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8
Assessing Exposures to Environmental Tobacco Smoke Using Biological Markers

Previous chapters have dealt with the formation and composition of tobacco sidestream smoke, its contribution to environmental tobacco smoke (ETS), and the conditions that govern the physicochemistry and toxicity of ETS. Personal monitoring of exposure and analysis of the respiratory environment enable us to estimate the level of toxic agents for individuals exposed to ETS. Studies on the uptake of smoke constituents by individuals and on the metabolic fate of such constituents can provide information relative to epidemiologic observations and the actual exposure levels of different populations.

Exposure to ETS may depend on several factors, including the number of smokers in an enclosed area, the size and nature of the area, and the degree of ventilation. Thus, optimal assessment of exposure should be done by analysis of the physiological fluids of exposed persons rather than by analysis of respiratory environment. The development of new biochemical methods enables us to obtain measurements of exposure to ETS by determining the uptake of specific agents in body fluids and calculating the risk relative to that of the exposure of active smokers. The uptake of individual agents from ETS can be determined by biochemical measures that have been developed for assessment of active smoking behavior, as long as these measures are sensitive and specific enough for quantitating exposure to such agents by nonsmokers.



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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects 8 Assessing Exposures to Environmental Tobacco Smoke Using Biological Markers Previous chapters have dealt with the formation and composition of tobacco sidestream smoke, its contribution to environmental tobacco smoke (ETS), and the conditions that govern the physicochemistry and toxicity of ETS. Personal monitoring of exposure and analysis of the respiratory environment enable us to estimate the level of toxic agents for individuals exposed to ETS. Studies on the uptake of smoke constituents by individuals and on the metabolic fate of such constituents can provide information relative to epidemiologic observations and the actual exposure levels of different populations. Exposure to ETS may depend on several factors, including the number of smokers in an enclosed area, the size and nature of the area, and the degree of ventilation. Thus, optimal assessment of exposure should be done by analysis of the physiological fluids of exposed persons rather than by analysis of respiratory environment. The development of new biochemical methods enables us to obtain measurements of exposure to ETS by determining the uptake of specific agents in body fluids and calculating the risk relative to that of the exposure of active smokers. The uptake of individual agents from ETS can be determined by biochemical measures that have been developed for assessment of active smoking behavior, as long as these measures are sensitive and specific enough for quantitating exposure to such agents by nonsmokers.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects BIOLOGICAL MARKERS IN PHYSIOLOGICAL FLUIDS Thiocyanate The hydrogen cyanide (HCN) absorbed from tobacco smoke is detoxified in the liver, yielding thiocyanate (SCN−). However, SCN− in serum and other biological fluids does not exclusively originate from inhaled tobacco smoke. Thiocyanate also can be derived from the diet (Haley et al., 1983; Jarvis, 1985). Before 1975, primarily two colorimetric methods were used for the manual determination of thiocyanate in biological fluids (Aldridge, 1944; Bowler, 1944). Subsequently, the automatic method by Butts et al. (1974) has found wide application in comparing physiological fluids from smokers and nonsmokers. It entails determination of thiocyanate by its reaction with ferric ions, which yield a color complex with maximal absorbance at 460 nm, the intensity of which can be measured in an autoanalyzer. In sera of nonsmokers, Butts et al. (1974) determined up to 95 µmol/L of SCN−. The critical value in differentiating between smokers and nonsmokers was 85 µmol/L of SCN−. In other investigations, 100 µmol/L of SCN− was found to be the critical level for serum (Junge et al., 1978) and for saliva (Luepker et al., 1981). This fact and the low concentrations of HCN in ETS (Hoffmann et al., 1984) explain why some investigators were unable to distinguish between nonsmokers exposed to ETS and those without any exposure to tobacco smoke (Hoffmann et al., 1984; Jarvis, 1985). Similarly, the mean serum level of SCN− in healthy pregnant women at term who were exposed to ETS (35.9 µmol/L) was not distinctly different from that in those without ETS exposure (32.3 µmol/L), nor was there a measureable difference in SCN− levels in the umbilical cords of the neonates (26 versus 23 µmol/L) (Hauth et al., 1984). In one study, it appeared that there was a trend toward higher thiocyanate levels in the saliva of nonsmoking children residing with smokers compared to the SCN− levels in saliva of children without ETS exposure, yet this trend was insignificant (Gillies et al., 1982). In a study of six volunteer nonsmokers exposed to a smoke-filled room for 4 hours, there was a significant increase in salivary SCN−. However, the SCN− values of the nonsmokers

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects exposed to ETS were not distinguishable from those nonsmokers free of tobacco smoke exposure (Pekkanen et al., 1976). In another study, mean serum thiocyanate levels were reported to be significantly higher (p<0.002) for children and adolescents with exposure to cigarette smoke at home (n=14; SCN−=97.3±45.4 µmol/L) than for those not exposed (n=10; SCN−=54.2±11.3 µmol/L). The authors of the latter study also reported a weak correlation between thiocyanate concentration and number of cigarettes smoked per family (Poulton et al., 1984). This study was criticized because some of the determined thiocyanate levels were within the range reported for heavy cigarette smokers. It is likely that there was deceptive reporting of adolescent smoking status (Jarvis, 1985). Based on the observations to date, the level of thiocyanate in saliva, serum, and/or urine is not useful as an indicator for the uptake of ETS by a nonsmoker. Carbon Monoxide and Carboxyhemoglobin Carbon monoxide (CO) in the body originates from endogenous processes as well as environmental sources. The endogenous production of CO is primarily a consequence of the breakdown of hemoglobin and of other heme-containing pigments. Healthy adults produce about 0.4 ml of CO per hour (0.5 mg/h; Coburn et al., 1964). This provides the major portion of CO that is found as carboxyhemoglobin (COHb) in nonsmokers. In nonsmokers without occupational exposure to CO, COHb ranges from 0.5 to 1.5% (National Research Council, 1981; Wald et al., 1981). The inhalation of CO from the environment is followed by an increase of the CO concentration in the alveolar gas and by diffusion from the gas phase through the pulmonary membrane into the blood. CO is complexed with blood to form COHb and, as such, is transported throughout the body. Complexing it with hemoglobin occurs with a strong coordination bond with the iron of heme, a bond that is about 200 times stronger than that with molecular oxygen. CO is only slowly released from the blood in the process of exhaling. In the case of nonsmokers who have been exposed to elevated levels of CO in the air for a few hours, the half-life of COHb lasts 2–4 hours (National Research Council, 1981). Monitoring of absorbed CO in the blood is done primarily by the analysis of CO in alveolar gas and by the analysis of COHb

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects in blood. The most widely used technique in the clinical laboratory is the determination of COHb with automated differential spectrophotometry (National Research Council, 1977). The determination of CO in exhaled air by standardized gas analyzers has been used less frequently. However, the portable “Ecolyzer” and other similar instruments have proved to be reliable instruments for the recent validations of the reported smoking habits among populations in field studies (Vogt et al., 1979). The data from both measurements, amount of CO in the alveolar gas and the concentration of COHb in blood, are well correlated. Theoretically, the slope of the graph relating the percent of concentration of COHb to alveolar CO should be about 0.155 at CO concentrations of 0–50 ppm. Most laboratory studies have confirmed this correlation experimentally (National Research Council, 1981). In the case of cigarette smokers who have inhaled puffs of smoke containing 20,000–50,000 ppm of CO, the correlation between exhaled CO and COHb is also in good agreement (r=0.97; Heinemann et al., 1984). The COHb levels are of value for comparing degrees of smoke inhalation. In a study of men aged 34–64 years, cigarette smokers had on the average 4.7% of COHb; cigar smokers, 2.9%; pipe smokers, 2.2%; and nonsmokers, 0.9% (Wald et al., 1981, 1984). However, measurements of exhaled CO or COHb are not valid indicators of chronic exposure to ETS. A study of 100 self-reported nonsmokers who were divided into four groups—without exposure to ETS, with little, with some, and with a lot—revealed no significant differences in measurements of expired CO (5.0–5.7 ppm; mean, 5.61±2.70 ppm) or COHb (0.80–0.94%; mean, 0.87± 0.67%) (Jarvis and Russell, 1984). This observation is also supported by a study of six nonsmoking flight attendants who served in the smokers’ section of a trans-Pacific aircraft. Preflight COHb levels were 1.0±0.2% and postflight levels (after serving round-trip) were 0.7±0.2% (Foliart et al., 1983). Heavily smoke-polluted environments can lead to elevated absorption of CO. This was shown for seven nonsmokers exposed for 2 hours in a pub, whose exhaled air revealed an average of 5.9 ppm of CO, a level that corresponds to the alveolar gas of a smoker after smoking one cigarette (Jarvis et al., 1983). Another study showed that twelve nonsmokers, sharing the nonairconditioned environment of a room with four smokers who smoked four cigarettes each within 30 minutes, had an COHb increase of the

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects same magnitude as that measured in a smoker after consuming one cigarette (Huch et al., 1980). Even though tobacco smoke is a major source for indoor air pollution, additional sources may contribute to increased CO concentrations in air and, consequently, to higher COHb levels in exposed subjects. Such sources include gas stoves, faulty furnaces, and space heaters (National Research Council, 1981). For example, kerosene heaters can be a major source for indoor pollution. Depending on the model and flame setting, kerosene space heaters generate up to 6.5 mg of CO per minute of operation (Leaderer, 1982). In summary, CO in alveolar air and as COHb in nonsmokers originates from endogenous processes as well as from environmental sources. ETS is an important pollutant of indoor environments; however, except for highly polluted settings, CO levels in exhaled air and COHb levels in the blood are not statisically significantly elevated following exposure to ETS, although acute short-term exposures from 3–4 hours may be detected if blood or expired air is sampled within 30 minutes of the end of exposure. In sum, however, measurements of exhaled CO and of COHb are not useful indicators of exposure to ambient ETS except in acute exposure studies in the laboratory. CO measures are a marker of gas-phase exposure to ETS. Nicotine and Cotinine Disregarding nicotine-containing chewing gum and nicotine aerosol rods as aids for smoking cessation, the presence of nicotine and that of its major metabolite, cotinine, in biological fluids is entirely due to the exposure to tobacco, tobacco smoke, or environmental tobacco smoke. The determination of nicotine and cotinine in saliva, blood, or urine of active and passive smokers is done primarily by gas chromatography (GC) with a nitrogen-sensitive detector and by radioimmunoassay (RIA). The GC method requires great precaution in order to avoid contamination by traces of nicotine from the environment or from solvents and/or equipment. This is of major importance for samples containing nicotine at levels <20 ng/ml of fluid, as is the case in nonsmokers exposed to ETS (Feyeraband and Russell, 1980). The GC method can be used to measure concentrations of nicotine as low as 1 ng/ml and concentrations of cotinine as low as 5 ng/ml

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects in samples of physiological fluids (Jacob et al., 1981). An experienced chemist can analyze up to 25 samples per day for nicotine and cotinine. The radioimmunoassays for nicotine and cotinine represent probably the most direct technique available. These assays have only low cross-reactivities with other naturally occurring metabolites of nicotine. The sensitivity of these assays is about 0.5 ng/ml for both nicotine and cotinine and has inter- and intra-assay variations of ±5% (Langone et al., 1973; Hill et al., 1983). An experienced biochemist with automated equipment can analyze up to 80 samples (plus 20 control samples) per day. So far, the RIA method has been used by a limited number of laboratories because it requires the synthesis of specific nicotine and cotinine derivatives for the generation of serum albumin conjugates and the raising of antibodies to these conjugates (Langone et al., 1973). In addition, the RIA method also requires careful drawing and handling of samples to avoid contamination. Table 8–1 presents results from the major studies on the uptake of nicotine by nonsmokers under acute exposure conditions. These data show that exposure to high levels of ETS in laboratories can lead to a significant uptake of nicotine. This uptake is clearly reflected in the concentrations of nicotine in plasma (up to 0.9 µg/ml for nonsmokers compared with a mean value of 14.8 µg/ml for smokers, an increase of 15-fold) and in urine (84 ng/ml for nonsmokers, compared with 1,750 ng/ml, a increase of 20-fold) (Russell and Feyeraband, 1975; Hoffmann et al., 1984). The significantly higher values for nicotine in the plasma compared to urine may be explained by the short initial half-life in smokers of 9 minutes and relatively short terminal half-life in smokers of 2 hours (Benowitz et al., 1982). Table 8–2 presents data for nicotine and cotinine uptake as measured in physiological fluids of nonsmokers exposed, to ETS under daily life conditions. With the exception of the report by Matsukura et al. (1984), the data demonstrate that the involuntary exposure of the passive smoker amounts to a few percent or less of the amount of nicotine that is inhaled by a cigarette smoker. Table 8–3 compares nicotine and cotinine levels as determined in one laboratory in plasma, saliva, and urine of nonsmokers with and without ETS exposure and of active smokers. This comparison shows that, generally, concentrations of nicotine and cotinine in plasma, saliva, and urine of nonsmokers exposed to ETS amount

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects TABLE 8–1 Nicotine Uptake by Nonsmokers Exposed to ETS Under Laboratory Conditions Authors ETS—Conditions No. of Nonsmokers Results Harke, 1970 Room—170 m3 7 Excretion in the urine (6 h after exposure)   (1) 11 smokers consumed 100 cigarettes during 2 h; no ventilation (30 ppm CO)   Nicotine: 10±6.8 µg/6 h Cotinine: 35±34.5 µg/6 h     (2) as (1)—but with regular ventilation (5 ppm CO) 7 Nicotine: 18±7 µg/6 h Cotinine: 19±9.4 µg/6 h   Cano et al., 1970 Room—66 m3         4 smokers and 2 nonsmokers   Excretion in the urine Nicotine, µg/24 h   (a) lived together for 5 days   Day 1—no smoking 0       Day 2—98 cigarettes smoked 35–44       Day 3—121 cigarettes smoked 50–61       Day 4—98 cigarettes smoked 62.5–70       Day 5—88 cigarettes smoked 47–50   (b) lived together for 4 days 2 Day 1—97 cigarettes; 15 µg nic./m3 23–34       Day 2—96 cigarettes; 22 µg nic./m3 22.5–58       Day 3—94 cigarettes; 35 µg nic./m3 47.5–69       Day 4—103 cigarettes; 33 µg nic./m3 32–65 Russell and Feyerabend, 1975 (1) Room—43 m3 12 Nicotine 9 smokers consumed 80 cigarettes and 2 cigars; no ventilation (38 ppm CO)   Before exposure: 0.73±1.6 µg/ml plasma After 78 min exposure: 0.90±0.29 µg/ml plasma 15 min after ending exposure: 80.0±58.7 ng/ml urine No experimental ETS exposure: 12.4 ng/ml urine 8.9 ng/ml urine   (2) Two groups measured after lunch 14 13 No experimental ETS exposure: 12.4±16.9 ng/ml urine 8.9±9.1 ng/ml urine

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects Authors ETS—Conditions No. of Nonsmokers Results Hoffmann et al., 1984 Room—16 m3 6 Time during exposure Nicotine Cotinine 4 cigarettes concurrently and continuously machine smoked for 80 min; 6 air exchanges/h (200 g nic./m3 20 ppm CO)   0 Saliva: Plasma: Urine: 3 ng/ml 0.2 ng/ml 17 ng/mg creat. 1.0 ng/ml 0.9 ng/ml 14 ng/mg creat. 80 min Saliva: Plasma: Urine: 730 ng/ml 0.5 ng/ml 84 ng/mg creat. 1.4 ng/ml 1.3 ng/ml 28 ng/mg creat.   Time following exposure   30 min Saliva: Plasma: 148 ng/ml 0.4 ng/ml 1.7 ng/ml 1.8 ng/ml 150 min Saliva: Plasma: Urine: 17 ng/ml 0.7 ng/ml 100 ng/mg creat. 3.1 ng/ml 2.9 ng/ml 45 ng/mg creat. 300 min Saliva: Plasma: Urine: 7 ng/ml 0.6 ng/ml 48 ng/mg creat. 3.5 ng/ml 3.2 ng/ml 55 ng/mg creat. aAbbreviations: creat., creatinine; nic., nicotine.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects to less than 1% of the mean values observed in physiological fluids of active smokers, even though some nicotine measurements in plasma give a higher reading (Jarvis et al., 1984). In a large-scale study of 839 nonsmokers (identified by their questionnaire response and also having a cotinine concentration of <20 ng/ml of saliva), cotinine levels increased with the number of smokers in the home for each of three age groups examined independently (<5, 6–17, and >18 years). The cotinine levels in saliva were found to be significantly associated with increasing number of smokers per household within each age group. The median salivary cotinine levels in adult smokers was 287 ng cotinine/ml (Coultas et al., 1986). Matsukura et al. (1984) report that cotinine in the urine of ETS-exposed nonsmokers reaches an average of 1.56±0.57 µg/mg of creatinine when 40 or more cigarettes per day have been smoked in the home of the exposed subjects. In the case of cigarette smokers, they found cotinine levels of 8.57±0.39 µg/mg of creatinine in urine. This study has been questioned because its findings of cotinine in urine of both active and passive smokers indicate levels substantially higher than those reported in other studies (Adlkofer et al., 1985; Pittenger, 1985) (see Chapter 12). Nicotine uptake by infants of cigarette-smoking mothers appears to be higher than is generally observed for the adult nonsmoker. The amount of cotinine excreted in the infant’s urine has been found to be correlated with the number of cigarettes smoked by the mother in the 24 hours preceding the measurement (Greenberg et al., 1984). The analysis of nicotine and cotinine in physiologic fluids can be misleading if made on very light smokers or nonsmokers who either sniff tobacco or are tobacco chewers or snuff-dippers. In the case of the very light smoker, nicotine and cotinine values may be similar to those of nonsmokers who had exposure to high levels of ETS (Russell and Feyerabend, 1975; Wald et al., 1984). In the case of individuals who use tobacco nasally, or orally, on a regular basis, the nicotine and cotinine values may approach those of heavy cigarette smokers (Russell et al., 1980; Russell et al., 1981; Palladino et al., in press). In both groups, the analysis of COHb will reveal that these subjects are light smokers or nonsmokers, respectively. However, nicotine and cotinine levels for such persons are clearly not valid for the determination of their exposure to ETS.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects TABLE 8–2 Nicotine Uptake by Nonsmokers Exposed to ETS Under Daily Life Conditionsa Authors Group of Nonsmokers No. of Nonsmokers Examined Results Russell and Feyerabend, 1975 Hospital employees     (urine collection 1 h after lunch)     ng/ml   (a) Group 1 14 Nicotine in urine: 12.4±16.9   (b) Group 2 13 Nicotine in urine: 8.9±9.1 Feyerabend et al., 1982 Hospital employees and outpatients     ng/ml   (a) nonexposed to ETS during the morning (self report) 30 Nicotine in the urine: 7.5±8.5     Nicotine in saliva: 5.9±4.4   (b) exposed to ETS during the morning (self report)   Nicotine in urine: 21.6±28.9     Nicotine in saliva: 10.1±9.7 Foliart et al., 1983 Flight attendants 6 Nicotine in serum: ng/ml   (San Francisco-Tokyo-San Francisco)   (a) before flight 1.6±0.8   (b) after flight 3.2±1.0 Wald et al., 1984 Hospital staff and outpatients     ng/ml   (a) nonexposed to ETS 22 Cotinine in urine: 2.0 (0.0–9.3)   (b) exposed to ETS (self report) 199 Cotinine in urine: 6.0 (1.4–22.0)   ng/ml Wald and Ritchie, 1984 (a) husbands of nonsmokers 101 Cotinine in urine: 8.5±1.3   (b) husbands of smokers 20 Cotinine in urine: 25.2±14.8 Jarvis et al., 1983 Employees in an office, sample collection at 11:30 a.m. (I) and 7:45 p.m. (II) (time between collections including 2-h stay in smoking “pub”) 7 ng/ml Before After     I II   Nicotine in plasma: 0.76 2.49   Nicotine in saliva: 1.90 43.63   Nicotine in urine: 10.51 92.63 Cotinine in plasma: 1.07 7.33 Cotinine in saliva: 1.50 8.04 Cotinine in urine: 4.80 12.94

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects   (Differences between I and II are statistically highly significant; p values range from <0.01 to <0.001) Jarvis et al., 1985 Nonsmoking school children (11–16-yr-old)   ng/ml   I. Neither parent smoked 269 Cotinine in saliva: 0.44±0.68 II. Only father smoked 96 Cotinine in saliva: 1.31±1.21 III. Only mother smoked 76 Cotinine in saliva: 1.95±1.71 IV. Both parents smoked 128 Cotinine in saliva: 3.38±2.45 Matsukura et al., 1984 472 nonsmokers     Urine collection in the morning (a) smokers in home 272 µg cot./mg creat. 0.79±0.1 (b) nonsmokers in home 200   0.51±0.09 Cigarettes smoked per day in home of nonsmokers   1–9 25 µg cot./mg creat. 0.31±0.08 10–19 57   0.42±0.10 20–29 99 0.87±0.19 30–39 38 1.03±0.25 >40 28 1.56±0.57 Unspecified 25 0.56±0.16 Greenberg et al., 1984 Infants under 10 months of age (not breastfed)     (a) not exposed to ETS 18 Urine ng nic./mg creat. 0 (0–59)   Urine ng nic./mg creat. 4 (0–145) Saliva ng nic./mg creat. 0 (0–3)   (b) exposed to ETS 28 Urine ng nic./mg creat. 53 (0–370)   Urine ng cot./mg creat. 351 (41–1,885) Saliva ng nic./mg creat. 12.7 (0–166) Saliva ng cot./mg creat. 9 (0–25) aAbbreviations: cot., cotinine; creat., creatinine; nic., nicotine.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects TABLE 8–4 Pooled Data of Various Biological Parameters Measured in Blood and Urine of Six Subjects After Periods of Nonsmoking, Passive Smoking, and Active Smoking Exposure COHb Plasma Cotinine Plasma Thiocyanate Mutagenicity in Urine Number of samples Mean±SE, % Number of samples Mean±SE, ng/ml Number of samples Mean±SE, µmol/L Number of samples Mean±SE, number of induced revertants/ml No smoking 12 0.57±0.04a 12 1.4±0.2 12 70.8±9.9 12 4.2±1.2 Passive smoking 12 0.55±0.05a 12 2.1±0.4b 12 71.8±9.9 24 5.8±1.0 Active smoking 12 3.38±0.54 12 54.4±11.4 12 70.7±10.2 24 6.4±0.8 aValues below the detection limit (0.5%) included as 0.4%. bDifference between nonsmoking and passive smoking values not significant but suggestive (p<0.10; t-test). SOURCE: Sorsa et al., 1985.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects appropriate method for estimating the genotoxic effect of the urine of ETS-exposed nonsmokers, the method itself and the diet of test subjects have to be standardized. Research in this area is needed, as are studies on the isolation and identification of the active agents in the urine of ETS-exposed nonsmokers. Adducts Formed in Passive Smokers upon Exposure to ETS Since about 1975, highly sensitive methods have been developed for the determination of protein- or DNA-adducts of environmental carcinogens and toxic agents in circulating blood. Methods probing these reactions for the toxic agents known to occur in tobacco smoke and ETS include determination of hemoglobin adducts of nitrosodimethylamine, methyl chloride, vinyl chloride, and benzene (National Institute of Environmental Health Sciences, 1984), as well as 4-aminobiphenyl (Green et al., 1984). DNA adducts with the smoke carcinogen, benzo[a]pyrene (BaP), have been described (Santella et al., 1985), and the tobacco-specific 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) leads to O6-methylguanine in DNA (Hoffmann and Hecht, 1985). RIA’s have been developed for quantitative determination of both the BaP-DNA adduct and O6-methylguanine (Perera et al., 1982; Foiles et al., 1985). So far, the method for the determination of the DNA adducts has been applied to the analysis of benzo[a]pyrene in smokers (Shamsuddin et al., 1985). In addition, the hemoglobin-4-aminobiphenyl assay has been used for the analysis of the blood of smokers (Tannenbaum et al., in press). In both cases, only a limited number of samples have been analyzed for these adducts. Nevertheless, the data appear encouraging. Another sensitive method for quantifying DNA adducts is the P32-postlabelling technique, which has been applied to human tissues (Gupta et al., 1982; Everson et al., 1986). Validation and quantitative determination of the uptake of tobacco smoke carcinogens is urgently needed. Assays of adducts of BaP, aromatic amines, and tobacco-specific nitrosamines with protein or DNA in the circulating blood are the most promising tests of exposure to tobacco smoke. Once such assays have been advanced to yield reproducible, informative methods in smokers, they may be subsequently refined to such sensitivities that they

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects will also furnish reliable data on such adducts in the blood of passive smokers. FUTURE NEEDS At present, the best method for quantifying human exposure to ETS is the assay of nicotine and cotinine in urine and possibly saliva. Nicotine and cotinine can also be determined in serum samples, but these samples require invasive techniques. In smoke-polluted environments, nicotine is present in the vapor phase as a free base, thus its uptake by the passive smoker may not be representative of the uptake of acidic and neutral smoke components from the vapor phase nor of any component in the particulate phase. Thus, future studies should be concerned with developing techniques to measure the uptake by the nonsmoker of various other types of tobacco-specific ETS components. This may include assays for the vapor-phase 3-vinylpyridine or flavor components that are indigenous to tobacco. Particulate-phase agents to be traced could include solanesol, tobacco-specific nitrosamines, and polyphenols such as chlorogenic acid or rutin. These components are likely to be found only in trace amounts in ETS, and, thus, only minute quantities would be found in the circulating blood of passive smokers, making the development of assays difficult. The development of new trace methods for quantifying the levels of some tobacco-specific materials in nonsmokers may require the identification of adducts formed between the ETS components and the proteins in blood. This approach would require the development of highly sensitive methods such as immunoassays (e.g., RIA, ELISA) or postlabelling with radioisotopes or other markers. The epidemiological studies on the effects of exposure to ETS by nonsmokers have to consider a number of non-ETS-related factors. This fact underlines the urgent need for the development of highly sensitive dosimetric methods for ETS-specific carcinogens that can be applied in field studies. SUMMARY AND RECOMMENDATIONS Passive smokers are exposed to trace amounts of toxic agents including tumor initiators, tumor promoters, carcinogens, and organ-specific carcinogens when inhaling ETS. The determination of thiocyanate, nicotine, and cotinine in body fluids such as saliva,

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects serum, and urine, as well as quantitication of CO in alveolar air and COHb in blood, has been useful for the assessment of the habits of individuals and groups of smokers of cigarettes, cigars, and pipes. Currently, for measuring the exposure to ETS by nonsmokers, nicotine and cotinine appear useful. In acute exposure studies, COHb can be a useful marker. Nicotine and cotinine, however, may not be directly related to the carcinogenic potential of the smoke. Indicators that are related to the carcinogenic risk are needed. To assess the risks involved in the exposure to carcinogenic agents from ETS, sensitive dosimetry methods for tobacco-specific compounds are urgently needed. During the last decade, immunoassays and postlabelling methods have been developed for tracing toxic and carcinogenic agents in circulating blood. These methodologies should be used for the development of dosimetry studies in nonsmokers exposed to ETS. Protein and DNA adducts may provide exposure measures that could be effectively used in epidemiologic studies. What Is Known Determinations of thiocyanate, nicotine, and cotinine in saliva, serum, and urine, as well as quantification of CO in alveolar air and carboxyhemoglobin in blood, have been shown to be useful parameters for the assessment of the habits of individuals and groups of active smokers of cigarettes, cigars, and pipes. However, in general, only nicotine and its metabolite cotinine have proven useful for measuring the exposure to ETS of nonsmokers. Assessment of average daily exposure on the basis of cotinine levels in saliva and urine is independent of the restrictions posed by variations of the half-life of nicotine in smokers and nonsmokers. The determination in urine of the amount of cotinine per milligram of creatinine should provide a more stable measure of recent environmental exposure to nicotine from ETS than cotinine without reference to creatinine, particularly when limited volumes of urine are available. It is likely that the exposure of nonsmokers to ETS increases the mutagenic activity of their urine over the activity observed in urine of the same nonsmokers when not exposed to ETS.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects What Scientific Information Is Missing The question of urinary hydroxyproline excretion in nonsmokers exposed to ETS is not settled. A study on the urinary excretion of aromatic amines in nonsmokers exposed to ETS is needed in order to correlate the total amounts of individual amines and their metabolites in the urine of nonsmokers exposed to ETS. Where exposure histories can be specified clearly, validation of the use of adduct assays to determine and quantify uptake of tobacco smoke carcinogens is needed. Information is needed on certain tobacco-specific constituents and their fate in the ETS-exposed nonsmoker, including solanesol, tobacco-specific nitrosamines, and polyphenols such as chlorogenic acid or rutin. Knowledge of the levels of nitrosothioproline following exposure to ETS as well as nitrosoproline is needed. Knowledge of the effects of diet is needed when interpreting results of the Ames bacterial assay for mutagenicity of the urine of ETS-exposed nonsmokers. Identification of the mutagenic agents in the urine of ETS-exposed nonsmokers needs to be made. Future studies should be concerned with methodologies that enable us to assay the uptake by the nonsmoker of various other types of ETS components that are tobacco-specific. New trace methods will have to be developed for dosimetry studies of carcinogens involving adducts (DNA and protein) and the development of highly sensitive methods such as immunoassays or postlabelling for other products. The epidemiological studies on the effects of ETS exposure in nonsmokers should consider a number of non-ETS-related factors. This fact underlines the urgent need for the development of highly sensitive dosimetric methods for ETS-specific carcinogens that can be applied in field studies. REFERENCES Adlkofer, F., G.Scherer, and W.D.Heller. Hydroxyproline excretion in urine of smokers and passive smokers. Prev. Med. 13:670–679, 1984. Adlkofer, F., G.Scherer, and U.von Hees. Passive smoking. N. Engl. J. Med. 312:719–720, 1985.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects Tannenbaum, S.R., M.S.Bryant, P.L.Skipper, and M.Maclure. Hemoglobin adducts of tobacco-related aromatic amines: Application to molecular epidemiology. In D.Hoffmann and C.Harris, Eds. Mechanisms in Tobacco Carcinogenesis (Banbury Report 23). Cold Spring Harbor, New York: Cold Spring Harbor Laboratories, in press. Tsuda, M., J.Niitusuma, S.Sato, T.Hirayama, T.Kakizoe, and T.Sugimura. Increase in the levels of N-nitrosoproline, N-nitrothioproline, and N-nitro-2-methylthioproline in human urine by cigarette smoking. Cancer Lett. 30:177–124, 1986. Vainio, H., K.Hemminki, and J.Wilbourn. Data on the carcinogenicity of chemicals in the IARC monographs programme. Carcinogenesis 6:1653–1665, 1985. Vogt, T.M., S.Selvin, and J.H.Billings. Smoking cessation program: Baseline carbon monoxide and serum thiocyanate levels as predictors of outcome. Am. J. Public Health 69:1156–1159, 1979. Wald, N.J., and C.Ritchie. Validation of studies on lung cancer in nonsmokers married to smokers. Lancet 1:1607, 1984. Wald, N.J., M.Idle, J.Boreham, A.Bailey, and H.Van Vunakis. Serum cotinine levels in pipe smokers: Evidence against nicotine as cause of coronary heart disease. Lancet 2:775–777, 1981. Wald, N.J., J.Boreham, A.Bailey, C.Ritchie, J.E.Haddow, and G.Knight. Urinary cotinine as marker for breathing other people’s tobacco smoke. Lancet 1:230–231, 1984. Yamasaki, E., and B.N.Ames. Concentration of mutagens from urine by adsorption with the nonpolar resin XAD-2: Cigarette smokers have mutagenic urine. Proc. Natl. Acad. Sci. USA 74:3555–3559, 1977.

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