Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects (1986)

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ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 133 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. 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 134 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 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 135 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 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 136 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 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 137 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 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 138 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 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 139 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 Nicotine: 10±6.8 µ g/6 consumed 100 h cigarettes during 2 h; no Cotinine: 35±34.5 ventilation (30 ppm µ g/6 h CO) (2) as (1)—but with 7 Nicotine: 18±7 µg/6 h regular ventilation (5 Cotinine: 19±9.4 µ g/6 ppm CO) h Cano et al., 1970 Room—66 m3 4 smokers and 2 Excretion in the urine Nicotine, µ g/24 h nonsmokers (a) lived together for 5 Day 1—no smoking 0 days Day 2—98 cigarettes 35–44 smoked Day 3—121 50–61 cigarettes smoked Day 4—98 cigarettes 62.5–70 smoked Day 5—88 cigarettes 47–50 smoked (b) lived together for 4 2 Day 1—97 cigarettes; 23–34 days 15 µg nic./m3 Day 2—96 cigarettes; 22.5–58 22 µg nic./m3 Day 3—94 cigarettes; 47.5–69 35 µg nic./m3 Day 4—103 32–65 cigarettes; 33 µ g nic./ m3 Russell and (1) Room—43 m3 12 Nicotine Feyerabend, 1975 9 smokers consumed 80 Before exposure: 0.73±1.6 µg/ml plasma cigarettes and 2 cigars; After 78 min exposure: 0.90±0.29 µg/ml no ventilation (38 ppm plasma CO) 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 14 No experimental ETS exposure: 12.4±16.9 measured after lunch 13 ng/ml urine 8.9±9.1 ng/ml urine 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 140 Authors ETS—Conditions No. of Results Nonsmokers Hoffmann et Room—16 m3 6 Time during exposure Nicotine Cotinine al., 1984 4 cigarettes 0 Saliva: 3 ng/ml 1.0 ng/ml concurrently and Plasma: 0.2 ng/ml 0.9 ng/ml continuously Urine: 17 ng/mg 14 ng/mg machine smoked creat. creat. for 80 min; 6 air 80 min Saliva: 730 ng/ml 1.4 ng/ml exchanges/h (200 Plasma: 0.5 ng/ml 1.3 ng/ml g nic./m 3 20 ppm Urine: 84 ng/mg 28 ng/mg CO) creat. creat. Time following exposure 30 min Saliva: 148 ng/ml 1.7 ng/ml Plasma: 0.4 ng/ml 1.8 ng/ml 150 min Saliva: 17 ng/ml 3.1 ng/ml Plasma: 0.7 ng/ml 2.9 ng/ml Urine: 100 ng/mg creat. 45 ng/mg creat. 300 min Saliva: 7 ng/ml 3.5 ng/ml Plasma: 0.6 ng/ml 3.2 ng/ml Urine: 48 ng/mg creat. 55 ng/mg creat. aAbbreviations: creat., creatinine; nic., nicotine. 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 141 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. 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 142 TABLE 8–2 Nicotine Uptake by Nonsmokers Exposed to ETS Under Daily Life Conditionsa Authors Group of Nonsmokers No. of Nonsmokers Results Examined Russell and Hospital employees Feyerabend, 1975 (urine collection 1 h ng/ml after lunch) (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 ng/ml outpatients (a) nonexposed to ETS 30 Nicotine in the 7.5±8.5 during the morning urine: (self report) Nicotine in saliva: 5.9±4.4 (b) exposed to ETS Nicotine in urine: 21.6±28.9 during the morning Nicotine in saliva: 10.1±9.7 (self report) Foliart et al., 1983 Flight attendants 6 Nicotine in serum: ng/ml (San Francisco-Tokyo- (a) before flight 1.6±0.8 San Francisco) (b) after flight 3.2±1.0 Wald et al., 1984 Hospital staff and ng/ml outpatients (a) nonexposed to ETS 22 Cotinine in urine: 2.0 (0.0–9.3) (b) exposed to ETS (self 199 Cotinine in urine: 6.0 (1.4–22.0) report) ng/ml Wald and Ritchie, 1984 (a) husbands of 101 Cotinine in urine: 8.5±1.3 nonsmokers (b) husbands of 20 Cotinine in urine: 25.2±14.8 smokers Jarvis et al., 1983 Employees in an office, 7 ng/ml Before After sample collection at I II 11:30 a.m. (I) and 7:45 Nicotine in plasma: 0.76 2.49 p.m. (II) (time between Nicotine in saliva: 1.90 43.63 collections including 2- h stay in smoking “pub”) 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 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 143 (Differences between I and II are statistically highly significant; p values range from <0.01 to <0.001) Jarvis et al., 1985 Nonsmoking school ng/ml children (11–16-yr-old) 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 0 (0–59) creat. 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 53 (0–370) creat. Urine ng cot./mg 351 (41–1,885) creat. Saliva ng nic./mg creat. 12.7 (0–166) Saliva ng cot./mg creat. 9 (0–25) aAbbreviations: cot., cotinine; creat., creatinine; nic., nicotine. 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 144 TABLE 8–3 Approximate Relations of Nicotine as a Parameter Between Nonsmokers, Passive Smokers, and Active Smokersa Nicotine/Cotinine Nonsmokers without ETS Exposure Nonsmokers with ETS Exposure Active Smokers No.=46 No.=54 No.=94 Mean Value % of Active Mean Value % of Active Mean Value Smokers' Value Smokers' Value Nicotine (ng/ml) 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/ml) in plasma 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 aDifferences between nonsmokers exposed to ETS compared with nonsmokers without exposure: *p<0.01; **p<0.001. SOURCE: Jarvis et al., 1984. Cotinine elimination in the plasma of nonsmokers exposed to ETS was reported to be slower than cotinine elimination in the plasma of active smokers. Cotinine elimination from urine was also significantly slower. In a study of 10 chronic smokers and 4 nonsmokers experimentally exposed to ETS, the half-life of elimination of cotinine from plasma was 49.7 hours in nonsmokers and 18.5 hours in smokers (Sepkovic et al., 1986). Disappearance of cotinine from urine was also significantly slower in nonsmokers than in chronic smokers (32.7 hours versus 21.9 hours). These preliminary data need to be considered when using cotinine to quantify the dose in nonsmokers exposed to ETS. In summary, the determination of nicotine and, especially, of cotinine in saliva, blood, and/or urine of nonsmokers exposed to ETS represents at present the most appropriate assay for estimating long-term (average daily) exposure. However, venipuncture needed to get serum samples is often impractical, if not impossible. The use of saliva for nicotine and cotinine assays, despite some advantages, also has certain inherent weaknesses, such as uncharacteristically high readings immediately after heavy ETS exposure and the need to wait several hours after exposure for the 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 145 cotinine concentration to stabilize (Hoffmann et al., 1984). Saliva is a particularly erratic source on which to make nicotine measures. Urinalysis for cotinine is the preferred method for assessment of long-term ETS exposure, because the sampling is noninvasive, the excretion rate of cotinine is only slightly dependent on the pH of urine, and assessment of the average daily exposure on the basis of cotinine levels is independent of the restrictions posed by variations of the half-life of nicotine in smokers and nonsmokers (Beckett et al., 1971; Klein and Gorrod, 1978). Creatinine—Reference Compound for Urine Analysis Urine sampling does have some associated problems. Often it is impractical to collect 24-hour urine samples for the analysis of biological markers of direct exposure to tobacco smoke or to ETS unless undertaken under strict medical supervision, such as in a metabolic ward. In this case, the ratio of biological markers to creatinine is often used to allow for variations in fluid intake (and excretion) (see Table 8–1). Creatinine excretion varies from person to person, but the daily output for each individual is almost constant from day to day. Urinary creatinine bears a direct relation to the muscle mass of the individual. The milligram amount of creatinine excreted during 24 hours per kilogram of body weight is often expressed as the creatinine coefficient. The coefficient varies from 18 to 32 in men (total excretion 1.1–3.2 g/day) and from 10 to 25 in women (total excretion 0.9–2.5 g/day). The coefficient is largely independent of variations in diet, since creatinine in healthy persons is of endogenous origin. In older people, the daily output of creatinine may decrease to 0.5 g/ day. In cigarette smokers, urinary output of creatinine in men appears to decrease with greater number of cigarettes smoked per day (Adlkofer et al., 1984). However, this finding needs to be confirmed. Based on the variations in daily creatinine excretions in the urine, one has to be aware of the limitation of the factor “amount of biological marker per milligram of creatinine.” In a study with 15 adult male cigarette smokers, the daily creatinine excretion varied between 1.0 and 2.5 g and the cotinine excretion between 1.3 and 13.1 mg (Hoffmann and Brunnemann, 1983). However, in certain cases, such as with healthy infants, the daily variations in urinary excretion are rather small. Thus, the measured nanograms of 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 146 cotinine per milligram of creatinine in urine reflect the inhalation of environmental nicotine from ETS rather well (Greenberg et al., 1984). For the determination in urine, creatinine is complexed with picric acid and the resulting red color is measured spectrophotometrically, a task now predominately done with an autoanalyzer (Faulkner et al., 1976). Although the determination of cotinine in urine without reference to creatinine has resulted in meaningful data in some studies, the standardized cotinine levels per unit of creatinine may give a more stable measure of ETS exposure—particularly when limited urine samples must be used. Hydroxyproline Inhalation of nitrogen dioxide causes degradation of lung collagen and elastin (Kosmidar et al., 1972; Hatton et al., 1977). This degradation results in elevated urinary excretion of hydroxyproline (Lewis, 1980). It is thus possible that the NO2 in tobacco smoke, and even NO2 in ETS, has the same lung-damaging effect as pure NO2. Kasuga et al. (1981) reported two studies in which healthy cigarette smokers excreted significantly more hydroxyproline than healthy nonsmokers and exsmokers. In the case of 6- to 11-year-old children of smoking parents, Kausga et al. (1981) found elevated hydroxyproline levels in the urine. Because of the relatively low concentration of NO2 in ETS (see Chapter 2), this finding was unexpected. Adlkofer et al. (1984) were unable to confirm this finding in a study of 23 nonsmokers exposed to ETS. At present, the question of quantitative aspects of urinary hydroxyproline excretion in nonsmokers exposed to ETS is not settled. It will require additional studies before this compound and its ratio to creatinine can be used as indicators for the degree of ETS exposure. N-Nitrosoproline N-nitrosoproline (NPRO) in urine reflects endogenous formation of nitrosamines, many of which are known animal carcinogens (Preussmann and Steward, 1984; Vainio et al., 1985). NPRO appears neither to undergo metabolism in mammals nor to alkylate 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 147 cellular macromolecules. NPRO is considered to be nonmutagenic and noncarcinogenic and is excreted nearly quantitatively in urine. It has been shown that endogenous formation of NPRO is significantly increased in cigarette smokers (Hoffmann and Brunnemann, 1983; Ladd et al., 1984; Scherer and Adlkofer, in press). The increase is probably due to the high concentrations of nitrogen oxides in tobacco smoke that serve as nitrosating agents and the elevated concentration of thiocyanate in smokers that catalytically enhance the endogenous formation of nitrosamines such as NPRO. These effects are absent in nonsmokers without ETS exposure. In one 5-day study, four male nonsmokers with controlled diets were exposed to known degrees of ETS for three periods of 80 minutes each on day 3 and day 4. Their 24-hour urine voids were analyzed for NPRO and for cotinine. While the cotinine levels in the urine of these nonsmokers increased from 5–7 ng/ml to 215–360 ng/ml, the NPRO excretion did not significantly change (Brunnemann et al., 1984). In another controlled study with 10 nonsmokers exposed to ETS containing 45 ppb of NO2, 400 ppb of NO, and 22 ppm of CO, urinary output of NPRO was also not elevated while COHb had increased significantly (Scherer and Adlkofer, in press). Although these two studies require confirmation and should include analytical assessment of nitrosothioproline (NTPRO) (Tsuda et al., 1986), another endogenously formed nitrosamine, at present neither NPRO nor NTPRO measurement in urine can be used to indicate exposure to ETS. Aromatic Amines During the burning of cigarettes, 20–30 times more aromatic amines are released into the sidestream smoke than are present in the mainstream smoke (see Chapter 2). Although at this time there is a lack of analytical data, it may be assumed that indoor environments that are strongly polluted with ETS contain measurably higher amounts of aromatic amines than ambient air without tobacco smoke pollution. Preliminary data indicate that free aniline and o-toluidine, serving as surrogates for aromatic amines, are increased, although not significantly, in the 24-hour urine voids of cigarette smokers (3.1±2.6 µg and 6.3±3.7 µg) compared with nonsmokers (2.8 ±2.5 µg and 4.1±3.2 µg) (El-Bayoumy et al., in press). The 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 148 next step requires the assay of the metabolites of aniline and o-toluidine in the urine of both smokers and nonsmokers. A study of the urinary excretion of aromatic amines in passive smokers would be indicated only if the total amounts of individual amines and their metabolites in smokers' urine are found to be significantly increased. GENOTOXICITY OF THE URINE The evaluation of the genotoxicity of urine in nonsmokers with ETS exposure must consider the possibility of confounding effects, because DNA modifiers may be present in urine as a consequence of dietary intake or as a secondary result of the activity of infectious agents in the urine of the host. Nevertheless, urinary constituents may be DNA modifiers, because the inhaled agents are known or suspected mutagens or because the inhaled agents lead to the formation of such biologically active compounds. Since 1975, the most widely used assay for genotoxicity of human urine is the determination of mutagenicity in bacterial-tested strains with and without activation by enzyme-induced liver homogenate. In 1977, Yamasaki and Ames reported the presence of mutagens in the urine of cigarette smokers, thus suggesting a correlation between mutagens in smokers' urine and increased risk for bladder cancer. Since publication of these data, other studies have reported an association of urinary mutagens that are active in bacterial tester strains with cigarette smoking (International Agency for Research on Cancer, 1986), but not all results from these studies have been consistent. One reason for the divergent findings could be the influence of dietary factors on the mutagens in the urine of smokers (Sasson et al., 1985) and, perhaps also, nonsmokers exposed to ETS. Three studies have attempted to explain the possible mutagenic activity of the urine of nonsmokers exposed to ETS. In one study, fractions and subfractions were isolated by high-pressure liquid chromatography (HPLC) from the urine of five passive smokers. Upon metabolic activation by S9 liver homogenates from rats pretreated with 3-methylcholanthrene, these materials were mutagenic in TA-bacterial tester strains (Putzrath et al., 1981). It appeared that these mutagens are a complex mixture of urinary 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 149 components in the polar lipophilic subfractions. Due to a lack of diet control, these results are ambiguous. In a second assay of urine for bacterial mutagenicity, 8 male nonsmokers (25 and 35 years of age) were placed in a poorly ventilated room (10 m3) with 10 smokers for an 8-hour period (Bos et al., 1983). The 12-hour urine samples of the nonsmokers were collected before, during, and after exposure to ETS. Metabolically activated concentrates of the urine samples were analyzed for mutagenic activity in the tester strain, TA 1538. Urine samples collected directly after exposure to ETS were significantly more mutagenic (relative activity: 3.9±1.0) than urine samples of the same nonsmokers prior to (3.1±0.7) or long after ETS exposure (2.5±0.5). In the third study, six women who were medical students were exposed to ETS in a 10-m 3 exposure chamber on 2 consecutive days for one 3-hour session in the mornings and a 2-hour session in the afternoons. During these sessions, three of the women smoked a total of 30 cigarettes per day of a low-yield filter-tipped brand (5.4 mg tar, 0.4 nicotine, 4.6 mg CO); the other three women did not smoke. After 3 days without exposure and without cigarette smoking by any of the women, the exposure was repeated with reversal of the roles, so that those who had previously been nonsmokers now were smokers, and vice versa. The CO concentration in the chamber averaged 3.0 ± 0.9 ppm. The uptake of smoke was assessed by determination of COHb, cotinine, and thiocyanate in the plasma. Urine samples were collected at the end of the daily smoking periods. Urine was concentrated according to Yamasaki and Ames (1977) and tested for mutagenicity with tested strain TB98 using rat liver homogenate for metabolic activation (Sorsa et al., 1985). As is evident from the data in Table 8–4, COHb values for nonsmokers and passive smokers were indistinguishable, while there was a trend for higher plasma cotinine values in the passive smokers. The authors observed an increase in the mutagenicity of the urine of passive smokers during the period of study. The differences observed were not significant. On the basis of presently available data, it is likely that the exposure of nonsmokers to heavy ETS increases the potential for metabolically activated genotoxic activity of their urine above and beyond the mutagenic activity that is observed in urine of the same nonsmokers before and long after exposure to ETS. However, before validating the Ames bacterial assay for mutagenicity as an 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 150 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 Mean Number Mean Number Mean Number Mean±SE, of ±SE, % of ±SE, of ±SE, of number of samples samples ng/ml samples µmol/L samples induced revertants/ ml No 12 0.57 12 1.4±0.2 12 70.8 12 4.2±1.2 smoking ±0.04a ±9.9 Passive 12 0.55 12 2.1 12 71.8 24 5.8±1.0 smoking ±0.05a ±0.4b ±9.9 Active 12 3.38 12 54.4 12 70.7 24 6.4±0.8 smoking ±0.54 ±11.4 ±10.2 aValues below the detection limit (0.5%) included as 0.4%. b Difference between nonsmoking and passive smoking values not significant but suggestive (p<0.10; t-test). SOURCE: Sorsa et al., 1985. 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 151 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 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 152 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, 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 153 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 1. 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. 2. 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. 3. 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. 4. 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. 

ASSESSING EXPOSURES TO ENVIRONMENTAL TOBACCO SMOKE USING BIOLOGICAL MARKERS 154 What Scientific Information Is Missing 1. The question of urinary hydroxyproline excretion in nonsmokers exposed to ETS is not settled. 2. 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. 3. 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. 4. 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. 5. Knowledge of the levels of nitrosothioproline following exposure to ETS as well as nitrosoproline is needed. 6. 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. 7. Identification of the mutagenic agents in the urine of ETS-exposed nonsmokers needs to be made. 8. 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. 9. 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. 10. 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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