sample="quota" bates="517262932" isource="rjr" decade="1990" class="ui" date="19920810" A Review Of Long-Term Inhalation Studies With Cigarette Smoke In Laboratory Animals. Christopher R.E. Coggins, Ph.D., DABT Research & Development, R.J. Reynolds Tobacco Co., Winston-Salem, NC 27106. Abstract A recent review (IARC,1985) Is this truly "recent"? How about "prior" or "earlier or "early" or...has stated there is sufficient evidence that inhalation of cigarette smoke as well as application of tobacco smoke condensate cause cancer in experimental animals This is not consistent The data presented in this earlier review on inhalation studies are very limited ( less than 20 papers). The present document constitutes an exhaustive review of the literature on long-term inhalation studies with cigarette smoke. Based on the evidence accumulated during this review, the conclusion is reached that the IARC evaluation of "sufficient evidence" is incorrect. (for inhalation only?) Introduction A number of inhalation studies have been performed with cigarette smoke and experimental animals. This article will review s the work that has been published over the last 20 years on inhalation studies with cigarette smoke. The most commonly used animals are were rodents: rats, mice, hamsters (Chinese, Syrian-Golden and European), guinea pigs, and rabbits. There is relatively little published work on In addition, few inhalation studies to have been published which used larger animals such as primates anddogs (see below for a discussion on tracheostomy) in dogs.) and primates Th e is review will consider s the individual studies used with each of the main species, with the induction of pulmonary neoplasia as the ndpoint of sole concern. Within the different species various strains have been used (particularly for mice). Reviews of the earlier literature are available (UICC, 1976; Wehner, 1983; Pepelko, 1984). The present document does not include short-term exposures . Rather, it restricts restricting the review to those studies with durations of at least several months. The decision on whether to include ,or not include a study or not in such a review has already been discussed (Pepelko 1984), ; the decision it varying primarily with the species of experimental animals being used. There are several key areas that were will be examined in each of the studies: the cigarettes used, the smoke chemistry data, the types of inhalation exposures (including the availability of deposition data), and the similarity (if any) of the histopathological changes produced with those reported in human smokers. Cigarettes: smoke chemistry The cigarettes used by many of the groups in this review were unfiltered and had very high yields of "tar" and nicotine. They are, thus, substantially different from cigarettes commercially available today. A typical cigarette used is the University of Kentucky 1R1 reference cigarette, made in 1969, which has a "tar" yield of 34.3 mg and a nicotine yield of 2.16 mg. In contrast, t The latest reference cigarette from the University of Kentucky, the 1R5F, has a sophisticated filter which results in a "tar" yield of 1.6 mg and a nicotine yield of 0.16 mg. Few of the studies made measurements on the chemical composition of the smoke generated, relying instead on nominal dilutions of published yields of reference cigarettes. Inhalation Technology Most of the papers with rodents experiments used nose-only exposure systems. Some of the smoking machines used single cigarettes , in an attempt to mimic human smoking (Guerin et al., 1979; x Griffith &Standafer, 1985). Others used large numbers of cigarettes on a rotating carousel, whereby a constant stream of smoke is generated and distributed (Baumgartner & Coggins, 1980). Some of the larger devices allowed the re-breathing of exhaled smoke by animals "downstream" (Henry et al., 1985). The manner in which the mainstream smoke was generated also varied. In only a few of the inhalation studies were deposition data presented. It is well known that animals when exposed to cigarette smoke are capable of varying degrees of breath-holding (Coggins et al., 1982; Coggins, 1985), a problem that is particularly pronounced in non-continuous or "bolus" exposures. Minimally, measures of blood carboxyhemoglobin (COHb) should be included to demonstrate that smoke was in fact inhaled, ideally , accompanied by measures of the deposition of constituents of the smoke particulate phase , such as plasma x nicotine and cotinine. Some of the more complete studies used radiolabelled markers , thereby providing definitive data on the amounts of deposition of "tar" in the lungs of the experimental animals. Large numbers of animals died during the experiment, some of the deaths being related to the method by which the animals were restrained during these smoke exposures. In particular, large numbers of animals had neck lesions. This was also noticed in the sham-exposed animals. The sham animals died in a significant rate from around 80 weeks of exposure; approximately 60% of these animals died from neoplastic diseases and 40% of non-neoplastic diseases. The major non-neoplastic diseases were pneumonia, nephritis and conditions were no major disease was found. The major neoplastic diseases observed were hematic tumors, sarcomas, fibrosarcomas, lung adenocarcinomas, liver carcinomas and mammary carcinomas. The only lung cancers observed were diagnosed as alveolar adenoma carcinoma. No squamous cell carcinomas were found. A total of 19 of 978 smoke-exposed mice and 7 of 651 sham-exposed mice were observed with alveolar adenocarcinomas. The difference between these groups was not statistically significant. The data were analyzed in the CTR book in a number of different ways; under no circumstances could a statistical significance between sham and smoke-exposed groups be noted. At no time in this study was the incidence in the smoke-exposed mice higher than that in the sham-exposed animals. An analysis was made using actuary tables. Using this technique, the results showed that there was no difference in the incidence or latency of alveolar adenocarcinomas between the smoke-exposed and sham-exposed mice. Although no differences were observed between sham and smoke-exposed mice in an experiment using good dosimetry with chronic exposures, the authors conclude that the 2R1 cigarette smoke has week carcinogenic activity. The statistics are quite clear and showing that there is no such difference between smoke-exposed and sham animals. A full analysis of the histopathology noted in the study is missing. There is no accurate description of the alveolar adenoma carcinoma. Additional problems with this study include the unknown effects of the inoculation of the Sendai virus, and also the physical restraint causing large numbers of mortalities in the study. Holt et al. (1976) exposed Balb/c and C57B1 mice to smoke from high-"tar" (c. 16 mg) or low "tar" (c. 6 mg) cigarettes for 7-8 minutes per day, 5 days per week, for up to 36 weeks. No figures are presented for CO yields of the 2 cigarette types. Measurements were made of blood COHb: concentrations approached 30% in the C57B1 mice exposed to smoke from the high "tar" cigarette, but were only around 9% in the low "tar" cigarette. Concentrations in the Balb/c mice were around 10% for both cigarette types. These COHb concentrations were accompanied in some cases by leukopenia. Mice exposed to smoke from the high "tar" cigarettes exhibited alterations in humoral immune responsiveness. However, cell-mediated immune responsiveness to bacterial and tumor-specific antigens were depressed similarly in animals exposed to low or high "tar" cigarettes. Standard necropsy and histopathology techniques were used. Histological findings included marked signs of persistent bronchitis, which are apparently more noticeable in the high "tar" groups than in the low "tar" groups or in controls. The alveolar parenchyma of some animals apparently showed interstitial pneumonia. However, no data are presented on the comparative incidences of this change. No information on disease status was presented, although the authors hypothesize that the pneumonia may have resulted from infection. There was no report of any neoplastic change. Keast and Ayre (1980) exposed C57B1 and Balb/c mice to high "tar" (16 mg per cigarette) or low "tar" (5 mg per cigarette) filtered cigarettes for eight minutes per day, for up to 30 weeks. No measurements of the smoke composition were made, nor were any estimates made of the dosimetry by plasma nicotine or blood COHb. Phagocytic and degradative properties of the liver and spleen were assessed using opsonized radioactive sheep erythrocytes, injected intravenously into naive or immune animals. The results demonstrated that both the high and low "tar" exposure of susceptible and resistant mice often modify systemic clearance mechanisms and decrease the ability of the liver to trap circulating antigen with concomitant variations in the rate of antigen breakdown. These results were thought to provide at least a partial explanation of the observed delays in antibiotic production by tobacco smoke-exposed mice, and to indicate that a genetic tobacco smoke susceptibility may exist, either thorough variations in the ability to metabolize tobacco smoke toxins or direct immunological susceptibility to tobacco smoke toxicity (possibly emitted through vapor phase compounds). Chronic cigarette smoke inhalation failed to alter plasma lipid or protein levels. No significant differences were seen in total plasma cholesterol or lack of protein cholesterol concentrations measured at four intervals over a period of one year. No details are presented in this study of necropsy or of subsequent histopathology. Rogers et al. (!988) exposed baboons to the smoke from more than 40 2RI reference cigarettes (37 mg of tar and 1.6 mg of nicotine) per day for periods of up to 3.3 years. A total number of 30 animals was used, with sub-groups of animals placed on an atherogenic diet. An estimate was made of blood COHb concentration: values were approximately 11/2% in the smoke-exposed animals and 0.5% in controls. The animals had mean puff volumes of 47 ml and had longer puffs than the FTC standard. A number of different parameters were examined in this study, including hematologic variables and atherosclerotic lesions. The latter were obtained through histologic sections of the abdominal aorta. There were no differences between smokers in controls in the extent of fatty streaks or in the prevalence of fibrous plaques. The necropsy results were restricted to cardiovascular system; however, it is clear that most examinations were made of all tissue. Cigarette smoking for up to three years did not increase the extent of diet-induced experimental atherosclerosis. The authors concluded that it was unlikely that this failure was due to the use of an ineffective method of smoke exposure. One of the criticisms that had been made of cigarette smoke inhalation studies in primates is that because of the complicated anatomy of the nasal passages, only small amounts of materials may reach the lungs of the smoke-exposed animals. This has been shown to be not the case for baboons, through work performed by Rogers et al. (1981). In this work, very similar to that described by Rogers et al. (1988), baboons were exposed to 14 C-labelled dotriacontane delivered in a manner providing extensive deposition of particulates. Lungs of these passively exposed animals were lavaged so that the efficiency of recovery of the lavage procedure could be determined. A second phase of this work was to expose nine baboons to actively-smoked labelled cigarettes followed by the lavage of the lungs of these animals to recover 14C-labelled dotriacontane. The total amount of particular matter present in the lungs was estimated using the efficiency factor previously determined. Smoking baboons retained and average of 9% of the total cigarette particular matter, in proportions similar to that retained by other animal smoke inhalation models. In this work, no data were given on the percentage lung deposition of the cigarette particulate matter content. However, the authors were confident that is should be possible to increase the fraction considerably to values of approximately 10%. Sopori et al. (1985) exposed 11 adult macaque monkeys to either a low does (human equivalent of one pack per day) or high dose (human equivalent three packs per day) of high tar, high nicotine reference cigarette smoke for four to eight years. Animals were exposed seven days a week, 30 and 90 minutes per day. The inhalation protocol was carried out twice per day using the University of Kentucky 2RI reference cigarettes. No details of blood COHb or plasma nicotine were reported, nor was any mention made of necropsy or subsequent histopathology. Parameters of immunological response were compared to those seen in six non-smoked controlled animals. The results suggested that cigarette smoking does not significantly affect the responses of spleen cells to mitogens. However, spleen cells subjected to the high dose of smoke demonstrated a reduction in their natural killer cell mediated activity. RATS Probably more inhalation experiments have been performed with this species than with any other.