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infants were on similar diets (they were not breastfed), the influence of nutrition may not play as great a role in the case of these infants but differences in the rates of uptake and metabolism of nicotine and/or the urinary excretion rates of cotinine were certainly established. The finding of relatively high uptake of ETS, as determined by nicotine/cotinine concentrations in the urine of infants, is in line with the observation that infants of smokers have higher rates of respiratory infections than infants in nonsmokers' homes (56). I Analytical data on nicotine and cotinine in physiological fluids of nonsmokers can be misleading in a few cases. These pertain to the very light smokers and those nonsmokers who either chew tobacco or use oral snuff. It is possible, though rare, that the very light smoker shows nicotine/cotinine levels approaching those of passive smokers with extremely high ETS exposure. When used in combination with cotinine measurements, COHb analyses can help to differentiate between the two groups. In regular consumers of snuff or chewing tobacco, cotinine levels are comparable to those found in cigarette smokers while thiocyanate levels and COHb values remain low (57). The determination of nicotine and cotinine in hair has been tried in an attempt to differentiate between active and passive smokers (58). This determination revealed higher nicotine concentrations in the hair of smokers than in the hair of ETS- exposed nonsmokers and documented the absence of cotinine, the major metabolite of nicotine, within the hairshaft of nonsmokers. Hair sampling for determining ETS-exposure of nonsmokers deserves as yet more thorough investigation. In summary, in the hands of experienced biochemists, the determination of nicotine and, especially, of cotinine in saliva, serum and/or urine in involuntary smokers represents a reliable, specific method for assaying the level of uptake of ETS by nonsmokers. The choice of biological fluid for the quantitation of cotinine depends upon the question asked. For the evaluation of changes in smoking behavior, serum or urine are preferred while saliva is sufficient to determine whether or not a proband is a smoker (59). For studies of ETS exposure, it is often impractical to collect serum by venipuncture, and since nicotine concentration in saliva can be extremely high immediately following ETS exposure, several hours must pass before the concentration of cotinine in saliva is stabilized (30). Also, when large numbers of probands are to be evaluated, it is preferable to avoid invasive procedures which might discourage participation and possibly bias the results. Measurements of cotinine in urine and saliva have been successfully used to quantitate ETS exposure in large 100

machine- smoked under identical conditions. Since the consumer of the low- yield filter cigarettes is likely to smoke more intensely, a larger portion of the tobacco column is burned during smoking of this type of cigarette than is burned during smoking of nonfilter cigarettes. Therefore, a somewhat lower yield of SS is expected from the low-yield cigarette smoked bv the consumer than is obtained by its standardized machine smoking. The exposure of nonsmokers to the effluents of burning tobacco products usually occurs after considerable dilution of these air pollutants. This is well substantiated by analyses of the air in enclosed spaces polluted by tobacco smoke (10,11). A. Biological Markers in Physiological Fluids The exposure of nonsmokers to ETS can be assessed with the help of questionnaires, by estimating the dose from the chemical analysis of smoke-polluted air, by personal monitoring of ETS components and/or by measuring the uptake of individual smoke components in physiological fluids of individuals during or after exposure. The last and most promising method will be discussed in this chapter. The degree of exposure to ETS depends on several factors, including length of time spent in a smoke-polluted area, the number of smokers within this area, the size and nature of the space, the degree of ventilation and the respiratory rate of the exposed individual. Thus, optimal assessment of ETS exposure is achieved by analysis of physiological fluids of exposed individuals rather than by analysis of the respiratory environment. New biochemical methods enable us to quantify exposure to ETS by determining the uptake of certain smoke contstituents (or their metabolites) in biological fluids. An ordinary requirement for such biochemical measurements is the availability of highly sensitive methods. These should be specific enough for quantitating exposure without interference by other factors. 1. Nicotine and Cotinine. Disregarding accidental or occupational exposure to tobacco (12,13), or the use of nicotine-containing chewing gum or nicotine aerosol rods as aids for smoking cessation (14), the presence of nicotine and of its major metabolites in physiological fluids is entirely due to the exposure to tobacco, tobacco smoke, or ETS. Nicotine and its major metabolite, cotinine, in saliva, blood or urine of active smokers and of passively exposed nonsmokers are primarily determined by gas chromatography (GC) with a nitrogen-sensitive detector, and by radioimmunoassay (RIA) (15-17). An HPLC method which has been developed for quantitation of cotinine in plasma or saliva of 96

populations. Cotinine excretion in urine is independent of pH, while nicotine excretion is greatly influenced by it. At values above pH 6.0, resorption of nicotine from the urine occurs especially during longer residence time in the bladder. Cotinine is not subject to resorption and, as far as it has been investigated, 3'-hydroxycotinine, a second major nicotine metabolite, is also not affected (60). Quantitation of cotinine in spot urine samples can have methodological problems relative to the volume of urine excreted in any given time period as well as dilution effects. The ideal standard for evaluation of cotinine excretion in urine would be the analysis of a 24-hour urine sample. Since this is impractical in epidemiological studies, random urine samples are usually collected at the time a questionnaire is administered. In this case, the ratio of cotinine to creatinine in a given sample is often used to allow for differences in urine dilution. Urinary creatinine excretion is usually constant from day to day for a given individual, but it does vary among individuals. As a reflection of muscle mass it is generally excreted at about 1 g per day (men, 1.1 to 3.2 g/day7 women, 0.9 to 2.5 g/day). In older persons, the excretion of creatinine may decrease to 0.5 g/day. Low levels of creatinine may also be found in dehydrated infants; this necessitates caution in the expression of ng cotinine/mg creatinine in a random sample (35). A recent study with pre-school children has shown that cotinine/creatinine ratios in passively exposed children 'track' over several weeks and reflect questionnaire data on exposure (61). 2. Carbon Monoxide. Carbon monoxide (CO) is formed during the combustion of organic matter including the burning of a tobacco product. It is also produced in vivo during metabolic processes. Endogenous CO results primarily from the breakdown of heme-containing proteins such as hemoglobin. In nonsmokers who are not exposed to industrial pyrolysis products or vehicle emissions, the baseline levels of Co, present in the bloodstream as carboxyhemoglobin (COHb), are generally below 1.5% of the total hemoglobin. Persons exposed to heavy vehicle emissions can have COHb levels up to about 2.5%. In cigarette smokers, COHb levels were found to average 5.74 in a study of 450 smokers (62) with little difference being noted between smokers of high- or low-yield products. This value is similar to that of 4.7% found in middle aged men in a study by Wald et al. (63). Carboxyhemoglobin levels are not good indicators of ETS uptake, due to the fact that CO exposure is not limited to tobacco smoker in addition, the measurement of COHb is relatively insensitive. A study in England did not find significant differences in COAb levels in subjects reporting no exposure, some exposure, or a lot of exposure (64). This was confirmed by 101

Most importantly, differences in the elimination times of cotinine from urine preclude a direct extrapolation to "cigarette equivalents of smoke uptake" by comparing the levels of cotinine excreted by active and passive smokers. This has been discussed by some investigators (10). Table 3 includes comparisons of nicotine and cotinine in physiological fluids of nonsmokers with or without ETS exposure, and of active cigarette smokers in England (41). Data on the uptake of nicotine by involuntary smokers from additional studies are summarized in Table 4 (29,42-54). Most of these studies demonstrate that nicotine and cotinine levels in physiological fluids of involuntary smokers generally amount to 1 percent and reach maximally a few percent of the amounts determined in active cigarette smokers. Data by Matsukura et al. from Japan on the other hand, show exceptionally high levels of cotinine in the urine of passive smokers. This may be due to several factors including differences in the design of studies (26). Aside from differences in methodology.one cannot rule out that differences in the uptake and metabolism of nicotine which have been observed in various population groups, and diet may be partially responsible for the exceptional data reported in the Japanese study (47). In fact, a recent finding indicates that the urinary excretion rates of Japanese smokers were significantly different from those determined in adult cigarette smokers in Europe and North America (55). This requires further thorough investigation. Survey data on exposure at home, in the workplace and on social occasions were collected from 319 employed probands and were correlated with levels of cotinine in a random urine sample. Mean urine/cotinine/creatinine levels were higher for women than for men possibly due to differences in creatinine excretion between the sexes. It is also noteworthy that 94% of the women were employed indoors. Higher levels of urinary cotinine were noted in both men and women who lived with a smoker than in those subjects who did not report living with a smoker (13.3±2.4 vs 5.1±0.4 in men and 13.9±1.9 vs. 5.(L0.6 in women). Differences in the prevalence of exposure at home existed between sexes (males 13.5% vs. females 29.2%). Levels of cotinine found across different exposures indicate that home exposure has a more pronounced effect on urine cotinine than does workplace exposure (Table 5; N.J. Haley et al., unpublished data). The nicotine uptake by infants due to ETS exposure, caused by smoking mothers or caretakers, appears to be higher than that observed in adult passive smokers. The amount of cotinine excreted in the urine of the infants was correlated with the number of cigarettes smoked by the mother, or caretaker, during the 24 hours preceding the measurement (33). Since all of the 99

FIGURES AND TABLES FOR CEAPTER 7 CHAPTER 8 ABSORPTION OF SMOKE CONSTITUENTS BY NONSMOKERS Dietrich Hoffmann PhD, Klaus D. Brunnemann MSc, and Nancy J.Haley PhD American Health Foundation, Valhalla, New York 10595 i INTRODUCTION Exposure to environmental tobacco smoke (ETS) occurs at the worksite, in public places, and in private homes. ETS is a composite of efflUents-generated in various ways during the burning of tobacco products. The major source for ETS is sidestream smoke (56) which is formed during smouldering of cigarettes, cigars and pipes between the taking of puffs. Minor contributions to ETS are made by those pollutants of the mainstream smoke (MS) that are exhaled after inhalation of each puff by the active smoker. The smoke escaping into the air from the burning cone and from the mouthpiece of a tobacco product during and after puff-drawing is another minor contributor, and there is some diffusion of MS gas phase components through the cigarette paper into the environment. In the laboratory, MS and SS are generated under standardized conditions by machine smoking (1,2). While these conditions enable us to compare the yields of individual smoke constituents from various brands of cigarettes, cigars and pipe tobacco, they do not fully reflect the patterns of smoking by humans (3,4). The consumer's intensity of puff-drawing and inhaling of the smoke is profoundly influenced by the nicotine content of the MS (4,5), and smoking intensity is highest when cigarettes with perforated filter tips are being smoked (6). The SS release is governed by the velocity of air currents around the burning cone; thus, higher air flow generates higher yields of most SS components. Even though a major reduction of mainstream smoke yields of the sales-weighted average cigarettes has occurred during the last three decades, (U.S. cigarettes declined from 35.5.mg tar in 1954 to 12 mg tar in 1983, 7), the SS emissions of smoke constituents were not significantly reduced (8,9). The data in Table 1 emphasize this with a comparison of the yields of a select group of toxic compounds in the MS and SS of four types of U.S. cigarettes. These cigarettes were 95

TABLES AND FIGURES FOR CHAPTER 8 ~s Gn O 116 ob W CII N

of these releases a keto alcohol (compound 5; Fig. I; 100). A highly sensitive GC-MS method has been developed to facilitate the detection of a derivative of compound 5. Refinement towards further increased sensitivity of the method should lead to a dosimetry assay allowing determination of the uptake of the carcinogenic TSNA by passive smokers. FUTURE NEEDS The absorption of tobacco-specific smoke constituents from ETS has been demonstrated through analyses of nicotine and its major metabolite, cotinine in the body fluids of exposed nonsmokers. Less tobacco-specific markers have also been measured in exposed populations; however, the results were ambiguous in regard to the quantitative uptake of ETS. There is a need to provide information about the uptake and disposition of carcinogenic constituents by individuals exposed to ETS in acute and chronic situations. Analyses to be fully developed and applied to passive smokers will include measurements of adducts of genotoxic smoke constituents covalently bound to DNA or hemoglobin. These techniques have been developed for benzo(a)pyrene, 4-aminobiphenyl, ethylene, and tobacco-specific N- nitrosamines. It is not known whether or not all of these methods can be made sufficiently sensitive to monitor the uptake of tobacco-specific components from ETS. Nicotine in ETS is predominantly present in the vapor phase of the smoke rather than bound to the aerosol particles. In order to measure the uptake of carcinogens and toxins residing in the particulate phase of ETS, deposition studies must be developed with specific markers. Particulate phase constituents which could be quantitated include tobacco-specific N- nitrosamines, polyphenols, such as the immunoactive compound rutin, or the tobacco-specific solanesol.(101) However, the levels of these compounds are expected to be low so that development of suitable methodology calls for highly sensitive detection methods. ACIQiOWLEDGEMENTS We thank Ilse Hoffmann and Bertha Stadler for editorial assistance. Our studies are supported by Grants No. CA-29580, CA-44377 and CA-32617 from the National Cancer Institute. 106

smokers (18) has not been applied to urine analysis even though the analysis of this biological fluid appears to have the greatest potential for evaluation of nicotine uptake by nonsmokers. A recently published, highly sensitive method for determining nicotine in plasma by HPLC with dual electrochemical detection (2 ng/ml) has not as yet been applied to physiological samples of involuntary smokers (19). Another emerging analytical method for the determination of nicotine or cotinine is the enzyme-linked immunosorbent assay (EISSA; 20). While the latter two methods appear to be suitable for assays in smokers, they have not yet attained the sensitivity necessary for evaluation of uptake of ETS obtained in current GC or RIA analyses. Trans-3'-hydroxycotinine has been found to be the most abundant nicotine metabolite in the urine of active smokers (21), however, it is difficult to quantitate this compound. Since the compound is not readily soluble it has to be transformed into a heptafluoro derivative prior to GC detection (22). The levels of 3'-hydroxycotinine in plasma have been found to be much lower than those of cotinine in the same smokers although the renal excretion of 3'-hydroxycotinine has been reported to be greater (23). Despite its abundance in urine of smokers, this compound has not yet been applied to the analysis of ETS uptake by nonsmokers. The GC and RIA methods are most widely used for assaying nicotine and cotinine in active as well as in passive smokers, primarily because•of their specificity and sensitivity, and because the needed instrumentation is available in most modern laboratories. Chromatographic methods, especially those using GC with nitrogen-phosphorus detectors (detection limit 0.1 ng/ ml fluid; 16), or a mass-spectral detection system, offer greatest specificity and high sensitivity; however, they require expensive instrumentation and technical expertise and they are rather labor intensive. Since the air as well as glassware in laboratories may contain traces of nicotine, the chromatographic methods require the utmost precautions to avoid contamination of samples. The RIA techniques are operationally simpler, less expensive and require smaller samples (detection limit 0.35 ng/sample: 17). More than 50 nicotine metabolites and structurally-related molecules have been tested as inhibitors of nicotine and cotinine antigen-antibody reactions; none of them interfer with the RIA (24). Nevertheless, the potential for cross-reactivity with unknown endogenous components exists. The fact that, upon analysis, thousands of samples obtained from nonsmokers in the US and UK have been found to be negative, indicates that diets and drugs commonly used in these two countries do not pose problems of interference. There is good correlation between results obtained by GC and RIA analysis for plasma cotinine concentrations (r-0.99; 25). 97

TABLE 13. Estimated average nonsmokers' exposures to RSP from ETS at home and at work.(Repace and Lowrey, 1985) The concentrations are calculated for model home and workplace microenvironments and are weighted by average respiration rates and time budget-studies for percent of time spent at home and at work by male and female nonsmokers. The typical nonsmoker is estimated to be exposed to from 0 to 14 mq of RSP from ETS per day, with an average exposure-of 1.5 mg/day. --------------__-_------_-~ ------------------------------Ar Lifestyle: Daily Average Probability of Being Exposed /Rounded Values) Modeled Daily Average Exposure (mg) Daily ProbabilityWeiBheed At work and at home: °'a 63 x 62 >< 39 2.27 0.89 Neither at work nor at home: eo 37 x 38 - 14 0.00 0.00 At home but not at work: ne 62 x 37 : 23 0.43 0.10 At work but not at home: We 63 x 38 : 24 1.82 0.44 Tocal: % l00 1.43 The average nonexclusive probability of a nonsmoker being exposed to ETS at work is estimated as 63%; the probability of not being exposed at work is 37%, the nonexclusive probability of being exposed to ETS at home is estimated as 62%; the probability of not being exposed at home is 38%. ----°------------------------------------°------------------- 92 n

also be increased in involuntary smokers (77,78). The data for NPRO in the urine of a limited number of involuntary smokers were not different from NPRO data for nonsmokers without ETS-exposure. A carefully designed study with a larger number of passive smokers may prove that the average value for NPRO or, more likely, for NTPRO is higher in ETS- exposed nonsmokers than in those without ETS-exposure. However, it is unlikely that the determination of N-nitrosamino acids in urine would ever lead to an assay for personal dosimetry of ETS- exposure. 6. Aromatic Amines. The sidestream smoke of cigarettes contains significantly larger quantities of aromatic amines than the mainstream smoke. For example, the MS of a nonfilter cigarette contains 0.36 ug aniline and 0.16 ug of 2-toluidine, whereas the SS of the same cigarette releases 10.8 ug of aniline and 4.1±3.2 ug of 2-toluidine (79). The urine of cigarette smokers contains somewhat higher amounts of aromatic amines than the urine of nonsmokers. The 24-hour urine void of cigarette smokers contains 3.1±2.6 ug aniline and 6.3±3.7 ug 2-toluidine, while the urine of nonsmokers contains 2.8±2.5 ug aniline and 4.1±3.2 ug 2-toluidine (80). The levels of metabolites of these aromatic amines are expected to be markedly higher in the urine of smokers than of nonsmokers. Confirmation of the significance of this difference would encourage the development of analytical dosimetry for evaluation of the impact of ETS-exposure on urinary excretion of the metabolites of aromatic amines. 7. Thioethers in Urine. Cigarette smokers excrete higher amounts of thioethers than do nonsmokers (81). A study of 26 cigarette smokers showed mean urinary thioether values of 4.3±0.4 mmol/mol creatinine compared to an equivalent mean value for 10 nonsmokers of 2.8±0.2 mmol/mol creatinine (82). In another study nonsmokers were placed on a controlled diet and were subjected to 8-hr ETS-exposure at two levels of concentration. Prior to ETS exposure 10 nonsmokers excreted 40.0±15.4 umol thioethers/24 hrs. The levels rose to 53.9±22.8 umol after exposure to ETS dose 1 (10 ppm CO). At a higher dose level (20-22 ppm CO), pre-exposure values were 69.3±36.3 and post- exposure levels 90.7±44.8. The 10 cigarette smokers who smoked 20 cigarettes each during 8 hrs in order to provide the ETS pollution in the chamber showed an increase of thioether excretion from 89.1±24.8 to 136.1±38.9 umol/24 hrs (67). In other words, the urinary thioether excretion of the passive smokers in this study increased up to 45% and, in the case of the active smokers with the same ETS exposure it increased about 50- 65%. These findings require confirmation but they appear to indicate that the thioether analysis of the urine will most likely not be suitable for the determination of the ETS uptake by involuntary smokers. 103

I An interlaboratory comparison of data from 11 laboratories in 6 countries has demonstrated that GC and RIA techniques can reliably quantitate nicotine and cotinine in urine and plasma samples. An excellent correlation of laboratory methods was observed in plasma samples and in urine samples to which cotinine had been added as a tracer. However, in urine samples without tracer, the RIA values for cotinine were found to be slightly higher than those observed by GC. This could be due to a cross reaction of the antibody with another compound present in urine, or the discrepancy could arise from a loss of urinary cotinine during GC extraction. The former explanation is more likely to apply here. All methods have led to perfect distinction between nonsmokers and active smokers (26). Table 2 presents data from model studies on the uptake of ETS by nonsmokers under acute exposure conditions (27-30). The main purpose of these assays was to develop the methodology for field studies and to compare the uptake of nicotine from environments with various degrees of pollution and different types of pollutants under controlled conditions. It has been shown that the equilibrium of nicotine between vapor phase and particulate phase of ETS depends greatly on the concentration and pH of the emitted smokestream (31) and, thus, influences the uptake of nicotine by inhalation. After repeated exposure to ETS under controlled conditions, such as twice daily 80-minute exposure on 3 consecutive days to the diluted pollutants of 4 concurrently smoked cigarettes (32), the measurements in 4 nonsmokers have shown that except for nicotine in the saliva, the physiological fluids do not reflect maximal concentrations of nicotine and cotinine until at least 24 hours later. This observation has led to comparisons of the elimination of cotinine in smokers and nonsmokers exposed to ETS (33). In the first study, the half-life (t1/2) of cotinine elimination from plasma of smokers was 18.5 hours; in the case of passive smokers, it was 49.7 hours. The corresponding disappearance (tl/z)of cotinine from the urine took 21.9 hours and 32.7 hours, respectively. In a second assay, five cigarette smokers were asked to abstain from tobacco use for 5 days and were then given nicotine gum for three days. After another 8 days of abstinence from nicotine, the volunteers were exposed to sidestream smoke. At this point, the cotinine elimination (tj/Z) from urine (ng/ml) by smokers took 15.4 hours, by nicotine gum users 18.2 hours, by 8-day exsmokers 27.5 hours, and by the never-smokers 25.6 hours (33). These findings suggest that the residence times of nicotine, cotinine and other tobacco alkaloids, are likely related to the route of nicotine uptake as well as to possible differences in metabolism between smokers and nonsmokers. The longer elimination time for cotinine in nonsmokers has been confirmed by other study groups (35-37), however, the observation has also been challenged (38,39). A longer residence time of nicotine metabolites in nonsmokers could conceivably increase the possibility of endogenous formation of carcinogenic N-nitrosamines (40). 98

REFERENCES 1. Pillsbury, H.C., Bright, C.C., O'Connor, K.J., and Irish, F.W. Tar and nicotine in cigarette smoke. J. Assoc. Offic. Anal. Chem. 52:458-462, 1969. 2. Dube, M.F. and Green, C.R. Methods of collection of smoke for analytical purposes. Recent Advan. Tobacco Sci. Jt: 42- 102, 1982. 3. Herning, R.I., Jones, R.T., Bachman, J., and Mines, A.H. Puff volume increases when low-nicotine cigarettes are smoked. Brit. Med. J. 283: 187-189, 1981. 4. Haley, N.J., Sepkovic, D.W., Hoffmann, D., and Wynder, E.L. Cigarette smoking as Risk for Cardiovascular Disease. Part VI. Compensation with nicotine availability as a single variable. Clin. Pharmacol. Ther. 38: 164-170, 1985. 5. Chamberlain, A.T. and Higgenbottam, T.W. Nicotine and cigarette smoking: An alternative hypothesis. Med. Hypotheses 17: 285-297, 1985. 6. Kozlowski, L.T., Frecker, R.C., Khouro, P., and Pope,M.A. The misuse of "less hazardous" cigarettes and its detection: Hole-blocking of ventilated filters. Am. J. Publ. Health 70: 1202-1203, 1980. 7. Federal Trade Commission. Report of tar and nicotine con- tent of the smoke of 208 varieties of domestic cigarettes, 1954-1983. U.S. Govt. Printing Office, Washington, DC, 1983. 8. Great Britain Laboratory of the Government Chemist. Report of the Government Chemist, 1981. Her Majesty's Stationery Office, London, p. 109, 1982. 9. Toxic and carcinogenic agents in undiluted mainstrea$ smoke and sidestream smoke of different types of cigarettes. Carcinogenesis 8: 729-731, 1987. 10. National Research Council. "Environmental Tobacco Smoke. Measuring Exposures and Assessing Health Effects." National Academy Press, Washington, DC, 1986. 337 pp. 11. U.S. Surgeon General. "The Health Consequences of Involuntary Smoking." U.S. Dept. Health and Human services. DHHS (CDC) 87-8398, 1987, 359 pp. 107

others (65) and also by a controlled chamber assay (61). One study in which significant elevations of COHb were found used controlled exposure to tobacco smoke at a level of 25 ppm CO for 8 hours. This intense exposure resulted in an average increase of COHb levels by 2.5% (85). However, such results are not applicable to free-living situations in field studies (67). 3. Thiocyanate. Smoke is detoxified in the liver to thiocyanate (SCN-). Measurement of SCN- has been used to differentiate smokers from nonsmokers or, as mentioned earlier, in combination with nicotine-cotinine assays to distinguish smokers from chewers of tobacco. Thiocyanate can also be derived from the diet, cruciferous vegetables being an excellent source (68). The specificity of SCN as a marker of tobacco smoke inhalation is poor and it is generally difficult to distinouish light smokers from nonsmokers. This lack of specificity makes SCN- unsuitable for the evaluation of ETS uptake by nonsmoking subjects. 4. Hydroxyproline. Japanese investigators have studied the excretion of hydroxyproline in persons exposed to ETS as well as in active smokers and in persons exposed to high levels of air pollutants (69). The rationale for these studies is that the inhalation of nitrogen dioxide causes degradation of lung collagen and elastin which results in urinary excretion of hydroxyproline. The investigations of the Japanese group suggested an elevated excretion of hydroxyproline by children of smoking parents as well as by wives of smoking husbands, active smokers, and individuals exposed to vehicle emissions. Since NOx levels in ETS are relatively low by comparison to mainstream smoke or vehicle emissions (56,70,71), such increased elimination of hydroxyproline in passively exposed persons seemed surprising. In fact, another group of investigators has been unable to confirm this finding (72). Additional investigations, under controlled exposure conditions and field studies are needed before this compound can be properly evaluated as a marker for ETS uptake. 5. N-Nitroso-Amino Acids. The occurrence of endogenous nitrosation reactions in cigarette smokers has been demonstrated in several studies. This phenomenon entails the risk of endogenous formation of carcinogenic N-nitrosamines. Endogenous formation of N-nitrosamines has been proven by urinary excretion of the noncarcinogenic N-nitrosoproline (NPRO), N- nitrosothioproline (NTPRO), and N-nitrosomethylthioproline (NNTPRO). Whereas the average excretion of NPRO in nonsmokers amounted to 2.0±1.5 ug/24 hrs, cigarette smokers excreted an average of 7.0±4.0 ug/24 hrs (73-77). In the case of NTPRO, the average urinary excretion by nonsmokers (ug/24 hrs) was 5.9, that by cigarette smokers 8.7 and that of NMTPRO was 5.6 and 8.5, respectively (75). Only two studies have explored the possibility that endogenous formation of N-nitrosamino acids may 102

B. Genotoxicity of Physiological Fluids Several studies have explored the possibility that physiological fluids of cigarette smokers contain significantly higher amounts of genotoxic agents than those of nonsmokers (81). The most extensive data base in this field has shown significantly higher mutaqenicity in the Salmonella thvohimurium assay of urine of cigarette smokers compared to those of nonsmokers. Since the original study by Yamasaki and Ames in 1977 (83) at least 20 investigations have shown that the urine of cigarette smokers is significantly more mutagenic than the urine of nonsmokers w#o are not ex#osed to genotoxic agents in occupational environments. But it has also been shown that the mutagenicity of the urine of smokers can be effected by diet (84). It has further been surmized that exposure of nonsmokers to ETS may lead to increased urinary excretion of mutagens. Of the 6 published studies in which the urine of passive smokers was tested for mutagenicity with the Ames test, 3 showed increased activity and 3 showed no increase or, at the most an insignificant increase in mutagenic activity (81,85-87). It appears likely that the presently widely used methodology by Yamasaki and Ames (83) can be significantly refined (86,88). This may then enable investigators to assay the urine of involuntary smokers for their exposure to genotoxic agents or their precursors due to exposure to ETS. C. Adduct Formation of Carcinogens in Passive Smokers. Measurements in physiological fluids of nicotine and its major metabolite, cotinine, have been shown to be objective indicators of the uptake of ETS. It appears also that, upon refinement of the methodology, the assay for mutagenicity of the urine will reflect the uptake of genotoxic ETS constituents by nonsmokers. However, these assays will not reflect an individual's response to specific ETS carcinogens. That information is best obtained by assessing levels of macromolecular adducts with carcinogens or their metabolites. Development of such assays is based an examining the mechanisms of metabolic activation and detoxification of tobacco smoke carcinogens. 1. Benzo(a)pyrene. In the case of active smokers, adducts of at least 4 types of tobacco carcinogens or procarcinogens have been studied. These adducts are formed by reaction of specific metabolites of tobacco smoke constituents with DNA and/or hemoglobin. Benzo(a)pyrene (BaP), a carcinogenic representative of the polynuclear aromatic hydrocarbons in tobacco smoke is known to be metabolized to bay region diol epoxides (e.g. 7,8- dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP). Such diol epoxides can bind to DNA in human tissues and lymphocytes. Antibodies developed against the major BPDE-DNA adduct have been used to 104

methylbutyramide. Methods Enzymol. 84: 628-640, 1982. 25. Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Van Vunakis, H., and Jarvik, M.E. Plasma nicotine and cotinine concentrations in habitual smokeless tobacco users. Clin. Pharmacol. Ther. 30: 201- 205, 1981. 26. Biber, A., Scherer, G., Hoepfner, I., Adlkofer, F., Heller, W.-D., Haddow, J.E., and Knight, G.J. Determination of nicotine and cotinine in human serum and urine: an interlaboratory study. Toxicol. Lett. 35: 45-52, 1987. 27. Harke, H.P. Zum Problem des Passiv-Rauchens. Muench. Med. Wochenschr. 112: 2328-2334, 1970. 28. Cano, J.P., Catalin, J., Badre, R., Duma, C., Viala, A., and Guillerme, R. Determination de la nicotine par chromatographie en phase gazeuse. II. Appl. Ann. Pharm. France 28: 633-640, 1970. 29. Russell, M.A.H. and Feyerabend, C. Blood and urinary nicotine in nonsmokers. Lancet 1: 179-181, 1975. 30. Hoffmann, D., Haley, N.J., Adams, J.D., and Brunnemann, K.D. Tobacco sidestream smoke. Uptake by nonsmokers. Prev. Med. 13: 608-617,1984. 31. Eudy, L.W., Thome, F.A., Heavner, D.L., Green, C.R., and Ingebrethsen, B.J. Studies on the vapor-particulate phase distribution of environmental nicotine by selective trapping and detection methods. Proc. 79th Ann. Mtg. Air Pollution Control Association, Minneapolis, June 22-27, 14 p., 1986. 32. Hoffmann, Brunnemann, K.D., Haley, N.J., Sepkovic, D.W., and Adams, J.D. Nicotine uptake by nonsmokers exposed to passive smoking under controlled conditions and the elimination of cotinine. Proc. 4th International Conference on Indoor Air Quality and Climate, Berlin, "Indoor Air '87^, Volume 2: 13- 17,1987. 33. Greenberg, R.A., Haley, N.J., Etzel, R.A, and Loda, F.A. Measuring the exposure of infants to tobacco smoke. New Engl. J. Med. 310: 1075-1078, 1984. 34. Haley, N.J., Sepkovic, D.W., Louis, E.T., and Hoffmann, D. Absorption and elimination of nicotine by smokers, nonsmokers and chewers of nicotine gum. In: The Pharmacology of Nicotine, Rand, M.J. and Thurau, K., eds., IRL Press, Washington, DC, 1988, pp. 20- 21. 35. Goldstein, G.M., Collier, A., Etzel, R., Lewtas, J., and Haley, N.J. Elimination of urinary cotinine in children exposed to known levels of sidestream cigarette smoke. Proc. 4th 109

48. Jarvis, M.J., Russell, M.A.H., Feyerabend, Eiser, J.R., Morgan, P., Gammage, P., and Gray, E.M. Passive exposure to tobacco smoke: saliva cotinine concentrations in a representative population sample of nonsmoking school children. Brit. Med. J. 291: 927-929, 1985. 49. Luck, W. and Nau, H. Nicotine and cotinine concentrations in serum and urine of infants exposed via passive smoking or milk from smoking mothers. J. Pedriatr. 107: 816-820, 1985. 50. Pattishall, E.N., Strope, G.L., Etzel, R.A., Helms, R.W., Haley, N.J., and Denny, F.W. Serum cotinine as a measure of tobacco smoke exposure in children. Am. J. Dis. Children 139:1101- 1104, 1985. 51. Schwartz-Bickenbach, Schulte-Hobein, Abt, Plum, C., and Nau, H. Smoking and passive smoking during pregnancy and early infancy: effects on birth weight, lactation period, and cotinine concentrations in mother's milk and infant's urine. Toxicol. Lett. 35: 73-81, 1987. 52. Sepkovic, D.W., Axelrad, C.M., Colosimo, S.G., and Haley, N.J. Measuring tobacco smoke exposure: clinical applications and passive smoking. Presented at the Both Ann. Mtq. Air Pollution Control Association 1987, New York, NY, Abstr. 87-80-2, 1987. 53. Jarvis, M.J., McNeill, A.D., Russell, M.A.H., W4est, R.J., Bryant, A. and Feyerabend, C. Passive smoking in adolescents: One year stability of exposure in the home. Lancet 1: 1324-1325, 1987. 54. Coultas, D.B., Howard, C.A., Peake, G.T. Salivary cotinine levels and involuntary tobacco smoke exposure in children and adults in New Mexico. Am. Rev. Resp. Dis. 136: 305-309, 1987. 55. Muranka, H., Higashi, E., Itani, S., and Shimiza, Y. Evaluation of nicotine, cotinine, thiocyanate, carboxyhemoglobin, and expired carbon monoxide as biochemical tobacco smoke uptake parameters. Int. Arch. Occup. Environ. Health 60: 37-41, 1988. 56. U.S. Department of Health and Human Services. "The Health Consequences of Involuntary Smoking". A report of the Surgeon General. DHHS (CDC) 87-8398, 1986, 359 p. 57. Palladino, G., Adams, J.D., Brunnemann, K.D., Haley, N.J., Hoffmann, D. Snuff-dipping in college students: a clinical profile. Milit. Med. 151: 342-346, 1986. 58. Haley, N.J. and Hoffmann, D. Analysis of nicotine and cotinine in hair to determine cigarette smoker status. Clin. Chem. 31: 1598-1600, 1985. 59. Sepkovic, D.W. and Haley, N.J. Biomedical applications of ill

95. Bryant, M.S., Skipper, P.L., Tannenbaum, S.R., and Maclure, M. Hemoglobin adducts of 4-aminobiphenyl in smokers and nonsmokers. Cancer Res. 47: 602-608, 1987. 96. Wynder, E.L. and Hoffmann, D. "Tobacco and Tobacco Smoke. Studies in Experimental Tobacco Carcinogenesis." Academic Press, New York, NY, 1967, 730 p. 97. Binder, H. and Lindner, W. Bestimmung von Aethylenoxyd im Rauch garantiert unbegaster Zigaretten. Fachliche Mitt. Oesterr. Tabakregie 13: 215-220, 1972. 98. International Agency for Research on Cancer. "Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs, Volume 1-42." IARC Monogr. Suppl. 7: 1987, 440 p. 99. Tornqvist, M., osterman-Golkars, S., Kautiainen, A., Jensen, S., Farmer, P.B., and Ehrenberg, L. Tissue doses of ethylene oxide in cigarette smokers determined from adduct levels in hemoglobin. Carcinogenesis 7: 1519-1521, 1986. 100. Hecht, S.S., Carmella, S.G., Trushin, N., Spratt, T.E., Foiles, P.G., and Hoffmann, D. Approaches to the development of assays for interaction of tobacco-specific nitrosamines with hemoglobin and DNA. IARC Sci. Publ. 89: 121- 128, 1988. 101. Benner, C.L., Bayona, J.M., Caka, F.M., Tang, H., Lewis, L., Crawford, J., Lamb, J.D., Lee, M.L., Lewis, E.A., Hansen, L.D., and Eatouqh, D.J. Chemical Composition of Tobacco Smoke. 2. Particulate Phase Compounds. Environ. Sci. Technol. 23: 688-699, 1989. 115

12. Saxena, K. and Scheman, A. A suicide plan by nicotine poisoning: A review of nicotine toxicity. Vet. Hum. Toxicol. 27: 495-497, 1985. 13. Gehlbach, S.H., Williams, W.A., Perry, L.D., Freeman, J.H., Langone, J.J., Peta, L.V., and Van Vunakis, H. Nicotine absorption by workers harvesting green tobacco. Lancet 1: 478- 480, 1975. 14. Pomerleau, O.F. and Pomerleau, C.S. "Nicotine Replacement - A Critical Evaluation". Pro#r. Clin. Biol. Res. 261: 1-317, 1988. 15. Fayerabend, C. Determination of nicotine in physiological fluids by gas chromatography. IARC Sci. Publ. 81: 299307, 1987. 16. Feyerabend, C. and Bryant, A.E. Determination in physiological fluids by gas chromatography. IARC Sci. Pubi. 81: 309-316, 1987. 17. Van Vunakis, H., Gjika, H.B., and Langone, J.J. Radioimmunoassay for nicotine and cotinine. IARC Sci. Publ. 81: 317-330, 1987. 18. Machacek, D.A. and Jiang, N. Quantification of cotinine in plasma and saliva by liquid chromatography. Clin. Chem. 32: 979- 982, 1986. 19. Chien, C-Y., Diana, J.N., and Crooks, P.A. Determination of nicotine in plasma by high performance liquid chromatography with electrochemical detection. LC-GC ¢: 53-55, 1988. 20. Bjercke, R.J., Cook, G., Rychlik, N., Gjika, H.B., Van Vunakis, H., and Langone, J.J. Stereospecific monoclonal antibodies'to nicotine and cotinine and their use in enzyme- linked immunosorbent assays. J. Immunol. Methods 90: 202-213, 1986. 21. Neurath, G.B., Duenger, M., Orth, D., and Pein, F.G. trans-3'-hydroxycotinine as a main metabolite in urine of smokers. Internatl. Arch. Occup. Environ. Health 59:199-201, 1987. 22. Neurath, G.B., Pein, F.G. Gas chromatographic determination of trans-3'-hydroxycotinine, a major metabolite of nicotine in smokers. J. Chromatog. Biomed. Appl. 415: 400-406, 1987. 23. Adlkofer, F., Scherer, G., Jarczyk, L., Heller, W.D., and Neurath, G.B. Pharmacokinetics of 3-hydroxycotinine. In: The Pharmacology of Nicotine. M.J. Rand and K. Thurau, eds. IRL Press, Washington, DC 1988, pp. 25-28. 24. Langone, J.J. and Van Vunakis, H. Radioimmunoassay of nicotine, cotinine, and gamma-(3-pyridyl)-gamma-oxo-N- 108

cotinine quantitation in smoking related research. Am. J. Public Health 75: 663-664, 1985. 60. U.S. Department of Health and Human Services. "The Health Consequences of Smoking - Nicotine Addiction". A report of the Surgeon General, DHHS (CDC) 88-8406, 1988, 618 p. 61. Mumford, J.L., Forehand, L., Burton, R., Lewtas, J., Hammond, S.K., and Haley, N.J. Serum and urine cotinine as quantitative measures of passive tobacco smoke exposure in young children. Proc. 4th Internatinal Conference on Indoor Air Quality and Climate, Berlin, "Indoor Air '87", Volume 2: 18-21, 1987. 62. Hill, P., Haley, N.J., and Wynder, E.L. Cigarette smoking: carboxyhemoglobin, plasma nicotine, cotinine and thiocyanate vs. self-reported smoking data and cardiovascular disease. J. Chron. Dis. 36: 439-449, 1983. 63. Wald, N., Idle, M., Smith, P.G., and Bailey, A. Carboxyhemoglobin levels in smokers of filtered and plain cigarettes. Lancet 1: 110-112, 1977. 64. Jarvis, M.J. and Russell, M.A.H. Measurement and estimation of smoke dosage to nonsmokers from environmental tobacco smoke. Eur. J. Respirat. Dis. (Suppl) 133: 68-75, 1984. 65. Jarvis, M.J. Uptake of environmental tobacco smoke. IARC Sci. Publ. 81: 43-58, 1987. 66. Hoffmann, D., Brunnemann, K.D., Adams, J.D., and Haley, N.J. Indoor air pollution by tobacco smoke: model studies on the uptake by nonsmokers. Proc. 3rd International Conference on Indoor Air Quality and Climate, Stockholm, "Indoor Air", Volume 2: 313-338, 1984. 67. Scherer, G., Westphal, K., Hoepfner, I., Adlkofer, F., and Sorsa, M. Biomonitoring of exposure to potentially mutagenic substances from environmental tobacco smoke. Proc. of the 4th International Conference on Indoor Air Quality and Climate, Berlin, "Indoor Air '87", Volume 2: 109-114, 1987. 68. Haley, N.J. Axelrad, C.M., and Tilton, K.A. Validation of self-reported smoking behavior: biochemical analysis of cotinine and thiocyanate. Am. J. Publ. Health 73: 1204-1207, 1983. 69. Kasuga, H., Matsuki, H., Osaka, F., and Inoue, M. The study on the relationship between urinary hydroxyproline and creatinine ratio from the viewpoint of public health. Tokai J. Exp. Clin. Med. 4: 343-351, 1979. 70. Guerin, M.R. Formation and physico-chemical nature of sidestream smoke. IARC: Sci. Publ. 81: 11-24, 1987. 112

assess its presence in surgical specimens of lung tissue, in human placenta, and in peripheral blood lymphocytes (89-91). Evidence for the presence of such adducts in samples from smokers has been ascertained but significant differences between smokers and nonsmokers have not been observed. 2. Aromatic Amines. 4-Aminobiphenyl and 2-naphthylamine are the known tobacco smoke constituents which are most likely to contribute to the increased risk of bladder cancer of cigarette smokers. The mechanisms by which these compounds are metabolically activated and produce DNA adducts in the bladder epithelium have been extensively studied (92). These studies have shown that the corresponding hydroxylamines are key intermediates in DNA and protei-n modification. The hydroxylamines also react with hemoglobin, in the case of 4- aminobiphenyl, a sulfinic acid amide of the beta-cysteine (93- 95). This adduct.readily releases 4-aminobiphenyl upon treatment with dilute acid. A method was developed to analyze the released 4-aminobiphenyl by gas chromatography with detection by negative ion chemical ionization mass spectrometry (95). Application of this method to smokers showed that adduct levels were higher than in nonsmokers, and decreased upon smoking cessation. The method may be further refined for assessing the uptake of carcinogenic aromatic amines from ETS by nonsmokers. 3. Ethylene. This volatile unsaturated hydrocarbon is present in both mainstream smoke (200-400 ug/cigarette) and sidestream smoke of cigarettes (96). Cigarette smoke contains also traces of the carcinogenic ethylene oxide (7.0 ug/cigarette; 97,98). Upon absorption, ethylene is metabolized to the reactive ethylene oxide. The latter binds to cellular macromolecules and to hemoglobin. The alkylated valine is cleaved off of the isolated hemoglobin and the derivatized hydroxyethylvaline is analyzed by GC-MS. Cigarette smokers showed significantly higher hydroxyethylvaline levels (389±138 pg/g hemoglobin) than nonsmokers (58±25 pg/g; 99). So far the method has not been applied to estimates of exposure of involuntary smokers to the procarcinogen ethylene. 4. Tobacco-Specific N-Nitrosamines. During tobacco processing and during smoking tobacco alkaloids give rise to tobacco-specific N-nitrosamines (TSNA). The nicotine-derived N- nitrosamines N'-nitrosonornicotine (NNN) and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are powerful carcinogans. They occur in relatively high concentrations in cigarette mainstream smoke (NNN, 0.12-3.7 ug/cigarette; NNK, 0.08-0.77 ug/cigarette) and sidestream smoke (NNN, 0.15-1.7 ug/cigarette; NNK, 0.2-1.4 ug/cigarette; 40). These agents are metabolically activated by aipha-hydroxylation, leading to a highly reactive intermediate which forms DNA adducts and protein adducts (Fig. I). Metabolic activation of NNN and NNK also leads to the formation of hemoglobin adducts. Acid or base hydrolysis 105

International Conference on Indoor Air Quality and Climate, Berlin, "Indoor Air '87", Volume 2: 61-67, 1987. 36. Etzel, R.A., Greenberg, R.A., Haley, N.J., and Loda, F.A. Urinary Cotinine excretion in neonates exposed to tobacco smoke products in utero. J. Pediatr. 107: 146-148, 1985. 37. Scherer, G., Westphal, K., Sorsa, M., and Adlkofer, F. Quantitative and qualitative differences in tobacco smoke uptake between active and passive smoking. In: "Indoor and Ambient Air Quality", R. Perry and P.W. Kirk, eds., London, 1988. pp 189-194. 38. Benowitz, N.L., Kuyt, F., Jacob, P., Jones, R.T. III., and Osman, A.-L. Cotinine disposition and effect. Clin. Pharmacol. Ther. 14: 604-611, 1983. 39. Jarvis, M.J.,.Russell, M.A.H., Benowitz, N.L., and Feyerabend, C. Elimination of cotinine from body fluids. Am. J. Publ. Health 78: 696-698, 1988. 40. Hecht, S.S. and Hoffmann, D. Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9: 875-884, 1988. 41. Jarvis, M.J., Tunstall-Pedoe, H., Feyerabend, C., Vessey, C., and Saloojee, Y. Biochemical markers of smoke absorption and self- reported exposure to passive smoking. J. Epidemiol. Comm. Health 38: 335-339, 1984. 42. Feyerabend, Higgenbottam, and Russell, M.A.H. Nicotine concentrations in urine and saliva of smokers and nonsmokers. Brit. Med.J. 284: 1002-1004, 1982. 43-. Foliart, D., Benowitz, N.L., and Becker, C.E. Passive absorption of nicotine in airline flight attendants. (Letter) New Engl. J. Med. 308: 1105, 1983. 44. Jarvis, M.J., Russell, M.A.H., and Feyerabend, C. Absorp- under natural conditions of exposure. Thorax 38: 829-833, 1983. 45. Wald, N.J., Boreham, A., Bailey, A., Ritchie, C., Haddow, J.E., and Knight, G. Urinary cotinine as marker for breathing other peoples tobacco smoke. Lancet 1: 230-231, 1984. 46. Wald, N.J. and Ritchie, C. Validation of studies on lung cancer in nonsmokers married to smokers. Lancet 1: 1607, 1984. 47. Matsukura, S., Tominato, T., Kitano, H., Seino, Y., Hamada, H., Uchihashi, M., Nakajima, H., and Hirota, Y. Effects of environmental tobacco smoke of urinary cotinine excretion in nonsmokers. New Enql. J. Med. 311: 828-832, 1984. 110

71. Repace, J.L., Indoor concentrations of environmental tobacco smoke: field studies. IARC Sci. Publ. 81: 141-162, 1987. 72. Adlkofer, F., Scherer, G., and Heller, W.D. Hydroxyproline excretion in urine of smokers and passive smokers. Prev. Med. 13: 670-679, 1984. 73. Hoffmann, D. and Brunnemann, K.D. Endogenous formation of N- nitrosoproline in cigarette smokers. Cancer Res. 43: 5570- 5574, 1983. 74. Ladd, K.F., Newmark, H.L., and Archer, M.C. N-nitrosation in smokers and nonsmokers. J. Natl. Cancer Inst. 73: 83-87, 1984, 75. Tsuda, H., Nutsume, J., Sato, S., Hirayama, F, Kakizoe, T. and Sugimura, T. Increase in the levels of N-nitrosoproline, N- nitrosothioproline, and N-nitroso-2-methylthioproline in human urine by cigarette smoking. Cancer Lett. 30: 117-124, 1986. 76. Lu, S.H., Ohshima, H., Fu, H.M., Tian, Li, F.M., Blettner, M., Wahrendorf, J., and Bartsch, H. Urinary excretion of N- nitrosamino acids and nitrate by inhabitants of high- and low-risk areas for esophageal cancer in Northern China: endogenous formation of nitrosoproline and its inhibition by vitamin C. Cancer Res. 46: 1485-1491, 1986. 77. Scherer , G. and Adlkofer, F. Endogenous formation of N- nitrosoproline in smokers and nonsmokers. Banbury Rpt. 23: 137- 147, 1986. 78. Brunnemann, K.D., Scott, J.C., Haley, N.J., and Hoffmann, D. Endogenous formation of N-nitrosoproline upon cigarette smoke inhalation. IARC Sci. Publ. 57: 819-828, 1984. 79. Patrianakos, C. and Hoffmann, D. Chemical studies on tobacco smoke LXIV. On the analysis of aromatic amines in cigarette smoke. J. Anal. Toxicol. 3: 150-154, 1979. 80. El-Bayoumy, K., Donahue, J.M., Hecht, S.S., and Hoffmann, D. Identification and quantitative determination of aniline and toluidine in human urine. Cancer Res. 46: 60646067, 1986. el. International Agency for Research on Cancer. "Tobacco Smoking, IARC Monogr. 38: 1986, 421 p. 82. Heinonen, T., Kytoniemi, V., Sorsa, M., and Vainio, H. Urinary excretion of thioethers among low-tar and medium-tar cigarette smokers. Internatl. Arch. Occup. Environ. Health 52: 11-16, 1983. 83. Yamasaki, E. and Ames, B.N. Concentration of mutagens 113

from urine by adsorption with the nonpolar resin CAD-2: cigarette smokers have mutagenic urine. Proc. Natl. Acad. Sci. U.S.A. 74: 3555-3559, 1977. 84. Sasson, I.M., Coleman, D.T., LaVoie, E.J., Hoffmann, D., and Wynder E.L. Mutagens in human urine. Effects of cigarette smoking and diet. Mutat. Res. 158: 149-159, 1985. 85. Scherer, G., Westphal, K., Biber, A., Hoepfner, I., and Adlkofer, F. Urinary mutagenicity after controlled exposure to environmental tobacco smoke (ETS). Toxical. Lett. 35: 135-140, 1987. 86. Mohtashamipur, E., Mueller, G., Norpoth, K., Endrikat, M., and Stuecker, W. Urinary excretion of mutagens in passive smokers. Toxicol. Letters 35: 141-146, 1987. 87. Husgafvel-Pursiainen, K., Sorsa, M., Engstrom, K., and Einistoe, P. Passive smoking at work: biochemical and biological measures of exposure to environmental tobacco smoke. Int. Arch. Occup. Environ. Health 59: 337-345, 1987. 88. Ling, P.I., Lofroth, G., and Lewtas, J. Mutagenic determination of passive smoking. Toxicol. Lett. 35: 147-151, 1987. 89. Harris, C.C., Vahakangas, K., Newman, M.J., Trivers, G.E., Shamsuddin, A., Sinapoli, N., Mann, D., and Wright, W.E. Detection of benzo(a)pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers. Proc. Natl. Acad. Sci. U.S.A. 82: 6672-6676, 1985. 90. Everson, R.B., Randerath, E., Santella, S.A., Cefalo, R.C., Avitts, T.A., and Randerath, K. Detection of smoking-related covalent DNA adducts in human placenta. Science 231: 54-57, 1986. 91. Perera, F.P., Poirier, M.C., Yuspa, S.H., Nakayama, J., Jaretzki, A., Curnen, M.M., Knowles, D.M., and Weinstein, I.B. A pilot project in molecular cancer epidemiology: determination of benzo(a)pyrene-DNA adducts in animal and human tissues by immunoassays. Carcinogenesis 3: 1405-1410, 1982. 92. Beland, F.A. and Kadlubar, F.F. Factors involved in the induction of urinary bladder cancer by aromatic amines. Banbury Rpt. 23: 315-326, 1986. 93. Neumann, H.G. Analysis of hemoglobin as a dose monitor for alkylating and arylating agents. Arch. Toxicol. 56: 1-6, 1984. 94. Green, L.C., Skipper, P.L., Juresky, R.J., Bryant, M.S., and Tannenbaum, S.R. In vivo dosimetry of 4-aminobiphenyl in rats via a cysteine adduct in hemoglobin. Caneer Res. 44: 4254-4259, 1984. 114

NO0 0 ~~ I (OJ - " NN CH3 NNK p N'O I ) N OH N~CH C OH Z ~ ON ~ ~ N N-0 f2 0 N-0 - ' crN~COZEf , C~0 N-NOM `v J N 3 N 1 4 Figure I. Metabolic activation of 4-(methylnitrosamino)-t-(3- pyridyl)-t-butanone (NNK) and N'-nitrosonornicotine (NNN) to intermediates which bind to DNA and protein.

8'7808355 Table 2 continued. No. of ETS-Conditions Passive Results Investiqators Smpkers Room - 16 m3 6 4 ciqarettes con- currently and con- tinuously smoked for B0 min; 6 air exch./hr. (200 q nicotine/m3; 20 ppm CO) Time during exposure Nicotine Cotinine Hoffmann et `1., 1984 (nq/ml) (nq/ml) (30) 0 Saliva 3 1.0 Plasma 0.2 0.9 Urine 17 14 80 min. Saliva 730 1.4 Plasma 0.5 1.3 Urine 84 28 Time following exposure d 30 min. Saliva 148 1.7 .~ .a Plasma 0.4 1.8 .i 150 " Saliva 17 3.1 Plasma 0.7 2.9 Urine 100 45 300 " Saliva 7 3.5 Plasma 0.6 3.2 urine 48 55 +Nicotine and cotinine were measured in urine as ng/mg creatinine.

87808361 Table 4 continued ... Nonsmoker Group Number of Nonsmokers Results Reference Cotinine/Urine (ng/mg creatinine) Neonates and infants No. No. Schwartz- exp'd I exp'd II Bicken- a) Mother smokes, bach et. breastfeeds 20 12 (1756) 0 -3520 8(935) 486-2440 al., (51) b) Mother smokes, feeds bottle 16 4 (47) 0 - 160 12 (107) 0- 341 c) Father smokes 18 10 (0) 8 (0) 0- 308 d) No exposure in the home 15 9 (0) 6 (0) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

87808359 Table 4 continued ... Number of Nonsmoker Group Nonsmokers Results Reference Neonates and infants Nicotine (ng/mg creatinine) Cotinine Luck and a) No exposure 10 (0) 0 - 14 (0) 0- 56 Nau, (49) (4-8 days old) b) Exposure via breast feeding 19 (14) 5 -110 (100) 10-555 (3-8 days old) c) Passive smoking (2.5-6 months old) 10 (35) 4.7-218 (327) 117-780 d) Exposure via breast feedinq and passive smokinq 9 (12) 3.0- 42 (550) 225-870 (1-12 months old) ~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - .i Infants (aqe 3-15 months) Catinine/ Serum (ng/ml) Pattishall .i a exposure in the h et al., ome (50) Black infants a) no exposure 9 1.0 (1.8732.38) Pattishall et al., 1985 b) passive smoking 15 4.0 (5.27*3.50) (51) White infants a) no exposure 9 0.0 (0.22f0.44) b) passive smokinq 5 0.4 (0.9011.30) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

87808362 Table 4 continued .. . Nonsmoker Group Number of Nonsmokers Results Reference Municipal workerp Cotiine/Urine (nq/mq creatinine) Sepkovic I. ETS exposure in t he et al., (52) a) workplace no exposure 25 4.5*0.6 b) liqht expsoure 126 6.6*0.6 c) moderate exposure 84 7.2*0.8 d) heavy exposure 32 8.411.3 II. a) ETS exposure in home no exposure the 77 6.1f0.8 b) liqht exposure 83 6.7f0.6 c) moderate exposure 71 7.8*1.1 d) heavy exposure 34 7.6t1.3 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - School girls (11-16 yrs) ETS exposure in the home a) neither parent smokes b) father smokes only c) mother smokes only d) both parents smoke 104 1.1*0.5 76 2.00.6 40 3.2*0.8 110 5.0*1.0 Jarvis et al., (53) Continued ...

Table 1. Toxic and tunorigenic agents in KS and SS Cigarette Smoke Smoke Constituent streama A(NF) B (F) C (F) D (PF) Tar MS 20.1 15.6 6.8 0.9 (mg) S5 22.6 24.4 20.0 14.1 --------------------------------------------- Nicotine MS 2.04 1.50 0.81 0.15 (mg) SS 4.62 4.14 3.54 3.16 --------------------------------------------- CO MS 13.2 13.7 9.5 1.8 (mg) SS 28.3 36.6 33.2 26.8 Catechol MS 41.9 71.2 26.9 9.1 (yg) SS 58.2 89.9 69.5 117 --------------------------------------------- BaP MS 26.2 17.8 12.2 2.2 (ng) 55 67.0 45.7 51.7 44.8 --------------------------------------------- Ammonia MS 76.0 19.4 34.0 40.4 (Yg) SS 524 893 213 236 ------°------------------------------------- NDMA MS 31.1 4.3 12.1 4.1 (ng) 5S 735 597 611 685 --------------------------------------------- NPYR MS 64.5 10.2 32.7 13.2 (mg) SS 117 139 233 234 NNN MS 1007 488 273 66.3 (ng) SS 857 307 185 338 --------------------------------------------- NNK MS 425 180 56.2 17.3 (ng) SS 1444 752 430 386 a Abbreviations: NF, nonfilter cigarette; F, filter ciqa- rette; PF, cigarette with perforated filter tip; BaP, benzo- (a)pyrene; NDMA, N-nitrosodimethylamine; NPYR, N-nitrosopyr- rolidine; NNN, N'-nitrosonornicotine= NNK, 4-(methylnitros- amino)-1-(3-pyridyl)-1-butanone. GO ~I (D O 00 111 b W C!1 W

87808360 Table 4 continued ... Number of Results Reference Nonsmoker Group Nonsmokers nusbands of a) nonsmokers 101 b) smokers 20 Cotinine/llrine (ng/ml ) 8.S• 1.3 25.2t14.8 Wald and Ritchie, (46) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Nonsmokers Cotinine/Urine (ng/mg creatinine) Matsukura a) nonsmokers at home 200 0.5 *0.09 et al., b) smokers at home 272 0.79*0.1 (47) Ciqarettes smoked day in home of nonsmokers; Cotinine/Urine (tug/mg) creatinine) 1- 9 25 0.31t0.08 10-19 57 0.42*0.1 20-29 99 0.87*0.19 x 30-39 38 1.03*0.25 .a > 40 28 1.56*0.57 ~ ~ unknown 25 0.56f0.16 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Infants (<10 months, Nico tine/Urine Cotinine/Urine Greenberq not breastfed) (ng/mg creItinine) (ng/mg) et al.; a) not exposed to ETS 18 0 (0-59) 4 (0-125) (33) b) exposed to ETS 28 53 (0-370) 351 (41-1,885) School children (11-16 yrs) Cotinine/Saliva (ng/ml) a) Neither parent smoked 269 0.44f0.68 Jarvis et b) Only father smoked 96 1.31t1.21 al., (48) c) Only mother smoked 76 1.9511.71 d) Both parents smoked 128 3.3832.45 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Continued ...

87808357 Table 3. Approximate Relations of Nicotine as a Parameter Between Nonsmokers, Passive Smokers, and Active Smokersa (41) Nonsmokers without ETS Exposure Nonsmokers with ETS Exposure Active Smokers No. = 46 No. = 54 No. = 94 Nicotine/Cotin % of ine Mean Value Active Smokers' Value % of Mean Value Active Smokers' Value Mean Value Nicotine (ng/m l) in plasma 1.0 7 0.8 5.5 14.8 in saliva 3.8 0.6 5.5 0.8 673 in urine 3.9 0.2 12.1* 0.7 1,750 Cotinine (ng/m in plasma l) 0.8 0.3 2.0* 0.7 275 in saliva 0.7 0.2 2.5** 0.8 310 in urine 1.6 0.1 7.7** 0.6 1,390 a0iff.erences between nonsmokers exposed to ETS compared with nonsmokers without exposure: *p<0.01; ** p<0.001.

8'7808363 Tahlr 4 continued ... Nonsmoker Group Number of Nonsmokers Results Reference Children and adults 529 males Cotinine/Saliva (ng/ml) Coultas 768 females smokers in family et al. (53) none one > two a) <5 years old 0.0 (0.0-2.5) 3.8 (0.0-6.1) 5.4 (3.2-7.7) b) 6-12 years old 0.0 (0.0-2.1) 2.0 (0.0-3.8) 5.2 (1.5-7.0) c) 3-17 years old 0.0 (0.0-2.0) 2.9 (0.0-4.9) 4.1 (2.7-7.6) d) 18-29 years old 0.0 (0.0-2.6) 0.0 (0.0-5.8) 0.0 (0.0-4.4) e) 30-64 years old 0.0 (0.0-2.7) 1.9 (0.0-4.5) 4.4 (1.8-11.0) f) > 65 years old 0.0 (0.0-2.6) 3.6 (0.0-6.5) 0.0 *Numbers in parenthesis median values.

Tab 4. 8'7808358 ' Uptake of nicotine by nonsmokers exposed to ETS under daily life conditions Nonsmoker Group Number of Nonsmokers Results Reference Nicotine/Urine (ng/ml) Ilospital personnel 14 12.4*16.9 Russell and (78 min in smoke- 13 8.9*9.1 Feyerabend (29) filled room) Hospital personnel and outpatients Nicotine/Saliva (ng/ml) a) no exposure to ETS 26 5.9 7.5 Peyerabend b) exposed to ETS 30 10.1 21.6 et al. (42) Fliqht attendants Nicotine/Serum (ng/ml) a .a 6 pre fl qht: 1.6*0 8 Foliart et al .i . post flight: 3.2t1.0 (43) . .a Office workers 7 C~o~~n~te__n~t[~m~l Nicotine (ng) Cotinine (nq) Jarvis et al. a) 11:30 a.m. sample sal~Tva'- a)1.90 b)43.63 a)1.50 b)8.04 (44) b) 7:45 p.m. sample serum 0.76 2.49 1.07 7.33 after 2 hr stay urine 10.57 92.63 4.80 12.94 in pub Hospital staff and Cotinine/Urine (ng/ml) Wald et al. - - outpations (45) a) no exposure to ETS 22 2.0 (0.0 - 9.3 b) exposed to ETS 190 6.0 (1.4 -22.0) - - - - - - - - - - - - - - - - - - - - - - " - - - - - - - - - - - - - - - - - - - - - - - - - Continued ...

8'7808356 Table 2. Uptake oE nicotine by nonsmokers exposed to ETS under controlled conditions ETS-Conditions Room - 170 m3 (11 smokers) (a) 100 ciqarettes were smoked during 2 hrs; no ventilation (30 ppm CO) (b) same conditions as above (a) but with ventilation (5 ppm CO) No. of Passive Smokers Results Investiqator(s) Urinary excretion 7 Nicotine: 10f6.8 pq/6 hrs. Cotinine: 35t34.5 pg/6 hrs. 7 Nicotine: 18t7 uq/6 hrs. Continine: 1939.4 pq/6 hrs. Room - 66 m3 (4 cigarette smokers) (a) Day 1, nonsmokinq 2 " 2, 98 ciq's smoked " 3, 121 " " " 4, 98 w w " 5, 88 w (b) Day 1, 97 " " 2 " 2, 96 w 3, 94 w w 4, 103 Room - 43 m3 9 smokers consumed 12 80 ciqarettes + 2 ciqars no ventilation (3B ppm CO) Nicotine/Urine (pq/24 hrs) Cano et al. (28) 0 - 0 35 - 44 50 - 61 62.5 - 70 47 - 50 23 - 34 22.5 - 58 47.5 - 69 32 - 65 Nicotine/Plasma (Uq/m1) Russell and Before exposure: 0.73~1.6 Feyerabend After 78 min. exposure: 0.9t 0.29 (29) Nicotine/Urine (ng/ml) 15 min~ aEter exposure: 80.0*58.7 continued ...