves nickel, beryllium, isopropyl oil and pollutants present in mines. Very few workers are employed in industries with other potentially carcinogenic substances. In Table 10, all workers who may have potential contact with lung cancer inducing chemicals have been grouped into the category of "Obvious Occupational Contact". The size of this group is likely to be substantially larger than individuals having "significant" and "real"

15. Chen, J.K.; Jin, B.; Yi, F.;, Wu, Z.L. and Du, Y.X. (1992) Measurement of unscheduled DNA synthesis and micronuclei formation in human fetal tracheal epithelium following exposure to BaP metabolites. Manuscript in preparation. I I i I I 16. Zhan, D.J.; Chen, J.K.; Jin, B.; Yi, F.; Wu, Z.L. and Du, Y.X. (1994) Detection of point mutation of codon 12 of H-ras oncogene in human fetal bronchial epithelial cells treated with BPED by polymerase chain reaction. Manuscript in preparation. 17. Kapitulinik, J., et al, (1977) Nature (London) 266, 378. 18. Yu, S.Y., et al. (1991) Study on mutagenicity of size fractionated air particles. Chinese Journal of Preventive Medicine 25(2): 70-74. 19. Li, X.M., et al., (1989) Carcinogenicyt and organic fraction of indoor inhalable particle. Chinese J. Preventive Medicine 23(6): 358-260. 20. Liang, C.K., et al. (1987) Kuming mice skin tumor initiating activity of extracts of inhalable particles in indoor air. The Chinese J. Preventive Medicine 21(6): 316-318. 21. Guan, N.Y., et al. (1990) A study of carcinogenicity of extracts from different size particles in air. The Chinese J. Preventive Medicine 24(1): 9-12. 22. Wang, G.X., et al. (1992) Multivariate analysis of causal factors included cooking oil fume and others in matched case-control study of lung cancer. The Chinese J. of Preventive Medicine 26(2): 89-91. 23. Zhang, P.C., et al. (1990) The determination of methylated purines (06-mGua, m7Gua) in liver and lung of mice. Chinese Journal of Preventive Medicine 24(3):136-138 24. Heinonen, O. P., (1994), N. Engl. J. Med. 330:1029-1035 ~ ' ~ ~ N O tb _L -4 - 15 - tb cNp , O I V

Table 9. Distribution of 5,546 Lung Cancer Deaths By Occupation in Gunagzhou, China (1980-1988) Job Male Female No. % No. % Office worker 884 21.1 129 6.5 Salesclerk 206 4.9 102 5.2 Engineer 116 2.8 6 0.3 Teacher 82 2.0 68 3.4 Waiter/Waitress 79 1.9 38 1.9 Doctor 55 1.3 33 1.7 Others 103 2.5 25 1.3 Farmer 69 1.6 40 2.0 Homemaker 37 0.9 494 25.0 Cargo Handler 400 9.5 68 3.4 Construction Worker 315 7.5 38 1.9 Machinist 299 7.1 38 1.9 Chemist 265 6.3 161 8.2 Cook 190 4.5 73 3.7 Driver 167 4.0 10 0.5 Foundry Worker 129 3.1 40 2.0 Handicrafter 121 2.9 125 6.3 Stoker 59 1.4 9 0.5 Lathe Operator 55 1.3 35 1.8 Other Worker 560 13.4 444 22.5 Total 4191 100.0 1976 100.0 -20- I I I I I I I I I I I I I I I

References 1. Huang, S.H. (1988) A survey on cigarette smoking in Guangzhou resident. Acta Academiae Medicine Guangzhou 16: 6-13. 2. Du, Y.X., et al. (1988) The occupational analysis of lung cancer deaths in Guangzhou. Acta Academiae Medicine Guangzhou 17(1): 69-74. 3. Du, Y.X., et al. (1991) Atmospheric pollution and human lung cancer. Lung Cancer Vol. 7 (supplement) P.2 4. Du, Y.X., et al. (1990) Indoor air pollution and woman lung cancer. The Fifth International Conference on Indoor Air Oualitv and Climate. Vol. 1: 59-64. 5. Gao, Y.T., et al. (1987) A case-control study of female lung cancer in Shanghai. Guangzhou Second Svmposium on Lung Cancer Research. P. 7 6. Dai, X.D., et al. (1991) The risk factors for lung cancer in women. Lung Cancer, Vol.7 (supplement) P.3. 7. He, H.Z., et al. (1991) A case-control study on risk factors of lung cancer. Lung Cancer, Vol. 7 (supplement) P.7. 8. Huang, L.F., et al (1991) Trend study of global atmospheric monitoring in Guangzhou, Guangzhou Health and Antiepidemic Station, Guangzhou, China. 9. Yan, L.Y. (1988) Application of the remote sensing technique in the study of the vegetation ecoline and air pollution in the city of Guangzhou, Environmental Monitoring Center, Guangzhou, China. 10. Du, Y.X., et al. (1979) The summation index of atmospheric quality, Information of Guangzhou Medical College 4, 10-16. 11. Wu, Z.H. (1987) The investigation of the indoor and outdoor concentration of 222Rn, 220Rn, and their daughters in Guangzhou city. Guangzhou Second Svmposium of Lung Cancer Research, P.34 12. Li, X.M., et al. (1985) Mutagenicity of total suspended particles from five large cities of China. Journal of the Institute of Health 14:23-26. 13. Eatough, D.J., et al. (1989) The chemical characterization of environmental tobacco smoke. Proceeding of the International Symposium at McGill University P. 3-39. 14. IACR Monograph on the Evaluation of Carcinogenic Risks to Humans (1987) supplement 7: 17- 74. -14- i I

Table 1. Trend of World Age-Adjusted Death Rate of the Five Leading Carcinomas in Guangzhou, China (1976-1989) Carcinorna Sex Regression Equation Regression Coefficient P-value Lung M y=1.472x-2870.7 1.4720 <0.001 F y=0.4199x-811.4 0.4199 <0.01 Liver M y=0.3454x-647.3 0.3454 >0.05 F y=0.0849x-158.4 0.0849 >0.05 Stomach M y=-0.0957x+200.9 -0.0957 >0.05 F y=-0.0255x+57.7 -0.0255 >0.05 Nasopharynx M y=-0.3309x+668.5 -0.3309 <0.05 F y=-0.1572x+316.7 -0.1572 <0.05 Esophagus M y=-0.3355x+676.3 -0.3355 <0.01 F y=-0.1164x+223.7 -0.1164 <0.05 Table 2. Correlation Between Atmospheric Pollution and Lung Cancer Death Rates in the Four Districts of Guangzhou Liwan Yuexiu Dongsban Haizhu Control AP Index (1972-1979) 2.49 1.68 117 1.64 0.57 AP Index (1982-1990) 0.898 0.721 0.47 - 0.246 Pollution statusa (1984) Severe/ heavy Heavy/ medium Mediutn/light Medium/light Clean Mutagenicity of TSPb 7600 6600 6100 - - LC Death Rate (1976-1987) 37.94 35.99 30.79 31.5 <20.00 a b Pollution status was monitored by remote-control aerial sensors, Mutagenicity was ascertained by Ames test and is based on the number of reverse colonies/100 m3. I I 1 -16- I

contact with cancer inducing agents. Thus, lung cancer cases which are truly attributed to occupational exposure should comprise no more than 15 % of the total lung cancer cases, which is substantially below the numbers observed in most highly industrialized cities. Judging from occupational SMR, the incidence of lung cancer in blue collar workers is significantly higher than office workers and professionals, suggesting that the probability of occupational exposure to cancer inducing substances does play a part in lung cancer development. 4. Diet and Nutrition Some epidemiological and laboratory studies show retinoids to play an important role in preventing the development of lung cancer. Doll(23) proposed that the risk for developing squamous cell carcinoma in lung cancer cases is significantly increased when smoking is combined with a deficiency of vitamin A. Our case-control studies show that vegetable intake is a protective factor for lung cancer. In laboratory investigations, preliminary experiments using bronchial epithelial cells show that a deficiency of retinoids in the culture media is accompanied by squamous cell transformation simultaneous with increased B(a)P-DNA adduct formation. Both cellular and molecular changes can be readily reversed by the addition of retinoids. Since fresh vegetables and fruits are readily available in Guangzhou, vitamin A deficiency is unlikely, except in rare cases of individuals having extreme food habits. However, conflicting results were reported by O.P. Heinonen et al.(24). In a randomized, double-blind placebo-controlled study performed on 29,133 male smokers supplemented daily with alpho- tocopherol (50 mg per day) and beta-carotene (20 mg per day) for five to eight years, no reduction in the incidence of lung cancer was observed. The possibility was also raised that these supplements may actually have harmful as well as beneficial effects. Conclusion 1. Major differences exist between males and females with regard to lung cancer risk factors. In males, cigarette smoking and occupational exposure play an important role, whereas in females, indoor air pollution, derived from cooking fuel and household coal consumption, is more important. These risk factors were confirmed in case-control studies. Moreover, exposure to ETS, a history of respiratory disease, and general living conditions were not risk factors for nonsmoking females. In nonsmoking males, contact with toxic substances and occupational exposure were risk factors. intake of vegetables is a protective factor for lung cancer in both males and females. 2. In terms of the cell types associated with lung cancer deaths, a significant difference was found between males and females. In males, squamous cell carcinoma constitutes the major type, whereas in females, adenocarcinoma is by far the most frequent cell type. When the effects of cigarette smoking are excluded, the frequency of adenocarcinoma shows an increase in both males and females and is more clearly demonstrated in the latter. In smoking females, the proportion of squamous cell carcinoma is lower than that of adenocarcinoma, suggesting that some factors must exist contributing to the high incidence of adenocarcinoma. I I I I -12-

Table 3. Average Indoor and Outdoor Levels of SOZ, NOX, TSP, and B(a)P in Guangzhou (1984-1985) I I I I SO2(kgIM3) NOX(µg/M3) TSP(fegJM3) B(a)P(µg/100M3) Indoor 190±80 70±30 210±70 1.30±0.98 Outdoor 80±20 40±10 200±30 0.50±0.26 Table 4. Daily Seasonal Changes in Indoor SOZ(µg/M3) and NOX (µg/M3) in Guangzhou, China (1984-1985)a Time (Hr) 7:00 9:00 11:00 13:00 15:00 17:00 19:00 S02 Spring 163 145 168 157 167 216 231 Summer 144 118 131 123 131 157 174 Autumn 174 141 167 135 110 179 173 Winter 251 217 262 235 231 342 420 NOX Spring 63 72 73 61 69 80 79 Summer 67 64 64 55 51 73 70 Autumn 73 67 73 60 54 73 69 Winter 106 95 96 78 80 118 153 a Twenty families were included in the survey. -17- I

Table 7. Influence of Construction Materials on Indoor Radioactivity Levels I I i I I I I I I I I I Wall Floor Radon (BqlM3) Tltoron (Bq/M3) Green Brick Brick 18.6 t 4.7 47.3 t 16.2 Cement 13.8 23.5 Red Brick Brick 18.4 t 3.8 42.3 t 16.1 Cement 17.8 f 4.8 29.6 t 14.5 Table 8. A Comparison of Radioactivity Levels Between Coal-Burning and Gas-Burning Kitchens Fuel Radon (Bq/M3) Thoron (Bq/M3) Coal-burning 18.6 ± 4.1 42.5 ± 19.9 Gas-buming 16.6 ± 5.1 28.3 ± 13.3 P-value >0.05 <0.01 N 0 ~ V 00 N -19- i t

Table 5. A Comparison of the Concentrations of TSP, TSP-B(a)P, SD, SD-B(a)P and Urine B(a)P Between Coal-Burning and Gas-Burning Kitchens (1986-1987) Briquette Coal- Burning Kitchen Liquefied Petroleum Gas- Burning Kitchen Totaf suspended No. ± D No. ± SD P-Value Particulate (µg/M3) 37 322±131.0 27 188.0±6.70 <0.01 TSP-B(a)P (µg/100 M3) 21 11.9±9.3 21 2.2±1.8 <0.01 Sedimentary dust(gm1M2/month) 37 11.9±8.4 24 5.4±2.9 <0.01 SD-B(a)P(p.g/M2/month) 28 11.1t8.4 12 2.2±1.7 <0.01 Housewives urine-B(a)P (ng/1) 24 4.0±1.8 20 2.8t1.5 <0.05 I di Table 6. A Comparison of Indoor and Outdoor Levels of Radon, Thoron and Their Daughters in Guangzhou (1984-1985) Radon (Bq/M3) Radon Daughter (10-8 UM3) Thoron (BqfM3) Thoron Dau hter (10-8 I/M~) Indoor 17.8t2.1 5.84f0.72 37.0±7.2 6.94t1.06 Outdoor 13.3t2.1 4.86t0.33 14.5t2.6 4.72t0.62 GB4792-84 3300.00 19.0 75.0 57.0 11 - 18 _ t ! I I I I

1 I I I I I I I I I I I Table 10. A Comparison of Exposure Levels in Males and Females Among 5,546 Lung Cancer Deaths in Guangzhou, China (1908-1988) Enposure Job Male Female No~_ ___-__~ _ _ __ Na No. % No. % % Chemist 265 7.5 161 10.7 Obvious Cook 190 5.4 514 14.6 73 4.8 243 16.1 Stoker 59 1.7 9 0.6 Cargo Handler 400 11.3 68 4.5 Construction 315 8.9 38 2.5 Worker Probable Machinist 299 8.5 1486 42.1 38 2.5 254 23.5 Driver 167 4.7 10 0.7 Foundry Worker 129 3.7 40 2.7 Handictafter 121 3.4 125 8.3 Lathe Operator 55 1.6 35 2.3 Office Worker 884 25.1 129 8.6 Sales Clerk 206 5.8 102 6.8 Engineer 116 3.3 6 0.4 None Teacher 82 2.3 1528 43.3 68 4.5 910 60.4 Waiter/Waitress 79 2.3 38 2.5 Doctor 55 1.6 33 2.2 Farmer 69 1.9 40 2.6 Homemaker 37 1.0 494 32.8 Total 3528 100.0 1507 100.0 _21_ I

2081783002

Table 14. Relationship Between Smoking History and "Occupation SMR" in Lung Cancer Deaths Male Female Occupation Rank Smoker Nonsmoker Occupation Rank Smoker Nonsmoker Office Worker 12 11 11 Homemaker 12 11 11 Cargo Handler 11 13 6 Office Worker 10 8 9 Construction 10 9 6 Chemist 10 8 9 Machinist 9 8 5 Salesclerk 9 8 8 Chemist 8 7 4 Cargo Handler 8 7 5 Salesclerk 7 6 4 Teacher 7 6 6 Cook 6 5 2 Cook 6 5 5 Engineer 5 4 3 Waitress 5 3 4 Teacher 4 2 1 Construction 4 3 1 Waiter 3 2 2 Machinist 3 2 1 Homemaker 2 I 0 Doctor 2 1 1 Doctor 1 0 0 Engineer 1 0 0 K 68 44 62 60 P value <0.01 >0.05 <0.01 <0.01 -24- 1 I I I I I

I I I I 1 Table 15. Relative Risks and Estimated Death Rates for Smoking and Coal Fumes Exposurea PART A RRb (95% CI) P-value Male Smoking 3.53 (2.44-5.11) <0.001 Coalfumesexposure 0.89 > 0.05 Female Smoking 1.93 (1.30-2.87) <0.01 Coal fumes exposum 2.21 (1.164.2t) <0.01 PART B RR° (95% CI) Estimated death Amibutable death Audbuted rare (100,000) rate (100,000) risk Male Non-sm + Non-ex 0.26 13.6 - - Smoking + Non-ez 5.15 (2.77-9.57) 68.5 55.2 4.15 Non-sm + exposed 1.50 (0.69-3.27) 19.9 6.6 0.50 Smoking + exposed 4.29 (2.33-7.88) 56.8 43.2 3.29 Female Non-sm + Non-ex 0.50 12.3 - - Smoking + Non-cx 1.00 12.3 0.0 0.0 Non-sm + exposed 1.56 (0.574.25) 19.2 6.9 0.56 Smoking + exposed 2.89 (1.09-7.65) 35.5 23.2 1.89 a A case-control study involving 849 cases of lung cancer deaths b Obtained using the method of Mantel-Haenszel c Obtained by stratification analysis w , L7 0 00 I ~ V 00 -25- N eC I c0 a V

I 1 I I I I I I I I I I Table 18. The Relationship Between Occupation SMR and Lung Cancer Cell Type Malt Pemale Occupation Squamous Cell Ca. Adcn. Ca. Squamous Cell Ca. Aden. Ca. Machinist 613 190 - 400 Chemist 597 500 250 476 Cook 525 69 282 395 Cargo handler 423 218 333 820 Consvuction 414 226 256 238 Office Worker 160 179 142 137 Salesclerk 196 48 155 270 Teacher 78 185 213 198 Doctor 191 169 - 103 Engineer 92 178 - 130 WaitedWaitress 48 - 88 Homemaker 85 - 142 137 N O O i -4 tb N -27- ~ tD I

I Table 19. Air Pollution (Indoor and Outdoor) and Lung Cancer Cell Type Cell Type Male Coal Fumes Exposure Female Coal Fumes Exposure Yes No Yes No No. % No. % No. % No. % Squamous Cell Ca. 273 56.76 177 60.00 73 27.76 10 50.00 Small Cell Ca. 39 8.11 17 5.76 22 8.37 1 5.00 Adeno Ca. 116 24.12 69 23.39 131 49.81 7 35.00 Large Cell Ca. 8 L66 7 2.37 2 0.76 0 0.00 Others 45 9.36 25 8.48 F 13.31 2 10.00 Total 481 100.00 295 100.00 26 100.00 20 100.00 X2 - 2.3998 P>0.05 X2 = 3.9398 Cell Type House Surrounded With Palludon Source House Surrounded With Pollution Source Yes No Yes No No. % No. % No. % No. % Squamous Cell Ca. 147 59.76 278 57.68 22 25.58 52 29.05 Small Cell Ca. 20 8.13 34 7.05 8 9.30 14 7.82 Adeno Ca. 59 23.98 lll 23.03 42 48,84 91 50.84 Large Cell Ca. 0 0.00 12 2.49 1 1.16 1 0.56 Others 20 8.13 4-, 9.75 13 15.12 21 11.73 TOTAL 246 100.00 482 100.00 86 100.00 179 100.00 X2 = 7.0280 P>0.05 X2 = 0.9687 28_ I I I I I I i I I I I I I

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I Table 16. Comparison of Cell Types Between Smoker and Nonsmoker Among 1,093 Lung Cancer Death Cases Male Female Cell Type Smoker Nonsmoker Total Smoker Nonsmoker Total No. % (1) (2) (3) (4) (5) (6) Squamous Cell Ca. 438 59.03 25 40.32 463 57.59 51 36.17 33 22.30 84 29,07 Small Cell Ca. 56 7.55 2 3.23 58 7.21 12 8.51 12 8.11 24 8.30 Adeno Ca. 170 22.91 22 35.48 192 23.88 57 40.43 83 56.08 140 48.44 Large Cell Ca. 14 1.89 1 1.61 15 1.87 1 0.71 1 0.67 2 0.69 Others 64 8.62 12 19.36 76 9.45 20 14.18 19 12.84 39 13.50 Total L 742 100.00 62 100.00 804 100.00 141 100.00 148 100.00 289 100.00 Squamous Cell carcinoma: (1) (2) P<0.01; (4) :(5) p<0.01 Adcnocarcinoma : (1) : (2) p<0.05; (4) : (5) p<0.01 Table 17. Relationship Between Smoking Index and Lung Cancer Cell Types Among 845 Cases (M: 712, F: 133) Male Smoking Index Cases Squamous Small Cell Adeno Ca. Large Cell Ca. Others Cell Cancer Cancer >200 21 33.33 4.76 52.38 0.00 9.53 200- 65 50.77 12.31 20.00 4.61 12.31 400- 79 50.63 5.07 35.44 0.00 8.86 600- 547 63.25 7.68 19.38 1.83 7.86 Female < 200 16 18.75 12.50 50.00 0.00 18.75 200- 23 39.13 13.05 34.78 0.00 13.04 400- 23 43.48 4.35 43.48 0.00 8.69 600- 71 38.03 8.45 39.44 1.41 12.67 Male(4 x 2) EC: p<0.01 AC: p<0.0001 Female (4x2) EC: p>0.05 AC: p>0.05 I ' I I I -26- I I

I Table 11. Crude Death Rate and Standard Mortality Ratio (SMR) of Lung Cancer by Occupation in Guangzhou, China (1982) No, of Popslaqooa No. of Deaths6 Dash Raros (per 100,000) SMRC Oo.vpatim Tmel M F Tadt M F Tobl M F Tad3 M F Homemeker 72377 5345 66032 80 6 74 112.1 112.3 112.1 920+ 328 1078+ Clicmitl 39549 17757 21792 42 26 16 106.2 146.4 73.4 866+ 880+ 842+ MachinA 46533 31454 3509 43 41 2 924 103.4 13.3 694+ 769+ 294 CargoHandler 36566 29042 7524 44 37 7 120.3 127.4 93.0 530+ 490+ 1051+ Cook 27339 12348 14991 29 23 6 106.1 186.3 40.0 509+ 588+ 335+ Canshuctlon 30071 19774 10297 26 26 0 86.5 131.5 0 417+ 491+ 0 Sales Clerk 50319 17363 32956 34 21 13 67.6 121.0 39.5 319+ 300 360+ OtTrce Worker 123803 93455 30348 93 79 14 75.1 84.5 46.1 166+ 155+ 266+ Teachcr 32628 14632 17996 13 5 8 39.8 34.2 44_5 142 73 352+ Engineer 43294 33426 9868 14 13 1 32.3 38.9 I0.I 100 100 120 Docior 27256 7796 19460 3 2 1 11.0 25.7 5.1 58 66 47 WairerlWairrest 82367 27261 55106 13 7 6 I5.8 25.7 10,9 51 45 73 Taal 611102 309653 30M49 434• 286 1" 71.0 92.4 49.1 Thcre wcrc 601 cucs of lung cancer dcaihv m 1982. Thc occupations of orficr 167 deaW arc drWer. IuMicrafmrsmker. ~exWc workcr. fomdry worker, Imhe offerabq PriN[q falrr6c.... ar. Table 12 The Population Attributable Risk (PAR) for 12 Occupations in Guangzhou, 1982. Age las been sbrtlartlaad ro 5MR 100 for Ihe total popdaricm of Gwog>JSOU. P<0.01 au om r«nsus o eop e s eya ¢ o Dan from Ihe deah cause registry of Guangrlsou. 1982 SMR - No. of obscrvcd deaihs No. of expecred dcadu. Job PAR (%) Job PAR (%) Homemaker 9.9 Salesclerk 3.11 Office Worker 9.64 Construction 2.8 Cargo Handler 5.51 Teacher 0.46 Chemist 5.02 Engineer 0.07 Machinist 4.84 Doctor -0.94 Cook 3.44 Waiter/Waitress -2.25 _22_ I I I I I I I I I I

i I I I I I I I I I I I A I I I I I Table 20. Comparison of Risk Factors for Lung Cancer in Males and Females Male Smoking rate among Guangzhou residents Smoking rate among lung cancer deaths Coal fumes exposure rate among LC deaths Cigarette per day among LC deaths Smoking index among LC deaths RR for smoking RR for coal fumes exposure RR for husband smoking (nontumor controls) RR for husband smoking (mmor controls) LC age-adjusted death rate (1985) Estimated death rate for smoking Estimated death nte for coal fumes exp. Correlation between LC death rate and cigarette sales Correlation between LC death rate and coal consumption of 23 Chinese cities Correlation between LC death rate and latitude of 23 Chinese cities The variates had been introduced in conditional logistic regression equation, case-control study on nonsmoker The highest occupation SMR Cell type Squamous Cell Ca. Adeno ca. Nonsmoker Smoker Smoking index and cell type Comparison of cell type constituent ratio between ETS and non-ETS among nonsmoker Comparison of cell type constituent ratio between coal fumes exposure and nonexposure 43 % 93 % <28% Md=25.6 Md=670 3.54, P<0.001 0.90, P>0.05 52.2 68.5 19.9 r=0.88, P<0.01 r=0.16, P>0.05 r=0.08, P>0.05 Low vegetable intake Chemical exposure Chemist 57.6 % 23.8 % EC = AC EC > AC X2=13.8, P<0.01 X2=0.66, P>0.05 X2=2A0, P>0.05 -29- Female 4% 93 % >80% Md=20.9 Md=632 1.93, P<0.01 2.21, P<0.0t 1.19, P>0.05 1.00, P>0.05 24.7 12.3 19.2 r=0.71, P>0.05 r=0.41, P<0.05 r=0.49, P<0.05 Low vegetable intake Chemical exposure Indoor air pollution Kitchen area narrow Homemaker 29.1 % 48.4 % EC < AC EC = AC X2=278, P>0.05 X2=1.34, P>0.05 X2=3.92, P>0.05 N O O ~ V O W O 0 ~ I

i I I I 1 I I I I I I I I I i I a I ASPECTS OF THE EPIDEMIOLOGY OF LUNG CANCER IN SMOKERS AND NONSMOKERS IN THE UNITED STATES Geoffrey C. Kabat Albert Einstein College of Medicine Bronx, New York, USA Abstract While it is well-established that smoking is the predominant risk factor for lung cancer, it is clear that factors other than smoking and occupational exposure must play a role in some lung cancers, and particularly adenocarcinoma. Data from a large, hospital-based case-control study are used to examine the association of smoking-related risk factors (amount smoked, filter status, mentholation, and differences in smoking habits between blacks and whites) and selected factors other than smoking (environmental tobacco smoke, previous primary cancer and radiotherapy, reproductive and endocrine factors, and body mass index) with lung cancer. Although smoking shows a dose-response relationship with all major lung cancer cell types, the strength of the relationship is weaker for adenocarcinoma, suggesting that other risk factors must play an important role for this cell type. In both blacks and whites of both sexes, odds ratios for lung cancer increased with increasing cumulative tobacco tar intake and decreased with years since quitting smoking. Use of mentholated cigarettes was not associated with lung cancer, relative to use of nonmentholated cigarettes. Exposure to environmental tobacco smoke in childhood was associated with a doubling of the odds ratio in female never smokers; however, exposure to a husband's smoking showed no increase in risk. A history of a reproductive primary and a history of radiotherapy were each associated with a four-fold increase in risk in female nonsmokers. An association of lean body mass with lung cancer was observed in current smokers, ex-smokers, and female never smokers. These results are discussed in the context of existing studies. In conclusion, variation in lung cancer rates between populations may be due to: 1) differences in effective exposure to tobacco smoke carcinogens, 2) differences in exposure to other independent risk factors for lung cancer, or 3) differences in factors which modify the effect of tobacco smoke, including differences in host susceptibility and metabolism of carcinogens. The Magnitude of the Problem Introduction Lung cancer, which in the early twentieth century was a rare tumor in the United States, is now the leading cause of cancer death in the United States and has recently become the most common tumor worldwide (1,2). The American Cancer Society estimated 170,000 new lung cancer cases in 1993, accounting for 17% of cancer incidence in males and 12% in females, and 149,000 deaths from lung cancer, or 34% of cancer deaths in males and 22% in females (1). Between 1930 and 1989 the lung cancer mortality rate increased from 4 to 73 per hundred thousand in males and from 3 to 30 in females (1). In recent years the lung cancer death rate in males has begun to level off and decline, but in females it has continued to rise at a steep rate. During this I

I I I  I I I I I I I I I I It should also be pointed out that although a previous history of a tobacco-related cancer was inadmissible, a previous history of other, nonsmoking-related tumors was acceptable. The age range of subjects was 20-80. All subjects were interviewed in-person in the hospital by trained interviewers using a standard questionnaire containing a detailed history of tobacco use and questions on usual occupation and occupational exposures, alcohol consumption, demographics, and other factors. Within this large study, a substudy of lung cancer in lifetime nonsmokers was initiated in 1983 with a primary focus on environmental tobacco smoke. In the substudy, for each nonsmoking lung cancer case interviewed, 3 control patients who were lifetime nonsmokers and were matched on age (± 5 years), sex, race, hospital, and date of interview (within 2 months) were sought. Subjects were considered lifetime nonsmokers if they had never consumed as much as 1 cigarette per day for a year, or had smoked fewer than 365 cigarettes over their lifetime. The inclusion of detailed questions regarding the initiation of smoking early in life and amount smoked provides a basis for excluding ex-smokers who quit decades prior to diagnosis but have smoked more than this minimum amount. In the substudy, detailed questions were asked about exposure to other people's smoking in childhood in the home, in adulthood both at home and at work, and in social situations and transportation. For each smoker in the household and for each job held for at least one year, subjects were asked about the intensity and duration of the exposure. Exposures were examined individually and in the aggregate for different periods of life. Over the period 1969-1991, the questionnaire was modified a number of times (there are 4 major distinct versions in this time period). While some items remained constant (core smoking history items), others were amplified and new items were added. For this reason, in the analyses I will present, the time period will vary for different analyses. For the period 1985 to 1991, a number of questions were added to the questionnaire. These included: a brief food-frequency questionnaire designed to estimate intake of dietary fat and of vitamin A; a prior history of radiotherapy (yes/no), the site treated, the diagnosis requiring treatment; and a history of (a nonsmoking related) cancer. Results Amount Smoked Smoking-Related Factors . In an analysis of 2,085 male and 1,012 female lung cancer cases and two times as many matched  controls, we noted a dose-response relationship with level of smoking for Kreyberg I (including squamous cell, small cell, and large cell lung cancers) and Kreyberg II (including adenocarcinoma, bronchiolar, and ' alveolar cell carcinoma) in current smokers of both sexes (7). Odds ratios were consistently higher for Kreyberg I(Ki) compared to Kreyberg II (KII). The OR for smokers of 41 + cigarettes per day reached 64.1 for KI compared to 28.4 for KII in males. The corresponding ORs in females were 88.7 and 20.1 N ~ (Table 1). O O ~ -4 tp ~ -3- W O tn I

  ' 4. I I 5. I 6. I 7. I I I I ~ ~ ~ ' I I I Adenocarcinoma appears to be the most common cell type in female nonsmokers. This is observed both in the West and in China and Japan. Risk factors for lung cancer in nonsmokers remain to be elucidated. Environmental tobacco smoke and radon are unlikely to explain the majority of these cancers, which account for approximately 10,000 cases per year in the United States. While there is some suggestive evidence of an association of reproductive or endocrine factors with lung cancer, this relationship needs to be characterized in much greater depth. There is a need for studies of gene-environment interactions. For example, it has recently been reported that PAH-DNA in 63 male smokers were inversely associated with serum vitamin E levels. Furthermore, the association between adducts and vitamin E was significant in subjects lacking the gene for the detoxifying enzyme glutathione S-transferase M1 (GSTM1) but not in those with the gene present (59). Comparisons of the histology of lung cancer and time trends, as well as differences in exposure patterns (both to tobacco and other lung cancer risk factors) between the United States and China could be highly instructive. 1

Among males, the proportion of never smokers was 2% for Kreyberg I and 5 % for Kreyberg II and among females the proportion of never smokers was 6% and 19%, respectively. Filter Versus Nonfilter Cigarettes Use of filtered cigarettes was associated with reduced risk for Kreyberg I lung cancer in both sexes, with an apparent trend with increased duration of smoking filtered cigarettes (Table 2). Only the odds ratio for male switchers who smoked filter cigarettes for at least 10 years was statistically significant (7). There was little indication of a reduction in risk for Kreyberg H lung cancer. Mentholation Black males have approximately 50% higher lung cancer incidence compared to white males in the United States (8). Furthermore, blacks tend to favor mentholated cigarettes. While combusted menthol does not appear to be carcinogenic, it has been suggested that use of mentholated cigarettes may be associated with a different exposure to tobacco smoke constituents, either because these cigarettes have higher average tar-yields or, additionally, because they are smoked differently, possibly due to menthol's anesthetic properties (9). For this reason, we undertook an analysis of use of mentholated versus nonmentholated cigarettes among current smokers (10). Detailed information on specific brands of mentholated cigarettes was available in our dataset for the period 1985-1990, and therefore the analysis is limited to this period. No significant association was observed between either short-term (1-14 years) or long-term (15 + years) menthol users relative to smokers of nonmentholated cigarettes, after adjustment for other variables. For specific histologic types of lung cancer, there was no indication of an association with menthol usage (Table 3). We also failed to observe a consistent association of mentholated cigarette use with esophageal or oral-pharyngeal cancers (11,12), two other cancers which have a higher incidence in blacks compared to whites. Differences in Smoking Habits Between Blacks and Whites We compared smoking habits of blacks and whites in a total of 23,011 case and control patients enrolled in the American Health Foundation study between 1980 and 1990 (13). Blacks of both sexes among cases and controls were more likely compared to whites to be current smokers and less likely to be ex-smokers. Furthermore, blacks were approximately three times more likely to be light (<20 cigarettes per day) versus heavy smokers (> 20 cigarettes per day). This association did not differ according to cigarette preference, degree of inhalation, or quitting. The association of race and light smoking was present in both current and ex-smokers. Sociodemographic or smoking-related characteristics did not appear to explain this difference in smoking habits. Possible differences in the association of smoking with lung cancer between blacks and whites prompted us to carry out a case-control analysis utilizing all lung cancer cases in these two ethnic groups enrolled in the study between 1979 and 1990. In all four race-sex groups, a dose-response was observed with lifetime tar intake (Table 4). However, the odds ratios for blacks were somewhat higher than those of whites at a given exposure level. For example, the adjusted OR for extreme quartiles of tar intake in males were 4.3 (95% CI 3.6-5.2) in whites and 5.7 (9546 CI 3.0-10.9) in blacks. A reduced risk of lung cancer was seen among quitters compared to current smokers, but the effect of quitting was somewhat -4- I I I I I I I I I I I I I ' '

I I I I I I I I I I I I I I 1 I more pronounced among whites of both sexes (Table 5). The distribution of histologic types, while differing between males and females, was virtually the same in blacks and whites of the same sex. Risk Factors other than Smoking Environmental Tobacco Smoke in Lifetime Nonsmokers In the substudy of lung cancer occurring in lifetime nonsmokers, the majority of cases and controls reported a history of exposure to ETS (i.e. at least 1 year) in childhood or adulthood, either at home or at work. Table 6 gives the distribution of exposure in different settings. Only 7-15% of subjects reported no exposure to ETS (i.e. for at least 1 year) in childhood, adulthood in the home, or at work. In males, the OR was elevated for having a spouse who smoked (1.6, 95% CI 0.7-3.8) (Table 7). Excess risk was limited to those whose spouse smoked 11+ cigarettes per day (OR = 7.5, 95 % CI 1.4-41.4), however the numbers were very small. The OR was elevated for those whose spouse smoked in the bedroom. In females, there was no indication of an association with exposure to spousal smoking, in spite of the larger sample size (OR = 1.1, 95% CI 0.6-1.9). When duration of ETS exposure (smoker-years) was examined (Table 8), females in the highest tertile of smoker-years in childhood had twice the risk of lung cancer (OR = 2.2, 95 % CI 1.1-4.5) and the linear trend across increasing tertiles was significant (p = 0.02). Previous Cancer and Radiotheranv For the period 1985-1990, we had additional questions including previous history of cancer and history of radiotherapy. In male never smokers, neither a history of a previous primary cancer nor a history of radiotherapy was significantly associated with lung cancer, however, the numbers of exposed cases were small (14). In female never smokers, after adjustment for age, education, hospital, ETS exposure, and body mass index, both a history of a reproductive primary and a history of radiotherapy were significantly associated with lung cancer (OR = 4.9; 95% CI 1.4-17.7, and OR = 4.4; 95% CI 1.3-15.1, respectively). When female never smokers cases were limited to 32 cases of adenocarcinoma of the lung, the adjusted OR for having a previous reproductive primary was 4.0 (95 % CI 0.9-17.6) and that for having a history of radiotherapy was 4.3 (95% CI 1.1-16.6) (Table 9). The OR for lung cancer among women who had both a history of radiotherapy and a previous reproductive primary, relative to those who had neither, was 18.8 (CI = 2.2-160.7). In female never smoker cases, the mean age at diagnosis of the first primary was 47 years (SD 12.9) and that of the lung primary was 62 years (SD 14.4). Histologic information was available for only four of the seven reproductive cancers (Table 10). No association of previous primary cancer or of history of radiation treatment with lung cancer was observed among ever-smokers of either sex. When attention was restricted to long-term ex-smokers (those who had quit more than 20 years prior to diagnosis), the adjusted OR for those reporting a previous reproductive primary was 1.3 (95 °fi CI 0.3-7.0) and that for a history of radiation therapy was 3.3 (95 % CI 1.0-11.7). No interaction was observed between radiotherapy and previous cancer in ever- smokers. -5- N 0 Co ~ V 00 Ca 0 0 V I

I I 59. Grinberg-Funes, RA; Singh, VN; Perera, FP et al. Polycyclic aromatic hydrocarbon-DNA adducts in smokers and their relationship to micronutrient levels and the glutathione-S-transferase I M1 genotype. Carcinogenesis (in press). I I I I I I I I I I I I I I I I

t rr . 1950s, which may be associated with inhalation of smaller particles and their deposition in the periphery . of the lung, where adenocarcinomas are thought to arise. I I I i I I I I The weaker association of smoking with adenocarcinoma compared to squamous and small cell carcinomas implies that other factors must play a proportionately greater role in adenocarcinoma. Black-White Differences Our data, and those from other studies, indicate that black smokers are less likely to quit smoking and that those who smoke, smoke fewer cigarettes per day compared to white smokers. Blacks had somewhat higher odds ratios for lung cancer compared to whites at given level of tar exposure and the reduction in the odds ratio associated with quitting was generally smaller in blacks compared to whites; however these differences were modest, and the design of the study precludes a direct comparison of whites and blacks. In a separate study, we found no elevation in lung cancer risk among smokers of mentholated cigarettes compared to smokers of nonmetholated cigarettes. Our results regarding black-white differences in smoking habits are compatible with a greater effect of smoking in blacks compared to whites. This could be explained by use of higher tar cigarettes by blacks. Recent work demonstrates that smokers of mentholated cigarettes have higher serum cotinine levels compared to smokers of nonmentholated cigarettes (personal communication from Dr. Pamela Clark). An alternative explanation of a greater lung cancer "response" in blacks for a given level of tobacco exposure is that the metabolism of nicotine may differ between blacks and whites, since, for a given level of smoking, blacks have been reported to have higher levels of serum cotinine levels compared to whites (27). Another possibility is that independent risk factors for lung cancer or effect modifiers of smoking account for the excess incidence in black males (28). Potential factors include: occupational exposures, diet, and altered genetic susceptibility for lung cancer (29-31). Environmental Tobacco Smoke , While our study was of modest size, we attempted to improve the quality of the exposure information by asking detailed questions about exposure in different settings throughout life. These , included: the average number of hours per day of exposure to different smokers in the household; whether the spouse smoked in the bedroom; and how the respondent rated each exposure. In addition, unlike many of the larger studies, all cases and controls were interviewed in person at the time of . diagnosis, thus eliminating the need to make use of proxy respondents. I Our data show a modest association of ETS exposure in childhood with lung cancer in women, but no suggestion of an association with a husband's smoking. The problem here is that bias could exert a greater role in recall of exposure in childhood than in recall of adulthood, and particularly, spousal exposure. ~ Since adenocarcinoma is the most common form of lung cancer in never-smokers, one would expect that if ETS is a detectable risk factor for lung cancer in nonsmokers that one would see the N _ strongest relationship for adenocarcinoma. Wynder and Goodman (32) have hypothesized that ETS may 00 , i V tb _ '] - 4f O t0 I

I I I I I I 1 I I I I I I I I I Table 1. Association of amount smoked per day with Kreyberg I and II lung cancer by sex. hiales Ifi ICII OR CI OR Cl Never smoked 1.0 -- 1.0 --- 1-10 cpd 13.3 8.4-21.0 2.4 1.3-4.6 11-20 cpd 15.8 10.7-23.4 8.4 5.8-12.2 21-30 cpd 29.6 19. 8-44.2 15.4 10.2-23.1 31-40 cpd 37.7 25.6-55.5 11.1 7.5-16.5 41 + cpd 64.1 43.1-95.2 28.4 18.3-44.0 Fesria(es Never smoked 1.0 - 1.0 - 1-10 cpd 6.6 3.9-11.0 3.1 1.8-5.1 11-20 cpd 18.2 12.4-26.5 4.5 3.2-6.3 21-30 cpd 26.5 16.9-41.5 9.4 6.1-14.4 31-40 cpd 95.2 60.7-149.2 13.4 9.0-21.1 41+ cpd 88.7 52.3-150.3 20.7 11.2-38.2 KI = Kreyberg I HII = Kreyberg II Current smokers only. N O ~ ~ ! O O C.W i -17- ~ I

I period no other major tumor shows an increase in incidence remotely comparable to that of lung cancer, although the decline in stomach cancer is almost as dramatic. Differences in age-adjusted incidence rates for specific histologic types of lung cancer by sex, race, and calendar time period suggest that different histologic types may have different etiologies. In white men in the United States, rates of adenocarcinoma and small cell carcinoma increased over the time period 1969-1988, while the rate of squamous cell carcinoma decreased. In white women, all major types showed an increase (3). Squamous cell carcinoma is still the predominant histological type among males, whereas adenocarcinoma predominates among females (3). Among nonsmokers, the proportion of adenocarcinoma is greater than in smokers (3), and particularly in females, reaching 76% in one large series of lung cancer cases in nonsmoking women (4). While cigarette smoking and specific occupational exposures have been well established as important risk factors for lung cancer, there is increasing recognition among epidemiologists that smoking and occupational exposures cannot explain all of the variation in lung cancer incidence within countries and between countries, and that other factors must play a role either as independent risk factors or as modifiers of the effect of smoking (5,6). Some issues that remain to be elucidated include: 1) the high rates of lung cancer in Chinese women, who have a low prevalence of smoking; 2) the higher incidence of lung cancer in black American males in the United States; 3) etiologic factors for adenocarcinoma other than smoking; and 4) risk factors for lung cancer in lifetime nonsmokers; and 5) factors that account for the increase in the incidence of adenocarcinoma. In this paper, I will focus on a number of risk factors for lung cancer, including certain aspects of smoking (amount smoked, filter use, mentholation, and black-white differences in smoking habits) and the role of certain factors other than smoking (including environmental tobacco smoke (ETS), a history of radiotherapy and of previous cancers, reproductive and endocrine factors, and body mass index). In addition to presenting data from our studies, I will refer to other relevant studies and attempt to suggest directions for further research. Methods Since 1969 the American Health Foundation has been conducting an ongoing, hospital-based, case-control study of tobacco-related cancers in a number of U.S. cities. The objectives of the study were to examine in depth the role of smoking in its various parameters as well as other environmental and personal factors in the etiology of cancers of the lung, larynx, esophagus, oral cavity and pharynx, bladder, kidney, and pancreas. Cases enrolled in the study were newly-diagnosed, histologically-confirmed primary cancer of the above-mentioned sites. For each case, a hospitalized control was sought who was matched on age (±5 years), sex, race, hospital, and date of admission. Controls were patients with conditions not thought to be associated with smoking. Thus, smoking-related malignancies as well as nonmalignant smoking-related conditions (such as chronic bronchitis, emphysema and peripheral vascular disease) were excluded. However, controls and cases could have a history of nonmalignant smoking-related conditions. -2- I I I I I I I  I I I I I

References Boring, CC; Squires, TS and Tong, T. Cancer Statistics, 1993. CA Cancer J. Clin. 43:7-26, 1993. 2. Parkin, DM. Trends in lung cancer incidence worldwide. Chest 96:5S-8S, 1989. 3. Devesa, SS; Shaw, GL and Blot, WJ. Changing patterns of lung cancer incidence by histological type. Cancer Enidemiol. Biomarkers Prev 1:29-34, 1991. 4. Fontham, ET; Correa, P; Reynolds, P et al. Environmental tobacco smoke and lung cancer in nonsmoking women: A multicenter study. J. Amer. Med. Assoc. 271:1752-1759, 1994. 5. Axelson, 0; Davis, DL; Forestiere, F et al. Lung cancer not attributable to smoking. Ann. NY Acad. Med. 609:165-178, 1990. 6. Kabat, GC. Recent developments in the epidemiology of lung cancer. Sem. Surg. Oncol. 9:73- 79, 1993. 7. Kabat, GC and Wynder, EL. The effect of low-yield cigarette smoking on lung cancer risk. Cancer 62:1223-1230, 1988. 8. Ries, LAG; Hankey, BF and Edwards, BK. Cancer Statistics Review, 1973-1988. National Cancer Institute. NIH Pub. No. 91-2789, 1991. 9. Sidney, S; Tekawa, I and Friedman, GD. Mentholated cigarette use among multiphasic examinlees, 1979-86. Am. J. Public Health 79:1415-1416, 1989. 10. Kabat, GC and Hebert, RJ. Use of mentholated cigarettes and lung cancer risk. Cancer Res. 51:6510-6513, 1991. 11. Hebert, JR and Kabat, GC. Menthol cigarette smoking and oesophageal cancer. Int. J. Enidemiol. 18:37-44, 1989. I I I I I I I I I 12. Kabat, GC and Hebert, JR. Use of mentholated cigarettes and oropharyngeal cancer. ' Epidemiology 5:183-188, 1994. 13. Kabat, GC; Morabia, A and Wynder, EL. Comparison of smoking habits of blacks and whites in a case-control study. Am. J. Publ. Health 81:1483-1486, 1991. 14. Kabat, GC. Previous cancer and radiotherapy as risk factors for lung cancer in lifetime nonsmokers. Cancer Causes and Control 4:489-495, 1993. I I 15. Knekt, P; Heliovaara; Riisanen, A et al. Leanness and lung cancer. Int. J. Cancer 49:208-213, N 1991. oojo, ~ -12- o' ~ A I

I r I I I ~ I I I I I I I I I I I I An alternative explanation compatible with the results reported here is that endocrine factors may play a role in the development of lung cancer. The fmding of an association of a previous reproductive primary with lung cancer in female nonsmokers is consistent with reports, based on tumor registry data and one prospective study, indicating an increased incidence of second primaries of the lung in women who had a first primary of the breast, endometrium, or other reproductive sites (46-48). Only one of those studies took smoking history into account. Annegers and Malkasian (48) noted that six cases of lung cancer (compared with 1.2 expected) occurred among 526 patients with endometrial cancer who were followed for at least 10 years. The mean interval between diagnosis of endometrial cancer and lung cancer was 14.8 years, comparable to that observed in our data (15 years; see Table 10). All five of the six lung cancer cases who had adenocarcinoma were nonsmokers. The possibility that endocrine factors may play a role in the development of lung cancer is raised by: (1) the greater proportion of nonsmokers and of adenocarcinomas among female compared to male lung cancer cases (24,25); (2) the presence of steroid receptors in some lung tumors (49-52); (3) the greater than expected incidence of lung cancer among female survivors of a primary of the reproductive organs (46-48); (4) an association of short menstrual cycle and late age at menopause with lung cancer (53); (5) an association of estrogen replacement therapy with lung cancer (54,55); and a more frequent than expected family history of reproductive cancer among female lung cancer cases compared to controls (56). Gao et al. (53) noted that women with late menopause were at increased risk of adenocarcinoma of the lung and that the OR increased with decreasing length of the menstrual cycle, with a 3-fold excess among women who had shorter cycles. In a study of adenocarcinoma of the lung in women, Wu et al. (57) reported a decreased OR for lung cancer in women who had taken oral contraceptives compared to those who had never used oral contraceptives (OR for <2 yrs = 0.9, 95 % CI 0.5-1.6; OR for > 2 yr = 0.4 (95 % CI 0.2-0.8). Cases and controls did not differ significantly in their use of estrogen replacement therapy. Taioli and Wynder (55) examined reproductive history and endocrine factors in relation to adenocarcinoma of the lung. Of the reproductive variables, an early age at menopause, < age 40 years, was associated with decreased risk of adenocarcinoma (OR = 0.3; 95% CI 0.1-0.8). Use of estrogen replacement therapy (ERT) was associated with adenocarcinoma (OR = 1.7, 95 % CI 1.0-2.8 for ever users relative to nonusers). There was a significant interaction between smoking and ERT. Relative to those who neither took ERT nor smoked, the OR for women who smoked and used ETR was 32.4 (95 % CI 15.9-665.3) and among women who only smoked, the OR was 13.1(95 R6 CI 6.8-25.2). Women who took ERT but never smoked had an OR for adenocarcinoma of 1.0 (95 % CI 0.3-3.8). At present, the existing evidence for a role of hormones in adenocarcinoma of the lung is limited and circumstantial. Some of the studies (i.e. Adami et al•) did not adjust for smoking or distinguish between histologic types. Further studies are needed that would obtain in-depth information on reproductive and endocrine factors as well as differences in serum and urinary estrogen levels between lung cancer cases and controls. N 0 00 ~ co W 0 -9- _' ~ I

Body Mass Index Several prospective studies have reported an inverse association between body mass index and lung cancer (15). Few of these studies, however, adjusted for cigarette smoking. We analyzed data on 3,607 lung cancer cases and 9,681 controls interviewed between 1981 and 1990 in order to determine whether the reported association could be due to confounding by smoking status, amount smoked, or other factors (16). Patients in the study were asked their weight five years prior to diagnosis. Separate analyses were carried out by smoking status and by sex. After adjustment for covariates, odds ratios for lung cancer by levels of body mass index, taking > 28 as the referent, showed an increasing linear trend with decreasing body mass index for current smokers and ex-smokers of both sexes and for female never smokers (Table 11). The association of leanness with lung cancer did not vary by level of other variables, including age, education, lifetime tar intake, alcohol intake, or race. In order to verify that the association was not due to overrepresentation of overweight subjects among the controls, we excluded diagnoses associated with obesity (cancers of the breast, endometrium, ovary, and gallbladder; fractures; back problems; arthritis; diabetes; and endocrine and metabolic disorders). This had no effect on the results. Comparison of the distribution of body mass index in the control group to that of the general U.S. population showed that both groups were similar. Discussion Association of Cigarette Smoking with Different Histologic Types In 1962, Kreyberg (17) classified lung cancer into two groups. Group I consisted of squamous cell, large cell, and small cell carcinomas, which he hypothesized were primarily due to exposure to external factors that had recently increased in prevalence and to which men were primarily exposed. Group II was heterogeneous, consisting of adenocarcinomas, bronchiolar (alveolar cell) carcinomas, and several other types, which Kreyberg hypothesized to be due to different etiological factors, possibly including developmental abnormalities, virus infection, and other factors of constant prevalence to which males and females were equally exposed. While Kreyberg's classification provides a valuable working hypothesis, it needs to be modified in the light of more recent epidemiological data. Studies examining the relationship of smoking to different histologic types show that smoking is associated with all 3 major types and shows a dose-response relationship (18-22). However, the magnitude of the association with amount smoked is, in most but not all studies (19), considerably stronger for squamous and small cell carcinoma than for adenocarcinoma. This tendency appears to be consistent between studies done in the United States and in China (23). The largest and most thoroughly analyzed study (19) indicates that duration of smoking is a more important variable distinguishing cell types than amount smoked. Duration of smoking showed a dose-response for all cell types, but the magnitude of the association was greater for squamous cell carcinoma than for adenocarcinoma. Part, but not necessarily all, of the explanation for the weaker association of smoking with adenocarcinoma is the fact that the proportion of never smokers among adenocarcinoma cases is larger compared to other cell types (19,24,25). Yang et al. (26) have suggested that the increase in adenocarcinoma of the lung may be explained in part by secular changes in cigarettes, specifically the increasing use of filter cigarettes starting in the -6- I I I I I I I I I I I , I I I

 I Table 2. Association of Slter status with Kreyberg I and II lung cancer by sex. Males KI I{Il. OR CI OR CI Nonfilter only 1.0 Switchers (1-9 yrs) 0.8 Switchers (10+ yrs) 0.7 Filter only 0.7 Nonfilter only 1.0 Switchers (1-9 yrs) 1.0 Switcher (10+ yrs) 0.7 Filter only 0.6 1.0 -- 0.6-1.2 1.0 0.6-1.5 0.5-0.9 0.8 0.5-1.2 0.4-1.3 0.9 0.4-1-1.5 Femeles ` - 1.0* - 0.5-2.0 0.4-1.4 1.0 0.8-1.3 0.3-1.4 1.0 0.6-1.5 Adjusted for cigarettes per day, age, inhalation, and years of education. *Referent category includes nonfilter only and switchers 1-9 years. I I I I I I I I I I ' ' , O 1 Co ~ -4 O W , -18- O N O I I

I I I I Table 3. Adjusted ORs for specific histologic types of lung cancer due to menthol use among current smokers (males and females combined). Histologic type OR 95% Cl I I , I I I I I I I I I Squamous cell carcinoma (N=268) Menthol 1-14 yr 1.2 0.8-1.8 Menthol 15+ yr 0.9 0.6-1.4 Small cell carcinoma (N=131) Menthol 1-14 yr 0.8 0.4-1.5 Menthol 15+ yr 0.9 0.5-1.5 Large cell carcinoma (N=106) Menthol 1-14 yr 2.0 0.7-5.4 Menthol 15+ yr 0.8 0.3-2.6 Adenocarcinoma (N=400) Menthol 1-14 yr 1.0 0.7-1.4 Menthol 15+ yr 1.0 0.7-1.4 Adjusted for: age, sex, duration of smoking, race, years of education, inhalation, and body mass index. IV , O O i V O , -19- W 0 N ~ I

Body Mass Index While increased body mass index is a risk factor for certain cancers, e.g. post-menopausal breast cancer and endometrial cancer, several studies indicate an inverse association with other tumors (including those of the lung, larynx, esophagus, bladder, and stomach) (58). Since these cancers are associated with smoking and since smokers tend to be leaner than nonsmokers, it is crucial to adequately adjust for smoking. The large numbers of cases and the detailed information on smoking history permitted us to stratify by smoking status and to adjust finely for cumulative tar intake in current and ex-smokers. Such adjustment did not reduce the magnitude of the association, but, rather, slightly increased it. Furthermore, the strongest association of leanness was observed in women who never smoked (Table 11). Self-reported weight 5 years prior to diagnosis was used in computing body mass index in order to minimize any effect of weight loss due to disease in the period immediately preceding diagnosis. However, when body mass index based on weight 1 year prior to diagnosis was used, the results were unchanged.. Further studies are needed to confirm whether there is in fact an association of lean body mass with lung cancer risk independent of smoking status and amount smoked and whether such an association is with leanness which is not a consequence of disease. If this association is not due to confounding or to weight loss due to disease, possible explanations include: 1) an association of leanness with decreased levels of nutrients that may be protective or with increased levels of dietary risk factors; 2) an association of leanness with increased metabolic rate and with accelerated cell turnover in the lung. Conclusions 1. Differences in smoking habits and tobacco products may contribute to observed differences in lung cancer incidence between populations. It is important to quantify lifetime tobacco intake (taking into account the tar-yield of cigarettes smoked throughout life, as well as amount smoked at different periods of life and duration of smoking, inhalation, etc.). It is also necessary to examine different histologic types separately, since the association with smoking differs by histologic type. 2. In addition to differences in tobacco smoke exposure, differences in lung cancer rates could be due to: 1) differences in host susceptibility, including the metabolism of carcinogens; 2) differences in exposure to independent risk factors; or 3) interactions of smoking with other risk factors or protective factors. 3. Since there is consistent evidence that cigarette smoking is not as strong a risk factor for adenocarcinoma as it is for squamous and small cell carcinoma, other factors must play a major role in the etiology of adenocarcinoma. -10- ' I I I I I I I I I I I I I

1 I I I I I I I I I I I Table 5. Odds Ratios for Lung Cancer by Duration of Quitting Smoking, by Race and Sex. Ma1e.s . Whites Blacks OR' 95% GI OR" 95% CI Current smokers 1.0 - 1.0 -- Ex-smokers 0.4 0.3-0.4 0.4 0.3-0.7 1-10 0.6 0.5-0.7 0.7 0.4-1.1 years 11-19 0.3 0.2-0.4 0.2 0.1-0.5 years 20+ 0.2 0.1-0.2 0.3 0.1-0.6 years Females Current smokers 1.0 -- 1.0 -- Ex-smokers 0.3 0.3-0.4 0.6 0.3-1.0 1-10 0.5 0.4-0.6 0.6 0.3-1.3 years 11+ 0.2 0.2-0.3 0.4 0.1-1.1 years ' Adjusted for age (continuous), years of education (continuous), number of cigarettes per day (continuous), time period (1979-1980, 1981-1984, 1985-1990), and hospital (Sloan- Kettering/other). ~ O 00 i , -4 ~ W O , -21- N w ,

have a greater effect on adenocarcinoma than on other cell types because the volatile components in sidestream smoke may be able to penetrate to the periphery of the lung. Several areas of inconsistency should be noted in studies of ETS and lung cancer. First, studies carried out in the United States have yielded contrasting results regarding an association with spousal smoking. The largest study, by Fontham et al. (4), reported an OR of 1.29 (95% CI 1.04-1.60) for having a smoking husband, with an increasing trend with increasing pack-years of exposure, reaching an OR of 1.79 (95 % CI 0.99-3.25) for nonsmoking women who had > 80 pack-years of exposure. Stockwell et al. (33) also noted a significantly increased OR (1.6, 95% CI 0.8-3.0) for exposure to a husband's smoking and an OR of 2.4 (95 % CI 1.1-5.3) for > 40 smoker-years in adulthood. Brownson et al. (34) reported a borderline elevated odds ratio for the highest level of cumulative exposure only (OR for > 40 pack-years of exposure from all household members = 1.3, 95 % CI 1.0-1.8). In contrast, other studies, including those by Janerich et al. (35), Wu et al. (36), and the present study provide no evidence for an association with spousal smoking. Second, there is inconsistency among studies with regard to the relationship of ETS to specific cell types. Fontham eta l. (4), whose large series of nonsmoking female lung cancer cases was predominantly adenocarcinoma (76%), showed a dose-response relationship for this type. Several studies, however, noted an association of ETS with squamous or small cell carcinoma but not with adenocarcinoma (37,38); two studies showed stronger associations with squamous cell carcinoma (or all types other than adenocarcinoma) than with adenocarcinoma (39,33); and one study reported an association only for "other/mixed" cell types, of which the numbers were small (34). Third, several studies indicate an association of ETS exposure in childhood with lung cancer (33,35), whereas others offer no support for an association (4,34). While an association of ETS exposure with lung cancer in never smokers has compelling biological plausibility and potentially important health implications (40,41), these inconsistencies as well as those from studies in other countries, point up the difficulties in quantifying ETS exposure throughout life and in detecting what on average among those passively exposed may be a small excess increment in risk. Radiation Treatment. Previous Renroductive Pritnarv, and Hormonal Factors Radiation treatment and a history of a previous reproductive cancer were so highly correlated among the female cases in our data that it was not possible to examine the effect of one factor independent of the other. These results are based on small numbers of never smokers and need to be confirmed in larger studies. Their interest lies in suggesting that endocrine-related tumors, or their risk factors or treatment, may increase the risk of lung cancer in nonsmoking women. Radiation is an established lung carcinogen (42-44), and radiation therapy, particularly for a previous breast cancer could have contributed to subsequent lung cancer (45). However, only three of the seven never-smoked lung cancer cases with a history of radiotherapy had a first primary of the breast. And the association of radiation treatment with lung cancer was comparable for those who reported radiation directed at the abdomen and neck. -8- I I I I I I I I I I I I I 1 I I I

i t 30. Caporaso, NE; Tucker, MA; Hoover, RN etal. Lung cancer and the debrisoquine metabolic phenotype. J. Natl. Cancer Inst. 82:1264-1272, 1990. 31. Racial variation in the distribution of Ha-ras 1 alleles. Molecular Carcinogenesis 4:265-268, 1991. 32. Wynder, EL and Goodman, MT. Smoking and lung cancer: Some unresolved issues. Enidemiol. Rev. 5:177-207, 1983. 33. Stockwell, HG; Goldman, AL; Lyman, GH et al. Environmental tobacco smoke and lung cancer risk in nonsmoking women. J. Natl. Cancer Inst. 84:1417-1422. 34. Brownson, RC; Alavanja, MCR; Hock, ET et al. Passive smoking and lung cancer in nonsmoking women. Am. J. Public Health 82:1525-1530. 35. Janerich, DT; Thompson, WD; Varela, LR et al. Lung cancer and exposure to tobacco smoke in the household. N. Enel. J. Med. 323:632-636, 1990. 36. Wu, AH; Henderson, BE; Pike, MC et al. Smoking and other risk factors for lung cancer in women. J. Natl. Cancer Inst. 74:747-751, 1985. 37. . Pershagen, G; Zdenek, H and Svensson, C. Passive smoking and lung cancer in Swedish women. Am. J. Epidemiol. 125:17-24, 1987. 38. Dalager, NA; Pickle, LW; Mason, TJ eta l. The relation of passive smoking to lung cancer. Cancer Res. 46:4808-4811, 1986. 39. Garfinkel, L; Auerbach, 0 and Joubert, L. Involuntary smoking and lung cancer: A case-control study. J. Natl. Cancer Inst. 75:463-469, 1985. 40. U.S. Environmental Protection Agency. Respiratory health effects of passive smoking: Lung cancer and other disorder. (EPA/600/6-90/006F). Washington DC, US EPA, Office of Research and Development RD-689, Dec. 1992. 41. National Research Council. Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects. Washington DC: National Academy Press, 1986. 42. Kohn, HI and Fry, RIM. Radiation carcinogenesis. N. Engl. J. Med. 310:504-511, 1984. 43. National Research Council, Committee on the Biological Effects of Ionizing Radiation. The Effects on Populations of Exposure to Low Levels of Ionizing Radiation. Washington, D.C.: National Academy of Sciences, 1980. 44. Court Brown, WM and Doll, R. Mortality from cancer and other causes after radiotherapy for ankylosing spondylitis. Brit. Med. J. 2:1327-1332, 1965. -14- I I I I I I I I I I I , I I I I I

 ' Table 4. Odds Ratios for Lung Cancer by Cumulative Tar Intake among Ever-Smokers of Cigarettes, by Race and Sex. Males Whites Blacks OR* 95m CI OR* 95% CI Quartiles of Tar Intake 1 1.0 -- 1.0 -- 2 1.9 1.6-2.2 2.3 1.4-3.7 3 2.9 2.5-3.5 5.0 2.9-8.5 4 4.3 3.6-5.2 5.7 3.0-10.9 Females 1 1.0 -- 1.0 - 2 2.3 1.8-2.9 2.1 1.0-4.2 3 4.5 3.5-5.8 5.1 2.4-11.5 4 5.3 4.1-6.8 12.8 4.3-38.7 *Adjusted for age (continuous), years of education (continuous), time period (1979-1980, 1981- 1984, 1985-1990), hospital (Sloan-Kettering/other), current smoker/ex-smoker. -20- I I I I I I I I I I I I I I

I I I I I I I I I I I I I I 16. Kabat, GC and Wynder, EL. Body mass index and lung cancer risk. Am. J. Enidemiol. 135:769- 774, 1992. 17. Kreyberg, L. Histological lung cancer types. A morphological and biological correlation. Acta. Pathol. Microbiol. Scand. Sunnl. 157:1-92, 1962. 18. Weiss, W; Boucot, KR; Seidtnan, H et al. Risk of lung cancer according to histologic type and dosage. J. Amer. Med. Assoc. 222:799-801, 1972. 19. Lubin, JH and Blot, WJ. Assessment of lung cancer risk factors by histologic category. JNCI 73:383-389, 1984. 20. Damber, LA and Larsson, LG: Smoking and lung cancer with special regard to type of smoking and type of cancer. A case-control study in north Sweden. Br. J. Cancer 53:673-681, 1986. 21. Morabia, A and Wynder, EL. Cigarette smoking and lung cancer cell types. Cancer 68:2074- 2078, 1991. 22. Brownson, RC; Chang, JC and Davis, JR. Gender and histologic type variations in smoking- related risk of lung cancer. Epidemiology 3:61-64, 1992. 23. Gao, YT; Blot, WJ; Zheng, W; Fraumeni, JF and Hsu, CW. Lung cancer and smoking in Shanghai. Int. J. Epidemiol. 17:277-280, 1988. 24. McDuffie, HH; Klaassen, DJ and Dosinan, JA. Determinants of cell type in patients with cancer of the lungs. Chest 98:1187-1193, 1990. 25. Kabat, GC and Wynder, EL. Lung cancer in nonsmokers. Cancer 53:1214-1221, 1984. 26. Yang, CP; Gallagher, RP; Weiss, NS et al. Differences in incidence rates of cancers of the respiratory tract by anatomic subsite and histologic type: An etiologic implication. J. Natl. Cancer Inst. 81:1828-1831, 1989. 27. Wagenknekt, LE; Cutter, GR; Haley, NJ et al. Racial differences in serum cotinine levels among smokers in the Coronary Artery Risk Development in (Young) Adults Study. Am. J. Public Health 80:1053-1056, 1990. 28. Hebert, JR. Differences in biological responses to cigarette smoking remain unexplained (letter). Am. J. Public Health 81:1679-1680, 1991. 29. Stellman, SD. Interactions between smoking and other exposures: Occupation and diet. Banbury Rept 23: Mechanisms in Tobacco Carcinogenesis. Cold Spring Harbor Laboratory, 1986, pp. 377-395. N O 00 - ~ ~ -13- V O Gf I O ~ ch

I I I 45. Neugut, Al; Robinson, E; Lee, WC et al. Lung cancer after radiation therapy for breast cancer. Cancer 71:3054-3057, 1993. I 46. Harvey, EB and Brinton, LA. Second cancer following cancers of the breast in Connecticut, I 1935-82. NCI Monograph 68:99-112, 1985. Curtis RE, Hoover RN, Kleinerman RA, et al. Second cancer following cancer of the female genital system in Connecticut, 1935-82. NCI Monograph 68:113-137, 1985. 47. Ewertz, M and Mouridsen, HT. Second cancer following cancers of the female breast in I 48. Denmark, 1943-80. NCI Monograph 68:325-329, 1985. Annegers, JF and Malkasian, GD. Patterns of other neoplasia in patients with endometrial carcinoma. Cancer 48:856-859, 1981. I 49. Chaudhuri, PK; Thomas, PN; Walker, MJ et al. Steroid receptors in human lung cancer cytosols. Cancer Lett. 16:327-332, 1982. I 50. Kobayashi, S; Mizuno, T; Tobioka, N et al. Sex steroid receptors in diverse human tumors. GANN 23:439-445 1982 I 51. , . Beattie, CW; Hansen, NW and Thomas, PA. Sex steroid receptors in human lung cancer. Cancer Res. 45:4206-4214, 1985. I 52. Cagle, PT; Mody, DR and Schwartz, MR. Estrogen and progesterone receptors in bronchogenic carcinoma. Cancer Res. 50:6632-6635, 1990. I 53. Gao, Y-T; Blot, WJ; Zheng, W et al. Lung cancer among Chinese women. Int. J. Cancer 40:604-609, 1987. I 54. Adami, H-O; Persson, I; Hoover, R et al. Risk of cancer in women receiving hormone replacement therapy. Int. J. Cancer 44:833-839, 1989. I 55. Taioli, E and Wynder EL. Endocrine factors and adenocarcinoma of the lung in women. J. Natl. Cancer Inst. 86:869-870, 1994. I 56. Sellers, TA; Potter, JD and Folsom, AR. Association of incident lung cancer with family history of female reproductive cancers: the Iowa Women's Health Study. Genet. Epidemiol. 8:199-208, 1991. I 57. Wu, AH; Yu, MC; Thomas, DC et al. Personal and family history of lung disease as risk factors for adenocarcinoma. Cancer Res. 48:7279-7284, 1988. I 58. Kabat, GC; Chang, CJ and Wynder, EL. The role of tobacco, alcohol use, and body mass index in oral and pharyngeal cancer. hrt. J. Eoidemiol. (in press). N 0 I 00 i V 00 -15- W I 0 i -1 I

Table 6. Prevalence of Reported Exposure to Environmental Tobacco Smoke' in Different Settings and Periods of Life among Never-smoking Lung Cancer Cases and Controls, 1983-90. Males Females Cases Controls Cases ' Controls tN=411 N=117 (N=69) _I8 Childhood only 22.0 Adulthood-home only 2.4 Work only 17.1 Childhood + adult- 7.3 hood-home Childhood + work 17.1 Adulthood home + 7.3 work All three 14.6 No exposure 12.2 18.8 7.3 9.6 2.6 5.8 10.1 14.5 10.1 8.0 7.7 29.0 21.4 22.2 13.0 5.4 2.6 8.7 11.8 16.2 18.8 20.3 15.4 7.3 13.4 *In order for a family member or job to qualify as a source of exposure, the subject had to report being exposed to an average of I cigarette, pipe, or cigar per day for at least one year. -22- I I I I I 1 I I I I I I I t '

I I I I I I I I , I I I I I I I I I Table 7. Association of Spousal Smoking with Lung Cancer in Lifetime Nonsmokers, 1983-90' Ca99~ _. Controls. . . C)dds:RAUds. . 95% CL.. Me)es Spouse smokes: No 28 79 Yes IN 19 Amount smoked by spouse(s)': 1.0 1.6 0.7-3.8 None 28 79 1.0 - i-l0 cigt/day 5 17 0.7 0.2-2.2 11+ cigtlday 5 2 7.5 1.4-01.4 Spouse smokes in bedroom: No' 5 14 1.0 -- Yes 5 5 5.0 0.7-35. Spouse smokes in bedroom: No/rionsmoker 33 93 1.0 - Yes 5 5 2.7 0.7-9.8 Femsles- .. . Spouse smokes: No 26 71 1.0 --- Yes 41" 102' 1.1 0.6-1.9 Amount smoked by spouse(s)': None 26 71 1.0 - 1-30 cigt/day 17 50 0.8 0.4-1.6 11+ 12 28 1.1 0.5-2.3 Spouse smokes in bedroom: No' 17 39 1.0 -- Yes 13 32 1.1 0.5-2.2 Spouse smokes in bedroom: No/nonsmoker 54 141 1.0 - Yes 13 32 1.1 0.5-2.2 * Limited to those who were ever-married. $ Adjusted for age and years of education (as continuous variables) and type of hsopital (cancer center vs. other). @ Includes 1 cigar/pipe smoker. # Referent group has no exposure in adulthood at home; exposure status based on weighted average number of cigarettes $ Spouse smokes/ed but not in bedroom. & Includes 12 pipe/cigar smokers. + Includes 24 pipe/cigar smokers. -23- I

I I Table 8. Association of Smoker-Years in Childhood, Adulthood, and at Work with Lung cancer in Never Smokers, 1983-1990. Odds Ratio" 95% Confidence Interval Smoker-ye_ars/i ob-years Males In childhood: Low 1.0 -- Intermediate 1.0 0.4-2.4 High 1.4 0.6-3.4 In adulthood: Low 1.0 Intermediate 2.0 High 1.5 At work: Low Intermediate 'High In childhood: Low Intermediate High 1.0 1.7 2.2# 0.7-6.0 0.5-4.9 0.4-2.9 0.5-3.1 0.8-3.6 1.14.5 In adulthood: Low 1.0 --- Intermediate 1.3 0.7-2.6 High 1.1 0.6-2.3 At work: Low 1.0 - Intermediate 0.9 0.4-2.1 High 1.6 0.6-2.8 'Ajusted for age and years of education (as continuous variables) and type of hospital (cancer center vs. other). "P-value for test for linear trend = 0.02. -24- I I I , I I I I I I I I I I

I I I I I I I I I I I I I I I I I I Table 9. Association of History of a Reproductive Primary and of Radiotherapy with Lung Cancer in Female Never-Smokers, 1985-1990. N N Adiusted, Cases Cotrtrols Odds Ratio 95% CI All lung cancer cases (N=46*) Previous reprod. ca. No 39 126 1.0 Yes 7 5 4.9' 1.4-17.7 No 39 126 1.0 --- Yes 7 5 2.9b 0.7-12.7 Hist. radiotherapy No 39 Yes 7 126 6 1.0 4.4` 1.3-15.1 No 39 126 1.0 Yes 7 6 2.2c 0.5-9.2 Adenocarcinoma (N=31*) Previous reprod. ca. No 27 126 1.0 -- Yes 4 5 4.0' 0.9-17.6 No 27 126 1.0 Yes 4 5 1.91 0.3-11.2 Hist. radiotherapy No 25 126 1.0 - Yes 6 5 4.3' 1.1-16.6 Yes 6 125 2.9c 0.6-13.7 One subject was dropped from logistic model due to missing data. ' Model including age (continuous), years of education (continuous), hospital (cancer center/other), lifetime environmental tobacco smoke exposure score (upper tertiles/lowest tertile), body mass index (> 28, <28). b Model including history of radiotherapy in addition to above variables. ` Model including previous reproductive cancer in addition to variables in a. -25-

I I Table 10. Age and Histology of First and Second Primary Cancers in Female Never-Smokers, 1985-1990. I I Previous Lung Cancer Reproductive Cancer Age' . Histology Age" Histology breast/uterus NOS` 57/60 breast 40 breast 41 breast 46 endometrium 69 cervix 32 genital 41 ductal/-- _d intraductal 57 adenoca. a 76 adenoca. adenoca. 75 adenoca. ° 41 non-small cell squamous 46 squamous 66 large cell 74 adenoca. ------------------------------------------------ 'mean=47 b mean = 62 NOS = not otherwise specified ° - = not available I I ' I I ' I I I I I I I -26- I I

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 1 I I I I I I I I I I I I I I I I Table 11. Association of body mass index with lung cancer in current, ex-, and never smokers, by sex, 1981-1990. Body mass index Adjusted OR Males 95% CI Females Adjusted OR 95%. Cl Current 8mokers' > 28 (referent) 1.0 - 1.0 - 25-27.9 1.2 1.0-1.5 1.2 0.8-2.0 22-24.9 1.5 1.2-1.9 1.7 1.2-2.4 <22 2.0* 1.5-2.5 Ex-smokers" 2.0' 1.4-2.7 > 28 (referent) 1.0 - 1.0 - 25-27.9 1.1 0.9-1.4 1.4 0.9-2.2 22-24.9 1.2 1.0-1.5 1.1 0.7-1.7 < 22 1.3" 1.0-1.8 Never snzokers` 1.5` 1.0-2.2 > 28 (referent) 1.0 - 1.0 -- 25-27.9 0.8 0.4-1.5 1.9 0.9-6.5 22-24.9 0.5 0.3-1.1 2.4 1.3-4.2 <22 0.91 0.4-2.1 2.9* 1.6-5.Q p value for linear trend = 0.0001. p value for linear trend = 0.02. + p value for linear trend = 0.2. " p value for linear trend = 0.4. a Adjusted for age, education, kilograms of tar, inhalation, race, hospital, time period, alcohol intake, and history of chronic lung disease. " Adjusted for above variables and years since stopping. Adjusted for age, education, race, hospital, time period, alcohol intake, and history of chronic lung disease. -27- I

I I I ' I I ' I I I I I I I I I I I RISK FACTORS FOR LUNG CANCER AMONG NONSMOKERS WITH EMPHASIS ON LIFESTYLE FACTORS Gao Yu-tang Shanghai Cancer Institute, Shanghai, China Abstract Exploration of risk factors for lung cancer among nonsmokers, in particular among females, has attracted the attention of cancer researchers in China for a considerable period of time. Lung cancer in females in some large Chinese cities is characterized by a relatively high incidence (although there is a relatively low smoking prevalence among females in the general population) and also by a high percentage of adenocarcinomas (1,2). Based on results of two population-based, case-control studies, it has been estimated that the population attributable risks (PARs) due to smoking for female lung cancer in Shanghai and Shenyang were 0.24 (95% CI: 0.19-0.29) and 0.37 (95% CI: 0.29-0.44), respectively (1,2), suggesting that the majority of female lung cancer cases (about 75% in Shanghai and 60% in Shenyang) cannot be attributed to smoking. The causes of lung cancer in nonsmoking females remain to be explained. . This review summarizes results of studies performed in China, focusing on risk factors for lung cancer among nonsmokers. I. Indoor air pollution 1. Coal burning The effect of coal fumes from heating or cooking in poorly ventilated houses on lung cancer risk has drawn the attention of numerous Chinese environmental scientists. A study in Xuanwei County, China (where extraordinarily severe indoor air pollution due to burning smoky coal is known to exist) showed a good correlation between indoor air pollution [as measured by benz(a)pyrene (B(a)P) concentration] and high lung cancer mortality rates (r=0.778; P < 0.01). In the same study, no relationship was found between female lung cancer risk and tobacco smoking or exposure to environmental tobacco smoke (3,4). Polycyclic aromatic hydrocarbons (PAH), well-known human carcinogens, were found in the indoor air from combustion of coal. It is noteworthy to mention, however, that indoor environmental conditions in high-risk areas, such as Xuanwei County, are exceptional, since the average B(a)P concentration in houses without chimneys located in Xuanwei was found to be extraordinarily elevated and it has been reported to be as high as 626.9 µg/100 m'. A study in Shenyang and Harbin involving household conditions typically found in Northeast China (2,5), showed that the risk for lung cancer was 30-50% higher among women who spent most of their lives in homes heated by coal and who used coal as the primary cooking fuel. The effects of indoor air pollution due to burning coal, were better correlated with squamous and oat cell carcinoma than for adenocarcinoma of the lung, for which the effects were similar to those of cigarette smoking. The frequent use of coal-burning stoves in Shenyang was estimated to contribute to 10-20 % of the lung cancer cases (2). I

I of smoking with pollution or a delayed effect of smoking due to differences in smoking histories between residents of these three areas. III. Other relevant risk factors 1. Diet and nutrients A review paper pointed out that consumption of vegetables, in particular those rich in S-carotene, may reduce risk for lung cancer (17). In Hong Kong, an association between vegetable intake and a reduced risk for lung cancer was observed among non-smoking women (18). Few studies in China have addressed the relationship between diet and lung cancer. The relationship between diet and lung cancer was studied in male residents in a mining cottununity in Yunnan Province (19). The "cases" consumed less protein-rich foods and vegetables than did controls. The relative risk for lung cancer across increasing quartiles of meat consumption were 1.00, 0.67, 0.72, and 0.46 (P for trend < 0.01). The relative risks for lung cancer across increasing quartiles of consumption of dark-green, leafy vegetables were 1.00, 0.62, 0.52, and 0.41 (P for trend < 0.01). A similar trend was observed in a population- based, case-control study in Shanghai (6), in which the risks for lung cancer were found to be lower among those with reduced consumption of carotene-rich foods. No effect on risk was found for consumption of retinol-rich foods. In Shenyang, a more frequent intake of retinol and carotene-containing foods did not protect against lung cancer in smokers or nonsmokers (2). 2. History of lung diseases In most case-control studies of lung cancer in China, it was found that a history of lung diseases such as tuberculosis, pneumonia, and emphysema were associated, to varying degrees, with an increase in risk for lung cancer. Smoking, which is usually associated with both chronic lung disease and lung cancer, was adjusted for in the analysis of the data from these studies. After adjusting for smoking, the excess risk for lung cancer in association with history of lung diseases persisted (5-7, 20). A retrospective cohort study of tuberculosis patients registered in the Shanghai TB registry since 1972 was carried out during 1987-89 to test the hypothesis that an association exists between lung cancer and pulmonary tuberculosis (21). A total of 30,373 cases of pulmonary tuberculosis (born before January 1, 1957 and residing in urban Shanghai) were followed until 1986. The standardized mortality rates (SMRs) for lung cancer (calculated to be 1.38 (95% CI: 1.19-1.61) and 2.73 (95% CI: 1.98-3.66) in males and females, respectively) were statistically significant. When the risk was adjusted for smoking, the adjusted SMRs for lung cancer were 1.72 (95% CI: 1.11-2.53) in males and 2.79 (95% CI: 1.79- 4.14) in females. Thus, the elevated risk for lung cancer among tuberculosis patients was independent of smoking. Neither INH treatment nor exposure to X-rays explained the higher risk. Considering that chronic diseases of the respiratory tract are prevalent among the Chinese population, the elevated risk for lung cancer associated with these diseases and their contribution to total risk for lung cancer should not be neglected. -4- I I I I I I I I I I I I I I I I

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I I 2. Volatile substances from some vegetable oils from wok cooking at high ' temperature. Another suspected risk factor for lung cancer is the volatile substances generated from cooking oils heated at high temperatures. This is supported by both epidemiologic and laboratory studies. A large-scale population-based, case-control study conducted in urban Shanghai (6) showed that lung cancer risk was increased with the use of rapeseed oil. Specifically, different levels of reported eye irritation experienced during cooking (used as a subjective variable to represent the severity of exposure to cooking vapors) were associated with an excess lung cancer risk. Controls were women who used soybean oil but never or rarely reported eye irritation. The few women who never cooked were excluded. The overall increase in lung cancer risk associated with rapeseed oil use, compared with soybean oil use, was 1.4 (95% CI.• 1.1-1.8). The patterns of risk were similar for squamous/oat cell cancer and adenocarcinoma of the lung. After adjusting for eye irritation, a 60% higher risk for lung cancer was observed among women who reported considerable or somewhat smoky conditions in their homes when cooking. This was considered as another rough measure of exposure to cooking vapors as well as the efficiency of household ventilation. In addition, the odds ratios (ORs) increased with the number of different dishes prepared by stir frying, deep frying, or boiling. No significant case/control differences were found in regard to the type of fuel used for cooking in the Shanghai study. A large-scale case-control study of female lung cancer carried out in Shenyang and Harbin (5) showed that, in addition to tobacco smoking, the following variables had a significant effect on risk for lung cancer. (P < 0.05) These appeared in the regression model in the order shown: deep frying, eye irritation, pneumonia, household tuberculosis, burning kang, self-reported occupational exposure to burning fuel, passive smoking from any household member, and heated brick wall/floor. It is interesting to note that the two variables related to cooking (deep frying and eye irritation) appeared in the model as the first and second most significant variables. Using a multivariate analysis of a case-control study of lung cancer in Nanjing (7), both squamous cell carcinoma and adenocarcinoma of the lung were significantly associated with cooking vapors; similar ORs were obtained for both types of lung cancer. In addition, coal stoves used for heating in the winter and non-gaseous fuel were also associated with an increase in lung cancer risk, although only for the squamous cell type. Researchers at the Shanghai Cancer Institute performed a number of laboratory studies on the genotoxicity of heated cooking oil vapors (8,9). They repeatedly observed that condensates of volatile emissions from rapeseed and soybean cooking oils were genotoxic in short-term tests, including the Salmonella mutation, the SV40 forward-mutation, the sister chromatid exchange, and the mouse bone marrow micronucleus assays. The mutagenic potential of volatile emissions from rapeseed oil was markedly greater than that of volatile emissions from soybean oil in the Salmonella mutation assay. In another study (10), volatile emissions from soybean oil (collected in a cold trap) also increased mouse bone marrow micronuclei, which was consistent with the results of similar studies done by the Shanghai Cancer Institute. In the same study, peanut oil and lard condensates were not mutagenic, irrespective of the assay used. -2- I I I I I I I I I I I I

I 1 , 14. I 15. , 16. I 17. I 18. I 19. I 20. I 21. I 22. ' , 1 ' I I I I International Agency for Research on Cancer. IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Vol. 38, 303-308. Tobacco Smoking. Lyon: IARC (1986). Lee, P.N. (1992) Environmental tobacco smoke and mortality. Basel (Switzerland): Karger. Deng, J.; Gao, Y.T.; Wang, Z.X. et al. (1992) Shanghai Tumor 12, 258-260. Smoking, general air pollution and lung cancer. A prospective cohort study among 210,000 adult residents. Steinmetz, K.A.; Potter, J.D. (1991) J. Enidemiology. Cancer Causes and Control 2, 325-357. Vegetables, fruit, and cancer. Koo, L.C. (1988) Nutr. Cancer 11, 155-172. Dietary habits and lung cancer risk among Chinese females in Hong Kong who never smoked. Swanson, C.A.; Mao, B.L.; Li, J.Y. et al. (1992) Int. J. Cancer 50, 876-880. Dietary determinants of lung cancer risk: results from a case-control study in Yunnan Province. Liu, Q.; Wu, M.X. (1987) Shanghai Tumor 7, 256-257. Smoking, ventilation of houses, and lung cancer-A matched case-control study among residents in Guangzhou. Gao, Y.T.; Zheng, W.; Jin, F. et al. (1992) Supplement to J. Epidemiol. (Japan Epidemiological Association) 2, S-82-88. Retrospective cohort study on the association of lung cancer with pulmonary tuberculosis. Zheng, W.; Gao, Y.T.; Sun, L. (1988) Shanghai Tumor 8, 119-121. Relationship between menstruation, reproduction, and lung cancer. N 0 00 ~ V ]_ CO W O W V

I I I I I I I I I I I I I I I I I I Two other laboratory studies also provided evidence of the genotoxicity of rapeseed oil vapors (11,12). A dose-dependent induction of rat tracheal epithelial cell transformation was shown by infusing condensates of rapeseed oil (at doses up to 1.5 mg/kg) into rat tracheas (11). The formation of DNA adducts resulting from cold-trapped condensates of rapeseed oil was studied with 'ZP-post-labeling techniques using a butanol enrichment procedure and conditions that amplify adduct detection. These studies showed that the condensate could react with calf thymus DNA to form DNA adducts without S9 (enzyme fraction) activation. A total of six spots were identified on thin layer plates. No spots were evident when the unheated rapeseed oil or the solvent were used as controls. These results suggest that rapeseed oil condensates contain some electrophilic compounds that could react with DNA directly to form adducts (12). Collectively, these laboratory findings give support to the epidemiologically-based hypothesis that exposure to volatile emissions from some types of cooking oil partially contributes to an elevated risk for lung cancer in females. Since the genotoxicity of rapeseed oil condensates disappeared (or decreased significantly) with the addition of butylated hydroxyanisole (BHA) or with hydrogenation, it may be suggested that oxidation and pyrolysis of unsaturated fatty acids in cooking oils contribute to the observed genotoxicity (8,9). Consistent with such an idea, rapeseed and soybean oil are known to contain linolenic acid, which has 3 double bonds and, hence, is easily oxidized at high temperature to produce pyrolysates. Condensates of linolenic acid have shown high mutagenicity in the Ames test. Moreover, condensates from peanut oil, which were initially non-mutagenic, became mutagenic when the peanut oil was first supplemented with linolenic acid, implying that linolenic acid plays an important role in the mutagenicity of condensates derived from cooking oil (8,13). 3. Environmental Tobacco Smoke (ETS) Despite the "trendy" suggestion that passive smoking contributes in some unexplained fashion to a slightly elevated risk of lung cancer (14), results of investigations in China on the relationship between ETS exposure and lung cancer risk were inconsistent and equivocal. Odds ratios for lung cancer in nonsmoking wives in relation to exposure to ETS from husbands were 2.16 (95% CI_• 1.03-4.53), 1.19 (95 % CL• .82-1.73) and 0.79 (95 % CI: .62-1.02) in Tianjing, Shanghai, and Shenyang and Harbin combined, respectively (15). The effects of exposure to ETS on lung cancer risk are difficult to evaluate due to uncertainties in the methodology of investigation. 11. General air pollution The contribution of air pollution in general as a possible contributing risk factor for lung cancer has been proposed for a number of decades by Chinese environmental scientists. Evidence supporting such a proposal, however, has been lacking. To evaluate the effect of general air pollution, and at the same time give consideration to other relevant important risk factors such as smoking, a prospective cohort study was carried out in three residential areas of Shanghai with substantially different levels of general air pollution (16). About 220,000 male and female adult residents in these areas were involved in the study. Information on smoking from each subject was obtained. Results of a five-year follow-up showed that there was no discernible effect of general air pollution on risk for lung cancer among male and female nonsmokers, but the risk for lung cancer was higher in urban smokers than in smokers residing in suburban areas and on the coast. Such a difference might reflect either an interactive effect -3-

I I I I I I I miners for which the attributable risk for radon-related lung cancer among nonsmokers would be about 12% based on a multiplicative model and over 30% based on a submultiplicative model between radon and smoking (27). Consumption of high levels of saturated fat and a history of prior lung diseases, especially pneumonia, were major contributors to population risk in this series. The etiologic link between saturated fat and lung cancer has been explained in only a few other studies so that a cautious interpretation of the high PAR seems warranted. Nonetheless, it seems prudent to assume that dietary factors could contribute to lung cancer risk, as they do other chronic diseases such as coronary heart disease, and thus a person should strive to reduce saturated fat and increase fruit and vegetable in their diets. -7- N O O V O W O ~ b

I I I I I I 3. Menstruation In a population-based, case-control study of lung cancer in Shanghai (6,22), an unexpected observation was that the risk for lung cancer was higher among women with shorter menstrual cycles. The association existed primarily for adenocarcinoma and showed a strong dose-response relationship. Additionally, among females age 55 and older with natural menopause, the risk for adenocarcinoma showed an increase with the total number of lifetime menstrual cycles. A study in Shenyang and Harbin suggests that the risk for lung cancer was positively associated with the age at which menopause occurs (5). Additional studies are needed to clarify the relationship between menstruation and risk for lung cancer in females. IV. Concluding remarks , Different parts of China have different proportions of lung cancer cases that cannot be attributed to smoking. Indoor air pollution, including coal burning in homes with poor ventilation, volatile ~ emissions from cooking oils, and environmental tobacco smoke, have been the focus of attention as potential risk factors for lung cancer among nonsmokers. I I I I I I I I One study in Shenyang estimated that coal burning may contribute to 10-20% of lung cancer cases. Both epidemiologic and laboratory studies support the notion that volatile emissions generated by heating rapeseed and soybean oil may contribute to an increase in the risk for lung cancer, especially among Chinese women (whose cooking practices often involve heating oil to high temperatures). Results of case-control studies on the effects of exposure to ETS are ambiguous and inconsistent. The idea that general air pollution contributes to an increase in lung cancer risk cannot be confirmed based on a cohort study of nonsmokers in Shanghai. Although occupational factors also increase the risk for lung cancer in highly industrialized cities, their contribution to lung cancer risk, as measured by population attributable risk (PAR), is relatively small. An association between history of lung disease and lung cancer risk, even after adjusting for smoking, was shown in most epidemiological studies performed in China. Since chronic lung diseases are prevalent among Chinese people, the significance of previous lung diseases in relation to risk for lung cancer can not be overlooked. A number of studies show that infrequent consumption of fresh vegetables, especially those rich in carotene, increases the risk for lung cancer. The effect of menstruation on risk for lung cancer in females deserves further investigation. N 0 ~ , 00 V 00 W ~ -5- c.~a N I

I References 1. Gao, Y.T.; Blot, W.J.; Zheng, W. etal. (1988) Int. J. Enidenriol. 17, 277-280. Lung cancer and smoking in Shanghai. 2. Xu, Z.Y.; Blot, W.J.; Ziao, H.P. et al. (1989) J. Natl. Canc. Inst. 81, 1800-1809. Smoking, air pollution and the high rates of lung cancer in Shenyang, China. 3. Mumford, J.L.; He, X.Z.; Chapman, R.S. eta l. (1987) Science 235, 217-220. Lung cancer and indoor air pollution in Xuanwei, China. 4. He, X.; Chen, W.; Liu, Z. et al. (1991) Environ. Health Perspec. 94, 9-13. An epidemiological study of lung cancer in Xuanwei County, China: current progress, case- control study on lung cancer and cooking fuel. 5. , Wu-Williams, A.H.; Dai, X.D.; Blot, W.J. et al. (1990) Br. J. Canc. 62, 982-987. Lung cancer among women in Northeast China. 6. Gao, Y.T.; Blot, W.J.; Zheng, W. et al. (1987) Int. J. Canc. 40, 604-609. Lung cancer among Chinese women. 7. Wang, G.X. et al. (1992) Chin. J. Prev. Med. 26, 89-91. Multivariate analyses of causal factors including cooking oil fumes and others in a matched case-control study of lung cancer (in Chinese). 8. Qu, Y.H.; Zu, G.X.; Zhou, J.Z. et al. (1992) Mutat. Res. 298, 105-111. Genotoxicity of heated cooking oil vapors. 9. Qu, Y.H.; Xu, G.X.; and Gao, Y.T. (1993) Carcino¢enesis. Teratogenesis and Mutagenesis 5, 59-62. Indoor pollution in the kitchen and lung cancer (in Chinese). 10. Liu, Z.Q.; Zhu, Z.G.; Wang, X.S. (1991) J. Environ. & Health 4, 10. Mutagenesis of smoke from cooking oils in the kitchen (in Chinese). 11. Wang, H. et al. (1992) Shanghai Tumor 4, 131. Transformation of epithelial cells of trachea of rats induced by condensates of rapeseed oil (in Chinese). 12. Wu, Y.Q. et al. (1992) Shanghai Tumor 12, 255. Investigations of DNA adducts of cold trapped condensates from rapeseed oil by 32P-post labelling techniques (in Chinese). 13. Qu, Y.H.; Xu, G.X.; Gao, Y.T. etal. (1990) Cereals and Fat 1, 45-48. Components relevant to mutagenicity of volatile condensates from cooking oils (in Chinese). -6- I I I I I I I I I I I I I I I I I I

27. Lubin, JH; Boice, JD Jr.; Hornung, RW; Edling, C; Howe, G; Kunz, E; Jusiak, RA; Morrison, HI; Radford, EP; Samet, JM; Tirtnarche, M; Woodward, A; Ziang, YS and Pierce, DA. Radon and lung cancer risk: A joint analysis of 11 underground studies. NII-I Publication No. 94-3644, 1994. N O O ~ -4 O W O -10- ~ I I I I I I Z I I I

I I ! I I I I I I I I I I I 1 I AITRIBUTABLE RISK OF LUNG CANCER IN NONSMOKING WOMEN Michael C.R. Alavania*, Ross C. Brownson**, Jacques Benichou* Christine Swanson* and John D. Boice, Jr.* * Epidemiology and Biostatistics Program, National Cancer Institute, Bethesda, Maryland, USA ** Department of Community Health, Saint Louis University School of Public Health, St. Louis, Missouri, USA Abstract Back rg ound In 1992, approximately 13,000 lung cancers occurred in nonsmoking U.S. women, but the etiology of these cancers is not well understood. Methods A population-based, case-control study of incident lung cancer among nonsmoking women in Missouri was conducted between 1986 and 1992. The study included 618 lung cancer cases and 1402 population-based, age-matched controls. Information on lung cancer risk factors was obtained by personal interview, or next-of-kin interviews (36% and 64% respectively). Year-long radon measurements were also sought in every dwelling occupied for the previous 5-30 years. Population attributable risks (PAR) for specific risk factors were computed for all subjects, for lifetime nonsmokers, for long-term ex-smokers, and by histologic cell type. Results The mean age of lung cancer diagnosis was 71 years, and nearly 50% of the lung cancers were histologically confirmed adenocarcinomas. Almost 40% of all lung cancers among lifetime nonsmokers and almost 50% of lung cancers among all subjects could be explained by the risk factors under study. Dietary intake of saturated fat and nonmalignant lung disease were the two leading identified risk factors for lung cancer among lifetime nonsmokers in Missouri, followed by environmental tobacco smoke, and occupational exposures to known carcinogens. Although an association with domestic radon exposure was not clearly demonstrated, it could be estimated that the PAR is less than about 5%. A similar pattern of risk was identified among former smokers, but in this group the lingering effect of a history of smoking was also very important. Along with saturated fat intake, the combined effect of previous active and passive smoking even after 15 years of active smoking cessation was responsible for more lung cancer than any other risk factor under study. A history of lung cancer among first degree relatives was a risk factor for exsmokers but not for lifetime nonsmokers. I

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Table 2. Lung Cancer Cell Types in Missouri Women, By Smoking Status: 1986-1991 Adenocarcinoma 73 219 292 62 Squamous cell carcinoma 17 10 27 6 Small cell carcinoma 9 25 3 Bronchoalveolar 2 17 19 4 Other cell types* 39 79 118 25 No pathologic confirmationt 46 104 150 Total 186 432 618 100 *Including those not otherwise specified and unknown cell types. tHistologic material not available for these cases. -12- 090£8L680Z r rM+r W a.r rn MIM rmr rre imMMM "..N

I I I Table 1. Sociodemographic Characteristics of Nonsmoking Women With Lung Cancer and Controls at the Time of Cancer Diagnosis: Missouri, 1986-1991 - ~ases n= ) . ontro s n= - haracteristic No. % No. %a ge at interview (years) 55 46 7 103 7 55-64 85 14 233 17 65-74 193 31 457 32 > 74 294 48 609 43 ducation < 12 240 39 536 38 12 228 37 477 34 > 12 121 20 355 25 Unknown 29 5 34 2 Marital status Married 292 47 752 54 Widowed 269 44 537 38 Separated 3 < 1 6 < 1 Divorced 28 5 59 4 Never married 26 4 47 3 Unknown 1 < 1 rurrent Missouri drivers license (<65 years old) Yes 118 90 335 > 99 No 13 10 1 < 1 Pealth Care Finance Registration (z65 years old) Yes 487 100 1,066 100 No 0 0 0 0 I I 1 I I I ~moking history Never 432 70 1,168 83 Former (> 15 years nonsmoker) 186 30 234 17 IVext-of-kin interviews (n = 396; 64%) Spouse, resident with study subject 105 17 0 Next-of-kin other than spouse, resident with study subject25 4 0 Daughter or son, nonresident with study subject 173 28 0 Sister or brother, nonresident with study subject 43 7 0 Other relative, nonresident with study subject 56 9 0 I

Conclusion Nearly forty percent of lung cancer cases among lifetime nonsmokers could be prevented if identified diet, occupational and general environmental factors were controlled. Genetic or familial factors seem to be most important to former smokers (and possibly to current active smokers) with little excess risk being seen among lifetime nonsmokers. The etiologic link between some of these factors (i.e., saturated fat and domestic radon) has not been examined in many other studies so a cautious interpretation of the population attributable risks presented for these exposures seems warranted. Introduction Cigarette smoking is by far the major cause of lung cancer, accounting for more than 80% of the 145,000 lung cancer deaths that occur each year in the United States. Lung cancer in nonsmokers, however, is also important and may account for more deaths than any other cancer except colon and breast in women and colon and prostate in men (1). Between June 1, 1986 to April 1, 1991, 19 k of all female lung cancer cases in Missouri occurred among nonsmokers (2). Despite its large public health impact, the etiology of lung cancer among nonsmokers is poorly defined. In this population, we previously determined the risk of various factors for lung cancer in a large, population-based, case-control study of lifetime nonsmokers and former smokers who had ceased smoking for at least 15 years (2-7). Here we present population attributable risk estimates to characterize, to the extent possible, the proportion of lung cancer that might be caused by each of the identified risk factors. Methods Population The study design and methods have been described previously (2-7). Briefly, white nonsmoking women 30-84 years of age who were residents of Missouri between June 1, 1986 and June 1, 1991 were eligible for inclusion. Lifetime nonsmokers consisted of those women who had not smoked more than 100 cigarettes or used any other tobacco products for more than 6 months in their lifetime. Former smokers were defined as women who ceased using all tobacco products 15 or more years prior to interview. Of the 3,475 women with lung cancer reported to the Missouri Cancer Registry, 650 were eligible for this study of whom 618 (95 %) agreed to participate. In addition to the registry-reported diagnosis of lung cancer, tissue slides were reviewed for histologic verification for 468 (76 %) of the cases by a panel of respiratory pathologist (10). A population-based sample of white, nonsmoking women control subjects were selected by frequency-matching on age from driver's license files provided by the Missouri Department of Revenue and for those over age 65, from lists of Missouri women provided by the Health Care Financing Administration (11). A total of 1527 nonsmoking control women responded to the initial screening interview; 1402 (92%) agreed to enroll in the study. Information on residential history, passive smoking exposure, family history, occupation, diet, previous lung disease or prior active smoking history was obtained from a structured questionnaire -2- I I I I I I I I I I I I I I I I I

l ~ ~ 14. U.S. Department of Health and Human Services. Reducing the Health Consequence of Smoking:  25 Years of Progress. A Report of the Surgeon General. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control, Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, DHHS Publication No. (CDC) 89-8411, 1989. I 15 World Health Organization, Overall Evaluation of Carcinogenicity: An Updating of IARC . Monograph. Volume i to 42, Supplement 7 International Agency for Research on Cancer, Lyon, 1987. I 16. Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders U.S. I 17. Environmental Protection Agency Washington, DC, 1992. Fontham, ETH; Correa, P; Wu-Williams, A et al. Lung cancer in nonsmoking women: a multicenter case-control study. Cancer Enidemiol. Biomarkers Prev. 1991;1:35-43. 18. Ernster, VL. The epidemiology of lung cancer in women. Annals of Epidemiology 1994;4:102- 110. I 19. Byers, TE; Graham, S; Haughey, BP et al. Diet and lung cancer risk: Findings from the Western Diet Study. Amer. J. Enidemiol. 1967;125:351-363. I 20. Jain, M; Burch, JD; Howe, GR et al. Dietary factors and the risk of lung cancer: results from a case-control study, Toronto, 1981-1958. hu. J. Cancer 1990;45:287-293. I 21. National Research Council. Health risks of radon and other internally deposited alpha-emitters. I 22. BEIR IV. Washington, DC: National Academy Press, 1988. Pershagen, G; Akerblom, G; Axelson, 0; Clavensjo, B; Damber, L; Desai, G; Enflo, A; Lagarde, F; Mellander, H; Svartengren, M and Swedjemark, GA. Residential radon exposure and lung cancer in Sweden. N. Enyl. J. Med. 1994;330:159-64. I 23 et al. Indoor radon and lung cancer in China. JNCI JD Jr ZY; Boice Xu Blot WJ . . , , , ; 1990;82:1025-30 ! . 24. Schoenberg, JB; Klotz, JB; Wilcox, HB et al. Case-control study of residential radon and lung cancer among New Jersey women. Cancer Research 1990;50:6520-24. I 25. Brownson, RC; Alavanja, MCR and Hock, E. Reliability of passive smoke exposure histories I 26. in a case-control study of lung cancer. Int. J. of EQdemiol. 1993;22:804-08. Dockery, DW; Pope, A; Xiping, X; Spengler, JD; Ware, JH; Fay, ME; Ferris, BG and An association between air pollution and mortality in six U.S. cities. N. E FE iezer S N O 00 . , p Med. 1993;329:1753-59. s V I I 9- OD W O A V I

I References 1. Schneiderman, MA; Davis, DL and Wagener, DK. Lung cancer that is not attributable to smoking. Letter. JAMA 1989;261:2635-6. 2. Alavanja, MCR; Brownson, RC; Boice, JD and Hock, E. Preexisting lung disease and lung cancer among nonsmoking women. Amer. J. E i~demiol. 1992;136:623-632. 3. Brownson, RC; Alavanja, MCR; Hock, Et and Loy, TS. Passive smoking and lung cancer in nonsmoking women. Amer. J. Public Health 1992;82:1525-1530. 4. Brownson, RC, Alavanja, MCR and Chang, JC. Occupational risk factors for lung cancer among nonsmoking women: a case-control study in Missouri (United States). Cancer Causes Control 1993;4:449-454. 5. Alavanja, MCR; Brown, CC; Swanson, C and Brownson, RC. Saturated fat intake and lung cancer risk among nonsmoking women in Missouri. J. Natl. Cancer Inst. 1993;85:1906-1916. 6. Brownson, RC; Alavanja, MCR; Berger, E and Chang, JC. Family history of cancer risk of lung cancer among nonsmoking women in Missouri. Amer. Journ. Epidemiol. (In review). 7. Alavanja, MCR; Brownson, RC; Lubin, JH; Brown, C; Berger, E and Boice, JD. Residential radon exposure and lung cancer among nonsmoking women. J. Natl. Cancer Inst. (In Press). 8. Bruzzi, P; Green, SB; Byar, DP; Brinton, LA and Schairer, C. Estimating the population attributable risk for multiple risk factors using case-control data. Amer. J. Epidemiol. 1985; 122:904-914. 9. Benichou, J and Gail, MH. Variance calculations and confidence intervals for estimates of the attributable risk based on logistic models. Biometrics 1990;46:991-1003. 10. Brownson, RC; Loy, TS; Ingram, E; Myers, JL; Alavanja, MCR; Sharp, DJ and Chang, JC. Histologic types of lung cancer among nonsmoking women: pathologic review and survival patterns. Cancer (In review). 11. Martin, G; Alavanja, MCR and Zahm, SH. Department of Health and Human Services epidemiology research 1989 data users conference proceedings. Baltimore, MD: Health Care Finance Administration, 1989:181-186. (HCFA publication no. 03293). 12. Steinmetz, KA and Potter, JD. Vegetables, fruit and cancer: Epidemiology. Cancer Causes Control 1991;2:325-357. 13. Nero, AV; Schwehr, MB; Nazaroff, WN and Revzan, KL. Distribution of airborne radon-222 concentrations in U.S. homes. cience 1986;234:992-997. ~ I I I I N O I tp ' ~ -4 tp W 8- ~ ~ ~

I I t I I I I I I I I I I I nonsmoking women in Missouri. Little difference in risk was experienced between long-term ex-smokers and lifetime nonsmokers, or between adenocarcinoma and other cell types. Exposure to environmental tobacco smoke (ETS) (>_40 pack-years) from a smoking spouse was experienced by one-fifth of all women in our study. The thirty percent excess relative risk among these women was responsible for approximately 6% of all lung cancers in this population (Table 3). This number rose to 8% in lifetime nonsmokers. Other sources of ETS might increase the population attributable risk even further but the Missouri Women Health Study was unable to assess the effect of ETS in most public places. A small additional increment of risk might be expected if a more comprehensive assessment of ETS related risk could be made. Ten percent of all nonadenocarcinoma cases could be attributed to spousal sources of ETS while only about 1% of the adenocarcinoma cases could be attributed to ETS. The combined effect of previous active smoking and passive smoking was responsible for 22 % of lung cancer in this population, and the figure rose to 30% for nonadenocarcinoma cell types. Working with asbestos or pesticides or in dry-cleaning facilities was associated with a moderate excess risk of lung cancer (OR=2.0). However, since exposure to these substances or workplace environments was uncommon in Missouri (approximately 5% of the female population) it was responsible for only about 5% of all lung cancer among nonsmokers. Both adenocarcinoma and nonadenocarcinoma cases were equally affected by these occupational factors. A family history of lung cancer among first degree relatives resulted in a small increased risk of lung cancer (RR = 1.4). Approximately 10% of the controls in our study population had such a history resulting in a population attributable risk of 4%. It should be noted, however, that the risk was not uniformly distributed, rather most of the risk was associated with former smokers (OR=3.9, not shown in table) and no excess risk was observed among lifetime nonsmokers (OR= 1.0, not shown in Table 3). A family history of lung cancer was about equally common in both adenocarcinoma and nonadenocarcinoma cases. Only 6% of the women in Missouri had a history of radon exposure exceeding 4pCi/L that spanned a 25-year period. This pattern of radon exposure is similar to that observed in the United States as a whole (13). In Missouri the mean radon level found in homes was 1.6pCi/L. In our study interviewing living cases resulted in a slightly more elevated estimated risk of lung cancer associated with domestic radon exposure than did interviewing next-of-kin. The reason for this discrepancy is unclear but we based our attributable risk computation on the experience of cases who were interviewed while still alive. This decision resulted in a larger radon associated lung cancer risk. For those living in dwellings with over a 4pCi/1 exposure the excess risk was 60%, resulting in an (nonsignificantly elevated) attributable risk of 4% in nonsmoking Missouri women, with little difference in risk found between lifetime nonsmokers and long-term ex-smokers. Seven percent of adenocarcinoma cases were associated with radon exposure but no excess risk was found among nonadenocarcinoma cases. N 0 00 ~ -4 00 -5- W ~ W I

Discussion Overall, 48% of all lung cancers among current nonsmokers could be attributed to a history of smoking, saturated fat intake, nonmalignant lung disease, environmental tobacco smoke, occupational exposures especially to asbestos, pesticides or dry-cleaning environments, and a family history of lung cancer. In Missouri domestic radon exposure in excess of the EPA action level was associated with a small nonsignificant additional risk of lung cancer. For lifetime nonsmokers 36% of all lung cancer among nonsmokers could be attributed to these nonsmoking risk factors. The amount of evidence from other studies supporting the association between these factors and lung cancer varies greatly and thus cautious interpretation is warranted. The strongest etiologic links identified involved a history of active smoking (14), and occupational exposures to carcinogens such as asbestos (15), while causal relationships are strongly suspected for environmental tobacco smoke (16,17), and a family history of lung cancer (18). Evidence from other studies supporting the etiologic association of saturated fat intake (19,20) and domestic radon exposure (i.e., z4pCi/L)(21-24), on the other hand, is not yet adequate and is in need of additional investigations. Strengths and Weaknesses The major strengths of our investigation include the evaluation of incident cases of lung cancer in a population-based setting, the relatively large number of nonsmoking women available for study and the comprehensive effort to ascertain domestic radon measurements in homes occupied by the study subjects during a 30-year period prior to enrollment in the study. Finally, we conducted a pathology review of cases, which enhances our histologic-specific findings. The potential weaknesses of this study included the use of self-reported data on previous lung disease, family history of lung cancer, passive smoking, diet and a history of active smoking. Moreover, we had no information on exposure to ambient air pollution which has been associated with lung cancer in certain industrial urban centers. Although we could not eliminate these potential weaknesses from the current study, a second interview conducted in a sample of cases and controls suggested that the reporting of nonmalignant lung disease and smoking was highly reproducible (25). Although air pollution is likely to be an independent risk factor for lung cancer (26), it is not likely to seriously confound the results reported in this paper. Conclusion Cessation of cigarette smoking remains the most constructive action to reduce the occurrence of many serious chronic diseases, including lung cancer. Even among long-tetm former smokers, 17% of their lung cancers could be attributable with some confidence to their prior habit. Smoke inhaled involuntarily by a nonsmoking spouse also could account for nearly 7% of lung cancers. In contrast, other exposures among nonsmoking women appear less important, such as occupation and domestic radon. Occupational risks are low because women of this generation were unlikely to work in hazardous jobs with toxic exposures. This will likely change in the future as more employment opportunities have opened for women for most occupations. While radon exposures in underground mines are clearly carcinogenic (20), the picture is not as clear for domestic radon (21-23). Making the most liberal assumptions in our data about possible radon risks, however, it is estimated that the PAR is likely less than 5 45. This percentage is much lower than that estimated by extrapolation of risks from underground -6- I I I I I I I

considered a more accurate source of information (for nonmalignant lung disease) and because they provided an upper limit of risk (for radon exposure). Results Most women in our series developed lung cancer after the age of 70 years, were married, and had completed high school (Table 1). There were few differences between the 618 cases and 1,402 controls in any of the demographic characteristics evaluated. However, the proportion of former smokers (women who had quit smoking more than 15 years previously : median period of cessation=26 years), was about twice as high among lung cancer cases (30 percent) as among controls (17 percent). Pathologic material from 468 cases was available for review. Adenocarcinoma was the most frequent lung cancer cell type (62 percent), followed by squamous cell carcinoma (6 percent), bronchoalveolar adenocarcinoma (4 percent), small cell carcinoma (3 percent), and all other cell types combined (25 percent) (Table 2). Women in the upper half of the saturated fat consumption continuum were at a seventy percent excess risk of lung cancer compared to women in the lower half. This excess relative risk translates into a population attributable risk of approximately 22 % since the exposed population in this case, constitutes 50% of the total population. We estimate that reducing the saturated fat intake below the 50th percentile (i.e., in this study estimated to be 18.8 grams/day) would be the single most effective action identified to reduce lung cancer incidence in a nonsmoking female population in Missouri (Table 3). Further reducing the saturated fat consumption to below the 20th percentile would reduce the risk of lung cancer even more, the PAR for saturated fat consumption above the 20th percentile being 48% (not shown in Table (3)(5). Both life-long nonsmokers and long term ex-smokers achieved a similar degree of benefit from a reduction in saturated fat intake. Fruit and/or vegetable consumption, which has been found to have a beneficial effect of reduced lung cancer incidence in some smoking and nonsmoking populations (12), did not have a measurable impact on lung cancer risk in this study. The population attributable risk of saturated fat intake was slightly higher among nonadenocarcinoma cell types (25 %) than adenocarcinoma (19%). The picture of risk seems to change, however, when more extreme saturated fat intakes are compared. The relative risk of lung adenocarcinoma was much greater than the risk for nonadenocarcinoma when extreme quintiles of intake of saturated fat are examined (5). Even after 15 years of smoking cessation, former smokers were at over twice the risk of lung cancer (OR=2.3) as were lifelong nonsmokers. This lingering risk to former smokers accounted for approximately 17% of all lung cancers in this population (Table 3). If all ex-smokers (including those who quit smoking 1-15 years) were included in this study the percent of risk attributed to a history of smoking would have increased substantially. Prior active smoking was associated with 22% of the nonadenocarcinoma compared to 13 % of adenocarcinoma. Based on in-person interviews only, a history of nonmalignant lung disease such as pneumonia, asthma and tuberculosis was associated with a significant excess lung cancer relative risk of 50% overall and in lifetime nonsmokers, but only 30% in long-term ex-smokers in our study. This was slightly more than when next-of-kin interviews were also included. Nonmalignant lung disease occurred in over one- third of the women in our control group and was associated with 16% of all lung cancer among -4- I I 11 I I I I I I I I I I I

I i 1 I 1 I I I I I I I I I I i I I administered by a trained telephone interviewer. Next-of-kin interviews were conducted for 64 percent (n=396) of the cases and none of the controls. Current residential radon concentrations were measured by placing two alpha track detectors in each dwelling occupied for at least one year by the study subject during the preceding 30 years in the state of Missouri. One detector was placed in the bedroom and the other in the kitchen for 12 months. Extensive quality control procedures were implemented to assure reliable radon measurements (7). Odds Ratio and Attributable Risk Estimation Unconditional logistic regression was used to estimate adjusted odds ratios. The risk factors under study were saturated fat intake, history of active smoking, previous nonmalignant lung disease, passive smoking, occupational exposure to carcinogens, family history of lung cancer and domestic radon. Each logistic model included the risk factor under study as well as those variables that were associated with a significant increase or decrease in lung cancer risk (2-7). Saturated fat intake was further adjusted to account for the caloric content of the daily diet (5). Namely, age (in five categories, 0-54, 55-64, 65- 74, 75-79, ? 80 years) and daily caloric intake (in five categories defined by quintiles of intake in the controls) were controlled for in all models, while saturated fat intake (in five categories defined by quintiles of intake in the controls), history of smoking (ever/never) and previous nonmalignant lung disease (ever/never) were controlled for in models where they were not already part of the exposure under study. Estimates of populations attributable risks (PARs) were obtained by using an approach based on unconditional logistic regression (8,9). By combining adjusted odds ratio estimates and the observed prevalence of the risk factor under study in the cases, this approach yields adjusted PAR estimates. The same logistic models were used for odds ratio and PAR estimation, therefore allowing one to adjust PAR estimates for the same factors as odds ratio estimates. Since both the odds ratios and the prevalence of exposure affect PARs, they are both tabulated (table 3). For smoking history, nonmalignant lung disease, occupation (use of asbestos, pesticides or working in the dry-cleaning industry), and a family history of lung cancer both the odds ratio and PAR were computed based on the comparison of ever vs. never exposed. For variables such as passive smoking, saturated fat intake and domestic radon, where exposure is ubiquitous, judgements had to be made to define exposure cut points along the exposure continuum that might be achieved as preventive measures in Missouri. For passive smoking the exposed group were women with >40 pack years of smoking from a spouse, while the unexposed group was for women with <40 pack years of exposure. For saturated fat intake which showed a significant monotonic dose-response effect (5) we compared the upper half of the exposure continuum with the lower half, assuming that a dietary modification of this extent might be possible. Finally, for domestic radon exposure, we estimated PAR by defining the exposed group as those subjects with a time-weighted-average (25 years) domestic radon exposure of 4pCi/L or greater (the current EPA action level). Cut points for each of these exposures were associated with a significant excess relative risk of lung cancer in our earlier study (3,5,7). For two variables, a history of nonmalignant lung disease and residential history (for radon exposure), odds ratios and PARs based on in-person interviews only were used because they were -3- I

I 1 I I I I 1 I I I I I I I I I I I Table 3. Age-Adjusted Lung Cancer Mortality Rates Among Nonsmoking Women Place Bombay, India 1979 Hawaii, Japanese 1982 Japan 1985 Hawaii, Hawaiians 1982 USA, White 1964 Hong Kong, Chinese 1983 Hawaii, Chinese 1982 Harbin 1987 Rate Source 3.3 Jussawalla et al. 1979 4.7 Hinds 1982 5.3 Tominaga 1987 6.2 Hinds 1982 9.4 Haenszel et al. 1964 13.8 Koo etal. 1985 14.1 Hinds 1982 14.8 Dai 1988 Table 4. Temporal Changes in Lung Cancer Mortality Rates in Females PAR' of Place Smoking eriod.' Age-Adjusted Average:.Increase Rate Rates Japan 13 % Harbin 24% I Shanghai 24% ° Population Attributable Risk 1950-1985 1986-1990 1963-1980 0.80-6.10 20.93-29.19 11.10-18.40 6.16 11.72% 3.21 Table 5. Percentage of Nonsmoking Female Lung Cancer Patients With Adenocarcinoma Place Harbin Hong Kong, Chinese Shanghai Patients . %. with adenocarcinoma 190 454 152 77.0 67.2 61.0 -5- I

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I I Table 1. Smoking Prevalence for Male Lung Cancer Patients Place England Hong Kong New York City San Francisco, White Singapore, Chinese San Francisco, Chinese Shanghai Aichi, Japan Malaysia Harbin Norway Study 99.5 99.0 98.1 98.1 96.6 92.8 92.0 91.4 89.0 88.0 87.9 Doll et al. 1952 Chan et al. 1979 Kabat et al. 1984 Green et al. 1982 MacLennan et al. 1977 Green et al. 1982 Gao et al. 1987 Shimizu et al. 1984 Menon et al. 1979 Dai et al. 1988 Kvale et al. 1983 Table 2. Incidence Rates and Smoking Prevalence for Female Lung Cancer Patients Place Hawaii, Hawaiians Harbin San Francisco, Chinese San Francisco, White Hawaii, Chinese Hong Kong Singapore, Chinese Shanghai Hawaii, Japanese India France Age-adjusted incidence rates '' Smoking prevalence 40.5 27.2 25.1 24.7 23.6 23.4 19.8 18.1 11.5 4.0 3.5 84.5 45.4 43.9 91.9 22.4 56.0 52.3 35.1 46.0 5.6 47.9 -4- I I I I I I I r I 0 NOo I I I I

I I I I I I 1 I 1 I THE ETIOLOGY OF LUNG CANCER IN NONSMOKSNG FEMALES IN HARBIN, CHINA Dai Xu-dong, Lin Chun-yan, Sun Xi-wei, Shi Yu-bo and Lin Ying ji Cancer Research Institute, Harbin Medical University Harbin, China Introduction Lung cancer is one of the most important types of malignancy in males, and is becoming increasingly common among females. A considerable amount of research conducted in different countries has focussed on studying the etiology of lung cancer. A number of epidemiological studies, conducted since the 1950s, have reported a close relationship between smoking and the incidence of lung cancer. Such an association, however, seems more directly applicable to males than females. Because Chinese women have a relatively low smoking prevalence yet have a higher incidence of lung cancer than might be anticipated, it seems possible that in females, some other risk factors are responsible for the increasing rate of lung cancer and may be more important than smoking. This report summarizes the results of a 1:1 population-based, case-control epidemiological study of nonsmoking females with lung adenocarcinoma, conducted in Harbin, China, designed to investigate the etiologic risk factors for lung cancer in nonsmoking females. Materials and Methods I Information analyzed in this report came from the data bank collected over the last several years by the Department of Cancer Epidemiology, Cancer Research Institute, Harbin Medical University. To be included as a subject for the study, a patient had to reside in the city of Harbin for more than 10 years, be between 30 and 69 years of age, never have smoked more than 100 cigarettes, and be diagnosed with primary lung cancer, with confirmation by pathology, between January 1, 1992 and December 31, 1993. For each subject, a population control was also selected. Control subjects were randomly selected from the city of Harbin, matched 1:1 with cases by age (± 5 years) and by nonsmoking status. The field staff conducted face-to-face interviews in the hospital or at home. An unconditional logistic regression model was used for data analysis. The administered questionnaire covered such parameters as education, marital status, residence history, income, fuel use, diets, cooking and heating practices, exposure to passive smoke, individual tumor history, and family tumor history. Results 1. According to data obtained in different countries, smoking prevalence is lower in female than in male lung cancer subjects. Many reports show that the smoking prevalence for males exceeds 90 %; we found a somewhat lower rate of 88 % in Harbin. The smoking prevalence in female lung cancer subjects in Harbin is 45.4%, which is slightly higher than the 35.1'Y reported for Shanghai. 00 i v 00 0 Lri 4f I

i I I I I I I I I I I I I I References 1. Gao, Y-T; Blot, W.J., etal. Lung cancer among Chinese women. Int. J. Cancer 1987; 40:604 2. Hinds, M.W., et al. Differences in lung cancer risk from smoking among Japanese, Chinese and Hawaiian women in Hawaii. Int. J. Cancer 1981; 27:297 3. Koo, L.C. et al. An analysis of some risk factors for lung cancer in Hong-Kong. Int. J. Cancer 1985; 35:149 4. Koo L.C. and Ho J. H-C. Worldwide Epidemiological patterns of lung cancer in nonsmokers. Int. J. Eoidemiol. 1990, 19 Supplement 1, 514-523 5. Mumford, J.L., et al. Lung cancer and indoor air pollution in Xuanwei, China. Science 1987; 235:217 6. Byers, T., et al. Dietary vitamin A and lung cancer risk. Amer. J. Epidemiol. 1987; 125:351 7. Lubin, J.H. and Blot, W.J. Assessment of lung cancer risk factors by histological category. L Natl. Cancer Inst. 1984; 73:383 8. Lam, T.H., et al. Smoking passive smoking and histological types in lung cancer in Hong Kong Chinese women. Br. J. Cancer 1987; 56:673 9. Koo, L.C. Dietary habits and lung cancer risk among Chinese females in Hong Kong who never smoked. Nutr. Cancer 1988; 11:155 10. Hirayama, T. Diet and cancer. Nutr. Cancer 1979; 1:67 11. Enstrom, J.E. Rising lung cancer mortality among nonsmokers. J. Natl. Cancer Inst. 1979; 62:755 i -4 O W 3_ O Clt Gf I

I Table 6. Logistic Regression Analysis for 120 Nonsmoking Female Lung Patients With Adenocarcinoma Factor B ~ OR . 05% CI . ~ Personal income per month in 1980 > =50 Average personal residcnce area > =6W Period of coal stove use in the bedroom 1-19 years > =30 years Period of heating by coal 1-24 years 25-34 years Exposure to coal dust > 10 years Fried and deep fried cooking >5 times per month Carrot consumption in 1980 >=65 times/year History of cancer in family -1.2523 0.286 0.090-0.903 0.0329 -1.7107 0.181 0.062-0.531 0.0019 1.4940 4.455 1.609-12.335 0.0040 2.9314 18.753 3.937-29.320 0.0002 1.7600 5.812 1.671-20.218 0.0057 1.5469 4.697 1.284-17,185 0.0194 0.9803 2.665 1.089-6.523 0.0318 2.2917 9.205 1.533-55.277 0.0152 -2.5954 0.075 0.018-0.307 0.0003 1.7646 5.839 1.581-21.568 0.0081 N O O j ~ 00 W O -6- ~ I I I I I I I I I I I I I I

I 2. A significant difference in worldwide lung cancer mortality rates has been observed for nonsmoking females. The lowest is 3.3/100,000, found in Bombay, India, and the highest is around 14.3/100,000, found in Chinese women in Hong Kong, Hawaii and Harbin. Time trends in the mortality rates for female lung cancer are very striking. In Hong Kong, where two-thirds of the female lung cancer cases are not attributable to smoking, the lung cancer mortality rate rose from 7.7 in 1961 to 29.3/100,000 in 1985. Likewise in Shanghai and Harbin, in which three-fourths of the female lung cancer cases are not attributable to smoking, the lung cancer mortality rate has also increased significantly over the years. 3. Previous studies have suggested that analysis of the histological types of lung cancer may provide clues to their etiology. Accordingly, the distribution of lung cancer histological types among nonsmoking females was determined in the present investigation. Our study found adenocarcinoma to account for 77% of lung cancer in nonsmoking females in Harbin, which is certainly consistent with several previously published findings and appears to be substantially higher than the 60% reported for Shanghai. 4. In order to examine the potential etiological factors for nonsmoking female lung cancer, a population-based, case-control study was conducted in Harbin, using 120 cases of nonsmoking female lung adenocarcinoma and the same number of nonsmoking female controls. An unconditional logistic regression model was used to analyze potential risk and protective factors. The results show the risk factors to be: period of coal stove use in the bedroom; period of heating by coal; exposure to coal dust for more than 10 years; practicing fried and deep fried cooking more than 5 times per month; and a history of cancer in the family. The protective factors are: personal income of more than 50 Yuan per month; average personal residence area of more than 6W in the last 20 years; carrot consumption of more than 65 times per year. Discussion The relationship between smoking and lung cancer has always been emphasized by epidemiologists. Many studies have shown an association between smoking and squamous cell carcinoma in males. Since in females the prevalence of smoking is relatively low, and because adenocarcinoma is the primary histological type in female lung cancer, it seems possible that risk factors exist for females which potentially play a more important role than smoking as the major cause of female lung cancer. I I I I I I I I I I I ~ ~ Based on data generated from epidemiological and experimental studies, several risk factors have been identified in the etiology of lung cancer in nonsmoking females. Koo and co-workers from Hong Kong indicate that the etiological factors for lung cancer in Chinese women may be related to some i unidentified environmental factors. Gao et al. from Shanghai point out the association between cookin g oil smoke and female lung cancer. Studies performed in Xuanwei, Yunnan Province, provide evidence for a relationship between smoke from indoor coal burning and the high rate of female lung cancer. Our ~ studies from Harbin, China, indicate that indoor air pollution from coal burning as well as lack o f vitamins are risk factors for female lung cancer. Additional studies are in progress to further analyze N these and other factors in order to fully expound the high rate of lung cancer in Chinese women. ~~ tp ~ -4 00 W , 2- Q CA ? I

s r IM r .r .. M r r .. .. .r .. M ..r .r r r O•• Table 3. Relative Risk and Population Attributable Risk for Lung Cancer Among Nonsmoking Missouri Women ~ . , Proportion of ~ ~ Controls With ~ Population Attributable Risk in Pcreent (95% Cl) Study Odds Ratio Risk Faciot Risk Factor Subieas ~ 95 C ~ (x100) ~. Alt Subiects UfUime Adenocarcinoma Nn cinoma Noasmokers ~ ~ . Samrated fat (518.8 grams vs. 218.8 grams) All 1.71 48.5 22.21 23.82 19.41 24.61 (1,2 to 2.4) (10.5 to 34.1) (10.0 to 37.7) (2.9 to 35.9) (9.9 to 393) History of active smoking, 15 or more years of All 2.33 17.5 1743 NA 13.33 21.53 smoking cessation (ex-smokcrs vs. lifnime (1.7 to 2.9) (11.6 to 22.3) (5.5 to 21.2) (13.5 to 29.6) nonsmokers) Nonmalignant lung disease (ever vs. never) living 1.54 35.2 16.04 15.73 14.3 17.74 cases (1.1 to 2.1) (3.4 to 28.6) (1.0 to 30.3) (-1.9 to 30,4) (-1.3 to 36.6) only Environmental tobacco amuke from spouse (24(l pack All 1.35 19.3 6.16 7.63 years vs. <40 pack years) (1.0 m 1.8) (-0.2 to 12.5) (0.3 to 14.8) Occupation, working with(in) asbestas, pesticides or All 2.06 4.9 5,16 5.53 drycleaning (ever vs. never) 1.3 to 3.2) (1.7 to 8.4) (1.6 to 9.5) 1.56 10.05 (-6.6 to 9.7) (1.5 to 18.5) 5.16 5.16 (0.6 to 9.7) (0.5 to 9.6) Family lung cancer history (ever vs. never) All 1,46 10.2 4.26 0.43 3.46 5.66 (1.0 to 2.1) (0.1 to 8.2) (4.4 to 5.3) (-1.9 m 8.6) (0.0 to 11.1) Darc.uic radon (25 years TWA 24pCi11) living 1.66 6.4 394 3.43 7.26 -0.76 caaea (0.9 to 2.9) (-1.5 to 9.3) (-2.8 to 9.7) (-0.3 to 14.7) (-7.7 to 6.2) only Smoting Exp (active or passive, 2 variables) (ever vs. All 1.83 33.1 22.23 NA 13.93 29.73 nerer) (1.4 to 2.3) (14.0 to 30.5) (2.5 to 25.3) (18.9 to 40.5) One or morc (ever vs. never) (6 variables for lifetimc All 2.21 80.5 48.17 36.17 50.27 45.93 nunamnkers, 7 variables otherwise) (1.5 to 3.2) (31.0 to 65.1) (15.4 to 56.7) (26.9 to 73.5) (23.3 to 68.6) Foomotes 1-7: 1. 2. 3. 4. 5. 6. 7. Adjusted for age at self-administered questionnaire (SAQ), history of active smoking, daily caloric intake and previous lung disease. Adjusted for age at self-administered questionnaire (SAQ), daily caloric intake and previous lung disease. Adjusted fur age at interview, daily caloric intake, previous lung disease and daily saturated fat intake. Adjusted for age at interview, history of active smnking, daily caloric intake and daily saturated fat imate. Adjuated for age s[ interview, daily caloric intake and daily saturated fat intake. Adjusted for age at interview, history of active smoking, daily caloric intake, previous lung disease and daily saturated fat intake, Adjusted for age and daily caloric intake at interview. -13- 4S0£81680Z

within the family, menstruation and pregnancy/child bearing, and job history. Completed questionnaires and relevant medical information were checked for accuracy by a supervisor, then coded and computerized. The Mantel-Haenszel method and multivariate logistic regression analysis were used to estimate the odds ratio (OR) for different risk factors and the statistical significance of their association with lung cancer. Population attributable risk (PAR) estimates were also computed for relevant risk factors. Results One hundred and thirty-five lung cancer patients were identified and interviewed. They were all lifetime nonusers of tobacco. None of them refused to be interviewed. More than half (57.2%) of the cases were diagnosed by tissue biopsy or cytology, and the other cases by medical signs and symptoms and repeated X-ray films. Among the cases diagnosed pathologically or cytologically, the distribution of histologic cell type was as follows: adenocarcinoma was the predominant cell type accounting for 54.5 % of the cases, 16.4% were squamous cell carcinoma, 20.4% were small cell or oat cell carcinoma, and 9.1 % were a mixture or undifferentiated carcinoma. An equal number of controls were also interviewed. The distribution by age and marital status was generally similar between cases and controls. Exposure to Passive Smoke Overall, no significant increase in risk was observed for exposure to passive smoke (also referred to as environmental tobacco smoke, ETS). Table 1 shows that for workplace exposure, the OR was 0.89 (95 % CI, 0.45-1.77). For exposure to ETS during childhood, the OR was computed to be 0.91 (95 % CI, 0.55-1.49). Exposure to ETS from a spouse who smoked was not associated with a significantly increased risk of lung cancer (OR = 1.11, 95% CI = 0.65-1.88). - 2 - N O O I I I I I I I I I I I I I I I I I

I Consistent with previous reports in Shanghai and Hong Kong, we did not find that the use of coal for cooking or heating increased the risk for lung cancer. In the univariate analysis, coal smoke was found to be a risk factor (Table 1) which subsequently disappeared when more detailed multivariate analysis was performed. In our study, previous lung disease was not a risk factor, in contrast to other findings in which previous lung disease, especially tuberculosis, increased the risk of lung cancer in nonsmoking women (12, 13). Conclusion In a case-control study of risk factors for lung cancer in nonsmoking women in the urban area of Shenyang, cooking practices and exposure to cooking oil vapor and a family history of cancer were found to be associated with a significant increase in risk, while other factors (such as exposure to environmental tobacco smoke and coal and kang use) did not show such an association. -6- ' I I I I I I I I I

I I I I I I I 1 I I 1 I I I I I Table 1. Association of different risk factors with lung cancer in nonsmoking women Variable Case Control OR 95% CI PAR (n-135) (n=135) % Workplace exposure to ETS 113 115 0.89 0.45-1.77 n.d. Childhood exposure to ETS 80 83 0.91 0.55-1.49 n.d. (exposure before marriage) Adult exposure 92 89 1.11 0.65-1.88 n.d. (to smoking spouse) Coal use 100 107 0.75 0.43-1.31 n.d Kang use 88 89 0.97 0.64-1.48 n.d Exposure to cooking oil vapor 77 35 3.79 2.29-6.27 42 Exposure to coal smoke 65 38 2.37 1.44-3.91 28 Previous lung disease 29 19 1.67 0.89-3.14 n.d. Family cancer history 19 9 2.29 1.01-5.17 8 To further investigate whether exposure to ETS was associated with lung cancer risk in nonsmoking women, the relative risk associated with the number of years living with a husband who smoked, or with the number of cigarettes smoked per day by a husband who smoked, was also calculated (Tables 2 and 3). No significant increase in lung cancer risk was found in either case. Table 2. Relative risk of lung cancer among nonsmoking women in relation to years lived with a smoking husband esrs lived wiTh.~kiughuahsnd Case (n-135 aatmt'(a=135 95%C <20 65 70 - >20 21 16 1.41 0.68-1.94 >30 32 32 1.08 0.58-2.00 >40 17 17 1.08 0.37-3.14 N O ~ 00 3 v 00 (J M 3 - N i

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I i I I I I 1 I I LUNG CANCER IN NONSMOHING CHINESE WOMEN: A CASE-CONTROL STUDY Wan Tian-jue, Zhou Ban-sen and Shi Jin-pu China Medical University, Shenyang, China Abstract The importance of risk factors for lung cancer in lifetime nonsmoking women was investigated in a case-control study in the urban area of Shenyang, China, between April 1992 and May 1994. One hundred and thirty-five newly-diagnosed lung cancer cases and an equal number of controls, matched for age and sex, were enrolled and interviewed by trained personnel who administered a standardized questionnaire. The histopathological cell type was predominantly adenocarcinoma (54.5 %), followed by small cell carcinoma (20%), squamous cell carcinoma (16.4%), and others (9.1%). The data were analyzed using the Mantel-Haenszel method and by multivariate logistic regression analysis. The odds ratio (OR) and confidence interval (CI) associated with cooking oil vapors and with family history of cancer were 3.79 (95r5 CI, 2.29-6.27) and 2.29 (95% CI, 1.01-5.17), respectively. No association was found between exposure to passive smoke, presence of previous lung diseases, and other variables. Introduction I 1 I I I I I ' I Study of the etiology and risk factors for lung cancer in nonsmoking women has received increasing worldwide attention in recent years (1). In China, several studies have been performed on the relationship between lung cancer and a number of risk factors. These studies have generated variable and equivocal results. Herein we report the results of a study aimed at exploring the influence of indoor air pollution, including exposure to passive smoke, and a number of other suspected risk factors, on lung cancer. Materials and Methods One hundred and thirty-five cases of newly diagnosed and eligible cases of primary lung cancer (according to the International Classification of Diseases, Ninth Revision, ICD-9 code 162, reference 2) were collected and identified in eighteen hospitals in the city of Shenyang between April 1992 and May 1994. All subjects, who ranged in age from 35-69 years and were lifetime nonusers of tobacco, were interviewed in person in the hospital by trained personnel within two weeks of diagnosis. Every case enrolled in this study was diagnosed by review of relevant medical records, chest X-ray and CT films, and cytologic and histologic slides (in 57.2% of the cases). All reviews were studied and confirmed by senior pathologists or clinicians. One hundred and thirty-five female controls, matched for age (± 5 years), were randomly selected from the general population located in urban areas of Shenyang. The number and age distribution of the controls were determined in advance based on the number and age distribution of reported primary lung cancer cases existing in the Liaoning provincial antiepidemic station between 1988-1989. Two controls in the appropriate age group were randomly selected. If the first control was absent or not eligible for interview during the study, the second control was accepted as the alternative. During the interview, a structured questionnaire was administered to obtain information on demographic characteristics, exposure to tobacco, dietary and cooking practices, the type of fuel used, general medical conditions, history of previous lung diseases, history of cancer (including lung cancer) I

1 ' Table 3. Relative risk of lung cancer among nonsmoking women in relation to the number of cigarettes smoked per day by smoking husband Number of cigsretms smoked pu day Ctse (nm 135) Control (a=135) OR. 1 95 %~ CI 0 43 49 - >1 4 13 0.35 0.11-1.12 > 10 45 38 1.35 0.75-2.45 >20 43 35 1.40 0.76-2.56 Cooking Practices Soybean oil is the oil used most often for cooking in Shenyang. When cooking practices that frequently or sometimes generate cooking oil vapor were compared with those that generate little or no cooking oil vapor, a statistically significant increased risk of lung cancer was observed, OR = 3.79, 95 % CI, 2.29-6.27. Likewise, exposure to coal smoke during cooking was associated with a significant increase in risk for lung cancer, OR = 2.37, 95% CI, 1.44-3.91 (Table 1). Family History of Cancer Family cancer history, which refers to history of lung cancer or other forms of cancer in next-of- kin relatives, is significantly associated with an increase in lung cancer, OR = 2.29, 95 % CI, 1.01-5.17 (Table 1). After stratifying subjects into a lung cancer group (group 1) and an other-than-lung cancer group (group 2), the association between family cancer history for each subgroup and risk for lung cancer in nonsmoking women was no longer statistically significant. A positive association, however, remains. The OR for group 1 was 3.64, 95% CI, 0.81-16.23 and the OR for group 2 was 2.12, 95% CI, 0.84- 5.35. Previous Lung Disease Previous lung diseases refer to tuberculosis, chronic bronchitis, pneumonia, emphysema, lung abscess, and asthma. The overall relative risk between previous lung disease(s) and risks for lung cancer in nonsmoking women was computed to have an OR of 1.67, 95 % CI 0.89-3.14. A history of tuberculosis alone produced an OR of 1.39, 95% CI, 0.94-3.04. Thus, no statistically significant association was observed between previous lung disease and risk of lung cancer in nonsmoking women. Coal Coal was the most common fuel used in Northeast China. However, in this study, its use was not associated with an increase in risk for lung cancer in nonsmoking women (OR = 0.75, 95% CI 0.43- 1.31). I I I I I I I I I I I i

a I I , I I I I I I I I References 1. Fontham, E.T.H. et al. (1991) Lung cancer in nonsmoking women. A multicenter case-control study. Cancer Epidemioloav 1, 35. 2. World Health Organization. International Classification of Diseases, Ninth Revision. Geneva, WHO, 1977. 3. Du, Y.XX et al. (1992) An epidemiological investigation of risk factors for lung cancer in Guangzhou, China. Guangzhou Third Symposium on lung cancer research. 4. Wang, F.L. et al. (1989) Analysis of risk factors for female adenocarcinoma in Harbin, China, J. of Preventive Medicine 23, 270. 5. Gao, Y.T. et al. (1987) Lung cancer among Chinese women. Int. J. Cancer 40, 604. 6. Wu-Williams, A. et al. (1991) Lung cancer among Northeast China. Br. J. Cancer 62, 982. y 7. Dai, X.D. et al. (1991) The risk factors for lung cancer in women. Lung_Cancer (Supplement) 7, 3. 8. Qu, Y.H. et al. (1986) An Ames test on the products of the history of cooking oil. Tumor 6, 58. 9. Wong, G.X. et al. (1992) Multivariate analysis of causal factor included cooking oil fume and others in matched case-control study of lung cancer. Chinese Journal of Preventive Medicine 2, 89. 10. Ooi, W.L. et al. (1986) Increased familial risk for lung cancer. J. Natl. Cancer Inst. 76, 216. 11. Osann, K.E. et al. (1991) Lung cancer in women: The importance of smoking, family history of cancer and medical history of respiratory disease. Cancer Res. 51, 4893. 12. Zhen, W. et al. (1987) Lung cancer and prior tuberculosis infection in Shanghai. Brit. J. Canc. 56,561. 13. Li, W.X. et al. (1989) A case-control study of female lung cancer at Xu Hui District in Shanghai. Chinese Journal of Preventive Medicine 2, 93. N O ~ . 00 -4 Lp W ~ 7 CD O O> I

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I I I I I 1 I I 1 I I I , I 1 I Kang "Kang" are made of brick and are often heated by burning coal in the winter. Prior to the modernization of China, kang were traditionally the most common form of bed as well as room heating device in the city of Shenyang. Use of kang was not significantly associated with lung cancer risk in nonsmoking women, OR = 0.97, 95% CI, 0.64-1.48 (Table 1). Multivariate Unconditional Logistic Regression Analysis When multivariate analysis was applied to all variables examined in this study, only two - oil vapor during cooking (OR = 4.02, 95 % CI, 2.38-6.78) and family cancer history (OR = 3.07, 95 % CI, 1.30-7.26) - were found to be statistically significant risk factors for lung cancer in nonsmoking women. Coal smoke, which was a risk factor in the univariate analysis, no longer appears in the equation. None of the other factors appear in the equation, indicating that they are not risk factors for lung cancer in this population. Discussion The relationship between cigarette smoking and lung cancer has been well established. The risk factors for lung cancer in nonsmoking women, on the other hand, are still unclear (1, 3-7) and are the primary focus of this investigation. Possible etiologic factors being considered include: exposure to ETS, coal smoke pollution, previous lung disease, family history of cancer, and others (Table 1). The results of our study suggest that exposure to cooking oil vapors and family history of cancer are the primary risk factors. In northeast China, soybean oil is the most commonly used oil for cooking. When cooking oil is poured into a cooking utensil and heated to a high temperature, the kitchen and living room may become smoky. Moreover, exposure to oil smoke often becomes unavoidable because of cooking practices frequently used by Chinese females. Gao et at. (5) and Wong eta l. (8) previously reported that the risk of lung cancer among women was increased by various modes of exposure to cooking oil vapors. It is therefore reasonable to suggest that cooking oil vapors play an important role in increasing the risk of lung cancer in nonsmoking women. The notion that lung cancer may be etiologically •related to cooking oil vapor is biologically plausible since extracts prepared from volatile condensates of rapeseed oil and soybean oil heated at 270°C show mutagenicity based on the Ames test (9), i.e., giving positive results when tested in S9-activated TA98 strain of Salmonella. Genetic factors also appear to be involved in the occurrence of lung cancer, as suggested by the increased risk associated with family cancer history in this study. Indeed, genetic make-up as well as the fact that families often share identical environments and are exposed to similar indoor air pollutants may contribute to the trend for lung cancer to cluster in a family (10). Whether exposure to ETS is a risk factor for lung cancer has been a subject of controversy (1, 3-7). In the present study, we found that exposure to ETS was not a risk factor for lung cancer. Neither exposure during childhood nor in adult life was associated with an increased risk of lung cancer in nonsmoking women. _ 5 - 1

I I Figure 2 reports the proportion of population controls with a low vegetable and fruit consumption (class 0) with relation to smoking habits. % of population controls 40 NS < 10 10- 19 20+ cigarettes/day Figure 2. Consumption of vegetables and fruit in relation to smotdng status. It is seen that the proportion of persons reporting a low frequency of vegetable and fruit consumption was higher among smokers, particularly among those smoking more than 20 cigarettes/day (p<0.005). For other food items, a larger proportion of persons smoking more than 20 cigarettes/day had a higher consumption of smoked/salted fish than nonsmokers (42.9 vs 26.3%, p<0.06). Coffee drinking habits are shown in Figure 3. % of population controls 75 50 25 0 NS <10 10-19 20+ , I I I I I I I I I cigarettes/day 00 ~ Consumption of coffee in relation to smoking status. Fi ure 3 ~ , W g . ~ -4- ~ ' I

a I I I I I I I I I I I I I LUNG CANCER, SMOKING AND DIET AMONG SWEDISH MEN Ra ng ar Rvlander*, Gdsta Axelsson*, Lars Andersson**, Tomi Liljequist* and Bengt Bergman*** * Department of Environmental Medicine, University of Gothenburg, Gothenburg, Sweden, ** The Pulmonary Clinic, North Alvsborg General Hospital, Trollhattan, Sweden, *** Institute of Lung and Heart Diseases, Sahlgren's hospital, Gothenburg, Sweden Abstract In a prospective case-control lung cancer study in the West of Sweden, the relationship between lung cancer, smoking and dietary factors has been investigated. Suspected cases were collected from pulmonary units at two central hospitals in the area investigated and population controls of the same age and sex were selected from registers. They were interviewed by specially trained nurses, using a food frequency questionnaire. The lung cancer diagnosis (ICD 7, 162.1) was made using data from the local cancer register. In an analysis based on 308 cases and 504 controls, a dose-related increase in lung cancer risk for smokers was found although no significant risk was found for males smoking 1-10 cig/day for less than 20 years. A lower consumption of vegetables was related to a higher risk, both for smokers and nonsmokers. A higher consumption of milk was related to an increased risk. Introduction It is common knowledge that an increased risk for lung cancer has been related to several different agents in the environment such as tobacco smoke, coke oven emissions and radon. The different incidence figures for lung cancer between different countries, also among nonsmokers, suggest that environmental agents can modify the risk. On a worldwide basis, food habits show large differences between different populations. There is overwhelming epidemiological evidence that dietary factors are related to decreased or increased risks for several different forms of cancer. A reduced risk for lung cancer related to the intake of vitamin A was first suggested by Bjelke (2). Since then, about 50 studies have been published and several of these have been analyzed in two major reviews (3,4). The findings are generally that fruit and vegetables are protective factors (9,13) and a high consumption of fat (15,18) and milk (14) increases the risk. S Against this background, a major reason for the variation in lung cancer incidence in populations in various parts of the world (12,20), could be differences in diet and other life style habits, which ~ influence the risk for lung cancer (5,7). As smokers deviate from nonsmokers in many lifestyle factors (19), these could be confounders in studies on smoking and lung cancer and need to be controlled for, N to obtain accurate risk figures. ~ co v tb w 0 , 0) 00 I

I I I 1 I I I I I I I I I I I I I I The data on milk consumption exemplifies the presence of natural risk factors in the diet. Similar results are reported from previous studies (14) and it has been suggested that the responsible agent is the fat in the milk (18). The dose-response for lung cancer and smoking demonstrated the expected dose-response relationship for number of cigarettes smoked and the number of years smoked. When the results were adjusted for vegetable intake, the odds ratios were almost unchanged. The number of years smoked was the most important dose determinator. The data did not demonstrate an increased risk among persons smoking less than 10 cigarettes/day and less than 20 years. The confidence levels in this group were, however, rather wide (0.25-3.38) and a larger material would be required to verify this finding. The findings in this study support a hypothesis of a balance between risk factors for a disease and protective factors. The eventual outcome of the balance between these factors determines the development of disease. The epidemiological implication of this and other studies is that investigations on lung cancer and environmental agents need to consider dietary factors as confounding agents, particularly as the consumption of risk or protective food items are different among nonsmokers and smokers. Acknowledgements This study was supported by the Swedish Cancer Foundation (contract 90-1137), the Jubilee Clinic Research Foundation, Gothenburg, Sweden, the Department of Community Medicine, Alvsborg County, Forschungsgesellschaft Rauchen and Gesundheit, MHB, Hamburg, Germany. a V 00 W O -7- ~ i

A prospective study has been undertaken in the West of Sweden with the aim to investigate the risk for lung cancer in relation to different environmental factors. The origin of the study was observations of an increased risk for lung cancer among tea drinkers (6,10,16,17), but the scope was extended to incorporate dietary factors of relevance for the Swedish population. This preliminary report from the study reports the data among males and describes some dietary characteristics for smokers and nonsmokers among population controls, and the risks associated with smoking, vegetable, and milk consumption. Materials and Methods Study base, cases and controls The study base comprises persons up to and including 75 years of age of Scandinavian origin and who were registered as residing in one of 26 municipalities in Goteborg and Bogus county and Alvsborg county in the southwest of Sweden. The municipalities were selected to represent the area from which patients with suspected lung cancer were referred to the pulmonary units at the regional hospitals. Routines were established for identifying suspect lung cancer cases at three (later two) hospitals in the region. Patients referred to the outpatient department at these hospitals, and who were suspected to have lung cancer, based primarily on changes detected on lung X-rays, were invited to take part in the study. A regular control was also made of in-patients at the hospitals to ensure that lung cancer cases in the study base who had been admitted directly to the wards were included in the project. Patients willing to participate in the study were contacted for an interview. Twice a year, a search for the patients was made in the regional cancer registry. They were finally classified as lung cancer cases only if they were present in the registry. To select population controls, a list of personal identification numbers of all suspected lung cancer cases in the study base was sent to the local tax authority. For each patient, the two persons within the respective areas of the two counties, who were of the same sex as the patient and were closest to the patients in the order of the personal identification number were selected. The first person was selected unless he was an immigrant in which case the second person was selected. If a control person was a non- respondent, a substitute was not selected. A search for the population controls was also made in the cancer register. Questionnaire The questionnaire included questions on smoking, environmental tobacco smoke (ETS), occupational exposures, conditions in the residential area (local air pollution) and dietary habits. The section on diet consisted of 37 questions divided into four blocks and covered the intake of over 80 food items. The frequency questions were "seldom or never", "once or twice/month", "once or twice/week", "daily or almost daily" and for some food items "several times/day...how many?" The questions referred to eating habits during the last year. -2- I I , I I I I I I I I I I I I I I I I

i I I i I I i 15. Shekelle, RB; Rossof, AH and Stamler J. Dietary cholesterol and incidence of lung cancer: The western electric study. Am. J. Epidemiol. 134:480-484, 1991. 16. Stocks, P. Cancer mortality in relation to national consumption of cigarettes, solid fuel, tea and coffee. Br. J. Cancer 24:215-225, 1970. 17. Tewes, FJ; Koo, L; Melsgen, TJ and Rylander, R. Lung cancer risk and mutagenicity of tea. Env. Res. 52:23-33, 1990. 18. Wynder, EL; Herbert, JR and Kabat, GC. Association of dietary fat and lung cancer. JNCI 79:631, 637, 1987. 19. Whichelow, MJ; Golding, JF and Treasure, FP. Comparison of some dietary habits of smokers and nonsmokers. Br. J. Addict. 1988, 83:295-304. I 20. Wynder, EL; Taioli, E and Fujita Y. Ecologic study of lung cancer risk factors in the U.S. and  Japan, with special reference to smoking and diet. Japan J. Cancer Res. 83:418-423, 1992. .. I I I I I I I I O 00 i -1 , -9- tb W O V O) I

I I It is seen that the odds ratios for lung cancer decreased with an increase in vegetable consumption. In the group with the highest consumption, the odds ratio was less than 0.5. No such relationship was found for fruit consumption (data not shown). Figure 6 shows the odds ratios for lung cancer in relation to consumption of milk. OR 4 3 I I I I I I 1- 2/month 1- 2/week daily sev times/d I Figure 6. Lung cancer odds ratio in relation to milk consumption. The figure illustrates that the odds ratio increased progressively, with an increase in milk consumption. Comments In the design of the study, we tried to minimize the influence of methodological errors by actively working for high participation rates and accurate descriptions of the personal characteristics. Regarding participation rates, several previous studies have reported between 65 and 75% and some studies even less than 50%. As it is known that risk factors are related to nonparticipation, the risk estimations in the present study are probably more accurate. The information on individual exposures was obtained in personal interviews. This secures more reliable information than that obtained through mailed questionnaires or through interviews with relatives - techniques which have been used in many previous studies. The results from this study confirm numerous previous reports that vegetables are protective against the risk for lung cancer (3,4,9). This related to nonsmokers as well as smokers. Regarding fruits, a protective effect could not be demonstrated. From a methodological point of view, this may reflect a smaller range in the consumption habits in the population studied as compared to previous studies where a protective effect has been found. On the other hand, a difference in consumption was found between nonsmokers and smokers which suggests that even the persons with a low consumption could have reached a level which gave them protection. -6- I I ' I , I 4

I I I 1 ' I , I I I I I I I I I The proportion of high consumers of coffee was significantly larger among 20+/day smokers than among controls. For other diet items, there were no significant differences between smokers and nonsmokers, either as a group or divided into different smoking classes. Figure 4 illustrates odds ratios for lung cancer risk among smokers, as compared to nonsmokers. OR agalnst non- smoker 120 50 20- 29 30-39 40-49 years smoked Figure 4. Lung cancer risk and smoking. A dose-response was present, both regarding number of cigarettes smoked and the number of years smoked. Among these variables, the strongest dose-response was found for the number of years smoked. For those who had smoked less than 10 cigarettes a day, and less than 20 years, no significant increase in risk as compared to nonsmokers could be found. Figure 5 reports the risk ratios for lung cancer and the consumption of vegetables, in terms of vegetable classes. 0 1 vegetable class , Figure 5. Lung cancer odds ratio in relation to vegetable class. ~ i V ~ -5 W O V N k

i I I I I I I I I I I I I , I I I I When analyzing the data, a vegetable index was formed by amalgamating the intake of carrot, tomato, cabbage, green pepper and lettuce. The consumption of each of these vegetables was weighted as 0 for seldom/never or once/twice per month, 1 for once/twice per week and 2 for daily/almost daily consumption. The sums were divided into three classes: 0-1 (vegetable class 0), 2-4 (class 1) and 5-10 (class 2). The lowest class thus indicates that the subject consumed not more than one of the five vegetables once or twice/week. A similar index was constructed for fruits. Interviews The interviews were performed by two nurses who had been employed and specially trained for the project. In most cases the interviews were made within a few days after the suspect cases had been identified at the hospital. Thus, the interview could generally be conducted before the diagnosis was established or before the patient's condition had become so serious that an interview could not be carried out. Interviews with controls usually took place at the department or at home within 4 to 8 weeks of the patient interview. Status otthe study The recruitment of patients started in January 1989. There were breaks each summer between June and September and also a break between May 1992 and February 1993. This paper describes the analysis of all male cases and population controls interviewed between January 1989 and June 1993. Of the 344 cases, 308 (90%) were interviewed and of 644 controls, 504 (78%) were interviewed. Statistical treatment of data For estimation of odds ratios, logistic regression models were fitted to the data with the EGRET software package for unconditional maximum likelihood estimation of the regression parameters. In all analyses, there was an adjustment for age, number of cigarettes/day, number of years smoked (continuous variables), marital status (four classes) and socioeconomic job classification (seven classes). Results Figure 1 reports smoking habits among cases and controls. cases controls Never smoker .'' Former smoker - Current smoker Figure 1. Smoking history. Nonsmokers represented 5% of the cases and 32% of the controls. -3- I

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I Conclusion Unless measures are taken to control cigarette consumption, deaths due to chronic disease, including lung cancer, will increase rapidly. -2- I I I I I I I I I I I I I I , '

I I I I I I I I I I I I I I I I I I significantly higher for squamous cell carcinoma cases (453) than for controls (417). Other parameters related to menstruation showed no appreciable differences between the two groups. A greater number of menstrual cycles could imply a greater frequency of elevated estrogen levels. Since estrogens are known to stimulate growth of cells, especially the epithelial cells, they may be a candidate for induction of squamous cell carcinoma. On the other hand, the total number of life-time menstrual cycles for the adenocarcinoma cases were not found to be higher than the controls. In fact, adenocarcinoma cases showing positive ERs and PRs actually had a later menarche and an earlier menopause. These observations suggest that sex hormone, as well as levels and functions of receptors may be involved in controlling the growth of lung cells. Our study found that the 162 adenocarcinoma cases had shorter menstrual periods than controls. The length of the menstrual period is often related to the regularity of the follicular cycles which in turn depends on the functions of the corpus luteum. A hyper-active corpus luteum function, for example, would shorten menstrual periods. Accordingly, it may be hypothesized that adenocarcinoma patients have a more active corpus luteum, compared to the controls. In conclusion, studies of female lung cancer risk factors must incorporate measurement of sex hormone levels, and assay of progesterone/estrogen receptor expression. Moreover, considerations must be given to the interrelationship of the hypothalamus-pituitary-ovary axis. Further case-control studies integrating assessment of endocrinological parameters and epidemiologic approaches are needed in order to understand the significance of sex hormone levels and receptor functions on female lung cancer. -5- ,

References 1. Stjernsward, J.; Stanley, K.; "Etiology, Epidemiology and Prevention," Lung Cancer 4 (Supp.): 11-14, 1988. 2. Zheng, W.; Gao, Y.T.; Sun, L.; "A Study of the Association of Menstrual History and Lung Cancer," Tumor 8(3): 150-152, 1988. 3. Gao, Yu-Tong; Blot, W.J.; Zheng, W., et al.; "Lung Cancer Among Chinese Women," Int_J. Cancer 40: 604-609, 1987. 4. Zheng, W.; Blot, W.J.; Liao, M.L., et al.; "Lung Cancer and Prior Tuberculosis Infection in Shanghai," Br. J. Cancer 56(4): 501-504, 1987. 5. Du, Y.X.; "Progress in Lung Cancer Prevention Research in Guangzhou," First Seminar of Guangzhou Research Center for Lung Cancer, Guangzhou, China, May 10, 1985. -6- I I I I I I I I I I I I I I I 1 I I I

I References 1. Alavanja, MCR; Brown, CC; Swanson, C and Brownson, RC. Saturated fat intake and lung cancer risk among nonsmoking women in Missouri. J. Natl. Cancer Inst. 85:1906-1916, 1993. 2. Bjekle, E. Dietary Vitamin A and human lung cancer. Int. J. Cancer 1975, 15:561-565. 3. Block, G; Patterson, B and Subar, A. Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutr. Cancer 18:1-29, 1992. 4. Fontham, E. Protective factors and lung cancer. Int. J. Epidemiol. 19:24-31, 1990. 5. Fraser, G; Beeson, L and Phillips, R. Diet and lung cancer in California seventh-day adventists. Am. J. Eoidemiol. 133:683-693, 1991. 6. Heilbrun, LK; Nomura, A and Stetnmermann, GN. Black tea consumption and cancer risk: A prospective study. Br. J. Cancer 54:677-683, 1986. 7. Hinds, MW; Stetttmertttatm, GN; Yang H-Y; Kolonel, LN; Lee, J and Wegner, E. Differences in lung cancer risk from smoking among Japanese, Chinese and Hawaiian women in Hawaii. Itrt. J. Cancer 27:297-392, 1981. 8. Jain, M; Burch, JD; Howe, GR; Risch, HA and Miller, AB. Dietary factors and risk of lung cancer: results from a case-control study. Toronto 1981-85. Int. J. Cancer 45:287-293, 1990. 9. Kant, AK; Block, G; Schatzkin, A and Nestle, M. Association of fruit and vegetable intake with dietary fat intake. Nutr. Res. 12:1441-1454, 1992. 10. Kinien, U; Willows, AN; Goldblatt, P and Yudkin, J. Tea consumption and cancer. Br_J. Cancer 58:397-401, 1988. 11. Knekt, P; Jarvinen, R; Seppanen, R; Rissanen, A; Aromaa, A; Heinonen, 0; Albanos, D; Heinonen, M; Pukkala, E and Teppo, L. Dietary antioxidants and the risk of lung cancer. Am. J. Epidemiol. 134:471-479, 1991. 12. MacLennan, R; Da Costa, J; Day, NE; Law, CH; Ng, YK and Shanmugaratnam, K. Risk factors for lung cancer in Singapore Chinese, a population with high female incidence rates. Int. J. Cancer 20:854-860, 1977. I I I I I I I I I I I I I I 13. Marchand, LL; Yoshizawa, CN; Kolonel, LN; Hankin, JH and Goodman, MT. Vegetable consumption and lung cancer risk: A population-based case-control study in Hawaii. J. Natl. I Cancer Inst. 81:1158-1164, 1989. 8 00 14. Mettlin, C. Milk drinking, other beverage habits and lung cancer risk. hat. J. Cancer 43:608- 612, 1989. -8-

I I I I I I I I I I I I I I I I A STUDY OF ASSOCIATION OF FEMALE SQUAMOUS CELL CARCINOMA AND ADENOCARCINOMA IN THE LUNG AND HISTORY OF MENSTRUATION Liao Mei-lin, Wang Jian-hwa, Wang Hwei-min, Ou Ai-qin, Wang Xiao jun and Long Wan-qing Shanghai Chest Hospital, Shanghai, China Abstract 181 cases of female squamous cell carcinoma and adenocarcinoma of the lung and 187 normal female controls were age-matched to compare the menstrual history of the two groups. The Epi-infor program, the Chi-square test, or the Bartlett test for homogeneity of variance, were used to evaluate possible statistical significant differences existing between the two groups. Parameters related to menstrual history included: age of menarche, menstrual cycle, number of days of menstrual period, amount of menstrual flow, menstrual pain, breast bloating/tenderness, and total number of inenstrual cycles prior to menopause or diagnosis of lung cancer. The results show that squamous cell carcinoma cases have a higher total number of menstrual cycles than controls, raising the possibility that estrogen may play a role in the induction of squamous cell carcinoma. Since adenocarcinoma cases were found to have shorter menstrual periods than controls, it may be proposed that activity of the corpus luteum is related to the occurrence of adenocarcinoma. Progesterone (PR) and estrogen (ER) receptor levels were also measured in 21 surgical specimens of adenocarcinomas. A positive ER and PR receptor expression was correlated with later menarche and earlier menopause. Biological implications of these findings must be further investigated. Introduction The association of smoking and lung cancer is well known. According to a 1989 World Health Organization report, over 97% of the nations of the world held the view that a history of smoking probably accounted for 80% of the lung cancer cases, especially in the ones involving squamous and small cell carcinomas(1). On the other hand, with regard to the etiology of lung adenocarcinoma, risk factors other than smoking have been proposed and identified, especially in nonsmoking females. A 1988 Shanghai population-based epidemiologic study reported that the development of female lung adenocarcinoma was associated with menstrual and reproductive history, thus raising the possibility that female lung cancer may be related to female sex hormones. The present study compares data on menstrual history using female lung cancer cases in the Shanghai Chest Hospital and controls drawn from healthy current or retired hospital employees. A detailed questionnaire was administered in order to obtain information on menstrual history. The data were analyzed to determine whether an association possibly exists between menstrual history and lung cancer in females. Our results may contribute to a better understanding of the etiology of squamous cell carcinoma and adenocarcinoma of the lung. Materials and Methods N O co ' From January 1993 to June 1994, the files of all the in-patients and part of the out-patients of the j Chest Hospital were randomly screened. Females, between 37-77 years of age, with cytologically and ~ histologically confirmed lung squamous cell carcinoma and adenocarcinoma were identified and used as V ~ tb ,

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I I I I I I .1 I I I I I I I I I COMBINED ANALYSIS OF CASE•CONTROL STUDIES OF SMOKING AND LUNG CANCER IN CHINA Yu Shun-zhane and Zhao Ning Shanghai Medical University, Shanghai, China Introduction Despite the fact that adverse health effects of smoking have been well known for many years, smoking continues to increase in China. According to a survey of 0.5 million people, the smoking rate for males ?20 years old was 68.9%. A large percentage of the population smoke and 1,400 billion cigarettes are consumed annually. In some cities and counties lung cancer is the leading cause of death. Methods By surveying and screening the literature, 15 case-control studies on active smoking and 3 case- control on passive smoking were selected and analyzed. The total numbers of lung cancer cases were 6,085 and there were 6,328 controls. Using meta-analysis, we applied the fixed and random effects models to test for their heterogeneity according to (Peto, DerSimonian and Laird), the pooled Odds Ratios (ORs), and 95% Confidence Intervals (95%CI). The pooled Population Attributable Risk (PAR) was calculated by the method of Levin and Bruzzi, and the Mantel test was used for trend. Results 1. The proportion of smokers among the lung cancer cases and controls was 69.09% and 31.15 % respectively. 2. The pooled OR (smoking vs nonsmoking) was 2.19 (95%CI 2.03-2.37) and the pooled PAR was 33.64%. There were no significant differences between males (OR=3.01, 95 %CI: 2.63-4.46) and females (OR=2.32, 95 %CI: 2.02-2.66). According to exposure rates, PAR were 56.84% for males and 33.10% for females. 3. The number of cigarettes smoked, the smoking duration, and the age of beginning to smoke were correlated with an elevated Odds Ratios of risk for lung cancer. There was a significant trend for amount of cigarette consumption: for example the OR=1.00 for nonsmoking, OR=1.24 for < 10 cig./day, OR=2.19 for 10-19 cig./day, and OR=4.47 for z 20 cig/day. Chi-square (X2) for trend was 223.13 (P<0.01). 4. Smoking is associated with squamous cell carcinoma (OR=4.79, 95%CI 4.02-5.70) but not adenocarcinoma (OR= 1.02, 95%CI: 0.87-1.20). , 5. Although passive smoking has been suggested to be an important risk factor for lung cancer, p the OR was 1.004 (95 %CI: 0.74-1.85) and, therefore, not statistically significant, and the PAR was only j 0.16% in this data set. -1 tb 0 0 tn I

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a I I I I I I I I I I I I I I I I I of breast bloating/tendemess was also significantly different between these two groups (variance = 30.84, degree of freedom = 1, P = 0.000032). Specifically, the adenocarcinoma group had only slight and infrequent "breast bloating/tenderness" and history of menstrual pain. Likewise, statistically significant differences were also found between the two groups with regard to the amount of menstrual flow (P < 0.005). No apparent differences were found in the total number of menstrual cycles, age of menopause and age of menarche between the two groups. (Table 1) 3. Comparison of Menstrual History Between Squamous Cell Carcinoma and Adenocarcinoma Cases. There is no difference between the age of the two groups. The mean menarche ages were 14.79 and 15.488 respectively (P < 0.05), suggesting an earlier menstrual onset for squamous cell carcinoma cases compared to the adenocarcinoma cases. There was no apparent difference in the age of menopause, the length of menstrual period, menstrual cycle, the amount of menstrual flow, and menstrual pain between the two groups. The total number of menstrual cycles was higher for squamous cell carcinoma cases (453.2) than for adenocarcinoma cases (413.3). The difference was statistically significant (P < 0.05). (Table 1) Table 1. Relationship Between History of Menstruation and Female Pulmonary Squamous Cell Carcinoma and Adenocarcinoma Ademcutivmu' C.nmot P. VaLe . F,H P F,H P F,H P Curu 19 181 187 A8e ef inemrche 14.5'/9 15.488 15.3n5 6.085 <0.05 1.858 >0.05 2.197 >0.05 MeemW cycle 27.895 28.895 28.802 1.369 >0.05 0.211 >0.05 1.602 >0.05 Lrngih of 5.368 4.852 5.289 3.211 >0.05 0.053 >0.05 8.703 <0.01 AgeofMrnppwae 50.500 49.273 48.774 1.672 >0.05 2.436 >0.05 1.025 >0.05 Nmnber of IaW 453.176 413.319 413.269 5.014 <0.05 4.818 >0.05 0 >0.05 Amawr m Light 2 26 21 Hary 0 38 52 Mad'~ 17 98 114 Pmnevwal Never 10 104 110 Occas1® 5 35 13 LigM 2 20 42 Medvm 1 1 9 Huvy 1 2 13 Meuswil Pam Nevu Il 108 125 Occasica 5 29 13 L~c I 1a 29 Med'oun 1 3 7 Hary 'Number of life-time meustrual cycles = [(age of menopause age of inenarche) x3651 meastioal cycle (in days) -3- I

cases. These included 22 surgical cases in which the surgically-resected specimens were also analyzed for female sex hormone (progesterone (PR) and estrogen (ER)) receptors. Controls, aged 35-78, were drawn from healthy current and retired employees who were determined by physical examination to be free of tumors or other serious disease. Controls were matched for age (± 5 years) with cases. Standardized questionnaires were filled out by trained professionals for both groups. Inquiries were made concerning the following items: age of inenarche; menstrual cycle (in days); length of menstrual periods; whether menstrual flow was heavy (as to affect physical activities), medium (without affecting activities, although the amount was not scanty), or light (basically no awareness that there was a flow); degree of premenstrual breast bloating/tenderness (scored as never, occasional, slight, medium or strong); and menstrual pain (scored as never, occasional, light, medium or strong). Because the ages of menarche and menopause do not completely show the cyclic nature of endocrine functions/changes in females, a formula was used to calculate the menstrual cycle index (MCI) according to the following equation: MCI = (age at which menopause occurs or age at which lung cancer was diagnosed - age of menarche) x 365 + menstrual cycle (in days). The data, from the three groups, i.e., squamous cell carcinoma, adenocarcinoma, and controls, were entered into Fox Base and then analyzed for statistical significance by using the Epi-infor software, or by applying the chi-square (XI) test, or the test for homogeneity of variances according to Bartlett. Data which showed P < 0.05 in the Bartlett test, hence indicative of an abnormal distribution of data points, were further analyzed for statistical significance by the Kruskal-Wallis H value method. These data giving a P of greater than 0.05 in the Bartlett test, and hence indicative of a normal distribution, were evaluated for statistical significance by the ANOVA F method. Results A total of 368 females were analyzed. These included 181 lung cancer cases (19 squamous cell carcinoma and 162 adenocarcinoma) and 187 controls. Comparison of Menstrual History Between Squamous Cell Carcinoma Cases and Controls. The ages of both groups showed a similar normal distribution and there was no statistically significant differences between the two groups (P > 0.05). With regard to menstrual history, no difference was found on age of menarche, length of menstrual period, menstrual cycle, degree of premenstrual breast bloating/tenderness, menstrual flow, menstrual pain, and age of menopause (each with P > 0,05). However, when the total number of menstrual cycles prior to menopause or to diagnosis of lung cancer (MCI) was tested for homogeneity of variance, a mean value of 453.17 was obtained for cases, compared to 413.3 for controls. Since the homogeneity of variances test showed normal distribution of data points, the two groups were evaluated for statistical significance by ANOVA. A P value of less than 0.05 was obtained indicating that squamous cell carcinoma cases have a higher total number of menstrual cycles than the controls. 2. Comparison of Menstrual History Between Adenocarcinoma Cases and Controls. The ages of both groups also showed a normal distribution (P > 0.05). The adenocarcinoma cases, however, had shorter menstrual periods (mean = 4.85 days) than the controls (mean = 5.29 days) with a medium value of 5 days. When these values were tested for homogeneity of variance, a P value of less than 0.005 was obtained indicating high statistical significance. In addition, the frequency/severity -2- I I I I I I , I I I I I I I I I I I I

I 4. Analysis of Estrogen and Progesterone Receptors and History of Menstruation in the 22 Surgical Cases of Female Squamous Cell Carcinoma and Adenocarcinoma. Of the 22 surgical cases analyzed, 21 were adenocarcinomas and only one had squamous cell carcinoma. Positive estrogen receptors (ER) were found in 16 cases (72.7% of total cases), 15 of which were adenocarcinoma and 1 was squamous cell carcinoma. Fourteen cases (representing 63.6% of total cases and 66.6% of adenocarcinoma cases), all adenocarcinoma were also found to be positive for progesterone receptors (PR). The relationship between sex hormones and the history of menstruation is illustrated in Table 2. Table 2. A Comparison of Menstrual Histories of 21 Female Adenocarcinoma Cases with Positive and Negative of Hormone Receptors +. No. EM01- ~ S' C. 15 71.4 6 2A.6 14 66.6 7 33.4 Meruche (Age) 15.1 13.8 <0.05 14.2 <0.05 Numbv af D.y, of Memuv.l Pulod 5 5 >0.05 5 5 >0.05 MemuW CSCIe(Days) 28.0 28.6 <0.05 28.1 2&4 <0.05 Menywtt (Aye) 45.7 48.4 <0.05 47.3 49.8 <0.05 Adenocarcinoma cases with positive ER and PR had later menarche than those with negative ER and PR, P < 0.05. The age at which menopause occurred was also earlier for the adenocarcinoma cases with positive ER and PR than those with negative ER and PR, P < 0.05. There was no difference in the number of days of the menstrual period or menstrual cycle, and no difference in breast bloating/tenderness, menstrual pain and amount of menstrual flow. Discussion In attempting to understand the etiology of lung cancer in females, an effort has been made to study risk factors other than smoking. For example, deficiency of vitamin A being associated with the occurrence of lung cancer has been supported both by epidemiological research and in animal experiments. Many studies in China have explored nonsmoking risk factors, such as cooking fumes(3), history of tuberculosis(4), burning of coal(5), and menstrual history(2). This study is a statistical analysis of the menstrual history of 181 female lung squamous cell carcinoma and adenocarcinoma cases and 187 age-matched normal female controls. The purpose was to find out whether similarities or differences existed in the menstrual history between the squamous cell carcinoma and adenocarcinoma cases and whether the occurrence of female lung squamous cell carcinoma/adenocarcinoma is related to the status of sex hormones. The results of our study show that the total number of menstrual cycles prior to menopause or before lung cancer was diagnosed were -4- I I I I I , I I I I I I I I

passive smoking (P<0.001). No significant differences were found between the cases and controls when exposure was at ages 23-30 and 31-69 years. When stratified by smoking status, the risk was significantly increased for the ever-smokers who were exposed at ages 22 or younger, likewise, the risk also increased for the nonsmokers who were exposed under the age of 15 years. Conclusions The findings of this study suggest that (1) household passive smoking, particularly that occurring during childhood, increases the risk of female lung cancer, and (2) the effects of PS on lung cancer, and possibly on other health conditions, should be assessed by different periods of exposure. -2- N 0 tp ~ ~ w 0 CO (D I I I I I I I I I I I I I I I I I I I

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increase of 4.7 %. The lung cancer mortality rate for Guangzhou is the third highest in China, lower only than that for Chongquing and Shanghai. The lung cancer death rate is equal to the death rates for liver, stomach, and laryngeal cancers combined, making it the highest among all cancers.(1) Now that smoking is established as one of the most important risk factors of lung cancer(2), the other risk factors warrant our further attention. Consequently, the authors undertook a comparative analysis of the lung cancer risk factors of different cell types in both men and women in a case-control study from 1990 to 1993. Materials and Methods 1. Study Subjects. The cases were drawn from current inpatients in the city of Guangzhou from the five affiliated hospitals of Sun Yat-sen Medical University, Ji-nan Medical College and Guangzhou Medical College, whose diagnoses of primary lung cancers had been pathologically confirmed. The controls consisted of non-malignant tumor patients who were hospitalized during the same time period at the same hospitals as the cases, and who were found to be free of any lung diseases. The cases and controls were matched for sex, place of residence, educational level, age (± 5 years) and were required to be either Guangzhou natives or residents of Guangzhou for over 20 years. 2. Survey Items. Items included in the survey were general health conditions, history of lung disease, family history of tumors, smoking status, passive smoking (at home and at work), dietary habits, kitchen ventilation, use of oral contraceptives, socio-psychological factors (Eysenck's Personality- Stress Inventory items). 3. Methods of Analysis. The data were subjected to single-factor logistic regression analysis with X2 test, conditional multi-variate logistic regression analysis, and analysis by log-linear model. The analyses of data were performed on the AST-386 computer with the SAS 6.03 software. Results 1. Gender and Patholo¢v Tvoe. The 390 primary lung cancer cases, aged 32-78 with an average age of 56.6, had a male to female ratio of 2.9:1. The overall squamous cell carcinoma to adenocarcinoma ratio was 1.2:1, with a predominance of squamous cell carcinomas among the male cases (squamous cell carcinoma:adenocarcinoma = 1:0.5) and a majority of adenocarcinomas among the female cases (squamous cell carcinoma:adenocarcinoma = 1:2.7). (See Table 1) -2- I I I I 1 I I I I I I I I I I

a I I I 1 I I I I I I I I I I I I I Table 3. Results of Conditional Multi-variate Logistic Analysis Data Regression Coefficient Standardized Regression Coefficient OR 95% Cl X? p X18 0.94 2.96 2.57 1.37-4.80 8.76 <0.01 X20 1.24 5.00 3.45 2.12-5.61 25.00 <0.005 X27 0.94 2.47 2.57 1.21-5.43 6.10 <0.05 X30 1.06 4.10 2.88 1.74-4.77 16.81 <0.005 X48 0.58 2.26 1.79 1.08-2.97 5.12 <0.05 X54 0.52 2.58 1.68 1.13-2.48 6.66 <0.01 X71 0.55 2.31 1.73 1.09-2.75 5.34 <0.05 4. Log-Linear Model Analysis. The results of two-factor, one-stage analysis showed significant interaction between history of chronic bronchitis/emphysema, history of tuberculosis, passive smoking, smoking and lung cancer. Interactions were also found between history of chronic bronchitis/emphysema and history of tuberculosis, between history of chronic bronchitis/emphysema and smoking or passive smoking, between history of tuberculosis and passive smoking. No significant interactions, however, were found in the three-factor or more-than-three-factor, two-stage analysis, indicating the interactions among them were not enough to affect the interactions between history of chronic bronchitis/emphysema, history of tuberculosis, smoking, passive smoking and lung cancer. (See Table 4) -5- I

I I I I I I I I I 1 1' I Table 1. Comparison of Pathology Types in Male and Female Lung Cancer Sex Squamous Cell Carcinoma Adenocarcinoma Large and Sma1I Cel1 Cancer - Other Tbtai Male 150 (51.6) 81 (27.8) 29 (10.0) 31 (10.6) 291 Female 21 (21.2) 57 (57.6) 5 (5.1) 16 (16.1) 99 Total 171 (43.8) 138 (35.4) 34 (8.7) 47 (12.1) 390 * Percentages are given in parentheses. 2. Comparison of Single-Factor Analysis Results. By single-factor conditional logistic regression analysis, at the single-side a = 0.05 level, the following 11 risk factors were identified as risk factors for lung cancer: family history of tumors, family history of lung cancer, history of chronic bronchitis/emphysema, history of tuberculosis, history of other lung diseases, smoking, passive smoking at home, passive smoking in the workplace, being professional drivers, use of oral contraceptives, and consumption of pickled and cured food. Six or seven of the above items were identified as risk factors for both male lung cancer and lung squamous cell carcinoma. Female lung cancer was found to be associated with four risk factors, i.e. family history of lung cancer, smoking, passive smoking in the home, and the use of oral contraceptives (XZ = 5.00-6.56, P < 0.05). The only significant risk factor for adenocarcinoma was a history of chronic bronchitis/emphysema (X2 = 4.69, P < 0.05). (See Table 2) -3- I

I I I I I 1 I 1 I I I I I I I I I A CASE-CONTROL STUDY OF CHILDHOOD AND ADOLESCENT HOUSEHOLD PASSIVE SMOKING (PS) AND THE RISK OF FEMALE LUNG CANCER Wane Fu-lin¢*, Edgar J. Love** and Dai Xu-dong* * Heilongjiang Institute for Cancer Research, Harbin, China ** University of Calgary, Calgary, Canada Summary Backeround Exposure to environmental tobacco smoke, i.e., passive smoking (PS), has been a public health concern. This type of exposure has been suggested to be associated with many illnesses, such as cancer (especially lung cancer), cardiovascular disease, respiratory system signs and symptoms, etc. However, how to measure this exposure and its effects on health remains a key issue. Most previous studies estimated PS from only the husband or the spouse, which is far from accurate. More importantly, the effects of early life exposure to tobacco smoke on health during adulthood have not received much attention. Only a few studies have considered the importance of exposure to tobacco smoke in early life on risk of cancer in adulthood; several studies did not find any association between lung cancer and nonsmoking women exposed to tobacco smoke during childhood. Methods This study, using household exposure to tobacco smoke as an estimate of PS, was done to evaluate the risk of female lung cancer from PS, and focused especially on exposure during childhood and adolescence. The 1:1 paired case-control study was conducted in Harbin, China during 1985-87. We personally interviewed 114 female primary lung cancer cases, aged 30 to 69 years, and their hospital- based controls, using an established questionnaire. The controls were noncancer patients, selected from the same hospital as the cases, and matched for age (± 5 years), residential area and lifetime smoking status. There were 59 pairs who smoked and 55 pairs who never smoked. Information on PS was collected by residence for each of the following five periods: 0-6, 7-14, 15-22, 23-30 and 31-69 years. Results We found that the risk of female lung cancer in relation to PS varied by exposure period and by sources of sidestream smoke. For the "0-14 years" exposed group, the exposure was mainly from parents. A higher percentage of the cases had exposure from mothers (38.5 %) than the controls (26.4%). For the older age exposed group, husbands were the major source of sidestream smoke and accounted for more than two-thirds of the exposure for both cases and controls. Further analyses show that when exposed under the age of 14 years, the risk of lung cancer was significantly increased for household exposure to maternal smoking (odds ratio, OR=2.70, 95% CI=1.49-4.88), but not for exposure to paternal smoking (OR= 1.40, 95% CI= 0.79-2.50). The risk was the highest in the group who were exposed under the age of seven (OR=3.46, 95 % CI=1.80-6.65), but was also significant when exposed at ages 7-14 (OR=3.08, 95% CI=1.62-5.57) and 15-22 (OR=3.10, 95% CI=1.52-6.31). Moreover, as long as exposure occurred under the age of 23 years, the OR increased with amount of exposure to I

Table 4. Log-Linear Model Analysis of Primary and Interactional Effects R.isk Factors df X2 P a (Lung Cancer) 1 4.35-5.32 0.0370-0.0211 b (History of Tuberculosis) 1 53.49-57.00 0.0001 c (History of Chronic 1 27.46-31.33 0.0001 Bronchitis/Emphysema) d (Smoking) 1 22.18-22.90 0.0001 e (Passive Smoking) 1 20.50-21.08 0.0001 a b 1 5.70-8.92 0.0168-0.0028 a c 1 8.94-22.70 0.0028-0.0001 a d 1 4.91-8.14 0.0267-0.0043 a e 1 11.92-12.02 0.0006-0.0005 c b 1 13.83-17.13 0.0002-0.0001 c d 1 11.83-13/27 0.0006-0.0003 c e 1 4.17-4.54 0.0411-0.0330 b c 1 7.19-7.87 0.0073-0.0050 a b c 1 0.31 0.5770 a b c d 1 0.27 0.1317 a b c d e 1 0.01 0.9292 G (Test of Likelihood) 25 28.04-30.32 0.3060-0.2124 -6- I I I I I I I I I I I

I I Table 2. Lung Cancer Risks By Sex and Pathology Type (OR value) Sex Pathology Type Risk Factors. TohaF mate Female Squamous . Adenocarcinoma Cell . Catciuoma.. Family History of Tumor 2.66* 2.90* 2.00 Family History of Lung Cancer 3.796 2.00 739* History of Chronic Bronchitis/ Emphysema 3.64* 3.24* 4.45* 2.27" History of Tuberculosis _ 3.06* 3.46* 1.33 13.50* History of Other Lung Disease 2.87* 3.70* 1.20 6.50* 1.02 Smoking 3.56* 3.47* 4.006 4.66* 2.22 Passive Smoking. in the Home 1.91* 1.02 2.54A 3.50* Passive Smoking in the Workplace 1.90 2.10 1.78* Professional Driver 4.00" 3.67A Use of Oral Contraceptives 3.00" 3.00" 1.79~ Diet of Pickled and Cured Food 1.614 * P < 0.01 A P < 0.05 3. Primary Effects Model. The risk factors screened out by single-factor analysis were again fit to the primary effects model for a multi-variate logistic regression analysis. The final coefficients brought into the equation were, in sequence, X18 (history of tuberculosis), X20 (history of chronic bronchitis/emphysema), X27 (family history of tumor), X30 (smoking), X48 (passive smoking in the home), X54 (passive smoking in the workplace), X71 (diet including pickled and cured food). (See Table 3) The combined OR for all the risk factors were: ln OR = 0.94 X18 + 1.24 X20 + 0.94 X27 + 1.06 X30 + 0.58 X48 + 0.52 X54 + 0.55 X71. I I I I I i I ~ I I I I I N G ~ ~ ~ -4 ~ O CD ~ -4- -P ~ t

I is estimated that 93% of the female lung cancer patients have a history of passive smoking (87% are exposed to environmental tobacco smoke (ETS) at home and 63% are exposed to ETS in the workplace). Passive smoking should not be overlooked as a risk factor for female lung cancer (OR = 2.5 95% CI 1.3-5.1, X2 = 6.56 P < 0.05). Family history of lung cancer appears to be more closely associated with female lung cancer (OR = 1.4 P< 0.05) than with male lung cancer (OR = 2.0 P> 0.05). Studies of family clusters of lung cancer have revealed that women have a higher susceptibility to the genetic factors of lung cancer(8,9). Our study has found some association of female lung cancer with the use of oral contraceptives (OR = 3.0 95 % CI 1.1-8.3, X2 = 5.0 P< 0.05); but due to the small sample size of oral contraceptive users, (17.2% of cases, 7.1 % of controls), conclusions cannot yet be drawn. In this regard, we are attempting to increase the sample base for further investigation. Since 61.5% of the male lung cancer cases and 64.9% of the lung squamous carcinoma cases have had chronic bronchitis/emphysema, tuberculosis or other lung diseases, a history of lung disease was found to have a closer relationship with male lung cancer or lung squamous carcinoma (OR = 3.2-13.5, X2 = 14.71-29.58 P< 0.01). In regard to lung adenocarcinoma, which accounts for 35.4% of all lung cancers, besides a weak association with chronic bronchitis/emphysema, no associations with other risk factors have been confirmed. I I I I I I I  '  , ~ ~ ~ C ~ tp ~ -J W O t0 ~ -8- Cp ~ I

I I I I I I I I I I t I I i I I I I I A COMPARATIVE STUDY OF THE RISK FACTORS FOR LUNG CANCER IN GUANGDONG, CHINA Wang Shen¢-vone*, Hu Yi-ling*, Wu Yi-long**, Li Xin*, Chi Gui-bo*, Chen Ying*** and Dai Wen-shan* * Medical College of Jinan University, Guangzhou, China ** Tumor Hospital of Sun Yat-sen Medical University, Guangzhou, China *** People's Hospital of the Yue-xiu District of Guangzhou, China Abstract To compare the risk factors for lung cancer of different histopathologic types in both sexes, a 1:1 matched case-control study of 390 pairs was carried out in Guangdong Province. Female lung cancers appear to differ from male lung cancers in epidenriological characteristics, pathologic types and risk factors. The 291 male lung cancer cases were predominantly squamous cell lung carcinomas (squamous cell carcinoma:adenocarcinoma = 1:0.5), whereas the 99 female lung cancer cases were predominantly adenocarcinomas (squamous cell carcinoma:adenocarcinoma = 1:2.7). The age at which lung cancer was first diagnosed was lower for females than for males (P < 0.0001). Single-factor conditional logistic regression analysis showed an association of lung cancer with family history of tumor, family history of lung cancer, history of chronic bronchitis/emphysema, history of tuberculosis, history of other lung disease, smoking, passive smoking in the home, passive smoking in the workplace, being professional drivers, use of oral contraceptives, and consumption of pickled and cured food (P < 0.05). Further multi-variate logistic regression analysis showed that family history of tuberculosis, history of chronic bronchitis/emphysema, family history of tumor, smoking, passive smoking in the home, passive smoking in the workplace and consumption of pickled and cured food were independent risk factors for lung cancer. By log-linear model analysis, it was confirmed that lung cancer had significant interactions with chronic bronchitis/emphysema, passive smoking, history of tuberculosis and smoking. Smoking, however, could explain only 115 of the incidence of female lung cancers. Since 93% of the female lung cancer cases had a history of passive smoking, passive smoking was considered one of the important risk factors of female lung cancer. Family history of lung cancer and the use of oral contraceptives were somewhat related to lung cancer in women. Except for a weak relationship with history of chronic bronchitis/emphysema, adenocarcinoma was found to have no association with the other risk factors. Introduction Lung cancer has been on the rise every year in China since the 1980s. From 1982 to 1989, the 58 National Disease Detection Centers reported an increase of 14.1 %(urban increase 43.7%) in the lung cancer death rate, with an average annual increase of 2%. From 1990 to 1992, the 145 National Disease Detection Centers reported another increase of 9.3% (rural increase 21.3%), with an average annual This study was financially supported by the Science Committee of Guangdong Province, Department of Health of Guangdong Province and Overseas Chinese Scientific Research Foundation of the Ministry of Foreign Affairs N 0 00 i V tb W O l0 a

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 , I I 1. I 2. ' 3. I 4. ' 5. I 6. 7. I 8. , 9 I . References Du, Y.X.; Zha, Q.; Chen; Y.L. et al. "An epidemiologic study of risk factors of lung cancer in Guangzhou," Guangzhou 3rd symposium on lung cancer, Guangzhou, 1992, pp. 1-23. Zhao, N. and Yu, S.C. "Meta-analysis of smoking and lung cancer in China," Chinese J. of E12idemiol. 14(6): 350, 1993. McDuffie, H.H.; Klaassen, D.J. and Dosman, J.A. "Men, women and primary lung cancer - a Saskatchewan personal interview study," J. Clin. Epidemiol. 44(6): 537-544, 1991. Sobue, T.; Suzuki, T. and Naruke, T. "A case-control study for evaluating lung-cancer screening in Japan," Int. J. Cancer 50: 230, 1992. El-Torky, M; El-Zeky, F. and Hall, J.C. "Significant changes in the distribution of histologic types of lung cancer," Cancer 65(10): 2361-2367, 1990. Osann, K.E.; Anton-Culver, H.; Kurosaki, T. and Taylor, T. "Sex differences in lung-cancer risk associated with cigarette smoking," Int. J. Cancer 54(1): 44-48, 1993. Alavanja, M.C.; Brownson, K.C.; Boice, J.D. and Hock, E. "Preexisting lung disease and lung cancer among nonsmoking women," Am. J. Epidemiol. 136(6): 623-632, 1992. Jin, Y.T. and He, X.Z. "An analysis of lung cancer family clusters in Xuan-wei," Chinese J. of Preventive Medicine 27(6): 329, 1993. Shaw, G.L.; Falk, R.T.; Pickle, L.W. et al. "Lung cancer risk associated with cancer in relatives," J. Clin. Epidemiol. 44(4-5): 429-437, 1991.  ~ I O ~ ~ -4 ' 00 . 9 W O cD cfl I

I I I I I I I .1 I Discussion Differences were found between male and female lung cancers in respect to epidemiologic characteristics, histopathologic types or risk factors. The age of diagnosis for lung cancer in females was obviously lower (X ± SE = 53.6 ± 1.0) than in males (X ± SE = 57.7 ± 0.5 P< 0.001). The average age of lung cancer occurrence in both men and women in China is 7-8 years earlier than what has been reported from abroad(3,4). Since the cases of female lung squamous carcinoma and the male lung adenocarcinoma, in proportion to all lung cancers had increased marked by from 1/10 in 1964 to 1/4 in 1985, and because the ratio of male lung squamous carcinomas has shown a slight decrease, while the ratio changes in female lung adenocarcinomas have fluctuated, the ratio of occurrence of lung squamous carcinoma and adenocarcinoma in males and females have changed from the original 9:1 and 1:10 to 1.6:1 and 1:1.5(5). The male to female ratios of lung squamous carcinomas and lung adenocarcinomas in this study are 2.4:1 and 1:2.1 respectively. (See Table 5) Table 5. Ratio of Lung Cancer Pathologic Types in Men and Women (Male:Female) Reference Counny Lwng Cancer Pathologic Type - : Squsmous Cell _Adenpcarcinpma .SSne/l.Cell - _Largr Cell Carcinoma Catcinoma Can:inOn>a 1 E1-Torky(5) U.S.A.* 9.3:1 1:10.3 1:1.5 0.8:1 1.6:1 1:1.5 1:1.8 4.1:1 2 Osann(6) U.S.A. 1.5:1 1:1.2 1:1.2 1.1:1 3 Sobue(7) Japan 2.3:1 1:1.7 1:0.7 1.6:1 4 Du, Y.X.(1) China 2.0:1 1:2.0 1:1.2 2.7:1 5 Wang (this China 2.4.t 1:2.1 1:0.5 2.0:1 report) * 1964 and 1985 data. ~ Smoking is a common risk factor for lung cancer in both men and women, but at a different level of significance (male OR = 3.5, 95% CI 2.1-5.8, X2 = 25.99 P < 0.01; female OR = 4.0, 95% CI 1.3-12.0, X2 6.05 P < 0.05). According to reports from abroad, about 9-22% of female lung cancer ~ patients and 1-13% of male lung cancer patients are nonsmokers(7). In our study, 83% of the female cases and 10% of the male cases are never-smokers. A Chinese researcher is of the opinion that "since smoking can only explain 1/4 of the female lung cancer cases, as a result, it cannot explain the increase ~ of female lung cancer incidence." (Gao, Y.T., Shanghai Tumor Research Institute, 1990). Another researcher thinks that "the rapid rise of female lung cancer in Taiwan cannot be explained by smoking alone and female lung cancer seems to be unrelated to smoking." (Yang, S.B., Medical College of ' Taiwan University, 1993). In Guangdong, very few smokers are women (4-8%), but as many as 70% N , of the men over 30 years of age are smokers. (Sun Yat-sen Medical University, 1994). As a result, it o~~o V CO ' 7 O <O V I

I I I I I I I I I I I also confirmed the mutagenicity of the contents of cooking fume. Our study found cooking fumes to be a common risk factor for both squamous carcinoma and adenocarcinoma in the lung. The respective relative risk was 3.8138 and 2.9943 and the respective PAR was 51.56% and 46.99%. Therefore, control of cooking oil-food pyrolysis and improvement of kitchen exhaust systems should be emphasized. At present, many urban residents of China still use coal and charcoal as fuel for daily living, and in winter coal stoves are used for heating. These are primary causes of indoor air pollution. Our study shows a close relationship between coal stove use and lung squamous cell carcinoma. Family tumor history is also a common risk factor for both squamous cell carcinoma and adenocarcinoma of the lung. Those with family history of tumor should avoid being exposed to other risk factors. Chronic bronchitis is related to the occurrence of pulmonary adenocarcinoma with RR value of 2.4124, and PAR value of 0.1724. Therefore, timely treatment of respiratory disease may help to reduce incidence of pulmonary adenocarcinoma. ~ ~ N O CD ~ ~ 00 -5- W ~ 0 ~ tr ~

References 1. National Environment Protection Bureau, "Environment of China 1991," J. of Chinese Environment 6(2): 1992. 2. Whittemore, A.S. "Estimating Attributable Risk from Case-Control Studies," Am. J. Epidemiol. 117: 76-85, 1983. 3. Walter, S.D. "Effects of Interaction, Confounding and Observational Error on Attributable Risk Estimation," Am. J. Epidemiol. 117: 598-604, 1983. 4. Bruzzi, P., et al. "Estimating the Population Attributable Risk for Multiple Risk Factors Using Case-Control Data," Am. J. Epidemiol. 122: 904, 1985. 5. Levin, M.L. "The Occurrence of Lung Cancer in Man," Acta. Intern. Cancer 19: 531-41, 1953. , I I I I I I I I I I

Data Collection The standardized questionnaire utilized information in the literature and incorporated features uniquely found in the local population and environments. Table 1 contains coding information methods and assigned values of data. Methods of Analysis Squamous carcinoma and adenocarcinoma risk factors were analyzed by conditional logistic regression; the risk factors were then subjected to analysis to estimate attributable risk(2-4). Table 1. Coding and Value Assignment of Data v.da6k . Focroc ~ ... .. : A+/1~1Dn vuiu Xl Sml®g mdex Amamt ma4ed (clgercua pcr d.y) x ycan of mol 20 X2 Degee of mhilatim' qg No: 0; SlWlavr: 1-Medium; 2-Deep: 3 , 30 Pasdvc nmolmig Nn: 0; Ya: I X4 Omryxtlooil expm¢e to cool®g Bme No: 0; Ya: 1 X5 Hubry of b[aoAd" No: 0; Yes: I X6 Humry of mCereWais Na: 0; Ya: I X7 Fwily - lti4ay No: 0; Yes: I X8 L'n•hy ryece avaage Ovmg am m lau 20 yon X9 Type of fwA in the tme (fisl "mdex)'• Noo-eolid fuel: 0; 3olid Nel: I XI0 Cod >ture for wmmr Icatiqg No: 0: Ya: I Xl1 Oil emnmpem m caok'mg eonS.p6oo per pecsm pa moNh X12 2GrcLm cooi®g fume po1WGm No: 0; Ya: I X13 Regder cmwmpim of fried food No: 0; Ya: I X14 Cooking m0ex Avenge times of cookmg per week Note: • Shallow: exhale by mouth; Medium: exhale by nose; Deep: swallow smoke. *• Based on data of last 20 years. Results Multivariate risk analysis A multivariate analysis was performed by conditional logistic regression model. Risk factors for pulmonary squamous carcinoma and pulmonary adenocarcinoma were analyzed respectively, using one- side test with a=0.05. Results are shown in Table 2 and Table 3. -2- I I I I I I I I I I I

I I I I I I 1 I I I I i I I I I ANALYSIS AND ESTIMATES OF ATTRIBUTABLE RISK FACTORS FOR LUNG CANCER IN NANJING, CHINA Shen Xiao-bine*, Wang Guo-xiong*, Huang Yuan-zhu**, Xiang Long-sheng* and Wang Xing-he* *Nanjing Railway Medical College, Nanjing, China **Nanjing Medical University, Nanjing, China Abstract This is a case-control study of 83 cases of primary pulmonary squamous cell carcinoma and 180 cases of primary pulmonary adenocarcinoma in Nanjing. Multivariate conditional logistic regression analysis shows five risk factors for pulmonary squamous carcinoma. These were: smoking, cooking fume pollution, family tumor history, type of fuel used in the home, and use of coal stove for heating in winter. The relative risks (RR) for these five risk factors were 1.03 (95 % CI, 1.00-1.06), 3.81 (95 % CI, 1.06-13.73), 5.61 (95 % CI, 1.23-25.79), 4.97 (95 % CI, 0.8-30.88), 3.72 (95 % CI, 0.88-15.71), and the respective population attributable risks (PAR) were 0.6823, 0.5156, 0.2772, 0.5465, 0.3611. The four risk factors of pulmonary adenocarcinoma were smoking, cooking fume, chronic bronchitis, family tumor history. The respective RRs were 1.01 (95% CI, 1.00-1.03), 2.99 (95% CI, 1.68-5.34), 2.49 (95 % CI, 1.68-5.34), 4.77 (95 % CI, 1.93-11.83, and the respective PARs were 0.1987, 0.4699, 0.1763, 0.1844. The combined PAR of the five risk factors for pulmonary squamous cell carcinoma was 0.9431 and the combined PAR of risk factors for pulmonary adenocarcinoma was 0.7895. Among malignant tumors, lung cancer has become one of the most threatening to human health. The lung cancer death rate in China shows an apparent rising trend in recent years.(1) Squamous cell carcinoma and adenocarcinoma are the two major histopathological types of lung cancer. To examine the risk factors for squamous cell carcinoma and adenocarcinoma we have conducted a matched case- control study of risk factors for pulmonary squamous carcinoma and adenocarcinoma in Nanjing. Selection of Cases Materials and Methods Diagnosed primary lung cancer cases were obtained from Nanjing Municipal Hospitals from 1986 to 1993 and were Nanjing residents for longer than 20 years. A total of 83 pulmonary squamous carcinoma and 180 adenocarcinoma cases was included in the study. Selection of Controls Healthy controls were selected from over-20 year, tumor-free Nanjing residents. They were matched 1:1 with cases by sex, age (± 5 years), nationality, and street address. O I N O ~ -4 O ~ O ~ I

I I I 1 I I 1 I I 1 Table 2. Results of Conditional Logistic Regression Analysis in Pulmonary Squamous Carcinoma Group s1aMUA Fira.of . . . . ReB~~. ' . Regrcasim Relativc . Fscbr CoeB'icies Coeffici<K. . P Velec - Risle 95A Cl Cooking fwsa 1.3386 0.6536 0.0203 3.81 1.0G13.73 Smahg udex 00334 0.0132 0.0056 1.03 1.01-1.06 Family e®ar biswry 1.7275 0.7767 0.0132 5.61 1.29-25.79 Fuel type 1,6027 0.9324 0.0428 4.97 0.8130.88 Cwl Sbve fm healmg 1.3134 0.7352 0.0370 3.72 0.88-15.71 Table 3. Results of Conditional Logistic Regression Analysis in Pulmonary Adenocarcinoma Group sumudEraeof... . . R<gnalbn ReStMWrt ....: . PactOr CocRSeiept . . /bel&!ett ' . P Va6M ReWAv9 KkR .. 43% Q " Smokmg mdex 0.0123 0.0065 0.0300 1.01 1.000.1.03 Claan'a: brmchiue 0.9133 0.3A11 0.0037 2.49 128d.ee Coolung 6mes 1.0967 0.2956 0.0001 2.99 L68S.34 Family nmma hisrory 1.5622 0.4621 0.0004 4,77 1.93-11.83 Estimate of population attributable risk After various risk factors were identified, the RR of the factors was calculated and the respective PAR and SAR were estimated. (Table 4) I I I - N O O i V O CA) I O w 1

Table 4. Population Attributable Risks of Lung Cancer Risk Factors Pwmmary sq~ Cea caremcros . Puunmaxjr naeuocntr6mn..-.. .. MumticoP N®ber.of Facox [<vd RR d.w B1l. ,.Catte. ...~ PAtt. . Smo¢in8 Wcx Mexnueleot 1.0340 83 0.6829 1.0124 ISO 0.1987 Coolmg Fm¢s 0 1 25 1 53 1 3.8138 58 0.5156 2.9943 127 0.4699 Chraoic 0 1 12] Bnmchtrix 1 2.4925 53 0.1763 Family T~ 0 1 55 1 138 Hiurory 1 5.6135 28 0.2R2 4.7693 42 0.1844 Coel S[we fm 0 1 42 H•,tb~g 1 3.7187 41 0,3611 FS~eI Ndex Meamcemeot 4.9665 83 0.5465 SAR• 0.9431 0.7895 Syudutlc PopWatieu Atln'buable Risk Population attributable risk (PAR) is a measure of the relative risk of a given factor. For a given risk factor, PAR depends on the relation risk of the magnitude of exposure to that factor.(4,8) As seen in Table 4, the main risk factors for pulmonary squamous cell carcinoma were smoking, type of fuel used in the home, cooking fumes, coal stove use for winter heating, and family tumor history; the risk factors for pulmonary adenocarcinoma were, in their order of significance, cooking fume, family tumor history, chronic bronchitis and smoking. Discussion Most studies show that smoking has different effects on various types of lung cancer. Our study shows that smoking is a major risk factor for squamous cell carcinoma in the lung, with a PAR of 68.23 %. But the PAR of smoking for pulmonary adenocarcinoma was 15.6%. Passive smoke was not found to be one a risk factor. In a separate case-control study involving 70 nonsmoking females with adenocarcinoma, exposure to passive smoke from >20 cigarettes/day had a relative risk of 0.85, 95% CI 0.26-2.74 (data not shown). Cooking fumes are the vapor-phase product of cooking and result from pyrolysis of cooking oil and food under high heat. Since Chinese traditionally cook with high temperatures, cooking fumes are one of the major indoor pollutants. We have tested the chemical composition of cooking fumes for their genetic toxicity and found them to contain benzo(a)pyrene and benz(a)thracene. Toxicology experiments -4- I I I I I I I I I I 1 I J

I I 1 t I I I I I I I I DIET AS A CONFOUNDER OF THE ASSOCIATION BETWEEN AIR POLLUTION AND FEMALE LUNG CANCER: HONG KONG STUDIES ON EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE, INCENSE, AND COOKING FUMES AS EXAMPLES Linda C. Koo, J.H-C. Ho Cancer Research Laboratory Hong Kong Anticancer Society Nam Long Hospital, Hong Kong Introduction A comparative study of Chinese, Japanese, White, and Hawaiian women residing in various localities in the Pacific Basin indicated that the lung cancer incidence rate among nonsmoking Chinese in Hong Kong, Shanghai, or Hawaii ranged from 15.2 to 20.5/100,000, whereas that for women of other ethnic origins residing in the same localities only ranged from 7.3 to 7.5/100,000 (1). Some clues helping to explain this "mystery" of the unusually high incidence of lung cancer among nonsmoking Chinese females in such warm climate communities as Hong Kong, Singapore, or Hawaii (2) can be obtained from epidemiological statistics and studies. From the 1992 edition of Cancer Incidence in Five Continents (3) it can be noted that Hong Kong's world standardized female lung cancer incidence rate of 32.6/100,000 is among the highest in the world. This high incidence is a phenomenon of recent decades since mortality data from the Hong Kong Cancer Registry indicates that female lung cancer deaths increased from 7.7/100,000 in 1961 to 23.3/100,000 in 1990 (4). However, the lung cancer mortality rate of Chinese females from Guangdong province in China, the origins of most Hong Kong Chinese, was only 2.9/100,000 for the period 1973-75 (5). A further factor that has been identified from epidemiological studies in Hong Kong (6) and Singapore (7) is that within the Chinese ethnic/dialect groups, Cantonese females are about two to three times more likely to have lung cancer as those of Chiu Chow origins, even when they live in the same city. ~ These statistics indicate that Chinese women, especially those of Cantonese origin, share a common environmental exposure which they retain when they migrate overseas. The fact that , nonsmoking overseas Chinese women still have higher lung cancer rates than other ethnic groups living in the same area, would seem to rule out the influence of outdoor ambient air pollution as an important etiological explanation. In terms of indoor home sources of air pollution, the contribution of cooking and ' heating equipment would also seem to be minor since these factors are not unique to Chinese homes. Moreover, unlike the female lung cancer studies in cold climate areas in China such as Yunnan (8) and Manchuria (9), where the use of smoky coal stoves for heating can produce such concentrated fumes that , one can barely see one's outstretched hand when standing in such rooms,, the problems of Chinese women in Hong Kong and overseas communities is not one of air pollution from coal space heaters. The trends in mortality data from Hong Kong indicating a tripling increase over 30 years also suggest that the N I factor(s) are due to a recently introduced exposure(s) or changes in traditional exposures which are related o to contemporary Chinese cultural habits. The question we'd like to pose is: Is it mostly due to inhaled ~ or ingested substances? ~ ta O 00 I

Methods Over the last 14 years, we have been doing a series of epidemiological studies to narrow down possible factors affecting lung cancer in nonsmoking women in Hong Kong: *1981-3 retrospective case-control study of 200 female lung cancer patients and 200 district matched controls (10-12). *1985 cross-sectional study measuring personal exposures to nitrogen dioxide and prevalence of respiratory symptoms among 362 primary school children and their 319 nonsmoking mothers (13, 14). * 1988 measured the concentration of airborne carcinogens in the kitchen and living room of 33 working-class homes by 24 hour sampling in each room and analyzing the effects of emission and ventilation sources on the concentration of 7 polycyclic aromatic hydrocarbons (PAH) including benzo(a)pyrene (15). * 1993-4 cross-sectional telephone survey of 500 women on their dietary habits, exposure to environmental tobacco smoke, and prevalence of respiratory symptoms. Although the design and specific objective of each of the four Hong Kong epidemiological surveys was different,, we collected data on ETS in all four studies, and in the early three we gathered data on incense burning and exposure to cooking fumes. Data on dietary habits was only gathered in the 1981-3 case-control study and the 1993-4 telephone survey. Further details on the methodology in each study are provided in the references. Results Environmental Tobacco Smoke: Exnosure levels: Estimates of Hong Kong women's exposure to ETS varies with how it is defined. In terms of the person/place/time paradigm in epidemiology, it can be defined as simply the smoking habits of the current and/or ex-husband(s) or all family members, whether the home and/or workplace is considered, if current and/or past exposures are counted, and if only cigarettes or all forms of tobacco smoke are included (11). These complications in definition were addressed in our 1981-3 case-control study where we identified four possible exposure categories if we looked at the place of exposure (i.e- home, work, home+work, and none) (16), and another four categories if we looked at time of exposure (i.e. childhood, adulthood, both, none) (10). Generally, however, most researchers are defming it as the presence of a smoking spouse at home. Using this common definition, 60% (53 out of 88) of the nonsmoking female lung cancer patients and 49% (67 out of 137) of the district controls were exposed (10). In contrast to this older population of women with a mean age of 59, the 1985 study of 362 primary school children and their mothers (mean age 38) estimated that 36 % of the households had a smoking father/husband (13). -2- I I I I I I I I , I I I I 1 I

I I I I I I I I I I I I I 1 These data indicate that as smoking patterns vary by age group and time, one must be cautious in assuming that levels of exposure measured at one point in time of a subject's lifespan are representative of her lifetime dose. When smoking occurs at home, the amount of exposure and the overall effects of exposure may differ. In our 1985 study of personal levels of exposure to nitrogen dioxide (NO2) among primary school children and their mothers, increased smoking by the father/husband resulted in slightly increased N02 among the children but decreased NOz among their mothers (14). In another detailed analysis of life history correlates of ETS among the nonsmoking controls from the case-control study, it was found that the lifetime hours or years of exposure among wives with husbands who were heavy smokers, i.e. > 20 cigarettes/day, were not significantly higher than those with husbands who smoked less, i.e. 1-20 cigarettes/day (11). Yet this is a common assumption in studies on ETS and health effects, that there is a positive correlation between the number of cigarettes smoked by the husband and the amount of cumulative lifetime ETS exposure by the wife or other household members. The actual amount of ETS pollution that a wife with a smoking husband may inhale is also an issue that needs further investigation. From our NOZ personal monitoring study in 1985, where increased smoking at home was weakly associated with reduced levels of NOZ among the 319 mothers, we suggested that this was because when smoking occurred at home, the mothers would increase ventilation, thus resulting in reduced overall pollution from the cigarettes as well as other sources (14). This pattern was supported by our 1988 24-hour site monitoring study measuring the concentration of 7 polycyclic aromatic hydrocarbons (PAH) in the airborne particulates in 33 Hong Kong homes (15). As shown in Figure 1, increased cigarette smoking in the living room resulted in reduced concentrations of PAH in airborne dust. Raure 1 Effects of Clgarette Smoke on LMna Room Concentrations of PM None Inw Hph N- 19 6 e 0 14 b11 M~~py~ 0 S16 ibIDO MwvMwiw.Uwmf4V/~+•b.cfyqP.NR~.bwm0* • ~/tMWb~~aa.eara+~dMmm~wmwbrxo...dm.eama.~1.ww. ... rqr.eaa.aa a,om~.wmwiaw.wwd...eanea~erwa.nca eo:.e.: rcc wm is'~'iqO I I ' N O Co , s -4 00 , -3- i o I

With normal levels of smoking in homes, and given the subtropical climate, these data suggest that the Hong Kong inhabitants' response to smoking is to increase ventilation by opening windows and doors. The result is that the concentration of all indoor air pollutants, even those from other emission sources like cooking fires and incense, are diluted. This effect is especially apparent at low levels of smoking in the living room (1-2 cigarettes or 3-15 minutes of cigarette smoke per day). However, at higher levels of smoking, the effects of the extra ventilation activities as a response to perceived ETS can be overwhelmed so that cigarette smoke can be a major source of indoor air pollution where there is continuous or high levels of smoking, as in commercial environments like bars or discos. The behavioral response to ETS exposure, leading to overall dilution of air pollution in homes may also be more apparent in communities like Hong Kong where highly dense living conditions mean that people cannot move to another room to avoid cigarette smoke, so they do something about it. Other exposure study results also support the finding that the Hong Kong Chinese woman's contact with ETS is not unusually high when compared with women in other countries. In a 10-country, 13-site comparative study of urinary cotinine/creatinine ratios (cotinine is a metabolite of nicotine) the levels among Hong Kong Chinese women ranked 4th whereas those for Shanghai women were the lowest of all, ranking 13th (17). In fact Hong Kong's ratios would have been considerably reduced if the absolute cotinine levels had been compared instead of being adjusted by creatinine. From studies in Western populations, the usual range of creatinine excretion is 1.0 to 2.5 g/day (18) but among our Hong Kong subjects, 75 % had levels less than 1.0 g/day and the mean level among our 101 subjects was 0.77 g/day (SD=0.48). Our very low creatinine levels meant that cotinine adjustment by it would result in very high cotinine to creatinine ratios. We have no explanation for our abnormally low urinary creatinine levels, although it is known that it is increased by meat consumption (19). Lung cancer risk: As discussed above, assessing lifetime exposure to ETS is complicated because of the need to account for changing environments and lifestyles throughout the lifecycle of a subject. Defining exposure doses is also intricate because of different types of smoking methods, smokers, degree of ventilation, etc. This is especially pertinent in case-control studies on lung cancer, since the patients and controls are older subjects. In our previous analyses on the risk of ETS to lung cancer, we used such summary measurements of lifetime dose as total years, total hours, hours/day, and cigarettes/day (i.e. sum of the number of cigarettes/day smoked by each household member weighted by the years of exposure from that smoker). We also investigated whether a combination of such variables as hours/day plus years of exposure, or age at first exposure plus years of exposure led to dose-response relationships. In all these analyses, we could not establish a statistically significant association between lifetime ETS exposure and lung cancer risk among women who had never smoked (10). Stratification of the tumor data by histological type, lobar location of the primary tumor, and whether the tumor was proximally or peripherally situated suggested a weak possibility that peripheral tumors of the squamous or small cell type that were located in the middle or lower lobes might be affected by ETS. However, only 8 of our 88 nonsmoking cases had this combination of tumor characteristics, this number is too small to do statistical analyses (10). -4- , ' I I I I I I I I 1 I I I I I I I I

I I 1 25. I 26. I 27. I I I I I I I I I I I 28. -A ' O ~ W / 15- rv . N Ger, L.P.; Hsu, W.L.; Chen, K.T.; Chen, C.J. Risk factors for lung cancer by histological category in Taiwan. Anticancer Res. 1993; 13:1491-500. Gao, G-Y.; Blot, W.J.; Zheng, W.; Ershow., A.G.; Hsu, C.W.; Levin, L.I.; Zhang, R. and Fraument, Jr., J.F. Lung cancer among Chinese women. Int. J. Cancer 1987; 40:604-9. Koo, L.C. Environmental tobacco smoke and lung cancer: Is it the smoke or the diet? Present and Future of Indoor Air Duality, C.J. Bieva, Y. Courtois, M. Govaerts, eds. Amsterdam: Elsevier Science Publishers. 1989. p.65-75. Koo, L.C.; Ho, J. H-C. and Li, K.W. Similarities in dietary risk factors among nonsmokers with bronchitis and lung cancer. Proceedings of the First Congress of the Asian Pacific Society of Respirology. Tokyo: Asian Pacific Society of Respirology. 1988. p.54-7. N O i

I to report exposure to air pollution, or be more sensitive to its occurrence than controls. This may be especially true when cases are asked about recent exposures to air pollutants. In contrast to the Shanghai study on current exposures to cooking, our 1981-3 study asked about duration of cooking in years. Our results indicated that increasing years of cooking significantly reduced lung cancer risk among nonsmoking women (Figure 5) (27). Women who cooked for more than 25 years had their risk decreased by more than 60%, and the trend was highly significant (p<0.001). Hpure 5 tq. aeeaun a,e Y.pcalewaYt ane,p n.v.e.niaa.a Nmp I(enpwhm.n r +.n 1 o-w nau 41+ 1fBmf af CodOq ~ M~b~m~.~.hMn.vtlclnoFYNF) 9m.a~I~iu1SN Diet: ~ hend u~l .1. The relationship between duration of cooking and dietary patterns was more complicated. A comparison of those who cooked for shorter vs. longer durations among 88 nonsmoking cases indicated no significant differences in frequency of consumption of 17 food items when adjusted by age and years of education. However, among the controls, women who cooked more than 25 years were less frequent consumers of fresh fish (p=0.005) and foods containing retinol (0=0.006) and calcium (p=0.01), but were more frequent drinkers of alcohol (p = 0.04), than those who cooked for < 25 years. Again, these dietary patterns are consistent with lung cancer risk factors in Hong Kong(12). Discussion From epidemiological studies on risk factors for lung cancer among Chinese women, at least two different patterns are emerging. Among Chinese women living in colder climates in China, e.g. Manchuria and northern Ytnman, the influence of heavy doses of indoor air pollution from smoky coal fires is apparent (8,9), although diet cannot be excluded as an additional factor in those populations. For southern Chinese who are not exposed to air pollutants from space heating, and for overseas Chinese in developed urban communities, outdoor air pollution and fumes from heating and cooking appliances are -10- I I I I I I I I I I I I I I

I among nonsmokers in Hong Kong, cured meat and alcohol were associated with increased risk and milk and fresh fruit with decreased risk (12). Thus the pattern of dietary habits associated with ETS exposures in Hong Kong coincides with their independent risk for lung cancer in nonsmoking women. In an international comparative study of dietary correlates of ETS in Hong Kong, Sweden, United States, and Japan, there was a generally consistent pattern that among all these countries nonsmoking wives with smoking husbands were less likely to eat fresh fruit and vegetables than wives with nonsmoking husbands (22). Another study among women in Hawaii found intakes of betacarotene and cholesterol to be inversely associated with ETS exposures (23). Thus diet can be a significant confounder of the association between ETS and various diseases in a variety of industrialized urban cross-cultural settings. Incense: Exposure levels: From our 1985 survey measuring personal exposures to NOZ by the use of monitor badges, incense was identified as the most important emission source in the time that increased NO2 exposures among the mothers (14). From our 1988 study measuring the concentration of 7 PAH compounds in the airborne particulates in Hong Kong homes, incense was also found to be the major contributor to PAHs, like benzo(a)pyrene, in the living room, as shown in Figure 2 (15). These studies suggest that incense is a major source of gaseous and particulate air pollution in Hong Kong homes. Rpure 2 Effects of Incerse Smoke an uvYip Room Concentratbns of PAH M o 1-2 3.24 ,~bumed..3tlcft + Mt.~.4ara.aaw.~~Y~amimo.lNfo.~wYw. sn.csrnnria.iaw On any given day, about half the homes in Hong Kong will burn incense (24). Estimates on population exposure levels to incense increase with the age of the woman. From our 1985 survey, 48 % of the mothers (mean age 38) burned incense at home (14), whereas among the nonsmoking controls of the 1981-3 case-control study, 77% of this older cohort with mean age of 59 said that they burned it. -6- , I I I I I I I I I r I I I I I

I I I I I I I I I I I I I I I I Overall, the possibility that ETS exposure explains the unusually high lung cancer incidence among nonsmoking Chinese women in Hong Kong is unlikely. Hong Kong Chinese women do not have extremely high ETS exposures compared to women living in other regions in the world (17). Our studies in Hong Kong have also shown that female lung cancer patients who have never smoked were significantly younger, by 7 years, than patients who had ever-smoked (20). In fact, all of our female patients under 40 years of age had never smoked (2). Since the concentration of noxious gases and particulates in mainstream smoke is 100 to 100,000 times higher than the inhaled concentration of such agents in room-diluted sidestream smoke, depending on the particular agent being measured (18), and the active smoker is also a passive smoker by inhaling the sidestream smoke of her own and others' cigarettes, it would be more logical that among lung cancer patients, passive smokers would be older than active smokers. In fact, the very young age of nonsmoking female lung cancer patients in Hong Kong suggests that the suspected agents are either introduced very early in life, have a stronger carcinogenic effect than active smoking, and/or result in lower exposure to protective agents. Diet: From the controls used in the 1981-3 case-control study, we identified 136 women who had ever-married and never-smoked to see whether there were other lifestyle variables being correlated with having a smoking husband. After analysis of 97 quantifiable variables that ranged from other exposures at home, personal habits and recreational activities, cooking and heating fuels, health histories and consumption of medications,, etc., dietary habits were the most significant variables correlated with ETS exposures. Wives with nonsmoking husbands consumed more cruciferous vegetables, carrots, beans/legumes, fermented bean products, milk, and home-cooked soup than wives with smoking husbands. On the other hand, wives with smoking husbands were significantly more likely to consume pickled vegetables, chili, and alcohol than wives with nonsmoking husbands, with their greater consumption of salted fish of borderline significance (p=0.09) (11). According to the U.S. National Research Council's 1982 report on Diet. Nutrition, and Cancer the former foods are generally protective of cancer and the latter potential inducers of cancer (21). More recently, from 1993-4, we conducted a telephone survey of women to study the relationship of dietary preferences with ETS exposures. From among 500 interviewed subjects, 232 were currently married, of which 67 had a currently smoking husband and 165 did not. This cohort of women, with a mean age of 44, is younger than the 1981-3 control subjects mentioned above. Each subject was asked how often she consumed a list of 9 food items and whether she liked or disliked eating a list of 14 food items on a 5-point scale. Wives with smoking husbands significantly preferred eating meat, chili, salted fish, cured meats, seafood sauces, and alcohol more than wives with nonsmoking husbands. On the other hand, the latter group significantly preferred eating soybean curd and milk, and were more frequent consumers of fresh fruit. From these two studies we can see some similar dietary habits correlated with ETS exposure although their ages are different and the studies were done about 10 years apart. Nonsmoking wives with smoking husbands tended to eat poorer diets by consuming more salted fish, cured meats, and alcohol, and were lower consumers of fresh fruit and milk. In a previous report on diet and lung cancer risk -5- I

qT 2081783123

I I I I I I I I I I I I I I Lune cancer risk: The high exposures to incense among Hong Kong women, is likely to be a major contributor to airborne carcinogens in the home. Figure 3 shows the lung cancer relative risk (RR) estimates from the 1981-3 case-control data. The subjects included 189 cases and 197 controls who had ever been married. Increasing years of incense exposure did not affect lung cancer RR in nonsmokers. However in smokers, increasing incense use resulted in significantly decreased RR. At the highest level of use, i.e. 40-70 years, the lung cancer risk among smokers was only 17% of those who did not burn incense at all. Although the data shown are adjusted by demographic variables and smoking, similar results were obtained in the unadjusted RR. Rgure 3 Incense Smoke and Wna Cancer Risk ,~_ • pw.e a nP. r,.. nw., arqna,aec~.y •• M~ u ~o.. qs LIYr.IMaeco, 9a,o•:IAIdOSCwMdBFWy These findings were unexpected. However, it is interesting to note that a case-control study of female lung cancer in Taiwan also found that incense burning was associated with significantly reduced risk for lung cancer (25). In the Taiwan study on female adenocarcinoma cases, the RR for burners of incense at the highest level, i.e. 14+ times per week, was about a fourth of that among the nonburners. Diet: Since incense burning in Hong Kong is done for the purpose of communicating with gods or ancestors, it is representative of a traditional lifestyle that may be correlated with other traditional behaviors. As diet was found to be an important factor affecting lung cancer risk in Hong Kong women (12) , we studied the relationship of diet with incense burning. When the ever-smoked female lung cancer patients were stratified by their incense burning habits (+/-), incense burners were found to consume more fresh fish, retinol, and dim sum (Cantonese pastries), and less alcohol and chili than those who did not burn incense. In our previous study of diet and lung cancer risk, the former items were associated with reduced risk and the latter with increased risk for lung cancer in nonsmokers (12). Thus the significantly reduced risk for lung cancer among smoking female lung cancer patients who burned incense may have been due to its correlation with traditional food items that are protective of cancer. -7- I

Cookine Fumes: Exoosure levels: The overwhelming majority of Chinese women have been exposed to cooking fames for some part of their adult life. From the 1985 study, 19 out of 319 mothers did not cook (6%), and among the 400 cases and controls in the 1981-3 study, 22 did not cook (6%). This leads us to discuss the types and sources of pollutants in cooking fumes (from the fire and/or from the cooked food), and whether significant amounts of fumes are inhaled by Chinese women. From our 1985 study on nitrogen dioxide exposures, such indices of cooking exposure such as the number of meals cooked at home and the frequency of frying had no effect on the children's or mothers' NOZ levels. On the other hand, the type of fuel used for cooking significantly affected the mother's NOZ levels. Mothers whose cooking stoves were fueled by liquid petroleum gas had significantly higher levels, and those who had piped gas had significantly lower NO2 levels (14). From our 1988 fixed site monitoring study of kitchen levels of PAH, we found that the total duration of time that cooking fires were lit (none of the homes had electric stoves), was associated with significant increases in benzo(a)pyrene and all 7 PAH compounds as shown in Figure 4. However, when we investigated the effects of different cooking methods on airborne PAH levels, frying and stir-frying generally led to reduced concentrations of PAH in dust. This is probably because women, sensing such oily fumes being generated, tried to disperse them by increasing ventilation (15). Electrically powered ventilation fans installed on kitchen windows were found to significantly reduce air pollution levels from both the personal NOZ measurements in 1985 and the fixed site monitoring in 1988. Figure 4 Tha Fffarte nf Stnva Firn< nn ICitrhrn PAH lavalc PAH ng/m dust t j ril.eBAP ' 1e.1 Y0. oY e.e ~ 15-30 31-60 61-90 91-360 Minutes Stove fire • M7Wt~i bf W41. wBf bfpr ql. er. aMnN qhTfn ~IMan. uf. nI Hnlw~ rmHlrtlne Hn. IaL.niity ef iMMi~ Wrn1nO e rna u1 Mr~~d kncfa eoerf. Scurc. x Ceo .t .1. n/N The results of the two sets of data, one monitoring personal exposures to air pollutants and the other a fixed site monitoring study, show the importance of doing both types of measurements to -8- t I I I I I I I I I I I I I I I

1 I I I I I I I I I I I I I I I I I interview, and 2 who refused an interview. Diagnosis of all cases were confirmed by histological review of tissue specimens obtained from surgery or bronchoscopy. Among the 102 who completed the interview, 78 were males and 24 were females. Interviews were also performed with 306 controls. Table I shows that the distribution of cases and controls was similar with respect to sex, age, education, and term of residency in Fuzhou. Table 1. Distribution of cases and controls by sex, age, education, and term of residency in Fuzhou Sex 78 76.5 234 76.5 Male Female 24 23.5 72 23.5 Age (yr) 14 13.7 42 13.7 35-49 50-59 27 26.5 81 26.5 60-- 61 59.8 183 59.8 Education 18 17.6 58 19.0 No school Primary school 31 30.4 102 33.3 Secondary school 26 25.5 76 24.8 Beyond high school 27 26.5 70 22.9 Years of residency in Fuzhou 24 23.5 67 21.9 30-39 40-49 25 24.5 56 18.3 50-75 53 52.0 183 59.8 -3- I

associated with lung cancer risk in nonsmokers (20). We can therefore see a similar pattern emerging, that diet seems to provide the link between lung cancer risk and its association with such air pollutants as ETS, incense, and cooking fumes, as well as chronic bronchitis. In conclusion, to answer the question we posed in the introduction, we feel that dietary factors may play a major role in lung cancer risk among nonsmoking Hong Kong Chinese women. Dietary habits can fulfill the five criteria that we have listed above: 1). specific Chinese foods are associated with risk (e.g. cured meats like Chinese sausage, pressed duck, cured pork); 2). these foods are specialties of Cantonese cuisine; 3). Chiu Chow cuisine does not include these foods; 4). these foods are also sold and eaten among overseas Chinese communities (especially in Chinatowns); and 5). meat was traditionally expensive and eaten rarely. With urban money-based economies replacing an agrarian economy where previous peasants would mostly grow and eat what they produced, the consumption of meat products would increase as people move from rural to urban communities. With possibly alcohol and chili consumption acting as promoters, cancer initiation can be caused by the N-nitroso compounds found in cured meats, salted fish, and other preserved foods. This possibility is further supported by the protective effects associated with the lowered lung cancer risks from fresh fruit and vegetables, which contain antioxidants like vitamin C or carotenoids. Additionally, the reduced risk associated with fresh fish and milk may be related to their retinoid content and/or calcium contact (12). Thus the particular diets of Hong Kong Chinese women are correlated with their exposure to specific air pollutants, and the quality of those diets were found to coincide with their specific risk relationship with lung cancer. Therefore, dietary factors may help explain the unusually high lung cancer rates among nonsmoking Chinese women in Hong Kong, Singapore, Hawaii, and other overseas communities. I I I I I I I I I ! ' cc ~ -4 00 W -1. ' -12- i to , ,

13. Koo, L.C.; Ho, J.H-C.; Matsuki, H.; Shimizu, H.; Mori, T. and Tominaga, S. A comparison of the prevalence of respiratory illnesses among nonsmoking mothers and their children in Japan and Hong Kong. Am. Rev. Respir. Dis. 1988; 138:290-5. 14. Koo, L.C., Ho, J.H-C.; Ho, C-Y.; Matsuki, H.; Shimizu, H.; Mori, T. and Tominaga, S. Personal exposure to nitrogen dioxide and its association with respiratory illness in Hong Kong. Am. Rev. Respir. Dis. 1990; 141:1119-26. 15. Koo, L.C.; Matsushita, H.; Ho, J.H-C.; Wong, M.C.; Shimizu, H.; Mori, T.; Matsuki, H. and Tominaga, S. Carcinogens in the indoor air of Hong Kong homes: Levels, sources, and ventilation effects on 7 polycyclic aromatic hydrocarbons. Environ. Tech. 1994; 15:401-18. 16. Koo, L.C.; Ho, J.H-C. and Saw, D. Is passive smoking an added risk factor for lung cancer in Chinese women? J. Exp. Clin. Cancer Res. 1984; 3:277-84. 17. Riboli, E.; Preston-Martin, S.; Saracci, R.; Haley, N.J.; Trichopoulos, D.; Becher, H.; Burch, J.D.; Fontham, E.T.H.; Gao, Y-T S.; final, K.; Koo, L.C.; Le Marchand, L.; Segnan, N.; Shimizu, H.; Stanta, G.; Wu-Williams, A.H. and Zatonski, W. Exposure of nonsmoking women to environmental tobacco smoke: a 10-country collaborative study. Cancer Causes and Control 1990; 1:243-52. 18. National Research Council. Environmental Tobacco Smoke: Measuring Exposures and Assessina Health Effects. Wash. D.C.: National Academy Press. 1986. 19. National Research Council. Diet and Health: Implications for Reducing Chronic Disease Risk. Wash. D.C.: National Academy Press. 1989. 20. Koo, L.C.; Ho, J.H-C. and Lee, N. An analysis of some risk factors for lung cancer in Hong Kong. Int. J. Cancer 1985; 35:149-55. 21. National Research Council. Diet. Nutrition, and Cancer. Wash. D.C.: National Academy Press. 1982. 22. Koo, L.C.; Rylander, R.; Kabat, G.; Tominaga, S. and Kato, I. Dietary correlates of passive smoking in Hong Kong, Sweden, USA, and Japan. htt. Epidemioloeical Association Regional Scientific meeting in Asia-Pacific Reeion May 9-11, 1991. Program and Abstracts. Nagoya, Japan. p. 123. 23. Le Marchand, L.; Wilkens, L.R.; Hankin, J.H. and Haley, N.J. Dietary patterns of female nonsmokers with and without exposure to environmental tobacco smoke. Cancer Causes and Control 1990; 2:11-6. 24. Hills, P. The Household Energy Transition in Hong Kong. Hong Kong: Centre of Urban Planning and Environmental Management, University of Hong Kong. 1991. p.67-71. -14- N O O I I I I I I I I I I I , I I I

 ' I I I I I I I I I I I I I I I I INDOOR BURNING COAL AIR POLLUTION AND LUNG CANCER - A CASE-CONTROL STUDY IN FUZHOU, CHINA Luo Ren-xia*, Wu Bin**, Yi Ying-nan** and Lin Ru-tao** * Fuzhou Senior Medical School, Fuzhou, China ** Fujian Medical College, Fuzhou, China Abstract A case-control study on risk factors for lung cancer was carried out in Fuzhou, China. One hundred and two newly-diagnosed primary lung cancer cases in urban Fuzhou (78 male and 24 female cases) were matched with 306 population-based controls. The primary histological types were adenocarcinomas (57 cases, 55.9%) and squamous cell carcinomas (39 cases, 38.2%). Controls were obtained from the general population by random, stratified sampling and consisted of noncancer cases matched for sex, ethnicity and age. Cases and controls were interviewed by trained professionals using a standardized questionnaire. Information was obtained on: smoking habit, living conditions, history of respiratory diseases, air pollution, and forty other variables. The data were evaluated by conditional logistic regression analysis. The major risk factors for lung adenocarcinoma were: indoor air pollution from burning coal, chronic bronchitis, and high economic income. These conclusions were based on the following model, which was derived from unconditional logistic regression analysis: Ln Px/Q, = 1.7923 X, + 1.4122 X6 + 0.9263 X5 X,:indoor burning coal air pollution X6:chronic bronchitis XS:high economic income When the same analysis was applied to lung squamous cell carcinoma, the following results were obtained. Ln P,/Q, = 2.6486 X, + 1.1160 X2 + 1.9647 X3 + 1.5104 X4 + 1.5678 X5 X,:indoor burning coal air pollution X2:number of cigarettes per day X3:deep smoke inhalation X,:passive smoking history before 20 years of age X,:high economic income Thus, risk factors for lung squamous cell carcinomas were: amount of cigarettes smoked per day, deep smoke inhalation, passive smoking history before 20 years of age, burning coal indoors, and high economic income. I

I I I I I . 2. I 3. I 4. I 5. I 6. I 7 I . 8. I 9. I 10. I 11. I 12. i References Shimizu, H.; Wu, A.H.; Koo, L.C.; Gao, Y-T and Kolonel, L.N.. Lung cancer in women living in the Pacific Basin area. Natl. Cancer Inst. Monoer. 1985; 69:197-201. Koo, L.C. and Ho, J. H-C. Worldwide epidemiological patterns of lung cancer in nonsmokers. Int. J. Eoidemiol. 1990; 19:S14-S23. Parkin, D.M.; Muir, C.S.; Whelan, S.L.; Gao, Y.T.; Ferlay, J. and Powell, J. eds. Cancer Incidence in Five Continents, Vol. VI. Lyon: IARC Scientific Publications No. 120. 1992 Hospital Authority, Hon¢ Kong Cancer Registry. Hong Kong Government. (published and unpublished statistics). Editorial Committee. Atlas of Cancer Mortality in the People's Reuublic of China. Beijing: China Map Press. 1981. Koo, L.C. and Ho, J-H-C. Chronic bronchitis, lung cancer, and nasopharyngeal cancer in Hong Kong. In: Eoidernioloev and Prevention of Cancer, R. Sasaki, K. Aoki, eds. Nagoya, Japan: University of Nagoya Press. p.131-6, 1990. MacLennan, R.; Da Costa, J.; Day, N.E.; Law, C.H.; Ng, Y.K. and Shanmugaratnam, K. Risk factors for lung cancer in Singapore Chinese, a population with high female incidence rates. Int. J. Cancer 1977: 20:854-60. Mumford, J.L.; He, X.Z.; Chapman, R.S.; Cao, S.R.; Harris, D.B.; Li, X.M.; Xian, Y.L.; Jiang, W.Z.; Xu, C.W.; Chuang, J.C.; Wilson, W.E. and Cooke, M. Lung cancer and indoor air pollution in Xuan Wei, China. Science 1987; 235:217-20. Xiao, H.P. and Xu, Z.Y. Air pollution and lung cancer in Liaoning Province,, People's Republic of China. J. NatL Cancer Inst. Monogr. 1985; 69:53-8. Koo, L.C.; Ho, J.H-C.; Saw, D. and Ho, C-Y. Measurements of passive smoking and estimates of lung cancer risk among non-smoking Chinese females. Int. J. Cancer 1987; 39:162-9. Koo, L.C.; Ho, J.H-C. and Rylander, R. Life-history correlates of environmental tobacco smoke: A study on nonsmoking Hong Kong Chinese wives with smoking versus nonsmoking husbands. Soc. Sci. Med. 1988; 26:751-60. Koo, L.C. Dietary habits and lung cancer risk among Chinese females in Hong Kong who never smoked. Nutr. Cancer 1988; 11:155-72. # N O O ~ , -13- v O .Wa N ' O

I I I I I I I I I I I I ' I I I Table 4. Odds ratios for squamous cell carcinoma associated with intensity and duration of cigarette smoking Smoking Cases Controls OR 95%-CI Average number of cigarettes/ day 0 5 51 1.0 1-19 3 39 1.2 0.1-10.0 20-29 22 23 24.6 4.2-145.7 30- 9 4 38.7 5.2-290.2 Duration/years 0 1-29 30+ 5 51 1.0 6 21 5.7 1.0-32.9 28 45 12.5 2.8-55.4 Table 5. Odds ratios for squamous cell carcinoma according to age when smoking began and years since cigarette smoking stopped Smoking Nonsmoker Age-started >_ 40 20-39 <_19 Years since stopped z10 Cases Controls 5 1 OR' 95% Cl 51 1.0 8 3.1 0.3-40.2 19 38 10.5 2.3-48.6 14 20 12.4 2.7-57.7 1 10 2.0 0.2-23.1 < 10 33 56 15.9 3.1-82.2 Passive smokine. We regard those who were exposed to smoking parents or other family members before 20 years of age as positive passive smokers. Passive smoking before 20 years old was associated with squamous cell carcinoma (OR=2.4, 95% CI 1.1-5.1). Indoor burning coal air oollution. In this investigation, we regard smoke generated in the living room while cooking with coal as a positive index of indoor burning coal air pollution. Indoor burning coal air pollution was associated with an increased risk of lung cancer (OR= 7.6, 95% CI 3.7-15.7). -5- I

I I Table 2 shows the distribution of histological types of lung cancer among the 102 cases. Adenocarcinoma (55.9%) and squamous cell carcinoma (38.2%) were the most frequent types. Table 2. Distribution of pathological types of lung cancer cases I I I Pathological types Males Females Total.. ' Squamous cell carcinoma 36 46.2 3 12.5 39 38.2 Adenocarcinoma 38 48.7 19 79.2 57 55.9 Oat cell carcinoma 2 2.6 1 4.2 3 2.9 Unknown 2 2.6 1 4.2 3 2.9 Smoking. Smokers were identified as those who had ever smoked cigarettes on a regular basis for six months or longer. Cigarette smoking was reported in 79.5 % of the males, but only in 12.5 % of the females. In controls, the percentage of smokers in both males and females was significantly lower (61.5% and 2.8%). Overall, smokers had a 2.7-fold increase risk of lung cancer. Table 3 shows that smoking was significantly associated with an increased risk for squamous cell carcinoma (OR= 10.9; 95 % CI 2.5-47.9), but not for adenocarcinoma (OR=1.5; 95% CI 0.7-3.0). Table 4 shows that the OR of squamous carcinoma increased both with amount usually smoked per day and with the duration of smoking. Moreover, the later the age at which smoking started, the lower the associated risk with squamous cell carcinoma. Similarly, the risk of squamous carcinoma declined markedly with an increase in the number of years since smoking ceased (Table 5). Deep smoke inhalation was associated with an increased risk of squamous cell carcinoma (OR=10.4; 95% CI 3.6-30.6). Table 3. Odds ratios for specific types of lung cancer associated with smoking hTonsmoker ;' Stnoker. OR 959'o CI Controls 160 146 1.0 - Cases 37 65 2.7 1.5-5.0 Squamous cell 5 34 10.9 2.5-47.9 carcinoma Adenocarcinoma 29 28 1.5 0.7-3.0 -4- I I I I I I I I I I I

unlikely to explain their ?2 times higher lung cancer incidence among nonsmokers. Our studies and discussions on risk factors in Hong Kong are more applicable to this latter group of Chinese women. From our introduction we identified certain clues about the etiological risk factors: I I I I I I I I I I I I I I 1). It is not a Western import, but is characteristically Chinese. 2). Urban Cantonese are highly exposed. 3). Those of Chiu Chow origin are not or are less exposed. 4). Overseas Chinese, most of whom are Cantonese, continue these habits. 5). It is a new Chinese habit of recent decades, or due to a change in exposure of some traditional practice(s). Among the three air pollutants we have studied, ETS does not conform with any of the above five criteria. Air pollution from incense fulfills the first, second, and fourth criteria, but not the others. Moreover, although incense was found in our NO2 and PAH monitoring studies to be a major source of air pollution in Hong Kong homes, its effects on lung cancer risk resulted in no or lower relative risk estimates in Hong Kong and Taiwan. And finally, for fumes associated with cooking, we must distinguish between pollutants from the cooking fire and that emanating from the cooking food. For the cooking fires, only criteria two and four and are fulfilled, although our NO2 and PAH studies have verified that there are measurable increases in these pollutants from cooking fires. For the oily fumes from cooking food, criteria three and five are not fulfilled. Moreover, our case-control study of lung cancer indicated that increasing years of cooking reduced risk for lung cancer by more than 60%. It is generally assumed that air pollution is not beneficial to respiratory health. Yet our epidemiological data indicates that increasing incense smoke and cooking fumes reduced risk for lung cancer (among smokers for the former and nonsmokers for the latter). To explain these unexpected findings, we studied dietary correlates of exposure to all three air pollutants. We have shown that for each air pollution exposure, the quality of the diet among the exposed coincided with their lung cancer risk patterns, i.e. poorer for ETS exposed, better with incense exposure, and relatively better for the cases with more cooking exposures (because it was significantly poorer among the controls). From a previous study of diet and lung cancer risk among nonsmokers (12), increasing consumption of fresh vegetables, carrots, fresh fruit, fresh fish, tofu and foods containing retinol, calcium, and vitamin C were associated with reduced risk for lung cancer whereas cured meats, chili, and alcohol increased risk. It is interesting, and probably no coincidence, that the women with ETS, incense, or cooking fume exposures generally had dietary patterns corresponding with how that air pollutant was related with lung cancer risk. Although the general pattern was that fresh foods reduced risk and preserved foods increased risk, the most consistent discriminating foods associated with these air pollutants was that fresh fish was protective, and chili and alcohol increased risk. It is also noteworthy that these dietary patterns were similar to an analysis studying the association of diet habits with symptoms of chronic bronchitis among 137 nonsmoking controls from the 1981-3 case- control study (28). In that analysis, nonsmoking women with symptoms of chronic bronchitis were more likely to eat cured meats, pickled vegetables, chili, and alcohol, and less likely to eat legumes, fresh fruit, and milk. We had previously established that increasing years of chronic bronchitis was significantly N 0 00 ~ V 00 ca ~ ~ CD I

! I I I I I I I I I I I I I I Table 7. Conditional logistic regression model parameters Factor oe ctent STD error U-va ue -va ue Squamous ce carcinoma Indoor burning coal 2.6486 1.1906 2.2245 14.1 0.0261 air pollution (Xl) Amount of cigarettes 1.1160 0.4200 2.6572 3.1 0.0079 per day (X2) Deep smoke 1.9647 0.8028 2.4472 7.1 0.0144 inhalation (X3) Passive smoking 1.5104 0.7058 2.1400 4.5 0.0324 history before 20 years old (X4) High income (X5) 1.5678 0.6631 2.3645 4.8 0.0181 Adenocarcinoma Indoor burning coal 1.7923 0.5728 3.1288 6.0 0.0018 air pollution (Xl) High income (X5) 0.9263 0.3481 2.6612 2.5 0.0078 Chronic bronchitis 1.4122 0.5814 0.4290 4.1 0.0151 (X6) The main effects of lung squamous cell carcinoma was established as: Ln P,/Q, = 2.6486 X, + 1.1160 X2 + 1.9647 X3 + 1.5104 X, + 1.5678 X5 Xi:indoor burning coal air pollution X2:amount of cigarettes per day X3:deep smoke inhalation X4:passive smoking history before 20 years of age Xs:high economic income The main effects of lung adenocarcinoma was established as: Ln Px/Qx = 1.7923 Xi + 1.4122 X5 + 0.9263 X6 X,:indoor burning coal air pollution XS:high economic income Xb:chronic bronchitis -7-

I I Discussion I The results show that the major risk factors for lung cancer in Fuzhou were: amount of cigarettes smoked per day, deep smoke inhalation, passive smoking history before 20 years of age, burning coal indoors, and high economic income. I An indoor air contamination monitor in Fuzhou showed that the concentration of SO2 and NOz was higher. The pollution in the kitchen generated by coal combustion was more serious (2). According I to a study in Shanghai (3), the contact quantity of B(a)P per person in rooms where coal is used as fuel is equal to the amount of B(a)P in 20 cigarettes per day. Indoor air pollution was found to be one of the risk factors for lung cancer in Guangzhou (4). A great quantity of coal has been used as cooking fuel I since 1959 in Fuzhou, the mortality of lung cancer in Fuzhou has increased rapidly since 1983 and has coincided with the latency period for lung cancer (more than 30 years). I Cigarette smoking increased significantly the risk of lung squamous cell carcinoma, but there was no significant association with lung adenocarcinoma. The findings are generally in line with studies of lung cancer in other parts of the world (5-7). I After adjusting for smoking and other confounding factors, chronic bronchitis is still associated with lung cancer and included in main effect model. This result supports the hypothesis that chronic I bronchitis is one of the risk factors for lung cancer. The association between high income and lung cancer must be further investigated. I Conclusion Our research supports the hypothesis that smoking and indoor air pollution are the major risk I factors for lung cancer in Fuzhou. The increase in the use of burning coal indoors and smoking were associated with lung cancer mortality in a major city in southern China. I I I I N I O tp i ~ 00 W I ~ -8- ~ I I

I I I I I I I I I I I I I I I I I I understand the dynamics of air pollution exposures in humans. People do not stay in one room, nor do they passively breathe in air pollution from emission sources over which they may have some control. When there is a noticeable source of air pollution, be it stir-frying or cigarette smoke, windows and doors will likely be opened to dispel such pollutants. When a passing vehicle spews out black soot on to the breathing zone of a pedestrian, she will frequently cover her nose and stop breathing. Thus the measurements of air pollution from fixed site monitoring studies may bear little relationship to the exposures of free ranging human beings. From our 1988 study of 33 female household heads, their mean duration of staying in the kitchen was 1.4 hours per day (15). This relatively short period of time may explain why cooking activities were not found to significantly affect personal NOZ levels among the 319 mothers in the 1985 study. It is normally assumed that there is a linear relationship between emission levels and personal inhalation of such pollutants. Our data from Hong Kong seem to indicate that this association can vary depending on people's reactions to the emission source. Unlike workplace or commercial indoor environments, inhabitants at home have more control over their air circulation and ventilation patterns. When an inhabitant perceives air pollution coming from a noticeable source, she can increase ventilation so that not only is the pollution from the perceived source reduced, but the accumulated pollution from other sources is also significantly reduced. This was found in our 1988 PAH study for such emission sources as gas powered water heaters, cigarette smoke, and stir-fry cooking whereby increasing emissions from these sources were associated with significantly reduced airborne PAH levels (15). By contrast, when boiling, steaming, or stewing activities took place, there was a dose-response increase in kitchen PAH levels. These variations in PAH levels by specific cooking activities are examples of the results of the importance of human perception. Unlike the large amount of oil and fumes generated from stir-frying; the latter cooking activities do not produce such noticeable air pollutants. Consequently women are less likely to actively increase ventilation in the kitchen. On the other hand some concentrated emission sources may be less affected by ventilation, as was found for incense, or be too overwhelming to be diluted by increased ventilation, as was found for the total time that the stove fire was lit (15). Lung cancer risk: In a case-control study of female lung cancer in Shanghai, China, Gao et al.(26) found that lung cancer RR increased when more meals were cooked by stir-frying, deep frying, and boiling. Shanghainese women who reported eye irritation and house smokiness when cooking also had increased RR. Although the authors postulated that exposure to oily cooking vapors may be etiologically related to lung cancer, it is interesting to note from their RR estimates that the most significant fmdings among the cooking methods was that from boiling, which does not produce oil vapors. Moreover, their study asked about current cooking habits, and the relevance of such current exposures to lung cancer etiology, which has a latency period of several decades, may be questionable. The possibility of reporting bias among cases is also a problem in all studies on respiratory diseases where exposure assessments are done by questionnaires. This is because of lay perceptions that respiratory diseases are caused/affected by inhalation of air pollutants. Thus cases may be more likely -9- I

I I I I I I I I I I I I I I I I I I References Breslow, N.E. and Day, N.E. (1980) Statistical methods in cancer research. Vol. 1- the analysis of case-control studies, p. 32. 2. Zheng, Z.Q. et al. (1989) Journal of Fuiian Medical College 23(1), 24. 3. Huang, Y. et al. (1985) Shan¢hai Environment Science 1, 26. 4. Liu, Q. (1987) Shanghai Tumor 7 (6), 256. 5. Damber, L.A. et al. (1986) Smoking and lung cancer with special regard to type of smoking and type of cancer. Br. J. Cancer 53 (5), 673. 6. Benharnou, E. et at. (1987) Lung cancer and women: Results of a French case-control study. Br. J. Cancer 55(1), 91. 7. Cha, Q. et al. (1992) Relationship between smoking and lung cancer cell type. Journal of Guangzhou Medical College 20 (1), 5. -9- I

I The results showed that the major risk factors for lung cancer in Fuzhou were: burning coal indoors, smoking, passive smoking history before 20 years of age, chronic bronchitis, and high economic income. Cigarette smoking significantly increased the risk of lung squamous cell carcinoma, but it had no significant association with the risk of lung adenocarcinoma. In summary, our research supports the hypothesis that smoking and indoor air pollution are the major risk factors for lung cancer in Fuzhou. Burning coal indoors and smoking were associated with lung cancer mortality in a major city in southern China. Introduction Cancer is the leading cause of mortality in Fuzhou, China. Among the various types of cancer that were followed in Fuzhou over the years, a downward trend has been observed in the death rates associated with stomach, esophageal, and cervical cancers. In direct contrast, there has been a marked increase in the mortality of lung cancer, making it now among the leading causes of cancer deaths. To investigate risk factors which may be associated with the elevated mortality of lung cancer, a case-control study was performed. In this report, we describe fuu3ings from such a study. Methods Lung cancer cases involved Fuzhou residents who were identified from a special reporting system designed to cover all incidence of lung cancer in hospitals located in urban Fuzhou. All were newly diagnosed, histologically confirmed primary lung cancer cases (according to code 162 described in the International Classification of Diseases, Ninth Revision) and were collected over a 1.5-year period during 1990-91. Controls were randomly selected by sex and age from the general population of urban Fuzhou, using a 2-stage sampling procedure. In the first stage, 20,000 people were randomly sampled from the 760,000 general population in urban Fuzhou, by means of a neighborhood census roster kept by a committee in each neighborhood. They were further grouped by sex and age (35-75 years). For every case, four controls were chosen randomly from the same sex and age group, and one of the four controls was used as a match for the case. Cases and controls were interviewed by trained personnel using a standardized questionnaire. The interviews were conducted usually in the subject's home. Information was obtained on smoking habits (active and passive), demographic variables, occupation, living conditions, air pollution, history of respiratory diseases, and forty other variables. The odds ratio (OR), a measure used to evaluate the relationship between various risk factors and lung cancer, was estimated by conditional logistic regression analysis (1). Results A total of 124 lung cancer cases were identified over the study period, of which 102 (82.3 %) completed the interview. Excluded were 12 who had died, 8 who were too ill to participate in the -2- I 1 I I I I I I I I I I I I I I I I

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I Lung diseases. Participants were asked about history of lung disease diagnosed at least three years prior to the time of the investigation. Table 6 shows ORs of 3.6 (95 % CI 1.8-7.2) and 2.5 (95 % CI 1.2-4.7) for chronic bronchitis and pulmonary tuberculosis, respectively, as significant risk factors being associated with lung cancer. Table 6. Odds ratios for lung cancer in relation to history of bronchitis and pulmonary tuberculosis Cases Controls OR 95%: CI : Chronic bronchitis No 83 286 Yes 19 20 1.0 3.6 1.8-7.2 Pulmonary tuberculosis No 86 283 1.0 Yes 16 23 2.4 1.2-4.7 Mental stress. Mental stress (such as being denounced at a public meeting or having relatives that died of an accident) was associated with lung cancer (OR=2.3, 95 % CI 1.3-3.9). Income. High economic income was associated with an increased risk of lung cancer (OR=2.9, 95 % CI 1.84.6). Dust. Persons who had a history of contact with dust or coal dust or other industrial dust in workplace had an elevated risk of lung cancer (OR=1.8, 95% CI 1.1-3.1). Conditional logistic resression analysis. Based on the analyses of individual factors, a conditional logistic regression model was constructed to evaluate simultaneously the effects of multiple variables on the risk of squamous cell carcinoma and adenocarcinoma, respectively. Included in the model were indicator variables for smoking, passive smoking, indoor burning coal air pollution, economic income, lung disease, alcoholic drink, dust and mental stress. There were 5 variables in the conditional logistic regression model for squamous cell carcinoma and 3 variables in the conditional regression model for adenocarcinoma. By the log-likelihood ratio of statistic (Crz=63.42 on 5 degrees of freedom and G2=23.75 on 3 degrees of freedom, respectively), we conclude that the fit of the models is good (P-value of both less than 0.01). -6- I I I I I I I I I I I 1 I I I I I I

I I I I I I I I I I I I I I I I I I THE EFFECT OF BETA-CAROTENE ON LUNG CANCER Lai Bai-tang and Wan Hui Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China Introduction As a pro-vitamin A, beta-carotene is present in abundance in green peppers, carrots, and pumpkins. A re-examination of evidence from prospective and case-control studies allowed Peto et al. to hypothesize in 1981 that dietary beta-carotene (B-C) has a preventive role against cancer. More recently, studies on beta-carotene in rats and mice have shown that beta-carotene has a protective action against tumors induced by different carcinogens at various sites including the skin, oral cavity, salivary gland, colon and bladder. Results from epidemiologic studies have also indicated that the concentration of beta-carotene in the serum of patients suffering from lung cancer is much lower than normal persons. The relative risk of lung cancer among subjects with low beta-carotene intake was significantly elevated. The purpose of this study was to investigate the effect of beta-carotene on lung cancer in vivo and in vitro. Materials and Methods 1. The Effect Of Beta-Carotene On Colony Forming Ability Of Lung Cancer Cells. The human large lung cancer cell line 801 was obtained from Hospital 301 in Beijing and was maintained in glass culture bottles containing Dubecco's Minimal Essential Medium (DMEM) and 10% newborn calf serum. The 801 cells were plated in polystyrene petri dishes at a density of 500 cells per dish and then cultured in a CO2 incubator. Beta-carotene was dissolved in dimethylsulfoxide (DMSO) and diluted to 3.125 µg/ml and 6.25 µg/ml. The two different concentrations of beta-carotene were then added into the dish-plated cells. After treating cells for 24, 48, 72 hr, the medium was changed to one without beta-carotene. After 14 days the dishes were stained with Giemsa stain. Colonies containing more than 10 cells were counted. The colony forming unit (CFE) was defined by the equation: CFE= No. of colonies formed x 100% No. of cells plated (500) ~ 0 ~ i -4 00 W ~ W A I

I Table 3. The Effects of Beta-carotene on DNA and RNA Synthesis in Lung Cancer Cell DNA Mean ineotpomtion on , , - RNA Mean 5ncerpottitlort ,~ . cpm3N-thymidine(Tdr) cpm3N-ruidine(Udr) Fipcriment DM50 comrol . B-C. ' DMSO cunttul .. i1-C . 1 100 78 118 78 128 (1l0) 90 (873) I52 (154) 64(81) 102 94 162 102 II 55 50 173 103 73 (67.3) 56 (53) 158 (I71) 108 (105) 63 50 183 104 [II 72 56 236 194 72(70) 62(50) 258 (246) 104 (178) 60 58 248 178 O for mean cpm of groups Table 4. Effect of Beta-carotene on DNA Synthesis of DNA in Isolated Lymphocytes Incorparatlan of mcan 3A tbymitline (RIr) Expuimant Db151)cbnorol -&C . 1 72 102 88(81) 90(82) 82 62 78 74 II 120 170 312 (249) 3l4 (248) 318 258 I11 170 164 188 (100) 188 (172) 140 166 O for mean cpm of groups N ~ 3 v ~ - 6 - ~ W I 1 I I I I I I 1 I I I I I I I

I I I I I I I I I I I I I I The inhibitory effect increased with the time that the cells were exposed to beta-carotene. Beta- carotene at 12.5 µg/ml was shown to completely inhibit CFE. The results indicated that beta-carotene has a concentration-and-time-dependent inhibitory effect on growth of lung cancer cells. 2. The Inhibitory Effect Of Beta-Carotene On Spontaneous Metastasis Of Murine Pulmonary Adenocarcinoma. TA795 murine adenocarcinoma used in our experiment is a tumor with high malignancy. It is very likely to metastasize to the lungs of inbred T739 mice. We counted metastatic lesions in the lungs of the mice fed B-C and in control groups. The results are shown in Table 2. When the metastatic lesions in lungs of mice in the B-C group were compared with the controls, a 42-68% decrease in spontaneous lung metastasis of LA795 murine pulmonary adenocarcinoma was observed in the treated group (p<0.01). 3. The Effect Of Beta-Carotene On DNA And RNA Synthesis In Lung Cancer Cells. Table 3 shows the results of three separate experiments with similar results. The incorporation of 3H-thymidine (TDR) or 3H-uridine (UDR) in the "B-C-treated" cells was significantly decreased, (p<0.05). Compared to controls there was no inhibitory effect of B-C on the synthesis of DNA and RNA in human lymphocytes (Table 4). 4. The Inhibitory Effects Of Beta Carotene On RAS Gene Expression In Lung Cancer Cells. The Neo-ras cells (with high expression ras genes) were stained by monoclonal antibody against p21H-ras-HRP bound to RAM-IgG. There were many dark-blue precipitates under the membrane of the Neo-ras cells. When the cells were exposed to beta-carotene for 24 hr, the dark-blue precipitates were apparently decreased. This suggests that expression of the ras gene in Neo-ras was inhibited by B-C. Discussion In recent years, it has been reported that beta-carotene inhibits the development of animal tumors induced by many carcinogens. A relationship between beta-carotene concentration in serum and the relative risk for lung cancer has been suggested by epidemiologic studies. Stabelins et al. surveyed 2,874 men from 1971 to 1978 and measured beta-carotene in their serum. Among the 533 mortalities in the 12 year study, 204 died of cancer (lung cancer 68, stomach cancer 30, colon cancer 17, all other malignancies 99). Interestingly, the mean concentration of B-C in the serum of men who died from cancer was significantly lower than in the survivors. The mean beta-carotene concentration in the serum of 2341 survivors was 0.428 µmol/L, but was 0.217 µmol/L in 68 lung cancer cases, 0.274 µmol/L in 20 stomach cancers, and 0.342 µmol/L for all the other cancer groups. The relative risk for lung cancer of subjects with low beta-carotene (less than 0.23 µmol/L) was significantly elevated (p < 0.05). In fact, when B-C in serum is lower than 0.28 µmol/L, the incidence of cancer will collectively increase by 1.74- 2.26 times. The present study provided evidence for an inhibitory effect of B-C on human lung cancer in vivo N 0 and in vitro. Joel et al. found an inhibitory effect of B-C on cancer cells at high concentrations in vitro. co ~ ~ 00 - 3 - W ~ W M I

I 2. The Effect Of Beta-Carotene On Spontaneous Metastasis In Mice With Adenocarcinoma. LA795 mice with lung adenocarcinoma were purchased from Tanzing Medical Pharmaceutical Institute. Tumors were taken from LA795 mice to make a Ix107/ml cell suspension. Two-tenths of a ml cell suspension (2 x 106 cells) were transplanted into T739 mice using the sub-cutaneous route. Tumors were taken from the mice when they had grown to 1 cm in size. The "treated" mice had been on diets containing B-C (25 mg/100g) for 2 weeks before injection of the tumor cells and continued on the same diet for 2 weeks after the resection of tumors. In order to compare the treatment group with controls, the lungs were taken from the mice four weeks after tumor resection. The metastatic lesions in the lungs were counted under a steromicroscope, and the percentage of decrease in the number of lesions in the treatment group was calculated. 3. The Effect Of Beta-Carotene On Synthesis Of DNA and RNA In Human Lung Cancer Cells And Lymphocytes. A 1 x 105/ml suspension of 801 cells was plated into 96 wells in polystyrene plates. Each well contained 1 ml suspension of 801 cells. After 24 hr, the culture medium was replaced by a medium containing 25 µg/ml beta-carotene using dimethyl sulfoxide (DMSO) as a solvent control, [3H]-thymidine (Tdr) or [3H]-uridine (Urd) (1 µCi/ml) was added into each well for two hr. The cpm (counts per minute) value of cells in each well was determined with a 210G scintillation counter. The average value of the counts in wells was considered as the value of a group. The incorporation of label into human lymphocytes was counted using the same method. 4. The Effect Of Beta-Carotene On Expression Of RAS Gene In Cells This study utilized the Neo-ras cell line with a high expression of the ras gene (a 3T9 cell line transfected with ras genes obtained from Australia Biological Institute). 1 x 104 cells/ml were plated into 24 polystyrene dishes. Each well contained 1 ml medium (1 x 104 cells). On the next day, the medium was replaced by one containing beta-carotene (12.5 µg/ml) using DMSO as a solvent control. After 24 hr, the coverglasses (with cells) were removed, dried in air, and fixed with cold acetone. The cells were stained with monoclonal antibody against p21 II-ras-horseradish-peroxidase RAM-IgD and examined under a microscope. Results 1. The Effect Of Beta-Carotene On Colony Forming Ability Of Lung Cancer Cells The inhibitory action of beta-carotene at different concentrations (6.25 µg/nil and 3.125 µg/ml), and different treatment time (24, 48, 72 hr) on colony formation of 801 cells was investigated. The CFE of 801 cells exposed to beta-carotene at a concentration of 6.25 µg/ml for 24 hr was inhibited significantly. (Table 1). - 2 - I I I ' I I I I I 1 I I I t

I Table 1. Odds Ratio (OR) Linear Trend Test for Lung Cancer by Histologic Subtypes (Controlling for Cigarette Smoking) According to Levels of Dietary Carotene Intake < 104 > 3282 13 1.00 11 1.00 3 1.00 62 2960-3282 15 2.04 9 1.45 3 1.86 35 < 2960 20 4.54 8 2.15 2 2.06 21 (Pxl trend=0.00) (PX2 trend=0.14) (PXZ trend=0.30) z 104 > 3282 4 1.00 8 1.00 8 1.00 20 2960-3282 9 5.00 6 1.67 9 2.50 9 < 2960 16 12.33 4 1.67 8 3.33 6 (PXZ trend=0.00) (PXz trend=0.43) (PXz trend=0.04) 77 46 33 156 Adjusted OR(95% CI) 100 1.00 1.00 2.61(1.81-3.78) 1.52(0.92-2.51) 2.21(1.34-3.63) 6.14(3.05-12.35) 1.98(0.88-4.47) 2.82(1.47-5.39) (PXZ trend=0.00) (PxZ trend=0.10) (PXZ trend=0.04) 1. Daily cigarette consumption X no. of years smoked. 2. Unit: µg/day I I I I I I I I I I I I I I NI 0 tp ~ v tp W ~ I -2- N I I

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I ' I I I I I I I I I I I I , Table 1. The Effect of Beta-carotene on Colony Forming Ability of Lung Cancer Cells (colony numbers/dish) Dose (µgtml) . . . . Treatment 3.125 6.23 . . - A2.5 Tmte (hr) . . . .. . DM50 CFE 13-C CFE DMSO CFE B-C - CEE- . DMSO .:.~ CFE. B-. CF6: Control (9b) (%) (%) (%) . (%) . C.. t%F . 2A 44 42 41 15 36 0 41 (43.3) 0.6 47 (42.0) 05 40 (39.0) 7.9 17(14) 2,8 31 (30.0) 6.1 0 0 (0) 45 39 38 t0 39 0 48 55 45 51 27 39 0 52 (53) 10.6 51 (50.3) 10 45 (47) 9.4 26 (25.7) 5.l 33 (31.3) 6.2 0 0 (0) 52 55 45 24 32 0 72 55 62 47 8 20 0 42 (50.7) 11.34 59 (54.7) 10.9 45 (44.7) 8.9 8(8.7) 1.7 15 (18.5) 3.8 0 0 (0) 55 43 42 10 20 0 O for mean colony numbers of each dish Table 2. The Inbibitory Effects of Beta-carotene on Spontaneous Lung Metastasis in T739 Mice Experiment Group Nmnber Body Lung Nmnber Mean . ~ Decrease . of Wcight Weight of Nmnb[33 i peftewa8e Animals Animals of of wl[b metzstatie poststatie wmors/tota) - tuwors (%). mice I DMSO 17 27.2 0.34 14/17 35.2 B-C 17 27.3 0.34 13/17 11.2° 68 II DMSO 24 24.2 0.19 24/24 50.1 B-C 24 24.1 0.26 20/24 29° 42 ~ a p<0.01 N O i V CO W ~ C.f CO I

I When lung cancer cell line SK-MES was exposed to beta-carotene at 78 µmol/L or 300 µmol/L, a 70- 80% decrease in cell density was noted. In our experiments, we have observed a lower concentration of beta-carotene to exert an inhibitory effect on lung cancer cells. When 801 cells were exposed to beta- carotene at 6.25 µg/ml, the ability of the cells to form colonies was inhibited. Complete inhibition was seen at 12.5 µg/ml. LA795 murine lung adenocarcinoma is a highly malignant lung cancer. When LA795 tumors were transplanted into T739 inbred mice by subcutaneous, muscular or peritoneal injection, cells of the tumor could spontaneously metastasize to the lung. In our experiments, when T739 tumors grew to 1 cm diameter and were then resected, a 42-68% (p<0.01) decrease in spontaneous lung metastasis was observed in mice fed a diet supplemented with beta-carotene (25 mg/100 diet). From these results, it can be hypothesized that beta-carotene can be used to prevent relapse and metastasis in postoperative patients with lung cancer. Santamaria et al. used beta-carotene to prevent relapse of 15 cases with cancer after operation. Results show that survival of patients with cancer (including lung cancer, colon cancer, bladder cancer, head and neck cancer) was longer in those who used B-C. The mechanism of action of beta-carotene on cancer cells has been studied. Okuzumi found that in cells exposed to 10 µg/nil beta-carotene for 4 hr, N-myc gene expression was decreased. Our studies show that beta-carotene at 12.5 µg/ml can decrease Ras gene expression which in turn raises the possibility that p21, product of the Ras gene, is associated with lung cancer cell multiplication. Another possible mechanism of the action of B-C, suggested by Soda et al., is the stimulation of the immune system. Volunteers given beta-carotene (180 ml/day) for two weeks were found to have elevated okT4, okT3 lymphocytes as well as an increase in serum beta-carotene. In our studies we also observed an increase in a T4/T8 ratio in the serum of volunteers taking B-C. Conclusion 1. Beta-carotene at a concentration of 6.25 µg/ml was shown to inhibit significantly the colony forming efficiency (CFE) of cultured human lung cancer 801 cells. Complete inhibition occurred at 12.5 µg/ml. 2. A 42-68 % decrease in spontaneous lung metastasis of TA739 murine pulmonary adenocarcinoma on T730 inbred mice was observed when the mice were fed a diet with beta-carotene (25 mg/100g diet) 3. The synthesis of DNA and RNA in 801 cells was decreased (p <0.05) after treating the cells with beta-carotene for 24 hr. 4. Expression of p21 Ras gene in Neo-ras cells was inhibited by beta-carotene. 00 ~ ~ - 4 - rp W I I ' I I I I I 1 , I I I I I I

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i I I I I I I I I I I I I I I I A MATCHED CASE-CONTROL STUDY OF THE RELATIONSHIP BETWEEN BETA-CAROTENE INTAKE AND LUNG CANCER Tan Ai-Jun, He Shang-pu, Huang Ming-xi, Li Guo-Guang and Shi Lu-yuan Tongji Medical University, Wuhan, China In order to evaluate the relationship between dietary intake and serum level of beta-carotene and lung cancer risk, a 1:1 matched case-control study of 156 newly histologically diagnosed primary lung cancer patients and 156 general patients (without tumors, respiratory, or other related diseases) was conducted in Wuhan. Three ml of venous blood were collected, and all cases and controls were asked to participate in a nutritional assessment and to answer a food frequency questionnaire containing 64 items on 11 kinds of foods. Subjects were instructed to recall dietary habits during the year prior to their first recognition of any symptoms associated with their diseases. Nutrient intake from foods was calculated from the annual intake of various food items by reference to a food composition database. The findings showed that there was a statistically significant difference between dietary intake of beta-carotene in cases and controls (2877.13 ± 393.43 vs 3445 ± 430.98 µg/day), and serum level as well (25.69 ± 5.57 vs 32.26 ± 5.02 µg/dl). Using conditional logistic regression analysis, an inverse association was found between dietary intake and serum concentrations of beta-carotene and lung cancer risk. When controlling for the potential confounding effects of cigarette smoking and alcohol consumption, a significant linear trend of lower dietary carotene intake and higher lung cancer risk was observed. N O _L V O W ' 3 ~ ~ '

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I 13. Miyamma, Y.; Tawa, R.; Koizumi, A.; Uehara, Y.; Kurishita, A.; Sakurai, H.; Kamiyama, S. and Ono, T., 1993. Effects of Energy Restriction on Age-associated Changes of DNA Methylation in Mouse Liver. Mutation Res., 295:63-69. I I I I I I I I I I I I I I '

a I I I I ' I I I I I I Table 2. Odds Ratio (OR) Linear Tread Test for Lung Cancer by Histologic Subtypes (Controlling for Alcohol Consumption) According to Levels of Dietary Carotene Intake Index' i Intake2 I No.. L OR I No. I OR . . I No. I OR I No. <25 > 3282 9 1.00 9 1.00 5 1.00 46 2960-3282 12 1.92 3 0.48 5 1.44 32 <2960 16 2.92 3 0.55 9 2.96 28 (FIX2 trend =0.02) (P~~ trend =0.31) (PX2 crend= 0.06) z 25 > 3282 8 1.00 18 1.00 5 1.00 30 2960-3282 14 3.75 11 1.31 2 0.86 14 < 2960 18 11.25 2 0.56 7 7.00 6 (PXZ trend =0.00) (Px2 trend =0.81) (PXz trend= 0.01) 77 46 33 156 Adjusted OR(95 % CI) 1.00 1.00 1.00 2.59(1.7 1-3.93) 0.91(0.76 -1.87) 1.19(1.06-1. 32) 4.86(2.4 3-9.72) 0.55(0.17- 1.78) 4.10(1.69-9.9 2) (PX' trend=0.00) (PXz trend=0.10) (PXz trend=0.04) I I I I 1. Alcohol consumed per day X drinking years. 2. Unit: µglday tv 0 co , s -4 3 C* W .p ~ W

I Table 1. Hypomethylation of H- and K-Ras Gene in Ad Libitum-Fed and Dietary Animals I AD LIB (0) DR (0) K-RAS I H-RAS increased decreased AD LIB refers to animals allowed to eat ad libitum without any restriction of calories. DR refers to animals placed on a 40% restriction diet of the ad libitum fed. O-denotes that this study was conducted on aged animals. I I I I I Table 2. 1 Gene Amplification and Gene Expression in Dietary Restricted Animals Amplification Expression H-ras H-ras AD LIB (0) increased increased DR (0) decreased decreased AD LIB refers to animals allowed to eat ad libitum without any restriction of calories. DR refers to animals placed on a 40% restriction diet of the ad libitum fed. O-denotes aged animals. -6- I I I I I I I I I I

a I I I I I I  ' , ~ I I I I I Figure 1. Ha-ras Gene Amplification in the Pancreas of BNF Rats. Figure 1 demonstrates increased gene amplification of the H-ras gene in young, middle age and old rat pancreatic acinar cells. It shows the decrease in gene amplification in DNA from old dietary restricted animals as a function of age. Dietary restriction in female rats (CR-F) show a pronounced inhibitory effect on gene amplification. A=AL-M, young; B=CR-M, Middle; C=CR-F. 1200 1000 o »r 800 ~ L o CL 00 0 U a 400 N ~ 200 YOUNG MIDDLE AGE OLD N ~ ~ -4 ~ 5- ~  W I

I I I I I I I I I I I I I I I I I I MODULATION OF MOLECULAR MECHANISMS BY DIETARY RESTRICTION IN RATS Beverly D. Lyn-Cook, Jin Bo and Ronald W. Hart Nutritional Modulators of Toxicity Program, National Center for Toxicological Research, Jefferson, Arkansas, U.S.A. Abstract Dietary restriction, which is 40% reduction in caloric intake of the ad libitum-fed animals, is known to modulate a number of pathological diseases, as well as, extend the life-span of a number of animals. Studies in our laboratory have shown that dietary restriction modulates epigenetic and genetic mechanisms which may contribute to the etiology and progression of a number of diseases, including cancer. Epigenetic mechanisms, such as the methylation status of specific cellular proto-oncogenes, are modulated by dietary restriction. The proto-oncogenes found in the ras family are known to be activated by point mutations or hypomethylation of CpG sites at critical points on the gene. Dietary restriction reduces hypomethylation and accumulation of mutations that results as a function of age in the exocrine pancreas of rats. Dietary restrictions decrease gene amplification of specific genes as a function of age. Gene amplification often occurs as a result of normal aging and metabolism. The p53 suppressor gene could become an oncogene upon mutation. It is also known to be modulated by dietary restriction. The wild-type p53 is known to suppress the growth of transformed or initiated cells. Mutated forms of the p53 gene have been associated with a number of human cancers. The mutational spectrum of the p53 gene ranges over a large area of the gene, however four known hot spots have been identified to be associated with exposure to certain classes of chemicals or carcinogens. Modulation of these mechanisms - epigenetic, point mutation frequencies or amplification by dietary restriction may play an important role in the ability of dietary restrictions to prevent or delay the formation of diseases such as cancer. Gene Expression and Dietary Restriction Cancer and other degenerative diseases are often associated with cell proliferation. Dietary restriction decreases the rate of cell proliferation in vivo and in vitro(1,2). Cell proliferation is often associated with activation of certain proto-oncogenes such as those found in the ras family(3,4). The gene product of the ras family is known to function as a G-protein and play a role in the signal transduction pathway. Activation of the signal transduction pathway generates second messengers in the cell which exert other effects that lead to proliferation. Dietary restriction is known through epigenetic and genetic means to decrease or delay ras activation which would lead to decreased cell proliferation(5). The expression of proto-oncogenes was initially linked with dietary restriction by Nakamura and co- workers(6), who showed that c-myc expression in cells from B6C3F1 mouse liver followed a circadian pattern and that the expression in dietary restricted animals was always suppressed relative to their ad libitum fed-controls. I

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' ' Methylation and Dietary Restriction Epigenetic factors such as methylation may modify gene structure by modifying the interaction of transcriptional factors which bind to specific regions on the gene(7). The loss of methylation (hypomethylation) may result in a change in the fidelity of the DNA. The loss of methylation may result from a number of factors including changes in methylation metabolism. Alteration in methylation metabolism may be due to a decrease in availability of methyl donors to various macromolecules as a result of deficiencies in certain lipotropes and molecules such as methionine, choline, vitamin B12 and folic acid(8). A decreased intake of these nutrients is known to promote a hypomethylated environment(9). Hypomethylation of cellular DNA may also result from an impairment of enzymes such as methyltransferase, which is known to catalize the post-synthetic methylation of DNA. Recently, studies have shown that the activity of inethyltransferases is dependent upon the presence of the trace metal, zinc(10). Our laboratory recently has shown a correlation between the hypomethylation of the H- and K-ras genes and marginal zinc deficiency in the rat exocrine pancreas(11). Gene Amplification and Dietary Restriction Amplification of cellular genes was first encountered in mammalian cells that had acquired a resistance to chemotherapeutic agents(12). However, amplification of specific proto-oncogenes has been found as an occasional feature of a number of tumors. Our laboratory has demonstrated that dietary restriction delays or decreases amplification of the H-ras gene as a function of age and gender in the exocrine pancreas of rats. The dietary restriction effect is more pronounced in the exocrine pancreas from female rats. Conclusion Pathological diseases, such as cancer, have many causes many of which may act through a common pathway via damage to cellular DNA. The three molecular mechanisms for carcinogenesis described in this manuscript-epigenetic, gene amplification and mutations-are summarized in Tables 1 and 2. Our studies, along with others (13), have shown that dietary restriction decreases hypomethylation of proto-onogenes, particularly the H-ras gene. Figure 1 shows the effect of age, dietary restriction, and gender on H-ras gene amplification in rat pancreatic acinar cells. Amplification and expression of the H- ras gene in aged and dietary restricted animals is decreased relative to their ad libitum counterparts. Dietary restriction in animals has provided a model that suggests low intake of calories to have profound protective effects against tumor formation. Such a paradigm also contributes to our understanding of the molecular mechanisms which may contribute to the development of tumors. However, establishing dietary restriction criteria as possible measures used in human studies need further investigation. Noninvasive methods must be developed to monitor dietary restriction effects on humans if such they are to be used as a tool in cancer prevention or as cancer therapy. Investigations must be carried out to determine if dietary restriction may play a role in the initiation or progression stage of carcinogenesis. N O 00 ~ N 00 -2- W ~ A T I I I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I I I References 1. Hass, B.S.; Hart, R.W.; Gaylor, D.W.; Poirier, L.A. and Lyn-Cook, B.D., 1992. An In Vitro Pancreas Acinar Model for Testing Modulation Effects of Caloric Restriction and Aging on Cellular Proliferation and Transformation. Carcinoeenesis 13:2419-2425. 2. Roebuck, B.D.; Baumgartner, K.J. and MacMillan, D.L., 1993. Caloric Restriction and Intervention in Pancreatic Carcinogenesis in the Rat. Cancer Res., 53:46-52. 3. Barbacid, M., 1987. Ras Genes. Annu. Rev. Biochem., 56:779-827. 4. Krengel, U.; Schlichting, L; Scherer, A.; Schumann, R.; Frech, M.; John, J.; Kabasch, W.; Pai, E.F. and Wittinghofer, A., 1990. Three-Dimensional Structures of H-ras p21 Mutants: Molecular Basis for Their Ability to Function as Signal Switch Molecules. Cell 62:539-548. 5. Hass, B.S.; Hart, R.W.; Lu, M.H. and Lyn-Cook, B.D., 1993. Effects of Caloric Restriction in Animals on Cellular Function, Oncogene Expression and DNA Methylation in Vitro. Mutation Research 295:281-298. 6. Nakamura, K.D.; Duffy, P. H.; Lu, M.H. and Hart, R.W., 1990. Hepatic Myc Proto-Oncogene Expression is Reduced and Possibly Correlated With Body Temperature in Fasted Peromyscus Leucopus Mice. Age 13:27-31. 7. Borrello, M.G.; Pierotti, M.A.; Tamborini, E.; Biassoni, D.; Rizzetti, M.G.; Pilotti S. and Della Porta, G. 1992. DNA Methylation of Coding and Non-Coding Regions of the Human H-ras Gene in Normal and Tumor Tissue. Oncogene 7:269-275. 8. Poirier, L.A.; Zapisek, W.F. and B.D. Lyn-Cook, 1990. Physiological methylation in carcinogenesis. In: Mutation and the Environment, Ed. Wiley-Liss, Inc. 9. Zapisek, W.F.; Cronin, G.M.; Lyn-Cook, B.D. and L.A. Poirier, 1992. The Onset of Oncogene Hypomethylation in the Livers of Rats Fed Methyl-Deficient, Amino Acid-Defined Diets. Carcinogenesis 13:1869-1972. 10. Bestor, T.H. 1992. Activation of Mammalian DNA Methltransferase by Cleavage of a Zn Binding Regulatory Domain. EMBO J. 11(7):2611-2617. 11. Lyn-Cook, B.D.; Ellwood, K.; Bo, J.; Roebuck, B.D, and Hathcock, J.N., 1994. Increased Expression of the Multidrug Resistance (MDR) Gene in Rat Pancreas of Rats Fed a Marginally Zinc-Deficient Diet. Proc. of American Association of Cancer Research. 35:3288. 12. Schimke, R.T., 1984. Gene Amplification in Cultured Animals Cells. Cell 37:705-713. N0 ' 00 ~ ~ 00 3_ ~ ~ I

I I I I I I I I I I I I I I I Table 2. Effect of PHITC on NNK-induced RTE cell transformation Groups NNK PHITC CFE% TF % (mg/kg.bw) (mmol) Control 0 0 1.578 1.16 NNK 30 0 1.492 3.60 NNK/PHITC 30 0.71 1.276 1.51 * *P<0.01 (compared with NNK group) Table 3. Effect of EGCG on B(a)P-induced RTE cell transformation Groups B(a)P EGCG CFE% TF% (mg/kg.bw) (mg/kg.bw) Control 0 0 1.57 0.68 B(a)P 25 0 1.46 5.23 B(a)P/EGCG 25 600 1.46 1.73* *P<0.01 (compared with B(a)P group) Table 4. Altered growth factor dependence of B(a)P-transformed RTE cell line SFM SFM-BPE CFE% 9.4t 1.2 0.95 t0.02* SFM-BSA SFM-EGP 6.5 t 1.0* SFM-B:B..E 10.6f1.5 0.0±0* PD/D 0.73±0.03 0.42±0.05 0.68±0.04 0.73±0.03 0.0±0 *P<0.01 (compared with SFM group) SFM: growth factors modified serum free medium , BPE: bovine pituitary extract BSA: bovine senun albumin EGF: epidermal growth factor N ' B.B.E.: BPE, BSA and EGF O CFE: colony forming efficiency 3 PD/D: population doubling/day ~ i U1 ~ _3- '

I I I I I I I I I I I I 1 I I I I I TRANSFORMATION OF TRACHEAL EPITHELIAL CELLS AND THE ROLE OF TRANSFORMING GROWTH FACTOR (TGF) AND P53 IN THE LUNG CANCER PROGRESSION Wang Hong, Cheng Shu jun, Lin Li-nun, Chen Lei, Guo Shu-pin, Fen Ji-nong, Han Nai jun and Sun Han-xiao Cancer Institute, CAMS and PUMC, Beijing, China Although lung cancer is one of the most common cancers in the world, little is known yet about genetic changes associated with its development. To facilitate the study of the genesis of lung cancer, we have developed a number of experimental models including a rat tracheal epithelial (RTE) cell transformation system and two SV40 T-antigen immortalized human bronchial epithelial (HBE) cell lines. The purpose of this study was to examine the potential of a carcinogen to induce neoplastic transformation and its chemoprevention. We investigated the role of growth factors, especially transforming growth factor (TGF), and p53 tumor suppressor genes in the lung cancer progression. 1. Carcinogen induced neoplastic transformation and its chemoprevention Cigarette smoking condensate (CSC), tobacco specific nitrosamine NNK, B(a)P, and coal tar pitch (CTP), which were considered as potential etiological factors for human lung cancer, were tested in the in vivo - in vitro RTE cell transformation system. Carcinogens were given by intratracheal instillation, RTE cells were then isolated and examined in culture for the presence of preneoplastic variants. The results showed that CSC (Table 1), NNK, B(a)P, and CTP can significantly increase the transformation efficiency (TF) of RTE cells. Squamous cell carcinoma arose in nude mice after they were inoculated with the serially subcultured transformed cells. Since 6-phenythyl isothiocyanate (PHITC) and epigallocatechin-3-gallate (EGCG) were considered as potential lung cancer preventive agents, their effects on RTE transformation were tested. The results showed that PHITC (Table 2) and EGCG (Table 3) inhibited the RTE cell DNA alkylation and preneoplastic transformation induced separately by NNK or B(a)P, and may be useful in lung cancer chemoprevention. 2. The role of TGF-a, TGF-(3, and p53 in neoplastic transformation of airway epithelium Most cancers develop in multiple stages. The RTE cell transformation system, which is suited to define multistages during transformation, is useful to explore mechanisms involved in the neoplastic transformation of airway epithelium. By a clonal growth assay, altered responsiveness of neoplastically transformed RTE cell lines to selected growth factors was determined. The results (Table 4) showed that transformed RTE cells lost their growth dependence on the addition of epidermal growth factor (EGF), but still required bovine pituitary extract (BPE) and bovine serum albumin (BSA) to be present for effective cell proliferation. I

I I Overexpression of the TFG-a protein was detected by inununocytochemistry in transformed (preneoplastic and neoplastic) RTE cell lines, SV40 T antigen immortalized HBE cell lines and non-small- cell lung cancer cell lines indicating that increased TGF-a expression is an early event in the multistage process of neoplastic transformation, and may play an important role in the lung cancer progression. EGF independence in the transformed RTE cells could conceivably be related to the overexpression of TGF-a which is known to share structural and functional homology with EGF. The colony forming efficiency (CFE) of normal primary and preneoplastic cells was inhibited to varying degrees by the conditioned medium (CM) prepared from preneoplastic and neoplastic RTE cells (Table 5). The inhibition was blocked by a TGF-(3, neutralizing antibody (Table 6). In contrast, the CFE of neoplastic RTE cells was not affected by the CM (Table 5). These data implied a paracrine role for TGF-01 in the RTE cell transformation. Southern blot analysis showed TGF-/3, to be amplified in a SV40 T-antigen immortalized HBE cell line, a lung squamous carcinoma cell line, and a lung adenocarcinoma cell line. In addition, the structure of the TGF-R, gene may also be altered in a small-cell lung cancer cell line. Taken together, these data strongly suggest that TGF-01 plays an important role in the airway epithelium transformation. p53 expression was also studied in these experiments. Partial deletion of the p53 gene was found in the NNK- and MNNG-, but not in B(a)P-transformed RTE cell lines, suggesting that deletion of the p53 gene is an important but not a necessary event in the RTE cell transformation. When a mutant p53 gene was transfected into NNK-treated preneoplastic RTE cells, cell transformation was observed. Transfection of a mutant, but not wild type p53 gene increased TGF-01 expression and its paracrine inhibition on normal RTE cells (Table 8). Wild type p53 also repressed the proliferation of preneoplastic RTE cells (Table 7). Alteration of TGF-a was not found in either the wild type p53 or the mutant p53 transfected RTE cells. It has been reported that activation of oncogenes or inactivation of tumor suppressor genes are involved in lung cancer development. In this study, we found that transfonning growth factor, TGF-a and TGF-,Q,, and p53 tumor suppressor gene play an important role in the lung carcinogenesis. Further studies will be required to define the relationships between TFG-a, TGF-01 and p53 gene expression. Table 1. Transformation of RTE cells by CSC CSC (mg/kg.bw) CFE% TP% 0 1.38 0.6 9 1.11 1.54* 17.5 1.01 2.75* *P < 0.01 CSC: cigarette smoking condensate CFE: colony forming efficiency -2- TF: trarsfotming &equency N 0 CO i V 00 w ~ cn tW I I I I I I I I I I I I I

I I I I I I I I I I I I I I Table 7. Colony forming efficiency of wild and mutant p53 gene transfected RTE cell lines Group CFE% NNK21 NNK21p53WT NNK21p53MT 6.60t0.6272 5.44±0.4307* 7.13±0.2622 *P G 0.05 (compared with NNK21 group) NNK21: NNK transformed RTE cell line NNK21p53WT: wild type p53 gene transferred NNK21 cell line NNK21p53MT: mutant type p53 gene transferred NNK21 cell line Table 8. Effect of CM harvested from wild or mutant p53 gene transfected RTE cell lines on the proliferation of primary RTE cells Source of CM primary RTE+CM primary RTE-+CM ' CFE % PTND Control 1.120±0.0794 0.69±0.04 NNK21 0.483 t 0.0252 0.56 t 0.01 NNK21p53WT 0.55±0.0608 0.60±0.03 NNK21p53MT 0.297±0.0569* 0.49±0.04 *PG0.01 (compared with NNK21 CM group) ' 0 00 -4 1 3 00 W i -5' CD I

' 1 Table 5. Effect of conditioned medium prepared from different cell lines on the CFE of primary, preneoplastic and neoplastic RTE cell lines I I Source of CM primary RTE NNK16 NNK47 .B(a)P39 CFE% CFE% CFE% CFE% I Control CM 0.99±0.04 8.94t0.18 9.84±0.56 9.90±0.48 NNK15CM 0.02±0.09* 3.07±0.28* 9.46±0.14 9.43t0.09 I NNK45CM 0.038t0.002* 3.66±0.08* 9.85±0.01 9.85t0.43 B(a)P37CM 0.017±0.004* 3.30±0.18* 9.83t0.26 9.75±0.38 I * P<0.01 CM: conditioned medium NNK15, NNK16: NNK induced preneoplastic transformed RTE cell lines NNK45, NNK47: NNK induced neoplastic transformed RTE cell lines B(a)P37, B(a)P39: B(a)P induced neoplastic transformed RTE cell lines I I Table 6. Bloclung of the inhibition of the conditioned medium from B(a)P41 on the proliferation of primary RTE cells by the TGF-,Gi neutralizing antibody I I primary RTE CFE% Relative CFE I SFM 2.62 t 0.06 100 % SFM+B(a)P41CM 0.82±0.04 31.3% I SFM+B(a)P41CM+TGF-/31 Ab (6µglml) 1.19±0.07 45.4% SFM+B(a)P41CM+TGF-/3, Ab (15µg/ml) 1.73±0.03 66.0% I SFM: modified serum free medium O O ~ I I V O W I ~ tT tn -4- I I

I ' I I I I I I I I I I I I I I I I with 12-o-tetra-decanoyl-phorbol-13-acetate (TPA, 10 pg/ml) for two weeks. DNA was isolated from cells by standard techniques, and used as a template for PCR amplification of H-ras sequence. The PCR-primers used to amplify codon 12 of H-ras genes are shown in Fig 2(6,7). PCR was performed at 97° C to denature the DNA for 5 min, at 72° C to anneal the primers for 1.5 min and at 93 and 550 C for 1 min at each temperature for primer extension. After amplification, H-ras point mutations were subsequently detected by the restriction fragment length polymorphism (RFLP) method with the use of the restricted enzyme Hpa II. The PCR product was digested with the restriction enzyme Hpa II. DNA fragments were electrophorezed on 6% polyacrylamide gel. Gels were stained with ethidium bromide and photographed on a UV transilluminator. Results HPLC analysis was performed after B(a)P was metabolized by microsomes isolated from human fetal liver and lung cells. The result indicated that three derivaties of dihydrodiolbenzo(a)pyrene [9, 10- diol-B(a)P, 7, 8-diol-B(a)P, 4 5-diol-B(a)P], two metabolites of hydroxybenzo (a) pyrene [9-OH-B(a)P, 8-OH-B(a)P] and one product of quinonebenzo (a) pyrene [quinone-B(a)P] were formed upon incubation with microsomes from either human fetal liver or lung cells. I

I by mutations induced in the DNA damage introduced by B(a)P metabolites. Among human lung tumors, point mutations of ras oncogenes may exist in 50% of lung adenocarcinomas. Most of point mutations are also at codon 12. This indicates that point mutation of ras oncogenes at codon 12 has a close relationship with the initiation of lung cancer. In our transformation test of human bronchoepithelial cells, the point mutation of the H-ras oncogene at codon 12 was found, despite the fact cells showed no significant morphological change. The initiation of point mutation of oncogenes was earlier than the transformation in cell morphology. The point mutation of oncogenes may be regarded as a sensitive indicator of cell transformation or an early stage of chemical carcinogenesis in human lung cancer. -8- I I I I I I I I I I I I I I I

i I I I I I I 1 I I I I I I I I I I BIOASSAYS OF BENZO(A)PYRENE AND LUNG CANCER Wu Zhon -g liang*, Chen Jia-kun*, Zhan De jin*, Jin Bo*, He Ling*, Du Ying-xiu* and Joseph M. Wu** * Guangzhou Institute for Chemical Carcinogenesis, Guangzhou Medical College, Guangzhou,China ** Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, USA Introduction Benzo(a)pyrene (B(a)P) is a ubiquitous environmental contaminant generated by combustion of substances such as coal, tobacco and other organic chemicals. Possible human exposure occurs through a number of routes including inhalation of polluted atmospheres and cigarette smoke. Epidemiological studies have shown a close relationship between human lung cancer and exposure to B(a)P(l). B(a)P is a procarcinogen that requires metabolic activation to exert its mutagenic and carcinogenic effects(2,3). The metabolism of B(a)P has been studied in detail and the mutagenicity or carcinogenicity of B(a)P metabolites have been examined in a variety of prokaryotes, eukaryotes and experimental animals. These studies have shown that the amount and type of metabolic activity for biotransformation of B(a)P differs markedly among species as well as among the tissues of a particular species. Thus, it is difficult to extrapolate the results from animals and cells to the humans because of inter- and intra- species variability. In this study, human fetal broncho-epithelial cells (HFBE) cultured in vitro were used as an assay system to investigate the genotoxicity of the metaboltes of B(a)P for a better understanding of the role of B(a)P in human lung cancer initiation. Materials and Methods Materials Anti-7,8-dihydrodiol-9,10-epoxybenzo(a)pyrene (anti-BPDE), syn-7,8-dihydrodiol-9,10- epoxybenzo(a)pyrene(syn-BPDE), 9-hydroxybenzo(a)pyrene(9-OH-B(a)P), 3-hydroxybenzo(a)pyrene (3-OH-B(a)P) and 7,8-dihydrodiolbenzo(a)pyrene (7,8-diol-B(a)P) were purchased from commercial sources. MCDB 153 medium, restriction enzymes, reagents used for culturing broncho-tracheal epithelial cells and oncogene analysis were obtained from Sigma Chemicals Co. All other reagents were purchased in China. Cell cultures and preparation of liver and lung microsomes 1. Bronchoepithelial cell cultures. Tracheobronchial tissues from an abortive fetus were cut into small pieces (2x2 mm) and seeded onto cover glasses coated with rat-tail collagen. The coverslips were placed in tissue culture plates. Cells were cultured in MCDB 163 medium supplemented with 0.1% fetal bovine serum, insulin (10 µg/m1), epinephrine (10 µg/m]), hydrocortisone (0.72 µg/ml), epidermal growth factor (2.0 µg/ml), transferrin (5 µg/ml) and antibiotics and incubated at 37° C in a humidified atmosphere of 5% CO2. The medium I

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l.-r Is-2 L-3 I.-a nln>P I.-1 ~j J Rab Liver ]s-l i.-2 1.-3 1s-4 a<n)r I.-2 /.-3 L-q gat Lung 1.-1 I.-2 ls-3 L~ lI F J I 0 Human retal Liver ilnman Fetai Lung Is-I: Ildielllfervne Is-2) Acenzphfhllene Is-7: Phrnnlhrrne Is-11 Pirene 9(a)N 9entn(a)pJrr.ne 9,19-d(n(a 9.19-0ihrJrmld(albevn(alpirene 1,1-Jiol ],d-9ihJdrwJdlolbrnm(alprrene I,S,-diol) /,SD(hplrallAln(benr.n(alplrene ]-011:7-IlrJrnlrhnnzu(a)prrnnn - 9-011~ 9-16drulrbeom(a)plrnne au(nnne-E(a)P~ Ouinoneben~a(a)p)rene Fr8.1 Reversed phase IIPI,C profile of B(a)P metabolites obtained from incubation of UWP wiLh microsomes (UV absorbance 254 nm ) 496£8L480Z m m r m m r = Iir m " t• m ~ M M ~ M M M

I I I I . References Brislow, L, et al.; Amer. J. Publ. Health, 1954; 44: 177 2. Huberma, E, et al.; Pro. Nat. Acad. Sci. (Wash), 1976; 73:607 I 3. Wood, AW, etal.; J. Biol. Chem., 1976; 251:4882 I 4. Lowry, OH, et al.; J. Biol. Chem., 1951;193:265 5. Fenech, M & Morley, A A; Muta. Res., 1985; 147:29 I 6. Capon, DJ, eta l.; Nature, 1983; 302:33 1 7. Minoru, Tada, et al.; Cancer Res., 1990; 50:1121 8. Autrup, H, et al.; Int. J. Cancer, 1980;25:293 I 9. Mivhsrl, H, et al.; Chem-biol. Iterations, 1988; 64:281 10 H i CC l I P h 1 . arr s, , eta .; n at ogenesis an Therapy of Lung Cancer, Harris, New York, Marcel Decker Inc, 1987;550 11. Heflich, RN, et al.; Biochem. Biophys. Res. Commum.. 1977;77:634 I 12. Kapitulnik, J, et al.; Cancer Res., 1978; Sept 38(9);2661-5 I 13. Veffery, AM.; Pharmac. Ther., 1985;28:237 14. Verina, DM, et al.; In: H.H Hlatt et al. eds, Origins of Human Cancer New York: Gold Spring I 15. Harbor Laboratory Press, 1977;PP639-658 Santos, E, et al.; Proc. Nat. Acad. Sci. USA, 1983;80:4679 I 16. Barbacid, M.; Ann. Rev. Biochem., 1987; 56:779 I 17. Guerrero, I, Pellicer, A; Mutat. Res. 1987; 185:293 I O 00 i 'V co , -9- ~ 0) I ol

I I I I I I I I I I I I I I I I I Treatment of HFBE cells with metabolites of B(a)P [anti-BPDE, syn-BPDE, 7, 8-diol-B(a)P, 9- OH-B(a)P, 8-OH-B(a)Pj resulted in induction of unscheduled DNA synthesis (UDS) in a concentration- dependent manner. (Table 1). Similar results were obtained with the broncho-epithelial cells isolated from different individuals indicating that no significant inter-individual variation existed. (Table 2). Each metabolite of B(a)P, except for Syn-BPDE, could enhance the micronucleus rate of HFBE cells; it was evident that there was a dose-response relationship. (Table 3). The result described above show that among the B(a)P metabolites studied, anti-BPDE had the most significant effect on either UDS or micronucleus formation in HFBE cells. These results demonstrated that metabolites of B(a)P can induce lesions in DNA which subsequently resulted in unscheduled DNA synthesis. Table 1. UDS in IIFBE cells induced by anti-BPDE (relative radioactivity, 'HP`C) Concentration Cell (µg/ml) HT-11-a HT-11-22 HT-11-12A 0.00 1.00 ± 0.20 1.00 ± 0.97 1.00 t 0.13 0.125 1.34 f0.60 1.18 0.86 6 5 1.25 0.25 0.250 1.43 t 0.23 1.81 t 0.18 1.65 t 0.46 0.500 2.36 0.88 8 2.55t0.17* 0 2.09 0.60 0.650 3.27 t 0.79* 3.20 t 0.19* 2.50 t 0.37 0.800 4.44 f 1.75* 6.18 f 0.23** 5.42 t 1.63* 1.000 1.88 0.21 1 2.07 f0.18 1.89 0.63 3 8 ± SD *P<0.05 ** P<0.01 Table 2. UDS in the same HFBE cells induced by B(a)P Metabolites (relative radioactivity, 'H/14C) Cnncentrauon (Izg/mt) an6-BPDE 0.000 1.00 ± 0.13 0.125 0.250 0.500 0.800 t SD 1.25 t 0.25 1.66 t 0.46 2.09 t 0.60** 5.42 t 1.83** P<0 05 syn-BPDE 7,8-dinl-B(a)P 1.00 0.22 2 1.00 0.09 9 0.98 0.44 4 1.04 0.09 8 1.33 t 0.33 1.29 t 0.05 1.46 t 0.29* 2.48 f 0.29"* 3.52 t0 57 P<0.01 1.33 ± 0.01 9-0H-B(a)P 3-011-11(a)P 1.00 0.02 2 1.00 0.04 4 1.66 t 0.40 - 1.70 027 7 1.08 0.18 8 1.20 1.16 6 1.37 0.25 5 N 3.57 t 1.17* 1.78 t 0.15• ~ 3 V 00 W -5- j rn f N I

I was replaced twice weekly. When outgrowths of cells radiated from the tissues to a distance of 0.5 cm, repeated transfer of explants to new coverslips was done to reinitiated cell cultures. Following their identification by immunohistochemical staining, epithelial cells were used in this study. 2. Preparation of liver and lung microsomes. Liver and lung tissues from a fetus were cut into small pieces, rinsed with 0.9 °lo sodium chloride solution and 50 ml of 50 mM sodium pyrophosphate. After the tissues were homogenized, the homogenate was centrifuged at 10,000 xg for 20 min. The supernatant was recentrifuged at 100,000 xg for 60 min. The pellated microsomes were stored at -70° C until ready for use. Microsomal protein was determined by the Lowry method(4). 3. Metabolism of B(a)P by microsomes The metabolism of B(a)P was studied in a 100 ml reaction mixture containing 50 mM Tris-HCI (pH 7.4), 0.3 mM magnesium chloride, 0.1 mM NADP+, 0.2 mM glucose-6-phosphate, 10 units of glucose-6-phosphate dehydrogenase, 100 mg of microsomal protein and 4 µM B(a)P. After shaking at 37° C for 60 min, the reaction was stopped by adding an equal volume of acetone. Materials in the organic phase were extracted twice with 1.5 volumes of ethyl acetate. To stabilize the metabolites, 1% triethylamine was added to the ethyl acetate fraction. The organic phase was dried with anhydrous sodium sulphate and the solvent was evaporated under reduced pressure. The residue was stored at -20° C or dissolved in methanol for analysis by HPLC. 4. Unscheduled DNA synthesis (UDS) The coverslips on which epithelial cells were growing were placed into liquid scintillation vials, treated with'"C-TdR (0.01 µCi/ml) for 72 hr., and then with'H-TdR (I µCi/ml) and B(a)P metabolites for an additional 24 hr. The cells on the coverslip were washed with 0.9% saline solution and treated with trichloroacetic acid and absolute alcohol. After drying at 60° C, radioactivity was measured with a Beckman LS6000SC liquid scintillation system. 5. Micronucleus test. The method for the micronucleus test used in this study was as described by Fenech and Morley(5). The epithelial cells cultured on the coverslip were exposed to the metabolites of B(a)P and cytochalasin B (3 µg/ml) for 24 hr. Micronuclei were scored in cytokinesis-blocked binucleus cells. The significance of the results was tested with the Poisson distribution method. Determination of point mutation of Ha-ras oncoeenes To determine point mutations on the Ha-ras oncogene, the polymerase chain reaction was used to amplify H-ras specific sequences present in DNA extracted from cells treated with the B(a)P metabolite, anti-BPDE. After outgrowths of the cells bordering the explants to a distance of 0.5 cm, anti- BPDE (1.5 µg/ml) was added to the medium. The medium was replaced by a fresh one 24 hr later. Cells were treated with anti-BPDE once a week for four weeks. Some cultures were subsequently treated I I I I I I I I I I I I '

10 µ1 of supernatant was used for the polymerase chain reaction (PCR). The specific primers for GSTµ gene selected from the known GSTmI cDNA sequence according to the homologous rat genomic DNA sequence(4) were prepared with a DNA synthesizer. A segment of about 250 base pairs covering exon 4 and exon 5 of the GSTg gene was amplified by PCR in this study (Fig 1). , E1 EZ E3 E4 ES E6 E7 E8 3, rr Fig. 1 Sketch map of human GSTµ gene and the segment of GSTµ nucleotide in this study ( t}). 3. PCR. PCR was performed in 100 µl reaction buffer containing 200 µM dNTP, 1 µM primers, 10 µl denatured DNA and 2 units of thermostable Taq polymerase using a heat block instrument (Techne). Thirty cycles of amplification involving a 1 min denaturation at 94°C, a 1.5 min annealing at 56°C and a 1 min extension at 72°C were performed. 4 Electrophoresis. The amplification products were separated on 2% agarose S (sea Kem) gels and identified under W light. The presence of GSTµ gene was identified by a clear band of GSTµ gene amplified products migrating to a position of 250 bp. No band was found if GSTp gene deletion had occurred. (Fig. 2). S t a a 4 e s 7 8 9 Fig. 2 The PCR amplification products of GSTfc nucleotide sequence between exons 4 and 5 using DNA samples from blood. No.1,2,4,7 showed GSTµ gene presence, and No.3,5,6,8,9 showed the gene absence. S: 100 bp DNA fragment ladder. -2- I I I I I I I I I I I I I I I

2081783174

I I I I I I I I I I I I I I I I Results 1. Table 1 shows the comparison of GSTµ gene deletion in lung cancer patients and controls. The results indicated that the GSTµ gene deletion rate in lung cancer patients was 71.4% which was significantly higher than the 51.9°k in controls. (p<0.005). Table 1. Frequency of the presence and absence of the GSTµ gene in lung cancer patients and controls Group Presence (%) ~ Absence (%) .. . . . . .. ~ . Total ~ ~ Lung cancer 50 (28.6) 125 (71.4) 175 controls 50 (48.1) 54 (51.9) 104 X'=10.37 P<0.005 OR=2.3 95%Cl 1.39-3.82 2. The stratified analysis of GSTµ gene deletion, according to the pathological types of lung cancer, indicated that in all squamous, adenocarcinoma and small cell carcinoma groups, the GSTµ gene deletion rate was markedly higher than that in controls. In the small cell cancer group, the deletion rate reached 77.5 % (see table 2). Table 2. Frequency of the presence and absence of the GSTµ gene in different pathological types of lung cancer Pmsence Absence Patholo8ic ----- ----- X, ~ • P . OR . .~. 95% Ct Cell Types . . Cases (%) CaSes (%)' .::.... Squamous cell carcinoma 22 (29.7) 52 (70.3) 5.78 <0.05 2,19 1.16d.15 Adenocan:inoma 19 (31.1) 42 (68.9) 4.31 <0.05 2.05 1.04d.04 Small cell carcinoma 9(22.5) 31 (77.5) 7.57 <0.05 3.19 1.40-Z29 3. Table 3 shows the stratified analysis of the GSTµ gene deletion rate, according to smoking status. The data show that the deletion rate of the GSTµ gene in lung cancer patients was significantly higher than in the controls, no significant difference was observed between the smoking and nonsmoking groups in either lung cancer cases or controls. 4. Both the patients and controls were divided into two age groups to analyze the rate of GSTµ gene deletion. The older age group included subjects above 50 years of age and the younger group included subjects below 50 years of age. The results showed that the frequency of GSTµ gene deletion in lung cancer patients in both the older group and the younger group was significantly higher than that in the controls. The stratified analysis in each group showed that in controls, the GSTµ gene deletion rate had no correlation with age, but in lung cancer patients, the deletion -3- I

I I I I I I I I I I I I i I I I I I Discussion B(a)P is a procarcinogen which requires metabolic activation to exert its carcinogenic effect. Activation occurs mainly in the liver. Fourteen kinds of metabolites may be formed by the metabolism of B(a)P. The majority of them are "not toxic," only a few metabolites have very significant biological activity. Metabolism of B(a)P by the lung has not been reported so far. Microsomal proteins from human fetal liver and lung are found to metabolize B(a)P into its ultimate carcinogenic fotms. These data are similar to those previously reported for human bronchoepithelial cells(8-10). In situ metabolism in lung tissues may be important in the initiation of cancer at these sites. The epithelial cells are of particular interest, since they are the first to be in contact with environmental contaminants. The ability of lung tissues to activate B(a)P may therefore be an important factor in the induction of lung cancer resulting from inhalation of air pollutants containing B(a)P, such as, tobacco smoke, cooking fuel, etc. In previous experimental studies, animals and their cells were used to detect whether B(a)P metabolites had potential harmful effects to lung tissue. The extrapolation to actual human situation of carcinogenesis based on studies in experimental animals and cells presents complex challenges because of inter-species differences. Human cells were used in the present study to avoid these shortcomings. Because the majority of human lung cancers originate from epithelial cells, it seems more reasonable to use human epithelial cells as target cells than animal cells or human fibroblasts. Using human epithelial cells may avoid inter-species differences and inter-tissue variability. Human fetal bronchoepithelial cells cultured in vitro were treated with each of the five metabolites of B(a)P. The results showed that anti-BPDE had the most significant effect in inducing UDS and enhancing the micronucleus formation. This finding was consistent with that published previously(I1). Kapituluik et al.(12) found that syn-BPDE did not induce tumor in mice. Thus, it is reasonable to conclude that anti-BPDE is the main carcinogenic metabolite of B(a)P, while 3-OH-B(a)P, 9-OH-B(a)P and 7, 8-diol-B(a)P are simply metabolic intermediates of B(a)P which must be metabolized further to form BPDE. Metabolic activation is the first step in the carcinogenesis process. Anti-BPDE can form a major DNA adduct by binding through its C10 position to the NZ of deoxyguanosine(13). It has been indicated that diol epoxide with the epoxyring located at the angular 'bay' region should be the most reactive, and therefore, likely to be the ultimate mutagenic and carcinogenic form of B(a)P(14). Binding of anti-BPDE to DNA may damage DNA and induce occurrence of UDS and MN. Anti-BPDE and Syn- BPDE are two metabolites of B(a)P which have different stereoscopic structures and possibly have differently biological effects. Since anti-BPDE has the most significant mutagenic effect in human cells, among the five metabolites of B(a)P, and mutagenesis is generally correlated with carcinogenesis, human fetal bronchoepithelial cells were treated continuously with anti-BPDE to further investigate its carcinogenesis by the determination of oncogene activation. The result indicated that cells grew normally and showed no morphological change. The point mutation at codon 12 of the H-ras oncogene in treated cellular DNA was detected by the polymerase chain reaction combined with RFLP analysis. It has been suggested that oncogenic activation occurs when any other aminoacid (except proline) is substitute in place of glycine as a result of a mutation in codon 12 of the ras gene(15). Point mutations in ras oncogene have been observed in human tumors of diverse origin and in a wide variety of carcinogen-induced animal tumors(16,17). These results further support the hypothesis that the ras oncogene is directly activated -7- I

I I I I I I I , I I I I I I carcinoma. No study has reported GSTµ gene deletion being significantly correlated with adenocarcinoma, especially in smokers. Our study shows that GSTµ gene deletion in the lung cancer patients were markedly higher than that in controls. All three pathologic types of lung cancer had elevated GSTµ gene deletions although the highest rate (77.5%) was seen in the small cell carcinoma group. However, when stratified groups by smoking, no apparent relationship between smoking and GSTµ gene deletion was found. These results suggest that there may be factors other than smoking which may have a potential association with lung cancer development. Since GSTµ has the highest specificity and activity for the bio-transformation of PAH and many environmental carcinogens (including the products of smoking and B(a)P belonging to the PAH family of compounds), to inactive metabolites, we can infer that the deletion of the GSTµ gene may be one of the most important host factors for susceptibility to lung cancer. Lafuente et al.(9) reported that susceptibility to cancer due to GSTµ gene deletion may manifest itself in an earlier age of cancer development and a more malignant form of carcinoma. In this study, when the groups were stratified by age, there was no relationship between GSTµ gene deletion and age found in the controls. However, in the lung cancer groups, the GSTµ gene deletion rate of the younger age group was 85.3 % which was markedly higher than that of the older group. Although the causes of lung cancer are becoming clear, the process of carcinogenesis is a complex interaction of multiple factors. These factors can include exposure to carcinogens, the degree of exposure, and the host conditions, all of which may converge in some yet-to-be-defined mechanism in causing the onset of cancer. The discovery of the GSTµ gene and its involvement in bio-inactivation of carcinogens provides an important lead for exploring host susceptibility to lung cancer at the molecular level. In this research, we used a case-control study to demonstrate that the GSTµ gene deletion rate in lung cancer patients was significantly elevated, compared to controls. A prospective cohort study involving GSTµ gene deletion assessment, in combination with data on exposure to environmental carcinogens may further clarify the role of GSTµ gene in lung carcinogenesis. -5- I

I I Table 3. Micronucleus formation in cytokinesis-blocked HFBE cells induced by B(a)P metabolites (%) Concentration` (ug/ml) ; anti-BPDE " svri-BPDE 9-OH-B(a)P 3-OH-B(a)P 4 4 4 9 8 23** 4 15* 39** 5 20** 50** 6 10 8 *P<0.05 **P<0.01 ***P<0.001 After being treated intermittently with anti-BPDE, HFBE cells showed no significant morphological changes. There were no cellular morphology changes characteristic of transformed phenotypes. The PCR-amplified H-ras oncogene fragments had a length of 145 bp including codon 12 of the H-ras oncogene. Two HPaII sites are present in wild type ras gene (one at the 25 bp position, the other at the 81 bp position) (Fig. 2), which could be lost as a result of mutations. Figure 2 shows that anti-BPDE induced point mutation at codon 12 of H-ras oncogene (Fig. 3) I I I I I I I I PlIMES 1 ~'CJS]tGTCGCAAAA]CGTTCi• l4WE[ C!'pGGGACACCQGlAGCAC]' Hpa I[ pem.r 1 / \ TD. 1] cca.v H-m. evcaC.ac / I ~]!!p I ~Ehp I pnna 2 I M62 TSe ®yyme Hn II cteavege ~~ PC& ampli6ed Ciegmenu of cT-ras ®mgene © a S ~a ra 's:+~ ~~ ~ "'~ u5g.3 Daa.vrm aC n ess ccd® 12 muted= uy Em u HFLF enaly®s DNA fnffi =evL ned enC-BPDF`¢mtai HFBE teIIa wm ampliCud . faa 40 eyekn with ~ ecd digestai wiih Hls IL FaDawmg digmCm[, xwft~ hagmmte wae eepm.amd thmugh eW/e paiYeac®da geL Cad= 12 mtIIffi$m ie mdi®trd by tye hepde O==ae®a'mg m i46 imne pnrse. ~nde vi®ule as 81 and 64 ®ee parze m~.>gmt wild type H-tm aIIrls Laoe 1 in P=ti~ omtvt '.mt}+ Pln®id PUCP.J 6.6, lan® 2®d ]0 ue FCR ymiuEy nwa nE®ei tn'EE ceTie. Ievm 3 is sr~darie, 250 agyx174. Hp II xeenietiGa ' :ngmmq lavea 6 Eo 9.nd ime 1 wca F'C8H pmfltuv fz>ffi veriwa a"'FHE I7AiA Cvtci with aad-EPDEM a-re. aodaa :2 mucnti®s ue eem m ttess la>va. -6- I I I I I I I

I I I I I I I I I I I I I I I I I I A RETROSPECTIVE LUNG CANCER MORTALITY STUDY OF PEOPLE EXPOSED TO INSOLUBLE ARSENIC SALTS AND RADON Liu Yu-tang and Chui Jin Institute of Occupational Medicine, Chinese Academy of Preventive Medicine, Beijing, China Abstract The incidence of lung cancer for workers in four mines who had been exposed to insoluble arsenic was found to be 290/105. A dose-dependent decrease in the incidence was associated with a reduction in the concentration of insoluble arsenic in the air. The content of arsenic in the lung of subjects exposed to insoluble arsenic was 51.4, which was 17 times higher than the 3.0 [G µg/g(d)] found in control groups. Moreover, the content of arsenic was found to correspond with the number of years working in the mine and with the incidence of lung cancer. Metabolic studies of arsenopyrite showed that it is converted to products such as arsenous acid, arsenic acid, methyl arsenate and dimethyl arsenate, which are identical to those generated from AsZ03. Although these metabolic products are formed at a lower rate, they nonetheless show that arsenopyrite should be considered as a carcinogen. Potential carcinogens such as As, Cr, Ni, Be, and Cd were evaluated in lung specimens of miners with lung cancer and compared with values obtained in controls by logistic regression analysis. Only As was found to be significantly associated with lung cancer. The concentration of As in lung tissues correlated well with the amount found in the air of the mining environment. A retrospective/prospective interference epidemiologic investigation performed over a 40 year period showed that the risk of radon was overestimated. After regulatory measures were implemented in the mines to control for exposure to radon, the value of radon was found to be RR/WLM = 0.17%, which was 9 times lower than the values estimated in the past. Introduction As and Rn are carcinogens commonly existing in nature in extremely low concentrations. In metal mines, As and Rn usually coexist. The arsenate in the deposit is mainly composed of arsenopyrite (FeAsS), which is not considered to be a carcinogen because of its low solubility. Consequently, the etiologic agent for cancer in mines is often attributed to Rn or its daughters. Indeed, the concentration of Rn in many uranium and non-uranium mines is very high, and may be elevated to 4-5 times the recommended working levels (WL). Despite the high levels of radon, the incidence of lung cancer is not increased in these environments. China is a country rich in nonferrous metal mines. High concentrations of insoluble arsenic and radon and its daughters are often detected in the air of many mines. High incidence of lung cancer is seen among many miners. I

We have been interested in determining the relationship between insoluble arsenic and lung cancer, and the possible confounding role of radon, which coexists with As in ore deposits, in the occurrence of lung cancer. There are at least two significant considerations in our studies: First, if Rn is proved to have significant carcinogenicity, then large sums of money must be invested in order to give protection from Rn. On the other hand, if the carcinogenic action of Rn proved to be minimal, then only efforts to protect from As are needed, representing a saving of 90%. Second, an insight to these relationships may provide useful leads for the prevention of lung cancer. Our studies show that for effective protection from Rn, positive pressure ventilation should be adopted, whereas wet operation with adequate ventilation affords good protection from As. Results Part 1. Epidemiologic Investigations of the Role of Insoluble Arsenic in Inducing Lung Cancer A. Research on Lung Cancer in Miners in Realgar Mines AszS; is the main composition of the ore. The solubility is 0.00005 % at 18°C water temperature. Al. Concentration of As in the Mine Air (as As): Arsenic concentration was measured on three separate occasions and the following results were obtained: 1973, n=6, 0.004-0.577 mg/m', average 0.23 mg/m' 1981, n=14, 0.003-0.166 mg/m', average 0.06 mg/m' 1988, n=8, 0.028-1.442 mg/m', average 0.32 mg/m3 A2. Epidemiologic Investigations A regressive-prospective cohort study was conducted with follow-up from January of 1972 to 1989 (1,2). The total prospective person years was 6,942. The total post-statistics person years was 6,566, representing a 5.5% loss to follow-up. In addition, 61 died during the cohort period, and 289 retired or moved. A3. Results (1) Causes of death: of the 27 cancer deaths, 16 were lung cancers, accounting for 59.2% of the total cancer deaths and raising the possibility that As is a risk factor for lung cancer. (2) Incidence of lung cancer: of the 6,566 person years, 17 were diagnosed with lung cancer. Sixteen deaths eventually resulted from these cases and only 1 survived. The incidence of lung cancer was calculated to be 258.8/105. 2 I I I I I I I I i I I I I I I I I I I

I 1 I I 1 I I 1 I I 1 I I THE STUDY OF CORRELATION BETWEEN C.STµ GENE DELETION AND SUSCEPTIBILITY TO LUNG CANCER Sun Gui-fan*, Pi Jing-bo*, Zheng Quan-mei** and Zheng Mei-zhen*** * Laboratory of Occupational Medicine, Department of Preventive Medicine, China Medical University, Shenyang, China ** Cancer Prevention Center of China Medical University, Shenyang, China *** Liaoning Cancer Hospital, Shenyang, China Introduction Studies have confirmed that smoking, air pollution and exposure to occupational carcinogens are the major risk factors for lung cancer. Research also suggests that polycyclic aromatic hydrocarbons (PAH) may be important carcinogens. In addition to these exogenous factors, some investigations have reported that host factors were also important in carcinogenesis. In 1981, Warholm eta l. (1) first isolated glutathione S-transferase classµ (GSTµ, EC, 2• 5• I• 18) from human liver, and demonstrated it to be the enzyme with the highest specificity and activity in the bio-transformation of certain types of carcinogens, especially PAH, to inactive metabolites, thus raising the possibility that GSTµ may play a crucial role in the prevention and suppression of carcinogenesis. The measurement of GSTµ in populations showed that only about 50% of people had the active GSTµ(2). In 1992, using a molecular biological technique, Brockmoller et al.(3) demonstrated that the inactivity of the enzyme was correlated with GSTµ gene deletion in certain populations. In order to study the relationship between GSTµ gene deletion and lung cancer susceptibility, we recruited 175 lung cancer patients with different pathological diagnoses and 104 healthy controls to detect the GSTp gene using the polymerase chain reaction (PCR) method. The results are reported below. Materials and Methods 1. Subjects. One hundred and seventy-five lung cancer patients in the Liaoning Cancer Hospital with confirmed pathological diagnoses were recruited as cases and 104 healthy residents living in the same area and with the same nationality were selected to be controls. Individual investigations included age, sex, occupation, smoking, family history, etc. 2. PreXaration of Genomic DNA and Primer. Three to 5 ml of blood were taken by venous puncture and transferred into polystyrene vials containing an appropriate amount of EDTA. Fifty µl of blood were taken and mixed with 0.5 ml TE buffer and centrifuged at 13,000g for 10 seconds. The supernatant was discarded and the pellet was washed once more with the TE buffer. The precipitate was finally suspended with 100 µl buffer (containing 50 tnmol/L KCI, 10-2ommol/L Tris-HCI, 2.5 mmol/L MgClz, 1% Laureth 12 at pH 8.3, 0.5% Tween 20 and 100 µl/ml proteinase K). The suspended solution was warmed at 56°C for 45 min, then at 95 °C for 10 min to inactivate proteinase K. The solution was centrifugated at 13,000g for 5 min,

I I rate in the younger group reached 85.3% which is significantly higher than that of the older group, 68.1 % (Table 4). Table 3. Comparison of GSTµ gene deletion between lung cancer patients and controls stratified by smoking GSTP(+) _..(S) GSTr(•) (x) ' GSPY(fl (Y) GSTP(.) (9) Smaki~ 36 (Z/.6) 89 (R.4) 14 (46,Y) 16 (53.3) 4.u3 <0AJ 2.29 LOY5.13 Nmsm~3iny __.. 16 (326) 36 (69.2) 36 (B.6) .,d (51.4) 4D2 0.05 2.13 1.02i.46 K2-O.IB P>U.05 22-0.03 P>0.05 Cmnpuism & GSTM lmm Eeletim Ddxeen fmotiy md mnsmakicg Table 4. Comparison of GSTµ gene deletion between lung cancer patients and controls stratified by age nsc Uiwq tmig Cuw 95% CI QtTr (-) ('L) GStP (-) (E) GSfx (1) . , .1A) G51'e (-)c..'_ (%) <Sp 5 (14.3) 29 (9S3) 28 (49.1) 19 (509) 9.4t <0AOOS 5.60 LlT16Jl 250 _.. . 45 (31.9) 96 (619) 22 (<6.W 25 (51.2) 3.0 005 1.91 1.013.A6 y2-3.91 P<0.05 x2-0.06 P>U.LLS CmnPuum d GSfM ~ 6e4tian hetwan elkr mC Ywngc Discussion With advances and new applications developed in molecular genetic techniques, especially the PCR method, Seidegard and Brockmo"ller confirmed that GSTµ genotypes were completely identical with phenotypes (i.e., the GSTµ activity was detected in the liver and other tissues of individuals carrying the GSTµ gene but not in individuals lacking the GSTµ gene)(3-5). Since genotypic characterization has the precision unmatched by other methods, application of the PCR technique for detecting the GSTµ gene becomes a most reliable method to determine whether the capacity for the broaynthesis of GSTµ is present. In this study, GSTµ gene in 175 lung cancer patients and 104 healthy controls was evaluated by this method. The results show that GSTµ gene deletion in lung cancer patients was as high as 71.4 % and was significantly higher than the rate in controls (OR 2.3, 95 % CI 1.39-3.82). Thus it can be inferred that GSTµ gene deletion is an important marker of the susceptibility of the host to lung cancer development. In 1990, Seidegard first reported that GSTµ gene deletion correlated with an increased risk of lung cancer(6), a finding which was subsequently confirmed by other studies (7,8). All published studies reported that the GSTµ gene deletion was most pronounced in small cell carcinoma and squamous cell -4- I I I I I I I I I I NI C 00 i -4 ta ~ °° I y I I I I I

I References 1. Warholm, M, etal., Purification of a new glutathione S-transferase (transferase µ) from human liver having high activity with benzo(a)pyrene-4, 5-oxide. Biochen. Biophys. Res. Commun 1981; 98(2):512-519 2. Brockmoller, J, et al. Genotype and phenotype of glutathione S-transferase class µ isoenzyme and in lung cancer and controls. Cancer Res. 1993; 53: 1004-1011. 3. Brockmoller, J, et al. Correlation between trans-stilbene oxide-glutathione conjugation activity and the deletion mutation in the glutathione-transferase class gene detected by polymerase chain reaction. Biochem. Pharmacol. 1992; 43: 647-650 4. Lai, H-C J, etal. Gene expression of rat glutathione S-transferase. J. Biol. Chem. 263:11389- 11395, 1989 5. Seidegard, J, et al. Hereditary difference in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion. Pro. Natl. Acad. Sci. USA 1988; 85:7298-7297 6. Seidefard, J, eta l. Isoenzymes of glutathione transferase (class µ) as a marker for the susceptibility to lung cancer: a follow-up study. Carcinogenesis 1990; 11:33-36 7. Nadachi, K, et al. Polymorphisms of the CYPIAI and glutathione S-transferase µ gene associated with susceptibility to lung cancer in relation to cigarette dose in a Japanese population. Cancer Res. 1993; 53:2994-2999 8. Nazar-Stewart, V, et al. The glutathione transferase polymorphism as a marker for susceptibility to lung carcinoma. Cancer Res. 1993; 53:2313-2318 9. Lafuente, A, et al. Human glutathione transferase µ(GSTµ) deficiency as a marker for the susceptibility to bladder and larynx cancer among smokers. Cancer Letters. 1993; 68:49-54 -6- I I I I I I I I I I I 1 I I

I B. Study of Lung Cancer in Tin Miners The main arsenate in the three tin mines (L. mine, M. mine, S. mine) is arsenopyrite whose solubility is 0.0005 % at the water temperature of 18oC. BI. The Average Concentration of As in the Air of Mines (as As) A total of 543 samples were used to assess As concentration. The average concentration of As in the air of mines was higher than 0.29 mg/m3 before 1950; 0.29 mg/m' in the 1950s; 0.022 mg/m3 in the 1960s; 0.015 mg/m' in the 1970s; and 0.010 mg/m' in the 1980s. These results showed that the concentration of As in the air of mines gradually decreased. B2. Epidemiologic Investigation Over one thousand cases of male subjects with lung cancer were analyzed in the tin mine study; 90% of these were exposed to insoluble arsenic before 1950. (1) Methods. Because of the limited database, a considerable amount of effort went into collecting cases and population controls as early as possible in order to obtain the CMR (crude mortality rate). In addition, a cohort of 751 persons who started working in the mines between 1960-1969 was established. Follow-up of the cohort continued until 1992. There was a 8.6% loss to follow-up. (2) Results. The CMR (crude mortality rate) was 290/105 in lung cancer cases in which exposure to As occurred before 1950s, as opposed to a CMR of 150/105 found in lung cancer workers who were exposed to As after 1950. In cases where exposure to As took place only in the beginning of the 1960s, the CMR was only 20/105. The gradual decrease of lung cancer mortality corresponded to the decrease of insoluble arsenic in the air in the mining environment and showed a dose-response relationship. B3. Etiologic Investigations (3-5) (1) The concentration of various carcinogens such as Cr, Ni, PAH, or possible carcinogens such as Be, Cd, in the mines were analyzed to further determine the cause of lung cancer. All had values below the threshold value, irrespective of whether the measurements were taken in the early or the late stage of the study. The only exception was the concentration of As (Table 2). I I I I I I I 1 I I I I I , N O Lp ~ ~ -4 tb 4- W s N CD ' , I

I I I I I I I I I I I I I I I I (3) Relative risk: post-statistics calculations showed RR=20.41, X2=15.49, P<0.01. (4) Standard mortality rate: the expected value was 0.3848, the observed value was 16. The standard mortality rate SMR was 41.58, P<0.01. The mortality of tumor in sites other than the lung was not significantly associated with As. A4. Research on Etiology It is well known that the incidence of lung cancer in smelters is associated with the presence of soluble arsenic. Because of the view that the arsenates (As2S3) in realgar mines does not dissolve, it is assumed not to accumulate in the body and accordingly presumed to have no association with lung cancer. Instead, Rn daughters have always been assumed to be the recognized agent for lung cancer. Measurement of Rn daughters in realgar mines, however, showed a value which was within the normal background level, making it unlikely that there existed a link between Rn daughter and the incidence of lung cancer among realgar miners. Accordingly, the concentrations of various carcinogenic agents in the mining air were measured; with inorganic carcinogens being quantified by ICP spectrography. In addition to showing the concentration of Rn to be within the normal background range, other suspected agents for lung cancer in humans, such as Cr, Ni, and possible carcinogens such as Be, Cd, were also found to be lower than the threshold value (Table 1). Table 1. The concentration of carcinogens in the mining air (mg/m') Measure Date N Carcinogens Minimum Maximum Average, 1988 8 As 0.0284 1.4422 0.3201 1988 8 Cr 0 0.0004 0.0003 1988 8 Ni (-) (-) 1988 8 Cd* 0.0002 0.0249 0.0047 1988 8 Be* 0 0.00001 0.00001 * Possible agents of human lung cancer. An attempt to examine the relationship between smoking and lung cancer yielded inconclusive results because of the few number of cases. Based on the number of cases that were actually collected, a non-significant association was found, with RR=3.007, Xz, - 1.54, P>0.05 IJ O , CO ~ -4 O W 1 3 V I

t I I I I I I I I I I I I I I I Table 2. Concentration of carcinogens/possible carcinogens measured at different periods of the investigation Carcinogen 1950 1960 Time 1970 1980 Cr mg/m' 0.010 0.008 0.003 0.001 Ni mg/m3 0.008 0.006 <0.001 <0.001 Be* mg/m' 0.003 <0.001 <0.001 <0.001 Cd* mg/m' 0.005 <0.001 <0.001 <0.001 PAH µg/m' 0.025 *Possible carcinogens (human lung cancer) (2) Metabolism of insoluble arsenic. The inorganic arsenic in ore mines is mainly composed of FeAsS, known as arsenopyrite, which is formed from FeAs03.2HZ0 by oxidation. This compound, as mentioned above, has extremely low solubility and was not considered a carcinogen for a long time. The biological properties of arsenopyrite have not been investigated before 1981. Our work in 1981 demonstrated for the first time that arsenopyrite can be dissolved and metabolized by rats. The metabolic products, namely, arsenous acid, arsenic acid, methyl arsenate and dimethyl arsenate, are the same as those formed from carcinogenic AsZOs. Compared to the soluble AszO3, however, arsenopyrite is metabolized at a much slower rate. Nonetheless, these results show that, despite its low solubility, inorganic As may act as a carcinogenic agent for lung cancer in miners. (3) Measurement of As contents in lung specimens. To further elucidate the involvement of arsenopyrite in the development of lung cancer, the target dose of As, which referred to the concentration of the putative causative agent actually found in the affected organ, was determined. The following results were obtained. The content of As in the lungs of 42 miners who were exposed to insoluble arsenic and developed lung cancer was 51.4 G µg/g (d). The content of As in the lungs of 3 miners exposed to insoluble arsenic without developing lung cancer was 6.2 G µg/g (d). The content of As in the lungs of 38 subjects with non-lung disease was 3.0 G µg/g (d). These results showed that the content of As in the lungs of As-exposed miners was 17 times higher than that of the control group. The amount of As accumulated corresponded with the number of years working in the mine and showed a dose-response relationship with the morbidity rate. tV O ~ V 00 W 3 ' -5_ V . <O !

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I In summary, although the insoluble arsenic is slowly dissolved and metabolized in the body, it is able to be accumulated over an extended period of time and therefore would be able to exert an effect in diseases, e.g., lung cancer, with a characteristic long incubation period. (4) Confounding factor analysis: In order to ascertain whether other carcinogens or possible carcinogens coexist with As in the target organ and, as a result, exert risks that are based on multi-factor interactions, a logistic regression analysis was performed. Methods and conditions. A total of 21 male miners with lung cancer, who had worked in the mines for between 6-42 years, were matched with 21 controls, i.e., subjects who had cancers in locations other than the lung. The variables consist of the generally recognized carcinogens such as As, Cr, Ni and possible carcinogens such as Be and Cd. They were represented by X1, X2, up to X5. A status of 0 was assigned to controls, while a status of 1 was assigned to the cases. EPIPAC software was used in calculation. The multifactor logistic regression analysis showed that, among the 3 carcinogens and 2 possible carcinogens considered, only As entered the model and reached statistical significance (Table 3). Table 3. Logistic Model variable BET EXP S.E.. BET/S.E. P As Xl 0.81678 0.01851 0.21725 3.7595 0.000170 Cr X2 0.11251 0.10113 0.87614 0.12841 0.897820 Ni X3 -0.29078 0.97134 0.10933 0.26597 0.790263 Be X4 0.15943 0.11728 0.27023 0.58998 0.555202 Cd X5 -0.66154 0.93558 0.58775 1.1255 0.260357 (5) Smoking and lung cancer. Of the 751 subjects that constituted the cohort, 85 % were smokers. Through a 29 year follow-up study, the incidence of lung cancer was only 20/105 and was well within the normal range. A note of emphasis was that miners usually smoked through a bamboo pipe and thus only breathed in smoke that had been water-filtered. Part 2. Epidemiologic Investigations of Rn daughters Rn daughters have long been considered a lung cancer inducing agent. A cumulative dose of 100 WLM of Rn daughters is often taken as a level which is correlated with an abnormally high incidence of lung cancer in China. The risk of Rn has also been estimated by using an ERR 0.015%/WLM, as recommended by BEIA IV (1988). -6- N O tb ~ N tp W ~ tb 0 I I 1 I I I I I I I I I I I

I REFERENCES 1. Lu, Y.T. et al. An epidemiological studies on occupational cancer. J. Hygiene Research 1980 9(4):10. 2. Lu, Y.T. et al. An epidemiological investigation on occupational cancer in workers exposed to arsenic. Chinese J. of Industrial Hygiene and Occupational Disease 1986 4(4):200 3. Lu, Y.T. etal. Chemical etiology research on lung cancer in Yjnxi miners. J. Hygiene Research 1980 9(4):15. 4. Lu, Y.T. et al. On a etiology of lung cancer in Yunxi miners. Chinese J. Industrial Hygiene and Occupational Disease 1987 5(1):20. 5. Lu, Y.T. et al. Etiological research on lung cancer excess occurrence in Yunxi miners. The selected papers of the symposium on occupational safety and Health in Asia-Pacific Region: 101. October 7, 1991, Beijing. 6. Lu, Y.T.; Chen Z. and Wang A.D. Metabolic study of insoluble arsenic. J. H iene Research 1981 10 (4):50. 7. Lubin, J.H. eta l. Radon and lung cancer risk. US Department of Health and Human Services. Public Health Service National Institute of Health NIH Publication No. 94-3644. - 10- I I I I I ! I I I I 1 I I I I I I I

I I I 2. I 3. I I 4. I 5. I 6. I I I I I I I I A 40 year interference epidemiologic investigation shows that the incidence of lung cancer in tin miners decreased in parallel with a reduction in the concentration of insoluble arsenic in the production environment, showing a causality between them. The so-called insoluble arsenate "arsenopyrite" is dissolved and metabolized in the body. Its metabolic products, i.e., arsenous acid, arsenic acid, methyl arsenate and dimethyl arsenate, are identical to those derived from the generally recognized carcinogen Asz03. Thus the arsenopyrite is proved to be a carcinogen. The incidence of lung cancer in miners showed a progressive yearly decrease, against a background in which the concentration of Rn daughters remained essentially unchanged. Even when the radiation accumulative exposure dose of Rn daughters averages 619.6 WLM, the incidence of lung cancer is only 20/103. A 40 year follow-up studies of a cohort demonstrates the carcinogenic role of Rn was over- estimated in the past. In determining the cause of lung cancer in miners who were simultaneously exposed to multiple factors including Rn, the RR of Rn daughters was calculated to be RR=0.17 % WLM, which was 9 times lower than the past estimation. ' 9- N O i -I tb W o0 I W

I investigations. Accordingly, mines with plentiful exposure to Rn daughters were selected for our study. Rn concentrations, people groups, patients with lung cancer, coexisting exposing factors were analyzed. Individuals who began to work in the mine in the 60s were included in the cohort. Observations were continued until 1992. B1. Radiation Accumulative Dose of Rn Daughters Since the installation of a ventilation system in the mines in 1976, the concentration of Rn daughters has been steadily declining: the concentration before 1976 averaged 3.1 WLM; the cumulative total averaged 5.4 WL from 1977 to 1985 (9 year period); the cumulative total averaged 2.7 WL from 1986 to 1991 (6 year period). B2. The Accumulative Calculation Result The year of 1965 was taken as the median of years of exposure to Rn. With that as a reference, the accumulative dose was calculated stepwisely. After 27 years, the radiation accumulative dose of Rn daughters received by each member in the cohort averaged 619.6 WLM. However, the incidence of lung cancer was only 20/105. B3. The Risk Assessment Even with an accumulative exposure dose of 619 WLM for a total of 27 years, the incidence of lung cancer was still within the normal range. Another group of workers in the same mine began to be exposed to Rn in the 50s and had a total exposure of 42 years. Their accumulative dose of Rn averaged 1120 WLM. According to information released by NIH in June, 1994 (7), the incidence was 43.4/105 after adjustment SMR 1.72, p<0.01. Suppose the accumulative exposure dose is 900 WLM, SMR 1.36, P>0.05. The RR carried out by BEIA IV (1988) is 1.5% WLM. Because the incidence of lung cancer ranges under the normal level under the accumulative dose 900 WLM, the former RR should be raised 9 times, that 1.5/9.0=0.17% WLM. In the new data released by NIH recently the RR claimed by BEIA IV (1988) has been altered greatly. Processing the data from China, ERR is changed into 0.5 % WLM (7). The result is a very close approximation to ours in 1990 (5). Summary 1. This paper investigates the incidence of lung cancer among miners in 4 mines who were exposed to insoluble arsenic. Long term exposure to high concentrations of As was associated with a high incidence of lung cancer. -8- I I I I I I I I I ' I I I I

I I I I I I I I I I I I I I I I I I Different concentrations of Rn daughters have been found in many mines, including uranium and non-uranium mines. Although many other carcinogens such as As, Cr, Ni, are also found in the same locations as Rn, emphasis has always been placed on Rn daughters. In our tin mine study, high concentration of Rn daughters and insoluble arsenate were both present. Thus to reduce the risk of exposure to these agents, reasonable, economic and effective preventive measures must be taken. Because As and Rn exert their harmful effects in different ways, different preventive measures are needed. To effectively discharge Rn in the mine, positive pressure high wind speed is needed, whereas protection from As is adequately achieved with wet operation and negative pressure low wind speed ventilation. From the economic point of view, energy consumption associated with Rn discharge is 90% higher compared to methods designed to minimize exposure to As. A. Regressive, Prospective Interference Epidemiologic Study The study is designed to test the relationship that may exist between changes in concentration of As and Rn in mine air and the incidence of lung diseases. Another objective is to study the separate role of these two agents in the etiology of lung cancer. The approach involved first surveying the carcinogenic capacity of Rn by epidemiologic methods, followed by estimation of the associated risk using a cumulative dosage method. The results of the 40 year regressive/prospective interference epidemiological study (regression for 25 years and prospection for 15 years) showed that the concentration of carcinogens and the dust in the mine air began to decrease in the middle of the 50s after wet operation began to be adopted to prevent pneumoconiosis. In the 60s, the wet operation became fully adopted; resulting in As being reduced from 0.29 mg/m' in the 50s to 0.015 mg/m' in the 70s. The radon daughters, being a naturally decaying product, were not expected to be affected by changes in the production mode to dry versus wet operation. Their concentration remained high (3.1 WLM L. mine) from the 50s to the middle of the 70s, during which period the incidence of lung cancer decreased from 150/105 in the 50s to 20/105 in the late 80s. Hence there is no epidemiologic evidence to support the existence of a link between the incidence of lung cancer and the concentration of Rn daughters. B. Radiation Accumulative Dose of Rn Daughters and Lung Cancer The relationship between Rn daughters as a causal agent of lung cancer is based on the radiation accumulative dose received by the workers. The method for calculating the cumulative dose of Rn daughters is amply illustrated in the literature. As mentioned above, the dose of Rn to induce lung cancer is suggested to be 100 WLM in China. Whether such a dose is supported by epidemiologic findings is unclear. Thus, in addition to examining the connection between Rn daughters and lung cancer, another objective of our study is to find out whether the alleged harmful accumulative dose of Rn matches with data provided by epidemiologic -7- I

a I I I I I I I I I I I I I I I LIFESTYLE, ENVIRONMENTAL POLLUTION AND LUNG CANCER IN CITIES OF LIAONING IN NORTHEASTERN CHINA Xu Zhao-vi*, Linda Brown,**, Pan Guo-wei*, Li Guang* and Feng Yi-ping* * Liaoning Public Health and Antiepidemic Station, Shenyang, China ** National Cancer Institute, Biostatistics Branch, Rockville, Maryland, USA Several studies were conducted in cities of Liaoning Province, one of the heavy industrial concentrated areas of China, to examine the effects of life-style factors and environmental pollutants on lung cancer causation. A case-control study involving 1249 lung cancer patients and 1345 population-based controls was conducted in 1985-1988 in Shenyang, the capital of Liaoning. Cigarette smoking was found to be the principal cause of lung cancer, accounting for 55% of the attributable fraction in males and 37% in females in this population. There was also a significant increase in lung cancer risk associated with an overall index of indoor air pollution due to coal-burning emissions. The population attributable risk (PAR) of indoor air pollution was 13 % for males and 17 % for females. Risks significantly increased for some occupations including workers in non-ferrous smelters (OR=2.6), chemical drug manufacturers (OR=3.0), glass and pottery industry (OR= 1.6). Studies in the Anshan iron-steel complex showed a significant excess of lung cancer for workers exposed to a variety of dusts. A standardized proportional mortality ratio (SPMR) study of 8887 deaths during 1980-1989 among male workers of the iron steel complex showed a 37% increase in lung cancer (1.37; 95% CI 1.28-1.45) compared to citizens of the city. A nested case-control study was then conducted in that complex. Six hundred and ten cases of lung cancer diagnosed during 1987-1993 and 959 randonily selected controls from 196,993 active and retired employees of the complex were interviewed. Records on monitors for dust and benz(a)pyrene from 1956-1992 were collected to calculate cumulative exposure for each person. Results suggest that all "dusty occupations" (exposure to metal or mineral dusts) increased the risk for lung cancer; the highest risk was among coke oven workers (OR=3.5) and fire resistant brick makers (OR=2.9). Significant dose-response patterns between cumulative total dust particles, cumulative total BaP, and lung cancer risk were observed. These findings suggest that smoking and environmental pollution account for elevated rates of lung cancer in cities of northeastern China. t N O tb i -4 ' co W i co M I

least 18 years of age, and working outside the home on a"regular" (ca. 8 am until 5 pm) shift at a minimum of 35 hours per week. Individuals were excluded from the study if they had "inappropriate" professions for such a study or membership in an advocacy group related to the objectives of the study (eg., no tobacco company workers or members of anti-smoking groups). Following the screening, the subject is assigned to a study cell. On the evening of Day 1 of the subject's involvement, the subject arrives at the test coordination site, and is rescreened to verify the accuracy of the telephone questionnaire. The subject then watches an instructional video with approximately 24 other participants and completes a "first visit" questionnaire concerning his/her lifestyle and details regarding the type of environment in which the subject works. The subject provides a saliva sample and receives his/her sampling systems, after being tested to insure that the subject can actually operate the sampling unit. On the morning of Day 2, the subject begins sampling with the workplace pump upon his/her arrival at work. The sampling apparatus consists of a sound-insulated pump (typically worn over the right shoulder and on the left hip) and a sampling head, containing both particulate and vapor collection devices which is worn in the subject's breathing zone. The subject also completes a workplace diary, recording various smells and observations concerning the use of products which may affect indoor air quality (eg., copying machines, correction fluids, coffee, cigarettes, etc.). Subjects are requested to remain at their work station during the lunch period. At the end of the workday, the subject turns off the workplace sampling pump, completes the workplace pump survey, dons the away-from-work pump (which is outfitted with a larger battery pack to afford sampling for a minimum of 18 hours), and returns home, conducting normal activities, such as shopping, dining, etc, on the way. The subject completes an away-from-work diary on an hourly basis. At bedtime, the subject takes off the pump, and sets it alongside of his/her bed, while the pump continues to sample. The next morning (Day 3), when the subject arrives at work, the away-from-work pump is turned off, and the home pump survey completed. After work that same day, the subject returns to the test coordination center with all of the "take home" materials, completes a last visit survey, provides a second saliva sample, and receives a $100 gratuity. Determination of Exposure Markers Particulate phase ETS air markers were collected on a Fluoropore membrane filter at a flow of approximately 1.7 L/min, while vapor phase markers were collected on XAD-4 resin cartridges (SKC Inc., Eighty Four, PA) at a flow of approximately 0.5 - 0.7 L/min, using a single air sampling pump. Particulate phase markers included respirable suspended particulate matter (RSP, 3.5 µm cut-off), solanesol, ultraviolet absorbing particulate matter (WPM), and fluorescing particulate matter (FPM). ETS gaseous phase markers included nicotine, 3-ethenylpyridine (3-EP), and myosmine. Briefly, RSP was determined gravimetrically (Conner, et al., 1990), and UVPM and FPM were determined by high performance liquid chromatography (HPLC) with UV and fluorescence detectors(Conner, et al., 1990), respectively. Solanesol was also determined using HPLC (Ogden and Maiolo, 1992). All of the vapor phase markers were determined using gas chromatography with thermionic specific (nitrogen selective) detection (Ogden, 1991). Levels of salivary cotinine were determined using radio-immunoassay (Davis and Stiles, 1993) -2- I I I I I I I I I I I I 1 I I I I

I I I I I I I I I I I I I 1 I I I I DETERMINATION OF PERSONAL EXPOSURE OF NONSMOKERS TO ENVIRONMENTAL TOBACCO SMOKE IN THE UNITED STATES Roger A. Jenkins, M.A. Palausky, R.W. Counts, M.R. Guerin, A.B. Dindal and C.K. Bayne Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Introduction In the United States, there is considerable controversy regarding the potential health effects related to exposure to environmental tobacco smoke (ETS). Authors of some epidemiological studies have concluded that there is a small but statistically significant increase in risk of contracting lung cancer to lifetime never-smoking women married to smoking spouses. Others authors have found no such statistically significant increases in relative risk (US EPA, 1992). To date, many studies attempting to quantify ETS exposure in the US population have had to rely on self-reports of exposure (Jenkins, et al., 1992), or extrapolations from determinations of area measurements of ETS levels in locations where cigarettes are actively being smoked (Oldaker, eta l., 1990; Leaderer and Hammond, 1991; Jenkins, et al., 1991; Colett, et al., 1992). Clearly, such is not the same as a direct determination of the exposure of nonsmokers to ETS. However, such direct determinations have been limited to relatively small study populations. The purpose of the study reported here is to directly determine ETS exposures of more than 1000 US nonsmokers. Experimental Study Design The study design consisted of recruiting approximately 100 individual subjects in each of 16 cities distributed geographically around the United States. To determine exposure, each individual wore a sampling pump during the work phase of his/her day, and another pump to collect samples from which to determine ETS exposure away from work. The sampling systems collected both particulate phase and gaseous phase components of ETS. While attempting to create an equally populated 2x2 matrix of subjects living in smoking or nonsmoking homes and working in smoking or nonsmoking workplaces of equal cell population, the difficulties of recruiting individuals living and working in smoking environments were such that the cells were unequally populated. Although all subjects were recruited on the basis of their nonsmoking status, salivary cotinine was used to assess actual smoking status, as well as the relationship between directly measured ETS exposure and salivary cotinine. Subject Recruiting and Itinerary Nearly all of the subjects were recruited through random telephone dialing or marketing research databases. Less than 10% of the subjects were recruited through mall intercept methods. During the initial contact, the subject was required to pass a screening questionnaire (administered by phone by a local marketing research firm). To be included in the study, individuals had to report themselves as not having used tobacco products in the last six months, nor using any form of nicotine patch or gum, be at I

Results and Discussion I I 1 i In Table 1 are presented 12 of the 16 urban areas around the United States for which data has been analyzed to date, and for which the results are reported in this manuscript. Samples were collected in these 12 cities from mid-May, 1993 to mid-December, 1993. Cities were chosen based on obtaining a good geographic distribution, weather during the time of year, logistics, lack of pervasive smoking restrictions, and likelihood of high quality field marketing survey research support. Table 1. Urban Areas Selected For Investigation and for which Results are Reported Knoxville, TN Boise, ID Columbus, OH Portland, ME Seattle, WA Buffalo, NY San Antonio, TX Baltimore, MD St. Louis, MO Fresno, CA Daytona Beach, FL Grand Rapids, MI In Table 2 are presented the initial cell assignment populations, based on the initial screening questionnaire results. The relative proportions of participants are indicative of the difficulty of recruiting individuals who live and work in situations where unrestricted smoking occurs. In many cities, thousands of telephone calls were required to locate relatively modest numbers of participants in Cells 1- 3. Table 2. Cell Populations For Cities 1-12 Based on Screening Questionnaire Assignment I I I I I I I I I . -bFumber of Subjects . ... . % of SuLject -po]iltlnnon Cell 1: Smoking Home/Smoking Workplace 121 10.4 Cell 2: Smoking Home/nonsmoking workplace 172 14.8 Cell 3: Nonsmoking home/Smoking workplace 222 19.0 Cell 4: Nonsmoking work and Nonsmoking Workplace 651 55.8 Total 1166 100 When compared with the US population as a whole (corrected for those greater than 18 years of age), extracted from the 1993 Statistical Abstract of the United States (U.S. Department of Commerce, 1993), our study population tended to be younger, have more years of formal education, have a higher median household income, and be comprised of a higher percentage of females. For example, about 68% of the subjects in Cities 1- 12 were female. This may be due to two general observations from marketing survey research: a) women are more likely to answer the telephone in a household; and b) a higher proportion of women are more likely to participate in such a study. When adjusted for the under-18 year old US population, the study is comprised of a slightly larger proportion of younger individuals.. This -3-

a I I I I I I I In Table 4 are presented similar data, but in the more conventional terms of a time averaged concentration of ETS components to which the participant is exposed. That is, the time averaged concentrations are equal to the sum of the concentration/time products for the workplace and away-from- work sampling systems, divided by the total time of measurement of the two sampling systems (ca. 24 hours). The conclusions from the data are the same as those for the data presented in Table 3, however. That is, individuals that work, live, and operate around smokers receive a substantially greater exposure to ETS, and the away-from-work venue appears to be the primary contributor. It is important to note that in general, the levels of ETS to which individuals are actually exposed are substantially lower than those which may be inferred from previous studies of ETS marker levels measured in specific areas over short durations. For example, in Guerin, et al., 1992, several studies of nicotine levels in offices are reported. For the most part, studies reported mean levels of 4 - 14 µg/m3. This compares with mean levels in this study of workplaces in which smoking is unrestricted of ca. 2.7 µg/m3, and median levels of 0.44µg/m3. In Figure 1 are com- pared the distributions of one- hour nicotine levels measured in a study of offices in which smoking was unrestricted in five cities (Oldaker, et at., 1990) with the 8-hour time weighted average levels of nicotine to which office workers were exposed in the 12 cities of our study. The mean one-hour nicotine level was 7.07 ± 8.42 µg/m3, compared with 3.15 ± 5.66 µg/m3 for the actual 8-hour exposure levels. Median values were 4.65 µg/m3 and 0.91 µg/m3, respectively. It is clear from the comparison in Figure I that the distribution of values is dramatically different at the lower concentration levels. There may be at least two explanations for this 60 ~~p, . 3 ~~.1-".I'~5'9 r-1~- 4 6 8 10 12 14 16 iB 20 >21 Nicotine, micrograms per cubic meter Smoking Office Levels ® Exposures in Offices Figure 1. Comparison of distributions of nicotine levels in smoking offices (1 hour measurements) and 8-hour exposure levels of non-smoking workers in smoking offices. difference. First, there has been a general trend in the US society for smokers not to consume their smoking materials in the presence of nonsmokers as much as in previous decades. A second obvious explanation is that nonsmokers, even in environments in which smoking is not restricted, spend only small amounts of time is areas where ETS levels are high. Individuals may believe that they are being exposed to the smoke of many cigarettes because they observe such around them. But in fact, due to either distance from the smoker or the relatively short time in the presence of significant quantities of smoke, the exposures received from the smoke are relatively small in most cases. This data also suggests that short term area measurements may actually overestimate worker exposure in many situations. -5-

Table 5. Relationship of Household Income and Airborne Concentration of Environmental Tobacco Smoke Markers Household Income Number of Median 24-hr Time Averaged Airborne Concentration, gg/m' ' (in Thousands) Participants 3-EP Nicotine Myosmine RSP. UVPM FPM Solanesol Less than 10 30 0.130 0.146 0.027 27.4 2.60 1.44 0.014 10 - 20 137 0.110 0.125 0.020 19.7 2.15 1.33 0.0032 20 - 30 183 0.068 0.078 0.014 17.3 1.68 0.964 0.0032 30 - 40 226 0.074 0.095 0.014 18.2 1.99 1.16 0.0032 40 - 50 195 0.042 0.047 0.010 17.3 1.63 0.978 0.003z 50 - 75 249 0.035 0.036 0.005 16.9 1.47 0.803 0.0032 75 - 100 66 0.024 0.020 0.002 13.9 1.27 0.659 0.0032 Greater than 100 44 0.019 0.027 0.0062 15.0 1.23 0.631 0.0032 'Analytical blank-corrected µg/sample/(Sampling time x Flow rate) = µg/m3 per Sample; [(µg/m3, Away from work sample x Hours, Away from work sample) +(µg/m3, Work sample x Hours, Work sample)]/(Hours, Away from work sample + Hours, Work sample) = Time Averaged µg/m' 2Actual value was nondetectable; one half of the limit of detection, in µg, and average flow rate, and a 24-hour time were used. 564£SL 6SOZ -8- M" r M Mm"'M ~ r MM W M M Mae M r

I may be due to the requirement that participants work at least 35 hours a week on a regular (ca. 8am - 5 pm) shift, which would tend to exclude individuals who are retired from the study. In general, the participants in the study had higher household incomes. For example, the annual US median household income is approximately $30,000, in contrast to approximately $40,000 for subjects in this study. This may be due to several factors. First, since subject recruiting is conducted by telephone, the selection method excludes those individuals who do not have telephones. Inclusion in the study required the participants to work at least 35 hours a week on a regular shift. A larger fraction of lower household income individuals may not work a full 40 hour work week. Also, higher income households often have two adult workers in the family, and since subjects living in smoking homes were required to be nonsmokers and live with a smoker, the smoking homes may have been selected from the higher income groups. With regards to the occupational distribution, the study contains a lower proportion of individuals in service occupations and those who work in factories. These individuals may have decided not to participate on the basis of safety or appearance concerns for wearing the air sampling pumps. As a result, the study contains a larger proportion of "white collar" workers, who may tend to be more highly paid. In Table 3 is presented a summary of the median exposures of individual participants segregated by those working in smoking and nonsmoking locations, as well as away-from-work settings which include either smoking or nonsmoking homes. (Except where otherwise noted, all of the smoke exposure data has been corrected for those individuals who can be clearly considered smokers: those with salivary cotinine values > 100 ng/mL have been excluded from these tabulations. However, some regular smokers are likely to have salivary cotinine levels which are an order of magnitude lower.) Note that the measured parameter in this table is actual exposure, defined as the average smoke marker concentration in µg/m3, multiplied by the time of exposure, and the estimated breathing rate, in L/min. (The rate of 20 ILmin was taken from the National Research Council report on Environmental Tobacco Smoke [1986].) First, it should be noted that only those individuals who reported consistent exposures (ie. reported observing - or not observing - tobacco products being smoked in their diaries, pump surveys, and last visit surveys) were included in this particular compilation. The justification for this is that many individuals work in locations where they report smoking is permitted, but where no actual tobacco products were observed to have been smoked. Thus, the assignment of such a facility as a "smoking" workplace, when the participant did not observe smoking taking place, seems incongruous, and clouds the interpretation of the data. The same argument can be used for assignment to a cell including a smoking home environment. From the data in Table 3, which are median values, it is clear that those individuals who live and work with smokers are exposed to substantially more ETS components than those who observe no cigarettes, pipes, or cigars being smoked around them. For example, median nicotine exposures for participants in Cell 1(smoking workplaces and an away-from-work categorization which included a smoking home) were more than 50 times greater than those who live, work, shop, and commute in a truly nonsmoking environment (Cell 4 subjects). That exposures to any discernable amount of ETS components occur in environments where no smoking is involved may be indicative of the ubiquitous nature of ETS at trace levels. A comparison of exposures of participants in Cells 2 and 3 provide an indicator of the greatest contributor to ETS exposure. Cell 2 is populated with participants that reported cigarettes being smoked in their presence outside of work, and reported no cigarettes being smoked within their sight or smell in their workplace. In contrast, the participants assigned to Cell 3 confirmed smoking occurring in their workplace, but not observing any cigarettes being smoked outside of work. The comparison indicates that Cell 2 participants are exposed (concentration times duration times breathing rate) to more than four times the amount of ETS componentsas Cell 3 participants, indicating that those locations outside the workplace are a much greater contributor to true ETS exposure. -4- N O O ~ v 00 W ~ c0 i I I I I I I I I I I I I I

Table 3. Comparison of Exposure of Individuals to Environmental Tobacco Smoke Markers Among Cells Away From Work Work Number of - 24-hr Time Averaged Exposure, Kg' . Cell Environment Environment Participants . 3-EP Nicotine Myosmine RSP UVPM FPM Solanesol 1 Smoking a Smoking c 119 Median 20.1 38.4 4.34 881 338 221 3.09 Mean 33.8 88.4 7.77 1211 662 513 13.6 95th %ile 113 252 18.4 3126 1926 1972 49.6 2 Smoking a Nonsmoking d 109 Median 10.3 15.2 L79 675 223 164 1.67 Mean 19.7 36.9 3.41 935 413 320 8.16 95th %ile 65.0 133 11.2 2308 1288 1070 27.9 3 Nonsmoking b Smoking C 163 Median 2.18 2.97 0.400 558 61.5 36.4 0.090 Mean 6.29 13.1 1.47 734 146 101 1.88 95th %ile 26.1 48.3 5.88 1664 529 400 9.23 4 Nonsmoking b Nonsmoking d 497 Median 0.593 0.671 0.024 412 29.9 15.0 0.0412 Mean E 7 E [ 9.09 0.301 492 49.6 31.1 0.134 95th %ile 612 6.90 0.926 1153 153 114 0.494 Noted observations of tobacco products on Home Diary, Home Pump survey, and Last Visit survey for home. Noted no observations of tobacco products on Home Diary, Home Pump survey, and Last Visit survey for home. Noted observations of tobacco products on Work Diary, Work Pump survey, and Last Visit survey for work. Noted no observations of tobacco products on Work Diary, Work Pump survey, and Last Visit survey for work. 'Analytical blank-corrected µg/sample/(Sampling time x Flow rate) = µg/m' per Sample; [(µg/m', Away from work sample x Hours, Away from work sample) + (µg/m', Work sample x Hours, Work sample)]/(I-Iours, Away from work sample + Hours, Work sample) = Time Averaged µg/m'; µg/m' x 1.2 m'/hour (National Academy of Sciences Inhalation Rate) x Hours = µg 'Acmal value was nondetectable; one half of the limit of detection, in µg, and average flow rate, and an 24-hour time were used. -6- £64£8LW0Z M a.. .. ar r..1"Im r-.r r r• .rr rIM .. iar .r r

WON r -M ms mi MM M M M M M ~li r r 00 Table 4. Comparison of Concentrations of Environmental Tobacco Smoke Markers to which Individuals have been Exposed Among Cells Away From Work Work Number of 24-hr Time Averaged Airborne Concentration, µg/m3 ' Cell Environment Environment Participants 3-EP Nicotine Myosmine RSP UVPM FPM Solanesol 1 Smoking a Smoking a 119 Median 0.800 1.46 0.161 32.0 11.9 7.67 0.113 Mean 1.20 3.10 0.275 43.3 23.6 18.3 0.483 95th %ile 3.92 8.81 0.642 116 67.5 57.0 1.77 2 Smoking a Nonsmoking b 109 Median 0.369 0.555 0.068 24.5 8.01 5.89 0.058 Mean 0.719 1.34 0.124 34.5 15.1 11.6 0.295 95th `~ile 2.45 4.79 0.4D1 84.3 46.2 37.5 0.982 3 Nonsmoking b Smoking a 163 Median 0.078 0.114 0.014 20.5 2.26 1.20 0.003 Mean 0.231 0.480 0.053 26.7 5.39 3.78 0.070 95th %ile 0.979 1.80 0.185 60.8 18.7 16.7 0.383 4 Nonsmoking b Nonsmoking b 497 Median 0.022 0.024 0.001 14.9 1.08 0.567 0.0032 Mean 0.059 0.350 0.011 18.1 1.83 1.15 0.005 95th %ile 0.221 0.251 0.035 41.5 5.49 3.97 D.018 a b Noted observations of tobacco products on Home Diary, Home Pump survey, and Last Visit survey for home. Noted no observations of tobacco products on Home Diary, Home Pump survey, and Last Visit survey for home. 'Analytical blank-corrected µg/sample!(Sampling time x Flow rate) = µg/m; per Sample; [(µg/m', Away from work sample x Hours, Away from work sample) + (µg/m', Work sample x Hours, Work sample)]/(Hours, Away from work sample + Hours, Work sample) = Time Averaged µg/m' 2Actual value was nondetectable; one half of the limit of detection, in µg, and average flow rate, and an 24-hour time were used. -7- b66£81480Z

From the data presented in Figure 2, one might infer incorrectly that the greatest exposure to ETS occurs in the workplace. However, such an inference is contrary to the data presented in Table 6, which is a comparison of actual exposures (ie., concentration multiplied by duration of exposure and average breathing rate) in micrograms, µg, for three groups of subjects: those that consistently reported observing cigarettes being smoked in their workplace, individuals who consistently reported cigarettes being smoked around them away from work, and those who reported cigarettes being smoked inside their residence in their away-from-work (home) diaries. In Table 7 are presented the TWA airborne concentrations of ETS markers used to compute the exposures. Using the markers nicotine, FPM, and 3-EP, the data indicate that median exposures to ETS are approximately five times greater away from work than in the workplace. The data also indicates that the more highly exposed individuals in each category are exposed to ETS to a greater extent away from work. For example, comparing the 80th percentile cut points for the three categories for nicotine and 3-EP, the exposure cut points for the away-from-work locations which include a smoking home are twice the quantities found in the smoking workplaces. An examination of the Figure 3. Nonwork Tobacco Products Reported on Home Diary. Percent of Total Tobacco Products Reported By Location. data in Table 7 indicates that there are two primary contributors to the greater exposures away from work. First, TWA levels of ETS components are typically two - three times as high, and the average length of exposure is twice as long away from work. O 'I I I I I I , 1 I -10- I I

2081783207

Conclusions I I I I I I I I 1 An initial evaluation of data regarding personal exposure of nonsmokers to environmental tobacco smoke constituents in 12 cities around the United States of America (USA) has been conducted. In general, the study population tends to be somewhat younger, more highly educated, and have a higher household income and proportion of females when compared to an age adjusted set of the population of the USA as a whole. This is believed to be due to the study inclusion requirement that subjects work a normal (for the USA) 40-hour work week in a job in which they are relatively stationary, and the necessity of recruiting volunteers. Across the study cells (smoking and nonsmoking workplaces and away-from-work locations), the median exposures to ETS constituents tend to decreasing with decreasing time spent in smoking venues. The levels of the exposures are less that those which might be extrapolated from short duration personal exposure or area measurements. This seems most likely due to nonsmokers not spending large amounts of time in the presence of smokers, either at work or away from work. Exposures away from work in the presence of smokers tend to be greater that those received at work when subjects work in the presence of smokers. This appears to be due to the higher ETS concentration levels encountered outside the workplace and the relative length of time that subjects spend in the two venues. As a group, the median salivary cotinine levels tend to track the median nicotine exposures for subjects whose true smoking status was confirmed to be nonsmoking. Finally, the rate at which current regular smokers misrepresent themselves to be lifetime never smokers was estimated to be ca. 3.6%, using a salivary cotinine level of z 10 ng/mL to establish nonsmoking status. -17-

I I I I I I I I There are some practical reasons why this and virtually any study which requires voluntary participation of the individual subjects can not be exactly representative of the population of the United States as a whole. One potential criticism of the study design is that it tends to exclude those individuals in lower socio-economic groups. However, the extent to which this may or may not affect the conclusions of the study is difficult to judge. One approach to determining the extent to which socio-economic status impacts study conclusions is to determine the extent to which smoke exposure is correlated with household income. In Table 5 is summarized the median 24-hour time averaged smoke marker levels as a function of self-reported household income. (Note that because the smoke marker levels are 24 hour averages, they are directly proportional to actual ETS exposure over the measured time period.) With the exception of solanesol, for which in many cases the concentrations were at or near detection limits, there is a definite inverse proportionality between household income and smoke exposure. The lowest income groups may receive two to seven times the exposure to ETS marker components as the higher income groups in the study. For example, the median time weighted average (TWA) level of 3-ethenylpyridine (3-EP) was 0.130 µg/m3 for those with household incomes <$10K, compared with 0.019 µg/m3 for those with incomes greater than $100K. Based on the ETS marker data from the first 12 cities, it appears that there may be an important difference between the perception of individuals' exposures to ETS (as judged by self-reported impressions of the number of cigarettes smoked around them) and the reality of that exposure (as judged by the actual levels of smoke constituents collected by the sampling pumps at and away-from work). In Figure 2 is summarized the location of all of the tobacco products to which individuals reported exposure. These data are taken from the last visit survey, which the individuals complete upon their return to the test coordination center in their city. (Note that this is essentially a recollection of the diary data, a count of all of the smoking products, regardless of a subject's classification, and not the data taken directly from the diary.) Greater than 50% of all of the tobacco products observations occur at work. About 25% of the observations are at home, and the remaining 25% are distributed among other locations. The away- from-work cigarettes (in this case, from Cities 1- 12) observed are presented according to a more detailed categorization in Figure 3. Nearly 60% of all of the cigarette "exposures" (actually observations of cigarettes being smoked around the subjects) during the Figure 2. Self-reported Number of Tobacco Products to which Subject was Exposed from Last Visit Survey. away-from-work sampling occur inside the private residence. 1.1 ~  ~ V co 9 '~ co ' W I

Table 11. Estimation of Misclassification Rate from Salivary Cotinine Level Salivary Cotinino Level, ng/mL Number of Subjects Claiming to be Never-Smokers Miselassif'itcation Rate, % Mean _ 100 7 1.82 Mean Z 30 10 2.60 Mean z 10 14 3.64 Start or Finish Level ? 10 20 5.20 Number of Study Participants for whom cotinine and demographic data were available: 1093 Assumes fraction of smokers in general population equals 28.1%, and that 90% of all smokers are "regular smokers." For the first 12 cities, salivary cotinine data and demographic data were available for 1093 subjects, so that the number of individuals whose salivary cotinine levels indicate that they are regular smokers is divided by this estimate of the number of smokers which would have been encountered to produce a misclassification rate. Obviously, the rate is highly dependent on the level of salivary cotinine which is chosen to indicate status as a smoker. Etzel (1990) has reviewed a variety of studies which have been conducted addressing cut-off points for assignment of individuals to smoking categories based on cotinine levels in physiological fluids, and has attempted to summarize the findings. Those subjects with salivary cotinine between 10 - 100 ng/mL were classified as infrequent smokers, or regular smokers with low nicotine intake. Those individuals with salivary cotinine > 100 ng/mL were classified as regular smokers. Those with salivary cotinine levels < 10 ng/mL were classified as probably having no nicotine use. In our study, data is acquired both before and after the exposure samples are taken (ie. "start" and "end" levels). Whether or not to assign individuals whose start or end salivary cotinine level is > 10 ng/mL, but whose mean of start and end levels are < 10 ng/mL, is debatable, and may not be appropriate. In Table 11, misclassification rates have been presented for four different cut-off points. Rates ranged from 1.8% to 3.6% for subjects whose mean of start and end level was ? 10 ng/mL. Note that these rates of misclassification are similar to those reported by Riboli, et al., 1990, who used urinary cotinine to assess the accuracy of self-representation of smoking status among nonsmoking women. -16- I I I I I I I I

I I I 1 I I I I I I I I I I ' I Table 6. Comparison of Exposures (µg) to Environmental Tobacco Smoke Markers of Participants Recording Cigarette Observations In Different Venues Exposure, µg (µghzt' x 1:2 z Time; hr)' Venue 3-EP Nicotine I4lyosmine .. BSP. UVPM ` FPM Solanesul Away Median 11.0 18.9 2.09 467 165 112 1.34 From Work Mean 20.1 46.7 4.13 738 380 294 7.92 n = 247 Low 0.0812 0.2132 0.0802 0.639 3.722 2.212 0.0412 Sampling time: High 352 2869 191 9647 7623 6443 292 Approximately 80th %ile 29.3 55.0 5.21 1015 539 405 10.3 16 hours 95th %ile 61.9 145 12.2 1927 1317 1081 28.7 Home Median 8.30 11.4 1.59 425 126 84.1 0.541 n = 303 Mean 16.9 39.0 3.46 669 321 248 6.54 Low 0.0812 0.2132 0.080' 0.639 3.722 2.212 0.0412 Sampling time: High 352 2869 191 9647 7623 6443 292 Approximately 80th %ile 25.6 47.3 4.58 937 485 320 7.60 16 hours 95th %ile 60.5 132 11.0 1742 1053 859 27.7 Work Median 1.77 2.76 0.367 229 38.8 24.6 0.107 n= 328 Mean 7.42 18.8 1.82 404 172 129 3.00 Low 0.0812 0.213~ 0.08W 106z 3.722 2.212 0.0412 Sampling time: High 123 288 29.1 6289 2779 2523 66.7 Approximately 80th %ile 10.2 28.0 2.78 557 222 170 2.85 8 hours 95th %ile 31.5 92.6 7.56 1233 803 628 15.9 Away From Work: Home: Work: Noted cigarettes on the Home Diary, Home Pump Survey, and Last Visit Survey Noted cigarette observations while inside the home on the home diary Noted cigarettes on the Work Diary, Work Pump Survey, and Last Visit Survey 'Analytical blank corrected µg/sample/(Sampling time x Flow Rate) = µg/m' per sample; µg/m3 x 1.2 m'/hour (National Academy of Sciences Inhalation Rate) x Hours = µg 2Acmal value was nondetectable; one half of the limit of detection in µg, an average flow rate, and an 8 or 16-hr time were used. N O ~ i -4 00 G,1 - 11 - ~ ~ 00 I

Table 10. Comparison of Salivary Cotinine Levels and Nicotine Exposure Segregation by Cell Classification I I I I I I I I I I I I I Cell # Away From Work Environment Work Environment Number of Participants Median Cotiitinel, ng/mL Median Nicotine Exposnrez> µg 1 Smoking3 Smoking4 95 1.62 42.3 2 Smoking3 Nonsmoking5 100 0,959 15.2 3 Nonsmoking6 Smoking4 152 0.360 2.85 4 Nonsmoking6 Nonsmoking5 481 0.162 0.682 'Cotinine results used in this calculation are the mean of Start and End determinations. 2Analytical blank-corrected µg/sample/(Sampling time x Flow rate) = µg/m3 per Sample; [(µg/m3, Away from work sample x Hours, Away from work sample) +(pg/m3, Work sample x Hours, Work sample)]/(Hours, Away from work sample + Hours, Work sample) = Time Averaged µg/m3; pg/m3 x 1.2 m3/hour (National Academy of Sciences Inhalation Rate) x Hours = µg 3To be included in the smoking "Away from Work" environment category, participants must have noted observations of tobacco products on their "Home" diary, "Home" Pump Survey, and on the Last Visit Survey for home. 4To be included in the smoking Work environment category, participants must have noted observations of tobacco products on their Work diary, Work Pump Survey, and on the Last Visit Survey for work. 5To be included in the nonsmoking Work environment category, participants must have noted no observations of tobacco products on their Work diary, Work Pump Survey, and on the Last Visit Survey for work. 6To be included in the nonsmoking "Away from Work" environment category, participants must have noted no observations of tobacco products on their "Home" diary, "Home" Pump Survey, and on the Last Visit Survey for home. -15- I

! 1 I I I I I I 1 I I 1 I I I I 12, Ogden, M.W.; Maiolo, K.C.; Oldaker, G. B. III and Conrad, F. W. "Evaluation of Methods for Estimating the Contribution of ETS to Respirable Suspended Particles," Indoor Air '90, Proceedings of the 5th International Conference on Indoor Air Quality and Climate, 2, 425-420; 1990. 13. Ogden, M.W., "Use of Capillary Chromatography in the Analysis of Environmental Tobacco Smoke," Capillary Chromatography -- The Applications, 1st ed., Chapter 5; 1991. 14. Ogden, M.W. and Maiolo, K.C. "Comparison of GC and LC for Determining Solanesol in Environmental Tobacco Smoke," LC-GC, 10, 459-462; 1992. 15. Risner, C.H., "The Determination of Scopoletin in Environmental Tobacco Smoke by High- Performance Liquid Chromatography," J. Lia. Chromatogr., 17:12, 2723-2736; 1994. 16. Riboli, E.; Preston-Martin, S.; Saracci, R.; Haley, N.J.; Trichopoulos, D.; Becher, H.; Butch, J.D.; Fontham, E.T.H.; Gao, Y.-T.; Jindal, S.K.; Koo, L.C.; Le Marchand, L.; Segnan, N.; Shimizu, H.; Stanta, G.; Wu-Williams, A.H. and Zatonski, W. "Exposure of Nonsmoking Women to Environmental Tobacco Smoke: A 10-Country Collaborative Study," Cancer Cause and Control, 1, 243-252; 1990. 17. U.S. Environmental Protection Agency (US EPA) "Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders." EPA/600/6-90/006F; 1992. 18. U.S. Department of Commerce, Statistical Abstract of the United States 1993, Available through the National Technical Information Service, Springfield, VA, 1993. O i V OD 19- N O O a

I I I I ' I I I I I I I I I In Tables 8 and 9 are reported the time averaged concentrations of selected ETS constituents in smoking and nonsmoking homes and workplaces measured by collecting samples with the individual sampling systems. (All of the values were taken from individuals who were confirmed nonsmokers, ie., with salivary cotinine levels < 10 ng/mL.) Note that in the case of the home levels, the values are necessarily low estimates, since individuals reported themselves at locations other than the home while the away-from-work pump was collecting samples, including commuting, shopping, going out to dinner, etc. Included in this comparison are only those samples from individuals which did not observe any tobacco products in use at those other away-from-work locations, so the estimates of ETS constituent levels in smoking homes are, in effect, "diluted" with air sampled at other away-from-work locations in which no tobacco usage was observed. However, the confounding influences of other venues are minimiz,ed. In the smoking homes, median time averaged levels of tobacco-related constituents tend to run about 20 - 30 times higher than those in nonsmoking homes. Median UVPM and FPM levels are also higher in smoking homes, but by not as large a proportion, perhaps due to other combustion-derived particulates being present. The differences between smoking and nonsmoking workplaces is somewhat smaller, although still considerable. Interestingly, in both homes and workplaces, the time-averaged levels to which the most highly exposed individuals are exposed (the 95th percentile levels) in nonsmoking venues are very similar to the median levels observed in smoking venues. This may be due in part to inaccuracies in self-reported tobacco product observation, or the fact that individuals may in fact not actually observe (see or smell) cigarettes smoked in their presence, but enter locations in which tobacco products were recently used. [Note that the levels reported in Tables 8 and 9, and the number of individual observations differ from those reported in Table 7, due to differences in inclusion criteria.] Table 8 Time Averaged ETS Constituent Levels* in Smoking and Nonsmoking Homes Determined Using Personal Sampling Systemsl . 16 hourltime Averaged Coitcentration, pglm' Nonsmoking 3EP Nicotine RSP UVPM FPM Solanesol Homea N = 725 Median: 0.0170 0.0197 15.2 1.10 0.561 0.0022 Mean: 0.0835 0.0941 20.2 2.41 1.64 0.0143 95th %ile: 0.231 0.271 47.2 7.28 4.95 0.0131 Smoking Homeb Median: 0.514 0.684 23.0 7.33 5.01 0.0585 N= 105 Mean: 1.15 3.35 37.1 18.9 14.3 0,386 95th %ile: 4.00 8.75 104 62.3 44.9 1.55 * Time averaged levels are measured over the total time that the sampling pump is operating, including both in the home and at other locations outside of the workplace. However, the majority of the time in which the sampling pump is operating is in the home. t Includes only those individuals with mean cotinine values less than 10 ng/mL. ~ Acmal value was nondetectable; one half of the limit of detection in µg, an average flow rate, and a 16-hr time was used. ' From participants who recorded no tobacco products (cigarettes, cigars, or pipes) on their home diary and recorded no tobacco products in other locations on their home diary. b From participants who recorded tobacco products while at home on their home diary, and recorded no tobacco products away from home on their home diary -13- I

REFERENCES 1. Collett, C.W.; Ross, J.A. and Levine, K.B., "Nicotine, RSP, and CO2 in Bars and Nightclubs," Environment International, 18, 347 - 352, 1992 2. Davis, R.A. and Stiles, M.F., "Determination of Nicotine and Cotinine: Comparison of GC and Radioimmunoassay Methods," presented at the 47th Tobacco Chemists' Research Conference, Gatlinburg, Tennessee, October 18 - 21, 1993 3. Guerin, M.R.; Jenkins, R.A. and Tomkins, B.A. The Chemistry of Environmental Tobacco Smoke: Comnosition and Measurement, Lewis Publishing, 1992 4. Etzel, R.A., "A Review of the Use of Salivary Cotinine as a Marker of Tobacco Smoke Exposure," Preventive Medicine, 19, 190 - 197 (1990)] 5. Conner, J.M.; Oldaker, G.B. III and Murphy, J.J. "Method for Assessing the Contribution of Environmental Tobacco Smoke to Respirable Suspended Particles in Indoor Environments," Environ. Technol., 11, 189-196; 1990. 6. Jenkins, P.L.; Phillips, T.J.; Mulberg, E.J. and Hui, S.P., "Activity Patterns of Californians: Use and Proximity to Indoor Pollutant Sources," Atmospheric Environment, 26A (12), 2141 - 2148; 1992 7. Jenkins, R.A.; Moody, R.L.; Higgins, C.E. and Moneyhun, J.H., "Nicotine in environmental tobacco smoke (ETS): comparison of mobile personal and stationary area sampling," Proceedings of the EPA/AWMA Conference on Measurement of Toxic and Related Air Pollutants, Durham, NC., 1991 8. Leaderer, B.P. and Hammond, S.K. Evaluation of vapor-phase nicotine and respirable suspended particle mass as markers for environmental tobacco smoke. Environ. Sci. Technol., 25, 770-777, 1991 9. National Research Council, Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects, National Academy Press, Washington, DC., 1986 10. Oldaker, G.B.; Perfetti, P.F.; Conrad, F.C., Jr.; Conner, J.M. and McBride, R. L. (1990) Results of surveys of environmental tobacco smoke in offices and restaurants. Int. Arch. Occua. Environ. Health, 99-104. 11. Ogden, M.W.; Maiolo, D.C.; Oldaker, G.B. III and Conrad, F.W., "Collection and Determination of Solanesol As a Tracer of Environmental Tobacco Smoke in Indoor Air," Environ. Sci. Technol., 23, 1148; 1989. -18- I I ' I I I I I I

Table 9. Time Averaged ETS Constituent Levels* in Smoking and Nonsmoking Workplaces Determined Using Personal Sampling Systemsl 8 hour time Averaged Concentration, µgFm3 Nonsmoking W k l a 3EP Nicotine RSP UVPM FPM Solanesol or p ace N= 730 Median: 0.0244 0.0264 13.0 0.914 0.449 0.0042 Mean: 0.0880 0.109 18.4 2.04 1.31 0.0154 95th %ile: 0.323 0.342 53.4 5.19 3.36 0.0285 Smoking W k l b Median: 0.134 0.200 22.8 3.112 2.20 0.0042 or p ace N= 379 Mean: 0.662 1.69 40.6 15.5 11.7 0.262 95th %ile: 2.79 7.66 114 75.0 58.9 1.51 t a b Time averaged levels are measured over the total time that the sampling pump is operating. Includes only those individuals with mean cotinine values less than 10 ng/mL. Actual value was nondetectable; one half of the limit of detection in µg, an average flow rate, and an 8-hr time was used. From participants who recorded no tobacco products (cigarettes, cigars, or pipes) on their workplace diary. From participants who recorded tobacco products on their workplace diary In Table 10 are presented data regarding the median levels of salivary cotinine of subjects for which cotinine levels were available (individuals with levels greater than 100 ng/mL have been excluded from the comparison), as a function of cell designation. The data reveal a systematic decrease in the cotinine levels as the nicotine exposures decrease. However, the levels are not strictly proportional. This may be due to the cotinine levels for the least exposed groups being very near the detection limits for the analytical method, or to influences from dietary intake of small quantities of nicotine. Computation of the rate of misclassification of regular smokers as nonsmokers is an important issue in many assessments of risks to nonsmokers of contracting lung cancer through exposure to ETS. Data acquired as part of this study can aid in an estimation of this misclassification rate, since a criterion for inclusion in the study is not having smoked or otherwise used tobacco products or nicotine patches or gum for at least 6 months prior to participation. In addition, all subjects are asked a number of questions regarding their former smoking status and habits. An important parameter in many risk studies is the fraction of current smokers who claim to be never-smokers. Since this study has only included nonsmokers, the number of smokers which "would have been encountered" must be estimated from the following equation: 1 S x S x R where N= number of subjects in the study S= fraction of individuals in the general population of the USA who smoke at all (ca. 28.1 %) R = fraction of smokers in the USA who are "regular" smokers (ca 90%) -14- I I I I I I I I I I I I I I I

Table 7 Comparison of Airborne Concentrations of Environmental Tobacco Smoke Markers to which Individuals Recording Cigarette Observations in Different Venues were Exposed 16-hr Time Averaged Airbome Concentrations, µg/m; t Venue 3-EP Nicotine Myosmine RSP. UVPM PPM Salanesol Away Median 0.616 1.11 0.116 27.5 9.26 6.60 0.079 From W k Mean 1.12 2.61 0.231 41.9 21.4 16.5 0.440 or Low 0.0042 0.0112 0.0042 0.035 0.361 0.078 0.0022 n = 247 High 19.8 162 10.8 517 409 345 15.7 80th %ile 1.68 3.18 0.301 59.4 35.2 24.2 0.622 95th %ile 3.32 8,13 0.649 105 76.4 58.8 1.68 Home Median 0.477 0.668 0.088 23.2 7.26 4.65 0.032 303 Mean 0.936 2.17 0.193 37.9 18.0 13.9 0.362 n = Low 0.0042 0.0112 0.0042 0.035 0.102 0.037 0.0022 High 19.8 162 10.8 517 409 345 15.7 80th %ile 1.53 2.65 0.263 55.0 25.8 17.9 0.469 95th %ile 3.13 7.34 0.628 97.8 62.7 53.7 1.50 8-hr Time Averaged Airborne Concentrations, µg/m3 t i 3-EP Nicotine Myosmine RSP UVPM FPM Solanesol Work Median 0.206 0.279 0.036 24.8 4.13 2.78 0.010 n = 328 Mean 0.823 2.07 0.197 43.2 19.1 14.5 0.344 Low 0.008, 0.0222 0.008= 11.02 0.3882 0.2312 0.0042 High 21.0 37.9 2.75 622 472 421 13.7 80th %ile 1.05 3.06 0.278 55.2 25.6 18.0 0.297 95th %ile 3.34 8.26 0.859 140 82.6 64.8 1.68 Away From Work: Home: Work: Noted cigarettes on the Home Diary, Home Pump Survey, and Last Visit Survey Noted cigarette observations while inside the home on the home diary Noted cigarettes on the Work Diary, Work Pump Survey, and Last Visit Survey 'Analytical blank corrected µg/sample/(Sampling time x Flow Rate) = gg/m3 per sample zActual value was nondetectable; one half of the limit of detection in µg, an average flow rate, and an 8 or 16-hr time were used. -12- I I I I I

I I I I I I I I 1 I 1 I I I BAYESIAN META-ANALYSIS, WITII APPLICATION TO STUDIES OF ETS AND LUNG CANCER Richard L. Tweedie, D.J. Scott, B.J. Biggerstaff and K.L. Mengersen Department of Statistics Colorado State University Fort Collins, Colorado, USA Abstract Meta-analysis enables researchers to combine the results of several studies to assess the information they provide as a whole. It has been used to give a systematic overview of many areas in which data on a possible association between an exposure and an outcome have been collected in a number of studies but where the overall picture remains obscure, both as to the existence or size of the effect. This paper outlines some innovations in meta-analysis, based on using Markov chain Monte Carlo (MCMC) techniques for implementing Bayesian hierarchical models, and compares these with a more well-known random effects (RE) model. The new techniques allow different aspects of variation to be incorporated into descriptions of the association, and in particular enable us to better quantify differences between studies. We apply both the classical and Bayesian methods to the current collection of studies of the association between incidence of lung cancer in female never-smokers and exposure to environmental tobacco smoke (ETS), both in the home through spousal smoking and in the workplace. We demonstrate that, compared with the RE model, the Bayesian methods (a) allow more detailed modelling of study heterogeneity to be incorporated; (b) are relatively robust against a wide choice of specification of such information; (c) allow for more detailed and satisfactory statements to be made, not only about the overall risk but about the individual studies, on the basis of the combined information. For the workplace exposure data set, the Bayesian methods give a somewhat lower overall estimate of relative risk of lung cancer associated with ETS, indicating the care that needs to be taken in using point estimates based on any one method of analysis. On the larger spousal data set the methods give similar answers. We also consider some of the other concerns with meta-analysis, such as consistency between different geographic areas (such as Asia and the United States), and show that Bayesian methods allow us to take into account the overall picture, thus improving the ability to estimate accurately in the subgroups; and publication bias, which we find with the spousal exposure data may lead to an inflated excess risk. I

I 1. Introduction In recent years there has been an enormous increase in the use of meta-analysis in many medical areas in order to obtain overall evaluations of association in areas where individual studies are equivocal [61]. With this has come a large number of discussion papers which assess the benefits, drawbacks and problems of these techniques (see for example [57, 19, 11, 58, 71]). Some of the most well-documented concerns are about the way in which data can be combined if the collection of studies is not homogeneous by design but is based on a variety of differently structured epidemiological cohort or case control studies [28, 51]. Some of these concerns are matters of judgment, and relate to such issues as differing aims of studies or differing study quality including control of confounders; others relate to the underlying variability in the information presented, and different statistical approaches have been developed to attempt to quantify this objectively. In the epidemiological literature a standard method of combining estimates of interest is via a "random effects" model, which attempts to allow for inter-study variation, perhaps due to uncontrolled covariates [58]. This has been argued to be preferable to an earlier "fixed effects" model which essentially assumes that any heterogeneity between studies is purely random (cf. [86, 81]) and hence is not modelled explicitly. The random effects model can be analyzed both in a frequentist or a Bayesian framework [58]. In the latter context it extends logically to hierarchical models such as those recently proposed by DuMouchel [16, 15] or Carlin [10]. In order to differentiate between the models we shall refer to the frequentist random effects model as the "RE model" and the hierarchical Bayes model, which is also formally a random effects model, as the Bayesian model. Details of these are given in the Appendix. Interpretations of the two types of statistical approach are different but the context should make the interpretations clear. Two advantages of the Bayesian approach are its greater flexibility in utilizing other (often prior) information or relationships, and the ability to make useful probability statements on the basis of all information. Moreover,new Markov chain Monte Carlo (MCMC) methods now allow analysis of models based on very general formulations of such prior information, which were previously thought to result in mathematical expressions too complex to be solved. Through their use a wider range of inferences can be made in a straightforward way [2], as we demonstrate here. In the Bayesian meta-analysis context, we will use MCMC to analyze such hierarchical models, without the need to approximate the solutions. Although we do not pursue them here, we note that there are alternative approaches to combining epidemiological studies, also using MCMC methods: a logistic-r model with additional unknown covariates is proposed in [3], and methods of multiple comparisons, proposed in the NRC Report [58, pp. 149-158] for detecting nonequivalence between populations, can also be approached through MCMC [55]. In this paper the Bayesian methods are not used, in the main, to describe "prior information" in any strong sense. Rather, one can view the models as describing in more detail the way in which the -2- I I I I I I I I I I I

I I I I I I I I I I I I , I I r I I 2.2 Spousal and workplace exposure to ETS Table 1 lists all studies known to us, through Medline and Cancerlink searches and reference to published reviews [18, 47, 491, which provide data relevant to a meta-analysis of the association between ETS and lung cancer in nonsmoking adults, using spousal smoking as the primary measure of exposure. This currently comprises 40 studies of which 3 are unpublished theses, and we given details of location and sex studied. 0 ~ i CO -5- tW ~ N I

! 1 I I I I I I I I I I I I I I I I studies might be heterogeneous, and this allows one to account more explicitly for greater variability in the underlying collection of studies than is done in the fixed or even the RE models. After describing the methods of analysis, we illustrate a Bayesian MCMC approach through an assessment of the overall association between incidence of lung cancer in female never-smokers and exposure to environmental tobacco smoke (ETS), or "passive smoking," both in the workplace and from spousal smoking. There have been many meta-analyses of the individual studies of ETS exposure associated with spousal smoking ([81, 17, 18, 74, 47, 56] and others) but there has been limited assessment of the current set of papers addressing general workplace exposure, apart from [47, 48, 4]. The results we give below can be compared with those we have derived using somewhat simpler methods in [56, 4], and those papers contain a more complete discussion of aspects we merely survey here. The ETS studies seem appropriate for meta-analysis for several reasons. Although the association between lung cancer and ETS is an issue of public and legal concern, there has been a tendency to extrapolate results for spousal smoking to the workplace area (cf. [62]). By utilizing all current information about workplace and spousal exposures explicitly, overall estimates of the relative risk relevant to the particular exposure and their variability may be constructed directly and compared across the two sources of exposure. Our main focus is on statistical methods in this paper. However, any meta-analysis involves choices, not just of the statistical methods, but of many other things: choice of relevant studies, data within those studies, and the preceding statistical analyses of these individual datasets. We assess each of these in the ETS application, and consider whether such choices can influence the outcome. We specifically comment below on (i) methods of accounting for homogeneity of studies, and whether the Bayesian methods give different inferences from RE models; (ii) the problem of comparability of data and study design so that the meta-analysis can be meaningfully interpreted: this includes the choice of subgroups of studies to include, and whether, say, to include studies from different countries, or case-control and cohort studies; (iii) the use of unadjusted or adjusted published data: the meta-analyst is often faced with the problem that some studies report estimates unadjusted for any covariates (often as 2 x 2 table), so any effects of covariates are obscured, while others report only estimates of relative risks adjusted for (usually different) covariates; (iv) the effect of publication bias, recognizing that failure to obtain all relevant studies, both published and unpublished, may result in a quite distorted meta-analysis. Clearly all of these are of concern in principle, but it is not obvious whether they will cause real problems in specific applications. Here we attempt to quantify the degree to which such issues might affect the meta-analyses of ETS studies in practice under the particular models we consider -3 I

Table 1. Studies which provide relative risk estimates associated with lung cancer for female nonsmokers exposed to ETS, as measured by spousal smoking and workplace exposure Study Case Control Studies 1 Akiba et at. [11 2 Brownson et at. [7] 3 Brownson et at. [6] 4 BuffLer et at. [8] 5 Chan and Fung [12] 6 Correa et al. [13] 7 Du et at. [14] 8 Fontham et at. [21] 9 Gao et at. [221 10 Garfinkel et at. [24] 11 Geng et at. [25] 12 Ger et al. [261 13 Huibte et aL. [33] 14 Inoue and Hirayama [34] 15 Janerich et at. [35] 16 Joecket [36] 17 Kabat [371 18 Kabat and Wynder [38] 19 Katandidi et aL. [39] 20 Koo et aL. [401 21 Lam T. et at. [42] 22 Lam Y. [437 23 Lee et at. [50] 24 Liu et at. [53] 25 Liu et al. [52] 26 Pershagen et at. [63] 27 Shimizu et at. [65] 28 Sobue et al. [67] 29 Stockwell et at. [69] 30 Svensson et at. [70] 31 Trichopolous at at. [73] 32 Vareta [77] 33 Wang at at. [82] 34 Wu et aL. [841 35 Wu-wiLLiams et al. [857 36 Ziegter et at. [87] Cohort Studies 37 Butter [9] 38 Garfinkel [23] 39 Hirayama [29, 30] 40 Hote et at. [32] Country JAPAN UNITED STATES UNITED STATES UNITED STATES HONG KONG UNITED STATES CHINA UNITED STATES CHINA UNITED STATES CHINA TAIWAN UNITED STATES JAPAN UNITED STATES GERMANY UNITED STATES UNITED STATES GREECE HONG KONG HONG KONG HONG KONG ENGLAND CNINA CHINA SWEDEN JAPAN JAPAN UNITED STATES SWEDEN GREECE UNITED STATES CHINA UNITED STATES CHINA UNITED STATES UNITED STATES UNITED STATES JAPAN SCOTLAND I ex F F F F F F F M+F F F F Workplace YES YES YES YES YES YES YES YES YES YES F F M+F M+F I I I I ! I I I I I I I I i O ~ v t)D W Gi I -6- 1

Table 3. Unadjusted RR, Bayesian shrinkage estimates and adjusted RR with 95% CI for female nonsmokers exposed to ETS through workplace smoking 8 1.12 (0.91-1.36) (1.01-1.24) 1.11 (0.94-1.32) 1.34 (1.11-1.74) 10 0.93 (0.55-1.55) (0.53-1.60) 1.07 (0.79-1.38) 0.93 (0.73-1.18) 15 N/A N/A N/A N/A 0.91 (0.80-1.04)* 17 1.00 (0.49-2.06) (0.46-2.21) 1.10 (0.78-1.45) 1.00 (0.49-2.06) 18 0.68 (0.32-1.47) (0.30-1.58) 1.05 (0.72-1.37) 0.68 19 1.39 (0.76-2.54) (0.73-2.67) 1.15 (0.86-1.54) 1.39 (0.76-2.54)** 20 0.91 (0.15-5.37) (0.08-6.95) 1.10 (0.77-1.51) 0.91 23 0.63 (0.17-2.33) (0.11-2.49) 1.07 (0.74-1.45) 0.63 (0.17-233) 27 1.18 (0.68-2.03) (0.66-2.09) 1.12 (0.84-1.46) 1.2 34 N/A N/A N/A N/A 1.3 (0.5-3.3) 35 1.22 (0.95-1.57) (0.94-1.58) 1.16 (0.95-1.41) 1.1 (0.9-1.6) OveraLL 1.12 (0.93-1.28) ~~ 1.10 (0.90-1.32) . *. Combined Ma[es and Fema(es Catcu[ated from [39, TabLe 2] campared to housewives as unexposed group The 40 studies in Table 1 comprise 30 studies as described by Lee [47, pp. 101-105j, and 10 more recent studies, most of which are also reviewed in the EPA Report [18]. Nine other related studies which do not provide data usable in our meta-analysis are not considered here; Lee [47] provides details of these. Having chosen the studies for inclusion, there is then a question of choice of data to be settled. Different values can be extracted from different parts of some studies: one could for example use different criteria for inclusion of subjects, such as inclusion of ex-smokers or cigar smokers, single or widowed subjects, surrogate respondents, or disease rather than death. In previous analyses [56] we considered these choices and found that they made little difference to this meta-analysis. We chose therefore to adopt the data tabulated in [47, Tables 3.13F and 3.13M1, which is well-documented [47, pp. 102-103]; fortunately, for the 10 more recent studies [6, 20, 35, 69, 53, 26, 36, 14, 52, 82] the abstraction of comparable data is straightforward. It is harder to assemble a coherent set of studies with sufficient data reported to enable meta- analysis of the relative risk of lung cancer associated with "general" exposure to workplace ETS. Table 1 also indicates that 11 of these papers contain statistics or data concerning exposure to ETS in the general workplace. These studies comprise, to our knowledge, all those currently published on this association, based on a Medline search and various reviews. Discussion and review of these studies may be found in Lee [47, p. 117-118] and [48, p. 37-41] (except the results based on [40, Table 2] which is not used there and [20] which is more recent) and we do not repeat details here. -8- I I I I I I I I I I I I I I I

I I I I I I I I I I I 1 I I I I We have omitted from our workplace meta-analyses published studies concerning occupation- specific environments such as passenger cabins in commercial airlines [60, 31] or the food service industry [66]; and also the study of Brownson et al [7) which relates to smokers and nonsmokers and only considers specific high lung-cancer risk occupations, since these are sufficiently different in design to violate the applicability of our models [58]. We have also only included studies for which the exposure is solely in the workplace, excluding those (Lam and Cheng [41] and Svensson et al [70]) which give relative risks for lung cancer when ETS is measured through exposure "at home or at work" or "at home and at work." 2.3 Exact and Approxitnate Analyses of Individual Studies Typically, studies report results either in "crude" or "unadjusted" from, as 2 x 2 tables, or as "reported" results, which may be adjusted in covariates as described by the individual authors. Ideally one would wish to construct a model with complete control of such covariates (eg [83]). Most often, however, the required information is not available in published epidemiological papers. Instead meta-analysis must be performed only on the basis of summary statistics. These statistical quantities of interest in the individual studies, which are later combined in our meta-analyses, are the point estimates and associated confidence intervals (CIs) of the relative risk (RR) of outcome in a population with some defined exposure (either spousal or workplace ETS in our examples), compared with outcome for an unexposed population. In Tables 2 and 3 we first provide analyses of the unadjusted data for the spousal and workplace studies respectively. A more detailed description of the methodology we use is relegated to the Appendix, and here we describe the notation and quantities needed to interpret these tables. We use the following notation throughout: we suppose that we have k studies, and that RRi = observed estimate of relative risk in study i Yi = log RRi, true log relative risk in study i, an appropriate estimate of (Var[Yi])'1. In the traditional setting for epidemiological studies, the empirical odds ratio provides a point estimate of the true relative risk for each study, and we use this throughout in this paper. Tables 2 and 3 also contain estimates of the individual parameters Bi and corresponding confidence intervals based on logit approximations to the variance, with an assumption of normality of the Yt which is known to be reasonable, at least for large individual sample sizes. As seen in these Tables we find that, compared with an exact method (also discussed in the Appendix) the logit method gives CIs that are perhaps 5-10 % too short; but for our purposes we will accept this level of accuracy -9- I

I 2. Data and Analysis of Studies on Exposure to ETS 2.1 Data Comparability and Bias Meta-analysis is designed to enable combination of results from studies which are comparable in outcome and exposure. The interpretation of comparability is a subjective and often difficult one. In order to paint an honest picture of the aims and applicability of any meta-analysis, we must first define the relevant measures of outcome and exposure with which we are concerned. The clinical outcome assessed in all of the ETS studies is death from "lung cancer." Several concentrate on or are dominated by one specific form of this disease (e.g., adenocarcinoma), and although some studies give data for different types of cancer, many others do not make such distinctions. Here we choose to combine RR estimates for all lung cancer types, but we are aware that the overall RR estimate may be based on individual RR's associated with quite different diseases in different studies. In order to identify studies with comparable exposures we primarily restrict the meta-analysis to the subset of all ETS studies of adults asserted to be never-smokers, with exposure to spousal smoking or workplace smoking the declared type of exposure to ETS. However, the relevant data are unavailable in a few "spousal" studies, and for these the restrictions are relaxed slightly to include other household exposure or long-time nonsmokers; see Lee [47] and the EPA Report [18] for further details. In choosing which studies to combine, we also need to consider the plausibility of comparing different subpopulations. Two obvious questions are whether there are gender or geographic differences. In accord with the practice in most individual studies and other meta-analyses of these data, we have analyzed males and females separately, and it is the latter that we report here. For males exposed to ETS in the workplace there is a comparable analysis in [4]. The geographic question seems more appropriately studied through a sensitivity analysis as in [18] and we do so in Section 3.3. It is also crucial in meta-analysis to attempt to collect all studies relevant to the relationship in question [27i. This involves collecting at least all published studies, if possible, and testing for the potential existence and influence of unpublished or uncollected studies. There is an insufficient number of studies of workplace exposure to decide if there might be missing infonnation due to publication bias. In contrast, for the spousal exposure studies detailed in the next section, it is possible to investigate completeness using funnel plots (see [511), and in Figure 1 of [56] there is a clear indication of the absence of small studies with negative (perhaps nonsignificant) estimates of effect. It does appear from this that there is indeed bias towards publication of raised relative risks, with perhaps 6-10 or so small but negative studies expected but not absent: this may impact on our overall results, and we comment on this in Section 4. Overall, our experiences with collating these data strongly reinforce those of Felson [19] and Chalmers [11]: data extraction and location is a nontrivial exercise, there are considerable problems in locating studies and relevant data within them, and there are many subjective decisions about data collection and analysis which need to be explicitly documented. We attempt to do this in the following sections. -4- I I I I I I I I I I I I I I

I 1 I I I I I I I I I I I I I I I Table 2. Unadjusted RR, Bayesian shrinkage estimates and adjusted RR with 958 CI for female nonsmokers exposed to ETS through spousal smoking 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 1.52 1.82 0.96 0.80 0.75 2.07 1.09 1.26 1.19 1.23 2.16 N/A 2.34 2.55 N/A 2.27 0.90 0.79 1.55 1.55 1.65 2.01 1.03 0.74 1.66 1.03 1.08 1.06 N/A 1.26 2.08 0.75 1.41 1.20 0.79 N/A 2.44 1.17 1.39 1.89 (0.87-2.63) (0.45-7.36) (0.77-1.20) (0.34-1.90) (0.43-1.30) (0.81-5.25) (0.64-1.85) (1.04-1.54) (0.82-1.73 (0.81-1.87) (1.08-4.29) N/A (0.81-6.75) (0.74-8.78) N/A (0.75-6.82) (0.46-1.76) (0.25-2.45) (0.87-2.83) (0.90-2.67) (1.16-2.35) (1.09-3.72) (0.41-2.55) (0.32-1.69) (0.73-3.78) (0.61-1.74) (0.64-1.82) (0.74-1.52) N/A (0.57-2.81) (1.20-3.59) (0.47-1.20) (0.54-3.67) (0.48-3.01) (0.62-1.02) N/A (0.58-10.22) (0.85-1.61) (0.97-1.98) (0.22-16.23) (0.85-2.n) 1.31 (0.33-8.90) 1.27 (0.77-1.21) 1.03 (0.32-2.21) 1.15 (0.42-1.35) 1.06 (0.75-6.06) 1.33 (0.62-1.93) 1.19 (1.14-1.40) 1.25 (0.80-1.77) 1.21 (0.80-1.92) 1.23 (1.03-4.56) 1.41 N/A (0.76-8.59) 1.34 (0.67-11.91) 1.32 N/A (0.68-8.28) 1.32 (0.44-1.88) 1.15 (0.22-2.83) 1.18 (0.83-2.92) 1.32 (0.86-2.77) 1.32 (1.144.39) 1.43 (1.04-3.92) 1.41 (0.38-2.88) 1.20 (0.31-1.92) 1.13 (0.68-4.14) 1.30 (0.59-1.80) 1.16 (0.62-1.89) 1.18 (0.73-1.55) 1.14 N/A (0.54-3.16) 1.24 (1.16-3.76) 1.45 (0.46-1.23) 1.02 (0.49-4.20) 1.25 (0-39-3.40) 1.23 (0.61-1.02) 0.92 N/A (0.38-12.55) 1.30 (0.84-1.64) 1.20 (0.96-2.04) 1.30 (0.21-8.96) 1.25 (0.96-1.83) 1.50 (0.9-2) (0.86-1.88) 1.68 (0.39-2.97) (0.84-1.23) 1.0 (0.8-1.2) (0.79-1.59) (0.73-1.41) (0.93-1.98) (0.86-1.60) (1.06-1-49) 1.29 (1.04-1.60) (0.92-1.57) About 1.4 (0.93-1.60) (0.99-2.06) N/A 1.18 (0.47-2.99) (0.92-1.98) 2.20 (0.8-6.6) (0.91-1.99) 2.25 (0.8-8.8) N/A 0.93 (0.55-1.57)* (0.91-1.99) (0.80-1.57) (0.79-1.68) (0.96-1.84) 2.11 (1.09-4.08) (0.96-1.81) 1.64 (0.87-3.09) (1.09-1.87) (1.01-2.01) (0.82-1.69) 1.00 (0.37-2.71) (0.75-1.57) (0.91-1.89) (0.83-1.54) 1.20 (0.7-2.1) (0.86-1.56) (0.88-1.46) 1.13 (0.78-1.63) N/A 1.6 (0.8-3.0) (0.87-1.73) About 1.5 (1.05-2.09) (0.72-1.34) (0.86-1.80) 1.20 (0.50-3.30) (0.84-1.76) (0.72-1.14) 0.7 (0.6-0.9) N/A (0.88-1.96) 2.02 (0.48-8.56) (0.94-1.50) 1.18 (1.01-1.68) 1.45 (1.02-2.08) (0.83-1.89) 2.41 (0.45-12.83)* Overall 1.20 (1.07-1.34) 1.22 (1-08-1.37) Results for sexes combined I

I I I I I I I I I 1 I I I I I I (i) For workplace exposure the following two groups of datasets were considered separately: only those studies of females which provided unadjusted RR estimates: these results can thus be directly compared with those of Tables 2 and 3; (ii) all studies, combining both males and females, giving a result directly comparable with that of Lee [48, Table 5]. Results are presented in Table 4. For dataset (a), under either model the combined point estimate for the females exposed to workplace smoking is 1.10-1.11. The dataset (b) includes both genders and indicates that the male studies are somewhat different in the sense that now ~2 = 0.017. The overall estimate of 1.07-1.08 is only marginally higher than that of Lee [48, Table 5] as we should expect since they differ only by two studies. The Bayesian methodology also enables us to assert that the posterior probability that the overall underlying relative risk is greater than 1.0 is 0.83-0.84 in both these cases. Table 4. Meta-analyses of Results (Adjusted where Available) of Workplace Exposure Studies °- . . . ayeslan ' e - o e RR (95X:GI) . RF. (95Y CI) f .. ~ Stutl T e . - (a) Females (9 Studies) 1.10 (0.89-1.32) 1.11 (0.96-1.29) 0.005 (6) Combined (14 Stadies) 1.08 (0.92-1.26) 1.07 (0.93-1.24) 0.017 For exposure to spousal smoking, we consider a different approach to the adjusted results, and indicate the effect of combining the case-control and cohort studies. Under a fixed effects model this is not advisable due to the inherent differences in the methodology. Here we are able to take that into account. We analyze against the totality of studies, consisting of adjusted RRs where given as in Table 2, and unadjusted RRs for other studies, thus using the maximum number of 35 case control and 4 cohort studies for females. Table 5 gives the results of analyzing this dataset. Again we note that the Bayesian and the RE models give very similar answers. The inhomogeneity in the studies is supported by a value of ~2 = 0.052, although the inclusion of the cohort studies in this case actually decreases ~2 slightly. -13- IV O 00 s V 00 W N N 0 I

I I I I t I I I I I I I I I In this formulation, the posterior distributions become quite complicated, leading DuMouchel [16] to make some (reasonable) approximations to normality for computational convenience. In contrast, in this paper we use simulation methods (specifically the Gibbs samples through the software package BUGS [68]) to carry out the analysis. As previously mentioned, these algorithms provide powerful computational tools for Bayesian analysis and release the user from restrictive assumptions about the distribution of the data and of prior information [2]. In the ETS case, for example, although the model (2) in the Appendix was considered appropriate, approximations to the posterior distribution were not needed, although comparison of our results here to those in [4] show that the Normal approximations of DuMouchel [16] are in fact very effective in this case. The Bayesian method, as implemented through MCMC software, also enables us to make inferences about the posterior probability that the overall relative risk is above 1.0, enabling more exact inferences to be made and thus more effectively enabling the meta-analysis to achieve one of its overall goals. It is equally possible to quantify statements such as P {overall US mean > 1} using this method, which is not a simple task in the RE models. In this paper we will show that the use of this more flexible description of the way in which relative risks are spread across studies can lead to small but possibly important differences in the overall conclusions made, and that these conclusions are essentially independent of how the prior distributions are chosen, so that in fact it is the data that are driving the conclusions. 3. Results 3.1 Analysis based on unadjusted relative risks The results of meta-analyses under both the RE and Bayesian paradigms are given as the "Overall" values at the bottoms of Tables 2 and 3. In the second-last column of Tables 2 and 3 we also give the estimates for the individual studies after "shrinkage" towards the overall mean through "borrowing strength" from the totality of studies. Note that these estimates have much tighter credible intervals than the original study estimates, since they are based on a combination of individual and overall study information. In Table 2, the overall Bayesian posterior mean estimate for spousal studies (1.22) is slightly higher than that of the logit-based RE model (1.20), although they are very much within each other's CI. For the spousal exposure studies we find P{µ > 1} = 0.9996, significant at well above the 5% level with this data. The values appear robust to some change in model choice. Under the Bayesian model, there were negligible changes to posterior distributions when input values for prior distributions were changed. Only when the degrees of freedom associated with the distributions of u2 and T2 or the entries in the matrix controlling the between studies variance were set at extreme and unreasonable values were there any real changes to posterior estimates. Other changes in prior specifications produced no effect at all. N O Co -L -4 00 I

here. (In [4] these methods are also compared with the results generated by Mantel-Haenszel methods [5, p. 141], [64], which are found to be typically less accurate again.) Even without 2 x 2 tabulations, reported results may be combined provided all the confidence intervals are also reported, through deriving a Normal-based variance estimate for the log relative risk estimate. This is the case for many of the studies in Tables 2 and 3. Note, however, that the different factors for which adjustment was made in each of the studies render it more difficult to be sure that like is being compared with like in such an analysis. In Table 2 we see that 28 of the 36 relevant studies with female respondents reported an increase in the unadjusted relative risk of lung cancer associated with spousal ETS exposure, with just 5 of these significantly different from 1.0 at the 95 % level. (Because we use both frequentist and Bayesian methods, it will be convenient to define the phrase "significantly different from 1.0" to cover either the situation in which there is a constructed 95 % confidence interval which does not cover 1.0, or a Bayesian 95% credible interval which does not cover 1.0: the context should make it clear which is meant.) In Table 3 we see that only 4 of the 9 relevant studies with female respondents reported an increase in the unadjusted relative risk of lung cancer associated with spousal ETS exposure, with just one of these significantly different from 1.0 (as indicated from the exact CI). Thus, as stated above, both of these collections of studies are certainly such that a simple interpretation is difficult and in which heterogeneity may well be a problem that both the RE and the Bayesian analyses can help to overcome. 2.4 Random Effects and Bayesian Approaches to Meta-analysis The RE model for meta-analysis is a natural starting point to describe a Bayesian methodology for meta-analysis. As described more formally in the Appendix, in the RE method we assume that there is a true underlying log RR over all studies, denoted µ, and that the observed log relative risks Yi for each study are from a distribution governed by qu,,antities Bi and ai2 which represent the true RR and within-study variability of study i, and a quanti~r~ which provides a measure of the between- or across- study variability. In the special case in which = 0, indicating homogeneity between studies, this RE model reduces to the well-known fixed effects model (see [86, 81] and others). In this non-Bayesian paradigm, µ, a and r are presumed fixed, and the Os are random variables with mean µ. In a general hierarchical Bayesian scheme [16], a12 and 72 are also random variables with (in our case) a x2 distribution, and these X2 distributions are in turn governed by parameters (degrees of freedom) dfQ and dfr which indicate how well the variance structures are assumed to be known. I I I I I I I I I The distributions of these quantities are specified a priori according to the application. It is standard practice to assume a "flat" or "uninformative" prior to µ as we do below, as even with a small " N  the combined data become relatively informative about the location of the effect-size number of studies, prior distribution" [10, p. 146]. The imposition of distributions on 0, a2 and 72 enables us to describe O 00 much more explicitly any underlying variability in the way the study outcomes are distributed. i -4 ~ W -10- CJ N ~ V , ~

I of subjects, such as the use of active instead of nonsmoking subjects in studies of exposure to spousal ETS (which is well-documented [75]) may account for 50% of the observed excess risk. But with all of these problems, meta-analysis is an increasingly common and useful practice and one that needs to be improved where possible. In this paper we approach one particular issue (study inhomogeneity) by trying to capture study differences in an expanded hierarchical model, and summarized a number of others from the earlier analyses in [56, 4]. These are all shown to make small but important differences in point estimates: on this data-set the overall excess risk for workplace exposure is shown to be increased slightly if the RE model is used rather than the Bayesian model, and in previous analyses of smaller data sets the increase has been considerably higher. Since none of these values are significantly different from each other, or from a null effect, this may still, of course, be a product of random variation in the data: but it does indicate that there can be considerable danger in ignoring the heterogeneity between studies. Even low observed excess risks, if used to generate attributable risk figures as in [62], can appear important: if they are inaccurate by 10% or 20% or 50%, as we have shown may be the case from choice of models or of data or because of publication bias, then very much more caution needs to be shown in using them than appears commonly to be the case. Appendix: Bayesian and Classical Hierarchical Models for Meta-Analysis in Epidemiology In order to implement the meta-analysis methods, we need to estimate the individual parameters e2 introduced in Section 2.4 and to calculate corresponding variances for these estimates. In our analysis we have used (a) Fisher's exact method [5, p.124], which gives a point estimate and a non-parametric confidence interval (CI) but no variance estimate for RRi; (b) The logit method [5, pp. 129-130], which gives a point estimate Yi and approximate variance Wi 1, with a corresponding confidence interval based on an assumption of normality which is known to be reasonable, at least for large sample sizes. A frequentist or classical random effects (RE) model (of which a fixed effects model is a particular case) for meta-analysis is an appropriate starting point to describe the classical and Bayesian I I I I I I I I I I I I I methods of meta-analysis. We consider the simple formulation O 00 Y=B+e -L ~4 B=Xµ+e, (1) W , in which Y=(YI,...,Yk)1 are the observed log relative risks for each study, B=(BI,...,Bk)1 are N N w the corresponding true log relative risks, e=(el,...,ek)I and e=(e1,...ek)1 are random errors, X -18- is ~ , I

problem appears to arise because the DuMouchel model does not permit the prior distribution of r2V to be sufficiently uninformative, so the data can not drive the posterior distribution to satisfactorily match the data. Future work is planned to consider a more appropriate way to model the variability of 0 1 µ,r. As noted by DuMouchel [16, p. 515], the particular prior distributions given here are chosen for convenience, so that the posterior distribution of 0 given Y is a mixture of multivariate Smdent-t distributions. For computational convenience, however, he suggests using a multivariate norntal approximation to the posterior, which can then be described through the posterior mean and covariance matrices. One of the advances in the present paper is the evaluation of this model using MCMC methods, which avoid the need for this approximation and also allow us to assess how much the results actually depend on some of these assumptions. In particular, the Gibbs algorithm was used in these analyses through the software package BUGS. A more restricted version of this hierarchical model is explored by Carlin [10], who more closely follows the RE version by taking V to be the the k x k identity matrix, C to be a diagonal matrix with the corresponding diagonal entries the (assumed known) variances of the individual observations Yl and u2 = 1, thus omitting the non-degenerate prior on o 2. Both µ and 72 are still unknown hyperparameters representing, as before, overall mean and between-study variance, respectively. In [4], both the DuMouchel method and the Carlin method are applied to the workplace exposure studies of Table 3. Both Carlin and DuMouchel suggest ways to examine the sensitivity of their respective methods to the assumptions made, and it is desirable to investigate the dependence of the posterior estimates of µ and 0 on the specifications of dfT and dfQ. Because of computational restrictions this was not done in [4], but the MCMC methods allow us to do this. Although sensitivity was not addressed for all of the analyses of this paper, the sensitivity analyses we conducted indicate that the only initial specifications which have any effect on the estimates are those for dfT and dfQ, and for meta- analyses such as these where the dataset is large, the changes were essentially negligible. Aclmowledgements The authors thank Professor P. Mielke for programs to do Fisher's exact methods, and the MRC Biostatistics Unit at the University of Cambridge for supplying a prototype of the BUGS software for the Bayesian analysis. This work was carried out with partial support from three tobacco companies: the conclusions here are however entirely those of the authors and should not be otherwise ascribed. -20- I I I I I I I I I I I I I I I

I The effect of the different methods is perhaps more noticeable in the analysis of workplace exposure than spousal exposure data. In using the RE approach to obtain an overall estimate of relative risk for females in workplace studies, the estimate of between-study heterogeneity is r2 = 0 so the RE and fixed effects models coincide, indicating that all studies may have a common true relative risk. (For males, in contrast, as shown in [4], there is indeed detectable between-study heterogeneity.) The RE estimate of the overall µ is then 1.12, although this is not significantly raised above unity. The overall (posterior) mean, based on the Bayesian model which does allow for between-study heterogeneity, is estimated to be 1.10. Both of these values are again well within each other's CIs: in this sense this is an insignificant difference. However, the excess risk which is often fed into calculations of attributable risk is some 20% higher for the RE model. The value of 1.10 is larger than the value of 1.07 calculated in [4], where it is also shown using a simplified formulation due to Carlin [10] that the posterior mean is estimated by 1.04. However, here we have added the recent result reported by Fontham et al [20]: using exactly the same data as in [4] we get a value exactly in accord with the DuMouchel approximation. These are slight discrepancies. Nonetheless they illustrate that the choice of model can play a serious role, for in using estimates of the overall effect, especially for values estimated as near 1.0 such as this one, a 10%-20% discrepancy in the excess risk makes a considerable difference in interpretation. Finally, we again quantify the posterior probability that µ> 1.0: for the workplace studies we find P{µ > 1} = 0.83, so that this is not significant even at the 10% level with this data. 3.2 Analysis using adjusted relative risks In any meta-analysis of published studies the role of covariates, either in the design or by adjustment in the analysis, raises questions of comparability. Unadjusted RRs may be quite appropriate for case-control studies since adjustments can often be assumed to have been made by matching and similar techniques in the design stage. Also, restricting the meta-analysis to data which have been adjusted seems extreme for case-control studies, since many studies do not report such adjusted values and would have to be ignored. For cohort studies, conversely, adjusted RRs are probably more appropriate; but of course, as with the case control studies, adjustments for the same covariates are not made on a common basis across studies. Our goal is fortunately rather more limited than making a final choice between the two types of estimates. We merely wish to see whether this problem of principle actually makes any practical difference in these particular meta-analyses. From each of Tables 2 and 3, we provide meta-analyses of the adjusted RR estimates where provided by individual studies, and unadjusted estimates for other studies. -12- i I I I I I I I I I I I I I I I

?npa,an3235

Table 5. Meta-Analysis of Results (Adjusted where Available) of Spousal Exposure Studies : > ayes. an e . e . - Stu T RR (95%CI) .. . : .. ..RR (95X CI) P2 e. ... _ ase ontro 1.22 (1.07-1.39) 1.22 (1.06-1.41) 0.061 Cohort 1.33 (1.03-1.78) 1.29 (1-02-1.64) 0 All 1.23 (1.09-1.39) 1.23 (1.08-1.39) 0.052 3.3 Choosing subgroups of studies Ensuring comparability an entail close examination of the data to identify appropriate subsets of studies for combination. As noted in the EPA Report [18], for this particular meta-analysis it may be sensible to consider the effect of grouping studies by geographic region, especially given the rather inexplicit nature of "exposure to ETS" and the way in which it might vary in different cultures. To illustrate the effect of geographic location we provide in Table 6 meta-analyses of two subgroups of studies of spousal smoking which may a priori be considered internally more homogeneous: Asian populations only (China, Japan, Hong Kong, Taiwan), comprising 15 case control and one cohort study; and U.S. studies only, comprising 11 case control and two cohort studies. Resulting overall unadjusted relative risks with logit variance estimates are again compared under both frequentist and Bayesian approaches. It can be seen that there are considerable differences between the two country groups with respect to both overall estimate and between-study heterogeneity (and that again there is 10%-15% difference between using RE and Bayesian methods). The relative risk estimate is significantly increased above 1.0 at the 5% level for the Asian studies, but for the U.S. studies we calculate the probability of the relative risk being above 1.0 as 0.92. Table 6. Meta-analysis of Asian and U.S. subgroups of studies of spousal exposure . . ayes an e - y Study T e RR (95X CI) .. . RR (95Y:CI) P2 stan tu 1es 1.25 (1.03-1.50) 1.25 (1.02-1.52) 0.067 U.S. Studies 1.13 (0.95-1.34) 1.11 (0.98-1.26) 0.003 One implication of the different RRs is that extrapolation of overall results to individual studies and from one country group to another may not be appropriate. It certainly highlights a need for further investigation of these differences, perhaps through a closer exploration of covariates or possible biases. Some recognized covariates in the association between lung cancer and exposure to ETS include diet and socioeconomic status. Possible biases include different underlying rates of lung cancer and misclassifica- tion of active smoking. There are many other breakups of the data that could be accomplished by the methods used here. For example, there has been considerable recent interest [54, 76, 18, 57] in accounting for the differing quality of studies in meta-analysis. The EPA Report [18] groups studies into four tiers based on a -14- I I I I I I I I I I I I I I I I I I

I ' I 85. I 86. I I 87. I I I I I I I I I I I I Wu-Williams, A.H.; Dai, X.D.; Blot, W.; Xu, Z.Y.; Sun, X.W.; Xiao, H.P.; Stone, B.J.; Yu, S.F.; Feng, Y.P.; Ershow, A.G.; Sun, J.; Fraumenti, J.F. Jr. and Henderson, B.E. Lung cancer among women in north-east China. Br. J. Cancer, 62:982-987, 1990. Yusuf, S.; Peto, R.; Lewis, J.; Collins, R. and Sleight, P. Beta-blockade during and after myocardial infarction: An overview of the randomized trials. Prog. Cardiovasc. Dis., 27:335- 371, 1985. Ziegler, R.G.; Mason, T.J.; Stemhagen, A. et al. Dietary carotene and vitamin A and risk of lung cancer among white men in New Jersey. JNCI, 73:1429-1435, 1984. -27- /

I i I I I I I I I I I 1 , I I I I I qualitative score. This is intended to take into account various design aspects and susceptibilities to common sources of bias and misclassification which may impact on the observed results. In Table 7 of [56] we show the effect of using only the studies in Tiers 1-2 and 1-3 in a classical model in order to avoid bias which may be inherent in the lower quality studies. For each gender, using only the "good quality" studies appears to give a more homogeneous collection, as indicated by the decreasing ~2 values with increasing "quality" although as also commented in [181, the "good quality" Tier 1-2 studies exhibited exactly the same point estimate of combined RR for females as does the complete set of studies. 4. Discussion 4.1 Effect of different statistical models In this paper we have compared a frequentist (random-effects) and a Bayesian approach to meta- analysis of epidemiological studies, and implemented the approaches for studies of the association of lung cancer with both workplace and spousal exposure to ETS. In general one would expect that the Bayesian methods would give a more explicit overall picture of the effect of variability in a collection of studies. The use of Bayesian methods was facilitated by the use of MCMC algorithms, which allow for more flexibility in the formulation of prior information and models, and for a wider range of inferences and comparisons through simulation. The workplace studies were analyzed using a similar Bayesian model in [4], but there approximations were used to enable direct analytical solutions; our results confirm the approximate analysis there. The spousal exposure studies have not been analyzed previously by Bayesian methods, although the results are very similar to those for RE models of the same data: the overall relative risk is 1.20-1.22 with a CI of (1.07,1.35) on either method. For workplace exposure, there is also a small but proportionally more noticable difference between the two estimates: the overall mean estimate under the RE model is [1.12 (0.93,1.28)] (essentially as noted by Lee [48]), higher than that obtained under the Bayesian formulation [1.10 (0.90,1.32)]. Use of the RE model thus gives a point estimate of excess risk which is 20% higher than for the Bayesian method. This effect of choice of approach is rather more marked before adding the recent Fontham study [20]: it was shown in [4] that a variation of almost 70% in excess risk (from 0.07 to 0.12) resulted by changing the model used. Such differences are almost certainly due to the different estimates of between-study variation. In this case we have an estimate of T2 = 0 in the classical model but the assumed non-zero across-study variability in the Bayes methods automatically down-weights individual study estimates with relatively small variances, so that they do not dominate overall esti-nates as much. Using MCMC methods, we are able to estimate the probability that the overall relative risk is greater than unity: for the workplace studies this is P{µ > 1.0} = 0.83. Taking into account variation between studies through the adoption of a Bayesian model is of considerable importance. Most previous meta-analyses of ETS spousal exposure studies have used simple -15- N O tp ~ N 00 W N N N s

fixed effects models (see [81, 59, 74, 18, 17]) and substantial conclusions have been based on them. We have shown that there is considerable indication of heterogeneity between studies and that moving to a Bayesian approach can give a more detailed approach to analysis of such data. 4.2 Use of appropriate studies One of the outcomes of this analysis is the ability to determine whether one can use spousal data to evaluate relative risks in the workplace. It is clear that this is a dangerous practice: the overall spousal relative risk estimated here is around 1.20. The overall workplace relative risk is estimated to be around 1.11. Thus the observed excess risk in one environment is only some 50% of the excess estimated in the other environment. In these analyses above we have deliberately ignored any need to adjust for covariates in the population. If the reported (and sometimes adjusted) data for spousal exposure are used, then there is little indication of any change in the estimate of relative risk. Similarly, the difficult choice between unadjusted and adjusted data seems immaterial in the case of workplace exposure, where adjustments up and down also seem to cancel. In the adjusted data there continues to be indication of substantial between-study variation. This warrants special attention in a meta-analysis, especially in applying overall results in subpopulations and individuals. In this example, the choice of subpopulations to be combined had very obvious effects. For example, the overall estimates vary considerably within different geographic areas: it is difficult not to conclude from Table 5 that in combining over the studies conducted in Asia and the U.S., one may well be ignoring meaningful differences. Again the excess risk in the current Asian studies is almost double that of the excess risk in the U.S. It is clear from these observations that quotation of point estimates alone, and their use as a basis for decisions, seems very unwise. A much more acceptable practice is to report the corresponding confidence intervals, taking explicit account of the between-study variation and, if necessary, reducing this variation through the adoption of more homogeneous subpopulations or expansion of the model to include relevant covariates. Detection of heterogeneity in a meta-analysis context becomes, of course, only the first step: it is then revealing for the analyst to investigate further the source of such variation, as we have tried to do in isolating study groups. 4.3 Publication and other biases Although we have not concentrated on it in this paper, the sensitivity of the combined point estimate and confidence interval to possible biases should be acknowledged and, where possible, explicitly taken into account in inferences based on the size and significance of the overall relative risk. In [56] -16- I I I I I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I I I I I a k x p design matrix, and µ is a p x 1-vector of parameters. We shall take X to be the k x 1-vector of I's, and so p to be a scalar parameter, representing the true underlying log RR over all studies. In (1) YI are u jed since they can be shown to be asymptotically Normal Y- N(O, WI), with WI = diag(Wl ,...,W~ ); accordingly we assume that B- N(Xµ, ~n, and hence that the ei are independent N(O,ai ) random variables, the ei are independent N(O,r2) random variables, and the ei and ei are mutually independent. A classical approach to meta-analysis using this model is widely used [58, 72]. Here µ,o2 and r2 are considered fixed parameters and 72 is estimated most commonly through an approximation proposed by DerSimonian and Laird (cf[58]). DuMouchel [16, 15] describes a general hierarchical Bayesian scheme, and this is the model we use here. This allows considerable flexibility in application, and can be viewed as a Bayesian generalization of the frequentist RE model. DuMouchel makes the following further distributional assumptions: YtB,o - N(O, o2C), a Z - XZ(dfQ)/dfQ, and (2) 9 ~ µ, r- N(Xµ, r2V9 k ' T - N(20,D (3) r Z - X (df7)/dfr where C and V are k x k observed and prior variance-covariance matrices respectively, and the degrees of freedom dfQ and dfT indicate how well C and V~ respectively, are known. (Note that for consistency with notation in (1) we have interchanged o2 and r` relative to [16], in accordance with notation in [15].) Implementation of this Bayesian model requires several initial specifications. We need to specify the matrix C describing within-study variability; here, since the studies are assumed to be independent, we take C as a diagonal matrix with individual logit estimates of variance of Yj on the diagonal. The specification of dfQ reflects our faith in these estimates in C. The average number of exposed cases from each study was used as a conservative estimate of the degrees of freedom dfa, with the average taken over those studies for which 2 x 2 tables are given. Specification of the matrix V and corresponding dfT similarly represents our prior beliefs. For most of the analyses the diagonal elements of V were taken to be the interstudy variability found in the corresponding frequentist analysis. In some cases this choice did not seem to allow the data to be modelled appropriately. In particular when the frequentist model reduced to a fixed effects model the initial choice of the diagonal elements of V were given a very small value (0.001), and then the individual study estimates were found to be almost identical, the posterior mean of 7 2 was large and the posterior distribution of 72 was very severely skewed to the right. To deal with this problem, different values were tried for the diagonal elements of V until it was found that the posterior mean of 72 was nearly one. This -19- 1

I I I I I 1 I 1 I I I I I I we showed that there is substantial indication of publication bias in the set of spousal exposure studies. It is calculated in that paper that the possible impact of this bias is to reduce the combined RR from 1.20 to 1.12 (95% CI of (1.01, 1.24)) using the RE model. Following the same approach of excluding the "high outliers" for which there appear to be no matching "low outliers", due perhaps to publication bias, we also get a combined RR of 1.11 (95% CI of (0.99, 1.24)) using the Bayesian analysis. Thus as much as 45 % of the observed excess risk could be due to publication bias. We must also note that there is a further well-known bias in the spousal studies, due to misclassification of smokers as non-smokers, which spuriously elevates the observed relative risk in this context. In using the results calculated here we should adjust both the overall point and interval estimates to allow for such a bias. Relevant methods were first developed in [81] and have been adopted widely [59, 17, 18, 44, 74]. The true extent of misclassification bias has been debated vigorously [44, 46, 45, 80, 79, 78, 18, 751; we will not repeat the arguments here. This systematic bias must be accounted for in a meta-analysis as in the analysis of a single study. Applying, say, the EPA Draft Review [17] estimate of an overall "spurious excess risk" of 0.12 due to this misclassification of smokers as nonsmokers to the estimates based on the RE model in Table 4, we would derive an estimated combined risk for all females reduced from 1.20 to 1.08, and the associated CI would also fall to around (0.96, 1.24). Based on the fmal EPA Report [18] in which adjustment for misclassification is made to individual studies prior to meta-analysis, correction of the combined estimate for this bias would not be as severe. It is interesting to note that an adjustment of around 0.10 would bring the spousal exposure relative risk to around the same level as that of the relative risk currently estimated for workplace exposure. 4.4 Overall comments Meta-analysis is often used simply to increase statistical power: that is, in effect to narrow the confidence limits around an estimate of effect, even if results are fairly consistent and clearcut. It can be used to greater advantage, however, in situations for which individual outcomes are difficult to interpret-- and is has become increasingly popular for this purpose [61]--or when excess risks are small or not significant in each study alone. It is important to realize that the impact of choice of method, selection of studies to be combined, and evaluation of bias, can be substantial, as we have seen in this one example. There are many problems with meta-analysis as a tool. We try to combine studies with different designs, of different quality, and from different areas. There may be consistent biases, either upward or downward, and these will flow from individual studies to an overall assessment. We have noted that in this data-set (and even more in the earlier analysis before the Fontham study [20] was released) the method of analysis may inflate the excess risk estimate by 20%; publication bias may account for almost 50% of the observed excess risk; addition of studies from other geographic areas may raise the U.S. excess risk by more than 100 %, and so their appropriateness needs careful consideration; misclassification -17- 1

I 42. Lam, T.H.; Kung, I.T.M.; Wong, C.M.; Lam, W.K.; Kleevens, J.W.L.; Saw, D.; Hse, C.; Seniveratne, S.; Lam, S.Y.; Lo, K.K. and Chan, W.C. Smoking, passive smoking and histological types in lung cancer in Hong Kong Chinese women. Br. J. Cancer, 6:673-678, 1987. 43. Lam, W.K. A Clinical and Epidemiological Study of Carcinoma of Lung in Hong Kong. PhD thesis, M.D. Thesis submitted to University of Hong Kong, 1985. 44. Lee, P.N. Misclassification of Smoking Habits and Passive Smoking: a Review of the Evidence. Springer-Verlag, Berlin, 1988. 45. Lee, P.N. Lung cancer and passive smoking (continued): Letter to the Editor. British J. Cancer, 64:200, 1991. 46. Lee, P.N. Lung cancer and passive smoking: Letter to the Editor. British J. Cancer, 63:161- 162, 1991. 47. Lee, P.N. Environmental Tobacco Smoke and Mortality. Karger, Basel, 1992. 48. Lee, P.N. An assessment of the epidemiological evidence relating lung cancer risk in never smokers to environmental tobacco smoke exposure. In Environmental Tobacco Smoke, pages 28- 70. Springer-Verlag, New York, 1993. ed. H. Kasuga. 49. Lee, P.N. Lung cancer and ETS: Is the epidemiologic evidence conclusive? In Joint Statistical Meetings in the ASA, IBS, SSC and IMS, August 1994. Toronto, Canada. 50. Lee, P.N.; Chamberlain, J. and Alderson, M.R. Relationship of passive smoking to risk of lung cancer and other smoking-associated diseases. Br. J. Cancer, 54:97-105, 1986. 51. Light, R.J. and Pillemer, D.B. Summing Up: the Science of Reviewing Research. Harvard Univ. Press, 1984. 52. Liu, Q.; Sasco, A.J.; Riboli, E. and Hu, M.X. Indoor air pollution and lung cancer in Guangzhou, People's Republic of China. Amer. J. Epidemiol., 137:145-154, 1993. 53. Liu, Z.; He, X. and Chapman, R.S. Smoking and other risk factors for lung cancer in Xuanwei, China. Im. J. Epidemiology, 20:26-31, 1991. 54. Longnecker, M.P.; Berlin, J.A.; Orza, Michele J. and Chalmers, Thomas C. A meta-analysis of alcohol consumption in relation to risk of breast cancer. JAMA, 260:652-656, 1988. 55. Mengersen, K. and Besag, J. Ranking and selection using MCMC. In Proceedings of the 3rd Schwerin Conference in Mathematical Statistics, Germany, September 1993. I I I I I I I I I I I I I I w -24.- ~ ' ,

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I I I I I I I I I I I , I I I I I 28. Hedges, L.V. and I. O1kin. Statistical Methods for Meta-analysis. Academic Press, 1985. 29. Hirayama, T. Non-smoking wives of heavy smokers have a higher risk of lung cancer: a study from Japan. British Medical Journal, 282:183-185, 1981. 30. Hirayama, T. Lung cancer in Japan: Effects of nutrition and exposure to ETS. In Lung Cancer: Causes and Preventions, pages 175-195. Verlag Chemie Weinheim, 1984. 31. Holcomb, Larry C. Impact of environmental tobacco smoke on airline cabin air quality. Environ. Technol. Letters, 9:509-514, 1988. 32. Hole, D.J.; Gillis, C.R.; Chopra, C. and Hawthorne, V.M. Passive smoking and cardiorespira- tory health in a general population in the west of Scotland. Br. Med. J., 299:423-427, 1989. 33. Humble, C.G.; Samet, J.M. and Pathak, D.R. Marriage to a smoker and lung cancer risk. Am. J. Public Health, 77:598-602, 1987. 34. Inoue, R. and Hirayama, T. Passive smoking and lung cancer in women. In M. Aoki, S. Hisamichi, and S. Tominaga, editors, Smoking and Health 1987, Proceedings of the 6th World Conference on Smoking and Health, Tokyo, 1988. 35. Janerich, D.T.; Thompson, W.D.; Varela, L.R.; Greenwald, P.; Chorost, S.; Tucci, C.; Zaman, M.B.; Melamed, M.R.; Kiely, M. and McKneally, M.F. Lung cancer and exposure to tobacco smoke in the household. New England J. Med., 323:632-636, 1990. 36. Joeckel, K.-H. Passive smoking--evaluation of the epidemiological findings (in German). VDI Reports 888, Association of German Engineers, 1991. Commission on Air Pollution of the VDI and DIN, Carcinogenic Substances in the Environment. 37. Kabat, G.C. Epidemiologic studies of the relationship between passive smoking and lung cancer. In Proc 1990 Toxicology Forum, pages 187-199, 1990. 38. Kabat, G.C. and Wynder, E.L. Lung cancer in nonsmokers. Cancer, 53(5):1214-1221, 1984. 39. Kaladidi, A.; Katsouyanni, K.; Voropoulou, N.; Bastas, G.; Saracci, R. and Trichopoulos, D. Passive smoking and diet in the aetiology of lung cancer among non-smokers. Cancer Causes and Control, 1:15-21, 1990. 40. Koo, L.C.; Ho, J.H.; Saw, D. and Ho, C. Measurements of passive smoking and estimates of lung cancer risk among non-smoking Chinese females. Int. J. Cancer, 39:162-169, 1987. 41. Lam, T.H. and Cheng, K.K. Passive smoking is a risk factor for lung cancer in never smoking women in Hong Kong. In Smoking and Health, 1987, Proceedings of the 6th World Conference on Smoking and Health, Tokyo, 1988. Eds. Aoki, M. and Hisamichi, S. and Tominaga, S. N 0 00 ~ V 00 23- W N W 0 I

I I I I I I I I I I I I I I I I I I 218 Exposure rate (cases) 74.7 % 292 143 Exposure rate (control) 49.0 % 292 .49 (3.78 - 1) PAR % = ------------------------ 57.7% (according the exposure rate of control) .49(3.78-1)+1 .747 (3.78 - 1) PAR % = _ ----.747----(3----.78------1)---+--1-- 76.5 % (according the exposure rate of disease) The OR for active smoking in males and females are shown in Table 3. Table 3. OR For Active Smoking in Males and Females Cases Smokers Nonsmokers 29 OR (m) _ ---- = 5.8 5 Controls. (female) Smokers.. Nonsmokers Smokers 63 29 53 73 5 2 22 45 73 OR(t)= --- = 3.32 22 95%CI=2.59-12.44 Exposure rate (cases) = 92.9% Exposure rate (controls) = 68.7% .929 (5.8 -1) PAR%= - -- --- .929 (5.8 -1) + 1 .687 (5.8 -1) PAR%= ------------------------ 95 % CI = 1.96 - 4.42 Exposure rate (cases) = 65.3% Exposure rate (cont.) = 38.9% .653 (3.32 -1) = 81.7% PAR%= - .653 (3.32 -1) + 1 .389 (3.32-1) = 76.7% PAR%= ------------------ = 47.4% -3- '

I I I 6. I 57. I 58. I 59. I 60. I 61. I 62. I 63. 64 _1 . I 65. I 66. I 67 I . 68. I 69. I I I Mengersen, K.L.; Tweedie, R.L. and Biggerstaff, B.J. The impact of method choice in meta- analysis. Australian J. Statistics. (Accepted subject to review). Mosteller, F. and Chalmers, T. Some progress and problems in meta-analysis of clinical trials. Statistical Science, 7:227-236, 1992. NRC Committee on Applied and Theoretical Statistics. Combining Information: Statistical Issues and Opportunities for Research. National Academy Press, Washington, 1992. NRC Committee on Passive Smoking. Environmental Tobacco Smoke. Measuring Exposures and Assessing Health Effects. National Academy Press, Washington, 1986. Oldaker, G. B. III and Conrad, F. C. Jr. Estimation of effect of environmental tobacco smoke on air quality within passenger cabins of commercial aircraft. Environ. Sci. Technol., 21, No. 10:994-999, 1987. Olkin, I. Meta-analysis: methods for combining independent studies. Statistical Science, 7:226, 1992. OSHA. Proposed Rule on Indoor Air Quality. Federal Register, 59 (65):15968-16039, 1994. Pershagen, G.; Hrubec, Z. and Svensson, C. Passive smoking and lung cancer in Swedish women. Am. J. Epidem., 125:17-24, 1987. Sato, T. Confidence limits for the common odds ratio based on the asymptotic distribution of the Mantel-Haenszel estimator. Biometrics, 46:71-80, 1990. Shimizu, H.; Morishita, M.; Mizuno, K.; Masuda, T.; Ogura, Y.; Santo, M.; Nishimura, M.; Kunishima, K.; Karasawa, K.; Nishiwaki, K.; Yamamoto, M.; Hismichi, S. and Tominaga, S. A case-control study of lung cancer in nonsmoking women. Tohuku J. Exp. Med., 154B:389- 397, 1988. Siegel, M. Involuntary smoking in the restaurant workplace. JAMA, 270, No. 4:490-493, 1993. Sobue, T.; Suzuki, R.; Nakayama, N. et al. Passive smoking among non-smoking women and the relationship between indoor air pollution and lung cancer incidence--results of a multicentre case controlled study. Gan to Rinsho, 36:329-332, 1990. Spiegelhalter, D.; Thomas, A.; Best, N. and Gilks, W. BUGS Manual 0.30. Technical report, MRC Biostatistics Unit, Cambridge, 1994. Stockwell, H.G.; Candelora, E.C.; Armstrong, A.W. and Pinkham, P.A. Environmental tobacco smoke and lung cancer in never smoking women. In Proc. 24th Ann Meeting Soc. Epidemiologic Research, 1991. -25-

I 70. Svenson, C.; Pershagen, G. and J. Klominek. Smoking and passive smoking in relation to lung cancer in women. Acta Oncologica, 28:623-639, 1989. 71. Thompson, S.G. and Pocock, S.J. Can meta-analyses be trusted? Lancet, 338:1127-1130, 1991. 72. Thompson, S.G. Controversies in meta-analysis: the case of the trials of serum cholesterol reduction. Statistical Methods in Medical Research, 2:173-192, 1993. 73. Trichopoulos, D.; Kalandidi, A. and Sparros, L. Lung cancer and exposure to ETS. Conclusion of Greek study. The Lancet, ii:677-678, 1983. 74. Tweedie, R.L. and Mengersen, K.L. Lung cancer and passive smoking: Reconciling the biochemical and epidemiological approaches. Br. J. Cancer, 66:700-705, 1992. 75. Tweedie, R.L.; Mengersen, K.L. and Eccleston, J.A. Garbage in, garbage out: Can statisticians quantify the effects of poor data? Chance, 7(2):20-27, 1994. 76. Uberla, K. Lung cancer from passive smoking: Hypothesis or convincing evidence? Int. Arch. Occup. Environ. Health, 59:421-437, 1987. 77. Varela, L.R. Assessment of the Association Between Passive Smoking and Lung Cancer. PhD thesis, Yale University, 1987. 78. Wald, N.J.; Cuckle, H.S.; Nanchahal, K. and Thompson, S.G. Response to letter from Dr Lee: Letter to the Editor. British J. Cancer, 64:201, 1991. 79. Wald, N.J.; Nanchahal, K.; Cuckle, H.S. and Thompson, S.G. Response to the letter from Dr P. Lee: Letter to the Editor. British J. Cancer, 63:163, 1991. 80. Wald, N.J.; Nanchahal, K.; Cuckle, H.S. and Thompson, S.G. Lung cancer and passive smoking: Letter to the Editor. British J. Cancer, 61:337, 1990. 81. Wald, N.J.; Nanchahal, K.; Thompson, S.G. and Cuckle, H.S. Does breathing other people's tobacco smoke cause lung cancer? Br. Med. J., 293:1217-1222, 1986. 82. Wang, F.-L.; Love, E.J.; Liu, N. and Dai, X.-D. Indoor air pollution and lung cancer in Guangzhou, People's Republic of China. Int. J. Epidemiol., 23:223-230, 1994. 83. Wolpert, R.L. and Warren-Hicks, W.J. Bayesian hierarchical logistic models for combining field and laboratory survival data. In Bayesian Statistics 4, pages 525-546. Oxford University Press, 1992. Eds Bernardo, J. M. Berger, J. O. Dawid, A.P., and Smith, A.F.M. 84. Wu, A.H.; Henderson, B.E.; Pike, M.C. and Yu, M.C. Smoking and other risk factors for lung cancer in women. JNCl, 74:747-751, 1985. -26- 1 I I I I I I ' I I I I I I I I I I

I I I I I I I I I I I I I I THE RELATIONSHIP BETWEEN SMOKING AND LUNG CANCER IN HUMANS Geng Guan-yi, Liang Zhong-hua, Xu Rui-heng, Liu Jing-ying and Shi Pei-ying Tianjin Medical University, Tianjin, China Abstract To attempt to clarify the relationship between smoking and lung cancer in humans, a case-control study was conducted. Ninety-nine male and 193 female lung cancer cases were included, matched 1:1 with controls on the basis of age, sex and area of residence. In males, the OR associated with smoking was 5.9 (95 % CI 2.65-13.50); ORs increased with the amount smoked. The average age of starting smoking was earlier for cases (22.4 years) than for controls (24.7 years). The OR for smoking was 3.31 (95% CI 1.96-4.42) in female cases. About 60% of cases were considered to be attributable to active smoking. The OR for lung cancer in a nonsmoking wife married to a smoking husband was 2.16 (95 % CI 1.03-4.53). 42 % of lung cancers in nonsmoking women were considered to be attributable to husband's smoking. ORs for passive smoking increased with the amount smoked by the husband, and with the length of time spent living with the husband. In total, about 60% of female lung cancer cases are believed to be attributable to active or passive smoking. The OR associated with occupational exposures was 3.1 (95% CI 1.58-6.02). The OR for cooking with coal (1x10° hours of exposure) was 1.54 (95% CI 1.20-1.96) and was 5.56 (95% CI 3.40- 9.10) for 4x10° hours of exposure, or approximately three hours per day for 37 years. The OR for pulmonary disease was 2.64. In conclusion, most lung cancer cases could be attributable to active or passive smoking in this population. Introduction Considerable controversy exists on the relationship between smoking and lung cancer, especially among females. Among all major cities in China, mortality rate of lung cancer in Tianjin is high; ranking second in males and first in females (28.3/105). We conducted a 1: 1 match case-control study to further investigate the relationship between smoking and lung cancer. Materials and Methods A total of 292 cases of lung cancer (99 male, 193 female) were analyzed. Of these, the majority p , (255 cases, 87.3 %) were confirmed histologically or cytologically. The other 37 cases were diagnosed ~ ~ to be lung cancer by CT, X-ray or bronchoscopy. All cases involved individuals residing in Tianjin for -4 more than 10 years. Cases were matched 1:1 with 292 controls, by age (± 2 years), sex, race, marital w w 0) I

I I status and location of home residence. The distribution of lung cancer histologic type is illustrated in Table 1. Table 1. Cell Type of Lung Cancer Cases Male . I pemate Cell Type No. % No. Group I(squamous cell and small cell) 42 42.4 83 43.0 Group II (adenocarcinoma) 32 32.3 73 37.8 Type unknown 25 25.3 37 19.2 Total 99 100.0 193 100.0 Results Age and residence distribution of the cases in this study is similar to other lung cancer studies conducted in Tianjin. The smoking rate of the controls is also typical of the population in Tianjin. Sutmnary of findings pertaining to active smoking The OR for active smoking and lung cancer is shown in Table 2. Table 2. OR of Active Smoking Controls Cases Smoker Nonsmoker Smoker Nonsmoker 116 102 27 47 102 3.78 OR 27 (102 - 27)2 ------------ 102 + 27 43.6 95%CI=2.17-4.30 P < 0.01 I I I I I I I I I I I I I O O V 0o -2- W N W V 1 I

.687 (5.8 -1) + 1 .389 (3.32 -1) + 1 I I I The OR for amount of smoking is shown in Table 4. I Table 4. OR and Amount of Smoking I No. of Cig./day OR (male) OR'(femaIe) I 0 1 1 I l- 1.66 1.47 10- 2.98 2.52 20- 14.78 6.27 I 30- 27.72 The OR for duration of smoking is shown in Table 5. I Table 5. OR for Duration of Smoldng I Year of smoking OR 95 % Cl I 0 1 1- 1.73 1.38 - 2.18 I 20- 3.00 2.17 - 4.16 40- 5.20 3.49-7.75 In analyzing the age at which smoking began, cases were shown to st controls (Table 6). art smoking earlier than I I ' ~ 4- i V N W tn I I

I 1 I I I I I I I I I I I I I I I I Table 6. Age in Which Smolting Began Male Female Cases 22.4 18.9 Controls 24.7 28.2 P < 0.05 < 0.01 OR was found to be higher among those who started to smoke at an earlier age (Table 7). Table 7. OR and Age Smolting Began Age starting to smoke OR Nonsmoker 1 _ 21 1.59 16-20 3.1 < 15 6.30 ~.61 In summary, the OR increased with the amount and with duration of smoking. Moreover, cases were shown to begin smoking at an earlier age than the controls. Summary of Studies in Nonsmoking Females Pertaining to Exnosure to Passive Smoke The OR of nonsmoking female cases being exposed to passive smoke from parents, siblings and colleagues was not significantly higher than the nonexposed group. However, exposure to smoke from husbands resulted in an elevated OR (Table 8). Table 8. OR of Passive Smoking in Females -5- I

I I 34 x 52 OR = ---------- = 41 x 20 2.16 95 % CI = 1.03 - 4.53 Exposure rate(cases) _ Exposure rate (control) = 34 34 + 20 41 63 % 44.1 % 41 + 52 .63(2.16-1) .441(2.16-1) PAR % = ----------------------- = 42.5 % PAR % _ ----------------------- = .63 (2.16 - 1) + 1 .441(2.16-1)+1 42.5 % Table 9 shows that OR of female lung cancer increased with number of cigarettes smoked per day by their husbands and with duration of exposure. Table 9. OR of Duration and Amount of Spousal Smoking Amount OR 95% CI Duratlon. OR of exp. of exp. (Yr) 95% CI- 0 (cig/d) 1 0 1 1- 1.4 1.12 - 1.76 1- 1.49 1.15 - 1.94 10- 1.97 1.42 - 2.72 20- 2.23 1.54 - 3.22 20- 2.76 1.85 - 4.10 40- 3.32 2.11 - 5.22 OR for active and passive smoking in relation to female lung cancer cases is shown in Table 10. Table 10. OR of Active and Passive Smoking Wives . .... --------------- - -- - -- - -- - -- - ---- --------- Nonsmoking Smoking Nonsmoking 1.0 3.32 (1.96 - 4.42) Husband Smoking 2.16 (1.03 - 4.53) 4.90 (1.8 - 9.5) -6- N O O i V 00 Gf N ? ~ ' I I I I I I I I I I ' I ' I I I

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a I I I I I I I I I I I I I I I I I If a smoking female has a husband who also smoked, the OR was 4.9. The exposure rate was 64.9% (137/211). PAR %=[0.649(4.9-1)] [0.649(4.9-1)1/ [0= 71.7%. For the 126 smoking female lung cancer cases 90.34 (126 x 0.717), and for the 67 nonsmoking female lung cancer cases 22.64 (67 x 0.338) were due to passive smoking. Therefore in Tianjin 58.5% (113/193) of female lung cancer may be attributed to smoking or passive smoking. Other Risk Factors for Lung Cancer in Females: Occupational Exposure OR of occupational exposure, e.g., textile workers, workers exposed to asbestos, benzene, etc., was 3.1, 95% CI = 1.58 - 6.02. Since the exposure rate was low in the Tianjin population (about 5%), the PAR% attributed to the occupational exposure was only about 9.5% and is much less than that due to smoking. Exposure to Cooking With Coal These results are shown in Table 11. Table 11. OR of Female Lung Cancer Due to Cooking With Coal Duration of Exposurc (hr) OR ° 95% CI 1 x 1 (1.5 r x 20 yr) 1. - 1. 2 x 10° (1.5 hr/d x 40 yr) 2.36 (1.66 - 3.34) 3 x 104 (2 hr/d x 42 yr) 3.62 (2.34 - 5.55) 4 x 10° (3 hr/d x 37 yr) 5.56 (3.40 - 9.10) The OR for exposure to coal fume for 2 x 104 hr, i.e., 1.5 hr per day for 40 years, was 2.36 which was nearly equal to the OR of passive smoking for 20 years. It is, however, much less than the OR for active smoking in females (3.32). Interactive Effects of Risk Factors for Lung Cancer in Females Multifactor analysis by conditional logistic regression method showed that the combination of active smoking, passive smoking, occupational exposure, history of lung disease and 4 x 10^ hr cooking with coal resulted in an OR of 50, 95 % CI = 13.7-185.3, in comparison with those without the above risk factors and cooking with coal for less than 3 x 10^ hrs. -7- I

I Discussion Due to the low rate of smoking among females, it seems likely that risk factors other than cigarette smoking exists for lung cancer. Because smoking rate is very high in men in all areas of China, it is conceivable that there is high exposure to passive smoke among females. A number of studies have addressed the effects of exposure to passive smoke, especially in relation to the incidence of lung cancer in nonsmoking females; the results, however, have been inconsistent and at times, contradictory, between studies. Hirayama showed data that suggest that the incidence of some cancers may be elevated in nonsmoking wives with smoking husbands than those with nonsmoking husbands. Similar results were obtained in our studies. Although cooking with coal (exposure to coal fumes) was one of the risk factors for lung cancer, exposure is expected to gradually decrease with the increased use of gas. The risk associated with occupational exposure is also expected to be small. These considerations, therefore, raise the possibility that the primary risk factors for female lung cancer are active and passive smoking (58.5 % female lung cancer was attributed to smoking). However, in the absence of actual exposure data in other areas, such conclusions must await further investigation in the future in other places in China. The PAR% of male lung cancer attributable to smoking was 76.7- 81.7, consistent with the notion that smoking was the main cause of male lung cancer. In conclusion, both active and passive smoking are important risk factors of lung cancer in Tianjin. The interactive effect among some risk factors increased the OR substantially. Therefore stopping smoking might significantly decrease the OR and conceivably also the incidence of lung cancer. While most other risk factors has a lower OR compared to smoking, the combined OR can also increase quite significantly if other risk factors are allowed to be combined with smoking. -8- I I I I I I I I I I I I I I I

I I I I I I I I I I I I I I SOME LIFESTYLE FACTORS IN HUMAN LUNG CANCER: A CASE-CONTROL STUDY OF 792 LUNG CANCER CASES Lei Yi-xiong, Chen Yong-zhong and Du Ying-xiu Department of Hygiene, Guangzhou Medical College, Guangzhou, China Abstract Eating habits and living conditions are fundamental lifestyle factors. Likewise, cigarette smoking, exposure to environmental tobacco smoke (ETS) and other indoor air pollutants are also commonly encountered in many societies. Many studies have examined the possible effects of these factors on the incidence of lung cancer and often produce different and at times conflicting results and conclusions. In order to investigate a relationship between some lifestyle factors and lung cancer, a case-control study involving all lung cancer deaths registered in 1986 was performed. The results show that among males, 92% of the cases and 76% of controls were smokers, implying that cigarette smoking is a primary risk factor for lung cancer in males. By contrast, among females only 60% of the cases and 30% of the controls were smokers, implying factors other than cigarette smoking must be involved in the development of lung cancer in females. As for the exposure to ETS, our results show that the risk of lung cancer in nonsmoking females was not increased by exposure to ETS. The studies of diet and eating habits show that the intake of vegetables and fruits could reduce the risk of lung cancer in males. Similar results have been reported elsewhere. Our studies also show that in the case of males the incidence of lung cancer was significantly increased in those that have a frequent intake of fried food. The positive association established from the studies between the intake of fried food and the risk of lung cancer could result from cooking practices and from inappropriate methods used in food preparation. With regard to the question of high protein diets, high fat diets, salty food and smoked food and their relationship to lung cancer incidence, different results have been observed. Our results show that no association can be demonstrated between the intake of foods mentioned and the incidence of lung cancer. Thus, it is not likely that lung cancer inducing carcinogens can be generated through the intake of food. ~ In addition, the positive association found to exist between the living index and the risk of lung cancer in females indirectly points to coal smoke or cooking practices generating indoor air pollutants and thereby contributing to a risk of lung cancer in females. However, many chemical carcinogens such as . B(a)P are known to be inducers for squamous cell carcinoma of the lungs, but in the case of females, , adenocarcinoma is found to be the predominant cell type. Therefore, the effect of lung air pollution on the incidence of lung cancer needs to be further investigated. ~ ~ 00 i V Do W ~ 1 A I

I Introduction Among all types of cancers, lung cancer is responsible for the highest death rate in the cities in many parts of the world. Although some understanding of the etiology of lung cancer has been gained, including the effects of cigarette smoking, our current knowledge still cannot adequately explain many observations about lung cancer. For example, the smoking rate is not necessarily lower for the peasants than for city dwellers, yet the peasants are known to have a lower lung cancer incidence than the urbanites. Conversely, although there are far fewer smokers among women than among men, the female lung cancer death rate is quite high, suggesting that there are other risk factors at play beside smoking. In the city of Guangzhou, the lung cancer mortality rate has risen dramatically in the decade between 1976 and 1986. For instance, the standardized mortality rate (SMR) for lung cancer increased from 25.6/100,000 (31.9 in males and 18.8 in females) in 1976 to 40.3/100,000 (55.8 in males and 23.9 in females) in 1986, representing an average annual increase of 1.57/100,000 over the 10-year period. To investigate the relationship between some lifestyle factors and lung cancer, a case-control study involving all lung cancer deaths registered in Guangzhou in 1986 was undertaken. The lifestyle factors surveyed and analyzed included active smoking, passive smoking, diet, living conditions, kitchen facilities, and exposure to coal smoke. Materials and Methods 1. Study population. All primary lung cancer deaths in Guangzhou in 1986 were investigated retrospectively in a case- control study. The cases were drawn from the 1986 lung cancer death records, routinely maintained by the local police stations. Deaths unrelated to primary lung cancer, or of those with less than 10-years' residency were excluded. Controls were selected from those with the same year of death, residence on the same street as the cases, but without a history of respiratory diseases or tumors and 1:1 matched for sex, age ( f 5 years). The reason for the selection of nonrespiratory or nontumor patients as controls was to exclude latent cases. The reason for the same-street residency was to exclude the potential confounding by outdoor air pollution. In 1986 there were 831 lung cancer deaths in Guangzhou, 792 of which were matched with controls (a 95.3% match from the total). They included 563 male pairs and 229 female pairs. 2. Method of investigation. Home interviews with spouses or cohabitating relatives of the decedents were conducted by trained interviewers. The questionnaires were standardized in terms of content, order and style and taken by the same interviewer from each pair of cases and controls and confirmed by data from existing hospital files. 3. Survey Contents. In addition to routine demographic information about both the cases and the controls, data were also obtained in five specific areas: -2- I I I I I I I I I I I I I I I I

 , I I I I I I I I I I 5. Diet. Food categories analyzed included: pork, beef, poultry, fish, egg and dairy products, leafy and nonleafy vegetables, fruits, fried food, preserved vegetables, salt-preserved fish and smoked and cured foods. The results show that the consumption of fresh vegetables and fruits have protective properties against lung cancer in males. On the other hand, fried food may contribute to the risk of lung cancer in males. No differences were shown for the remainder of the food categories. (Table 5) When data were stratified into "never" or "frequent" (almost daily) intake groups for comparison, then the correlations between frequent intake of vegetables and fruit and the decrease of male lung cancer rate became more pronounced. (Tables 6, 7) N 0 ~ 1 °° V 00 W ~ -7- C~Ir i ,

(B) Analysis of compatibility: The demographic characteristics obtained on cases and controls were first subjected to the X1 Test to ascertain compatibility. These include father's place of birth (Guangzhou or other); marital status (single, married, divorced, widowed, separated); education (illiterate, primary school, middle school, high school, technical school, or college education); occupation (professional, government official, clerk, businessman, service personnel, manual laborer, and others). (C) Stratification and analysis of factors: For bipartite variables the X2 value was calculated by the McNemar method. The odds ratio (OR) with a 95% CI were determined according to Miettinen. To obtain information on total exposure; the cumulative X2 was calculated by the RXC table for pooled theoretical value method and OR were calculated. 1. Quality of survey data. Results and Analysis Data from 272 samples, i.e. 8 pairs of cases and controls and 17 risk factors were taken by investigators A and B. As shown in Table 1 the data were accurate and reliable. Table 1. 272 Samples of Risk Factors Taken By Investigators A & B Investigator B Investigator A Yes No Total Yes 99 15 114 No 10 148 158 Total 109 163 272 Result of Kappa Test: K = 0.81, P < 0.001 2. Test of Balance. Uniformity exists for cases and the controls in sex, age of death, and street address. The distribution of the other demographic characteristics of the cases and the controls include father's place of birth, marital status, education, and occupation which was also distributed uniformly. No difference was found by the test of balance, which demonstrated good compatibility between the two groups (Table 2). -4- I I I 1 I I I I I I I I I I I I

Table 3. Distribution of Smoking Index in Cases and Controls Smoking Index Cases Controls OR 95% CI Tests M: 0 41 123 1.00 1.00-1.00 Test of hypothesis < 400 57 93 1.84 1.24-3.26 X2 = 77.71, P< 0.001 400-799 136 122 3.34 2.72-5.60 Test of Trend > 800 250 146 5.36 3.60-7.93 X2 = 77.33, P< 0.001 F: 0 85 147 1.00 1.00-1.00 Test of hypothesis < 400 29 26 1.93 1.70-3.02 X2 = 44.68, P< 0.001 400-799 33 16 3.57 2.45-5.11 Test of Trend > 800 61 19 5.55 3.21-7.22 X2 = 43.92, P< 0.001 Note: Smoking index = daily smoking rate times total years of smoking. 4. Passive smoking. To determine the effects of the husbands' smoking on their wives, the following factors were taken into consideration: spousal smoking, daily smoking rate, and years of smoking. These factors were examined to evaluate the effects of exposure to husbands' smoking on nonsmoking wives which are compared in both case and control groups in Table 4. The results did not show a significant relationship between the husbands' smoking status, or the daily number of cigarettes smoked, or the number of years of smoking and the occurrence of lung cancer in their wives. Table 4. Distribution of ETS Exposure Among Nonsmoking Cases and Controls Cases Controls OR 95% Confidence Level ETS exposure No 28 53 Yes 47 75 1.19 0.66-2.16 X2 =0.327 P> 0.05 Amount of exposure to ETS 0 28 53 1.00 1.00-1.00 (number of cigarettes) < 20 13 34 0.72 0.53-0.98 > 20 30 35 1.62 1.03-2.55 X2 = 4.308 P > 0.05 Length of exposure to ETS 0 28 53 1.00 1.00-1.00 (years) < 30 14 19 1.39 0.63-1.60 > 30 29 47 1.17 0.80-1.25 X2 = 0.652 P > 0.05 -6- I I I I I I I I I I I I

I I I I I I I I I I I I I I I I I I (A) Smoking history: Specifically, daily smoking rate (cigarette/day), age at which the person began to smoke, years of smoking, and smoking index (daily smoking rate times years of smoking). The smoking index was divided into four categories: 0, <400, 400-799, >800. In our analysis, we used the smoking index to compare the effects of smoking on the cases and the controls. (B) Exposure to ETS: In order to assess the effects of exposure to environmental tobacco smoke (ETS), the effect of active smoking must be first excluded. Since 92% of the male lung cancer cases were active smokers and since the workplace exposure to ETS cannot be accurately determined, the present survey concerned itself only with the effects of exposure to spousal smoking in non-smoking females. The daily cigarette smoking rate and the years of smoking by the husbands were used in the statistical analysis. (C) Diet history: Survey items included the consumption of pork, beef, poultry, fish, eggs, milk and dairy products, leafy and nonleafy vegetables, fruits, fried food, pickled vegetables, salted fish and smoked and cured foods. The consumption of these food items was stratified into the categories of: never, weekly, and daily. (D) Living conditions: Data concerning the following were obtained: old or new building; location of residence within the building (ground level, second, third, or fourth floor and above); interior dimensions including ceiling height (6, 9 or 12 feet) and average size of living area per person (18, 36, 54, 72 square feet per person); ventilation (excellent: 1; medium: 2; poor: 3); and use of insect repelling incense (never, occasional, average, and frequent). (E) Kitchen facilities and exposure to coal smoke/dust: Information obtained included average kitchen size (< 9, 9-18, > 18 square feet) and the type of cooking fuel used (coal, propane, wood). Information was also obtained regarding exposure to benzo(a)pyrene as pollutants generated by frying food, and the preference for cooking by frying. 4. Methods of analysis. (A) Quantitative data analysis. To estimate the reliability of the information collected, 1% of the total sample was randomly resurveyed. Consistency of data between the first and second survey measurements was evaluated by the Kappa test using the following equation. Po-Pe K = where 1 -Pe Po = consistency of observation agree. Pe = the value expected based on consistency obtained solely by chance. The significance of the Kappa test was evaluated by the Fleis 3-level assessment: 0.75..,1.00 excellent p co concordance 4 A 0 75 avera e concordance 0 01 A 3 l ki d ; . . g ; . .. . ac ng concor ance. i -4 CO W 3- A V I

I I I I I I I I I I I I I I I I I 1 Table 9. Analysis of Living Conditions Index Male Female Living Condition Index Cases Controls OR 95% CI Cases Controls OR 95% CI 8 or more 100 104 1.00 1.00-1.00 25 46 1.00 1.00-1.00 4 or more 126 112 1.17 0.89-1.54 64 46 2.56 1.394.70 1 or more 288 304 0.99 0.97-1.01 111 108 1.89 1.25-2.85 Male: X2 = 1.276 P > 0.05 Female: X2 = 9.199 P < 0.05 7. Kitchen facilities and exposure to coal fume and dust. For residents of Guangzhou, wood was the main fuel source in the 1950s. Coal began to be used as a fuel in the 1960s, and its usage has increased since that time. Beginning in the 1980s individual families began to use propane gas. The investigation of kitchen facilities, coal fumes and dust exposure in the current study showed the following: 91.9% of the families used coal as the major source of fuel during the past 20 years (with 46.2 % of them using wood simultaneously); only 4.6 % used propane and 3.5 % used other fuels. There was no significant difference between the cases and controls with respect to the type of fuel used. Although there was an apparent difference in the cooking activities between the male and female groups (37% of males and 86% of females regularly participated in cooking), no obvious difference was detected between cases and controls within each sex group. Neither was any appreciable differences found between the cases and the controls with regard to kitchen space, years of regular cooking activity, exposure to coal smoke/dust and whether frying was generally the preferred style of cooking. (Table 10) N 0 00 , V tb W -11- N UT I

Table 8. Distribution of Living Conditions of Cases and Controls Male Female No. % No. % No. % No. % of Cases of Controls of Cases of Controls Home New building 255 47.1 248 45.8 91 41.7 83 38.1 characteristics Old building 286 52.9 ~ 293 54.2 127 58.3 135 61.9 XT---7-0-182 P> 0.05 Xrt-::--0.612 P> 0.05 Floor within the Istfloor 220 45.4 211 43.5 78 42.6 88 48.1 building 2nd floor 129 26.6 132 27.2 51 27.9 50 27.3 3rd floor 64 13.2 68 14.0 29 15.8 27 14.8 4th floor and 72 14.8 74 15.3 25 13.7 18 9.8 above XT-=-0.371 P> 0.05 X-=-1.823 _P> 0.05 Ceiling height 2 or more 35 7.1 23 4.7 9 4.3 17 8.2 (M) 3 or more 302 61.3 310 62.9 119 57.2 107 51.4 4 or more 156 31.6 160 32.5 80 38.5 84 40.4 X2-=-2 638 P> 0.05 XT-=-3 196 P> 0.05 Living space 2 or more 165 30.3 137 25.1 58 26.5 66 30.1 (M2lperson) 4 or mom 122 22.4 132 24.3 59 26.9 50 22.8 6 or more 97 17.8 103 18.9 34 15.5 37 17.0 8 or more 160 29.4 172 31.6 68 31.1 66 31.1 XT 3.603 P> 0.05 XX = 1.416 P> 0.05 Room ventilation Good (I) 226 41.1 228 41.5 73 33.8 77 35.6 Medium (2) 208 37.8 230 41.8 93 43.1 87 40.3 Poor(3) 116 21.1 92 16.7 50 23.1 52 24.1 X2---3 883 P> 0.05 X7 = 0.346 P> 0.05 Burning of insect Never 193 34.9 205 37.0 97 43.9 75 33.9 repellant incense Occasional 168 30.4 150 27.1 57 25.8 72 32.6 Average 100 18.1 108 19.5 35 15.8 42 19.0 Frequent 92 16.6 91 16.4 32 14.5 32 14.5 XZ = 1.693 P> 0.05 XT= 5.195 P> 0.05 -10- i I I I I I I I I I I I

I I I I I I I I I I 1 I I ~ I I I I Table 6. Analysis of Fresh Vegetable Intake by Male Cases and Controls Cases Controls Not Regularly Regularly Total Not regularly 0 8 8 Regularly 30 518 548 Total 30 526 556 OR = 3.75 95 % CI 1.75-8.00 X2 = 11.605 P < 0.001 Table 7. Analysis of Fruit Intake by Male Cases and Controls Frequency Cases Controls Total Not regularly Regularly 78 125 92 250 170 375 Total 203 342 545 OR 1.36 95 % CI 1.04-1,78 X2 = 5.018 P < 0.05 6. Conditions of living quarters. To describe the characteristics of living quarter conditions, the following data were collected: building characteristics, floor within the building, ceiling height, average living space per person, room ventilation, frequency of burning of insect repellant incense. The results showed no obvious differences between the case and the control groups. (Table 8) However, when the living conditions index, i.e. average living area per person (M2/number of persons)/room ventilation (1, 2, 3), was used for a combined evaluation, it was found to be related to female lung cancer (Table 9), i.e., when the living conditions index was low the female lung cancer rate showed an upward trend. N O tb -L V -9- w N Cn W I

I I I I I I I I I I I I I I I I Table 2. Demographic Characteristics of Cases and Controls Male Female Cates Conttols Cases Controls Native province Guangdong Provlnce 465 454 179 182 Province other than Guangdong 32 43 12 9 X2 =1.745 P>0.05 X2 =0.213 P>0.05 Marital status Single 13 22 8 6 Married 476 473 147 139 Divnrced 14 8 1 4 Widowed 28 34 64 67 Separated 12 6 3 7 x2 =6.541 P>0.05 g2=2.158 P>0.05 Bducation Illiterve 47 44 100 107 Grade School 264 249 80 69 Middle Schoul 131 124 23 26 High School & Technical 66 81 15 16 College 36 46 4 4 x2=3.480 P > 0.05 X2 =1.258 P>0.05 Occupxtion Profasional 49 50 19 9 Government official 46 48 2 2 Clerical 32 46 4 4 Other 35 49 38 46 Business 41 56 12 13 Service Personnel 48 40 21 19 Laborer 270 232 75 78 x2 = 10.822 P> 0.05 2 = 4.532 P> 0.05 3. Analysis of smoking history. Among the 563 pairs of male cases and controls, the percentage of smokers was 92.5 % for the cases and 75.5% for the controls. Among the 229 female pairs, the smoking rate was 60.6% for the cases and 30.8% for the controls. The majority of smokers smoked cigarettes (68.2%); the next largest smoker group used roll-your-own cigarettes (28.3%); a few used water pipes (1.9%); and a very small number used pipes and cigars. Though little difference existed in the type of tobacco product used, both the male and the female cases had significantly higher smoking indexes and the test of trend also revealed an obvious dose-response relationship. (Table 3) These results support the view that smoking is an important risk factor in the incidence of lung cancer among residents of Guangzhou. N O -4 , O -4 O W , -5 A tG I

I I I 46. I 47. I I I I I I I I I I I Wang, X.Z. et al. "Experiments of mutagenicity in indoor and outdoor pollutants," Second Chinese Conference on Environmental Health, Nanjing, 1984. Abstracts. Ou, Z.L. eta l. "Relationship of coal-burning and lung cancer in housewives," Proceedings of Second Conference of Cancer Research, Guangzhou, 1987, p. 76-81. 0 , tb i V 0 -19- W N w ,

Table 5. Analysis of Dietary Habits of Cases and Controls Male Female No. % No. % No. % No. % of Cases of Comrola of Cases of Controls. Meat and fish Basically never 12 2.2 18 32 9 3.9 15 6.6 Average frequency 106 19.1 105 18,9 44 19.3 41 18.0 Almost daily 437 78.7 432 77.8 175 76.8 172 75.4 X0234 P> 0.05 X0.632 P> 0.05 Egg and dairy products Basically never 169 30.8 147 26.8 74 33.0 86 38.4 Average frequency 194 35.4 216 39.4 92 41.1 80 35.7 Almost daily 185 33.8 185 33.8 58 25.9 58 25.9 X0721 P> 0.05 X0737 P> 0.05 Leafy vegetables Basically never 30 5.4 8 1,4 2 0.8 3 1.3 Average frequency 45 8.1 54 9.7 23 10.1 25 11.0 Almost daily 481 86.5 494 888 203 89.0 200 87.7 X03.728 P< 001' XZ 0. 192 P> 0.05 Nonleafy vegetables Basically never 41 7.4 28 5.0 10 4.4 8 3.5 Average frequency 141 25.3 154 27.6 63 27.6 60 26.3 Almost daily 375 67.3 375 67.3 155 68.0 160 70.2 X0022 P> 0.05 X 0 375 P> 0.05 Fruiss Basically never 203 37.2 170 31.2 81 36.3 79 35.4 Average frequency 249 45.7 276 50.6 102 45.7 99 44.4 Almost daily 93 17.1 99 18,2 40 17.9 45 20.2 XZ 4.496 P> 0.05 X 0 364 P> 0.05 Fried Faod Basiwlly never 291 53.1 306 55.8 145 65.3 154 69.4 Average frequency 154 28.1 171 31,2 59 26.6 52 23.4 Almost daily 103 18.8 71 13.0 19 8.1 16 7.2 X0151 PcOQ05• X 0830 P>0.05 Prescrvcd vcgcnblcs Basically ncvcr 337 67.5 343 68.7 135 63.7 139 65.5 Average frequency 123 24.6 118 23.6 51 24.1 49 23.1 Almost daily 39 7.8 38 7.6 26 12.3 24 11.3 X1 I70 P> 0.05 X0178 P7-0 .05 Sab-preserved fish Basically never 323 60.0 336 62.5 125 55.6 139 61.8 Avenge frequency 152 28.3 146 27,1 75 33.3 67 38.2 Almost daily 63 11.7 56 10.4 25 11.1 19 8.4 X0789 P> 0.05 X0011 P> 0.05 Smoked and cured foods Basically never 335 62.5 358 66.8 152 69.4 155 70.8 Average frequency 162 30.2 134 25.0 52 23.7 56 25.6 Almost daily 39 7.3 44 8.2 15 6.9 8 3.7 X0713 P> 0.05 X0308 P> 0.05 ' St2tistically significant. 8 I I I I I I I I I , I

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Table 10. Analysis of Cooking Activity and Exposure to Coal Smoke Particles in Cases and Controls Male Female No. % of cases No. % of controls No. % of cases No. % of controls Kitchen space < 1 18 3.5 14 2.7 6 2.8 10 4.7 (M2/household) 1 or more 66 12.8 73 14.2 28 13.1 39 18.3 2 or more 431 83.7 -- 428 83.1 179 84.0 164 77.0 Xz = P> 0.05 X1 -=3 402 P> 0.05 0.863 Cooking activity infrequent 329 63.1 329 63.1 29 14.4 24 11.9 (years) <_ 20 83 15.9 90 17.3 28 13.9 32 15.8 5 40 79 15.2 72 13.8 83 41.1 78 38.6 > 40 30 5.8 30 5.8 62 30.7 68 33.7 XZ= P> 0.05 X2 = 1.171 P> 0.05 0.608 Exposure to coal smoke Regular 196 35.9 186 34.1 177 78.7 181 80.4 Infrequent 350 64.1 360 65.9 48 41.7 44 19.6 X2 = P> 0.05 Xl-= 0 219 P> 0.05 0.403 Cooking by frying Preferred 192 35.2 165 30.2 55 24.4 53 23.6 Average 177 32.4 210 38.5 93 41.3 102 45.3 Not preferred 177 32.4 171 31.3 77 34.2 70 31.1 - - - XT P> 0.05 X7 = 0.786 P> 0.05 4.960 Discussion Eating habits and living conditions are among some of the most fundamental lifestyle factors. Likewise, cigarette smoking, exposure to ETS and other indoor air pollutants are also common elements of daily living. Many studies both in China and in other countries, have examined the possible effects of these factors on the incidence of lung cancer. These studies often produce different, and at times conflicting, results and conclusions for diverse geographical localities. The present investigation has explored the relationship between these lifestyle factors and lung cancer among Guangzhou residents. Sampling error was minimized in this study by targeting the total number of lung cancer deaths in one year, 1986. Confounding by atmospheric pollution and by factors that could influence the latency of lung cancer was also reduced by applying rigid criteria in the selection of controls. -12- I I I I I I I I I I I I I I I I I I I

a I I I I I I I I I I I 1 I I I ' 1. Active smoking. Many studies aimed at understanding the etiology of lung cancer have suggested that cigarette smoking is an important risk factor for lung cancer (1). Results of this study show an obvious dose- response relationship between active smoking and the risk of lung cancer in both males and females, further substantiating the close relationship between smoking and lung cancer deaths in Guangzhou residents and strongly supporting the notion that cigarette smoking is etiologically associated with the pathogenesis of lung cancer (2-7). Of special note is the high smoking rate among males (92% of the cases and 76% of controls were smokers), which implies that cigarette smoking is a primary risk factor for lung cancer in males. By contrast, among females only 60% of the cases and 30% of the controls were smokers. Since as many as 40% of the female lung cancer cases do not smoke, factors other than cigarette smoking are like to be involved in the development of lung cancer in females. 2. Exposure to ETS. Considerable controversy exists regarding the relationship between exposure to ETS and lung cancer. Because the presence of ETS in most environments tends to be highly variable and cannot be accurately measured, this study has used the exposure to tobacco smoke from cohabitating spouses as a more reliable measure for exposure to ETS. Our results show that the risk of lung cancer in nonsmoking females was not increased by exposure to ETS. Moreover, there was no association between the incidence of lung cancer in nonsmoking females and the husbands' daily cigarette consumption and the number of years smoked. These results are in agreement with the report of Chan et al. in Hong Kong (8), Lee et al. in England (9), Wang eta l. in Harbin (10), Wu-Williams et al. in Shenyang (11) and He et al. in Xuanwei (12), a high female lung cancer incidence region. Although a number of studies have suggested a possible etiological link between exposure to ETS and the incidence of lung cancer in nonsmokers, three different schools of thought have emerged with regard to the involvement of ETS. These are discussed below. The first viewpoint is based on the studies of Garfinkel (13), Kabat et al. (14), Kalandidi et al. (15), and Janerich et al. (16), in which the following points were emphasized: (a) Methodological and design difficulties in quantifying ETS exposure and the lack of a dose-response relationship between exposure to ETS and incidence of lung cancer are generally acknowledged. (b) Concerns with conclusions that attempt to link ETS with an increase in the risk of lung cancer are raised. Moreover, the possibility that ETS exposure may be present as a risk factor for lung cancer only applies to situations where husbands are extremely heavy cigarette smokers. (c) There is often the suggestion that additional studies must be performed to more fully explore the effect of exposure to ETS. The second viewpoint is the strong allegation that ETS is a major risk factor for nonsmoking females and induces primarily adenocarcinoma of the lung, without any apparent effect on any other histological types of lung cancer (17-19). In the third school of thought ETS is proposed to be the primary inducer for squamous and small cell carcinoma of the lung (20-22). Since different histological lung cancer cell types are known to have distinctly different etiologies, the last two viewpoints are fundamentally opposite to each other. Delbert et al. (23) analyzed the results of 22 investigations on 108 chemicals which are known to be present in both mainstream and sidestream smoke, and concluded that the concentration of the majority of these chemicals is 66 % higher in sidestream smoke. Such a result may be explained by the -13- I

higher temperature known to exist in mainstream smoke which, therefore, causes a higher decay of the chemicals present. By contrast, Russell eta l. (24), and Reasor eta l. (25), proposed that the exposure to chemicals present in ETS represent only 0.001-1.5% of the chemicals present in mainstream smoke. Given the possibility that the chemical ingredients in ETS is at most 1/70th of that in active smoke and since years of smoking is required before lung cancer develops it seems reasonable to propose that the amount of time required for ETS to induce cancer far exceeds the life expectancy of living systems. In summary, the existing data do not support a relationship between exposure to ETS and lung cancer. To unequivocally confirm or refute a relationship between the two, more studies must be performed. 3. Diet and eating habits. Many epidemiologic and laboratory studies suggest a close relationship between dietary factors, social habits and malignant tumor formation in humans. Among the many factors known to promote tumor formation, 90% of them are thought to be attributed to a variety of environmental factors, with dietary factors accounting for 20-50% of the total (26-27). In addition to the presence of naturally occurring chemical carcinogens in the food, many of the potential carcinogens in food can be derived from contaminations, use of food additives, or as a result of cooking practices. (28) These illustrate the importance of dietary factors in relation to tumor malignancy in humans. Our studies show that the intake of leafy and nonleafy vegetables could reduce the risk of lung cancer in males, suggesting that these types of food could serve as protective factors for lung cancer. Similar results have been reported elsewhere. For example, in Norway, Japan and in the United States there have been many studies showing a relationship between a decrease in vitamin A in food and an increase in the risk of lung cancer (29-31). Hirayama (30) investigated (1965-1975) the dietary habits of 265,118 Japanese cases, aged 40 and above, and found that those that ate vegetables on a regular basis had a lower lung cancer risk by 50% when compared to the infrequent vegetable users. Byers et al. (32) observed that the concentration of vitamin A in vegetables and fruits could reduce the risk of lung cancer in light cigarette smokers or ex-smokers. Bond et al. (33) analyzed the concentration of vitamin A in a variety of foods and pointed out that vitamin A from plant sources may be more important than vitamin A from animal sources. A similar conclusion can be drawn from the present investigation, i.e. a more frequent intake of fresh vegetables and fruits could have beneficial effects in the development of lung cancer. Our studies also showed that, in males, the incidence of lung cancer was significantly increased in those who had a frequent intake of fried food (P < 0.05). Many studies have shown that cooking practices and inappropriate methods of food preparation could result in the formation of many carcinogens, e.g. meat that is overfried can result in the formation of polycyclic aromatic amines (27). Recent studies also showed that exposure of protein rich food to high temperatures, could induce the formation of chemicals that are highly carcinogenic (27). The positive association established from our studies between the intake of fried food and the risk of Iung cancer could have resulted from cooking practices and other inappropriate food preparation methods. With regard to the question of high protein and high fat diets and their relationship to lung cancer incidence, different results have been observed. Hinds et al. (34) observed that high-cholesterol diets increased the risk of lung cancer; by contrast, Byers et al. (32) did not observe such a relationship, but -14- N 0 , I I I I I I I I I I I I

I References IARC Monoeraph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Tobacco Smoking, (Lyon: IARC), Vol. 38, 1986. 2. Ochsner, A. et al. "Primary pulmonary malignancy treatment of total pneumonectomy: Analysis of 79 collected cases and presentation of 7 personal cases," Surg. Gynecol. Obstet. 68: 435, 1939. 3. Doll, R. and Hill, A.B. "Lung cancer and other cause of death in relation to smoking: Second report on mortality of British doctors," Br. Med. J. 2: 1071, 1956. 4. Royal College Physicians of London. Smoking and Health Now, (London: Pitman), 1971. 5. Public Health Service. The health consequences of smoking: A report to the Surgeon General, DHEW Publ. No. (HSM) 71-7513, 1971. 6. Doll, R. and Peto, R. "Mortality in relation to smoking: 20 years observation in British doctors," Br. Med. J. 2: 1525-1536, 1976. 7. Wynder, E.L. "Etiology of lung cancer reflections of decades of research," Cancer 20: 1332- 1339, 1972. 8. Chan, W.C. et al. "Lung cancer in non-smokers in Hong Kong," In: Cancer Camnaifin Grundmann, E. (ed.) (Gustav Fucher Verlag, Sturspart, New York), vol. 6, pp. 199-202, 1982. 9. Lee, P.N. eta l. "Relationship of passive smoking to risk of lung cancer and other smoking- associated diseases," Br. J. Cancer 54: 79-105, 1986. 10. Wang, F.L. et al. "Analysis of risk factors for female lung adenocarcinomas in Harbin," Chinese J. of Prevent. Med. 23: 270-273, 1989. 11. Wu-Williams, A.H. et al. "Lung Cancer Among Woman in North-east China," Br. J. Cancer 62: 982-987, 1990. 12. He, X.Z. eta l. "A case-control study on risk factors of lung cancer," Lung Cancer 17(Supplement): 2, 1991. 13. Garfinkel, L. "Time trends in lung cancer mortality among nonsmoker and a note on passive smoking," J. NatL Canc. Inst. 66: 1061-1066, 1981. 14. Kabat, G.C. et al. "Lung cancer in nonsmokers," Cancer 53: 1214-1221, 1984. -16- I I I I I I I I I I I I I I I I I I

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I I I 15. Kalandidi, A. et al. "Passive smoking and diet in the etiology of lung cancer among non- smokers," Cancer Cases and Controls 1: 15-21, 1990. I 16. Janerich, D.J. et al. "Lung cancer and exposure to tobacco smoke in the household " N. Enel. , J. Med. 323: 632-636, 1990. I 17. Wynder, E.L. et al. "Smoking and lung cancer: some unresolved issues," Enidemiologic Reviews 5: 177-207, 1983. I 18. Lam, T.H. et al. "Smoking, passive smoking and histological types in Hong Kong Chinese women," Br. J. Cancer 56: 673-678, 1987. , 19. Elizabeth, T.H., Fontham et al. "Lung cancer and nonsmoking women: A multicenter case- control study," Cancer Epidemiology, Biomarkers Prevention 1: 35-43, 1991. 20. Garfinkel, L. et al. "Involuntary smoking and lung cancer: a case-control study," JNCI 75: I 463-469, 1985. 21. Dalager, N.A. et al. "The relation of passive smoking to cancer," Cancer Res. 46: 4808-11, I 1986. 22. Pershagen, G. et al. "Passive smoking and lung cancer in Swedish women," Am. J. Epidemiol. I 125: 17-24, 1987. 23. Delbert, J., Eatough et al. "Environmental tobacco smoke," Proceedings of the International I 24. Symposium at NcGill University, Quebec, Lexington, 3-12, 1989. Russell, M.A.H. et al. "Use of urinary cotinine concentrations to estimate exposure and I mortality from passive smoking in non-smoker," Brit. J. Addict. 81: 275-281, 1986. 25. Reasor, M.J. et al. "Assessing exposure to environmental tobacco smoke: is it valid to I extrapolate from active smoking?," J. Smoking-Related Disorders 2: 111-128, 1991. 26. Diet Nutrition and Cancer National Academy Press Washington D.C. 1982 ' , , , , , . 27. "Dietary Mutagens," Environmental Health Persoect. 67: 1986. I 28. Ames, B.N. "Dietary carcinogens and anticarcinogens. Oxygen radical and degenerative diseases," Science 221: 1256-1264. I 29. Bjelke, E. "Dietary vitamin A and human lung cancer," tnt. J. Cancer 15: 561-565, 1975. 1N 30. Hirayama, T. "Diet and cancer," Nutr. Cancer 1: 67-81, 1979. O 00 I ~ 00 -17- Gf tU O> I ~ i

I I I I I I I I I I I I I I I I I t instead, observed a relationship between salty food and smoked food and the incidence of malignant tumor formation. Studies performed in China, Japan and Ireland have shown a close relationship between the intake of cured and smoked food with esophageal and stomach cancers but not lung cancer. Because no association can be demonstrated between the intake of cured or smoked food and the incidence of lung cancer in our study, it is unclear whether lung cancer inducing carcinogens can enter the body by ingestion of these foods. 4. Living conditions and exposure to coal smoke. Since approximately two-thirds of a person's life is spent indoors, the importance of air quality on human health is obvious (35). Based on reports from environmental monitoring stations, indoor air pollution is much more significant in China compared to developed countries. Such severe indoor air pollution may be related to the fact that coal is the primary source of cooking and heating fuel in the People's Republic of China (36-37). Our studies show that the shared living space and the condition of ventilation in the living quarters are closely associated with an increased risk of lung cancer in females. However, there was no distinct difference between the controls and the cases insofar as exposure to coal smoke or participation in cooking was concerned. On the other hand, the positive association between the living conditions index and the risk of lung cancer in females indirectly points to coal smoke or cooking practices as sources of indoor air pollutants which in turn contribute to a risk of lung cancer in females. Similar results were obtained by Wu et al. (38) in which the use of coal indoors was found to be a very strong risk factor for female adenocarcinoma. Studies by Leung (39) in Hong Kong showed a positive correlation between the incidence of lung cancer and the use of coal burning stoves. Maclennan et al. (40) investigated the lifestyle factors of Chinese females in relation to lung cancer and proposed that in Singapore the incidence of lung cancer in females may be associated with the method used for cooking. Likewise, Gao et al. (41) in a case control study in Shanghai suggested that indoor air pollution resulting from cooking process and the use of rapeseed oil was a very important risk factor for lung cancer in females in the city of Shanghai. Du et al. (1990) (42) studied the use of burning coal in the home and the incidence of lung cancer in females in Guangzhou and found a close correlation between the two. A similar result was obtained by Dai et al. (43) in Harbin. Moreover, He et al. (44) studied the extremely high incidence of female lung cancer in Xuanwei and found a close correlation between the incidence of lung cancer in females and the use of coal indoors. These results obtained in epidemiological studies were also supported by laboratory findings (45,46). Ou et al. (47) found that the concentration of benzo(a)pyrene was significantly higher in coal-burning kitchens as opposed to gas-using kitchens. Moreover, the concentration of benzo(a)pyrene was found to be elevated in the urine of housewives who used coal as opposed to those who used gas, demonstrating that carcinogens derived from indoor air pollutants can enter into the body. Although it is easy to understand why indoor air pollution is a more important risk factor for lung cancer in females than in males there are a number matters that cannot be totally explained by indoor air pollution alone. For example, benzo(a)pyrene is known to be an inducer for squamous cell carcinoma of the lung, however, in the case of females, adenocarcinoma is found to be the predominant cell type. Therefore, the effect of indoor air pollution on the incidence of lung cancer needs to be further investigated. -15- I

31. Shelleke, R.B. et aL "Dietary vitamin A and risk of cancer in the western electric study," Lancet 2: 1185-1190, 1981. 32. Byers, T.E. et al. "Diet and lung cancer risk: findings from the Western New York Diet study," Am. J. Epidemiol. 125: 351-363. 33. Bond, G.G. et al. "Dietary vitamin A and lung cancer: results of a case-control study among chemical workers," Nutr. Cancer 9: 109-121, 1987. 34. Hinds, M.W. et al. "Dietary vitamin A, carotene, vitamin C and risk of lung cancer in Hawaii,' Am. J. Euidemiol. 119: 227-237, 1984. 35. Spengler, J.D. et al. "Indoor air pollution: a public health perspective," Science 221: 9-17, 1983. 36. Zhan, X.M. "Epidemiological study of indoor air pollution in Guangzhou," Second Chinese National Conference on Environmental Health, Nanjing, 1984. Abstracts. 37. Ou, F. "Report on the investigation of indoor air pollution in Districts of Guangzhou," Second Chinese National Conference on Environmental Health, Nanjing, 1984. Abstracts. 38. Wu, A.H. et al. "Smoking and other risk factors for lung cancer in women," JNCI 74: 747- 751, 1985. 39. Leung, J.S.M. "Cigarette smoking, the kerosene stove and lung cancer in Hong Kong," Chest 71: 273-276, 1977. 40. Maclennan, R. et al. "Risk factors for lung cancer in Singapore Chinese, a population with high female incidence rates," Int. J. Cancer 20: 854-860, 1977. 41. Gao, Y.T. et al. "A case-control study of female lung cancer in Shanghai," Guangzhou Second Symposium on Lung Cancer Research, p. 7, 1987. 42. Du, Y.X. et al. "Indoor air pollution and women lung cancer," The Fifth International Conference on Indoor Air Quality and Climate, vol. 1, 59-64, 1990. 43. Dai, D.X. et al. "The risk factors for lung cancer in women," Lun Cancer acer 7(Supplement): 3, 1991. 44. He, H.Z. et al. "A case-control study on risk factors of lung cancer," Lune Cancer 7(Supplement): 7, 1991. 45. Yin, X.R. "Mutagenicity experimental study of the etiology of lung cancer in the high incidence area of Xuanwei," Second Chinese Conference on Environmental Health, Nanjing, 1984. Abstracts. I I I I I I I I I I I I I N O 00 I s 1 -4 00 ca N -18- N/ ~

i I I I I I I I I I I I I I I , I I HEALTH IMPACTS BY LIFESTYLE AND BEHAVIORAL FACTORS IN GUANGDONG, CHINA Zhou Jiong-liane, Liang Hao-cai, Wang Zhi-jin and Liu Oing Institute of Preventive Medicine, Sun Yat-sen University of Medical Sciences, Guangzhou, China In order to evaluate the relationship between health and lifestyle and behavioral changes due to rapid economic development, several epidemiologic studies were conducted in two developing cities (Guangzhou and Zhuhai) during the last ten years. The studies consisted of surveys on the impact of behavioral factors on deaths in the two developing cities. These studies also analyzed smoking in factories and in the countryside, smoking and its intervention measures among medical university employees and students, and the association of smoking, home ventilation and lung cancer. The main results were as follows: Zhuhai. 1. Unhealthy lifestyles and behavior were the major causes of death in Guangzhou and Both the Yuexiu district in Guangzhou and Zhuhai city have reliable death reporting systems and were selected for the surveys. A total of 1,104 deaths (1991) in Zhuhai and 893 deaths in Yuexiu were identified for home visits, during which a questionnaire was given to relatives by trained interviewers. According to Dever's Classification, the leading cause of death in both cities was shown to be "unhealthy lifestyles and behavior." (Table 1) This association was found in one-half of the cases and also was the leading cause of death for cerebral vascular disease, malignant tumors, respiratory disease, and heart disease. Table 1. Distribution or Four Factors in Seven Causes of Death (Male and Female) Cause of Death % of the Total Unhealthy Lifestyle & Behavior Human/Biological Factors Environmental Factors Medical Scrviccs Cerebral Vascular Disease 23.28 31.29 34.55 Z94 6.22 Malignant Tumors 19.93 60.65 33.08 8.76 2.51 Respiratory Disease 11.63 59.04 24.57 8.53 7.85 Accidents 8.04 32.59 1.86 54.04 11.18 Heart Disease 7.79 50.64 35.90 7,05 6.41 Digestive Disease 5.64 53.10 17.70 7.96 21.24 Miscellaneous 20.68 27.78 49.25 7.73 15.23 Total 100.00 49.03 3L32 10.70 8.84 I

A comparison to other data (Table 2) indicated that the contributions by unhealthy lifestyles and behavior in causing death appear to be similar to results obtained in a U.S. survey in 1977. Additionally, there was a 12% increase in deaths attributable to unhealthy lifestyles and behavior when the data for 1991-92 was compared to those for 1982-83. Table 2. Comparison of the Ratio of Four Factors in Causing Death (%) Location Unhealthy Lifeetyles & Behavior Human (Biological Factors) Environmental Factors Medical services Two Cities in Guangdong (1991-1992) 49.05 31.32 10.79 8.84 * 19 Cities & Towns in China (1982-t983) 37.3 32.1 19.7 10.9 ** U.S.A. Nationwide (1977) 48.9 23.2 17.6 10.3 By Liang Hao-cai From Reports of The Department of Health and Human Services, U.S.A. These data suggested that unhealthy lifestyles and behavior would produce most obvious health impact on the populations during economic growth. 2. Smoking appeared to be the leading unhealthy lifestyle and behavior in various populations which is not easily stopped. The nationwide smoking rate in China has been reported to be around 61 % for males and 7% for females. Although the smoking rate among the Guangzhou population was lower, the problems were still serious. In one village, most of the smokers had started to smoke as teenagers. Their stated motivation was that it was "refreshing." (41%). In one petrochemical plant known to have a good antismoking campaign, 195 out of 350 smokers (54.6%) were found to have stopped smoking but had started again. Surveys in universities showed that the lower the educational level of employees, the higher the smoking rate, e.g., 18% for those with university level education, and 66% for those with primary school education. Smoking as a risk factor was poorly recognized, e.g., 75.3% of the university students considered that smoking had nothing to do with health; 55.7% of the university employee smokers believed that smoking had both beneficial and harmful effects. 3. Smoking appeared to be the greatest risk factor for lung cancer, but other indoor pollutants should not be ignored. A case-control study of 203 cases of primary lung carcinoma from eight main hospitals in Guangzhou during 1983-1984 showed that by "conditional logistical analysis," the smoking level (in terms of number of cigarettes/day) had a large standardized regression coefficient value of 5.7728 and a high Odds Ratio of 3,2670, indicating a significant association between smoking and lung cancer risk. However, since indoor pollution due to cooking with coal was very frequent, the standardized regression coefficient value for pollution in room-kitchen area or in kitchens were also high (3.4123 and 2.644, respectively) and corresponded to Odds Ratios of 3.32 and 1.84, respectively. This suggested that working and living in a poorly ventilated room would be another unhealthy lifestyle and behavior related to lung cancer. ' ' I I I I I ' I I 1 I I I I -2- I '

a I I I I I I I LOW RISK EPIDEMIOLOGY AND GOOD EPIDEMIOLOGICAL PRACTICE Raanar Rylander Department of Environmental Medicine, University of Gothenburg, Gothenburg, Sweden Abstract This presentation reviews the methodological difficulties involved in low risk epidemiology. Important basic concepts relate to dose-response relationships in terms of threshold or the J-shaped curve. The possible errors in establishing exposure estimates are outlined, particularly in terms of dose descriptions, and good epidemiological practice is discussed. Finally, the responsibilities of the researcher in terms of the caution necessary in the interpretation of data, as well as the public health impacts of those interpretations, are delineated. Background I I I I I , I Investigations of risk factors are an important part of scientific efforts to assess relations between the environment and risks for disease. Risk assessment has a long tradition. As early as 3200 BC, the Sumerians had special priests - the Asipus - whose role was to evaluate risks. They aided kings, governors or individuals in evaluating risks, using a simple mathematical system based on yes and no. The sum of these directed marriage alliances, the purchase of property and other everyday events in society. Risk estimations are equally important today as during Sumerian times, and the results are still expressed in numbers. However the priests of our times are epidemiologists, toxicologists and statisticians, and the procedures followed to arrive at numbers for risk have become very complicated. Our risk estimations now deal with low numbers which adds to the complexity. Most of the large risks related to environmental agents have been defined and described - with an acknowledgment of an absence of preventive measures for some of them. In striving for good health, our attention has increasingly been directed toward low risk agents in the environment. The purpose of this presentation is to discuss some of the methodological difficulties related to studies of low risk agents and the interpretation of results, and to delineate some suggestions for good scientific practice in evaluating results. Low Risk Agents I I I Problems in low risk epidemiology have been dealt with in several publications and workshops (14,22-24,27). Wynder defined an increased low risk as up to 2 and a decreased low risk as down to 0.5 (24). The concept of the appreciation of risks at different levels is shown in Table 1. I

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 ' I I the data was corrected for day-care attendance, an important risk factor for respiratory disease in children (7), the risk disappeared. In spite of this, the summary contains a statement regarding the risk for infection resulting from ETS. Another responsibility of the researcher is to acknowledge his own limited knowledge. Understanding of the complex relation between environmental agents and disease develops continuously and what are today accepted ideas, such as the importance of diet for the risk of disease and the concept of special risk individuals, were not known some decades ago. Conclusion I I I I I I , I I I With prudence, caution and good epidemiological practice, epidemiology can, in spite of its inherent methodological problems, bring new knowledge to the understanding of disease and environment, with corresponding gains in preventive power and population health. Without these precautions, epidemiology can bring chaos and in the end, a mistrust of public health and environmental medicine. This would obviously bring about a severe delay in the progress in the field of prevention, a critical field in increasing the health of the population. -7- I

I I I I I I I I I I I I I I I The J-shaped curve is of particular interest for environmental exposures. It implies that a small dose of an agent decreases the risk, as compared to no dose at all. At higher dose levels, risk increases appear. The concept is illustrated by the following examples. Vitamin A in foods is a necessary nutritional item, whereas the intake of high doses of vitamin A is toxic and involves a risk for liver cancer. Alcohol in moderate doses decreases the adherence of platelets and reduces the risk for cardiovascular disease. At high levels, alcohol is toxic and increases the risk for cardiovascular death. The J-shaped dose-response curve probably also relates to air pollution. Low levels of irritation, such as are caused by respiratory infection or air pollution, seem to decrease the risk for IgE-related sensitization to inhaled allergens. There are also data suggesting that the risk for lung cancer is reduced by exposure to inflammatory agents. The Balance Concept The balance concept is an important consideration concerning the relationship between the environment and disease development. It is now understood that the development of disease caused by environmental agents seldom follows a direct cause-effect relationship. The body has a series of defense systems which deactivate many environmental agents or their metabolites. Particularly important is the P 450 enzyme system. Paradoxically, it may indeed be suggested, that of all the defense against the alien agents in our environment, the best ones are against carcinogenic substances, the reason being the large number of natural carcinogens that is present in the normal environment or produced in the body itself by bacterial metabolism in the gastrointestinal tract. An important part of the balance system is the defense brought about by foods. It is generally agreed that fruit and vegetables are important protective factors against cancer (1,3,4,8,10) and probably other diseases, such as atherosclerosis. A trace element such as selenium is also important, mainly in its capacity as an antioxidant. Implications for Low Risk Epidemiology A consequence of the dose-response and balance concepts referred to above is that low risk epidemiology must take into consideration the potential beneficial effects of a particular exposure as well as the presence or absence of protective factors. Particularly dangerous is the situation in which the factors influencing the risk covary with the agent studied. An example is smokers, from whom the exposure to tobacco smoke according to cigarettes per day is inversely related to the consumption of such protective factors as vegetables and fruit (12,13,19). Dose Errors The potential errors in low risk epidemiology are not different from those in epidemiology in , general, but there is a need for high precision in view of the normal random variation in a studied material. Extensive reviews of possible errors have been presented previously, in particular during a workshop reported by Wynder (23). Here remarks will be limited to the exposure description. N 00 i V O W , 3 V O I

assurance, data interpretation and verification. Among the different conditions, the request for data storage in a form that would make a later reanalysis possible is particularly important. Similar rules have been discussed in epidemiology and proposals have been made by different bodies including WHO (5, 16, 17, 18, 20), but no generally accepted standards have yet been presented. There is a need for this in low risk epidemiology. Even if such rules may initially hurt the pride of or seem self-evident to the knowledgeable scientist, experience gained in toxicology has demonstrated their usefulness. Table 2 illustrates some of the basic concepts to be defined in rules for good epidemiological practice. Table 2. Basic principles for good epidemiological practice Defined organizational structure Defined principal investigator Personnel qualified or trained for study Establishment of study plan Documentation of collected data Appropriate storage of data for later reanalysis Responsibility of the Researcher Put in one perspective, the sound use of epidemiological techniques remains the responsibility of the researcher. Initially, it is paramount to realize that analytical epidemiology is actually the weakest link in a chain of evidence relating an exposure to a disease (26). It is essential to consider evidence from other studies, including toxicology, exposure assessment and molecular biology. Associations found in epidemiological studies are not a proof of causality and the researcher should be aware of the many pitfalls involved in his own interpretation of his data as concerns causality. Wish bias is an important error in the interpretation of results of epidemiological studies (25), particularly in studies of low risk agents. The researcher is also responsible for the use of his data in public health practice. An overuse of some preliminary results or data supporting a paradigm hypothesis is not only unethical but also approaches a scientific fraud. Increasing the importance of small findings by multiplying a low risk with the number of persons in a population must only be done when the evidence is good and with careful caveat as to the uncertainties involved. Another example of erroneous reporting of results is a recent study on respiratory infection in children and different risk factors (2). The authors found an increased risk for exposure to ETS but when -6- I I I I I I I I I I I I I I I

Table 1. Assessment of different degrees of risk Risk Discovery > 10 Perceived by the population itself 9-2 Relation to exposure relatively easily established with epidemiological techniques <2 Severe methodological problems The table illustrates that high risks are appreciated without the interference of scientists - they may be required to give precise figures, but will not influence the general appreciation of the risk involved. A good example is the common knowledge, as cited by the Swedish scientist Linneaus, that exposure to dust was a major cause of death among granite workers in Dalecarlia in Sweden in the 16th century. The table further illustrates that the detection of low risks requires the involvement of well trained epidemiologists. Studies of this nature have become further complicated during recent years, by developments in toxicology having to do with new principles for dose-response relationships and an increased understanding of mechanisms for the development of diseases induced by environmental agents. These problems will be treated in the following. Dose-response Relationships The traditional concept of a dose-response relationship as it relates to environmental agents was a linear curve on which even small doses were shown to cause an effect. This concept was applied particularly to radiation and carcinogens, and it allowed toxicologists to work with high doses in experimental settings. Estimations of risks from low level exposures could be made from experimental and epidemiological observations of high dose levels. It is now becoming increasingly clear that two other relationships - the threshold concept and the J-shaped dose relationship - are more appropriate for describing the human reaction to environmental agents. The three different concepts for dose-response relationships are illustrated in Figure 1. a os o. I I I Figure 1. -2- 1 I I I I I I I I I I I I I I I

Exposure determinations can be made using questionnaires or biological markers. Biological markers describe susceptibility, internal dose or the biological effect. A major advantage of biological markers is that they reduce the risk for misclassification, which is a particularly important source of error in low risk epidemiology. However, biological markers are not available, for most substances particularly in relation to long term exposure, and questionnaires offer the only possible method for dose determination. As for study design, case-control studies are often the sole alternative, as exposure descriptions are poor or nonexistent in most health registers. Criteria for exposure assessment in case-control studies have recently been reviewed (6). When dealing with high risk factors, the dose description is less critical. While an exposure estimation error may cause the risk to vary between e.g. 7.8 and 8.9, the conclusion will be that an exposure is related to a substantial risk. For low risk agents, this error becomes crucial. This is illustrated in Figure 2. 20 dsk 15 10 0 ; i I I _-._ I . 0 3 6 9 dose Figure 2. An observed low risk on the borderline of statistical significance may entirely be the result of a poor exposure description (overestimation of dose) and the correct conclusion is that no risk exists. Alternatively, if the dose is underestimated, the risk may be larger and statistically significant. The problem increases in complexity when several risk factors are involved. Tobacco smoke, coke oven emissions, radon, asbestos, keeping pet birds and ETS have been identified as risk factors for lung cancer. They cause a risk at different levels, but nonetheless cause risk for the same disease. As several of these factors are interrelated, the dose for all of them must be described with equal precision to arrive at a conclusion concerning the risk for an individual factor. -4- I 1 I I I I ' I I I ' I I I

I References 1. Alavanja, MCR; Brown, CC; Swanson, C and Brownson, RC. Saturated fat intake and lung cancer risk among nonsmoking women in Missouri. J. Natl. Cancer Inst. 1993; 85:1906-1916. 2. Berg, AT; Shapiro, ED; and Capobianco, LA. Group day care and the risk of serious infectious illness. Am. J. Enidemiol. 1991; 133:154-163. 3. Bjelke, E. Dietary vitamin A and human lung cancer. Int. J. Cancer 1975; 15:561-565. 4. Block, G; Patterson, B and Subar, A. Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutr. Cancer 1992; 18:1-29. 5. Chemical Manufacturers' Association Epidemiology Task Group, Guidelines for good epidemiology practices for occupational and environmental epidemiology research. J. Occun. Med. 1991; 33:1221-1229. 6. Correa, A; Stewart, WF; Yah, H and Santos-Burgoa, C. Exposure measurement in case-control studies: Reported methods and recommendations. Enidemiol. Rev. 1994; 16:18-31. 7. Dahl, IL; Grufman, M; Hellberg, C and Krabbe, M. Absenteeism because of illness at day care centers and three-family systems. Acta Pediatr. Scand. 1991; 33:1221-1229. 8. Fontham, E. Protective factors and lung cancer. Int. J. Epidemiol. 1990; 19:24-31. 9. He, Y; Lam TH; Li LS; Du RY; Jia GL; Huang JY and Zheng JS. Passive smoking at work as a risk factor for coronary heart disease in Chinese women who have never smoked. Br. Med. J. 1994; 308:380-384. 10. Kant, AK; Block, G; Schatzkin, A and Nestle, M. Association of fruit and vegetable intake with dietary fat intake. Nutr. Res. 1992; 12:1441-1454. 11. Koo, LC; Ho JH; Saw D and Ho CY. Measurements of passive smoking and estimates of lung cancer risk among nonsmoking Chinese females. 1nL J. Cancer 1987; 39:162-169. 12. Margetts, BM and Jackson, AA. Interactions between people's diet and their smoking habits: the dietary and nutritional survey of British adults. Br. Med. J. 1993; 307:1381-1384. 13. Morabia, A and Wynder, EL. Dietary habits of smokers, people who never smoked and ex- smokers. Am. J. Clin. Nutr. 1990; 52:933-937. I I I I I 1 I I I I I , I I ~ 14. Rylander, R; Lebowitz, M and Peterson, Y. Assessing low risk agents for lung cancer: ~ methodological aspects. Int. J. Epidemiol. 1990; 19:S2-S87. ~. iV ~ W K) 8 v ' U I

a I I I I I I I I I I I I I I I I I It is particularly important to describe the exposure to a significant risk factor before analytical work on small risk factors is undertaken. An example of the need to control for the major risk factor can be taken from studies on ETS exposure and cardiovascular disease. Support for the hypothesis of covariation has been found in some epidemiological studies. Only one has controlled for dietary fat intake however, which is a major risk factor for cardiovascular disease (9). When fat intake was corrected for, the statistical significance disappeared in all but one study. Another illustration is the study of lung cancer and exposure to diesel exhaust among truck drivers as an occupational group. A slightly increased risk has been demonstrated, for both lung cancer and cardiovascular disease. The common interpretation is that these effects are caused by exposure to diesel exhaust. Recently, Alavanja et .(1) showed that fat is an important risk factor for lung cancer, with odds ratios as high as 11 in nonsmoking females. Fat is also a well-known risk factor for cardiovascular disease. In view of this, it can be hypothesized that the increased risk for lung cancer and cardiovascular disease among truck drivers is not due to the relatively low exposure to diesel exhaust but rather to dietary habits - eating high-fat foods at cafeterias during night shifts. Not until this factor has been controlled for can a final evaluation of the previous hypothesis be made. Paradigm Bias In the interpretation of low risk relationships, particularly those "on the borderline" of statistical significance, paradigm bias is important. An overview of the scientific literature suggests that a ruling paradigm, itself often based on only a small material, is difficult to overcome. The resistance is found among reviewers of journal, editors, colleagues and the public. This paradigm defense is particularly important for studies which report a negative finding in the perspective of a previous positive finding. Paradigm bias is also reflected in the number of articles that appear confirming a new result. When the data on cold fusion were published, there was initially quite a number of studies confirming the original observations. Not until several months later did the negative studies begin to dominate. Paradigm bias is also present in the judgment of data that do not support the paradigm. Such data are referred to as "strangely different," "based on different populations," resulting from poor techniques or simply ignored. A good example of paradigm bias is studies which cannot demonstrate an association between exposure to environmental tobacco smoke (ETS) and lung cancer. One of the first negative studies on ETS stems from Hong Kong, where a thorough investigation of the subjects was made that incorporated lifestyle factors such as consumption of vegetables (11). Comments on this study are that conditions in Hong Kong are not relevant for other societies when, in fact, a deviating set of data from a different society may have a much larger potential to assess the general validity of a fii nding for the very reason that conditions are different (29, 30). Good Epidemiological Practice In toxicology, the concept of good laboratory practice has existed for many years. These are a set of rules for the structure of the investigation, responsibility and quality control. While these rules do N 0 they represent an important basis for quality not exclude the possibility of errors or even frauds 00 , .a 5 I

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I I I 15. I 16. 17. I 18. I 19. I 20. I 21. I 22. I 23. I 24. I 25. I 26. , 27. I 28.  29. ' , ' Rylander, R. Environmental exposures with decreased risk for lung cancer? Int. J. Epidemiol. 1990; 19:S67-S72. Szklo, M. Design and conduct of epidemiologic studies. Prev. Med. 1987; 16:142-149. Stellman, SD. Confounding. Prev. Med. 1987; 16:165-182. Schlesslman, JJ. "Proof' of cause and effect in epidemiologic studies: criteria for judgment. Prev. Med. 1987; 16:195-210. Thompson, DH and Warburton, DM. Lifestyle differences between smokers, ex-smokers and nonsmokers and implications for their health. Psychol. Health 1992, 7:311-321. WHO. Guidelines on studies in environmental epidemiology. Environmental Health Criteria 1983; nr 27:1-351. Von Mutius, E; Martinez, FD; Fritsch, C; Nicolai, T; Roell, G and Thiemann, H-H. Prevalence of asthma and atopy in two areas of west and east Germany. Am. J. Resn. Crit. Care Med. 1994; 149:358-364. Wynder, EL. Weak associations in epidemiology and their interpretation. Prev. Med. 1982; 11:464-476. Wynder, EL. Workshop on guidelines to the epidemiology of weak associations. Pre. Med. 1987; 16:139-141. Wynder, EL. Guidelines to the epidemiology of weak associations. Pre. Med. 1987; 16:211- 212. Wynder, EL.; Higgins, ITT and Harris RE. The wish bias. J. Clin. Epidemiol. 1990; 43:619- 621. Wynder, EL.; Cohen, LA; Rose, DP and Stellman, SD. Dietary fat and breast cancer: where do we stand on the evidence? J. Clin. Epidemiol. 1994; 47:217-230. Wynder, EL. Epidemiological issues in weak associations. Int. J. Enidemiol. 1990; 23:1491- 1496. Wynder, EL; Taioli E and Fujita Y. Ecologic study of lung cancer risk factors in the U.S. and Japan, with special reference to smoking and diet. Japan J. Cancer Res. 1992; 83:418-423. Wynder, EL and Stellman, SD. The "overexposed" control group. Am. J. Epidemiology 1992; 135:459-461. N C 00 ~ ti 0* -9- N 4 6>

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I I I I I I I RECENT PROGRESS IN THE EPIDEMIOLOGY OF LUNG CANCER IN HUMANS Du Ying-xiu Guangzhou Medical College, Guangzhou, China Abstract Lung cancer has been on the rapid rise worldwide during the last three or four decades. In China, the death rate from lung cancer is the highest among all types of malignant tumors in the urban population. Smoking, indoor and outdoor air pollution, and certain occupational exposures have been recognized as the main risks of lung cancer. This has been confirmed by many epidemiological research and laboratory studies. However, the significance of such risk factors may vary between different sexes or for different areas. Smoking is an important risk factor of lung cancer in both men and women; however, a large number of female lung cancer patients are never-smokers, indicating potential important risk factors other than smoking. There is a great debate over the association of passive smoking and lung cancer. Currently, available information cannot sufficiently confirm that passive smoking is capable of lung cancer induction. The relationship of atmospheric air pollution and lung cancer has long been noted; however the relationship is complex and the investigation requires data from long-term studies. Indoor air pollution is an important risk factor for lung cancer in women in China, but this is rarely reported outside China. At present, li carcinogens and 5 industrial processes have been confirmed as causes of occupational lung cancer. With further etiology research, more lung cancer causes may be discovered. To date, several observed phenomena are still without explanation. For example, why is smoking not an important factor in lung cancer in farmers? Or, what is the reason for the high incidence of adenocarcinoma in women? The answers may require research in the pathogenic mechanism of lung ancer. Introduction I According to a World Health Organization (WHO) report, for the past few years, stomach cancer and cardiovascular disease have decreased, while lung cancer is on a rapid rise, globally(1). There are already 35 nations where lung cancer is the number one malignant tumor in men; other nations may see lung cancer also becoming the number one malignant tumor in women. According to forecasts based on available information, AIDS and lung cancer will be the two most frequent health threats to mankind in early 21st century(l). In 1980, the number of new lung cancer cases in the world were estimated to be 600,500 (including 66,300 in China). If effective measures of prevention are not adopted, this number could reach 2 million in the year 2000 and 5 million in 2025. In China, according to annual nationwide health statistics, during the seven years 1982-1988,the average annual death rate of China's 16 largest cities was 565/100,000 with little change in the last seven years (regression coefficient b = 0.011, P > 0.05). The total cancer death rate, on the other hand, was on the rise; the average of 100/100,000 in 1982 was increased to 125/100,000 in 1988 (b = 0.0117, P < 0.05), including the lung cancer death rate which not only constitutes 25°l0 of all cancers but also increased most rapidly, 25/100,000 in 1982, 32/100,000 in 1988 (b = 0.0151, P< 0.01). The swiftness with which the lung cancer death rate has risen is not often seen in other diseases; this inevitably causes great concern. I

Regarding the cause of lung cancer, according to Brunner(2) there is reason to believe that the global prevalence of lung cancer is caused by conditions of the modern society and the unhealthy lifestyles of the people. Smoking, indoor and outdoor air pollution, and certain occupational exposures are considered the three most important factors in the etiology of lung cancer. Selawry and Hansen(3) suggest that at least 80% of lung cancer can be attributed to chemical carcinogens. Thus, research into the mechanism of carcinogenesis will be the basis to prevent lung cancer. Active Smoldng and Lung Cancer That smoking can cause lung cancer has been confirmed. Doll and Peto's 20-year retrospective study of 34,440 British male doctors found the adjusted death rate for nonsmokers was 10/100,000; for noncigarette smokers 48/100,000; for 15-24 cigarettes per day smokers 127/100,000; for over 25 cigarettes per day smokers 251/100,000(4). The occurrence rate for lung cancer decreased by 11 % of the estimated occurrence within 15 years of smoking cessation, while no change occurred for the incidence of other tumors. These data strongly suggest the close relationship between smoking and lung cancer. Many case-control studies on the relationship between smoking and lung cancer have been conducted in many areas of China. For example, the Wuhan Medical College study reported a relative risk (RR) value of 5.33 for smoking and lung cancer; Liaoning Health Investigation Bureau reported a RR of 8.45; Nanjing Health and Antiepidemic Station reported a RR of 6.51 for smokers of fewer than 20 cigarettes per day, and RR as high as 17.95 for smokers of more than 21 cigarettes per day. In 1985, our study of 849 cases and controls of lung cancer in Guangzhou showed smoking had an important significance for both men and women(5). The RR for men, at a 95% confidence level, was 3.53 (2.44- 5.11, P < 0.01) and 1.93 (1.30-2.27, P < 0.01) for women. The reason for the lower relative risk for women was that many female lung cancer patients were nonsmokers, which in turn indicated that potential risk factors other than smoking existed for female lung cancer. Eatough reviewed world literature on the chemical composition of mainstream and sidestream tobacco smoke and found that among the 108 traceable chemicals, in addition to the six that had already been designated as carcinogens by IARC (2-naphthyalmine, 4-aminobiphenyl, benzo(a)pyrene, N- nitrosodimethylamine, formaldehyde, and acetamide), others may be potential carcinogens also(6). Zhan et al.(7) applied a metabolite of B(a)P, anti-BTBE, to bronchial epithelia] cells of human fetus and observed not only mutation at H-ras gene 12 but the damages also resembled the mutation phenomenon observed in human lung cancer specimens. Kapitulinik et al.(8) induced lung tumors in mice by anti- BTBE. Chen et aL(9) applied smoke aerosol to cells of human fetus and found that the cells underwent morphological transformation suggesting that smoking is associated with lung cancer. Additionally, Wu et al.(10) applied extracts from snuff tobacco and chewing tobacco to BALB/3T3 cells and found the cells underwent mutations and the cell growth from the transformed colony exhibited characteristics of neoplastic transformation. These findings indicate a possible carcinogenic effect of smokeless tobacco. In summary, whether based on epidemiological or laboratory research, evidence exists for the association of smoking and lung cancer. Smoking cessation is apparently an important measure to prevent lung cancer. - 2 - I I I I I I I I I I i I I I

I nonsmoking women married to smokers (when compared to those married to nonsmokers) is actually higher than the OR of 4.4 for active smokers (compared to nonsmokers). That passive smokers can have a higher risk of lung cancer than smokers is biologically implausible. In summary, it is reasonable to conclude that current data have not strongly proven an association existing between ETS and lung cancer. However, this does not mean that ETS is harmless to humans. The sidestream smoke of cigarettes contains many harmful substances which are apparently hazardous to health. Atmospheric Air Pollution and Lung Cancer Statistical data show that the 1988 death rate in China for large cities is 32.14 per 100,000 which is higher than for medium size and small cities, 17.00; which is, again, higher than for rural areas, 12.53. Two questions arise: 1. Is air pollution one of the causes of a higher death rate for urban populations than for rural populations? 2. There is little difference in the smoking rates of urban and rural population? Why is it that smoking does not appear as important to farmers' lung cancer? The atmospheric air pollution and lung cancer relationship has long been noted. Stocks studied lung cancer rates in various parts of Great Britain and reported that they are closely related to the local atmospheric deposit index, smoke index, population density, and atmospheric concentrations of benzo(a)pyrene (B(a)P), beryllium, molybdenum, vanadium and arsenic(16, 17). Blot analyzed regional lung cancer death rates in the United States and found higher male lung cancer death rates in locations where paper, chemical, petroleum, and locomotive manufacturing industries are located and that the death rate is also related to atmospheric air pollution(18). Blot and Xu conducted a case-control study in Shenyang and found more male and female lung cancer patients among those living near smeltering plants for many years(19). Wang et al.(20) studied the relationship of lung cancer regional distribution and industrial pollution in Shanxi Province. They found that lung cancer at a particular location in Shanxi is inversely correlated with its distance from an industrialized center but is directly associated with its degree of industrialization. The lung cancer death rate is also positively correlated with atmospheric air pollution. The direction of the spread of lung cancer from high-incidence areas is also related to prevailing wind directions, being higher in "down-wind" areas than "up-wind" areas. For example, the spread from Taiyuan as the focal center, the locations of high-incidence areas in Quingxu, Jiaocheng, Wenshui, Taigu and Yuci coincides with the prevailing wind direction of Taiyuan area. Since lung cancer is a slow developing disease, the atmospheric air pollution is under constant change, and the conditions of human exposure unstable, the determination of air pollution-lung cancer relationship must be based on information from long-term studies. In 1991 we compiled atmospheric monitoring data collected by Guangzhou authorities during a 17-year period (1972-1981) and calculated the air pollution index(21). We subjected the data and Guangzhou's lung cancer death rates during 1976- 1989 to further analysis. We found higher lung cancer death rates in districts with more serious air pollution. - 4 - 1 I I I I I II

It is understandable that indoor air pollution should be more important to female lung cancer than male lung cancer. A remaining unexplained phenomenon relates to the fact that whereas chemical carcinogens such as B(a)P in general induce squamous cell carcinoma, primarily adenocarcinomas have been observed in female lung cancer. Occupational Exposure and Lung Cancer The confirmation of the etiology of disease must be based on verification from epidemiologic studies and laboratory research. Based on this priticiple, Saracci(30) of IARC classified lung cancer causing industrial carcinogens and industrial processes into the following. 1. Lung cancer carcinogens with sufficient evidence: arsenic and arsenic compounds, asbestos, dichtoromethyl ether, 6-valence chromium, tar, mustard gas, coal smoke, talcum powder contaminated by asbestos fibers, vinylchloride, nickel and nickel compounds; 2. Industrial processes with sufficient evidence: aluminum production, coal gasification, tar production, charcoal production, hematite-refining and radon radiation, steel casting; 3. Lung cancer carcinogens with insufficient epidemiological information: vinyl cyanide, beryllium and beryllium compounds, cadmium and cadmium compounds, crystal silicone; 4. Unconfirmed potential lung carcinogens: dimethylsulfate, aluminum, mineral oil, formaldehyde and phenobarbital. In China, lung cancer among tin miners was the first to be noticed as an occupational lung cancer. Wu et al. (31) in their study of Yuennan tin mines found the workers' lung cancer death rate had increased yearly since the 50s, reaching 0.3% in the 70s. In order to control the incidence of occupational tumors, China established National Cooperative Occupational Tumor Working Groups, to conduct overall investigations into the relationship of certain occupations and lung tumor. For instance, the National Arsenic Workers Lung Cancer Working Group(32) surveyed ten refining plants in Shenyang, Shanghai, Yuennan and mines in Hunan, found arsenic workers had both higher lung cancer standard mortality rate (SMR) and higher relative risks (RR) than the control group. The crude lung cancer incidence rate was as high as 248/100,000. The Asbestos Working Group(33) conducted occupational tumor surveys in nine asbestos plants and found malignant tumors to be the number one cause of death (SMR = 2.19, P< 0.01), and lung cancer the number one among malignant tumors (SMR = 6.33, P< 0.01). The two death rates are higher than for the control group. The Chloromethyl Ether Working Group's(34) survey of 11 chloromethyl ether manufacturing or user plants found for all tumors an SMR of 336, P < 0.01, and lung cancer SMR of 1546, P< 0.0001. In addition, Na et al.(35) conducted a retrospective cohort study of four nickel plant and mining operations and found lung cancer risks to be statistically significantly elevated in nickel refining and finishing workers. Further surveys by Chen et al.(36) among hematite workers and by Zhang et al.(37) among asphalt workers, all support the view that occupational risk factors have an important and significant contribution in the incidence of lung cancer. In general, it is easier to establish the relationship of occupational exposure and incidence of lung cancer, because there is clear and accurate employment history to verify exposure. On the other hand, - 6 - 2081783292 I I I I I I

structures in early passages which could be indicative of adenocarcinoma phenotypes. The estrogen-lung cancer relation needs to be further investigated. Prevention There cannot be real prevention of a disease without studying its etiology. Research has shown smoking, indoor and outdoor air pollution and occupational exposure as the three most important factors for lung cancer. Therefore, promoting smoking cessation, eliminating indoor and outdoor air pollution and controlling occupational hazards are the most important measures in the prevention of lung cancer. Smoking is not only an important risk factor for lung cancer, but it is also one of the risk factors in tumors of the oral cavity, larynx, esophagus, cervix and kidney. China's smoking rate ranks tenth in the world and its total tobacco consumption ranks first. Ten percent of the world's cancer deaths occur in China. In Guangzhou, for the over-15 population, the smoking rate is 43% for males and 4% for females, with a trend towards a smoking increase among young people. Smoking cessation should, therefore, be an urgent matter at hand. The U.S. National Cancer Institute (NCI) reported a lung cancer rate in American males at 71/100,000 and for females at 20/100,000 in 1982. If smoking is not stopped, in 2025 male lung cancer deaths will reach 311/100,000 and females, 56.9/100,000. If the NCI smoking plan is adopted (decreasing smoking rate to 15°k, use of low-tar cigarettes), in 2025 the male death rate will be 31.2/100,000 and the female rate 13.4/100,000. In 1986 the European Action Against Tobacco Committee was established(40). Funds of over $20,000,000 per year were raised for various activities, such as control of the tar content of cigarettes, health warning labels on tobacco products, ban of direct or indirect advertising of cigarettes, setting cigarette pricing policy, antismoking education in schools, etc. China should also establish a stop-smoking policy according to its own circumstances. Home coal-burning may cause severe indoor air pollution. It has been proven that the carcinogens released as a result of coal-burning are able to enter the body. Therefore, elimination of indoor air pollutants will have important meaning for lung cancer in females. It is believed that with the wider use of gas, the lung cancer death rate among females will decrease. Regarding the relationship between atmospheric air pollution and lung cancer, long-term, wide- scale investigation and research is needed. Atmospheric pollution is mainly due to industrial and mining operations; the control and elimination of industrial pollution are important measures in the prevention of occupational lung cancer. Improvement of general health may be important in lung cancer prevention. Trials are in progress in the U.S. in which high-risk population take daily doses of 50mg of vitamin E and 20mg of (3-carotene(41). At the present, these are several questions with no satisfactory answers. For example, why is I I I smoking seemingly not as important a factor in the incidence of lung cancer for farmers? Or, what is the cause (or causes) of adenocarcinoma, especially its high incidence in women? All these await further research. -1 - 8 - 2081783294 1 I

a 1 I I I I I 1 I i the use of chemical substances by man is on the rise, both in quantity and in type. It has been reported the number of registered chemicals has reached over 5 million, with 60,000 of the chemicals in constant use, and with 200-1,000 new chemicals per year being added to the "use" list. Clearly, research of chemical carcinogenicity will be an important subject facing mankind, and the prevention of occupational lung cancer will be an important task. Etiology and Lung Cancer Cel1 Types Lung cancer is classified into 13 types 2nd several sub-types, by WHO, according to cytopathological appearance. The most frequently seen are squamous cell carcinoma, adenocarcinoma, small cell carcinoma and large cell carcinoma, with squamous cell carcinoma and adenocarcinoma making up 80% of all lung cancers. The reason why lung cancer by different causes are of different cell types must be explained by the mechanism of pathogenesis of the disease. Squamous cell carcinoma is formed by mutation of squamous metaplasia of epithelial cells lining the larger bronchial membrane near the hilus of the lung; it is the central type. Adenocarcinoma is formed by mutation of alveolar cells or the epithelial and glandular cells of the smaller bronchi; it is the peripheral type. According to epidemiological research(5): 1. The most frequent lung cancer in men is squamous cell carcinoma, followed by adenocarcinoma as the next most frequent. The reverse is true for women, in whom adenocarcinoma is the most frequent, followed by squamous cell carcinoma; 2. Smokers of either sex have a higher rate of squamous cell carcinoma than nonsmokers, and the cancer rate is further affected by smoking index (number of cigarettes smoked per day times years of smoking), with a higher smoking index being correlated with the higher rate of squamous cell carcinoma; 3. Among smokers, squamous cell carcinoma is higher than adenocarcinoma in males , while in females squamous cell carcinoma and adenocarcinoma are about equal; 4. When smokers are excluded (when comparison is made only among nonsmokers) squamous cell carcinoma and adenocarcinoma rates are similar in males, while in females the rate for adenocarcinoma is much higher than for squamous cell carcinoma. The above results show that smoking mainly induces squamous cell carcinoma and that the higher ratio of squamous cell carcinoma in males may be associated with smoking (the majority of male lung cancer patients are smokers) while in females, there may exist unknown factors for adenocarcinoma. Selawry and Hansen(3) in their analysis of the relationship of cell type and lung cancer etiology, noted that cancers associated with smoking, air pollution, occupational exposure and other environmental factors are chiefly squamous and small cell lung cancers. Such an association probably means that upon entering the lung, it is easier for carcinogens to settle in the larger bronchus and ultimately cause central type squamous cell carcinoma. That being the case, it is hard to conceive how the peripheral type adenocarcinoma and the central type squamous cell carcinoma would have the same underlying biologic mechanism(s). Kobayashi et aL(38) found that adenocarcinoma cells show a higher positive estrogen receptor than other lung cancer cell types, thus raising the possibility that estrogen uptake and/or function may be linked to development of adenocarcinoma. When human fetal bronchial epithelial cells were treated with DMNA and estrogen, cells were found to survive for 45 weeks, which was longer than cells not treated with estrogen or androgen(38). Moreover, the cells also showed the appearance of gland-like - 7 - I

I I I I I 11 I I I I I I I I I I I I Seminars in Surgical Oncology 9:73-79 (1993) Recent Developments in the Epidemiology of Lung Cancer GEOFFREY C. KABAL Pho From the Department ol Eoidemioiogy and Social Medicine. Albert Einstein College or Medicine, Bronx. New York Lung cancer is currently the leading cause of cancer death in the United States and also the most common tumor worldwide. Changes in the distribution of histologic types over the past two decades in the United States, as well as high rates of lung cancer in certain suboopula- tions, require explanation. While cigarette smoking and specific occu- patibnai exposures are firmly established as important risk factors for lung cancer. recent work provides evidence that other factors may play a role either as indeoenaent risk factors or as modifiers of the effect of smoking. This paper reviews the eptdemiology of lung cancer. with an emphasis on developments in the past decade. ' 1993 wiiey-Uss. Inc. KEY WORDS: lung neoplasms. smoking, environmental tobacco smoke. radon, diet, host factors INTRODUCTION The Magnitude of the Problem The United States is currently in the sixth decade of an epidemic of lung cancer. Lunz cancer is the leading cause of cancer mortality in both sexes. accounting for an estimated 146.000 deaths in the United States in 1992. or 34'% of cancer deaths in males and in femaies (1]. The American Cancer Society projects that 168.000 new cases will be diacnosed in 1992. ac- countme for 18% of new cancers in males and 12% in females [1]. Between 1930 and 1987 the age-adjusted lung cancer mortality rate increased from 4 to 74 cases per 100,000 in males and from 4 to 27 in females [1]. During the past few years. lung cancer incidence rates in males have begun to level orT, but those in females have continued to rise. It is clear from these ngures that lung cancer will remain a major public health problem for decades to come. While lung cancer incidence in males is approaching its peak in the United States. rates in developing coun- tries are increasing, presaging a globalization of the epidemic [2]. Lung cancer is already the most common tumor worldwide [2]. Differences in age-adjusted incidence rates for spe- cific histologic types of lung cancer by sez, race. and calendar time period suggest that different histologic types may have different etiologies. In white men in the United States, rates of adenocarcinoma and oat cell carcinoma increased over the period 1969-1988, while the rate o f sa, uam ous cell carcinoma decreased. In white women, all major types showed an increase [3]. Squamous cell carcinoma is still the predominant histological type among males. whereas adenocarci- noma predominates among femaies [33. Among non- smokers. the proportton of adenocarcutoma is ereater than in smokers [41,, and parttcuiarly so in females. reaching 78% in one series of lung cancer cases in nonsmoking women [5]. While cigarette smoking and specific occupational exposures have been rirmiv established as important risk factors for lung cancer, over the past decade there has been increasine recoenition that smoking and oc- cupauonal exposures may not explain ail of the varia- tion in lung cancer incidence within countnes and be- tween countnes, and that other factors may play a role either as independent risk factors or as modifiers of the edect of smoking. Some issues that remain to be eluci- dated include ( t) the high rates of lung cancer in Chi- nese women, who have a low prevalence of smoking; (2) the higher incidence of lung cancer in black Amen- Address reorint mqtssu to Gcodrev C. Kabat. Ph.D.. Albert Ein- stein College of Medinne. Deoarrment of Eoidcmiolo¢yand Sodal Belfer Bldg. Rm. 1307- 1300 Moms Park Ave.. Bronx. Medicine. NY 10461-1602. ' 7 1993 Wiiey-Liss, Inc.

16. Stock, P. "Cancer and Bronchitis Mortality in Relation Atmospheric Deposit and Smoke," Brit. Med. J. 1: 74-79, 1959. 17. Stock, P. "The Relation Between Atmospheric Pollution in Urban and Rural Localities and Mortality from Cancer Bronchitis and Pneumonia," Brit. J. of Cancer 14(3): 397-418, 1960. 18. Blot, W.J. "Geographic Pattern of Lung Cancer, Industrial Correlation," American J. of Epidemiology 14(3): 397-418, 1976. 19. Xu, Z.Y. and Blot, W.J. "Smoking, Air Pollution and the High Rates of Lung Cancer in Shangyang, China," J. of the National Cancer Institute 81(23): 1,800-1806, 1989. 20. Wang, J.S., et al. "Study of the Association of Industrial Pollution and Characteristics of Lung Cancer Distribution in Shanxi Province." (from exchange of information at conference) (In Chinese) 21. Du, Y.X. "Atmospheric Pollution and Human Lung Cancer," Lung Cancer 7(Supplement): 2, 1991. 22. Yu, S.Y., et al. "Study on the Mutagenicity of Atmospheric Particulates of Various Size," Chinese J. of Preventive Medicine 25(2): 70-74, 1991. (In Chinese) 23. Gao, Y.T. "Case-Control Study of Lung Cancer in Women," Proceedings of Second Lung Cancer Conference, Guangzhou, p. 17, 1987. (In Chinese) 24. Sun, X.W. "Risk Factors of Female Lung Cancer from Heating Fuel and Respiratory Disease," Chinese Tumor J. 13(6): 413-415, 1991. (In Chinese) 25. Wang, G.X., et al. "Multi-Variant Analysis of Cooking Fume and Other Risks in A Case- Control Lung Cancer Study," Chinese J. of Prevention Medicine 26(2): 89-91, 1992. (In Chinese) 26. Ye, Z. "Study of Environmental Factors of Lung Cancer in Housewives of Tianjin," Clinical Tumor in China 17(4): 195-198, 1990. (In Chinese) 27. Ou, X.L., et al. "Relationship of Home Use of Coal and Lung Cancer in Housewives," Proceeding of Second Lung Cancer Conference, Guangzhou, 1987, p. 76-81. (In Chinese) 28. Liang, C.K., et al. "Lung Cancer in Animals Induced by Breathing Coal and Wood Burning Smoke - An On-Site Study," Health Research 14(2): 16-22, 1985. (In Chinese) 29. Zhang, P.C., et al. O. "The Determination of Methylated Purines (06 M Gua and M7-Gua) in Liver and Lung of Mice," Chinese J. Preventive Medicine 24, 136-138, 1990. (In Chinese) l ' , I I I

14 Kabat can males in the United States: (3) etiologic factors for after quitting smoking; (6) the correlation between adenocarcinoma other than smoking: and (4) risk fac- prevalence of smoking and lung cancer mortality rates tors for lune cancer in lifetime nonsmokers. Recently in successive birth cohorts of men and women in the attention has been drawn to a number of new poten- United States: and (7) the induction of tumors in ex- tial risk factors for lung cancer. including: passive perimental animals following exposure to tobacco smoking. domestic radon exposure. diet. body mass smoke. index. alcohol consumption. reproductive factors and Following Krevbere's ciassincation (15], for a long exposure to exogenous hormones. and host suscepti- time it was generally accepted that smoking was only biiity. In addition. changes over the past 3-4 decades associated with squamous and oat cell carcinomas and in the type of cigarettes smoked in the United States not with adenocarcinoma. However. studies carried have been adduced as a possible explanation of the out in large case-control series indicate that. although observed increase in adenocarcinoma of the lung [6,7]. the magnitude of the association with smoking is This review does not aim to be exhaustive but rather smaller in the case of adenocarcinoma. nevertheless a to provide an overview of the epidemiology of lung dose-response relationship exists for this cell type as cancer with an emphasis on developments over the well [16,17]. A recent analysis of 87 cases of the rare past decade. Several topics. such as occupational ex- bronchioloalveolar carcinoma has shown a consistent posures and air pollution. are given less space than association with smoking [18]. Large cell carcinoma their importance warrants. and the reader is referred ais6 appears to beasso.ciated with smoking [17]. to comorehenstve reviews. Studies examining the use of filter versus nontilter Although most of the discussion below deals with cigarettes and cigarettes of reduced tarr nicotine yieids specinc exposures as independent risk factors. interac- generally indicate that there is a modest reduction in tions between various factors isuch as smoking. occu- the odds ratio for lung cancer associated with smoking pation. and diet) may be important in determining an these "less haiardous cigarettes." on the order of 20- individual's risk of lung cancer [8]. Classical examples 30% [l9]. In an effort to explain the higher lung cancer of interaction between risk factors for fun¢ cancer are incidence rate in black American males compared to the greatly enhanced effect of exposure to radon as whites. recent work has focused on differences in well as to asbestos in smokers compared to nonsmok- smoking patterns between blacks and whites (20] and ers [9,10]. . on the effect of mentholated versus nonmentholated cigarettes on lung cancer risk (21]. The latter study SMOKING showed no increase in the odds ratio for lung cancer in Since the publication of the first epidemioio¢ic stud- smokers of mentholated relative to smokers of non- ies linking cigarette smoking with lung cancer in 1950. mentholated cigarettes. the association has been connrmed in epidemioioeic Various estimates are available for the proportion studies carned out in manv countnes and has been of lung cancers titat are due to smoking-that is. the further buttressed by animal evidence of the car- proportion of lung cancers that would be eliminated if cmoeenicitv of tobacco smoke ( I f-13]. Tobacco is the smoking were totally eliminated. Estimates of the pro- most exhaustively studied human carcinogen. and the portion of lung cancer attributable to cigarette smok- evidence for a causal association is overwheimine. In ing in various develooed countries range from 83% to fact, one could say that the association of cigarette 94% in males and from 57% to 80% in females (12]. smoking with lunz cancer provides a model for the Smoking cessation among current smokers and pre- associauon of an environmental risk factor with a vention of smoking initiation starting in school-aee chronic disease. This model is explicitly formulated in children offer the best prospects for reducing the inci- Sir Bradford Hill's criteria for judging the causality of dence of lung cancer. Between 1965 and 1987, the an association [14]. Evidence for a causal association proportion of current smokers in the United States of smoking with lung cancer includes (1) the rarity of declined from 50?% to 31.7% in men and from 31.9% lung cancer in lifetime nonsmokers: (2) the large ma2- to 26.8% in women [11-22]. However, the reduction in nitude of the association. generally a 10-fold increased smoking prevaience has been greatest among the more risk for current smokers relative to never-smokers: (3) educated. particularly in men. and as a result smoking a dose-response relationship between amount is becoming increasingly a habit associated with lower smoked and the relative risk of lung cancer. which can socioeconomic status. Extensive health promotion re- exceed 40-fold in heavv smokers: (4) the fact that the search has focused on designing effective strategies to relative risk increases with duration of smoking and help smokers autt and to deveiop the "life skills" and earlier age of starting smoking; (5) the progressive self-esteem that enable children and young adults to reduction in the relative risk with increasing years resist taking up smoking [23]. 2081783280 I I I I I i 'I I ~ I I

I I I There are many types of carcinogens in the atmosphere, including benzo(a)pyrene and benzo(a)anthracene. Among them the most important is B(a)P. There are many sources of B(a)P, mainly from industrial and home coal-burning, automobile and airplane exhaust. It has been reported that burning of 1 kg of coal can produce 2.1 mg of B(a)P, and 100g of coal smoke contains as much as 6.4 mg of B(a)P. In locations where traffic flow rate is 540-1,050 car/hr, 0.79-3.25 µg/100M3 of B(a)P can be detected. Yu et al.(22) conducted research on the mutagenicity of particulates according to size (diameter) contained in the atmospheres of Beijing, Taiyuan, Wuhan, Shengyang and Xuanwei. They found all samples to be mutagenic, and in inverse relationship with size of the particulates. Particulates with diameter < 1.0 µm have the highest mutagenicity, and at the same time the < 1.0 µm size particulate are the easiest to be retained in the lungs. In summary, it can be confirmed that lung cancer is related to atmospheric air pollution. However, since the occurrence of lung cancer is affected by many factors, and since the conditions of atmospheric air pollution are subject to constant ongoing changes, the search for a quantitative relationship may not be realistic. Indoor Air Pollution and Lung Cancer 1 I I I In China, a high incidence of female lung cancer has been reported from Harbin, Shanghai, and Guangzhou. All have pointed to indoor air pollution, caused by coal-burning, as an important cause of lung cancer. Gao et al.(23) in Shanghai found indoor air pollution and the use of rapeseed oil for cooking to have significant effects on the occurrence of female lung cancer. Sun et al.(24) in Harbin found, after adjusting for smoking, that indoor coal stoves and fire pits for heating can increase the risk of female lung cancer. Wang et al.(25) in Nanjing found kitchen cooking fumes to be a cancer risk factor in both squatnous cell carcinoma and adenocarcinoma in the lung. Ye(26) in Tainjin found that, after excluding cigarette smoking as a factor, women who live in run-down one-story houses in close proximity to low boiler chimneys and fumes from workshops have higher risks of lung cancer. Ou et aL(27) found that coal-burning households not only have higher levels of suspended dust, suspended dust-B(a)P, sedimentary dust, sedimentary dust-B(a)P in the air than propane-burning households, but housewives in the former have higher urine B(a)P content. This is direct evidence that carcinogens in indoor air are capable of entering the body. Liang et al.(28) conducted an on-site study in Xuanwei County of Yuennan Province by letting mice and rats breathe air containing coal-burning smoke, wood burning smoke, or "unpolluted air" (control). After 15-19 months, the lung cancer rate of the coal smoke group was higher than that from the wood smoke group, and the wood smoke group was higher than that of the control group. The B(a)P concentration of kitchen air is related to methods of cooking, with frying and stir- frying meats producing the highest B(a)P concentration. It has also been reported that carcinogens that entered the body carried by food have been found in the lungs(29). The question of whether food-bome substances can cause lung cancer is worth noting. There are great differences in indoor air problems in China and in highly industrialized nations. The main fuel for cooking and heating is coal in China, electricity and gas in industrialized nations. Of course, there are indoor air pollution problems abroad, such as wall board and ceiling tile installations, harmful emissions from carpets and other allergenic particles. But these materials are not significantly related to lung cancer. In China, with increasing use of gas, the nature of indoor air pollution problem will change correspondingly. - 5 - I

I I 1 I I I I I I I I I I I Passive Smoking and Lung Cancer The relationship of environmental tobacco smoke (ETS) exposure and lung cancer is still being debated. In China, studies from Harbin, Shanghai, Guangzhou and Xuanwei all reported no association between smoking and female lung cancer. Studies from other countries have produced mixed results. Some found no relationship between the two; others, while finding ETS an important risk in female lung cancer, disagree on cell type. Some find an association of ETS and adenocarcinoma only, which is unrelated to lung cancer of any other cell type, others find ETS a risk for squamous cell carcinoma only. Since the etiology of a disease is closely related to cell types, the latter two groups actually hold opposing views on the effect of ETS. Lung cancer is characterized not only by having multiple risk factors but also by its long latency. The conditions of human exposure to ETS can also be complex. For these reasons, to ascertain the relationship between ETS and lung cancer, the research must include good controls for a number of factors, such as: 1. The study subjects must experience "true" exposure to ETS, i.e. other than being nonsmokers themselves, the study subjects' exposure to air pollutants and occupational exposure must be controlled; 2. Both the extent of the exposure to ETS and the active smoker's smoking status should be accurately measured; 3. Objective reference biological markers exist that can precisely reflect the exposure to ETS. Since these conditions cannot be simultaneously achieved, it is not surprising that the association of ETS and female lung cancer cannot be confirmed. We conducted a case-control study of nonsmoking lung cancer patients that included effects of the husbands' smoking on lung cancer of nonsmoking wives, which also analyzed the relationship of active and passive smoking with lung cancer cell tvpes(l1). We found no association between the two. Pershagen et al.(12) conducted a case-control study which surveyed 27,409 Swedish female nonsmokers by questionnaire. They found that when nonsmoking women were married to smoking husbands the RR (3.30) of squamous carcinoma for these women increased significantly (P < 0.05). It is noteworthy that in their 20-year follow-up, only a small number of lung cancer cases (67 total) were found, only 20 of which were squamous and small cell carcinoma. In order to explain why ETS only induces adenocarcinoma of the peripheral type and not the central type squamous carcinoma which is primarily induced by active smoking, Wynder et aL(l3) proposed the following hypothesis: when ETS passes through the nasal cavity, the vibrissae are able to block certain particulates, with the result that gaseous phase carcinogens in the sidestream smoke are able to penetrate deep into the lung, even deeper than active smoking, and thereby inducing peripheral type adenocarcinoma. This hypothesis invites discussion. Carcinogens in tobacco smoke have high vaporization temperatures: for example, 2-naphthylamine vaporizes at 3060C, 4-aminobiphenyl at 302oC, benzo(a)pyrene at 311OC and N-nitrosodimethylamine at 152oC. It is extremely unlikely that these chemicals will maintain their gaseous state in the ambient environment or in the body without coalescing into particulates, which will make their deep penetration into the lung less probable. Recently, Trichopoulos et al.(14) evaluated the effects of active and passive smoking by using the increase in squamous metaplasia and abnormalities of the bronchial and alveolar basal cells as evidence of EPPL (epithelial possibly precancerous lesions). By using an EPPL value of 60 as a baseline value, they found that nonsmoking women married to smokers had a higher EPPL value than those married to nonsmokers. This result was interpreted as evidence supporting the view that ETS can induce lung cancer. A number of findings in the same paper, however, are at variance with such a view. For example, the EPPL value for heavy smokers could be as low as 29; the OR of 6.0 for - 3 - I

I 30  . ~ 31. 32.  I 33. I 34. # I 35. 36. I 37. I ~ 38. 39. . 40. ~ 41. ~ I I I Saracci, R. "Environmental Carcinogenesis of Lung Cancer." Lung Cancer 4(Supplement): 17, 1988. Wu, Z.Z. "A Study of Chemical Causes of Lung Cancer in Tin-Mine Workers," Health Research 4: 15-20, 1980. (In Chinese) National Cooperative Working Group on the Investigation of Lung Cancer in Arsenic Workers. "An Epidemiologic Study of Occupational Lung Cancer in Arsenic Workers," Chinese J. of Occupational Disease 4: 200-203, 1986. (In Chinese) Cooperative Working Group on the Investigation of Occupational Tumor, Asbestos Specialty Group. "Retrospective Cohort Study of Occupational Tumor Among Asbestos Workers," Chinese J. of Occunational Disease 5(1): 29-31, 1987. (In Chinese) National Cooperative Working Group on the Investigation of Occupational Tumor, Chloromethyl Ether Specialty group, "Conclusions of the Investigation of Lung Cancer in Chioromethyl Ether- Exposed Workers," Chinese J. of Occupational Disease 4(4): 222, 1986. (In Chinese) Na, C.J., et al. "An Epidemiologic Study of Lung Cancer in Nickel Workers," Chinese J. of Occupational Disease 11(5): 261-264, 1993. (In Chinese) Chen, S.Y. "An Epidemiological Investigation of Lung Cancer Risks Among Hematite Miners," Chinese J. Industrial Hygiene and Occunational Disease 5: 26-29, 1987. (In Chinese) Zhang, Y.D. "A Retrospective Investigation of the Etiology of Malignant Tumor Mortality Among Asphalt Workers," Chinese J. Industrial Hygiene and Occupational Disease 4: 223-226, 1986. (In Chinese) Kobayashi, M., et al. Proceeding of the 4th World Conference on Lung Cancer, 1985. Du, Y.X., et al. "The Culture of Human Fetal Broncho-Epithelia Cell Treated With Carcinogens and Sex Hormones," Lung Cancer 7(7): 13, 1991. Tubiana, M. "The European Action Against Tobacco," Lung Cancer 7(7): 9-10, 1991. Greenwald, P., et al. "Chemoprevention of Lung Cancer: Problems and Progress," Lung Cancer 4, Supplement 21-24, 1988. IV O OD i V Cb W Is) tC V

76 Kabat of homes could pose a lung cancer hazard. Conditions in homes differ from those in mines, and it is possible that long-term exposure to the relatively low radon lcvels typical of dwellings may pose a greater hazard than expected based on linear extrapolation from the levels tvpical of mines [37]. In the absence of system- atic surveys of domestic radon levels in the United States. estimates of the number of persons with high exposure (defined by the U.S.E.P.A. as 4 pCi/ 1) can- not be made [37]. Epidemiological studies of residential radon expo- sure in relation to lung cancer have yielded mixeS results. Studies carried out among women in New Jer- sey [38] and China [39] detected little or no effect of domestic radon exposure, while a study from Sweden [40] provided evidence of an association as well as of an interaction between radon exnosure and cigarette smoking. These :inconsistenaes may be due to me- thodoloeic problems including: subiect mobilitv, er- rors in estimating exposure, and inadequate sample size [41]. A number of epidemioiogtc studies of domes- tic radon exposure are in progress. and it is hoped that these will help clarify some of the crucial issues. in- cluding (1) the level of radon exposure at which an increase in risk is observed. and (2) the nature of the interaction between radon exnosure and cigarette smoking (is it additive or multiplicative?). RADIATION In addition to excess lung cancer rates observed in miners exposed to alpha-radiation from radon and its oroeenv-increased risks oflune cancer have been re- ported in patients treated wnh radiation for ankylos- ing spondvlitis in the United Kingdom and in those exposed to radiation from the atomic bombs dropped on Japan [24]. DIET Epidemiologic studies of diet and lung cancer show, according to a recent, authoritative review. "a consist- ent substantial protective effect of dietary vitamin A intake from vegetable sources" [421. Some studies have found a stronger protective effect of all vegeta- bles, dark green vegetables. cruciferous vegetables. and tomatoes, than for beta-carotene specifically [43. 44], suggesting that other vegetable constituents, in- cluding other carotenoids (lutein. lycopenc) and in- doles. may be protective against lung cancer. The most widely accented mechanism underiying the apparent protective effect of vegetable consumption is the role of anti-oxidants, including beta-carotene- vitamin C, and vitamin E, in scavagine free-radicais [45]. The apparent protective effect of beta-carotene and other anti-oxidants in observational studies has led to controlled ciinical trials of vitamin A. beta-carotene- svnthetic retinoids. and vitamin E and selenium in persons at high risk of lung cancer, including smokers and asbestos-exposed workers [46.47]. Pilot studies have demonstrated feasibiiity. and full-scale interven- tion trials are in progress. Another aspect of diet which may play a role in lung carcinogenesis is that of fat intake. International cor- relation studies suggest that there is an association between fat intake by country and lung cancer inci- dence or mortality [48-50]. One study in particular [50] found that per capita supply of animal fat was stronely associated with lung cancer mortality rates. This finding appeared to be due to the interaction between cigarette smoking and animal fat consump- tion. The authors concluded that cigarette smoking mav have a lesser imoact on lung cancer mortalitv in populations with a low intake of saturated fat. Other ecological studies correlating tobacco consumption and lung cancer rates between different countnes or in subpopuiauons within a country [51.52] are consistent with a modifvine effect of fat intake on the association of smoking with lung cancer. Several case-control studies provide evidence of a positive association between dietary fat/cholesterol intake and lung cancer. The first of these studies, from Hawaii, showed a relative risk of 3-5 (95%confidence interval 1.7-7.2) in males for the highest level of die- tarv cholesterol intake versus the lowest level, after adiustment for smoking and other covariates [53]. Later studies have provided generally confirmatory results [54-57.44]. In a prospective study of middle- aged American men. dietary cholesterol was as- sociated with increased lung cancer after adjustment for smoking. age, intake of beta-carotene and fat: however. the association held oniv for cholesterol from eggs. not from other sources [57]. BODY MASS INDEX Eight prospective studies and one case-control study have noted an association between leanness and lung cancer[58.59]. The association does not appear to be explained by differences in smoking habits or to weight loss due to disease. One possible explanation is that leanness may be associated with decreased levels of nutrients that are protective or with increased levels of dietary risk factors [581. Further studies are needed to determine whether the association of low body mass with lung cancer is due to the influence of factors associated with leanness or to a biological effect of leanness itself. ALCOHOL CONSUMPTION A number of reports have suggested that alcohol consumption is associated with lung cancer indepen- dent of smoking [60,61]. However. a large case-control 2081783282 ~ I I  I

z I References I I , I I I 1. Stjersward, J. and Stanley, K. "Lung Cancer -- A Worldwide Health Problem," Lung Cancer 4(Supplement): 11-14, 1988. 2. Brunner, K.W. "Foreword of Fifth World Conference on Lung Cancer," Lung Cancer 4(Supplement), 1988. 3. Selawry, O.S. and Hansen H.H. "Lung Cancer, Cancer Medicine," pp. 1709-1744, 1982. 4. Doll, R. and Peto R. "Mortality in Relation to Smoking: 20 Years Observation in British Doctors," Brit. Med. J. 2: 1525, 1976. 5. Du, Y, X., et aI. "An Epidemiological Study of Lung Cancer Risk Factors in Guangzhou," Proceedings of Third Lung Cancer Conference in Guangzhou, 1-23, 1992. (In Chinese) 6. Eatough, D.J. "The Chemical Characterization of Environmental Tobacco Smoke," Proceeding of the International Symposium at MeGill University, 3-39. 7. Zhan, D.J., et al. "Study of Mutagenicity of H-ras Gene of the Bronchial Epithelial Cell of Human Fetus Induced By Anti-BPDE," J. of Health Toxicology 6(4), 1992. (In Chinese) 8. Kapitulinik, J., et al. Nature (London) 266: 378, 1977. 9. Chen, J.K., et al. "Study of the Effect of Cigarette Smoke Aerosol on Kidney Epithelial Cells of Human Fetus," J. of Health Toxicology 5(3): 143-146, 1991. (In Chinese) 10. Wu, Z.L., et al. "Study of the Effect of Smokeless Tobacco on the Mutagenicity of BALB73T3 Cells," J. of Health Toxicology 5(3): 167-169, 1991. (In Chinese) 11. Du, Y.X. "Exposure to Environmental Tobacco Smoke (ETS) and Lung Cancer in Women," Proceedings of Third Lung Cancer Conference, Guangzhou, 1992, p. 24-35. (In Chinese) 12. Pershagen, G., et al. "Passive Smoking and Lung Cancer in Swedish Woman," Am. J. Eoidemiol. 125: 17-24, 1987. 13. Wynder, E.L., et al. "Smoking and Lung Cancer: Some Unresolved Issues," Epidemiology Review 5: 177-207, 1983. 14. Trichopoulos, D., et al. "Active and Passive Smoking and Pathological Indicators of Lung Cancer Risk in an Autopsy Study," JAMA 268(13): 1697-1701, 1992. N C 15. Ministry of Health, People's Republic of China, Annual Report on National Health Statistics, ~ 1955. (In Chinese) V tb W IV - 9 - <O N I

I I I I I 1 I I 1 I I 1 EXPOSURE TO ENVIRONMENTAL TOBACCO SMOKE AND THE INCIDENCE OF LUNG CANCER -- A REVIEW Du Ying-xiu* and Joseph M. Wu** * Guangzhou Medical College, Guangzhou, China ** Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York It has generally been established that smoking is an important risk factor for lung cancer in both men and women, but the relationship between environmental tobacco smoke (ETS) and lung cancer is still being debated. While many studies have not reported a close relationship between ETS and lung cancer, others have reported an association between ETS and lung cancer in nonsmoking women. However, studies in the latter group cannot agree among themselves on the lung cancer cell types being associated with ETS; some studies reported that ETS is associated only with squamous cell carcinoma while being associated with ETS; others reported that ETS is associated with adenocarcinoma of the lung. The relationship between ETS and lung cancer is a complex one. This is because lung cancer is a disease with multiple risk factors and of long latency. To be able to determine the association of exposure to ETS with lung cancer, other lung cancer risk factors or confounders need to be adequately controlled. Moreover, due to the long lapse of time and the complexity of the conditions of ETS- exposure of the study subjects, it is also difficult to design a precise study. In addition, a biomarker that can accurately reflect the exposure to ETS is lacking at the present time. Perhaps all these elements have contributed to the failure to establish a clear conclusion whether there is a relationship between ETS and lung cancer. To provide an overview of the research on the relationship of ETS and lung cancer, this paper seeks to present a survey of the world literature on this subject to date, delineate some of the issues and problems inherent in the research, and make some suggestions regarding the directions such future research might take. Focus of ETS-Lung Cancer Research It is generally accepted that in the investigation of the etiology of a disease several steps must be taken. First, the epidemiologic approach is used to hypothesize a cause-effect relationship. Laboratory experiments follow in order to confirm the hypothesis developed by epidemiology. Finally, the epidemiologic method is again used to verify the laboratory results. In other words, any investigation into the disease etiology must rely on the mutual corroboration of epidemiology and laboratory research; the research on the relationship between ETS and lung cancer is, of course, no exception. To correlate epidemiologic and laboratory research, an important step is to identify a biomarker that can accurately reflect the exposure to ETS, and on which a dose-response relationship can be based. Theoretically, this biomarker should be a direct marker for the ETS-induced mutation of cells of the lung, or at least an indirect marker for the ETS-induced biochemical change and such change is believed to be related to lung cancer. However, to date, neither a direct nor an indirect biomarker has been established I

I I ' I I I ! I I I I Z I I Epidemiology of Lung Cancer 75 OCCUPATION had a significantly increased risk of lung cancer rela- Studies of occupational groups have shown that tive to the nonsmoking wives of nonsmokin¢ hus- occupational exposure to a number of agents is as- bands [30.31]. Since then over 30 studies of the issue of sociated with lune cancer. These include asbestos. environmental tobacco smoke (ETS) and lung cancer radon. poiycyclic aromatic hydrocarbons. mustard have appeared. A metanalysis carried out by the Na- gas, chloromethyl ethers, chromium. nickei, inorganic tional Research Council [32] in 1986 found that the arsenic. and vinyl chloride [24,25]. Other agents sus- summary relative risk for lung cancer of nonsmoking pected of being lung carcinogens are: acrylonitrile- omen whose husbands smoked, relative to nonsmok- exposures encountered by rubber workers. beryflium. ing women whose husbands were nonsmokers, was ferric oxide dust- lead- and cadmium (241. 134 (95% confidence interval 1.18-1.53). The identification of synergism between occupa- Epidemioiogic studies of passive smoking are con- tional exposure (radon. asbestos) and cigarette smok- fronted by a number of challenges: the rarity of lune ing (9,10] has important implications for both an un- cancer occurring in never-smokers: the greater dilu- derstanding of the biological mechanisms of lung tion of ETS compared to smoke inhaled by the active carcino¢enesis and for prevention. smoker, the difficulty of obtaining accurate exposure The proportion of cancer attributable to occupa- information by means of self-reports: the lack of a tional exposures has been a subiect of controversy biological marker for long-term exnosure: misclassiii- [26], and the limited availability oi accurate exoosure cation of smokers as nonsmokers: and the possibility of coniounding by other risk factors. including diet or. data on ootennally exposed workers makes any esn- mate of this proportion highly uncertain. Within these in places like China, exposure to cooking fumes [33]. limitations. an educated estimate of the proportion oi There is inconsistency among the existing studies as to lun¢ cancers attributable to occupational exnosure is the presenceiabsence of an association; the histologic l5% of male and 5% of female lun¢ cancer cases [271. type of lune cancer for which an association is ob- served: the sex in which an association is observed: AIR POLLUTION and the timing of exposure (i.e.. childhood versus adulthood) [34,35]. It has lon¢ been suspected that exposure to environ- In spite of these problems. given the chemical com- mental (as opposed to occupational) air pollution position of ETS and what is known about the effects might contribute to excess lung cancer incidence. of active smoking, it is biologically plausible that However, studies of air pollution and lung cancer arc heavy ETS exposure over long pertods, and perhaps complicated by the fact that air pollution is a compiex particularly in those exposed in childhood. can in- mixture that varies from place to place and over time: crease the risk of lung cancer. The largest study to date by the overwhelming effect of cigarette smoking; and ot lun¢ cancer in nonsmoking women indicates that bv movement of subjects both within and between women whose husbands smoked had an increased rel- different cities. The so-called "urban factor." !hat is. ative risk for lun_g cancer (odds ratio = 1.3; 95%con- the 1.5- to 2.0-fold greater lung cancer incidence in fidence interval 1.0-1.7) and for adenocarcinoma cities compared to rural areas. can be largely explained lodds ratio = 1.5: 95% confidence interval 1.I-2.0) by cigarette smoking and occupational exposure. Nev- after adjustment for socioeconomic variables [4]. A ertheless, studies of populations exposed to point siznincant trend in the odds ratio for adenocarcinoma sources of pollution, such as nonferrous smelters, sug- was seen with increasin¢ number of pack-years of the gest that even after adjtutment for smoking and occu- husband's smoking. In the highest exposure group pation. exposure to high levels of air poilution is as- (> 80 pack-yearsl the odds ratio was 1.7 (95% confi- sociated with increased lung cancer: and analytical dence interva10.8-3.5). Other ETS exposures in adult- studies exammine lung cancer risk by urbani rural res- hood(in the household. on the job. and in social set- idence indicate that. in both smokers and nonsmokers. tings) were also associated with increased risk of lung urban residence is associated with increased lung can- cancer. but exposure in childhood was not. cer risk [28,291. Tntu. while the overall contribution of air pollution is difficult to gauge, exposure to polluted RESIDENTIAL EXPOSURE TO RADON air is likely to account for a modest percentage of lung Based on studies of underground miners. it is firmly cancer incidence (27,291. established that exposure to relatively high levels of radon and its proeeny can cause lung cancer in hu- ENVIRONMENTAL TOBACCO SMOKE mans [363. Over the past decade. the detection of In 1981. two reports were published purporting to radon and its progeny in homes has led to public show that the nonsmokine wives of smoking husbands concern that exposure to lower levels of radon typical 2081783281

I I I I I ~ I I t I I I I 1 ® I I study [62] showed clearly that although alcohol drink- ing by itself was associated with lung cancer, after adjustment for smoking, the association totally disap- peared. Results from a prospective study of men of Japanese ancestry in Hawaii [63] indicated that the association of alcohol intake with lung cancer was reduced after adjustment for smoking but still retained marginal statistical significance. Because smoking is the overwhelming risk factor for lung cancer (with relative risks reaching 20.0. 30.0. and higher in heavy smokers), and since smoking and drinking habits are highly correlated. it is crucial to adequately adjust for lifetime smoking habits before drawing conclusions about an association of alcohol with lung cancer. REPRODUCTIVE FACTORS AND EXOGENOUS HORMONES The higher prevalence of adenocarcinoma oi the iung in women compared to men suggests a possible roie of endocrine factors ( menstrual history. reproduc- tive history. use of exogenous hormonesl in the devel- opment of this type of lung cancer. Several studies have reported observations which are consistent with a role of endocrine factors: the finding of steroid receptors in some lung cancers [64]; a higher-than- expected rate of lung cancer. pantcuiarly adenocarcn- noma- among 10 + year survivors of endometnal can- cer [65]; an apparent increase in the risk of lung cancer in women receiving potent estrogens as hormone re- placement therapy [66]; and a significantly increased risk of adenocarcinoma of the lung (after adiustment tor smoking) in Chinese women with short menstrual cycles I<'_6 days) [671. [Chinese women have high rates of lung cancer tpredominantly adenocaranomai in spite of a low prevalence of smqking, potnting to the importance of factors other than smoking.) More ex- tensive investigation of the relation of endocrine fac- tors to lung cancer is needed before any conclusions can be drawn. HOST FACTORS Indirect evidence of a genetic component in the eti- ology of lung cancer comes from the facts that ( I) not all smokers who reach the age of 80 develop lung :ancer. and (2) most carcinogens require metabolic activation and this is under genetic control. Analysis of famiiies of cancer cases and controls, pedigree anal- ysis, and studies of genetic markers have been carried out in an effort to identify a genetic factor [68-721. Looking at familial clustering of lung cancer and other diseases enables one to study the interaction between genetic endowment and environmental exposures, al- though famiiial aggregation does not prove the pres- ence ot a eenettc component. since smoking habits and Epidemiology of Lung Cancer 77 other environmental factors (diet. infectious diseasesl also are known to aggregate in families. However. lung cancer tends to cluster in families& even after adjustment for smoking habits [73.741. Furthermore. adenocarcinoma and alveolar cell carcinoma are more common in families with other cancers. acquired im- mune deficiencies. or heritable disorders of the lung [751. To date, studies of genetic markers of lung cancer risk. such as aryi hydrocarbon hydroxylase and de- brisoquine phenotype, have been inconclusive [76]. The findine, in four studies- that extensive metaboliz- ers of debrisoquine are at increased risk of lung cancer relative to poor metabolizers would appear to be the most promising evidence for a genetic component in lung cancer. However, no metabolic pathway linking debrisoquine metabolism and metabolism of known lung carcinogens has yet been identiued [76]. Researcn in the area of ¢enetic control of metabolic activation and detoxification or carnnogens is likely to make a major contribution to understanding the inter- action between exposure to carcinogens and host sus- cepttbility. OTI-IER RISK FACTORS A report from the Netherlands has suggested that keeping pet birds in the home may be an independent risk factor for lung cancer [77]. The adds ratio for lung cancer among keepers of pet birds was 6.7 (95% con- fldence intervat 2'-20.0) after adjustment for smok- ing and vitamin C intake. Of two more recent studies with larger sample sizes undertaken to confirm this finding. one showed a more modest association (78], while the other found no association with birdkeeptng in the home but did note a relattonship limited to keepmg pigeons outside the house_(79]. Further stud- ies which control in greater depth for lifetime smoking and which address possible sources of bias could shed light on the nature of this intnguing association. CONCLUSION Two points emerge from this brief review of the eoidemiolo¢y of lung cancer. First. although smoking, and secondarily occupation. are major established risk factors, other factors. inctudine other environmental exposures and host susceptibility are likely to play a role either as independent risk factors or synergisti- caily with smoking or occupational exposure- As noted in the introduction. the occurrence of lung can- cer in certain groups and recent changes in the distri- bution of histoiogic types represent a challenge to fur- ther research. Second. since smoking is the overwhelming risk fac- Lor for lung cancer and because of the discretionary nature of smoking, lung cancer is. to a large extent. a I

I that can accurately reflect the effects of ETS on lung cancer or even the effects of ETS on health in general. The lack of a biomarker appears to be a significant problem for the research at present. It is commonly known that there are four lung cancer cell types: squamous cell carcinoma, small cell carcinoma, adenocarcinoma and large cell carcinoma. The four cell types are differentiable by their origin, form, structure, function and site within the lung. More importantly, many studies show that different etiologies may induce different cell types in lung cancer. It has been reported that smoking induces central type squamous cell carcinoma. Since research has found little difference in the constitutors of mainstream and sidestream smoke, there is little reason to believe that if ETS is capable I I ! I of lung cancer induction, it must also induce primary squamous cell carcinoma. Therefore, any study of ETS exposure that makes no inquiry into the lung cancer cell type must be considered methodologically inadequate. ! In summary, the research in the relationship between ETS and lung cancer must consider the cell type, use precise biomarkers and adopt a methodology that correlates epidemiologic and laboratory These are the suggested focal considerations for a good study design results # 1 . . Difficulties in the Study of The Relationship Between ETS and Lung Cancer 1 As previously stated, the study of disease origin should seek corroboration between both i epidemiology and experimental research. In the studies of the ETS/lung cancer relationship to date, the majority of results are based on data from epidemiologic studies of women who report spousal smoking in the home. Common to these epidemiologic studies are several inherent problems: L Regardless of whether it is induced by active smoking, occupational exposure or air pollution, lung cancer is characterized by a long latency. In other words, lung cancer is the result of cumulative effects of carcinogens. If ETS can induce lung cancer, it is reasonable to assume its latency period is just as long if not longer than in the case of active smoking. During such a prolonged period of time, the ETS exposure data are likely to be variable and complex; and as a result, it is difficult to establish a true, quantitative dose-response relationship. 2. The etiology of lung cancer is multifactored. To ascertain the effects of ETS on lung cancer, in addition to working with only true nonsmoking study subjects (to avoid misclassification), possible effects due to occupational exposure and air pollution (or other confounders) must also be controlled. 3. In the investigation process, if the survey is conducted retrospectively or by correspondence, the data obtained may be difficult to control for accuracy. If the survey is based on hospital cases, in addition to screening for the subject disease, attention must be given to the accuracy of the reported smoking habits of the active smokers to whose cigarette smoke the study subjects are exposed. 4. Not only is the smoking status of active smokers likely to be nonconstant during a long period of time, it is also difficult to exclude self-imposed avoidance of exposure by the nonsmoker due to aversion to the smoker's cigarette smoke. I -2-

5. It is not only difficult to estimate exposure to ETS in the public places, but also in the home environments. If the living space is large, for example, the effect of ETS is more difficult to estimate. I I ' I I I I I I I 6. Even though B(a)P, dimethylnitrosamine and other carcinogens can be found in the sidestream smoke, these chemicals typically will induce squamous cell carcinoma centrally located in the lung. The sidestream smoke that enters the body is greatly diluted, less capable of penetrating deeply into the lung when compared with the mainstream smoke, and thus has less opportunity to cause adenocarcinoma in the periphery of lung. Those epidemiological studies that do not give consideration to the cell type are less likely to be reliable. 7. At present, a precise biomarker for ETS exposure has not been identified; neither is a personal exposure measuring device available. Thus, ETS exposure data are generally descriptive. In general, the acceptability of descriptive data is not without reservations. 8. Due to the long latency period in the development of lung cancer, some researchers have noted that it is very difficult to design an animal model to study the association of ETS and lung cancer.(31)(35) The above are some of the important yet difficult issues in the epidemiologic research; together they may have given rise to the inconclusiveness of a link between ETS exposure and the development of lung cancer. Review of Literature We have collected and reviewed the world literature since 1980 on the subject of the association of ETS (mostly from spouses) and nonsmoker lung cancer cases. Among the 33 studies, 15 did not find any ETS-lung cancer association. Seventeen of the 33 studies attempted to associate ETS exposure with cell types. (See Table 1). Among these, some suggest that, like active smoking, ETS induces primarily squamous cell carcinoma and small cell carcinoma (12, 14, 15, 19), while other consider ETS to be associated with adenocarcinoma (7, 20, 31). It is well known that squamous cell carcinoma is the result of squamation of the mucous epidermal cells of the larger bronchi near the hilus of the lung, and this cancer is mostly centrally located; whereas adenocarcinoma is formed by the mutation of mucous epidermal and glandular cells of the smaller bronchiole and is mostly located in the periphery of the lung. Analyses of active smoking, regardless of gender, show a relationship primarily with squamous cell carcinoma. Since similar chemical constituents of mainstream smoke can be found in sidestream smoke, why, then, is exposure to ETS mainly associated with peripheral type of adenocarcinoma? Wynder and Goodman (34) postulated that for the passive smoker, inhalation of sidestream smoke components through the nasal passages, because the vibrissae are capable of blocking and retaining certain particulates, allows certain gaseous carcinogens to reach deeper into the lung (even deeper than for the active smoker), i.e. to the periphery of the lung. This is the reason why ETS is capable of inducing adenocarcinoma typically in the periphery of the lung. This hypothesis is open to debate. It is well -3- N 0 ~ ~ V 00 W W C ~

I I ! I I I Recently, Hecht, et al. (33) reported that nonsmokers exposed to machine generated sidestream cigarette smoke had significantly higher urinary excretion of NNAL (a metabolite of lung carcinogen) after exposure than base line. This study only showed that the carcinogen in sidestream smoke, like many other air pollutants, can be taken up and metabolized by a nonsmoker, and really did not provide information on the pathogenesis of ETS exposure. Conclusions and Suggestions for Research Directions As mentioned above, because the study of the association of ETS and lung cancer is fraught with research difficulties, no definitive conclusions have been reached. Based on past experience, a well- designed study plan will be needed as the next step in the investigation of the possible association between the two. To this end, I present the following suggestions: 1. The methodology should require mutual corroboration from both epidemiologic and laboratory research. Only by doing so, can we ensure that the obtained results are complete and reliable. 2. In the investigation of the effects of ETS, biomarkers are of crucial importance. As in all chemical carcinogenesis, the same characteristics, such as a dose-response relationship which can be evaluated via a biomarker, must be present in the components of ETS. The biomarker can be at the cellular level, sub-cellular-, molecular- or biochemical level. I I I I 1 3. Worldwide studies have all reported female lung cancers as being largely adenocarcinomas, implying the importance of taking into account the close relationship between the causal factor and the cell type of lung cancer. Therefore, whether an association of ETS and adenocarcinoma can be confirmed or negated will have significant meaning in the final analysis of the carcinogenic effects of ETS. At the same time, the vigorous pursuit of the etiology of adenocarcinoma may also help to clarify the effects of ETS. 4. Because of the multifactorial nature of lung cancer, the study design must control for smoking, air pollution and occupational exposures when selecting study subjects. -5- tV O 00 ~ V O W W O W

known that chemicals that penetrate the lungs via the respiratory organs can be in the form of gas, vapor and aerosol which include smoke, fume and dust. Only those matters that remain gaseous under ambient temperature and pressure, such as 02 and C02, are capable of reaching the depth of the lungs in gaseous form. Others, such as vapor and aerosol, all have condensation nuclei. Only particulates of less than 5 µ in size, the so-called respirable particulates, can reach deep into the lungs. The volatile temperatures for the primary carcinogens in cigarette smoke are quite high: 2-naphthylamine (at 306°C), 4- aminobiphenyl (302°C), benzo(a)pyrene (311°C), N-mtrosodimethylamine (152°C) and acetamide (222°C), etc. It is difficult to imagine that such high temperatures would exist in the ambient environment or in the human body, to sustain the gaseous state of the chemicals, without quickly becoming aerosols or adhering to dust particles and entering into the body as such. It is, therefore, difficult to accept the hypothesis that ETS can induce adenocarcinoma in the periphery of the lung, because carcinogens in the sidestream smoke, inhaled in their gaseous state, via the nasal passage can penetrate deeper into the lung than the mainstream smoke inhaled by mouth. Studies that found an ETS-squamous cell carcinoma association have very few positive cases to report. For example, Garfinkel (12) in his 134 nonsmoking lung cancer cases and 402 colon cancer controls, found the husbands' smoking was an important factor in the wives' squamous cell carcinoma; but found only I 1 cases of squamous cell carcinoma, together with 87 cases of adenocarcinoma. Dalager (14) in his 48 lung cancer cases and 446 controls, found ETS, like active smoking, was an important factor in squamous cell carcinoma and small cell carcinoma; but reported only 4 cases of squamous cell carcinoma and small cell carcinoma as being associated with exposure to spousal smoking. Pershagen (19), in a correspondence survey of 27,409 nonsmoking Swedish women, found that wives of smoking spouses had a higher relative risk of squamous cell carcinoma and small cell carcinoma. Yet in 20 years he found only 20 cases of squamous cell and small cell carcinomas. Recently Trichopoulos (32), in an autopsy study, examined lung specimens for basal cell hyperplasia, cell atypia, and (in membranous bronchioles and bronchiolo-alveolar airways) mucous cell metaplasia, i.e. pathological entities that may be lung cancer risk indicators or epithelial, possibly precancerous, lesions (EPPL). They also measured the bronchial and mucous gland thickness (G) and the bronchial wall thickness (W) and used the ratio G/W (Reid Index) to evaluate the effects of active and passive smoking. Results show that when nonsmoking women are married to smokers, their EPPL and Reid Index are higher than those of nonsmoking women who are married to nonsmokers. Therefore, they support the view that ETS is a risk factor for lung cancer. In this study, there are 31 cases of nonsmoking women, among them, 17 are married to smokers, 13 to nonsmokers, with 11 cases being excluded from analysis due to inadequate information. The odds ratio (OR) for an EPPL score of 60 or more contrasted to an EPPL score of less than 40, was 4.4 for the active smokers (compared with nonsmokers), whereas among nonsmoking women the OR was 6.0 for those married to smokers (compared with those married to nonsmokers). In other words, exposure to spousal smoking carries a higher lung cancer risk than engaging in active smoking. The result is indeed puzzling. In the same study 4 heavy smokers among the lung cancer cases were reported; their respective EPPL values are 29, 53, 142 and 253, with an average value of 119; but the standard deviation is as high as 88. The value of EPPL for heavy smokers was as low as 29. Thus, the significance of using an EPPL value lower than 40 as an indicator of lung cancer risk can be open to question. I I I I I I I I I I I I , OP~ 0 i V O ! W W O N -4-

I I I I I I I I I I I I I I I I i 44. Jain M. Burch JD. Howe GR. Miller AB: Dietarv factors and risk of lung eancer. Results from a casetonuol studv, Toronto. t981-85. 1nt 1 Cancxr 45:549-553. 1990. 15. Packer JE. Mahood 1S. Mora-Arellano VO. et at: Free radicals and sineiet oxygen scavengers. Biochem Biophvs Res Commun 98:901-906. 1981. 46. Malone WF: Studies evaluating antioxidants and betataro- tene as chemopreventives. Am J Clin Nutr 53:305S-3135. 1991. 47. Omenn GS. Goodman GE. KJeinman GD. et at: The role of intervention studies in asartaimne Ihe contribuuon of die- tary factors in lung cancer. Ann NY Acad Sci 534:575-583. 1988. 48. Kolonei LN. Hankin JH, Lee J. et at: Nutrient intakes in relation to cancer tnndrnce in Hawaii. Br J Cancer 44:332- 339, 1981. 49. Wynder EL- Hebert JR. Kabat GC: Association of dietary fat intake and lung cancer. JNCI 79:631-637. 1987. 50. Xie 1, Lesaqre E. Kesseloot H: The rciauonship of animal fat intake. ctgarette smoking, and lune cancer. Cancer Causes Control 2:79-83, 1991. 51. Wynder EL-Taioli E. Fujita Y: Ecolo¢ic stuav of lung cancer risk factors in US and Japan, with spcnai ref erence to smoKUtg and diet. lanan J Cancer Rcs 83:418=23. 1992. ::. Taioii E. Nicolost A. Wvnder EL: Posstbic roic oi dlet as a host actor in me euotoey oi tobaceo-mduad tune cancec An eco- logtcal study in southern and nonhem ltaly. (nt 1 Eptdemtoi 20:611-614. 1991. 53. Hinds MW. Kolonel LN- Lee J. et ai: Dietarv choiesterol and lung cancer risk among men in Hawaii. Am I Clin Nutr 37. [92-193. 1983. 54. Goodman MT. Kolonel LN, Yoshizawa CN. et al: The effect of dictarv, cholesterol and fat on the risk of lung cancer tn Hawaii. Am J Epidemtoi 128:1241-1255. 1988. 55. Byers TE. Graham S. Haughey BP. et at: Diet and lung cancer: Findings from the Western New York Diet Studv. Am J Epidemtot 125:351-363. 1987. 56. Knekt P. Seppanen R. Jarnnen R. et at: Dietary cholesterol. fatty aads. and the risk of lung cancer among men. Nutr Cancer 16:267-275, 1991. 57. Shekelle RB. Rossof AH. Stamler J: Dietarv cholesterol and incidence of lung caneer. The Western Electric Study. Am J Enidemwi 134:480-484. 1991. :3. Knekt P. Heliovaara M. Rissanen A. et at: Leanness and lung cancer nsK. Int J Cancer 49:208-213. 1991. 59. Kabat GC. Wvnder EL: Bodv mass mdcx and lung cancr nsx. Am I Epidemtoi 135:769-774. I992. 60. Pouer JD. McMichael AJ: Alcohol. beer and lung cancer-A meamnaful reladonship? tnt J Epidemml 13:'-40-242. 1984. Epidemioiogy of Lung Cancer 79 61. Stockwell HG. Matanowski GM: A case-control studv of lung cancer in painters. J Occup Med 27:125-126. 1985. 62. Williams RR. Horm JW: Association of cancer sites with to- baceo and alcohol consumouon and soaoeconomtc status of patients: Interview study from the Third National Canmr Sur- vey. JNCI 58:525-547. 1977. _ 63. Stemmerman GN. Nomura AMY. Chvou PH- et at: Prostuc- tive studv of alcohol intake and laree bowel cancer. Digesc Dis Sci 35:1414-1420, 1990. 64. Cagle PT. Modv DR. Schwartz MR: Estrogen and progester- one reoeotors in broncbogentc carnnoma. Cancer Res 50: 6632-6635, 1990. 65. Anaeeers 1F. Malkasian GD: Patterns of other neonlasia in patients with endometriaL carctnoma. Cancer 48:856-859. 1981. 66- Adami H-O. Persson 1. Hoover R. et al: Risk of cancer in women receiving hormone repiaeemrnt therapy. Int 1 Cancer 44:833-839. 1989. 67. Gao Y. Blot W. Zheng W, et at: Lung cancer among Chinese women. Int J Cancer 40:604-609. 1987. 68. Wu AH. Yu MC. Thomas DC. et al: Personal and familv history of lung disease as risk factors for adenocarctnoma o[ the lung. Cancer Res 48:7279-7284, 1988. 69. Sellers TA. Bailev-Wilson JE. Elston RC. et at: Evidence of Menaeiian mhentance in the pamo¢enes¢ oi lung cancer. J J N C I 8 2:1272 - L2 7 9. 1990. -0. McDume HH: Clustenne of cancer m families of patients with prmtarv lune cancer. 1 Clin Epidcauol 44:69-71. 1991. "I. Lynch HT. &imberlin¢ WJ. Markvska SE. ct al: Genctta and smoiung-auomatcd canccrs: A study of 485 families. Cancer 57:1640-1646. 1986- 7'-. Ayesh R. Idle JR. Ritchie JC. et al: Metabolic oxidation phenotypes as markers for susceptibility to lung cancer. Nature fLond1.312:i69-170, 1984. "3. Tokuhata GK. Lilicnfeld AM: Familial aggregation of lung can¢r in humans. 1NCi 30.289-312. 1963. 74. Ooi WL. Eiston RC. Chen VW. et at: Increased familial risk for lung cancer. JNCI 7b:217-17~ 1986. 'S. Mulvihill !J: Host factors in human tumors: An example of cco-genencs in oncology. JNCI 57:3= , 1976. 76. Law MR: Genetic predisposition to lung cancer. Br J Cancer 61:195-206. 1990. 77. Hoist PA. Kromhout D. Brand R: Pet birds as an mdepenaent nsk factor for tune cancer. Br Med J 297:1319-1321. 1988. '8. Kohlmner L. Amtmeer G. Bartolomevnk S. ct at: Pet birds as an macoenocnt nsk factor tor lune cancer. Case-control studv. Br Med J 305:986-989. 1992. 79. Gardiner AJS. Forev BA. Lee PN: Avian cxoosure and bron- choeentc carcinoma. Br Mcd 1 305:989-992. 1992. I

Janerich et al. Spousal ETS not associated with an increased lung cancer risk; approximately 17% of lung cancers among nonsmokers estimated to be attributed to high levels of exposure to cigarette smoke during childhood and adolescence . Capewell et al. More nonsmokers had adenocarcinoma than smokers (42% v. 13%) and fewer had squamous cell carcinoma (32% v. 49%) or small cell carcinoma (15% v. 24%). He et al. No positive association of lung cancer and passive smoking was found. Fontham et al. A 30% increased risk of lung cancer was associated with exposure to ETS from a spouse, and a 50% increase was observed for adenocarcinoma of the lung. Trichopoulos et al. The lung cancer risk indicators (EPPI and Reid index) values were significantly higher among deceased nonsmoking women married to smokers than those married to nonsmokers. Hecht et al. Nonsmokers exposed to sidestream cigarette smoke take up and metabolize a lung carcinogen, which provides experimental support for the pproposal that ETS can cause lung cancer. -8- Yes 28 I I ! f ! Yes 29 ~ No 30 ~ Yes 31 ~ No 32 ~ No 33 I I I I I 1 N O O -L N O W ta O I I S 01

Table 1. Studies of Spousal ETS and Lung Cancer in Nonsmokers Study Conclusions Garfinkel Very little, if any, increased risk of lung cancer Trichopoulos et al. Study of female nonsmokers with lung cancer other than adenocarcinoma. RR for married to < 1 pack/day smokers is 2.4; for > 1 pack/day smokers is 3.4. Hirayama Nonsmoking wives of heavy smokers have a higher risk of lung cancer. Chan & Fung No association between ETS and female lung cancer. Correa et al. More studies are needed to demonstrated the role of ETS in the development of lung cancer. Garfinkel More studies are needed to demonstrate the role of ETS in the development of lung cancer. Kabat & Wynder No effect; need further investigation. Hirayama et al. The RR of lung cancer in nonsmoking wives were 1.00, 1.36, 1.42, 1.58 and 1.91, when husbands were nonsmokers, ex- smokers, or daily smokers of 1-14, 15-19, or 20, or more cigarettes daily, respectively. Koo et al. Passive smoking was not found to be associated with a significant increase in risk for lung cancer. Wu et al. Spousal ETS has a slightly elevated, but insignificant, RR for female adenocarcinoma. Sandler et al. Elevated risks were seen for several specific cancer sites and were not limited to lung cancer. Garfinkel et al. A logistic regression analysis showed a significant positive trend of increasing risk with increased exposure to the husband's smoking at home. Lee et al. Among lifelong nonsmokers, passive smoking was not associated with any increase in risk of lung cancer. Dalager et al. The elevated risk associated with spousal smoking was restricted to squamous and small cell carcinoma (OR=2.9, 95%C1 0.9-9.3). Cell ! Type Examined? Ref I Yes 1 Yes 2 ~ No 3 1 Yes 4 I No 5 No 6 ~ Yes 7 ~ No 8 ~ Yes 9  Yes 10 I No 11 I Squamous cell Adeno- carcinoma 12 I No 13 Yes 14 r 3 -4 -6- 00 w w O

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I I I 1 I Akiba et al. ETS exposure may increase the risk of lung cancer among nonsmokers. Blot & Fraumeni et al. Long term exposure to ETS increases the risk of lung cancer, however etiologic role of passive smoking needs to be evaluated. Humble et al. Never-smokers married to smokers had about a two-fold increase of risk of lung cancer. Koo et al. RR based on the husbands' smoking habits showed no apparent increase. Pershagen et al. RR=3.3 for squamous cell and small cell carcinoma in woman married to smoker. Lam et al. Among never smoking woman, RR for passive smoking due to a smoking husband was significantly increased. Lee Bias caused by misclassification of smoking habits coupled with between-spouse smoking habit concordance can completely explain reported apparent excesses in lung cancer risk in nonsmokers married to smokers. [noue and Hirayama Passive smoking has come to be suspected as the possible causative factor of lung cancer in woman. Shimizu et al. Elevated RR of lung cancer was observed for ETS from mother (RR=4.0) and from father (RR=3.2). No association was observed between the risk of lung cancer and smoking of husband or ETS exposure at work. Koo ETS and lung cancer might be just a matter of 'smoke', and the real culprit may be what is cooking over the fire as the etiological factors accounting for the excessively high lung cancer rate. Svensson et al. Only 38 cases had never been regular smokers and the risk estimates for exposure to ETS were inconclusive. The high RR of small cell and squamous cell carcinoma associated with smoking may have implications for risk assessments regarding passive smoking. Wu-Williams et al. The lowered risk associated with a spouses who smoked was seen. Kalandidi et al. Marriage of a nonsmoking woman to a smoker was associated with an increased risk of lung cancer (RR=2.1, 95% CL=1.1-4.1). -7- Squamous 15 and small cell carcinoma No 6 No 17 Yes k8 Yes 19 Yes 20 No 21 No 22 No 23 No 24 Yes 25 Yes 26 No 27

adenocarcinoma cells is higher than other lung cancer cell types (Kabayashi). Still others have suggested that female lung cancer may be related to use of oral contraceptives (Wang Sheng-yong). Physiologists have long recognized that the lung is not only an air exchange organ but also has endocrine functions. Further research into the relationship between estrogen disturbance and lung cancer, especially in relation to adenocarcinoma which is known to have mucus secreting characteristics, is urgently needed. Research aimed at examining endocrine disturbances in relation to lung cancer must proceed in parallel with the research on the cause of the rapid increase of lung cancer. Without the understanding the etiology of a disease, the effective prevention of the disease cannot proceed. Research into the etiology of lung cancer should be considered as the key to halt the rapid rise of lung cancer. -6- I I I I I I I I I I I I I I I

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I 15. Akiba, S.; Kato, H. and Blot, W.J.; "Passive smoking and lung cancer among Japanese women," Cancer Research 46: 4804-4807, 1986. 16. Blot, W.J. and Fraumeni, J.F.; "Passive smoking and lung cancer," Journal of National Cancer Institute 77(5): 993-1000, 1986. 17. Humble, C.G.; Samet, J.M. and Pathak, D.R.; "Marriage to a smoker and lung cancer risk," American Journal of Public Health 77(5): 598-602, 1987. 18. Koo, L.; Ho, J.H. -C.; Saw, D. and Ho, C. -Y.; "Measurements of passive smoking and estimates of lung cancer risk among nonsmoking Chinese females," International Journal of Cancer 39: 162-169, 1987. 19. Pershagen, G.; Hrubec, Z. and Svensson, C.; "Passive smoking and lung cancer in Swedish women," American Journal of Eoidemiologv 125(1): 17-24, 1987. 20. Lam, T.H.; Kung, I.T.M.; Wong, C.M.; Lam, W.K.; Kleevens, J.W.L.; Saw, D.; Hsu, C.; Seneviratne, S.; Lam, S.Y.; Lo, K.K. and Chan, W.C.; "Smoking, passive smoking and histological types in lung cancer in Hong Kong Chinese women," British Journal of Cancer 56(5): 673-678, 1987. 21. Lee, P.N.; "Lung cancer and passive smoking association: an artifact due to misclassification of smoking habits," Toxicology Letter 35(1): 157-162, 1987. 22. Inoue, R. and Hirayama, T.; "Passive smoking and lung cancer in women" Smoking and health 1987, eds. M. Aoki, S. Hisamichi and S. Tominaga (Amsterdam: Excerpta Medica, 1988): 283- 285. 23. Shimizu, H.; Morishita, M.; Mizuno, K.; Masuda, T.; Ogura, Y.; Santo, M.; Nishimura, M.; Kunishima, K.; Karasawa, K.; Nishiwaki, K.; Yamamoto, M.; Hisamichi, S. and Tominaga, S.; "A case-control study of lung cancer in nonsmoking women," Tohoku Journal of Experimental Medicine 154: 389-397, 1988. 24. Koo, L.G.; "Environmental tobacco smoke and lung cancer: Is it the smoke or the diet?" Present and Future in Indoor Air Quality, Proceedings of the Brussells Conference 14-16, February 1989, ed. C.J. Bieva, Y. Courtois and M. Govaerts (Amsterdam: Excerpta Medica, 1989): 65-75. 25. Svensson, C.; Pershagen, G. and Klominek, J.; "Smoking and passive smoking in relation to lung cancer in women," Acta Oncoloeica 28(5): 623-629, 1989. 26. Wu-Williams, A.H.; Dai, X.D.; Blot, W.; Xu, Z.Y.; Sun, X.W.; Xiao, H.P.; Stone, B.J.; Yu, S.F.; Feng, Y.P.; Ershow, A.G.; Sun, J.; Fraumeni, J.F. and Henderson, B.E.; "Lung cancer among women in north-east China," British Journal of Cancer 2: 982-987, 1990. -10- 1 IV 0 I I I I I I I I I I I

I I I 27. I 28. , 29. ~ 30. , 31.  I 32. 33  . I 34. I I I I I 1 I Kalandidi, A.; Katsouyanni, K.; Voropoulou, N.; Bastas, G.; Saracci, R. and Trichopoulos, D.; "Passive smoking and diet in the etiology of lung cancer among nonsmokers," Cancer causes and control 1: 15-21, 1990. Janerich, D.; Thompson, W.D.; Varela, L.R.; Greenwald, P.; Chorost, S.; Tucci, C.; Zaman, M.B,; Melamed, M.R.; Kiely, M. and McKneally, M.F.; "Lung cancer and exposure to tobacco smoke in the household," The New England Journal of Medicine 323: 632-636, 1990. Capewell, S.; Sankaran, R.; Lamb, D.; McIntyre, M. and Sudlow, M.F.; "Lung cancer in lifelong nonsmokers," Thorax 46(8): 565-568, 1991. He, X.Z.; Chen, H.; Chen, W. and Chapman, R.S.; "A case-control study on risk factors of lung cancer in nonsmoking women," Lung Cancer supp. 7: 6, 1991. Fontham, E.T.H.; Correa, P.; Wu-Williams, A.; Reynolds, P.; Greenberg, R.S.; Buffler, P.A.; Chen, V.W.; Boyd, P.; Alterman, T.; Austin, D.F.; Liff, J. and Greenberg, S.D.; "Lung cancer in nonsmoking women: A multicenter case-control study," Cancer Epidemiolot+y. Biomarkers and Prevention 1: 35-43, 1991. Trichopoulos, D. et al.; "Active and passive smoking and pathological indicators of lung cancer risk in an autopsy study," Journal of American Medical Association 268(13): 1697-1701, 1992. Hecht, S.S.; Carmella S.G.; Murphy S.E. et al.; "A tobacco-specific lung carcinogen in the urine of men exposed to cigarette smoke," New England Journal of Medicine 329: 1543-1546, 1993. Wynder, E.L. and Goodman, M.T.; "Smoking and lung cancer: some unresolved issues," Epidemiologv Review 5: 177-207, 1983. I

I I I I I I I I I I I 1 I I I I i I Table 3. Comparison of Lung Cancer Cell Types Between Male/Female Smokers and Nonsmokers in Both Male and Female Cases* M ale Fe male Smoker Nonsmoker Smoker Nonsmoker No. % No. % No. % No. % Squamous cell carcinoma 438 59.03 25 40.32 51 36.17 33 22.30 Small cell carcinoma 56 7.55 2 3.23 12 8.51 12 8.11 Adenocarcinoma 170 22.91 22 35.48 57 40.43 83 56.08 Large cell carcinoma 14 1.80 1 1.60 1 0.71 1 0.67 Others 64 8.62 12 19.36 20 14.18 19 12.84 TOTAL 743 100.00 62 100.00 141 100.00 148 100.00 * A total of 1094 (male 805, female 289) cases were used. 1. Smoking and Female Lung Cancer. Smoking is generally recognized as an important risk factor for lung cancer in both men and women. However, the relative significance of smoking in female lung cancer seems to be lower than that for male lung cancer. Because a large number of female lung cancer patients are nonsmokers, the odds ratio (OR) for smoking in women is lower than the OR for men. When the cell types of smoking and nonsmoking male and female patients are compared, the results show that among male smokers, the rate of squamous cell carcinoma is decidedly higher than that of the adenocarcinoma, but among female smokers the rates are similar. Among nonsmokers, the squamous cell carcinoma and adenocarcinoma rates are similar in men, but the adenocarcinoma rate is much higher than the squamous cell carcinoma rate for women. (Table 4) Table 4. Comparison of Sqtramous Cell Carcinoma and Adenocarcinoma in Male and Female Smokers* Male Female Smoker Sqm (59.03) > Ade (22.91) Sqm (36.17) _ Ade (40.43) * A total of 1094 (Male 805, female 289) cases were used. The results clearly show that in men smoking may induce squamous cell carcinoma; but in women, there must be other risks for the high incidence of adenocarcinoma. -3- !

2. Exposure to ETS and Female Lung Cancer. This is a difficult subject for research which will yield accurate results. First, lung cancer has a long latency; it usually takes more than 10 years for active smokers to develop lung cancer. It is not unreasonable to assume that it may take even longer for the effects of ETS exposure to be observed, if ETS is capable of inducing lung cancer. During such a long latency, data and conditions of the nonsmokers' exposure, such as numbers of cigarettes smoked by the smokers, the extent of close contacts with the smokers, the conditions of the shared living space, can be dynamic and variable. Moreover, in order to obtain accurate results on the effects of ETS exposure on lung cancer, not only must the study subjects be truly nonsmokers, other risk factors or confounders, such as effects of air pollution and occupational exposures, must be excluded. All these elements are realistically difficult to control. Secondly, since the smoking-related lung cancers are known to be squamous cell carcinoma in the center of the lung, it follows that ETS-related lung cancers should also be of the same cell type, not the adenocarcinoma located in the periphery of the lung which is the prevalent cell type in female lung cancer. This contradiction notwithstanding, it needs to be noted that although the relationship between ETS and lung cancer has not been established at the present, ETS still should not be dismissed as a health risk. 3. Indoor Air Pollution and Female Lung Cancer. Reports from many areas of China have clearly demonstrated a significant relationship between indoor air pollution and lung cancer in women. A major source of indoor air pollution is coal-burning for cooking and heating. Our combined epidemiological survey and laboratory study demonstrated that female lung cancer is likely to be related to indoor air pollution, because indoor coal-burning increases B(a)P concentration in the indoor air. Housewives in coal-burning households are shown to have significantly higher levels of urine B(a)P than housewives in households using liquefied gas for cooking. Our correlated epidemiological studies also indicate that indoor air pollution is associated more with female lung cancer than with male lung cancer. In addition to coal-burning as a source of indoor air pollution, Gao and coworkers also reported a relationship between lung cancer and pollutants generated by certain cooking oil and cooking practices. (Table 5) -4- ? I I I I I I I I I I I I I I I I

I I I I I I I I I I I I I / I I 1 I ETIOLOGY OF LUNG CANCER IN WOMEN Du Yine-xiu Guangzhou Research Center for Lung Cancer, Guangzhou, China Epidemiological studies have shown that there are more male smokers than female smokers, yet female lung cancer rates remain very high. For example, for the over-age-15 population in Guangzhou, 65 % of the males, but only 5% of the females are smokers. (Table 1) Yet the lung cancer death rate for many years has maintained a male to female ratio of 1.8:1.0, implying that in female lung cancer there may be other risk factors beside smoking. Table 1. A Comparison of Smoking Rates with Male/Female Lung Cancer Death Rates Smoking Rate Lung Cancer Death Rate Male Female Relative Ratio Male Female Relative Ratio Guangzhou 65,000/105 5,000/105 13.0 45/105 25/105 1.8 In addition to active smoking, many studies have emphasized the importance of environmental tobacco smoke (ETS) as a risk factor for lung cancer. Since case studies have shown that over 50% of the female lung cancer cases are nonsmokers, leaving aside the question of whether ETS-exposure is actually related to lung cancer, it is hardly reasonable to attribute 50% of all nonsmoking female lung cancers to exposure to ETS. Clearly, further research on all the potential risk factors for female lung cancer is needed. It is generally accepted that the etiology of a disease is closely related to the mechanism of disease development; thus, in any etiology research, the methodology should include data obtained from both epidemiological and experimental results. Lung cancer mortality rates have risen dramatically in recent decades. One explanation is that lung cancer is caused by environmental carcinogens, since the influence of genetic factors is usually relatively constant and rarely triggers sudden changes in a relatively short time. Since there is an apparent difference in the relative distribution of cell types between male and female lung cancers, it seems possible that different mechanisms are involved in the induction of different histological types of lung cancer. At the same time, some studies have reported the tendency of lung cancer to be clustered in families. Whether the rapid rise in lung cancer death rate is due to external or to endogenous factors or, alternatively, to their interactions, remains to be investigated. There are as many as 13 cell types in lung cancer, the four most common of which are squamous cell carcinoma, adenocarcinoma, small cell carcinoma and large cell carcinoma, with squamous cell carcinoma and adenocarcinoma constituting more than 80% of the total lung cancer cases. These two malignant cell types have many different biological characteristics. (Table 2) I

' Table 2. Comparison of some Biological Characteristics between Squamous Cell and Adeno Cell Carcinoma of Lung Squamous Cell Adenocarcinoma Etiology Frequently seen in cigarette smokers and Frequently seen in women patients with chronic bronchitis Cell Origin Arises from bronchial epithelium and has Arises from mucous cell of the undergone squamous metaplasia bronchial glands or lung alveolar cell Morphology Cells are in sheets, cords and bundles The neoplasm is composed of columnar separated by varying amounts of vascular cells and usually with mucin-containing connective tissue vacuoles in many cells Tumor location Located in the larger bronchus near the Located in the smaller or smallest hilum and at central sites within the lung bronchus and at peripheral sites of lung Doubling times 100 days 187 days Stains Keratin + - Mucin - + K-ras oncogene Mutation in squamous cell needs further Mutation frequently seen in study, but never seen in small cell adenocarcinoma of lung carcinoma Biochemical characteristic Low serum cytokeratin-19 level High serum cytokemtin-19 level, capable of endocrine secretion Cell Membrane Receptors Low positive estrogen receptors High positive estrogen receptors The cell type differences in male and female cancer patients have clinical significance. Squamous cell carcinoma is the most frequently seen lung cancer cell type in males (approximately 55 % of the total cases), with adenocarcinoma being the next most frequent (approximately 25 % of the cases). (Table 3) The reverse is true in female patients: the most frequently seen cell type is adenocarcinoma (approximately 60% of the cases), with the next most frequent being squamous cell carcinoma (approximately 25 % of the cases). It is apparent, then, that in the investigation of the etiology of female lung cancer, the research focus should be on the adenocarcinoma. Furthermore, as many recent reports have indicated, proportion of lung adenocarcinoma continues to rise in lung cancers, emphasizing the vital importance of the cell type to the research in lung cancer. _2_ I I I I I I I I I I I I I I I I

a I 1 I I I I I I I 1 i I I REFERENCES 1. Garfinkel, L.; "Time trends in lung cancer mortality among nonsmokers and a note on passive smoking," Journal of the National Cancer Institute 66: 1061-1066, 1981. 2. Trichopoulos, D.; Kalandidi, A.; Sparros, L. and MacMahon, B.; "Lung cancer and passive smoking," International Journal of Cancer 27(1): 1-4, 1981. 3. Hirayama, T.; "Nonsmoking wives of heavy smokers have a higher risk of lung cancer: a study from Japan," British Medical Journal 1, 282: 183-185, 1981. 4. Chan, W.C. and Fung, S.C.; "Lung cancer in nonsmokers in Hong Kong," Cancer Campaign Vol. 6. Cancer Epidemiology, ed. E. Grundmann (Stuttgart: Gustav Fischer Verlag, 1982): 199-202. 5. Correa, P.; Pickle, L.W.; Fontham, E.; Lin, Y. and Haenszel, W.; "Passive smoking and lung cancer," The Lancet II: 595-597, 1983. 6. Garfinkel, L.; "Passive smoking and cancer -- American experience," Preventive Medicine 13(6) 691-697, 1984. 7. Kabat, G. and Wynder, E.; "Lung cancer in nonsmokers," Cancer 53: 1214-1221, 1984. 8. Hirayama, T.; "Cancer mortality in nonsmoking women with smoking husbands based on a large- scale cohort study in Japan," Preventive Medicine 13: 680-690, 1984. 9. Koo, L.; Ho, J.H.-C. and Lee, N.; "An analysis of some risk factors for lung cancer in Hong Kong," International Journal of Cancer 35(2): 149-155, 1985. 10. Wu, A.H.; Henderson, B.E.; Pike, M.C. and Yu, M.C.; "Smoking and other risk factors for lung cancer in women," Journal of the National Cancer Institute 74(4): 747-751, 1985. 11. Sandler, D.P.; Everson, R.B. and Wilcox, A.J.; "Passive smoking in adulthood and cancer risk," American Journal of Epidemioloev 121(1): 37-48, 1985. 12. Garfinkel, L.; Auerbach, 0. and Joubert, L.; "Involuntary smoking and lung cancer: a case- control study," Journal of the National Cancer Institute 75(3): 463-469, 1985. 13. Lee, P.; Chamberlain, J. and Alderson, M.R; "Relationship of passive smoking to risk of lung cancer and other smoking-associated diseases," British Journal of Cancer 54: 97-105, 1986. 14. Dalager, N.A.; Pickle, L.W.; Mason, T.J.; Correa, P.; Fontham, E.; Stemhagan, A.; Buffler, P.A.; Ziegler, R.G. and Fraumeni, J.F.; "The relation of passive smoking to lung cancer," Cancer Research 46(9): 4808-4811, 1986. -9-

I I I I I I I I I I 1 I I I I I I , Table 5. Comparison of Air Pollutants and Urine B(a)P Levels in Housewives Coolung with Coal or Propane Cooking With Coal Cooking With Propane Gas Coal/Propane S02 (µ/M3) 279 58 4.81 NOx (µ/M3) 3 76 63 1.21 CO (p/M ) 9,424 2,340 0.03 TSP (µ/M3) 332 188 1.77 SD (glM2/month) 12 5 2.40 B(a)P ((u/100M3) 11.9 2.2 5.41 Radon (Bq/M3) 18.6 16.6 1.12 Thoron (Bq/M3) 42.5 28.3 1.50 Urine-B(a)P (ng/t) 4.0 2.8 1.43 However, many questions remain unanswered: coal has been in use for cooking and heating and vegetable oil has been used for frying for many years; why, then, have lung cancer rates been on the rise only during the past 20 or 30 years? Environmental carcinogenic chemicals have been known to induce squamous cell carcinomas, but why are female lung cancers predominantly adenocarcinomas? Since indoor air pollution sources and characteristics are different for industrialized and developing countries, why have all nations experienced similar trends of higher female lung cancers, dominated by adenocarcinomas? 4. History of Respiratory Disease in Female Lung Cancer. Several epidemiological studies have indicated that lung cancer patients often have a history of bronchitis. This is easy to understand in the case of women, since in cooking, women are more likely to be exposed to smoke from burning-coal and other irritants generated by deep frying and stir-frying. A question that begs for our attention is that it has been reported that the disturbance of the microbial population may cause the metabolic disturbance of the intestine and lead the metabolized procarcinogens to be activated as ultimate carcinogens, which, in turn, can induce colon cancer. In the case of lung cancer, can large doses of antibiotics used to combat chronic bronchitis result in the disturbance in the microbial population in the lung, causing the procarcinogens in the lung to be activated as ultimate carcinogens? 5. Estrogen and Female Lung Cancer. Estrogen disturbance and female lung cancer may be a question worthy of our consideration. It has been reported that prophylactic use of estrogen for heart disease is correlated with an increase in lung cancer incidence. Some researchers consider early menarche, long menstrual periods, shortened menstruation cycles and delayed menopause as some of the risk factors of female lung cancer (Gao Yu- tang, Liao Mei-lin). Others have reported that the level of estrogen receptors on the surface of -5- t

I 78 Kabat preventable disease. In view of its preventability. ef- forts aimed at reducing the prevalence of smoking in developed countries should be intensined and new initiatives under the ae¢is of the World Health Oreani- zation undertaken to reverse the expansion of tobacco use in developing countries. ACKNOWLEDGMENTS The author is grateful to Dr. Emanuela Taioli for comments on the manuscript. REFERENCES I. Boring CC. Squires TS. Tone T: Cancer Statistics. 1992. CA 42:19-38. 1992. 2. Parkin DM: Trends in lune_ cancer incidence worldwide. Chest 96:5S-8S. 1989. 3. Devesa S5. Shaw GL. Blot WI: Changing patterns of tune cancer incidence by histolomcai tvpe. Cuncer Epidemml Bi- omarkers Prev: 1:29-34. 1991. 4. Stockwell HG. Armstrone AW. Lcavenon PE: Histooa- thology of tune canccri among smokers and nonsmokers in Florida. Int J Epidcmtol 191supol 11:548-S52. 1990. 5. Fontham ETH. Correa P. Wu-WiOiams A. et at: Lung cancer in nonsmoking women: A muiucemer case-conuot stud_v. Can- ccr Epidemtol Biomarkcn Prey 1:35-43.1991. 6. Wynder EL. Goodman MT: Smoking and lung cancer: Some unresolved tssues. Epidemtol Rev 5:177-207. 1983. 7. Yang CP. Gallagher RP. Weiss NS. et al: Differences in ma- dena rates of cancers of the resotratorv tract by anatomic substte and histologtc type: An etiologic tmpiicauon. JNCI 81:1828-1831. 1989. 8. SteBman SD: Interactions between smoking and other exoo- sures. ln Hoffmann D. Harrts C(cdsl: Banburv Reoon No. 23. "Mechantsms of Tobacco Caranogenests." Cold Spnng Har- bor. New York: Cold Spnne Harbor Laboratory. 1986. 377- 393. 9. Archer VE. Wagoner JK. Lundin FE: Uranium mining and nearette smoxm_e eriects on man. 1 Occup Med 15:204-211. 1973. 10. Hammond EC. Selikoff IJ. Seidman H: Asbesms exoosure. _ ctgarette smokm¢ and deam ates Ann NY Acad Sci 330-473- 19. Wynder EL. Kabat GC: Effect of low-vicid ciearette smoking on tune cancer risk. Cancer 62:1=-1230. 1988. :0. Kabat GC. Morabta A. Wynder EL: Comparison of smoking habits of blacks and whites tn a case-control study. Am 1 Public Health 81:1483-1486. 1991. 21. Kabat GC. Heben JR: Use of mentholated cigarettes and tune cancer risk. Cancer Res 51:6510-6513. 1991. '-L Resnicow K. Kabat G, Wynder EL: Progress in decreasing ctgarette smoking. in DeVita VT. Heiman S, Rosenberg SA ledsl: "Important Advances in Oncoiogy." New York: 1.B. Lippincott. 1991. 205-213. 73. Walter HL Vaughan RD. Wvnder EL: Primary prevention of cancer among children: Changes in eearetc smoking and diet after six years of interventton. JNCI 81:995-999. 1989. 24. Fraumeni JF. Blot WJ: Lung and pleura. In Schottenfeld D. Fraumeni JR (eds): "Cancer Epidemioloey and Prevenuon." Philadelphia: W.B. Saunders. 1982. 564-582. '_5. Tomatts L. Aiuo A. Wilboum J. Shuker L: Human carnno- gens so far identified. Jpn 1 Cancer Rcs 80:795-807. 1989. 36. Peto R. Schnetderman M: "Quantification of Occupational Cancer." Banbury Report No- 9. Cold Spring Harbor. New York: Cold Sprlne Harbor Laboratory. 1981. _. Doll R. Peto R: "The Causes of Cancer. Quantitative Esti- mates oi Avoidable Risks of Cancer in the United States Today." Oxford: Oxford University Press. 1981. Tomaus L: Outdoor atr pollution and tune canccr. Ann Oncol 2:265-267. 1991. =9. Persna8cn G. Simonato L: Epidemmlo0cai evidence on air pollunon and cancer. !n Tomaus L/ed): "Air Pollution and Human Cancer. European School of Oncology Monographs." Berlin: Spnnger-Verlag. 1990. 63-74. 30. Hirayama T: Non-smokmg wives of heavy smokers have a higher nsk of tung cancer. A study from Japan. Br Med J ?82:183-185. 1981. 31. Trichopoulos T. Kalandidi L. Sparros L. McMahon B: Lung cancer and passive smoking. Int J Cancer 27:1-4, 1981. National Arademv of Sdences: "Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effecu." Washtn¢ron, D.C.: Nauonai Academy Press. 1986. 33. Koo LC. Ho 1H-C. 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I Materials and Methods 1. Retrospective survey of lung cancer deaths. Guangzhou has a population of approximately 2 million, who live in four city districts and who are served by 63 local police stations. Each local police station keeps complete and systematic records of the residents, which include population characteristics such as the number, sex, age, occupation, street address of the residents, and the annual registration of births and deaths, (including time and cause of death). We have conducted retrospective surveys of all lung cancer deaths between 1980-1988 by interviewing the relatives of the decedents using standardized questionnaires. Surveys were conducted once each year (to cover lung cancer deaths that occurred in the previous year) by trained medical staff who also examined the hospital records prior to death. All data were entered into computers to establish a database. The data fields are as follows: name, sex, city, date of birth, date of death, prior native city, proximity to sources of pollution, education, marital status, religion, date of initial diagnosis of lung cancer, where diagnosed, level of diagnosis, method of diagnosis, cell type, tumor site, lung involved, metastasis, site of metastasis, medical history, smoking history, smoking members of household, family status, kitchen facilities, diet history, occupational history, family tumor history, relative giving the information, hospital record and cooperation of the relative providing the information. 2. Atmospheric pollution. The geographic area of Guangzhou is approximately 55 square kilometers, divided into the four districts of Liwan, Yuexiu, Dongshan and Haizhu. From 1972 to 1977, the Guangzhou Health and Antiepidemic Station established 30 atmospheric pollution sampling stations and tested the atmospheric content of S02 and sedimented dust (SD). SO2 (µg/m3) was analyzed using the hydrochloric-rosaniline method from seasonal samples, collected during 5 consecutive days, 3 samples a day, once every month. The samples were collected in glass containers, 15 cm diameter and 30 cm height, placed on roof-tops. The naturally sedimented dust was measured by weight in ton/km2/month. As part of the WHO's global monitoring plan, a reference station and four sampling stations were established in Liwan, Yuexiu, Dongshan, Haighu to study the total suspended particulates (TSP) and SO2 in 1981-1990. SO2 and TSP, continuously collected from the first to the 15th day of each month, were analyzed according to WHO's criteria.(6) The databases from these two tests were quite large. The following formula was used to calculate the air pollution index of various localities of Guangzhou. 1 n Ci API = E where: n i=1 Si O 00 n - air pollutants tested ~ Ci - concentration of air pollutants 00 Si - acceptable level of pollutant (according to government guidelines) W W I I I I I I , I I I I I I I I

I I I I , I I I I I I I I I I I I I Table 2. Comparison of Average Levels of SO2, NOX, TSP and B(a)P Between Indoor and Outdoor Air in Guangzhou (19841985) B(a)P SO (µ g/m3) NO (µ g/m3) TSP (µ g/m3) (µ g/10om3) Indoor 190 t 80 70 t 30 , 210 t 70 1.30 t 0.98 Outdoor 80 t 20 40 f 10 200 t 30 0.50 f 0.26 Three peaks of indoor SO2 and NOX were found, (7 a.m., 11 a.m. and 7 p.m.) which were also substantially higher in winter/spring months when doors/windows were generally closed, than in the summer or fall (Figure 5), suggesting that the primary source of indoor air pollution originated from cooking. Residents of Guangzhou depended on wood in the 50s, on coal during the 60s, and progressively switched to propane and gas in the 80s. In 1991, about 40% of the families in Guangzhou used gas. Thus, indoor air pollution was probably most pronounced from 1960 to 1990. Table 3 compares the indoor air pollutant levels in houses using gas as compared with those using coal. Not only is indoor air pollution higher in coal users' homes, housewives and family members of households using coal also have higher concentrations of benzo(a)pyrene in the urine. -5- I

I I I I I I I I I I I I INDOOR AND OUTDOOR AIR POLLUTION AND LUNG CANCER Du Ying-Xiu*, Huang Lan-fang**, Feng Zhen-zhi** and Feng 7ian-wei* * Department of Hygiene, Guangzhou Medical College, Guangzhou, China ** The Municipal Health & Antiepidemic Station of Guangzhou, Guangzhou, China Introduction In China, the urban lung cancer mortality rate is the highest of all cancers. In 16 Chinese cities, the mortality rate for all types of cancers rose from 100/100,000 in 1982 to 125/100,000 in 1986 (b = 0.0117, P < 0.05), with lung cancer not only accounting for 25 % of the total cancer deaths but also increasing at the fastest rate: from 25/100,000 in 1982 to 32/100,000 in 1988 (b = 0.0151, P < 0.01). The city of Guangzhou now has the third highest lung cancer death in China; only Chongqing and Shanghai have higher rates. In 1989, the five leading cancer-related standardized mortality rates (SMRs) in Guangzhou were: lung cancer 39.79/100,000, liver cancer 24.12/100,OOQ stomach cancer 9.67/100,000, nasopharyngeal cancer 6.07/100,000 and esophageal cancer 5.00/100,000. Notably, the lung cancer death rate was higher than the combined SMR for liver, stomach and nasopharyngeal cancers. Air pollution, smoking and certain occupational exposures are considered to be the three most important risk factors for lung cancer. However, their significance may vary due to locality or sex differences. In order to examine the potential risk factors for lung cancer in Guangzhou, we have undertaken an analysis of the relevant data collected during the last 20 years. This paper primarily discusses the relationship between indoor/outdoor air pollution and lung cancer. The association of atmospheric air pollution and lung cancer has long been noted. Stocks reported that the lung cancer incidence in various areas of Great Britain was related to the local atmospheric deposit index, smoke index, and population density.(1) A close relationship was found between lung cancer and 3, 4 benzo(a)pyrene, beryllium, molybdenum, vanadium and arsenic in the atmosphere(2). Blot and Fraumeni analyzed the distribution of lung cancer deaths in the United States and found higher lung cancer incidence in males in locations near paper manufacturing, chemical engineering, petroleum, and vehicle manufacturing plants, and concluded that lung cancer was associated with both atmospheric pollution and occupational exposures(3). Xu and Blot performed a case-control study in the industrial city of Shenyang and found that, in addition to smoking, lung cancer was related to indoor/outdoor air pollution, because a higher incidence of lung cancer was found in men and women living near refining facilities for long periods of time(4). The traditional coal-burning "kangs" appeared to be an important contributing factor to indoor air pollution. Moreover, they thought that smoking and indoor/outdoor air pollution were associated with squamous cell carcinoma and small cell carcinoma. In their 1987 case-control study in Shanghai, Gao etal.(5) found that indoor air pollution and the use of rapeseed oil for cooking had an important significance in female lung cancer. Our current study combines the atmospheric pollution data for the last 20 years, the indoor air pollution data from a 2-year study, and Guangzhou's lung cancer mortality records for the last 10 years in a retrospective case-control study in order to examine the relationship between indoor/outdoor air pollution and lung cancer.

! 1 (31.50/100,000), and Dongshan (30.79/100,000), suggesting a history of pronounced difference in lung cancer death rates by districts in Guangzhou. 2. Atmospheric pollution. Between 1972 and 1979, data obtained at 30 sampling stations were used to calculate the atmospheric pollution index (API). It was found that Liwan had the highest API (2.49), followed by Yuexiu (1.68), Haizhu (1.64) and Dongshan (1.17). For comparison, the "control" region had only an API of 0.57. Between 1982 and 1990, four sampling stations were established in order to provide 24-hour around the clock monitoring. Samples were obtained on 180 days each of the nine years and then analyzed. The results showed that Liwan had the most severe API (0.898), followed by Yuexiu (0.721), and lastly Dougshan (0.470). For comparison, the control district only showed an air pollution index of 0.246. The severity of atmospheric pollution was also studied in 1984 with an aero-remote sensing system which further substantiated the atmospheric pollution for Liwan to be severe/heavy, Yuexiu to be heavy/medium and Dongshan and Haizhu to be medium/light, and control to be clean. (Table 1) Table 1. Comparison of Atmospheric Pollution and Lung Cancer Death Rate in Four Districts of Guangzhou Liwan Yuexiu Dongshan Haizhu Control AP Index (1972-79) 2.49 1.68 1.17 1.64 0.57 AP Index (1982-90) 0.898 0.721 0.470 -- 0.246 Degree of pollution by aero severe/ heavy/ medium/ medium/ clean remote sensing (1984) heavy medium light light Lung Cancer death rate (1976-87) 37.94 35.99 30.79 31.50 -- Results of these surveys are also shown in Figures 2-4 which show clearly that atmospheric pollution for the past 20 years was the most severe in the Liwan district, followed by the Yuexiu district. This is highly correlated with the higher lung cancer death rates for these two districts. 3. Indoor air pollution. Indoor and outdoor air pollution studies carried out over a two-year period (1984-1985) showed that indoor air pollution was more severe than outdoor air pollution (Table 2). This probably reflected the fact that during this period of time most of the factories in Guangzhou were located outside the city and automobiles were rather scarce. 4 I I I I I I I I ' I I I I I I I I

I Table 3. A Comparison of the Concentrations of Some Air Pollutants and Urine-B(a)P Between the Coal-Burning and Gas-Burning Kitchens Briquette coal- butning kitchen Liquefied petroleum gas- burning kitchen Coal/gas SO2 (µ g/m3) 279 58 4.81 NOX (µ g/m3) 76 63 1.21 CO (µ g/m3) 9420 2340 4.03 TSP (µ g/m3) 332 188 1.77 SD (µ g/m3) 12 5 2.40 BaP (µ g/100 m3) 11.9 2.2 5.41 Radon (Bq/m3) 18.6 16.6 1.12 Thoron (Bq/m3) 42.5 28.3 1.50 Urine-BaP (ng/1) 4.0 2.8 1.43 11 4. Case-control study. The results of this study are shown in Table 4 which shows that cigarette smoking is the most significant risk factor for lung cancer in males and is less important for lung cancer in females. Indoor air pollution is the most significant risk factor in females and has no obvious association with lung cancer in males. -6- I I I I I I I I I I I I I I I I I ,

I I I I In December, 1984, the Guangzhou Municipal Science & Technology Committee organized an aero-remote sensing test for the city of Guangzhou. Atmospheric pollution was assessed by observing the effects of pollution on plants by the vegetation ecoline on air infrared color film and sychromonitoring. Levels of air pollution were classified as clean, light, medium, heavy or severe. 3. Indoor air pollution. I I I I I I I In order to examine indoor air pollution caused by coal fumes, a two-year continuous systematic study of indoor air pollution was conducted in 1984-1985. Five households from each of the four districts of Guangzhou, 20 in total, were randomly selected. The daily and seasonal variations of SO2 and NOx were tested 7 times daily with samples taken every two hours from 7 a.m. to 7 p.m. for 5 consecutive days, once during each season, i.e. four times in a year. Since the 1980s, residents of Guangzhou have been in the process of gradually switching over from coal to gas. In order to compare the indoor air pollution associated with either fuel, 5 coal-user households and 5 gas-user households were again selected from the four districts in 1986. The indoor air contents of 502, NOx, CO, TSP, SD, B(a)P, radon and thoron were tested. Moreover, the urine B(a)P levels of housewives in both coal and gas households were also tested. The housewives were all non-smokers with at least one year's experience in managing the households. S02 was analyzed by the hydrochloric-rosaniline method, NOx by the diphenylamine hydrochloride colorimetric method, CO by gas chromatography, TSP by weighing 4-hour continuous samples from ambient air collected using large flow samplers. SD was weighed after 15 days' exposure of flat dishes of 15 cm diameter placed 1.7 meters above ground. B(a)P was analyzed by paper chromatography fluorospectrophotometer and urine B(a)P content by subjecting polyurethane foam absorbed B(a)P (from urine volume above 2000 m]) to fluorimetric analysis. Radon and thoron samples were taken by DK-60 particulate samplers and examined by a FJ-13 a radiometer. 4. Case-control study. In 1985, there were 806 cancer deaths (531 males, 275 females) in Guangzhou. We matched the cases of lung cancer deaths with controls of noncancer deaths in the same year and in the same residential areas for sex, age (± 2 years), and obtained 659 pairs (82% of total number of cases); there were 143 male pairs and 216 female pairs. By using the Mantel-Haenszel method, we calculated the relative risks of smoking and exposure to coal dust at the 95% confidence level. Results 1. Distribution of lung cancer deaths by districts. ' Between 1980 and 1988, a total of 6,812 lung cancer deaths were registered at the 63 local police . stations of Guangzhou. After excluding nonprimary lung cancers and less than 10-year residencies, 5546 cases were obtained (3760 males, 1786 females; ratio 2.1:1). Based on police station records, the M ' regional distribution of lung cancer deaths was calculated and is shown in Figure 1. The highest lung 0 , 00 cancer rate (37.94/100,000) was in Liwan, and in descending order, Yuexiu (35.99/100,000), Haizhu -3 V O t 3- W  IV O I

I I I I ' I I 1 I I , I I I ' I Table 4. A Case-Control Study Among 659 Lung Cancer Deaths RR (95% CL) P-Value Male Smoking 3.53 (2.44 - 5.11) < 0.001 Coal fumes exposure 0.89 > 0.05 Female Smoking 1.93 (1.30 - 2.87) < 0.01 Coal fumes exposure 2.21 (1.16 - 4.21) < 0.01 Discussion The incidence of lung cancer is known to be higher in industrialized nations compared to developing countries. For a given country, lung cancer is more prevalent in highly industrialized zones, compared to agricultural regions and is also significantly higher in the city than in the country. These observations underscore the importance of atmospheric pollution as a significant factor for the induction of lung cancer. However, because the development of lung cancer is known to be associated with a multitude of risk factors, and with an extremely long latency, and because atmospheric pollutants are known to be complex and have dynamic and variable interactions with humans, studies that intend to explore the inter-relationship between atmospheric pollution and lung cancer require data generated by long-term observations. Moreover, the existence of lung carcinogens in the atmospheric pollution should also be clarified. The severity of atmospheric pollution is influenced by many factors, such as: population density, the nature and sophistication of industries in that locale, source and type of energy and number of automobiles, city planning and number of trees, geographic location, weather conditions, etc. At the present time in China, the primary source of atmospheric pollution comes from coal fumes generated by the use of coal for cooking and heating in the home. In northern China where coal is used for heating, the smoke is commonly vented to the outside by a chimney. This results in severe atmospheric pollution. In southern China, by contrast, chimneys are usually not available to vent the coal smoke. Instead, smoke from burning coal remains indoors, giving rise to severe indoor air pollution. We have investigated the relationship between atmospheric pollution in the last 20 years and the number of lung cancer deaths in the last 10 years. Our studies show that the districts of Guangzhou with the more severe atmospheric pollution are the same ones with the higher rate of lung cancer deaths, thus confirming the close relationship between atmospheric pollution and lung cancer deaths. Our case-control study further shows that in the case of females, the majority of whom are lifetime nonsmokers, indoor air pollution actually is a more important and significant risk factor than active smoking. N Lb ~ y 00 _7_ ~ A J

I Both cigarette smoking and coal-burning are known to produce benzo(a)pyrene. While most of the existing studies of the effects of benzo(a)pyrene are based on the use of animal cells and animal models, our recent studies show that benzo(a)pyrene is effectively biotransformed by microsomes prepared from human lung tissues. The metabolites, when incubated with human fetal tracheal epithelial cells, induce an increase in unscheduled DNA synthesis, promote the formation of micronuclei, and introduce point mutation in codon 12 of the H-ras oncogene. Taken as a whole, these results provide direct evidence for the ability of benzo(a)pyrene to induce human lung cancer. Additional studies are needed to show whether other carcinogens exist in atmospheric pollutants. Many other studies in China have demonstrated a close relationship between lung cancer and atmospheric and indoor air pollutants. Liang et al.(7) showed that although both coal and wood smoke contain potential carcinogens, coal smoke is regarded to have a higher carcinogenic potential than wood smoke. Wang et al.(8) showed that a high coal consumption index, indoor smog pollution and low ceiling height in the living quarters were major risk factors for lung cancer. Guan et al.(9) reported that extracts of air particles collected from Beijing, Taiyuan and Xuanwei all had potential carcinogens. Moreover, the smaller size particles were found to have a stronger potential for carcinogenicity. Ye et al.(10) showed that air particles collected in five Chinese cities (Beijing, Taiyuan, Wuhan, Shenyang and Xuanwei) were mutagenic based on the Ames and the SCE tests, with the highest lung cancer incidence area, Xuanwei, showing the most severe air pollution. Wang et al.(11) demonstrated that cooking oil fumes were a common risk factor for lung cancer and that winter heating by coal stoves was a risk factor for squamous cell carcinoma of the lung. In conclusion, while the relationship between atmospheric pollution and lung cancer has been established, some observations on this relationship remain to be explained. For example, while indoor air pollution due to coal burning has generally been regarded as a risk factor for lung cancer in nonsmoking females, coal has been in use in China for thousands of years, the question arises why the incidence of lung cancer has been rising only in the last 20 to 30 years. Moreover, atmospheric pollutants are thought to give rise primarily to squamous cell carcinoma; this appears to be inconsistent with the prevailing adenocarcinoma cell type commonly observed in nonsmoking females. The answers to these seemingly conflicting observations must, therefore, await additional studies in the future. -8- I I I I I I I I I I I I I

I I I Concentration of Air Pollutants (ug/rn7) 500 r 400 50~ Winter I I I I I 300 200 100 N nring . -~ '---••-- 0 SOs Spring SO a Summer SO, Autumn NO~ Winter -.-.t....... _.•_. O S NO: Surnmer ~ NO.Autumn 7:00 9:00 11:00 13:00 1 0-:00 17:00 19:00 Time (hr) Fgure 5. iNDOOR SO, AND NOx DURING DAYTIME O 00 IN FOUR SEASONS FOR TWENTY FAMILIES ~ IN GUANGZHOU, CHINA (1984-1985) -4 aD W W -L I I I I I I , , I

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I I i I I I , I I I I References Stocks, P. "Cancer and bronchitis mortality in relation to atmospheric deposit and smoke," Br. Med. J. 1: 74-79, 1959. 2. Stocks, P. "The relationship between atmospheric pollution in urban and rural localities and mortality from cancer, bronchitis and pneumonia with particular reference to 34 benzopyrene, beryllium, molybdenum, vanadium and arsenic," Br. J. Cancer 14(3): 397-418, 1960. 3. Blot, W.J. and Fraumeni, J.F. "Geographic patterns of lung cancer: industrial correlation," Am. J. Epidemiol. 103(6): 539-550, 1976. 4. Xu, Z.Y.; Blot, W.J.; Zhao-yi, Xu et al. "Smoking, air pollution, and the high rates of lung cancer in Shanyang," China J. of the National Cancer Institute 81(23): 1800-1806, 1989. 5. Gao, Y.T. et al. "A case-control smdy of female lung cancer in Shanghai," Second Symposium on Lung Cancer Research, Guangzhou, p. 7, 1987. 6. WHO. "Sulfur oxides and suspended particulate matter," Environmental Health Criteria 8, Geneva, 1979. 7. Liang, C.K. et al., "Kuming mice skin tumor initiating activity of extracts of inhalable particles in indoor air," Chinese J. Prev. Med. 21: 316-318, 1987. 8. Wang, F.L. et al. "Analysis of risk factors for female lung adenocarcinomas in Harbin," Chinese J. Prev. Med. 23: 270-273, 1989. 9. Guan, N.Y. et al., "A study of carcinogenicity of extracts from different size particles in air," Chinese J. Prev. Med. 24: 9-12, 1990. 10. Yu, S.Y. et al., "Study on mutagenicity of size fractionated air particles," Chinese J. Prev. Med. 25(2): 70-74, 1991. 11. Wang, G.X. et al. "Multi-variate analysis of causal factors including cooking oil fume and others in matched case control study of lung cancer," Chinese J. Prev. Med. 26(2): 89-91, 1992. O 00 ..a -4 ' 00 W ~ ! 9 I

~ r .. .. .. ... .. .. .. .. ... .~s .. .r .. .. r r .o 4081783329 Figure 3. TREND OF AIR POLLUTION INDEX IN GUANGZHOU (1982-1990) ~~ ::` LLHfI~r '' YUC%lU ~ 17 11 11 011 1.0 _ cO11 t:rO). ~~1.0 i l.U . . f , I o.s a 0.5 lm 199U ..~ 1905 1990 ~ 1965 1990 i 19t35 199U . .4.,. t . (~~y .~ ~ '"'.` Ilal.zhu N ~r. Air PolLritlnn InJex )h rn AP[- SO., T51' . LRl I

2081783328 1 l S It 11 ul,1,u Alr I'ul.lutlon Index C' Figure 2. DISTRI6UTION OF AIR POLLUTION INDEX IN CUANOZIIOU (1972-1979) CID TITl „ nr[ ; ' C1 i_[ sl 9 m s ~ .. ..:; :. ... .. .. .. .r .. .. .w r. .. .r.~;: ..: ~rr .~ +

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.. .. m .. .rm a. .m .m m .m w w .= m .. ..m m .. _ 2081783327 Figure 1. DISTRIBUTION OF LUNG CANCER DEATH RATE (pcr 100,000) IN GUANGZIfOU (1976-1983) 111111 :ra~, '[~ ~~~_ Llunn Yucatu Unuptslulu ~ " )~ . - II y.: I Ilfl)Z1111\ I.uug Cnncer DeoCh RnCe ( per 100,000 ) 0 - 21) - 30 - 60 - 50 - FM ® MEN win ®. lf. 1-.~r I?

208'1783330 .:; , ~ I Figum 4. ATMOSPHERIC POLLUTION MEASURED BY AERO-REMOTE SENSING TECHNIQUE (Decem6er 1984) ~r ,n. I ,1 `, I ~,' 61wan Cnl~trvl ' Yuexla Uvn tihiln ~ II \ {/'/~~ 11 i, l ~ +~. 0 Uerrec nC Atmospheric Pollution '` `~~ 1} 1 2 7 4 5 ~ m Mllllll BJ1Jlul9 ~ ! W~J ® t,~) C1.ean LiRIit lledLuni Ilea.•r Serere " m .m m .. .. .m w .m wrs wr .r. .m wm m w w m ..

I I References I 1. Y-T, Gao et al. Lung Cancer and Smoking in Shanghai. I Int. J. Enidemiol. (1988);17(2):277-280. 2. Samet, Jonathan et al. Cigarette Smoking and Lung Cancer in New Mexico. Am. Rev. Respir. Dis. (1988); 137:1110-3. I 3. Beckett, W.S. Epidemiology and Etiology of Lung Cancer. Clinics Chest Medicine (1993);14(1):1-15. I 4. Schlesselman, J.J. Case-Control Studies. Oxford University Press (1982). , 5. Hennekens, C.H. et al. Epidemiology in Medicine, Little Brown & Company (1987) 1 1 ' I I I I I N O ' ~ ~ ti C* w ' w .p O -8- I I

I 1 Using data from the Sino-MONICA-Beijing Project in the People's Republic of China from January 1, 1990 to December 31, 1991, Odds Ratios (OR), Attributable Risk (AR), and Population Attributable Risk (PAR) were measured in this study. Methods A case-control study of primary lung cancer patients (International Classification of Disease Ninth Revision, Code 162) in this study came from the monitoring system for the Sino-MONICA-Beijing Project during a period of 24 months. A three-level monitoring•system for the Sino-MONICA-Beijing Project was formally started on January 1, 1984 in six scattered urban districts and one rural county of Beijing. The WHO-MONICA Project is a worldwide monitoring system for cardiovascular disease from 1984 to 1993. From January 1, 1990 to December 31, 1993 the epidemiology of lung cancer was an integral part of Sino-MONICA-Beijing Project. The three-level monitoring system consisted of the following: a) The Beijing Heart, Lung and Blood Vessel Medical Center served as the coordinating Center (the first level); b) forty-two districts and regional hospitals formed the actual monitoring units (the intermediate level) and c) the residence committee and residence health stations were the basic units of the monitoring system which involved 335 units in urban areas and 188 villages in rural areas in 1990. There were 742,198 residents in the Sino-MONICA-Beijing monitoring system in 1990 (369,427 males and 372,771 females). There were 580,973 residents in urban areas and 161,225 in rural area. The cases in this study were adults aged from 18 to 80 years old. Controls were selected from the same residence committee or village of the general population of the monitoring system. All of the controls were of the same sex as the cases and their ages were within two years more or less than the cases. Interviews were sought with all living cases and controls. Family members were visited if the case was deceased. The OR, AR and PAR were the measures of the association between smoking and primary lung cancer. Results A total of 252 male and 151 female cases of primary lung cancer were identified over 24 months. Interviews were completed with 734 male and 417 female controls. (Table 1) I I I I I I I I I I I I I -2- 0 00 ~ ~ co W W W A I I I I

I Table 3. Indices of risks between cigarette smoking and lung cancer measured by OR, AR and PAR Indices of Risk Male Female Total 1. OR 2.84 3.92 -. 2.65 2. AR 64.8 74.5 62.3 3. PAR 55.5 40.5 46.0 Diagnosis of 68.2% of the lung cancers (403) were based on pathological examination of tissue specimens and/or based on cytology examinations. Table 4 shows the risk values measured by OR, AR and PAR in different cell types. According to the PAR, 87.3 % of squamous cell carcinoma and 44.5 % of SCLC and 14.3% of adenocarcinoma were attributed to smoking. Table 4. OR, AR and PAR for squamous, adenocarcinoma and SCLC associated with cigarette smoking Pathology andtor Cytology Eaam. No. of Cases OR AR PAR Squamous cell carcinoma 81 *12.18 91.8 87.3 Adenocarcinoma 112 #1.39 28.1 14.3 SCLC 55 **2.48 59.7 44.5 . .* P = 0.00000001 P=0.008 N P = 0.139 I I I I I I I I I I I I I , IV O O ~ ' V 00 W 4- W W O ' , I

I I I I I I I I I I I In this paper, the percentage between males (160 cases) and females (88 cases) was different for the principal types of lung cancer (248 cases). Among males, the percentage for squamous cell type was 79.0 %(21.0% in female), 69.1 % for small cell lung carcinoma and 51.8 % for adenocarcinoma. There also was a difference in the risk observed in different cell types. All of the principal types of lung cancer (squamous cell, small cell lung cancer and adenocarcinoma) were affected. OR (12.18), AR (91.8 %) and PAR (87.3) for squamous cell have been reported. The association between smoking and squamous cell type was the strongest (P=0.00000001). The association between smoking and SCLC was the second (P =0.008). The figures for SCLC were 2.48. 59.7% and 44.5%, respectively, and 1.39. 28.1% and 14% for adenocarcinoma, respectively. The correlation between smoking and adenocarcinoma was not statistically significant (P=0.139). Acknowledgements: Thanks to the Beijing Scientific Association and Beijing Bureau of Public Health as sponsors. I I I I I N O O ~ -4 00 I I _'j_ W W W CO

I I I I I I I I I I I I 1 I I I I I STUDY OF THE RELATION BETWEEN SMOKING AS A LIFESTYLE FACTOR AND LUNG CANCER IN BEIJING AREA OF CHINA Fan Ruo-lan*, Zheng Su-hua*, Wu Zhao-su**, Wu Zhao-ru*, Zhang Rui-song**, Cao Li-hua* and Li Yu-zhen* * Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China ** Beijing Heart, Lung and Blood Vessel Medical Center, Beijing, China A case-control study involving interviews with 403 (252 male and 151 female) primary lung cancer patients and 1151 (734 male and 417 female) population-based controls from the Sino-MONICA- Beijing Project (involving about 750,000 residents) showed that cigarette smoking, as a lifestyle factor, is the highest risk factor for lung cancer. The overall odds ratio (OR) was 2.65 (95 % CI 2.04-3.44) for all patients. The OR was 2.84 (95% CI I.90-4.28 for males and 3.92 (95% CI 2.59-5.94) for females. The Population Attributable Risk (PAR) was 55.5% for males and 40.5% for females. It is clear that risk trends rise with increasing smoking intensity, duration and degree of deep smoking (inhalation). About 70% of all patients were examined by pathological and/or cytological techniques. The association between smoking and lung cancer was the strongest in squamous cell carcinoma (PAR 87.3 %). Small cell carcinoma ranked second (PAR 44.5%). The correlation was not statistically significant for adenocarcinoma (PAR 14.3 % and P> 0.05). Introduction Disease patterns in China have changed greatly from the 1950s to the 1990s. The mortality rate from malignant neoplasms was 63.9/100,000 in the 1950s. It increased rapidly to 128.0/100,000 in the 1990s and became a major cause of death. The percentage of total deaths from cancer was 5.17% in the 1950s and it increased to 21.88 % in the 1990s. Cancer ranked as the leading cause of death in the 1990s; it was only the seventh cause of death in the 1950s. Lung cancer mortality increased in China from 1973-1975 to 1990. Nationwide survey figures for lung cancer mortality in 1973-1975 were 5.45/100,000; it increased to 32.89/100,000 in 1990. The percentage of deaths from lung cancer increased for all of China each year; the average annual increase rate was 11.9%. The increase in the lung cancer death rate is the highest among the selected sites of malignant neoplasms: 8.1 % for intestinal and rectal cancer, 5.1 % for breast cancer, 4.0% for liver cancer and 1.4% for stomach cancer. Only esophageal cancer decreased from 1973-1975 to 1990 with an average annual reduction rate of 3.3 %. Generally speaking, lung cancer mortality was higher in large cities such as Beijing, Shanghai, Tianjin and Guangzhou and along the east coast. The mortality from lung cancer in Beijing men was 29.6/100,000 in 1977-1978 (urban areas) and 20.6/100,000 in females. It increased to 33.0/100,000 in males and 23.3/100,000 in females in 1986. It is obvious that the trend is for lung cancer mortality, in Beijing, to continue to increase in recent years. Thus, it is important and necessary to study the etiology of lung cancer in the Capital city. W I

I I I I I I I I I I I I I I I I I I Table 1. The status of interviews with primary lung cancer and population-based controls in the Sino-MONICA-Beqing monitoring system (1990-1991) Sex Number of Lung Cancer Cases .. . Number of Controls . Male 252 - - - 734 - -- - Female 151 417 Total 403 1151 Table 2 shows the odds ratio from a case-control study to evaluate the relationship between cigarette smoking and lung cancer. The reported frequency of cigarette smoking in lung cancer patients (85.7% (216/216+36) in males and 54.3% (82/82+69) in females) was much higher than that in the controls. The corresponding figures were 67.8% (498/498+236) in males and 23.3% (97/97+320) in females. Smokers experienced an increased risk of lung cancer (OR 2.84 in males, 3.92 in females). Table 2. Odds Ratios from a case-control study of the relationship between cigarette smoking and lung cancer among monitoring system for Sino-MOIVICA-Beijing (1990-1991) Cigarette Smoking Male Case Control Female Case Control Total Case Control Yes 216 498 82 97 298 595 No 36 236 69 320 105 556 OR 2.84 3.92 2.65 95%CI 1.904.28 2.59-5.94 2.04-3.44 The results of the indices of risk measured by OR, AR and PAR are shown in Table 3. The AR of 64.8 % among males and 74.5 % among females was attributed to smoking. A PAR of 55.5 % for males and 40.5 % for females was calculated. -3- ~ 0 0 ~ -4 0o W W W tJf I

I I I I I I I I I I I I I I I I I I Table 1. Data Index and Assigned Value Variable Factor Assigned Value X1 Smoking index Amount of smoking (cigarettes/day) x years of smoking/20 X2 Smoke inhalation* No: 0; Shallow: 2; Medium: 3; Deep: 4 X3 Passive smoking exposure No: 0; Yes: 1 X4 Occupational exposure No: 0; Yes: 1 X5 History of chronic bronchitis No: 0; Yes: 1 X6 History of tuberculosis No: 0; Yes: 1 X7 Family tumor history No: 0; Yes: I X8 Crowded living conditions No: 0; Yes: 1 X9 Fuel use in the home (fuel Non-solid fuel: 0; Solid fuel (coal, charcoal, index)** etc. ): 1 X10 Coal stove used for winter No: 0; Yes: heating 1 X11 Amount of cooking oil used Fat consumption per person per month X12 Kitchen cooking fume pollution No: 0; Yes: 1 X13 Regular consumption of fried No: 0; Yes: 1 food X14 Cooking index Average times of cooking per week Note: * Shallow: exhale by mouth; Medium: exhale by nose; Deep: swallow smoke. ** Based on use in the last 20 years. The index represented average fuel used/year. Results and Analysis 1. Analysis of risk factors for pulmonary lung adenocarcinoma in men. Fourteen indexed variables were subjected to single-factor analysis by conditional logistic regression. Using a one-sided test with a = 0.05. Smoking index, occupational factors, history of chronic bronchitis, cooking fumes, and family tumor history were identified as the five significant variables for men. These were then further analyzed by the multi-variate conditional logistic regression -3- I

Introduction 1 The incidence of lung cancer in China has been on an apparent increase in recent years. According to data published in many parts of the world, ethnic Chinese women, known to have a low smoking rate, also have high incidence of lung cancer, primarily pulmonary adenocarcinoma(1,2). Numerous epidemiological studies on risk factors for adenocarcinoma have been conducted in different regions of China. To examine the risk differences in lung cancer due to sex difference, we conducted a pair-matched case-control study of 180 primary pulmonary adenocarcinoma cases in Nanjing during 1986-1993. Materials and Methods This study was based on the retrospective pair-matched case-control study method. Selection of Cases: Primary adenocarcinoma cases, confirmed by analysis of pathological sections or exfoliated cells, were drawn from Nanjing municipal hospitals during 1986-1993. All 180 cases were Nanjing residents of over 20 years, including 100 men and 80 women. Selection of Controls: Healthy controls were residents of Nanjing for 20 years and were randomly selected from the same neighborhoods as cases. They were matched 1:1 with cases by sex, age ( f five years), and street address. Data Collection: The standardized questionnaire utilized information in the published literature, and also incorporated distinct local conditions and population characteristics. Indices that appeared in the questionnaire and their quantitation are shown in Table 1. Method of Analysis: The information was first analyzed by the conditional logistic regression model. The identified risk factors were further analyzed for relative risks and population attributable risk(3-5). I I I I t I I I I I I I I I V , ~ W W 2 W - t

I I I I I I I I I I I I I I Tables 5, 6 and 7 show that risk trends increased with intensity (daily dose), duration and degree of inhaling. Table 5. OR for lung cancer associated with intensity (daily dose) of cigarette smoldng Mzle r<mLle . . . .. .. Tunl Nwvba of Cigmctle.clday rM Cadro] OR Cx.u Cwtral- DR Cam C®ttol OR 0 36 236 1.00 69 320 IAO 105 556 1.0 1 13 121 070 17 48 1.64 30 169 0.9 10 53 171 2.03 30 37 3.75 83 208 2.1 20 111 183 3,98 31 12 11.94 142 195 3.8 30 39 23 11.12 4 1 18.49 43 24 9.8 Table 6. OR for lung cancer associated with duration of cigarette smoldng Dmmen of Meic Frndc TUW Smoking by YeUs Cue CmtrN OR C.ce Ctmrcal OR Ctx Covtrol OR 0 36 236 1.00 69 320 1.00 105 556 I 1 29 135 1,41 8 15 2.47 37 150 1 30 44 122 2.36 19 23 3.83 63 145 2 40 143 2A1 3.98 55 59 8.38 198 300 3 ' ! N 0 00 , V Co W W ' S V !

a I I I I I I I I I I I I I I I I I ANALYSES OF SEX DIFFERENTIALS IN RISK FACTORS FOR PRIMARY LUNG ADENOCARCINOMA Shen Xiao-bin¢*, Wang Guo-xiong*, Xiang Long-sheng* and Huang Yuan-zhu** * Nanjing Railway Medical College, Nanjing, China ** Nanjing Medical University, Nanjing, China Abstract To analyze potential sex differences in risk factors for primary lung adenocarcinoma, a case- control study was carried out in Nanjing, China. One hundred and eighty cases (100 males, 80 females) involving Nanjing residents who had lived in an urban area for at least 20 years and who had been diagnosed with primary lung adenocarcinoma were analyzed. Age- and sex-matched controls were identified from healthy neighbors of cases. Information on possible exposure to risk factors for the past 20 years before diagnosis was obtained by trained interviewers and included: cigarette smoking index (average number of cigarettes smoked per day times number of years smoked), passive smoking, family history of lung cancer and other tumors, chronic bronchitis, pulmonary tuberculosis, occupational exposure to cooking oil fumes, home exposure to cooking oil fumes, cooking practices, housing conditions, types of domestic fuel (quantified by an index with a weighted average), heating from coal stoves in the winter, etc. Conditional logistic regression analyses were performed in order to identify risk factors and to estimate the relative risks (RR) of selected factors. Population attributable risk (PAR) estimations for various risk factors were also computed. The data suggest that exposure to cooking oil fumes in the home, chronic bronchitis, and family history of tumors are the most common risk factors for lung adenocarcinoma in both men and women. The relative risks (RR) of the above three factors for male adenocarcinoma were 2.84, 2.30 and 4.89, respectively. In females, the same three factors had RR of 3.20, 3.23, and 4.23, respectively. PAR of these factors were 42.78%, 20.36%, and 17.5%, in males, and 53.41 %, 14.68%, and 19.0%, in females. In addition to the three most common risk factors, cigarette smoking index is also a risk factor for male lung adenocarcinoma, with a RR of 1.01 and a PAR of 27.69%. For females, another risk factor is the use of a coal stove for winter heating, which produced a RR of 2.29 for female adenocarcinoma and a PAR of 17.59%. These data suggest that exposure to cooking oil fumes is a major risk factor for lung adenocarcinoma in the city of Nanjing and could conceivably, in part, account for the high incidence of lung adenocarcinoma in Chinese women. In separate studies, the mutagenicity of cooking oil fumes has been demonstrated. Taken together, it seems reasonable to propose that lung adenocarcinoma may be primarily induced by exposure to cooking oil fumes. Additional studies must be performed to further test this hypothesis. I

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I Table 7. OR for lung cancer associated with degree of inhalation of cigarette smoldng Degree Of Ma1e Fema(c 7op1 Inhalatirm Caae Cmtrnl OR Ca.u Contrul OR Gse Cootroi OR No amkmg 36 236 1.00 69 " 320 1.00 IOS 556- I. I" 7 89 0.52 14 25 2.59 21 114 0. I1"" E 61 171 2.34 9 27 1.54 70 198 1. IR'•• 36 72 3.28 5 7 3.30 41 79 2. SIWIow mialafioa "seuW geu m and an af mwlh Middle iWalaficn: "smolse" geu hn aM mLL of nose Oeep mhaledo¢ 'smoke' gets m and out of Nurax Discussion A case-control study is a suitable design for an epidemiological study of lung cancer. The investigation in this paper was carried out in the Sino-MONICA-Beijing Project. Cigarette smoking is very common in China. Nationwide random survey data show that smoking rates are 69.7% among men more than 20 years old and 8.2% in women more than 20 years old. The residents of the Sino-MONICA-Beijing Project are a defined population. Because both lung cancer cases and the population-based controls came from the defined residents, this can avoid the Berkson bias. The correlation between smoking and lung cancer was measured by OR, AR and PAR. The results were an OR 2.84 for males and an OR 3.92 for females in this paper. It is not clear why the OR in males was lower than that in females; further study is required. The risk of lung cancer increased with the daily dose (intensity) and duration of cigarette smoking and with the degree of inhalation. It is obvious that cigarette smoking, as a lifestyle, is the highest risk factor for lung cancer among Chinese men. The PAR among men (55.0%) is larger than that among women (40.5 %). By comparison, the PAR value for men in Shanghai (69.0%) was higher than that in Beijing men (55.5%). On the contrary, the PAR among women in Shanghai (24.0%) was lower than that in Beijing women (40.5%). But the PAR among American men (89.5 %) and among American women (85.5 %) was much higher than that in Chinese men and women. Perhaps cigarette smoking is less hatmful to Chinese than to people elsewhere. I I I I I I I I I I I I I I I O O R y 00 -6- 0 W 1 W O I

model, at a = 0.05. The four variables shown in Table 2 appeared as factors which were associated with adenocarcinoma. Table 2. Results of Analysis of Pulmonary Adenocarcinoma in Males by Conditional Logistic Regression Multi-Factor Model (a = 0.05) actor Regression Coefficient Standard Error of Regression Coefficient Value Relative Risk (RR) Smoking Index 00.0135 0.0069 0.0260 1.0136 Chronic bronchitis 0.8338 0.4111 0.0213 2.3021 Cooking vapors 1.0448 0.4235 0.0068 2.8428 Family cancer history 1.586 0.6338 0.0062 4.8856 2. Analysis of risk factors for female adenocarcinoma of the lung. Using the same method for females, at a = 0.05, single-factor analysis identified six variables, which were chronic bronchitis, history of tuberculosis, heating by coal stove, cooking fumes, fried food, and family tumor history. They were subjected to further multi-variate analysis, at a = 0.05. Four risk factors were identified (Table 3). Table 3. Results of Analysis of Female Pulmonary Adenocarcinoma By Conditional Logistic Multi-Factor Model (a = 0.05) actor Regression Coefficient Regression Coefficient Standard Error Value Relative Risk (RR) Chronic bronchitis 1.1736 0.6702 0.0399 3.2336 Coal Stove for heat 0.8278 0.4476 0.0322 2.2883 Cooking fumes 1.1625 0.4208 0.0029 3.1979 Family tumor history 1.4415 0.7235 0.232 4.2270 3. Estimate of attributable risks from male and female pulmonary adenocarcinoma risk factors. Attributable risks were calculated by multi-variate analysis of identified adenocarcinoma risk factors (Table 4). -4- I 1 I I I 1 I I I I I I I I I I

I I References I I. Waterhouse, J. et al. "Cancer Incidence in Five Continents," (4): 1982. I 2. IARC, Monoeraph on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (38): 1986. I 3. Whittemore, A.S. "Estimating Attributable Risk From Case-Control Studies," Am. J. Enidemiol. 117 76 85 1983 : - , . t 4. Walter, S.D. "Effects of Interaction, Confounding and Observational Error on Attributable Risk Estimation," Am. J. Epidemiol. 117: 598-604, 1983. I 5. Bruzzi, P. et al. "Estimating the Population Attributable Risk for Multiple Risk Factors Using Case-Control Data," Am. J. Enidemiol. 122: 904, 1985. ' I I I I -6- 1 I I I I I I

I I I I I ' I I I I I I I Table 4. Attributable Risks from Male and Female Lung Adenocarcinoma Risk Factors Mzle Fewl. F~ ~I Retxtlw Ruk Number of - C9sa Ai¢ibuuble R6t' ReWirx Risk Number of hles Attrlbuublc Risk smokfrg iMea Meuurement 1.0136 t0o 0.2769 Chmnic brorchius Caking fume Fsmily .vwr M1urory HeuinB by cml ame 0 1 0 1 0 1 0 1 1 2.3021 1 2.8428 1 4.8956 6E 36 34 66 78 22 ' 0].¢36 0.4278 0.1750 I 3]]36 1 3.I979 I 4,2270 I 2.4881 63 17 19 61 60 20 55 25 0.1468 0.5241 0.1909 0.1759 I CambiaA populs[ion .IVibuuble risk _..0.7898 0.76B7 Discussion The results of this study show the incidence of pulmonary adenocarcinoma in persons both sexes in Nanjing to have similar risk factors. These are cooking fumes, chronic bronchitis, and family tumor history. Among these, exposure to cooking fumes is most harmful. Cooking fumes are the product of pyrolysis resulting from cooking oil or food at very high temperatures. The Chinese traditionally cook with high heat, and the resulting cooking fumes are one of the most common indoor pollutants found in Chinese living quarters. We have studied the chemical composition of cooking fumes and its mutagenicity. Our results show that cooking fumes contain benzo(a)pyrene, benz(a)anthracene, etc. The mutagenicity of cooking fumes has also been shown in toxicology studies. The attributable risk of cooking fumes on the incidence of pulmonary adenocarcinoma in Nanjing residents were calculated to be 42.78 % for males and 52.41 % for females. Thus, exposure to cooking fumes may be one of the reasons for the high incidence of lung adenocarcinoma in Chinese women. The PAR of family cancer history on the incidence of adenocarcinoma was 0.175 for men and 0.1909 for women. Thus, people with a family history of tumor are in the high-risk group for lung cancer and should, if possible, avoid exposure to other risk factors. The respective PAR of chronic bronchitis was 0.2036 for men and 0.1468 for women, suggesting that people should seek early treatment of their respiratory disease in order to minimize the chance of lung cancer. The incidence of adenocarcinoma in males was related to smoking, with a PAR of 0.2769. However, no effect of passive smoking was found in this study. Lung adenocarcinoma in females was ~ associated with the use of coal-burning stoves. Since this is a traditional method used for winter heating, N the indoor air pollution it causes warrants attention. o 00 V t'to W W ' S_ 4 != O> I

I I I I I I I I I I I I I 1 I THE RELATIONSHIP BETWEEN HISTOLOGIC TYPES OF LUNG CANCER AND CIGARETTE SMOKING Zhou Bao-sen, He An-guang and Wang Tian jue China Medical University, Shenyang, China Abstract This paper discusses 1056 cases of lung cancer which were identified by pathological examination in the Department of Thoracic Medicine of the China Medical University from 1978-1994. The sections were stained in order to identify the specific type of lung carcinomas. In the 1056 cases diagnosed by histologic examinations, squamous cell carcinoma accounted for 516 cases (439 male and 77 female), adenocarcinoma for 345 cases (219 male and 126 female), small cell carcinoma for 128 cases (91 male and 37 female), large cell carcinoma for 46 cases (37 male and 9 female), and other types for 21 cases (18 male and 3 female). A relatively high proportion of cases in our study (101 cases, 9.76%) were under 40 years of age (73 male and 28 female). To correlate the number of cigarettes smoked per day, and the duration and index of smoking with lung cancer development as well as the histologic types of lung cancer (WHO classification), we retrospectively analyzed the data for 1035 inpatients with lung cancer and compared them with 116 (48 smoker and 68 nonsmoker) inpatients without malignant diseases by means of the Mantel-Haenszel Method. The results show that squamous cell carcinoma (total, 516 cases; 378 smoker and 138 nonsmoker), large cell carcinoma (total, 46 cases; 32 smoker and 14 nonsmoker), and small cell carcinoma (total, 128 cases; 83 smoker, and 45 nonsmoker), are correlated with cigarette smoking status. The computed odds ratio (OR) values are 3.88 (95% CI: 2.49-6.05, P=0.001), 3.24 (95% CI: 1.47-7.23, p=0.0001), and 2.64 (95% CI: 1.52-4.62, P=0.01), respectively. All three types have dose-response relations with the amount, index and duration of cigarette smoking. The results show that the OR increased both with the amount of cigarettes smoked per day and with the duration of smoking. The excess risk for the heaviest smokers was 4.84-fold for squamous cell carcinoma, 2.45-fold for small cell carcinoma and 4.35-fold for large cell carcinoma. By contrast, adenocarcinoma did not correlate with cigarette smoking (OR 1.22; 95% Cl: 0.78-1.92, P=0.35). Thus it may be concluded that the amount, duration and index of cigarette smoking are high-risk factors for squamous cell, small cell, and large cell carcinomas. Introduction This paper reports on data from a study that examined the pathology and epidemiology of lung cancer in northeast China, where there is a high incidence of lung cancer in youths. Specifically, we aimed to study the possible relationship between histologic type of lung cancer and cigarette smoking.  O ~ 00 a V i 00 W W ~ I

Table 2. The Relationship Between Lung Cancer and Cigarette Smoldng I I I I I I  I I I Csse Crntrd OR 95% CI P P or L'mc Tmd Smoker + 853 48 - 507 68 2.38 1.59-3.58 0.00 Male + 740 41 9wkcr - 275 36 2.36 1.43-3.89 0.00 Femzle + 112 7 S1noker 231 32 2.22 0.89-5.74 0.06 Smoke 0 507 68 I Amuua 1- 61 5 1.64 U.60d.80 0.30 10- 211 14 2.02 1.08-3,85 0.02 20- 581 29 2.69 1.68<.33 0.00 0.00 smoke 0 507 68 1 Ynrs 1- 170 12 0.91 0.54L53 0.71 20. 678 36 3.95 2.37-663 0.00 0.00 Smoke 0 507 68 1 Indcx 1- 432 25 2.32 1.41-3,84 0.00 11 500- 416 23 2.43 I 45d,08 0.00 0.00 The possible relationship between cigarette smoking and the histologic types of lung cancer were also analyzed by the Mantel-Haenszel method (Tables 3-6). The results suggest that squamous cell carcinoma (OR, 3.88, 95 %CI: 2.49-6.05, P=0.001), small cell carcinoma (OR, 2.64, 95 %Cl: 1.52-4.62, P=0.01), and large cell carcinoma (OR, 3.24, 95% Cl: 1.47-7.23, P=0.001), were correlated with cigarette smoking and showed dose-response relationships with the amount, index and duration of cigarette smoking. The excess risk for the heaviest smokers reached 4.84-fold for squamous cell carcinoma, 2.45-fold for small cell carcinoma and 4.35-fold for large cell carcinoma. There was no correlation between cigarette smoking and the incidence of adenocarcinoma (OR, 1.22, 95%CI: 0.78- 1.92, P=0.35). -3- I

I I I I t I I I I I I I I I ,i I 1 I Table 5. The Relationship Between Large Cell Carcinoma and Cigarette Smoking Case -. Cwtd . OR 95% CI . . P P ef L'me TrenC Smoker + 32 48 14 68 3.24 1.47-7.23 0A0 Male + 29 41 SmoYCr - 8 36 3.18 L19-8.T/ 0.01 Female + 3 7 ' Smdcer 6 32 2.29 0.34-14.86 0.31 Smoke 0 14 68 1 Amau¢ 1- 0 5 - 10- 6 14 2.08 0.59-7.I9 0.19 20- 26 29 4.35 1.86-10.29 0.00 0.00 Smoke 0 14 68 I Yeen 1- 6 12 2.43 0.67-8,63 0.12 20. 26 36 3.51 1.53-8,12 0.00 0.00 Smoke 0 14 68 I Index 1- 14 25 2.72 1.05-7.11 0.02 500. 18 23 3.80 1.51-9.65 0.00 0.00 Table 6. The Relationship Between Adenocarcinoma and Cigarette Smoking Case Cmvol OR 95% CL P Smoker + 161 48 184 68 1.22 0.78-1.92 0.35 Male + 131 41 Smoker 88 36 1.31 0.75-2.29 0.31 Frnah + 30 7 Smoker - 96 32 1.38 0.51-3.89 0.48 -5- K) O 00 .a V ~ W W tn W I

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Table 3. The Relationship Between Squamous Cell Carcinoma and Cigarette Smoking Ca3e Canrtol OR 95% Cf P P nf Une Tratl Smoker + 378 48 138 68 3.88 2.49-6.05 0.00 Male + 343 41 Smoker - 96 36 3.14 1,83-5.37 0.00 Female + 35 7 Smoker - 42 32 3.81 1.38-I0.93 0.01 Smokc 0 138 68 1 Amawl 1- 15 5 1.47 0.484.88 0.46 100. 78 14 2.75 1.39-5.48 0.00 20- 285 29 4.84 2.92-8.06 0.00 0.00 Smoke 0 136 68 1 Years 1- 60 12 2.50 1.21-5,17 0.00 20- 315 36 4.38 2.72-7.05 0.00 0.00 Smokc 0 136 68 1 Ldex 1- 161 25 3.22 1.88-5.56 0.00 500- 214 23 4.65 2.69-8.09 0.00 0.0D Table 4. The Relationship Between Small Cell Carcinoma and Cigarette Smoking Cese Canvd OR 95% CT P P af LNe Tsmd Smoker + 83 48 45 68 2,64 1.52-0.62 0.01 Mete + 74 41 Smoker 17 36 3,82 1.80-8.17 0.00 Femile + 9 7 Smoker 28 32 1.47 0.4b5.18 0.49 Smake 0 45 68 1 Amaw1 1- 11 5 3.32 0.98-11.88 0.03 10- 26 14 2,81 1.23-6.38 0.00 20- 47 29 2.45 1.29,4.65 0.00 0A0 Smokc 0 45 68 1 Yeers I- 2C 12 3,02 1.29-7,18 0.00 20- 60 36 2.52 1.39-0.59 0.00 0.00 Smoke 0 45 68 1 IMex 1- 58 25 3.51 1.84-6.70 0.00 500- 26 23 1.71 0.82-3.55 0.11 0.00 -4- N ~ i t 1 I I I I I I I

I I Discussion A histological analysis in 1035 cases of lung cancer collected in northeast China over a 6-year period shows the following distribution: squamous cell carcinoma 49.8%, adenocarcinoma 33.33%, small cell carcinoma 12.37%, and large cell carcinoma 4.44%. These results are largely in agreement with previously published results (1). In terms of age distribution, 9.76% of the cases came from the 40 year-old and younger group, 68.70% from the 41-60 age group, and 21.55% from the group age 60 and above. The overall ratio of male-to-female was 3.16, as compared to a ratio of 2.6 in the young-age group. Interestingly the proportion of small cell ckrcinoma was 27.73 % in the latter, as opposed to 12.37% in total cases, 11.53% in the 41-60 age group and 8.07% in the group age 60 and above. The incidence of adenocarcinoma has previously been reported in subjects younger than 35 years (2) and in the 45 year old age group (3). Results of this investigation also showed that smoking is the most significant risk factor for lung cancer in northeast China, affecting squamous cell, small cell and large cell carcinomas. The risk of lung cancer increased with the daily dose and duration of cigarette smoking, with the trend being the most significant for squamous and large cell carcinomas. These features are consistent with results of worldwide epidemiological studies of lung cancer and smoking (4). Thus, it is possible that cigarette smoking induces lung cancer in northeast China in a manner which is qualitatively and quantitatively similar to what has been reported in other parts of the world. Detailed analysis shows that cigarette smoking is a significant risk factor for male smokers and is associated with the incidence of squamous cell, small cell and large cell carcinoma in men. By contrast, among women, only squamous cell carcinoma is correlated with smoking. Since adenocarcinoma constitutes the predominant cell type in females living in northeast China and elsewhere (5-8), constituting as much as 50.6% of the histologic cell type in female lung cancer cases, it is unlikely that cigarette smoking is responsible for the high rates of lung adenocarcinoma among females in our study. Conclusion Our study reported a high proportion of lung cancer in youth in northeast China. Cigarette smoking was determined to be a major risk factor for squamous cell carcinoma, small cell carcinoma and large cell carcinoma. The incidence of adenocarcinoma is not correlated with cigarette smoking. I I I I I I I I , I I I I ~ ~ O W W N .P O O -6-

I Materials and Methods Of the 1360 cases of lung cancer enrolled in the Department of Thoracic Medicine at the China Medical University from 1978-1994, 1035 cases were confirmed by examination of the stained pathological specimens and typed in accordance with the WHO classification (1981). Other diagnostic information came from studies of chest X-ray films, CT, and clinical evaluations. The 116 controls with no malignant diseases were randomly selected from inpatients at the same hospital. Information on the history of cigarette smoking, daily cigarette consumption, and the duration of cigarette smoking was obtained from the medical records. Smoking index refers to years smoked times the amount of cigarettes consumed per year. The odds ratio (OR) and P values were calculated according to the Mantel-Haenszel method. Results Table 1 shows that among the 1035 cases of lung cancer diagnosed by histologic examinations, squamous cell carcinoma accounts for 516 cases (439 male and 77 female), adenocarcinoma for 345 cases (219 male and 126 female), small cell carcinoma for 128 cases (91 male and 37 female) large cell carcinoma for 46 cases (37 male and 9 female) and other types for 21 cases (18 male and 3 female). About 10% of the cases were subjects under 40 years of age (total, 101 cases; 73 males and 28 females). In the lung cancer cases, 72.9% of the males and 32.7% of the females were cigarette smokers. By comparison, 53.2% of the males and 17.9 % of the females in the control group were smokers. The potential relationship between lung cancer and cigarette smoking was analyzed by the Mantel-Haenszel method. A significant increase in risk was shown for lifetime cigarette smokers (OR 2.38, 95 %CI: 1.59- 3.58). In the case of males, the OR was 2.36 (95 % CI: 1.43-3.89) whereas in females an OR of 2.22 (95 % CI: 0.89-5.74) was obtained (Table 2). Table 1. Age and Sex Distribution in Relation to Hi.stology of Lung Cancer Alstnlo8ical Age Type Sex Tuul (%) M+F (%) 540 41~60 >60 Squamaus Cell Ca. M 34 299 106 439 (55.85) F 3 60 14 T! (30.92) 516 (49,86) Adrno. G. M 19 145 55 219 (27.80) F 13 90 23 126 (50.68) 345 (33.33) Small. Cell G. M 16 60 15 91 (11.58) F 12 22 3 37 (14.86) 128 (12.37) Tolil M 73 531 182 786 (75.94) F 28 180 41 249 (24.06) 1035 (100.00) M+F 101 711 223 1035 % 9.76% 68,70% 21.55% 100% -2- I I I I I I t I I I I I I

I I I I References He, A., et al. Study on the Difference of Histological Type and Age Distribution of Lung Cancer Between China and Japan. Journal of China Medical University 1989; vol 18 I 2. supplement: 31. Homb, L, et al. Adenocarcinoma of the lung in patients younger than 35 years. JAMA 1984; 252 (15):2007. I 3. Gian, C.R., et al. Lung Cancer in the young. Chest 1985; 87(4):456. I 4. International Agency for Research on Cancer. Tobacco Smoking. IARC Monographs on evaluation of the carcinogenic risk of chemicals to humans. Vol.38.pp. 203-44.Lyon. IARC, 1986. I 5. Gao, Y., etal. Lung Cancer and Smoking in Shanghai. International Journal of Epidemiology 1988; Vol. 17. No. 2:278. I 6. Kung, I, et al. Lung Cancer in Hong Kong Chinese: Mortality and Histologic Types 1973-1982 . Br. J. Cancer 1984; 50:149. I 7. Koo, L.C., et al. An Analysis of Some Risk Factors for Lung Cancer in Hong Kong. Int J _ . Cancer 1985;35:149. I 8. Maclennan, R, et al. Risk Factors for Lung Cancer in Singapore Chinese a Population with High , Female Incidence Rates. Int. J. Cancer 1977; 20:854. I I I I I N C ~ I ~ ~ -7- ca W I Ch <J1 I

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I Reference 1. Li Lie. "Histological Classification and Aetiology Discussion of Lung Cancer in Guangzhou City." Published in the First International Academic Discussion of Pathology. Zhu Hai, China. June, 1988. - 4 - I I I I 1 I I I I I I I I I

I I I I I I I I I I I I I I I I I I PROGRESSIVE CHANGES IN THE RELATIVE DISTRIBUTION OF DIFFERENT HISTOLOGICAL TYPES OF LUNG CANCER IN GUANGZHOU, CHINA Li Lie, Huang Shu-wei, Lu Zhen jie and Wan Guang-ai Department of Pathology, Guangzhou Medical College, Guangzhou, China Abstract A total of 1,048 lung cancer cases obtained from the First Affiliated Hospital of Guangzhou Medical College from 1978 to 1994 were reviewed in this paper. According to WHO's lung cancer classification, the proportion of squamous cell carcinoma (SCC) was 54.68%, for adenocarcinoma 32.44%, for small cell carcinoma 5.15 %, and for large cell carcinoma 1.91 %. These data on SCC and adenocarcinoma were compared to those in a former paper(1). In this comparative study, the total lung cancer cases were nearly the same. When the total number of cases collected in the 16-year period was grouped into three sub-periods (1978 to 1984, 1985 to 1989 and 1990 to 1994) and then analyzed, the percentage of SCC for the three sub-periods was 68.72 %, 57.99 % and 38.8 % respectively. On the other hand, the percentage of adenocarcinoma was 19.43%, 29.1% and 47.49%, for the same three sub- periods. These results showed that the rate of SCC decreased progressively during the past two decades while that of adenocarcinoma increased markedly and even surpassed the rate of SCC. There are statistically significant differences with regard to trend changes in both carcinoma types during the sub- periods (P <0.05 for SSC and P<0.005 for adenocarcinoma), thus confirming the assumption that there is a trend towards an increase of adenocarcinoma in the 16-year period. The increase of adenocarcinoma appears to be due to multiple contributing factors. Future studies of longer duration are needed to help to elucidate the relative role of these factors. Introduction Pathological studies are central to lung cancer research since they provide information on the etiology, prevention, and cure of lung cancer. In this report, the WHO's 1981 criteria for lung cancer histological classification was used to analyze 1048 lung cancer cases collected from 1978 to 1994. A progressive change in the relative distribution of squamous cell carcinoma and adenocarcinoma was observed over the 16-year period. Materials and Methods A total 1,060 lung cancer specimens, obtained by biopsy and during surgery, were collected in the First Affiliated Hospital of Guangzhou Medical College from 1978 to 1994. Based on the histological classification of lung malignant epithelial tumors issued by WHO in 1981, 1048 cases were classified and compared to data presented in a former paper (1). Twelve cases (including a carcinoid tumor, malignant lymphoma and adenoid cystadenocarcinoma) were excluded in the statistical analysis. The data collected in the 16 years, from 1978 to 1994, was grouped into 3 sub-periods - the first sub-period from 1978 to 1984 the second from 1985 to 1990 and the third from 1990 to 1994 - and then analyzed by sub-period. I

I I I I I I I I I I I I I I Discussion The rates of squamous cell carcinoma and adenocarcinoma in a total of 10481ung carcinoma cases are similar to those described previously (1). The incidence of squamous cell carcinoma declined while the incidence of adenocarcinoma increased significantly, confirming the hypothesis previously proposed. Epidemiological data showed that during the last two decades the incidence of lung cancer continues to increase; thus, as a consequence the mortality due to lung cancer also increased. Specifically, the incidence of lung adenocarcinoma has been rising significantly. More emphasis should be given to the epidemiology and etiology of lung cancer. From a pathological standpoint, more investigations on the relationship between histological types and environmental factors such as smoking as well as airborne pollution and sex are needed. From the point of view of the genesis of lung cancer, squamous cell carcinoma arises from the bronchial epithelium which has already undergone squamous metaplasia ie., repeated proliferation and destruction of cells accompanying chronic inflammation has already occurred. As such, pathological development of squamous cell carcinoma is considered to be a lengthy process. Adenocarcinoma develops in a much shorter time than squamous cell carcinoma since it can arise directly not only from the epithelium of the bronchial tree but from the glands along the bronchial wall as well. Thus, adenocarcinoma tends to occur in younger individuals. More sub-type classification of lung cancer could be beneficial for better understanding the multifactorial nature of lung cancer. - 3 - L

INDUCTION OF DNA-PROTEIN CROSSLINK IN RAT LUNG AND BLOOD BY THE CARCINOGEN NICKEL Lei Yi-xione*, Zhang Qiao** and Zhuang Zhi-xiong** I ** * Department of Hygiene, Guangzhou Medical College, Guangzhou, China Research Unit of Genotoxicology, Sun Yat-sen University of Medical Sciences, Guangzhou, China I I I I I I I I Abstract Nickel(II) compounds are common environmental contaminants and human carcinogens. One of the lesions associated with nickel(II) exposure is formation of DNA-protein crosslinks (DPC), but the biological significance of DPC by nickel(II) in vivo is presently poorly understood. In order to investigate the relationship between lung cancer and DPC induced by nickel compounds, and in an attempt to develop biomarkers for nickel exposure, we have used a rapid, simple and sensitive 1251_ postlabelling assay to detect the formation of DPC in white blood cells (WBC) and lungs from male Sprague-Dawley rats exposed intraperitoneally to nickel chloride (NiC12). The results show that 20 hr after the rats were treated with NiC12 at concentrations ranging from 10 to 30 mg/kg body wt. i.p., DPCs were found in white blood cells (WBC) and lungs in a dose-dependent manner. The formation of DPC in WBC and lungs was also observed following multiple exposure of rats to NiC12 (10 mg/kg, i.p. 3 weeks); the results were similar to those after a single dose. We consider that the DPCs found in rat lungs after NiCI2 treatment are possibly related to the carcinogenicity of nickel compounds. In addition, the DPC in the lungs and WBC may be used as biomarkers to quantitatively represent exposure to NiCl2 and genotoxic lesions induced from such exposure. In our DPC-induction studies, WBC were shown to be more sensitive than the lungs in responding to nickel; there also was a significant correlation in DPC between the two tissues, indicating that measuring DPCs in WBC may be a good surrogate for investigating human exposure of target tissues to environmental carcinogens or mutagens. Introduction t! DNA-protein crosslinks (DPC) are thought to be important genotoxic lesions induced by environmental contaminants and carcinogens such as UV light (1), y-radiation (2), aklylating agents f (3), formaldehyde (4), benzo(a)pyrene (5), and some metal compounds such as nickel (6),  chromate (7) and cis- or trans-platinum(II) diamine-dichlorides (8). These lesions, unlike DNA strand breaks and other readily-repaired DNA lesions are relatively persistent in the cells (9),(10). ~ Because they are poor repaired, DNA-protein complexes may be present during DNA replication and possibly cause a loss of important genetic material such as the inactivation of tumor suppressor genes (10),(11),(12). N - O .' 00 Nickel compounds are common environmental contaminants and human carcinogens. A v number of epidemiological and experimental studies have shown that nickel compounds cause lung cancer W = in both humans and animals (13). Recently, many studies with these agents have shown that they N I

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induce DNA-protein crosslinks, mostly in intact cells in vitro; in contrast, there are fewer reports in vivo (14),(15),(16),(17). The biological significance of DNA-protein crosslinks in vivo is poorly understood at present. In order to investigate the relationship between lung cancer and DNA- protein crosslinks induced by nickel compounds, and in an attempt to develop biomarkers of nickel exposure, we have used a new rapid, simple and sensitive 1251-postlabelling assay developed recently by Zhuang et al. modified from earlier report by Lin X eta l. (17) to detect the formation of DNA-protein crosslinks in vivo in white blood cells (WBC) and lungs from male Sprague-Dawley rates exposed intraperitoneally to nickel chloride (NiC12). Materials and Methods Chemicals Nickel chloride was purchased from Guangzhou Chemical Factory; sodium dodecyl sulfate (SDS) was purchased from SERVA; protein K was purchased from E. Merck; Tris was purchased from FARCO Chemical Supplies; urea was purchased from Promega Corporation; Na1251 was purchased from the Atomic Energy Institute, Chinese Academy of Science; Q-mercaptoethanol was purchased from FARCO Chemical Supplies; male Sprague-Dawley rats were obtained from the Center of Experimental Animal, Sun-Yat-Sen University of Medical Science. Animals Male Sprague-Dawley rats were randomly assigned to exposed and control groups using weight as a factor. Each group comprised 8 rats. In the acute exposure experiment, rats weighing 175-200g were given i.p. injections 0.5 ml of 0.9% NaCl solution containing NiCl2 at doses of 10, 20, 30 mg/kg body wt. Control rats were given i.p. injections of 0.5 ml of 0.9% NaCI solution. In the subacute exposure experiment, rats weighing 150-175g were given by i.p. injections of 0.9% NaCl solution containing NiC12 at 10 mg/kg (twice a week i.p.) for 3 weeks. Control rats were given by i.p. injections of 0.5 ml of 0.9% NaCI solution. DNA isolation After exposure, rats were sacrificed, the lungs were excised and 3-5 ml blood per rat was collected in a tube using heparin as an anticoagulant and the WBC were isolated by centrifugation. The lungs collected were homogenized. DNA was extracted from lung and WBC tissues as previously described (17) with some modification. Briefly, tissue pellets were lysed in 10 mM Tris, pH 8.0 containing 100 mM NaCI and 1% SDS. The homogenate was incubated with RNase (10 mg/ml) and proteinase K (300 µg/ml) following which DNA and DNA-protein complexes isolated by repeated extraction with phenol/chloroform and precipitation with ethanol. -2- I I I I I I I I I I I I I I I

Figure 1. Formation of DPC in Rats WBC and Lungs Following Single Exposure to Nickel Chloride DPC % of Oontrol 400 1 I I I I 0 m 0 6 10 Wn 25 30 36 NICIs (m0/k0) Table 2. Forwation of DNA-protein Crosslinks in Rats WBC and Lungs Following Multiple Exposure to NiC12 (10 mg/kg) NiCl2 (mg/kg) No Rats ODa XtSD (cpml}tg DNA) DPC%of Control WBC Control 8 1.81 1340 ± 181 100 NiCl 8 1.76 3310 ± 906 247** Lung Control 8 1.92 4347 ± 757 100 NiC12 8 1.89 6352 ± 1538 146* * P < 0.0.5 ** P<0.01 Compared with control (Student's t-test) A Comparison of DNA-protein Crosslinks Formed in WBC With Those in Lungs To determine DNA-protein crosslinks in WBC relative to target organs of nickel toxicity of DNA- protein crosslinks between WBC and lung are analyzed and correlated. The results showed that WBCs were more sensitive to nickel than lung tissue. In addition, there was a significant correlation between the amount of DNA-protein crosslinks in WBC and in lungs (r=0.97, P<0.05) (Fig. 2). -4- I I I I I I I I I I

the formation of DNA-protein crosslinks may be related to carcinogenicity of chemical carcinogens and since nickel compounds are well established as human and animal carcinogens, we consider that the DNA-protein crosslinks found in rat lungs after NiC12 treatment is possibly related to the carcinogenicity of nickel compounds. In addition, the DNA-protein crosslinks in lung and WBC may be biomarkers to represent quantitative exposure to NiC12 and the development of genotoxic lesions resulting from such exposures. A number of investigators have suggested the use of lymphocytes as targets for developing biomarkers of chemical exposure (19),(20). These cells offer a number of advantages. First, they are easily obtainable from humans and can be isolated in relatively high purity. Second, many lymphocytes are long lived in the body and therefore have the potential to be sentinels to past exposure. Third, lymphocytes are nucleated cells thus allowing the DPC to be formed. Recently, some studies have demonstrated the preferential accumulation of chemicals in lymphocytes as compared to other blood cells (21). In the present studies, WBC was found to be more sensitive to nickel inducing DPC formation than lung tissues. In addition, there was a significant correlation between DNA-protein crosslinks in WBC and in lung, indicating that DNA-protein crosslinks in WBC may be a good surrogate for investigating the exposure of human population to environmental carcinogens or mutagens. N O tb ~ V t70 W W Qi -6- -1 I i I I I I I I I I I

15. Klein, CB et al.; The rote of oxidative prosesses [sic] in metal carcinogenesis. Chem. Res. I I I Toxicol. 1991; 4:592-604 16. Kasprazak, KS; The rote of oxidative damage in metal carcinogenicity. Chem. Res. Toxicol. I 1991; 4:604-615 17. Lin, X etal.; Analysis of residual amino acid-DNA crosslinks induced in intact cells by nickel I and chromium compounds. Carcino eg nesis 1992; 13(10):1763-1768 18. Ciccarelli, RB et al.; Nickel carbonate induces DNA-protein crosslinks and DNA strand breaks in rat kidney. Cancer Lettersl8. 1981; 12:347-354 , 19. Perera, F; The Potential usefulness of biological markers in risk assessment. Environ. Health Perspect 1987; 76:141-145 I 20. Lucier, GW and Thompson, CL; Issues in lymphocytes be used as surrogate markers? Environ. Health Perspect. 1987; 76:187-191 I 21. Coogan, TP et al.; Distribution of chromium within cells of the blood. Toxicol. Aopl. Pharmacol. 1991; 108:157-166 I I I I I I , 0 , tp ~ ~ w w w , 0) -8- ' I

I I Results In the total of 1048 cases there were 573 squamous cell carcinomas (54.68%), 340 adenocarcinomas (32.44%), 54 small cell carcinomas (5.15%) and 20 large cell carcinomas (1.91%). The remaining 61 cases were adenosquamous carcinomas (5.82%). The histological types and their rates during different sub-periods are shown in Table 1. Table 1. Hlstologicaltypes of 1048 lung cancer cases and their rates during different sub-periods S-3teaciod/Cc 3Vc l97@-1?%4 19B0-I494~~ ~ .ToL4 ~ SquammrCeBM1+~.+ai-•• 145 (68.72%) 312 (57.99%) 116 (38.8%) 573 SvvaCellCaremom. 16 (7.58%) 25 (4.65%) 13 (4.34%) 54 Admoc.xmoma 41 (19.43%) 157 (29.18%) 142 (47.49%) 340 LuseCeU r•-...:..-. 9 (4.26%) 6 (1.12%) 5 (1.67%) 2U Mmo.qu.moueCucmoma 38 (7.06%) 23 (7.69%) 61 TnW 211 538 299 1048 The rates of squamous cell carcinoma and adenocarcinoma in the 1048 cases on the above table are similar to a previous publication (1). The rate of adenosquamous carcinoma, however, appears to have increased. The data in Table 1 are graphically illustrated below. M J00 80 60 40 20 a 19'/8-1084 1985-1990 1900 t99G - Squamous Cell CarcSnoma --AdBnoeareinuma As shown above, during the last 16 years squamous cell carcinoma, in proportion to the total lung cancers, has been decreasing progressively. The rate of adenocarcinoma, however, has markedly increased. A statistically significant difference was observed in the incidence of the two types of lung cancer for the three sub-periods (P < 0.05 for squamous cell carcinoma and P< 0.005 for adenocarcinoma). _ 2 _ t I I 1 I I I I I I I I I I

I I I I I I I I I I I I 1251-Radioactive postlabelling 125I-postlabelling of DNA-protein complexas was as described (17). DNA (100 µg) was sus ended in 100 µl of 2% SDS, 30% urea and 0.5 M Tris-HC1 pH 7.6 and mixed with 10 µCi of NaP25I and 5 µl of chloramine T solution (6 mg/ml) and incubated at room temperature for 2 min. The iodine was reduced by the addition of 10 µl of 20% 0-mercaptoethanol, and the DNA-protein complexes (with tyrosine labeled by iodination) was isolated by repeated (3x) p25cipitation with ethanol. The pellet was finally dissolved in 10 mM Tris pH 8.0. The unincorporated I I in the supernatant was discarded. The DNA samples were assayed for radioactivity in a ry-counter and their UV absorbance was measured at 260/280 nm. Efficiency of 1251-labelling was expressed as cpm/µg DNA. Results DNA-protein crosslinks induced by NiCI2 in tissues The ability of nickel chloride to induce DNA-protein crosslinks in rat WBC and lungs was analyzed using 1Z5I-postlabelling assay. The results showed that 20 hr after rats had been injected intraperitoneally with NiC12 ranging from 10 to 30 mg/kg body wt., DNA-protein crosslinks were found in WBC and lungs in a dose-dependent manner (Table 1 and Fig. 1). Similarly, the formation of DNA- protein crosslinks in the two tissues were also observed in rats exposed repeatedly to NiC12 at 10 mg/kg for 3 weeks (Table 2). Table 1. Formation of DNA-protein Crosslinks in Wlrite Blood Cell (WBC) and Lungs of Rats Following a Single Exposure to Nickel Chloride NiC12 (mg/kg) No Rats ODa X ± SD (cpm/µg DNA) DPC % of Control WBC 0 8 1.82 1281 t 256 100 10 8 1.76 2876 t 594 236* 20 8 1.75 4474 t 1455 367* 30 8 1.75 3192 + 1410 262* Lung 0 8 1.90 4063 t 658 100 10 8 1.90 6459 f 1144 159* 20 8 1.91 7804 t 1089 192* 30 8 1.90 5861 f 803 144* aThe ratio of optical density of samples measured at 260/280 *P<0.01 Compared with control (Student's t-test) 0 ao ~ V 00 W ~ W 3 p i

I I I , I I I I I I I I I I I I I I Figure 2. The Correlation of DPC Between WBC and Lungs Following a Single Exposure to NiC12 OPC (Fold of ecnhci) In lung Zff ~ y0.6768•0.3380x 0•0.97, P-0.051 1.6 t ~ OPC (FC/d cf eontrol) In W6C Discussion s The formation of DNA-protein crosslinks may be an important mechanism of chemical-mediated genetoxicity. Structural proteins that normally do not bind to DNA can become covalently crosslinked with DNA under the influence of certain chemicals, such as nickel and chromate compounds. The formation of inappropriate covalent DNA-protein crosslinks can disrupt gene expression and chromatin structure and can also lead to deletion of DNA sequences during DNA replication, since these lesions cannot be readily repaired (10),(11),(12). Previous studies have shown that DNA-protein crosslinks may be related to genetoxicity and carcinogenicity of chemical carcinogens. For example, Lam et al. (18) observed the formation of DNA-protein crosslinks by acetaldehyde in target tissues of the rat nasal cavity at concentrations similar to those that induced nasal cancer. Sugiyama et al. (10) suggested that the results from CaCrO4 induced DNA-protein crosslinks in Chinese hamster ovary (CHO) cells implicated the crosslinks as an important lesion that may be responsible for the cytotoxic and carcinogenic properties of chromate. Many studies with nickel compounds have shown that it can directly and indirectly induce DNA- protein crosslinks in vitro. Nickel(II) was thought to form stable protein-nickel(II)-DNA complexes, and a strong interaction between ttickel(II) and amino terminal residues and the imidazole group of histidine residues has been demonstrated (14). On the other hand, increasing evidence suggests that mckel(II) may generate reactive oxygen species (ROS), which may indirectly mediate DNA damage, protein oxidation and DNA-protein crosslinks formation (15),(16). The reports on the DNA-protein crosslinks in vivo by nickel compounds are fewer. Ciccarelli et al. (18) had detected DNA-protein crosslinks in kidney nuclei from nickel carbonate-treated rats using alkaline elution technique. They considered that the results tnight be related to the nephrotoxicity and carcinogenicity of nickel compound. However, the DNA- N protein crosslinks by NiClz in vivo are poorly understood. In our studies, DNA-protein crosslinks were 0 00 found in WBC and lung of rats treated with NiC12 in a dose-dependent manner. Moreover, multiple ~ exposure of rats to NiC12 also produced DNA-protein crosslinks in the two tissues. Due to the fact that 00 W W -5- I

 ~ ! , 1. , 2. ' 3. I 4. I 5. I 6. I 7. I I 8. 9. 10. I 11. I 12. I 13. I 14. . . . . . . 1988; 19(4):341-384 0 co a -~ V W i -7- W * Q1 tb References Smith, KC; Dose dependent decrease in extractability of DNA from bacteria following irradiation with ultraviolet light or visible light plus dye. Biochem. Biophys. Res Commun 1962; 3:157- 163 Fornace, AJ Jr and Little, JB; DNA crosslinking induced by X-ray and chemical agents. Biochem. Biophys. Acta. 1977; 477:343 Grunicke, H eta l.; Effect of alkylating antitumor agents on the binding of DNA to protein. Cancer Res. 1973; 33:1048-1053 Cosma, GN etal.; Growth inhibition of DNA damage induced by benzo(a) pyrene and formaldehyde in primary cultures of rat tracheal epithelial cells. Mutat. Res. 1988; 201:161-168 Christine, M et al.; DNA-protein crosslinks induced in a Hamster tracheal epithelial cell line by benzo(a) pyrene. Biochem. Biophys. Res. Commun. 1982; 109:1291-1296 Patierno, SR and Costa, M; DNA-protein crosslinks induced by nickel compounds in intact cultured manmialian cells. Chem. Biol. Interactions 1985; 55:75-91 Wedrychowski, A et al.; Chromium-induced crosslinking of nuclear proteins and DNA. J. Biol. Chem. 1985; 260:7150-7155 Banjar, ZM et al.; Cls- and trans-dianuninedichloroplatinum (II) -mediated crosslinking of chromosomal non-histone protein to DNA in Hela cells. Biochemistry 1984; 23:1921-1926 Oleinick, NL et al.; The formation, identification and significant of DNA-protein. Br. J. Cancer 1987; 55 (Suppl VIII):135-140 Sugiyama, M et al.; Characterization of DNA lesions [sic] induced by CaCrO4 in synchronous and asynchronous cultured mammalian cells. Mol. Pharmacol. 1986; 29:606-613 Costa, M; Molecular mechanisms of nickel carcinogenesis. Annu. Res. Pharmacol. Toxicol. 1991; 31:321-337 DeFlora, S and Watterhahn, KE; Mechanisms of chromium metabolism and genetoxicity. Life Chem. Res. 1989; 7:169-244 IARC; Monographs on the evaluation of carcinogenic risk to humans, Supplement 7, Lyon, France, 1987 Coogan, TP et al.; Toxicity and carcinogenesis of nickel compounds CRC Crit Res Toxicol W I

I I I I No statistically significant difference was found in RAL between males and females in the cases. However, in the controls, a significant difference was observed (Table 3) Table 3. RAL between males and females in the controls/cases Female Male Group Average S.D, Average S.D, t-test P-value Cases 1.36 1.10 2.01 1.33 1.61 > 0.1 Controls 0.79 0.46 2.06 1.96 5.66 <0.001* No statistically significant difference could be found between males and females in the controls after controlling for smoking. I I I I I I I I (3) Relationship between relative adduct labeling (RAL) and different modes of exposure: single risk factor analysis A. RAL and smoking. Adducts were higher in female smokers, compared to female nonsmokers; but were comparable to those found in male smokers (Table 4). Table 4. Analysis of RAL in smokers versus nonsmokers Smokers Nonsmokers Sex Average S.D. Average S.D. t-test P-value Female 2.26 1.46 1.01 10.73 2.45 <0.05* I Male 2.06 1.34 - No significant difference existed between the RAL of male and female smokers B. RAL and passive smoking. After controlling for smoking, no difference was found between the "exposed" versus the "unexposed" group, in either cases or controls (Table 5). I

I , (1) General information on samples analyzed , A total of 19 pairs of males and 18 pairs of females were analyzed. Ages of the cases and controls were 46.4±7.8 years and 45.9 f 9.0 years, respectively. Their residence was almost equally I distributed between rural (12 pairs, 32.4%), township (13 pairs, 31.5%), and the city (12 pairs, 32.4%). The distribution of respiratory disease in the controls is illustrated in Table 1. Table 1. Distribution of respiratory disease in controls I I Disease Number % of Total I Tuberculosis 18 48.7 Bronchitis 8 21.6 ! Tuberculosis +bronchitis 3 8.1 Pulmonary abscess 3 8.1 I Pulmonary cyst 3 8.1 Inflammatory pseudotumor 2 5.4 I (2) Relative adduct labeling (RAL) in cases and controls I Although RAL was higher in the cases (1.69.t 1.24 x 10-8) compared to the controls (1.45 t 1.56 x 10-8); the difference was not statistically significant (t = 1.05, p> 0.2). When the RAL was calculated on the basis of sex, a statistically significant difference was found between the females but not between the males (Table 2) I Table 2. Relative adduct labeling (RAL) in cases and controls I Sex Pairs Cases Controls S.D. T-test P-value I RAL1 RAL1 RAL F* 18 1.36 0.79 0.89 2.84 < 0.025 I M 19 2.01 2.06 1.71 -0.15 > 0.5 N O 1 ex ressed as (x10-8) tb ~ I p * V found to be statistically significant between cases and controls even after controlling for smoking 00 w ca V I ~ -4- 1 I

Table 8. Multiple regression analysis of risk factors for lung cancer Term Coefficient STD P Odds ratio 95% CI Recent exposure Smoking 1.789 1.22 0.142 5.982 0.5503 - 65.02 Passive smoking 0.240 0.53 0.652 1.271 0.4488 - 3.60 Occupational exposure -0.889 1.07 0.408 0.411 0.0502 - 3.37 Cooking 1.895 0.81 0.019* 6.650 1.3600 - 32.53 Air Pollution 1.820 1.01 0.072 6.173 0.8486 - 44.91 Cumulative exposure Smoking 2.459 1.12 0.033* 11.69 1.2120 - 112.70 Passive smoking 1.512 0.83 0.068 4.538 0.8960 - 22:98 Occupational exposure 0.311 0.96 0.747 1.365 0.2060 - 9.04 Cooking 3.124 1.32 0.018* 22.75 1.7260 - 299.70 Air pollution 2.864 1.38 0.038* 17.54 1.1680 - 263.40 P values of <0.025 or 0.05 means that there are significance. (7) Analysis of histologic types of lung cancer Table 9 shows that squamous cell carcinoma was the predominant cell type of lung cancer among males (47.4%) while adenocarcinoma was highest among females (44.4%). Table 9. Analysis of histologic types of lung cancer Total Sex No. Both 37 M 19 F 18 Squamous Cell Adenocarcinoma Small Cell % No. % No. % No. % 100 14 37.8 14 37.8 9 24.3 100 9 47.4 6 31.6 4 21.1 100 5 27.8 8 44.4 5 27.8 I 1 I I I I I I I I I I I I I ,

I I I I I I I , I I I I I I I I Table 7. Multiple linear regression analysis of risk factors for RAL Variables in the Equation Sex Variable B S.E. of B Beta T Sig T Total Recent smoking 62.17 10.60 0.545 5.86 0.000 (constant) 80.44 16.70 4.82 0.000 Male Recent smoking 58.05 15.45 0.484 3.76 0.0006 (constant) 54.72 36.05 1.52 0.1381 Female Recent smoking 86.60 19.06 0.615 4.54 0.0001 (constant) 90.58 12.31 7.36 0.0000 (5) Correlation and simple regression analysis for the "synthesis" index of exposure When the relationship between the "summed" indices ("synthesis" plus actual exposure) of the variables and RAL was analyzed in female cases and controls, a significant positive correlation was found. In the lung cancer cases, correlation coefficient r = 0.9566, p<0.005, the intercept a = 0.3868, and the regression coefficient b = 0.1689. In the case of the controls, r = 0.8055, p<0.05, a = 0.5277, and b = 0.0997. No such significant correlation was found in the males in either group (for cases, r = 0.8584, p<0.02, and for controls, r = 0.6052, p>0.05). (6) Multiple logistic regression analysis of risk factors for lung cancer (Table 8) These results show: (i) both recent exposure and cumulative exposure to cooking were risk factors for lung cancer, OR = 6.65 and 22.75, p<0.025, (ii) cumulative but not recent exposure to active smoking was a risk factor for lung cancer, OR = 11.69, p<0.05, and (iii) cumulative exposure to air pollution may be a risk factor for lung cancer, OR = 17.54, p<0.05. Other factors, e.g., exposure to passive smoke and occupational exposure, were not associated with lung cancer. K1 o ' tb ~ V 00 ' -7- tWa . ~ V I

i I I I I I I I I I I I I I I I I I C. Smokers were divided into active smoker, ex-smoker, and nonsmoker. An active smoker is an individual that smoked an average of at least one cigarette a day until time of surgery. An ex- smoker is an individual who has stopped smoking at least one month before surgery. A never-smoker is an individual who has never smoked or who smoked an average of less than one cigarette per day in one year. Recent smoking status was defined as follows: 0, nonsmoker or stopped smoking for over a month before surgery; 1, smoking on average 1-9 cigarettes per day; 2, smoking an average of 10-19 cigarettes per day; and 3, smoking at least 20 cigarettes per day. Smoking index (BI), defined as [cigarettes per day x smoking years], was also divided into the category of 0, nonsmoker; 1, BI<200; 2, BI 200-400; 3, BI>400. D. Passive smoking means contact with smokers at home, in the office, or in the workplace. Recent passive smoking situations were further categorized in the following manner: (i) Occasional exposure in which "0" was defined as no exposure in one month and "1" referred to contact with (1-9) x 2 cigarettes per day; and (ii) Constant exposure in which "2" indicated exposure to (10-19) x 2 cigarettes per day and "3" showed exposure to at least 20 x 2 cigarettes per day. Likewise, cumulative passive smoking was calculated based on the following: (i) Occasional exposure: with "0" showing no contact and "1" showing a cumulated total of <400; and (ii) Constant exposure: with "2" showing a cumulated total of 400-800 and "3" showing a cumulated total of > 800. E. Occupational exposure referred to contact with methermal, benzol, metal powder, asbestos, mist/dust, coal tar, nickel, chromium, arsenic etc. at work. On the basis of contact history, "recent" and "cumulative" exposure was distinguished by: "0" referred to no contact and "1" referred to having had contact. F. Cooking included setting up the coal burning for cooking as well as doing the actual cooking and stir-frying. "Recent" and "cumulative" exposure was defined as: "0"-no involvement with cooking or on average less than once per day; "1"-average of once per day; and "2"-average of twice or more per day. G. Air pollution referred to working or living near (approximately 1 km) a factory or a place capable of discharging mist and/or dust. "Recent" and "cumulative" exposure was distinguished in the following manner: "0" indicated a lack of air pollution, and "1" indicated the presence of air pollution. Results Among the 84 lung cancer samples collected in the Pathology Division, 8 samples were destroyed by temperature, 2 of the female samples were excluded since DNA could not be extracted from one of them, leaving a total of 74 samples for actual analysis of carcinogen-DNA adduct. V co W 3- W w I

I Table 5. Effect of exposure to passive smoke on RAL ' ' Lung Cancer* Controls I Passive smoke Average S.D. Average S.D. t-test P-value None+occasional 0.56 0.28 0.63 0.27 0.39 >0.05 I Constantly 1.21 0.78 0.77 0.53 1.46 >0.01 No significant difference was found between the "none +occasional" and the "constantly exposed" groups I (t=1.54, P>0.1). C. RAL and cooking. In females but not in males, significant differences were found I between cases and controls (Table 6). Table 6. Effect of Cooking on RAL I I Lung Cancer Controls Sex Average S.D. Average S.D. t-test P-value I Female* 1.36 1.10 0.73 0.73 0.34 <0.05 Male 1.83 1.34 2.13 2.20 1.49 >0.01 , No significant difference in females between cases and controls persisted even after controlling for smoking. D. RAL and others. After controlling for smoking, no difference could be found from I occupational exposure and from air pollution. A note of caution is that relatively small numbers were used in this study. I (4) Multiple linear regression analysis of the relationship between RAL and exposure to environmental agents The five variables of recent and cumulative exposures were analyzed by the multiple linear I regression model. After controlling for interaction between the factors, only recent smoking was found to be related to RAL in both males and females (Table 7). I I -6- I

ABSTRACTS 2081783382

2081783380

Analysis of Pathological Specimens. After surgery was completed, lung tissues from subjects who also completed the questionnaires were sent to the Pathology Division of Beijing Tuberculosis & Thoracic Tumor Institute. Specimens (approximately 1.5 x 1.5 x 3 cm) consisted of the lesion itself and the surrounding tissues. They were stored in liquid nitrogen until time of analysis. Molecular analysis of the stored samples was performed at the Molecular Toxicology Division, Institute of Industrial Health and Occupation Disease, Chinese Academy of Preventive Medical Sciences. Pathological diagnosis was done at the Pathology Division by first dividing the samples into those that had primary lung cancer (as defined by WHO recommended criteria published in 1981) and those that had other types of respiratory diseases. Molecular Analysis. To minimize bias, DNA was extracted from doubly blind coded samples by first digesting lung tissues with protease K, followed by extraction with chloroform/phenol. To determine the formation of carcinogen-adduct in target DNA, the extracted DNA was first digested with nuclease P1 (0.11 units/µl) and bacterial alkaline phosphatase (13 munits/µl) to yield [XpN+N+Pi]. The DNA digest was then labeled with [-y-32P]ATP (6,000 Ci/mmol, 3 µCi/µl) and polynucleotide kinase (0.24 units/µl), resulting in *pXpN. Unreacted labeled ATP was enzymatically converted to [ADP+3ZPi]. The total reaction mixture was then spotted on PEI-plates to generate a pXpN map based on resolution of different labeled spots and zones on PEI-plates. Radioactivity present in different zones and spots on the chromatogram was quantitated by scintillation counting The values obtained were converted to moles of adducts formed by using the specific activity ofs[32P-ATP] in the labelling scheme, applying the formula: [ry-32P]ATP (specific activity) =[cpm in pdAP]/[pmole dAP/spot x 32P decay coefficient] The relative adduct labeling value (RAL) of DNA adduct was obtained as follows: RAL = [cpm of adduct]/[32P ATP (specific activity)xDNA in spot] Statistics and Analysis. The Epi-info software was used for primary data entry and analysis. The student t-test was used to examine whether differences existed between cases and controls with respect to sex. The same t-test was also used to obtain analysis of variance and to check for differences between the various factors analyzed. Since medical data has a tendency to assume a skewed distribution, the primary data were converted into logarithmic terms and then reanalyzed, similar results were obtained. SPSS software was used for multiple liner regression and multiple logistic regression analysis. Risk Factors and Parameters Analyzed. A. Residential area was divided into four categories: city, township, rural, and industrial. "City" refers to municipality directly under the jurisdiction of the central or provincial government whereas "township" refers to all other places besides the "city." B. Environmental exposure refers to being exposed to any one of the following five conditions: active smoking, passive smoking, occupational exposure, cooking, and air pollution. In each of these cases, since there is a source for generating the "e