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3
In Vivo and In Vitro Assays to Assess the Health Effects of Environmental Tobacco Smoke

INTRODUCTION

Suitable methods for assessing the potential for adverse health effects resulting from exposure to environmental tobacco smoke (ETS) are limited by the complexity of the composition of the mixture. In vivo and in vitro assays are commonly used to establish carcinogenicity and in some cases to extrapolate risks to humans. For complex mixtures such as ETS, these assays may be done on the mixture itself or on individual chemical constituents. Many properties of ETS change as the smoke “ages” after its initial generation. Aging probably affects the bioavailability, as well as physicochemical characteristics, of the smoke.

As inhalation is the primary route by which humans are exposed to tobacco smoke, it is obviously the preferred method of administration in animal models for evaluating the toxicological properties of both cigarette smoke and ETS. While extensive inhalation studies have been performed on the toxicological properties of mainstream cigarette smoke (MS), far fewer studies have been performed on sidestream smoke (SS) and ETS. The selection of appropriate animal models requires familiarity with exposure systems, as well as with basic anatomical differences between the model and human respiratory tracts.

Methods other than inhalation, such as in vitro assays, have been developed for the evaluation of MS. A few of these methods have been applied to the assessment of the relative toxicological properties of SS versus MS. These methods are frequently criticized because of differences in the way the smoke constituents



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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects 3 In Vivo and In Vitro Assays to Assess the Health Effects of Environmental Tobacco Smoke INTRODUCTION Suitable methods for assessing the potential for adverse health effects resulting from exposure to environmental tobacco smoke (ETS) are limited by the complexity of the composition of the mixture. In vivo and in vitro assays are commonly used to establish carcinogenicity and in some cases to extrapolate risks to humans. For complex mixtures such as ETS, these assays may be done on the mixture itself or on individual chemical constituents. Many properties of ETS change as the smoke “ages” after its initial generation. Aging probably affects the bioavailability, as well as physicochemical characteristics, of the smoke. As inhalation is the primary route by which humans are exposed to tobacco smoke, it is obviously the preferred method of administration in animal models for evaluating the toxicological properties of both cigarette smoke and ETS. While extensive inhalation studies have been performed on the toxicological properties of mainstream cigarette smoke (MS), far fewer studies have been performed on sidestream smoke (SS) and ETS. The selection of appropriate animal models requires familiarity with exposure systems, as well as with basic anatomical differences between the model and human respiratory tracts. Methods other than inhalation, such as in vitro assays, have been developed for the evaluation of MS. A few of these methods have been applied to the assessment of the relative toxicological properties of SS versus MS. These methods are frequently criticized because of differences in the way the smoke constituents

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects are presented to the test system as compared with that which occurs in the human situation. Despite these limitations, the use of cigarette smoke condensate (CSC) from MS has provided insight into the relative carcinogenic potential of various constituents in the MS of cigarettes. Similar studies using suitable condensates from SS and aged ETS could provide additional data on the effects of ETS. IN VIVO ASSAYS ON ENVIRONMENTAL TOBACCO SMOKE Exposure Methods in Laboratory Research Several methods are available to evaluate the potential health effects of inhaled pollutants. Some common ones are whole-body exposure, head-only exposure, nose- or mouth-only exposure, lung-only exposure, or partial-lung exposure. Since the primary objective of an inhalation experiment is to determine the effects of the test substances or mixture on the respiratory system, it is preferable to eliminate or limit exposure through the skin or through ingestion (such as through contact with materials deposited on the fur or contaminated food and water). Three methods have been used to determine the amount of material deposited in the respiratory tract (Phalen, 1984): direct measurement, calculations using airborne concentrations and uptake models, and calibration of the exposure apparatus using tracer substances. Direct measurement requires analysis of major components and their metabolites in tissues as well as in urine and feces or measurement of the amounts of material in the inspired and expired air. Aside from calculating dose based upon particle aerodynamic size and physiological data on lung function of experimental animals, tracers can provide reasonable estimates of exposure. Inhalation exposure chambers are used for those studies in which whole-body exposure is desired. The ability to expose a large number of animals at one time and the absence of a need to restrain or anesthetize the animals are among the advantages in using this approach. There are, however, several major disadvantages. The animals are exposed through skin absorption and mouth ingestion and, in prolonged instances, by food and possibly water contamination. Animals tend to avoid exposure in such

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects chambers by huddling together or covering their noses with their own fur. Losses of particulate aerosols to the interior walls of the chambers are also frequently a problem. Head-only exposure systems eliminate many of these problems. The disadvantages of these systems are that the animal must be restrained and is stressed or anesthetized, and there is difficulty in forming an adequate seal. Nose- or mouth-only exposure systems further limit exposure to the oral cavity and the respiratory tract. Masks or the use of catheters in the nose are generally used with larger animals. Lung and partial-lung-only exposure systems such as endotracheal tubes are employed to bypass the upper respiratory tract and to directly expose the lung. Most of these methods require that the animal be anesthetized, which may alter normal respiration. Other disadvantages include disruption of normal airflow by the presence of tubes in the airways and the loss of normal humidification and thermal regulation of the inspired air caused by bypassing the upper respiratory tract. Intratracheal instillation is an alternative to inhalation for evaluating the effects of individual compounds on the respiratory system. While there are several advantages in employing this bioassay technique, it is also known that the distribution of test material to respiratory tissue may differ from that which would be obtained by actual inhalation exposures. Instillation of an aqueous suspension of radiolabeled particles resulted in a less uniform deposition than inhalation (Brain et al., 1976). Animal Models in Inhalation Studies The selection of an appropriate animal model for inhalation studies with potentially toxic agents is compounded by the fact that one of the major functions of the mammalian sensory apparatus is to limit the exposure to toxic agents either by altering breathing or by producing avoidance behavior (Alarie, 1973; Wood, 1978). Also, the selection of animal species and strains for inhalation exposure studies requires thorough evaluation. The use of several (at least three) animal species, several dose levels, and animals that metabolize the suspect toxin in a similar manner to humans is recommended for those studies that attempt to evaluate human hazards (Stuart, 1976). The appropriate animal model should have (1) a similarity to the human respiratory tract with

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects respect to anatomy, physiology, and susceptibility; (2) a life span appropriate for the proposed study; (3) a sensitivity to certain classes of toxic agents; (4) anatomical or physiological properties that could lead to increased precision in empirical measurements; (5) an existing data base; (6) a documented history of appropriate procedures; and (7) an adaptability for generating data that might be used for mathematically modeling the animal system and its responses to airborne particulates. Results of Inhalation Studies Inhalation studies on the carcinogenicity of MS have been performed on a variety of laboratory animals. The early studies with rodents have been previously reviewed (Wynder and Hoffmann, 1967; Mohr and Resnik, 1978). More recent studies verify these findings for several animal species exposed to whole smoke or MS. A few studies have exposed mice to the vapor phase of fresh MS, and one (see below) exposed mice to the vapor phase of flue-cured MS. Because commonly utilized filter systems do not remove many of the vapor-phase constituents, studies contrasting the effects of exposure to whole smoke with the effects of exposure to the gas phase should throw some light on the possible health effects of ETS. Male and female C57Bl mice (100 in each group) were exposed nose only for 12 minutes daily to the gas phase of smoke of cigarettes prepared from flue-cured tobaccos (Harris et al., 1974). The treated mice had lung tumors and emphysema, independent of the tumors, which were not found in control mice. A total of 219 C57Bl and 186 BLH mice were exposed to the gas phase of cigarette MS. The particulate matter was removed by passing the smoke through a Cambridge filter. The animals were exposed to the gas phase of 12 cigarettes for 90 minutes daily over 27 months. The percentages of mice with lung adenomas were 5.5% and 32% in the smoke-exposed C57Bl and BLH mice, as compared with 3.4% and 22% for their respective controls (Otto and Elmenhorst, 1967). Therefore, it appears that there are carcinogenic constituents in the vapor phase of the smoke. Using Snell’s mice, similar studies evaluated the toxicological properties of whole MS and the gas phase of MS. In these studies, the animals were housed in individual chambers during the exposure (Leuchtenberger and Leuchtenberger, 1970). There was

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects a significant difference (p<0.1) in the incidence of pulmonary tumors between the animals exposed to whole smoke and control animals. The difference was greater (p=0.005) for animals exposed to only the gas phase of cigarette smoke as compared with the same controls, so that the rate of tumors among the gas-phase-exposed animals was greater than among the whole-smoke exposed animals. In Vivo Bioassays Other Than Inhalation Alternative methods have been used to assess the relative chronic toxicity of cigarette MS in an attempt to reduce the cost and technical difficulties associated with inhalation experiments. The most common approach has been to use the CSC in bioassay procedures. In preparing the condensate, many of the volatile and semivolatile components are lost. In addition, it is not known how the aging of the CSC may affect chemical composition and biological activity. To date, only one study has examined the carcinogenic potential of the condensate of SS of cigarettes (Wynder and Hoffmann, 1967; International Agency for Research on Cancer, 1986). Cigarette “tar” from the SS of nonfilter cigarettes, which had settled on the funnel covering a multiple-unit smoking machine, was suspended in acetone and applied to mouse skin for 15 months. Fourteen of 30 Swiss-ICR mice developed benign skin tumors, and 3 had carcinomas. In a parallel assay of MS from the same source, a 50% CSC:acetone suspension applied to deliver a comparable dose of CSC to 100 Swiss-ICR female mice led to benign skin tumors in 24 mice and malignant skin tumors in 6. This indicates that the smoke condensate of SS has greater tumorigenicity per equivalent dose on mouse skin than MS “tar” (p<0.05; Wynder and Hoffmann, 1967). IN VITRO ASSAYS ON ENVIRONMENTAL TOBACCO SMOKE Several short-term bioassays have been performed to evaluate the genotoxicity of cigarette MS. These studies have been the subject of two recent reviews (DeMarini, 1981; Obe et al., 1984). While most of them have evaluated the effects of CSC, some have attempted to evaluate either the gas phase or the whole smoke.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects The most commonly employed assay for mutagenic activity employs various strains of Salmonella typhimurium. Whole smoke as well as CSC from four types of tobacco were found to be mutagenic in S. typhimurium TA1538 (Basrur et al., 1977). Recent studies have shown that SS is also mutagenic in a system where the smoke was tested directly on the bacterial plates (Ong et al., 1984). They support extensive assays performed on CSC that indicate that tobacco smoke has significant mutagenic potential and show that the particulate matter of SS is likely to be a significant contributor to the mutagenic activity of indoor air particulate matter (Bos et al., 1983; Lofröth et al., 1983). Thus, similar mutagenic activity for the CSC of SS would be expected. In another study (Lewtas et al., in press), condensate from air polluted with ETS for 10 hours was used in an assay employing S. typhimurium. The average indoor air mutagenicity per cubic meter was significantly correlated with the number of cigarettes smoked. Another in vitro assay measures the number of sister-chromatid exchanges (SCEs) in human lymphocytes. Valadand-Barrieu and Izard (1979) used a solution of the gas phase from cigarette MS. They showed that this solution induced a significant dose-related increase in SCEs. SUMMARY AND RECOMMENDATIONS Sufficient data are not available to assess the relative genotoxicity and toxicity of whole ETS. A few isolated reports have dealt with the genotoxicity of SS and ETS, and the relative toxicity of MS and SS. In order to evaluate ETS, it is suggested that in vitro genotoxicity assays in at least two systems should be done with ETS per se as well as with its particulate matter. These assays under controlled and, subsequently, under field conditions should not be limited to freshly generated ETS, but should also attempt to determine effects of various degrees of air dilution and aging. In a comprehensive analytical approach, data should be generated to determine systematically the concentrations of toxic and tumorigenic agents in various milieus with ETS. At the same time, it may be useful to examine the uptake of tobacco-specific agents as well as the mutagenicity of the urine of nonsmokers exposed to ETS. All of these measures should be considered in the context of detailed exposure histories.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects What Is Known The lungs of various species have different physiological properties, making each of them the experimental species of choice only for certain situations, depending on the objective of the research study. ETS and SS have been shown to be mutagenic in a system where the smoke was tested directly on bacterial plates. The extensive studies of MS can serve as a guideline for the evaluation of ETS. Many of the constituents in the smokes are similar. Despite the limitations of extrapolating from various bioassays to man, the use of CSC from MS has provided insight as to the contribution of various components to the carcinogenic potential of MS from cigarettes. In the only study reported to date using SS condensate, SS condensate was shown to be more carcinogenic than MS condensate. What Scientific Information Is Missing Only a few laboratory methods have been applied toward the assessment of the relative toxicological and genotoxic properties of SS generated from cigarettes and, more importantly, of ETS. Research is needed to clarify the appropriate methods for estimating genotoxicity and to isolate and identify the active agents in body fluids of ETS-exposed nonsmokers. Comparative inhalation studies with MS, SS, and ETS are still needed. Such assays, while not duplicating human exposure patterns, would provide more definitive information about the relative carcinogenic potential of SS in comparison to the MS of the same cigarettes. The aging of the atmosphere in which ETS occurs can have a profound effect on its chemical composition, physical characteristics, and overall biological effects. Therefore, studies of aged ETS are needed. Where exposure histories can be specified clearly, validation and quantitative determination of genotoxic markers for substances in ETS that also occur in the environment would be of value for measuring dose of ETS. In examining the effects of MS, many research workers have used condensates of the smoke painted on the shaved skin of mice.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects Similar work with skin painting has not been done with ETS and would be of value for assessing the differential toxicity of ETS and MS. In vitro assays are needed for estimation of the tumor promotion and cocarcinogenic effect of ETS. In vitro tests are quicker than in vivo tests, and enough material can not be collected to do in vivo tests. REFERENCES Alarie, Y. Sensory irritation by airborne chemicals. CRC Crit. Rev. Toxicol. 2:299–363, 1973. Basrur, P.K., S.McClure, and B.Zilkey. A comparison of short term bioassay results with carcinogenicity of experimental cigarettes, pp. 2041–2048. In H.E.Nieburgs, Ed. Prevention and Detection of Cancer, Vol. 1. New York: Marcel Dekker, 1977. Bos, R.P., J.L.G.Theuws, and P.Th.Henderson. Excretion of mutagens in human urine after passive smoking. Cancer Lett. 19:85–90, 1983. Brain, J.D., D.E.Kundson, S.P.Sorokin, and M.A.Davis. Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11:13–33, 1976. DeMarini, D.M. Mutagenicity of fractions of cigarette smoke condensate in Neurospora crassa and Salmonella typhimurium. Mutat. Res. 88:363–374, 1981. Harris, R.J., G.Negroni, S.Ludgate, C.R.Pick, F.C.Chesterman, and B.J.Maidment. The incidence of lung tumours in c5761 mice exposed to cigarette smoke: Air mixtures for prolonged periods. Int. J. Cancer 14:130–136, 1974. International Agency for Research on Cancer (IARC) Monographs: Evaluation of Carcinogenic Risk of Chemicals to Humans, Vol. 38, pp. 163–314. Tobacco Smoking. Lyons: IARC, 1986. 421 pp. Leuchtenberger, C., and R.Leuchtenberger. Effects of chronic inhalation of whole fresh cigarette smoke and of its gas phase on pulmonary tumorigenesis in Snell’s mice, pp. 329–346. In P.Nettesheim, M.G. Hanna, Jr., and J.W.Deatherage, Jr., Eds. Morphology of Experimental Respiratory Carcinogenesis. Proceedings of a Biology Division, Oak Ridge National Laboratory, Conference, Gatlinburg, Tenn., May 13–16, 1970. Washington, D.C.: U.S. Atomic Energy Commission, 1970. Lewtas, J., S.Goto, K.Williams, J.C.Chuang, B.A.Petersen, and N.K. Wilson. The mutagenecity of indoor air particles in a residential pilot field study. Atmos. Environ., in press. Lofröth, G., L.Nilsson, and I.Alfheim. Passive smoking and urban air pollution: Salmonella/microsome mutagenicity assay of simultaneously collected indoor and outdoor particulate matter, pp. 515–525. In M.D. Waters, S.S.Sandhu, J.Lewtas, L.Claxton, N.Chernoff, and S.Nesnow, Eds. Short-Term Bioassays in the Analysis of Complex Environmental Mixtures. III. New York: Plenum, 1983.

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Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects Mohr, U., and G.Resnick. Tobacco carcinogenesis, pp. 263–367. In C.C. Harris, Ed. Pathogenesis and Therapy of Lung Cancer. New York: Marcel Dekker, 1978. Obe, G., W.-D.Heller, and H.-J.Vogt. Mutagenic activity of cigarette smoke, pp. 223–246. In G.Obe, Ed. Mutations in Man. Berlin: Springer-Verlag, 1984. Ong, T., J.Stewart, and W.Z.Whong. A simple in situ mutagenicity test system for detection of mutagenic air pollutants. Mutat. Res. 139:177–181, 1984. Otto, H., and H.Elmenhorst. Experimentelle Untersuchungen zur Tumorinduktion mit der Gasphase des Zigarettenrauchs. Z. Krebsforsch. 70:45–47, 1967. Phalen, R.F. Inhalation Studies: Foundations and Techniques. Boca Raton, Florida: CRC Press, 1984. Stuart, B.O. Selection of animal models for evaluation of inhalation hazards in man, pp. 268–288. In E.F.Aharonsen, S.Ben-David, and M.A. Klingberg, Eds. Air Pollution and the Lung. New York: John Wiley & Sons, 1976. Valadaud-Barrieu, D., and C.Izard. Action de la phase gazeuse de fumée de cigarette sur le taux d’echanges des chromatides-soeurs du lymphocyte humain in vitro. C.R. Acad. Sci. (Paris) 288:899–901, 1979. Wood, R.W. Stimulus properties of inhaled substances. Environ. Health Perspect. 26:69–76, 1978. Wynder, E.L., and D.Hoffmann. Tobacco and Tobacco Smoke: Studies in Experimental Carcinogenesis. New York: Academic Press, 1967. 730 pp.