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10 Tobacco Smoke and Toxicology PHYSICAL AND CHEMICAL CHARACTERISTICS OF TOBACCO SMOKE obacco smoke is a complex mixture of toxicants and the chemical T properties changeârapidly in some casesâas smoke ages. Toxi- cants measured at one point in time may not be what the smoker actually experiences. It is estimated that there are more than 2,000 chemi- cal constituents of tobacco. Almost twice that number results when to- bacco is burned incompletely during smoking. Three kinds of smoke can be described, each differing in terms of toxicant concentration, size of particles, effects of temperature, and a host of other characteristics. Main- stream smoke (MS) is what emerges from the âmouthâ or butt end of a puffed cigarette. Sidestream smoke (SS) is what arises from the lit end of a cigarette, mostly between puffs. Environmental tobacco smoke (ETS), smoke present in air, consists of exhaled mainstream smoke and side- stream smoke. Smoking machines are used to analyze mainstream smoke. A set of parameters has been agreed on by various international organizations. These parameters are 35-cm3 puff volume, 2-second puff duration, once per minute puff interval, and smoking to a butt length of 23 mm for nonfiltered or 3 mm above the filter overlap for filter-tipped cigarettes. The âyieldsâ of toxicants in the MS are frequently reported by the stan- dard-setting organization. The two most well known organizations are the U.S. Federal Trade Commission (FTC) and the International Organi- zation of Standardization (ISO). The controversy regarding the standard 283
284 CLEARING THE SMOKE parameters and reporting of values is covered in other chapters of this report. Mainstream smoke is pulled through the mouth end of the cigarette and then through a âCambridge filter pad.â Aerosol particles in the smoke larger than 1 Âµm in diameter are trapped with 99% efficiency. The material is referred to as cigarette smoke condensate or total particulate matter (TPM). âTarâ is the weight of TPM minus nicotine and water. The material that passes through the filter pad is the gas or vapor phase of cigarette smoke. In general, the vapor phase consists predominantly of compounds with a molecular weight <60 and the particulate phase consists of com- pounds with a molecular weight >200. The yields of MS increase with successive puffs as the cigarette is machine-smoked due to the decrease in filtration provided by the ciga- rette rod itself. However, smoking behavior studies coupled with yield measurements suggest that yields remain consistent from first puff to last when assessed under real-life smoking conditions. When tobacco is heated, moisture and volatile material are distilled, and combustion leads to the generation of volatile gases and the residual, carbonized char. Char reacts with oxygen in the air during puffing and smoldering, producing volatile gases (carbon dioxide, carbon monoxide, and water) and the inorganic material known as ash. The highest temperature reached during the burning of tobacco is approximately 800ËC in the center of the burning zone during smolder. During puff, a solid-phase temperature of approximately 910ËC is reached at the burning zone periphery, while the gas temperatures are lower. They vary between 600 and 700ËC as the puff progresses. After the puff ends, solid-phase temperatures rapidly cool to approximately 600ËC. This greatly influences particle formation, particle size, and toxicant forma- tion. These temperatures contrast with that achieved with a newly mar- keted cigarette-like device, Eclipse, that combusts differently than con- ventional cigarettes and aerosolizes nicotine and glycerin. The chemical nature of MS changes as smoke ages. The burning zone generates a highly concentrated vapor that is drawn down the cigarette to form mainstream smoke. The vapor cools quickly (in milliseconds) due to diluting air. Less volatile compounds quickly condense, mostly in air- borne state. A combination of physical size and concentration affects both thermal and mechanical properties, which influence the number of par- ticles in smoke. Droplets of less than about 0.1 Âµm will attach to the tobacco through which they pass or to other particles, which continue on into MS. Particles with sizes around 1 Âµm are âfilteredâ out by depositing onto the tobacco surface. MS is a highly concentrated aerosol mixture. Smoke particles are liq- uid, consisting of approximately 20% water by volume. The particles vary
TOBACCO SMOKE AND TOXICOLOGY 285 from less than 0.1 to 1.0-Âµm diameter. The small size and high concentra- tion promote rapid coagulation, leading to decreased concentration and increased size of the resulting particles within less than a second. The size of particles also increases due to absorption of water, which is relevant for human smoking because of the high relative humidity of the human res- piratory tract. Sidestream smoke particles are smaller than MS particles initially. However, the aging of SS over a few minutes leads to an increase in particle size of ETS due to coagulation of particles and removal of smaller particles that attach to surfaces in the environment. Particle size in smoke is important, because it influences where within the respiratory tract a toxicant is deposited. Smaller particles, in general, deposit further down into the lungs. Inhaled particles of the size found in tobacco smoke would be pre- dicted to deposit mainly in the alveolar region of the lung. However, cigarette smoke-induced tumors are more prevalent in the bronchial re- gion, suggesting that smoke particles deposit higher up in the respiratory tract than would be predicted from the initial particle size. (Recent in- creases of adenocarcinomas in lower airways of smokers are hypothesized to be due to so-called smoking compensation of low-yield products. Smok- ers of these products inhale more deeply to increase their nicotine dose.) Mucociliary clearance of inhaled particles up the respiratory tract may also increase the dose of particles to the upper airways. More impor- tantly, the cloud-like nature of MS (see below) and the increased size of smoke particles on aging are responsible for this finding. Specific factors influencing the site of deposition in airways include coagulation of fresh smoke particles, absorption of water in the humid respiratory tract, hu- man breathing patterns, aerodynamic interactions between nearby par- ticles, electrostatic charge, and vapor deposition on airway walls (Dendo et al., 1998). Theoretical models of particle deposition predict that MS particles would have approximately 20% deposition in the respiratory tract. Measurements in humans suggest that deposition is actually much higher, from 50 to 95% (Phalen, 2000). The explanation for this high deposition rate is that cigarette smoke is so dense, that it acts as a cloud. Clouds are high concentrations of aerosol particles surrounded by relatively clean air. They behave as entities that are much larger than the individual components. In depositing, the cloud behaves as if it were a much larger particle, with an aerodynamic diam- eter of approximately 6 or 7 Âµm. This particle size (see Figure 10-1) results in high total deposition in the respiratory tract, with especially high depo- sition in the tracheobronchial region. Deposition in the respiratory tract is also influenced by the size of the person. Smaller individuals have greater tracheobronchial and less
286 CLEARING THE SMOKE 1 0.9 Total 0.8 Deposition Fraction 0.7 NOPL 0.6 TB 0.5 0.4 0.3 0.2 P 0.1 0 0.001 0.01 0.1 1 10 100 Particle Diameter (mm) FIGURE 10-1 Aerosol deposition curves. NOTE: applies to the Reference Man; NOPL=nasal oral pharyngeal laryngeal regions; TB=tracheobronchial region. SOURCE: Adapted from National Council on Radiation Protection and Measure- ments report #125, 1997, with permission from the National Council on Radiation Protection and Measurements. pulmonary deposition. Smaller individuals also have greater minute ven- tilation normalized for body weight, which is especially important for SS. This is hypothesized to be one reason infants might be more vulnerable to ETS. Importantly the small airway diameter of infants leads to high dis- position in the upper airways, where it can be an irritant (Phalen, 2000). Whole MS consists (by weight) of mostly air (nitrogen N2, oxygen O2, argon). The vapor phase constitutes approximately 20% by weight of the smoke, with the particulate phase accounting for approximately 5%. The majority of cigarettes sold today have filters to remove portions of the smoke. Cellulose acetate filters are used almost exclusively in the United States, whereas charcoal filters are popular in Japan (Norman, 1999). Cellulose acetate filters remove some of the particulate phase of the smoke but have little influence on the vapor phase. The efficiency of particle removal depends on particle size and is minimal at the number- average particle size found in cigarette smoke (about 0.3 Âµm diameter). In
TOBACCO SMOKE AND TOXICOLOGY 287 general, cellulose acetate or paper filters remove tar and nicotine particles in this size range with an efficiency of 40â50%. Charcoal filters influence the retention of vapor-phase components and are made by adding up to 60 mg of activated charcoal in a segment of the cellulose acetate filter. Factors that influence the retention of vapor-phase compounds include the amount of charcoal used, the activity of the charcoal (based on surface area and pore volume), and the smoke velocity through the segment. Low-molecular-weight compounds with low boiling points (less than 150Â°C), which are not retained by the cellulose acetate filters, are partially removed by the charcoal filters. Removal efficiencies vary with the com- pound but are reported to be from 30 to 90%. As an example, benzene may be removed at an efficiency of 67%. Vented filters are designed to improve filter efficiency by decreased smoke flow through and increased residence time in the filter. However, vent holes can readily be covered by the fingers of the smoker, who may be inclined to do this in order to get the maximum amount of nicotine during smoking. Under similar smoking conditions, filtered cigarettes will have lower MS yield relative to nonfiltered analogues. SS yields will not vary much, since they are reflective of tobacco weight burned during smolder. In general, more tobacco is consumed during smolder than during puffing. However, SS generally contains more alkaline and neutral compounds. SS smoke contains less or equal amounts of acids, phenols, and phytos- terols than MS. Differences are due to temperature and mechanisms of chemical transfer (release) from the unburned tobacco. The approximate chemical composition of MS is given in Table 10-1. The relative concentra- tion of specific constituents in MS versus SS tobacco smoke is shown in Table 10-2. The pH of cigarette smoke influences the degree of protonation of the active addictive chemical, nicotine. The free-base form of nicotine is fa- vored at a higher pH (more basic) and is more rapidly absorbed into the bloodstream than the mono- or diprotonated salt forms of nicotine that exist at lower pH (more acidic). Tobacco blends with a high sugar content produce a more acidic smoke; a basic cigarette smoke can be achieved by addition of ammonia. TOXICITY OF TOBACCO SMOKE The health effects of tobacco smoke in humans are well known from both clinical and epidemiological studies; such information is summa- rized in later chapters of this report. Animal studies of the toxicity of tobacco smoke are reviewed in the present section. The purpose of this review of animal models of tobacco smoke toxicity is to determine the potential usefulness of such models for assessing the toxicity of new and
288 CLEARING THE SMOKE TABLE 10-1 Approximate Chemical Composition of Whole Mainstream Smoke Constituent % by Weight Air } N2 62 O2 13 75.9 Ar 0.9 Vapor Phase } Water 1.3 CO2 12.5 CO 4 H2 0.1 CH4 0.3 Hydrocarbons 0.6 Aldehydes 0.3 Ketones 0.2 19.6 Nitriles 0.1 Heterocyclics 0.03 Methanol 0.03 Organic acids 0.02 Esters 0.01 Other compounds 0.1 Particulate Phase } Water 0.8 Alkanes 0.2 Terpenoids 0.2 Phenols 0.2 Esters 0.2 Nicotine 0.3 4.5 Other alkaloids 0.1 Alcohols 0.3 Carbonyls 0.5 Organic acids 0.6 Leaf pigments 0.2 Other compounds 0.9 NOTE: Ar=Argon; CH4=methane; CO=carbon monoxide; CO2=carbon dioxide; H2=hydrogen; N2=nitrogen; O2=oxygen. SOURCE: Dube and Green, 1982. Reprinted with permission from the authors.
TOBACCO SMOKE AND TOXICOLOGY 289 TABLE 10-2 Some Typical SS/MS Yield Ratios for Plain Cigarettes Substance MS Yield SS/MS Small Molecules Carbonyl sulfide 18-42 Âµg 0.03-0.1 HCN 160-500 Âµg 0.06-0.5 CO 10-23 mg 2.5-4.7 Hydrazine 20-43 Âµg 3 Methane 600-1000 Âµg 3.1-4.8 Acetylene 20-40 Âµg 0.8-2.5 Nitrogen oxides 100-600 Âµg 4-13 CO2 20-50 mg 8-11 H2O (gas phase) 3-14 mg 24-30 NH3 50-130 Âµg 40-170 N2 (generated) <10 Âµg >270 Neutral Heteroatom Organics Acetonitrile 50-130 Âµg 40-170 Benzonitrile 5-6 Âµg 7-10 Acetamide 70-100 Âµg 0.8-1.7 Methyl chloride 150-600 Âµg 1.7-3.3 Aldehydes, Ketones, Alcohols Acetaldehyde 0.5-1.2 mg 1.4 Propionaldehyde 175-250 Âµg 2.4-2.8 Acetone 100-250 Âµg 2-5 Acrolein 60-100 Âµg 8-15 2-Butanone ~30 Âµg 2.9-4.3 2-Furaldehyde 15-43 Âµg 4.9-7.4 Furfuryl alcohol 18-65 Âµg 3.0-4.8 Cyclotenea 3-5 Âµg 6-10 Pyranoneb 13-150 Âµg 0.1-1.2 Phytosterols Î²-Sitosterol 59 Âµg 0.5 Campesterol 43 Âµg 0.6 Cholesterol 22 Âµg 0.9 Phenols Phenol 60-140 Âµg 1.6-3.0 Cresols (o-,m-,p-) 11-37 Âµg 1.0-1.4 Catechol 100-360 Âµg 0.6-0.9 Hydroquinone 110-300 Âµg 0.7-1.0 Acids Formic acid 210-490 Âµg 1.4-1.6 Acetic acid 270-810 Âµg 1.9-3.9 3-Methylvaleric acid 20-60 Âµg 0.8-1.5 continues
290 CLEARING THE SMOKE TABLE 10-2 Continued Substance MS Yield SS/MS Lactic acid 60-170 Âµg 0.5-0.7 Benzoic acid 14-28 Âµg 0.7-1.0 Phenylacetic acid 11-38 Âµg 0.6-0.8 Succinic acid 70-140 Âµg 0.4-0.6 Glycolic acid 40-130 Âµg 0.6-1.0 Amines, Pyridines, Alkaloids Methylamine 12-29 Âµg 4.2-6.4 n-Propylamine 1.6-3.4 Âµg 2.8-3.8 n-Butylamine 0.5-1.5 Âµg 2.2-4.0 Aniline 360 ng 30 Pyridine 16-46 Âµg 6.5-20 3-Ethenylpyridine 11-30 Âµg 20-40 Methylpyrazine 2-5 Âµg 3-4 Pyrrole 16-23 Âµg 9-14 Nicotine 0.8-2.3 mg 2.6-3.3 Myomine 13-33 Âµg 4.0-7.5 Nicotyrine 4-40 Âµg 5-14 Anatabine 2-20 Âµg 0.1-0.5 2,3â-Bipyridyl 16-22 Âµg 2-3 Aza-arenes Quinoline 0.5-2.0 Âµg 8-11 Isoquinoline 1.6-2.0 Âµg 2.5-5 Benzo[h]quinoline 10 ng 10 Indole 16-38 Âµg 2.1-3.4 Hydrocarbons Isoprene 330-1100 Âµg 13-19 Benzene 36-68 Âµg 5-10 Toluene 100-200 Âµg 6-8 Limonene 15-50 Âµg 4-12 Neophytadiene 66-230 Âµg 1-2 Polynuclear Aromatic Hydrocarbons Naphthalene 2.6 Âµg 17 Pyrene 45-140 Âµg 2-11 Benzo[a]pyrene 9-40 Âµg 2-20 Anthracene 24 ng 30 Phenanthrene 77 ng 2-30 Fluoranthene 60-150 ng 11 Nitrosaminesc N-Nitrosodimethylamine 10-40 ng 10-50 N-Nitrosodiethylamine nd-25 ng 3-35 N-Nitrosopyrrolidine 6-30 ng 6-30 continues
TOBACCO SMOKE AND TOXICOLOGY 291 TABLE 10-2 Continued Substance MS Yield SS/MS N-Nitrosodiethanolamine 0-70 ng 1.2 Nâ-Nitrosonomicotine 0.2-3 Âµg 0.5-3 NNKd 0.1-1 Âµg 1-4 Nâ-Nitrosoanatabine 0.3-5 Âµg 0.3-1 Inorganic Constituents Cadmium 100 ng 4-7 Nickel 20-80 ng 0.2-30 Zinc 60 ng 0.2-7 NOTE: CO=carbon monoxide; CO2=carbon dioxide; HCN=hydrogen cyanide; H2O=water; N2=nitrogen; nd=not detected; NH3=ammonia; NNK=nitrosonornicotine ketone. aHydroxy-3-methyl-2-cyclopentanone. b5m6-Dihydro-3,5-dihydroxy-2-methyl-4H-pyran-4-one. cMuch of the data in the literature on the smoke levels of volatile and tobacco-specific nitrosamines may be in error, due to artifact formation on the Cambridge pad part of the smoke collection procedure (Caldwell and Conner, 1990). dNitrosonornicotine ketone or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. SOURCE: Reprinted, with permission from Davis, DL and Nielsen, MT eds. Tobacco: Pro- duction, Chemistry and Technology. Pp. 418. Copyright 1999 by Blackwell Science. existing products that claim to reduce harm from tobacco use. The possi- bilities and the limits of using animals to test for toxic effects related to tobacco use are discussed. The major adverse tobacco smoke-induced health effects that require evaluation because of their prominence in hu- mans are pulmonary inflammation, induction of lung cancer, chronic ob- structive pulmonary disease (COPD), cardiovascular disease, reproduc- tive and developmental effects, and the suppression of the immune system. In Vitro Toxicity Tests Toxicity tests that can be performed on cell systems in vitro have the advantage of being done rapidly and with relatively low cost. Such tests can be used to screen for general toxic properties of a chemical or a chemi- cal mixture, such as the cytotoxicity of the material or its ability to alter the genetic material, DNA. The cytotoxicity of a compound can be predic- tive of its ability to induce inflammation; the genotoxicity of a compound suggests its potential to induce cancer. The limitation of such tests is that the results are based on the response of single cell types and do not
292 CLEARING THE SMOKE include the influence of the whole-body system on the response. Never- theless, such tests can be valuable in excluding products from further development if they either are extremely cytotoxic or have a high poten- tial for producing mutations in DNA. Cellular screening assays should include benchmark materials of known cytotoxic or genotoxic potential (based on both in vitro and in vivo studies) for comparison to the test material and to allow better interpretation of the results. Cytotoxicity Tests Cytotoxicity tests (Balls and Clothier, 1991) are based on either pri- mary cultures or established cell lines from the target organ of interest. Dye exclusion or the release of cytoplasmic enzymes is used to measure damage to cell membranes. In dye exclusion tests, the ability of cells to exclude extracellular dyes such as trypan blue or neutral red is measured. Release of the cytoplasmic enzyme lactate dehydrogenase (LDH) is com- monly measured as an indicator of cell membrane damage. For specific purposes, the release of other enzymes, such as the hydrolytic enzymes of pulmonary macrophages, can be useful. The exclusion of the dye, neutral red, and the release of LDH have been recently used to compare the cytotoxicity of smoke condensates from standard cigarettes and a new tobacco-related PREP for which harm reduction was claimed (Eclipse Ex- pert Panel, 2000) Genotoxicity Tests Several in vitro tests designed to assay for the mutagenic potential of a chemical or mixture were recently reviewed in an International Work- shop on Genotoxicity Test Procedures (Lovell et al., 2000). Perhaps the most commonly used screening tool is the Salmonella typhimurium bacte- rial mutagenicity assay or Ames test (Ames et al., 1987). In addition to assays for point mutations, there are assays of clastogenic DNA damage as indicated by chromosomal aberrations, sister chromatid exchanges (SCEs), micronuclei formation (Hayashi et al., 2000), or single strand breaks via the Comet assay (Tice et al., 2000). Assays for specific chemical adducts to DNA can be used as a measure of dosimetry and, in a few cases, as predictors of adverse effects (Phillips et al., 2000). Measures of oxidized bases in DNA can be used to assay for oxidative stress (Cadet et al., 1998). In recent comparative potency studies comparing the genotoxicity of smoke condensates from standard and new tobacco- related PREP, a battery of in vitro assays included the Ames test, SCEs, and chromosomal aberrations (Eclipse Expert Panel, 2000).
TOBACCO SMOKE AND TOXICOLOGY 293 In Vivo Toxicity Tests Animals do not smoke cigarettes in the same manner as humans, and much effort has been expended in the past in animal studies to mimic human exposures to intermittent puffs of smoke. It is impractical to repli- cate all of the parameters of human smoking in animals. One problem is that rodents tend to hold their breath during puffs of irritating tobacco smoke and thus avoid receiving a high dose of smoke (Kendrick et al., 1976). Studies were conducted comparing three modes of exposure of rats to cigarette smoke: nose-only intermittent, nose-only continuous, and whole-body continuous (Chen et al., 1995; Mauderly et al., 1989). Plasma nicotine was higher by a factor of 3 in whole-body exposed rats compared to nose-only exposed rats. This suggests that dermal absorption and grooming as well as inhalation contributed to the dose of nicotine re- ceived in rats exposed in the whole-body mode. Urinary cotinine was not higher in the whole-body exposed group compared to the nose-only in- termittent exposure group but was higher by a factor of 1.5 compared to the nose-only continuous exposure group. This study demonstrated few significant differences in either smoke characteristics or biological effects among the three exposure modes. The biological effects thought to be related to chemical carcinogenesis (cell transformation, chromosomal damage, DNA adducts) and chronic lung disease (cell proliferation, in- flammation, respiratory function) were similar for all groups. Whole-body exposures were less labor intensive and less stressful to the rats (based on body weights) and avoided the reduction in breathing known to occur during puff-by-puff exposures. Thus, whole-body exposures may be use- ful as a method to achieve dosing of tobacco smoke in small laboratory test animals. ASSESSMENT OF POTENTIAL EXPOSURE REDUCTION PRODUCTS Lung Cancer Animals have not proven to be good models for the type of lung tumors induced by cigarette smoke in humans. Rodents tend to develop peripherally arising lung adenomas rather than centrally arising bron- chial tumors when exposed to chemicals. Exposure of animals to tobacco smoke has not often produced an excess of lung tumors of any type. In 1986, the International Agency for Research on Cancer (IARC, 1986) criti- cally reviewed animal studies on tobacco smoke; out of four rat studies judged to be adequate for analysis, only one yielded unequivocal evi- dence for tobacco smoke as a respiratory tract carcinogen. One problem
294 CLEARING THE SMOKE may be that rats build up carboxyhemoglobin faster than humans when exposed to the same level of carbon monoxide (CO) (Guerin et al., 1974; Silbaugh and Horvath, 1982). This results in their not being able to toler- ate the level of exposure to cigarette smoke that humans can. Other fac- tors undoubtedly contribute to this species difference in response to ciga- rette smoke. However, rodent models can be used to test the ability of tobacco products to cause alterations in DNA, and recent studies (dis- cussed below) indicate that the A/J strain of mouse shows promise as a model for in vivo carcinogenesis induced by tobacco smoke. Short-term exposure of rodents followed by analysis of DNA isolated from the lungs for DNA modifications, such as oxidative damage or methylation, can determine the ability of the product to damage DNA in vivo. Aberrant methylation of DNA can be used as a marker for early stages of oncogenesis in both rats and humans (Belinsky et al., 1998; Nuovo et al., 1999; Swafford et al., 1997). Oxidative damage to DNA is used to monitor oxidative stress (Halliwell, 1998; Loft et al., 1998). The A/J mouse strain, which is sensitive to induction of lung adenomas, has been used in a series of studies by Witschi to test for the carcinogenic potential of tobacco smoke and the effectiveness of chemopreventive mea- sures (Witschi et al., 1997a, 1999, 2000). The A/J mice exposed to 87 mg/m3 of environmental tobacco smoke for five months and allowed to recover for four months had a statistically significant elevation in number of lung tumors (Witschi et al., 1997a). The same strain of mice exposed similarly to filtered and unfiltered tobacco smoke suggested that the particulate phase was not required for carcinogenicity (Witschi et al., 1997b). In a chemoprevention study, acetylsalicylic acid, an agent known to protect against nicotine-derived N-nitrosaminoketone (NNK)-induced tumors in the same strain of mice, had no protective effect against tobacco smoke (Witschi et al., 1999). A second chemoprevention study indicated that a diet containing myoinisitol-dexamethasone was effective in preventing tobacco smoke-induced lung tumors but that agents known to protect against NNK-or polycyclic aromatic hydrocarbon (PAH)-induced tumors did not protect against tobacco smoke (Witschi et al., 2000). The committee concludes that these studies indicate that removal from tobacco smoke of single classes of carcinogens, such as nitrosamines or PAHs, may not be protective against the induction of lung tumors by smoke. These studies also suggest that the A/J mouse, used in âstop- startâ studies, shows promise as an animal model of value in screening for the potential of tobacco products to induce lung tumors. Future stud- ies should determine if the model is robust enough to be repeated in other laboratories. In recent comparative potency studies for a newly devel- oped tobacco-related PREP, the potency for smoke condensates to induce
TOBACCO SMOKE AND TOXICOLOGY 295 cancer was evaluated in 30-week dermal tumor-promotion studies in mice (Eclipse Expert Panel, 2000). Such skin painting studies provide informa- tion for hazard identification. Chronic Obstructive Pulmonary Disease An early response to any inhaled toxicant is pulmonary inflamma- tion, which if persistent may lead to more severe alterations in the struc- ture and function of the lung. Animal models can readily be used to detect and quantitate the pulmonary inflammatory response to inhaled compounds or mixtures. Analysis of bronchoalveolar lavage (BAL) fluid for cellular and biochemical indicators of inflammation has become a common tool for quantitation of the pulmonary inflammatory response of rodents to inhaled toxicants (Henderson, 1989), including tobacco smoke (Mauderly et al., 1989; Sjostrand and Rylander, 1997; Subramaniam et al., 1996). The differential cell count and the functioning of cells obtained by the BAL technique can be used to classify the type of inflammatory re- sponse. The biochemical content of BAL fluid can be used to detect the release of various cytokines and alterations in the pulmonary surfactant. Chronic obstructive pulmonary disease in the form of either emphy- sema, bronchitis, or both is a well-recognized sequela of cigarette smok- ing (Vial, 1986). Harm reduction strategies must take into account the degree to which this type of chronic lung disease is reduced in new prod- ucts. Animals models to study the degree of COPD induced by the use of new products have been suggested by the work of March et al. (1999a). F344 rats exposed to cigarette smoke over a two-week period showed enhanced pulmonary epithelial cell replication and alterations in axial airway mucosubstancesâchanges consistent with the development of chronic bronchitis (March et al., 1999a). Both B6C3F1 mice and F344 rats exposed to cigarette smoke over a longer period (7â13 months) were found to develop morphological evidence of emphysema. Mice developed more pronounced signs of emphysema than rats, and the condition progressed with time in mice (March et al., 1999b). In earlier studies, rats exposed for three months to sidestream smoke were reported to have emphysematous changes in the lung (Escolar et al., 1995). Comparative potency studies for a newly developed tobacco-related PREP made use of 90-day inhalation studies in hamsters and rats to test for inflammation as well as epithelial hyperplasia and metaplasia (Eclipse Expert Panel, 2000). The committee concludes that these studies indicate there are animal models that show promise for use in screening for development of COPD- like symptoms from the inhalation of new or existing tobacco products.
296 CLEARING THE SMOKE Cardiovascular Disease Cockerels and rabbits are two animal models that have been used to test for the cardiovascular effects of tobacco smoke. Penn and coworkers (1993, 1994) found that 16-week exposure of cockerels to tobacco smoke (2-3 mg/m3) increased the size of arteriosclerotic plaques in the aorta. Rabbits fed a cholesterol-rich diet and exposed for 10 weeks to 4 and 33 mg/m3 showed a dose-dependent increase in the size of arteriosclerotic lesions in the aorta and pulmonary artery, as well as increased stickiness of platelets (Zhu et al., 1994). C57BL/6 mice have also been used to test for the effect of inhaled pollutants on the induction of atherosclerosis and enhancement of arterial fatty deposits in animals fed a high-fat diet (Lewis et al., 1999). The committee concludes that such studies suggest animal models can be used to detect the potential for tobacco products to enhance the development of atherosclerosis. Immune System Dysfunction Smoking-related changes in the peripheral immune system in hu- mans include elevated white blood cell counts, increased cytotoxic or suppressor and decreased inducer or helper T-cell numbers, slightly sup- pressed T-lymphocyte activity, significantly decreased natural killer (NK) cell activity, lowered circulating immunoglobulin titers (except for IgE, which is elevated), and increased susceptibility to infection. Similar ef- fects have been observed in animals (Johnson et al., 1990; McAllister- Sistilli et al., 1998; Sopori et al., 1994), suggesting that animal models can be used to test for harm reduction to the immune system from use of new tobacco products or nicotine delivery devices. The major areas of interest are reduced host resistance to infections and tumors, suppression of the cellular and humoral immune system, and interference with macrophage cell function. The effect of tobacco smoke on the immune system of humans and rodents depends on the duration and level of exposure. In general, short- term, low-level exposures do not affect the immune system or may be stimulatory, whereas longer-term exposures (six months or more) or high levels of exposure are immunosuppressive. The committee concludes that this finding indicates that long-term animal studies are required to evaluate adverse effects of tobacco prod- ucts on the immune system. Animals exposed to cigarette smoke for extended periods are more susceptible than naÃ¯ve animals to tumor and infectious agent challenge. Mice exposed to cigarette smoke for six months or longer were more
TOBACCO SMOKE AND TOXICOLOGY 297 susceptible to intratracheally instilled Lewis lung or TKL5 tumor cells in terms of increased tumor growth, metastases, and early death than unex- posed mice (Chalmer et al., 1975; Thomas et al., 1974b). Such changes are not observed in mice exposed to cigarette smoke for short periods of time (days). Chronic exposure of mice to cigarette smoke results in increased susceptibility to infectious agents such as murine sarcoma virus (Thomas et al., 1974a) and influenza virus (Mackenzie et al., 1976; Mackenzie and Flower, 1979). Cellular immunity, as evaluated by phytohemagglutinin (PHA)- induced lymphoproliferative response or development of tumor-specific cytotoxic T cells, was initially increased but, on continued exposure, greatly decreased in mice exposed to cigarette smoke (Chalmer et al., 1975; Holt et al., 1975; Thomas et al., 1973). Lymphocytes from mice ex- posed chronically to tobacco smoke have a decreased response to the mitogen PHA and release factors that inhibit the cytotoxic activity of NK cells against tumor cells. T-cell suppression may be due to defective anti- gen processing or antibody production. The humoral immune response is also suppressed by chronic expo- sure of mice to tobacco smoke, while acute exposures may stimulate the humoral response. The primary and secondary antibody production by lymphocytes in the lung, lymph nodes, and spleen of mice exposed to tobacco smoke for longer than 26 weeks and challenged by inoculation with sheep erythrocytes was decreased (Thomas et al., 1975). Laboratory test animals can be used to demonstrate the ability of cigarette smoke to slow the mucociliary clearance of particles from the lung and to alter the function of pulmonary macrophages. This effect has been observed in humans (Bohning et al., 1982; Cohen et al., 1979) and animals (Mauderly et al., 1989). In the latter study, rats exposed for eight weeks to cigarette smoke were exposed one time to cerium144 dioxide particles. Smoking increased the half-time of the short-term clearance of these particles by 63% and long-term clearance twofold. The slowing of clearance of inhaled particles is an adverse health effect that should be considered in studies of tobacco product toxicity. Alveolar macrophages from rats exposed to tobacco smoke for six months have a decreased ability to phagocytize S. tuphylocoesus aureus (Drath et al., 1979; Huber et al., 1980). Alveolar macrophages from rats exposed to tobacco smoke for 36 days or longer had an increased ability to release reactive oxygen species, a property dependent on the particulate faction of the smoke and not the gases. Clearance of Pseudomonas aeruginosa from rodents exposed to cigarette smoke for 36 weeks was slower than in controls (Holt and Keast, 1977). The decreased ability to clear particles, including pathogens, and the increased release of reactive
298 CLEARING THE SMOKE oxygen species contribute to enhancement of inflammatory processes in the lung. Reproductive and Developmental Effects There are several reports that exposure of pregnant rats to either sidestream or mainstream tobacco smoke results in decreased birthweight of the pups (Leichter, 1989; Rajini et al., 1994; Reznik and Marquard, 1980; Witschi et al., 1994). Thus, animal models are capable of detecting tobacco smoke-induced growth retardation in utero. Rat models have also been used to demonstrate the adverse effects of maternal tobacco smoke expo- sure on lung maturation in utero (Lichtenbeld and Vidic, 1989), leading to an increase interstitial volume in the lung parenchyma. Another study demonstrated that postnatal rats exposed to tobacco smoke had reduced proliferation of their bronchiolar epithelial cells accompanied by increased levels of cytochrome P-450 enzymes (Ji et al., 1994). Studies in rodents have shown that rat pups exposed in utero to tobacco smoke have altered composition of pulmonary surfactant (Subramaniam et al., 1999). The committee concludes that such studies indicate the potential use- fulness of animal models to detect the interference of tobacco smoke prod- ucts on airway epithelial cell development. SYNERGISTIC EFFECTS WITH OTHER POLLUTANTS Occupational exposures to materials such as asbestos or radon daugh- ters have proven to have a synergistic interaction with tobacco smoke leading to greatly increased production of lung tumors in exposed work- ers who also smoke. Although rats are not good models for detecting the induction of lung tumors from cigarette smoke alone, rats exposed to both cigarette smoke and plutonium oxide particles clearly revealed the synergistic effects of cigarette smoke on the induction of lung tumors in combined exposures (Finch et al., 1998). In the past, it has not been cus- tomary for regulatory agencies to require testing for synergistic effects of a new product with other substances. The committee concludes that in the case of tobacco smoke, for which several synergisms are known, it would be wise to consider adding such a test to the standard regimen. MOLECULAR BIOLOGY TESTING TOOLS Recent advances in the area of molecular biology hold promise as future tools for toxicity screening. The technology for producing trans- genic mice allows one to gain gene functions, while the development of
TOBACCO SMOKE AND TOXICOLOGY 299 knockout mice allows one to delete gene functions (Arbeit and Hirose, 1999). At present these tools are better suited for mechanistic studies than for screening purposes, but in the future, genetically altered animals may become the standards for testing for specific types of toxicity, just as genetically altered Salmonella bacteria have become standards for testing the mutagenic potential of xenobiotics. DNA microarray chips, which consist of an array of thousands of specific cDNA sequences or genes on a chip, allow one to detect and quantitate messenger RNAs that are the transcription products of the specific cDNA samples on the surface of the chips. Thus, if one knows the specific genes that are upregulated in association with the onset of a dis- ease process, one could theoretically use the microarray technique to de- tect some of the earliest indicators that a disease process has begun (Nuwaysir et al., 1999). This type of tool should be invaluable in develop- ing rapid screens for early indicators of developing disease in exposed animals (or for clinical purposes in humans) in contrast to the long time frame required to detect indicators of established disease in laboratory animals. The field is developing rapidly, and some microarrays designed to detect squamous cell carcinomas of the lung have already been re- ported (Wang et al., 2000). Future research will be required to determine which genes are upregulated at different times during the progression of specific diseases so that microchip arrays can be designed as accurate and specific preclinical indicators of developing disease. SMOKELESS TOBACCO TOXICITY Smokeless tobacco products, traditionally, are differentiated into snuff and chewing tobacco; are not combusted but exert their effects by direct mucosal contact and consequent entry of toxicants into the bloodstream. Snuff is typically a finely ground tobacco product that is used orally or nasally. Snuff is manufactured in a variety of forms including moist, dry, and fine cut (Connolly et al., 1986). The oral tobacco form that is chewed or simply kept in the mouth is generally known as chewing tobacco. Chewing tobacco is also produced in different forms including plug, loose-leaf, and twist varieties (Connolly et al., 1986). (See Chapter 4 for a more in-depth description of smokeless tobacco products and use statis- tics.) Smokeless tobacco products are composed primarily of fire or air- cured dark tobacco (Wahlberg and Ringberger, 1999). The tobacco then undergoes an extended aging process that involves heating or fermenta- tion depending on the product. During production, various additives are used for the desired flavor and aroma. The chemical composition of smoke- less tobacco products varies due to differences in tobacco composition
300 CLEARING THE SMOKE and cut, additives, and curing or processing conditions. The differences are also found among countries for similar reasons. In Sweden, for ex- ample, moist snuff (snus) has a lower level of tobacco-specific nitro- samines (TSNAs) due to processing differences and lack of fermentation compared to snuff in other countries. Generally, the TSNA levels in both U.S. and Swedish products have decreased over the last decade second- ary to changes in processing methods, and TSNA levels in certain U.S. brands of snuff have approached the Swedish variety (Ahlborn et al., 1997; Wahlberg and Ringberger, 1999). The exact chemical composition of smokeless tobacco, as in tobacco smoke, is difficult to assess. The main target of exposure in the smokeless tobacco user is the oral cavity and the upper aerodigestive tract. The lower digestive tract, however, is exposed at a certain level because of the swallowing of snuff particles within saliva. Common carcinogens found in smokeless tobacco include TSNAs, PAHs (especially benzo[a]pyrene [BaP]), and polonium -210. The concentration of TSNAs in snuff ranges from 5,280 to 141,000 parts per billion (ppb), which is hundreds to thou- sands times higher than that allowed in other consumer and food prod- ucts (Connolly et al., 1986). TSNAs are thought to be important carcino- gens in humans and have been proven to be potent carcinogens in animal studies. Among the TSNAs, NNK and N-nitrosonornicotine (NNN) have proven to be the most important carcinogens in smokeless tobacco in Europe and North America (Hoffmann et al., 1987; Nilsson, 1998). Hecht et al (1986) showed that oral exposure to NNK and NNN in rats caused lung tumors as well as oral tumors at the site of exposure. A snuff user (10 grams of snuff per day) is exposed to 24-46 Âµg of TSNAs per day com- pared to a pack per day smoker who is exposed to, on average, 16.2 Âµg of nitrosamines (Hoffmann et al., 1995). Snuff use also exposes the user to trace amounts of lead, cadmium, and selenium (Hoffmann et al., 1987). Dark tobacco has a high level of nicotine, with 3.5-4.0% reported in certain brands (Wahlberg and Ringberger, 1999). In general the nicotine content per dose of smokeless tobacco product is higher than that of cigarettes, but the maximum serum nicotine levels are similar among all tobacco users (Benowitz, 1997). While there are interindividual differ- ences in nicotine absorption and metabolism, nicotine is absorbed more gradually from smokeless tobacco than from smoking, and blood concen- tration persists over a longer period of time and even overnight (NIH Consensus Report, 1986). Overall, smokeless tobacco users are exposed to a greater amount of nicotine because of continued slow absorption of nicotine up to an hour after the tobacco is taken out of the mouth as well as the more alkaline pH, causing nicotine to be present in its unprotonated form contributing to better absorption (Benowitz et al., 1988; Hoffmann and Djordjevic, 1997).
TOBACCO SMOKE AND TOXICOLOGY 301 Long-term smokeless tobacco use has been linked to oropharyngeal cancer (IARC, 1985; Mattson and Winn, 1989). The evidence has been more convincing for snuff than for chewing tobacco. As outlined in the 1986, National Institute of Health (NIH) Consensus Development Confer- ence on the Health Implications of Smokeless Tobacco, case reports and controlled studies have consistently reported tumor growth in the loca- tion of smokeless tobacco contact with mucosa or skin, resulting in a risk of oral cancer up to 4.2 times that of nontobacco users as reported in one influential study (Winn et al., 1981). The most common type of cancer attributable to smokeless tobacco is oral squamous cell carcinoma, but verrucous carcinoma has also been reported (Connolly et al., 1986). In contrast, recent epidemiological studies from Sweden have failed to con- firm a link between Swedish snus use and cancer (Lewin et al., 1998; Schildt et al., 1998). In a large population-based study looking at risk factors for squamous cancer of the head and neck, Lewin et al. (1998) found no increased risk with the use of Swedish snuff. Results of animal studies were initially mixed regarding the effects of oral, subcutaneous, or topical administration of smokeless tobacco in ro- dents (Main and Lecavalier, 1988; Pershagen, 1996). More recently, how- ever, as noted in a review of the health hazards of moist snuff by Ahlborn et al., (1997), there as been increased experimental support for the carcino- genicity of snuff. Surgically formed canals in the lips of rodents into which snuff and snuff extracts are placed have been used to more closely model the human practice of snuff dipping (Hoffmann and Djordjevic, 1997). Studies in rats and hamsters have shown a higher incidence of malig- nancy when there was exposure to both tobacco and herpes simplex type 1 virus or a cancer initiator (Ahlborn et al., 1997; Connolly et al., 1986). There has been inconclusive evidence linking snuff use to a variety of other cancers including prostate, pancreas, bladder, stomach, and kidney (IARC, 1985; Nilsson, 1998; Winn, 1997). Smokeless Tobacco Research Recommendations In terms of smokeless tobacco use as a strategy for harm reduction, more research is needed to investigate further some of the contradictory findings regarding the risk of oral cancer and cardiovascular disease. Swedish snus (lower TSNA and nicotine levels than American brands) should be evaluated as a possible harm reduction product since two re- cent epidemiological studies have suggested that it does not increase the risk of oral cancer and has favorable cardiovascular risk outcomes. More investigations into the cellular toxicity and genotoxic potential of smoke- less tobacco extracts are needed. Smokeless tobacco may be a valid substi- tute for cigarette smoking but would pose specific risks in certain groups
302 CLEARING THE SMOKE including pregnant women, those with inflammatory bowel disease, and those with established cardiovascular disease. Also, the population risks include concomitant smoking and adolescent use of smokeless tobacco as a gateway to cigarette smoking. The same types of animal studies used to evaluate the toxicity of inhaled tobacco smoke could be done to evaluate the toxicity of smokeless tobacco products, such as Swedish snus or snuff, with a change in empha- sis to the oral route of delivery. Based on known adverse health effects in humans, animal tests would be needed to evaluate the potential for smokeless tobacco to cause chronic inflammation or cancer in tissues of the oral cavity. Toxicokinetic studies would be required to determine other potential target organs for extracts of smokeless tobacco. Examples of in vitro studies include reports showing that smokeless tobacco causes pro-inflammatory changes in cultured endothelial cells (Furie et al., 2000) and activates the complement cascade, suggesting an inflammatory po- tential (Chang et al., 1998). Animal studies have been used to evaluate the potential of smokeless tobacco to induce oral cancer, as reviewed by Grasso and Mann (1998). Thus, the committee concludes that preclinical toxicity testing should be of value for assessing the potential adverse health effects from use of smokeless tobacco. GENERAL RESEARCH AGENDA AND RECOMMENDATIONS Toxicology studies, both in vitro and in vivo, provide the opportunity to evaluate the potential harm reduction offered by potential reduced- exposure products (PREPs). The comparative potency of the PREP can be determined in a series of preclinical studies that include both the PREP and the standard tobacco product that can be replaced by the PREP, par- ticularly tobacco-related PREPs (Figure 10-2). Such tests have recently been reported for a new cigarette-like product (Eclipse Expert Panel, 2000). The preclinical tests should include in vitro tests in both animal and hu- man cells to determine the cytotoxicity and the genotoxicity of the tobacco product to which humans will be exposed. Such a test must include dose- response studies to determine the amount of the exposure material re- quired to cause toxicity. Next, studies should be conducted in vivo in the best animal models available to determine the comparative potency of the PREP versus the standard product in producing: (1) pulmonary inflam- mation, (2) COPD, (3) cardiovascular disease, (4) reproductive toxicology, and (5) pulmonary neoplasms. If these preclinical studies indicate that the PREP is less potent than the standard tobacco product, clinical studies should be conducted to determine acute toxic effects, the toxicokinetic properties, or the adverse effects of the PREP in humans. The determination
TOBACCO SMOKE AND TOXICOLOGY 303 Clinical Epidemio- In Vivo Studies logical In Vitro Studies Studies In Humans Studies Animal Human Acute Chronic Cells Cells Animals Effects Effects Standard Tobacco â â â â â Product Modified Tobacco â â â â ? Product FIGURE 10-2 Preclinical studies for standard and new tobacco products. of human health effects from chronic use of the new product can only be inferred from comparisons of the results of comparative tests in the old and the new product, but cannot be determined directly. Thus the testing approach will allow the rejection of risk reduction claims for products that are as toxic or more toxic in preclinical tests compared to products already on the market; however, only after long-term use of the product by many people could it be determined if the chronic toxicity of the new product is less than that of the standard product. Based on the above information, it is clear that preclinical toxicity testing in vitro and in vivo can be done to assess the potential health effects of new products before they are released for human use. It is beyond the scope of the committeeâs task to recommend the specific set of toxicity tests that should be done on new or existing tobacco products. The committee recommends that a panel of experts be convened to deter- mine the specific set of toxicity tests and details of the testing regimens. Details to be considered include species and strains of test animals, dura- tion of tests, end points of interest, dose-response considerations, bio- markers of dosimetry and response, and standard comparison products to be tested as positive and negative controls. REFERENCES Ahlborn A, Olsson UA, Pershagen G. 1997. Health Hazards of Moist Snuff. September, 1996: National Board on Health and Welfare. Socialstyrelsen. Ames BN, Magaw R, Gold LS. 1987. Ranking possible carcinogenic hazards. Science 236(4799):271-280.
304 CLEARING THE SMOKE Arbeit JM, Hirose R. 1999. Murine mentors: transgenic and knockout models of surgical disease. Ann Surg 229(1):21-40. Balls M, Clothier R. 1992. Cytotoxicity assays for intrinsic toxicity and irritancy. Watson RR, ed. In Vitro Methods of Toxicology. Boca Raton: CRC Press. Pp. 37. Baker RR. 1999. Smoke chemistry. Davis DL, Nielsen MT, eds. World Agriculture Series; Tobacco Production, Chemistry and Technology. London: Blackwell Science. Pp 418-419. Belinsky SA, Nikula KJ, Palmisano WA, et al. 1998. Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci U S A 95(20):11891-11896. Benowitz NL. 1997. Systemic absorption and effects of nicotine from smokeless tobacco. Adv Dent Res 11(3):336-341. Benowitz NL, Porchet H, Sheiner L, Jacob P. 1988. Nicotine absorption and cardiovascular effects with smokeless tobacco use: comparison with cigarettes and nicotine gum. Clin Pharmacol Ther 44(1):23-28. Bohning DE, Atkins HL, Cohn SH. 1982. Long-term particle clearance in man: normal and impaired. Ann Occup Hyg 26(1-4):259-271. Cadet J, DâHam C, Douki T, Pouget JP, Ravanat JL, Sauvaigo S. 1998. Facts and artifacts in the measurement of oxidative base damage to DNA. Free Radic Res 29(6):541-550. Caldwell WS, Conner, JM. 1990. Artifact formation during smoke trapping: an improved method for the determination of N-nitrosamines in cigarette smoke. J Assoc Off Anal Chem 73:783-789. Chalmer J, Holt PG, Keast D. 1975. Cell-mediated immune responses to transplanted tu- mors in mice chronically exposed to cigarette smoke. J Natl Cancer Inst 55(5):1129-1134. Chang T, Chowdhry S, Budhu P, Kew RR. 1998. Smokeless tobacco extracts activate comple- ment in vitro: a potential pathogenic mechanism for initiating inflammation of the oral mucosa. Clin Immunol Immunopathol 87(3):223-229. Chen BT, Benz MV, Finch FL, et al. 1995. Effect of exposure mode on amounts of radiola- beled cigarette particles in lungs and astrointestinal tracts of F344 rats. Inhalat Toxicol 7:1095-1108. Cohen D, Arai SF, Brain JD. 1979. Smoking impairs long-term dust clearance from the lung. Science 204(4392):514-517. Connolly GN, Winn DM, Hecht SS, Henningfield JE, Walker B, Hoffmann D. 1986. The reemergence of smokeless tobacco. N Engl J Med 314(16):1020-1027. Davis DL, Nielsen MT. 1999. World Agriculture Series; Tobacco Production, Chemistry and Tech- nology. London: Blackwell Science. Dendo RI, Phalen RF, Mannix RC, Oldham MJ. 1998. Effects of breathing parameters on sidestream cigarette smoke deposition in a hollow tracheobronchial model. Am Ind Hyg Assoc J 59(6):381-387. Drath DB, Karnovsky ML, Huber GL. 1979. Tobacco smoke. Effects on pulmonary host defense. Inflammation 3(3):281-288. Dube MF, Green CR. 1982. Methods of collection of smoke for analytical puproses. Rec Adv Tob Sci 8:42-102. Eclipse Expert Panel. 2000. A safer cigarette? A comparative study. A consensus report. Inhalation Toxicology 12 (Supplement 5):1-48. Escolar JD, Martinez MN, Rodriguez FJ, Gonzalo C, Escolar MA, Roche PA. 1995. Emphy- sema as a result of involuntary exposure to tobacco smoke: morphometrical study of the rat. Exp Lung Res 21(2):255-273. Finch GL, Lundgren DL, Barr EB, Chen BT, Griffith WC, Hobbs, CH, Hoover, MD, Nikula, KJ, Mauderly JC. 1998. Chronic cigarette smoke exposures increases the pulmonary retention and radiation dose of 239Pu inhaled as 239PuO2 by F344 rats. Health Phys 75(6):597-609.
TOBACCO SMOKE AND TOXICOLOGY 305 Furie MB, Raffanello JA, Gergel EI, Lisinski TJ, Horb LD. 2000. Extracts of smokeless to- bacco induce pro-inflammatory changes in cultured human vascular endothelial cells. Immunopharmacology 47(1):13-23. Grasso P, Mann AH. 1998. Smokeless tobacco and oral cancer: an assessment of evidence derived from laboratory animals. Food Chem Toxicol 36(11):1015-1029. Guerin M, Maddox WL, Stokely J. 1974. Tobacco Smoke Inhalation Exposure: Concepts and Devices. Proceedings of the Tobacco Smoke Inhalation Workshop on Experimental Methods in Smoke and Health Research. DHEW Publication No. (NIH) 75-906. Pp. 31-48. Halliwell B. 1998. Can oxidative DNA damage be used as a biomarker of cancer risk in humans? Problems, resolutions and preliminary results from nutritional supplementa- tion studies. Free Radic Res 29(6):469-486. Hayashi M, MacGregor JT, Gatehouse DG, et al. 2000. In vivo rodent erythrocyte micro- nucleus assay. II. Some aspects of protocol design including repeated treatments, inte- gration with toxicity testing, and automated scoring. Environ Mol Mutagen 35(3):234- 252. Hecht SS, Rivenson A, Braley J, DiBello J, Adams JD, Hoffmann D. 1986. Induction of oral cavity tumors in F344 rats by tobacco-specific nitrosamines and snuff. Cancer Res 46(8):4162-4166. Henderson RF. 1989. Bronchoalveolar Lavage: A tool for assessing the health status of the lung. McClellan RO, Henderson RF, eds. Concepts in Inhalation Toxicology. New York: Hemisphere Publishing Corporation. Pp. 415-444. Hoffmann D, Adams JD, Lisk D, Fisenne I, Brunnemann KD. 1987. Toxic and carcinogenic agents in dry and moist snuff. J Natl Cancer Inst 79(6):1281-1286. Hoffmann D, Djordjevic MV. 1997. Chemical composition and carcinogenicity of smokeless tobacco. Adv Dent Res 11(3):322-329. Hoffmann D, Djordjevic MV, Fan J, Zang E, Glynn T, Connolly GN. 1995. Five leading U.S. commercial brands of moist snuff in 1994: assessment of carcinogenic N-nitrosamines. J Natl Cancer Inst 87(24):1862-1869. Holt PG, Chalmer J, Keast D. 1975. Development of two manifestations of T-lymphocyte reactivity during tumor growth: altered kinetics associated with elevated growth rates. J Natl Cancer Inst 55(5):1135-1142. Holt PG, Keast D. 1977. Environmentally induced changes in immunological function: acute and chronic effects of inhalation of tobacco smoke and other atmospheric contami- nants in man and experimental animals. Bacteriol Rev 41(1):205-216. Huber GL, Drath D, Davies P, Hayashi M, Shea J. 1980. The alveolar macrophage as a mediator of tobacco-induced lung injury. Chest 77(2 Suppl):272. IARC (International Agency for Research on Cancer). 1986. Tobacco Smoking: Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans. Vol. 38 ed. Lyon, France: IARC. IARC. 1985. Tobacco habits other than smoking; betel-quid and areca-nut chewing; and some related nitrosamines. IARC Working Group. 23-30 October 1988. IARC Monogr Eval Carcinog Risk Chem Hum 37:1-268. Ji CM, Plopper CG, Witschi HP, Pinkerton KE. 1994. Exposure to sidestream cigarette smoke alters bronchiolar epithelial cell differentiation in the postnatal rat lung. Am J Respir Cell Mol Biol 11(3):312-320. Johnson JD, Houchens DP, Kluwe WM, Craig DK, Fisher GL. 1990. Effects of mainstream and environmental tobacco smoke on the immune system in animals and humans: a review. Crit Rev Toxicol 20(5):369-395. Kendrick J, Nettesheim P, Guerin M, et al. 1976. Tobacco smoke inhalation studies in rats. Toxicol Appl Pharmacol 37(3):557-569.
306 CLEARING THE SMOKE Leichter J. 1989. Growth of fetuses of rats exposed to ethanol and cigarette smoke during gestation. Growth Dev Aging 53(3):129-134. Lewin F, Norell SE, Johansson H, Gustavsson P, Wennerberg J, Biorklund A, Rutqvist LE. 1998. Smoking tobacco, oral snuff, and alcohol in the etiology of squamous cell carci- noma of the head and neck: a population-based case-referent study in Sweden. Cancer 82(7):1367-1375. Lewis JG, Graham DG, Valentine WM, Morris RW, Morgan DL, Sills RC. 1999. Exposure of C57BL/6 mice to carbon disulfide induces early lesions of atherosclerosis and en- hances arterial fatty deposits induced by a high fat diet. Toxicol Sci 49(1):124-132. Lichtenbeld H, Vidic B. 1989. Effect of maternal exposure to smoke on gas diffusion capac- ity in neonatal rat. Respir Physiol 75(2):129-140. Loft S, Deng XS, Tuo J, Wellejus A, Sorensen M, Poulsen HE. 1998. Experimental study of oxidative DNA damage. Free Radic Res 29(6):525-539. Lovell DP, Yoshimura I, Hothorn LA, Margolin BH, Soper K. 2000. Report and summary of the major conclusions from statistics in genotoxicity testing working group from the International Workshop on Genotoxicity Test Procedures (IWGTP), March 1999. Environ Mol Mutagen 35(3):260-263. Mackenzie JS, Flower RL. 1979. The effect of long-term exposure to cigarette smoke on the height and specificity of the secondary immune response to influenza virus in a mu- rine model system. J Hyg (Lond) 83(1):135-141. MacKenzie JS, MacKenzie IH, Holt PG. 1976. The effect of cigarette smoking on susceptibil- ity to epidemic influenza and on serological responses to live attenuated and killed subunit influenza vaccines. J Hyg (Lond) 77(3):409-417. Main JH, Lecavalier DR. 1988. Smokeless tobacco and oral disease. A review. J Can Dent Assoc 54(8):586-591. March TH, Barr EB, Finch GL, et al. 1999a. Cigarette smoke exposure produces more evi- dence of emphysema in B6C3F1 mice than in F344 rats. Toxicol Sci 51(2):289-299. March TH, Kolar LM, Barr EB, Finch GL, Menache MG, Nikula KJ. 1999b. Enhanced pulmo- nary epithelial replication and axial airway mucosubstance changes in F344 rats ex- posed short-term to mainstream cigarette smoke. Toxicol Appl Pharmacol 161(2):171-179. Mattson ME, Winn DM. 1989. Smokeless tobacco: association with increased cancer risk. NCI Monogr (8):13-16. Mauderly JL, Chen BT, Hahn FF, et al. 1989. The effect of chronic cigarette smoke inhalation on the long-term pulmonary clearance of inhaled particles in the rat. Wehner AP, ed. Biological Interaction of Inhaled Mineral Fibers and Cigarette Smoke. Richland, WA: Battelle Press. Pp. 223-239. McAllister-Sistilli CG, Caggiula AR, Knopf S, Rose CA, Miller AL, Donny EC. 1998. The effects of nicotine on the immune system. Psychoneuroendocrinology 23(2):175-187. National Council on Radiation Protection and Measurements. 1997. Deposition, Retention, and Dosimetry of Inhaled Radioactive Substances. Report #125. Bethesda, MD. Nilsson R. 1998. A qualitative and quantitative risk assessment of snuff dipping. Regul Toxicol Pharmacol 28(1):1-16. NIH (National Institutes of Health). 1986. Health implications of smokeless tobacco use. NIH Consensus Statement 6(1):1-17. Norman A. 1999. Cigarette design and materials. Davis DL, Nielson MT, eds. Tobacco: Pro- duction, Chemistry, and Technology. Oxford: Blackwell Science Ltd. Pp. 353-387. Nuovo GJ, Plaia TW, Belinsky SA, Baylin SB, Herman JG. 1999. In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogen- esis. Proc Natl Acad Sci U S A 96(22):12754-12759. Nuwaysir EF, Bittner M, Trent J, Barrett JC, Afshari CA. 1999. Microarrays and toxicology: the advent of toxicogenomics. Mol Carcinog 24(3):153-159.
TOBACCO SMOKE AND TOXICOLOGY 307 Penn A, Chen LC, Snyder CA. 1994. Inhalation of steady-state sidestream smoke from one cigarette promotes arteriosclerotic plaque development. Circulation 90(3):1363-1367. Penn A, Snyder CA. 1993. Inhalation of sidestream cigarette smoke accelerates develop- ment of arteriosclerotic plaques. Circulation 88(4 Pt 1):1820-1825. Pershagen G. 1996. Smokeless tobacco. Br Med Bull 52(1):50-57. Phalen R. 2000. Physical-chemical characteristics of tobacco smoke. Open Meeting of the IOM Committee to Assess the Science Base for Tobacco Harm Reduction: Washington, DC. Phillips DH, Farmer PB, Beland FA, et al. 2000. Methods of DNA adduct determination and their application to testing compounds for genotoxicity. Environ Mol Mutagen 35(3):222- 233. Rajini P, Last JA, Pinkerton KE, Hendricks AG, Witschi H. 1994. Decreased fetal weights in rats exposed to sidestream cigarette smoke. Fundam Appl Toxicol 22(3):400-404. Reznik G, Marquard G. 1980. Effect of cigarette smoke inhalation during pregnancy in Sprague-Dawley rats. J Environ Pathol Toxicol 4(5-6):141-152. Schildt EB, Eriksson M, Hardell L, Magnuson A. 1998. Oral snuff, smoking habits and alcohol consumption in relation to oral cancer in a Swedish case-control study. Int J Cancer 77(3):341-346. Silbaugh SA, Horvath SM. 1982. Effect of acute carbon monoxide exposure on cardiopul- monary function of the awake rat. Toxicol Appl Pharmacol 66(3):376-382. Sjostrand M, Rylander R. 1997. Pulmonary cell infiltration after chronic exposure to (1->3)- beta-D-glucan and cigarette smoke. Inflamm Res 46(3):93-97. Sopori ML, Goud NS, Kaplan AM. 1994. Effects of tobacco smoke on the immune system. Dean JH, Luster MI, Munson AE, Kimber I, eds. Immunotoxicology and Immuno- pharmacology. Second Edition ed. New York: Raven Press, Ltd. Pp. 413-434. Subramaniam S, Srinivasan S, Bummer PM, Gairola CG. 1999. Perinatal sidestream ciga- rette smoke exposure and the developing pulmonary surfactant system in rats. Hum Exp Toxicol 18(4):206-211. Subramaniam S, Whitsett JA, Hull W, Gairola CG. 1996. Alteration of pulmonary surfactant proteins in rats chronically exposed to cigarette smoke. Toxicol Appl Pharmacol 140(2):274-280. Swafford DS, Middleton SK, Palmisano WA, et al. 1997. Frequent aberrant methylation of p16INK4a in primary rat lung tumors. Mol Cell Biol 17(3):1366-1374. Thomas WR, Holt PG, Keast D. 1973. Cellular immunity in mice chronically exposed to fresh cigarette smoke. Arch Environ Health 27(6):372-375. Thomas WR, Holt PG, Keast D. 1974a. Development of alterations in the primary immune response of mice by exposure to fresh cigarette smoke. Int Arch Allergy Appl Immunol 46(4):481-486. Thomas WR, Holt PG, Keast D. 1975. Humoral immune response of mice with long-term exposure to cigarette smoke. Arch Environ Health 30(2):78-80. Thomas WR, Holt PG, Papadimitriou JM, Keast D. 1974b. The growth of transplanted tumours in mice after chronic inhalation of fresh cigarette smoke. Br J Cancer 30(5):459- 462. Tice RR, Agurell E, Anderson D, et al. 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35(3):206-221. Vial WC. 1986. Cigarette smoking and lung disease. Am J Med Sci 291(2):130-142. Wahlberg I, Ringberger T. 1999. Smokeless tobacco. Davis DL, Nielsen MT, eds. World Agriculture Series; Tobacco Production, Chemistry and Technology. London: Blackwell Sci- ence. Pp 452-460.
308 CLEARING THE SMOKE Wang T, Hopkins D, Schmidt C, Silva S, Hougton R,Takita H, Repasky E, Reed SG. 2000. Identification of genes differentially over-expressed in lung squamous cell carcinomas using combination of cDNA subtraction and miroarray analysis. Oncogene 19:1519- 1528. Winn DM. 1997. Epidemiology of cancer and other systemic effects associated with the use of smokeless tobacco. Adv Dent Res 11(3):313-321. Winn DM, Blot WJ, Shy CM, Pickle LW, Toledo A, Fraumeni JF. 1981. Snuff dipping and oral cancer among women in the southern United States. N Engl J Med 304(13):745-749. Witschi H, Espiritu I, Maronpot RR, Pinkerton KE, Jones AD. 1997a. The carcinogenic potential of the gas phase of environmental tobacco smoke. Carcinogenesis 18(11):2035- 2042. Witschi H, Espiritu I, Peake JL, Wu K, Maronpot RR, Pinkerton KE. 1997b. The carcinoge- nicity of environmental tobacco smoke. Carcinogenesis 18(3):575-586. Witschi H, Espiritu I, Uyeminami D. 1999. Chemoprevention of tobacco smoke-induced lung tumors in A/J strain mice with dietary myo-inositol and dexamethasone. Car- cinogenesis 20(7):1375-1378. Witschi H, Lundgaard SM, Rajini P, Hendrickx AG, Last JA. 1994. Effects of exposure to nicotine and to sidestream smoke on pregnancy outcome in rats. Toxicol Lett 71(3):279- 286. Witschi H, Uyeminami D, Moran D, Espiritu I. 2000. Chemoprevention of tobacco-smoke lung carcinogenesis in mice after cessation of smoke exposure. Carcinogenesis 21(5):977- 982. Zhu BQ, Sun YP, Sievers RE, Glantz SA, Parmley WW, Wolfe CL. 1994. Exposure to envi- ronmental tobacco smoke increases myocardial infarct size in rats. Circulation 89(3):1282-1290.