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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