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I I I I I I I I I I I I I I MOLECULAR EPIDEMIOLOGIC STUDY OF COAL SMOKE-GENERATED ENVIRONMENTAL CARCINOGENS AND LUNG CANCER IN HUMANS Xie Huei-iiang, Chen Xiao jia, Wang Su-min and Zheng Su-hua Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China Introduction Animal studies show that the incidence of lung cancer is closely associated with the amount of carcinogens present. It is also known that most environmental agents exist as pro-carcinogens and must be further activated to exert their carcinogenic effect. For example, after gaining entry into the human body, the polycyclic aromatic hydrocarbons, including benzo(a)pyrene (BaP), must be enzymatically metabolized to an activated product, benzo(a)pyrene diol epoxide (BPDE), which in turn interacts covalently with the DNA to form the active carcinogenic macromolecular products, BPDE-DNA adduct. Relatively few studies - especially those that are population-based, however, have provided direct experimental evidence for the actual presence of such types of carcinogenic macromolecular products. In China, environmental pollution comes from coal smoke and cigarette smoking. Cigarette smoking has frequently been regarded as the culprit for the rapid worldwide rise in the incidence of lung cancer. Although lung cancer has also been increasing steadily in recent years in adult Chinese females, it is doubtful whether cigarette smoking can be offered as an explanation since less than 10% of the females are known to be cigarette smokers. Risk factors for lung cancer in nonsmoking females have traditionally been studied by epidemiologic methods. The advent of molecular biology has paved the way for the development of a branch of epidemiology, often referred to as molecular epidemiology. In this study, we have integrated the principles and the tools of molecular biology, with those derived from classical epidemiology, and have explored the influence of lifestyle factors on human lung cancer. Materials and Methods Subjects. In this case-control study, cases (20 males and 20 females) were obtained from primary lung cancer in-patients with lesions that could be excised, and matched by sex, age ( t 5 years) and residence (city, township, rural versus industrial settings) with 20 pairs of controls that consisted of in-patients without primary lung cancer and with resected lesions. Questionnaire. Prior to surgery, cases and controls were given two questionnaires which focussed on , environmental factors for respiratory disease, as well as on situations that related directly to the  respiratory disease itself. In addition to personal data, (such as subject's name, sex, place of birth, recent address), information was also obtained on the following: history of occupations (special attention being ~ given to possible occupational exposure to harmful substances), history of smoking, family history of lung . cancer, history of previous respiratory disease, conditions of residence (including type of building N material, oven use, heating equipment, cooking style, number of smokers in the family), and in the case 00 , of females, information on menstruation history, pregnancy, and delivery. s V tb W W , V i I

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 "exposed" condition, i.e., "exposure" is "synthesized," a "synthesis" index was added to the "exposure" index in the calculation of "total exposure." In addition, depending on the history of exposure, "recent," referring to exposure within the past month, and "cumulative" exposure was calculated separately in the single risk factor analysis. 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 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 , (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

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 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

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

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 I I I When the cases of males and females were combined and analyzed as a group, a significant difference was found between squamous cell carcinoma, adenocarcinoma, and small cell carcinoma, with respect to the relative adduct labeling (F = 3.56, p<0.05). Such differences were no longer present when males and females were analyzed separately, probably as a result of the small number of samples available for this study (Table 10). Table 10. RAL present in different histologic types of cancer Squamous Cell Adenocarcinoma Small-Cell* Sex Average %- Average % Average ' % F P Both 2.32 1.42 1.47 1.14 1.07 0.64 3.56 <0.05 M 2.67 1.39 1.63 1.18 1.08 0.40 F 1.67 1.36 1.35 1.18 1.06 0.84 Almost all of the small cell carcinoma cases had been treated by chemotherapy before surgery. Discussion In recent years, a number of highly sensitive methods, such as 32P-nucleotide postlabeling, synchronous scanning fluorescent spectrophotometry, mass spectrophotometry and immunoassays, have been developed for detecting carcinogen-DNA adducts in tissues of choice. In the case of 32P postlabeling, the sensitivity approaches the specific activity of the radionucleotides and is therefore particularly suitable for DNA-adduct analysis. Indeed, these methods have provided the basis for a field of science, often referred to as molecular epidemiology, which uniquely integrates classical epidemiological tools and molecular biology principles to explore the molecular basis of cancer, including lung cancer, in target tissues. In the present study, we have conducted a small-scale study to basically test the feasibility of 32P-postlabeling method for the analysis of DNA-adducts in lung specimens. No significant difference was found between the relative DNA-adduct labeling (RAL) between cases and controls, although there is a tendency for RAL values to be somewhat higher in the lung cancer cases. When the cases and controls were further analyzed on the basis of sex, a significant difference was found in females where RAL was shown to be higher in females. In addition, the elevated RAL was found to be positively and statistically significantly correlated with exposure to cooking, suggesting that cooking was a risk factor for lung cancer. In addition, RAL was also associated with exposure to cumulative active smoking and to air pollution as well. Conclusion Benzo(a)pyrene diol-epoxide-DNA adducts could be further developed into a marker for lung o cancer in Chinese females. ~ i V 00 W -9- W V t0 I