Arthur Penn

Inhalation of steady-state sidestream smoke from one cigarette promotes arteriosclerotic plaque development.

BackgroundA number of epidemiologic studies have suggested that every year environmental tobacco smoke (secondhand smoke) is responsible for tens of thousands of deaths, mostly from heart disease, in the United States. Environmental tobacco smoke is composed mainly (80% to 85%) of aged and diluted sidestream smoke. The remainder is exhaled mainstream smoke. Among the thousands of compounds that have been identified in environmental tobacco smoke are a number of carcinogens, including polynuclear aromatic hydrocarbon carcinogens, such as benzo(a)pyrene. We have demonstrated previously that a number of carcinogens, including benzo(a)pyrene, promote plaque development after injection into cockerels. There have been almost no studies showing a direct stimulatory effect of environmental tobacco smoke on plaque development. Recently we demonstrated that cockerels exposed to sidestream smoke for approximately 0.4% of their projected lifespan exhibited accelerated development of arteriosclerotic plaques.6 In that study, cockerels in specially designed inhalation chambers were exposed to the steady-state sidestream smoke from 5 cigarettes for 6 h/d for 16 weeks. This level of exposure is high but environmentally plausible. Statistically significant increases in plaque size were demonstrated in the smoke-exposed cockerels. Methods and ResultsIn the present study, exposure levels were decreased by a factor of 5. Thirty cockerels were exposed to the steady-state sidestream smoke from 1 cigarette for 6 hours per day for 16 weeks. The smoke was mixed with filtered air. Ten control cockerels were exposed to filtered air only. Levels of smoke surrogates, including carbon monoxide and total suspended particulates, were measured three times a day. Again, there was a statistically significant increase in plaque size in the smoke-exposed cockerels. To place these studies within a context of environmental relevance, levels of carbon monoxide were measured independently over 1 to 3 hours in four bars where there was heavy smoking. Measured carbon monoxide levels were as high or higher in the bars than they were in the exposure chambers during the 1-cigarette sidestream-smoke study. ConclusionsExperimental exposure to secondhand smoke at levels equal to or even below those routinely encountered by people in smoke-filled environments is sufficient to promote arteriosclerotic plaque development.

Inhalation of sidestream cigarette smoke accelerates development of arteriosclerotic plaques.

BackgroundEnvironmental tobacco smoke has been blamed for

1,3 Butadiene, a Vapor Phase Component of Environmental Tobacco Smoke, Accelerates Arteriosclerotic Plaque Development

-40 000 excess deaths from heart disease annually in the United States. As yet, no pathophysiological process that could be responsible for these deaths has been identified. Environmental tobacco smoke is composed mainly of aged and diluted sidestream smoke but also contains 15% to 20% exhaled mainstream smoke. Carcinogens, including nitrosamines and polynuclear aromatic hydrocarbons, are present in mainstream smoke and sidestream smoke. Carcinogen levels in sidestream smoke, unlike those in mainstream smoke, are not reduced in filtered cigarettes. The US Environmental Protection Agency has designated environmental tobacco smoke as a human (class A) carcinogen. In cockerels, subtumorigenic doses of polynuclear aromatic hydrocarbons carcinogens accelerate aortic arteriosclerotic plaque development. Methods and ResultsTo determine whether sidestream smoke inhalation affects arteriosclerotic plaque development, we exposed cockerels to sidestream smoke (n=30) or to filtered air (n=12) in inhalation chambers for 6 hours per day, 5 days a week from 6 to 22 weeks of age (0.4% of projected lifespan). Chamber levels of carbon monoxide, total suspended particulates, and nicotine were measured regularly during the exposures. The abdominal aorta from each cockerel was cut into 10 segments, and the plaque index (mean plaque cross-sectional area [mm2]/mean luminal circumference [mm]× 100) was calculated for each segment. There were no differences in plaque incidence or distribution between sidestream smoke-exposed and control cockerels; however, plaque indexes were significantly greater for sidestream smoke-exposed than control cockerels in all segments ConclusionsThus, relatively brief exposures to sidestream smoke early in life are sufficient to enhance arteriosclerotic plaque development.

Mutational events in the etiology of arteriosclerotic plaques

BACKGROUNDnOur recent results support predictions from epidemiology studies that thousands of excess heart disease-related deaths result yearly in the United States from involuntary exposure to environmental tobacco smoke (ETS). Limited exposures of cockerels to ETS significantly accelerate arteriosclerosis. Despite little direct in vivo support, tar fraction rather than vapor phase compounds are considered largely responsible for the plaque-promoting effects of cigarette smoke. Here, we evaluate the effects of two ETS components on plaque development: the vapor phase component, 1,3 butadiene, and the tar component, the tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). At relatively high doses, injected NNK is carcinogenic in rodents. Epidemiology studies have identified increased mortality from arteriosclerotic heart disease among black men working in the butadiene rubber industry. Neither butadiene nor NNK has been tested experimentally for a possible role in plaque development.nnnMETHODS AND RESULTSnCockerels inhaled butadiene (20 ppm; 16 weeks) or were injected biweekly with NNK (10 mg/kg, 16 weeks). Control cockerels were exposed to filtered air or were injected with the NNK solvent dimethylsulfoxide. Plaque incidence, prevalence, location, and size were determined double-blind. NNK had no significant effect on any of these measurements. In contrast, butadiene elicited a statistically significant increase in plaque size comparable to that seen after steady-state exposure to ETS from 5 cigarettes.nnnCONCLUSIONn(1) This study represents the first time that a single cigarette smoke component has been demonstrated to accelerate arteriosclerosis, at a dose that is environmentally relevant. (2) The plaque-promoting components of ETS may reside in the vapor phase. (3) The cockerel model should be valuable in understanding the mechanism underlying the reported increases in heart disease deaths among black workers in the butadiene rubber industry.

Inhalation of carbon monoxide does not accelerate arteriosclerosis in cockerels

The arteriosclerotic plaque is the lesion most often associated with cardiovascular disease, which is the leading cause of death in North America and Western Europe. Plaques are composed of cells (mostly smooth muscle cells but also macrophages and some lymphocytes) and formed elements (cellular debris, collagen, elastin, glycosaminoglycans, lipid droplets, cholesterol crystals and sometimes calcium deposits). Proliferation of smooth muscle cells is essential to plaque formation and development. Most theories of plaque development have viewed this proliferation as a secondary event following an initiating stimulus (e.g., endothelial injury). According to this view, the proliferating smooth muscle cells are otherwise identical to the large number of non-proliferating smooth muscle cells in the artery wall. The monoclonal hypothesis of plaque formation presents a fundamentally different view; namely, that the cell proliferation critical to plaque development follows the stable transformation of smooth muscle cells and that the plaques can therefore be viewed as benign smooth muscle cell tumors of the artery wall. Environmental agents, including viruses and chemicals that have been previously associated with cell transformation and tumorigenesis may therefore also contribute directly to plaque development. Data are provided from in vivo and in vitro studies in support of this proposition. Evidence is also presented that in standardized assays human and animal plaque DNAs elicit responses similar to those elicited by tumor DNAs. Thus, both plaque formation and tumorigenesis may share common mechanisms.

Evidence for strain-specific differences in benzene toxicity as a function of host target cell susceptibility

The effects of chronic exposure to moderate levels of carbon monoxide (CO) upon the augmentation of arteriosclerotic plaque development were investigated in a series of in vivo studies. Cockerels were exposed to carefully regulated CO levels in dynamic exposure chambers. The plaque volume percentage in the aortic walls of experimental and control animals was determined by point-counting. Chronic CO inhalation, at levels up to 200 ppm, did not stimulate arteriosclerotic plaque development (at 200 ppm CO, carboxyhemoglobin (COHb) levels 10 min after exposures ended were 11-12%). When administered concomitantly with cholesterol feeding, CO did not augment plaque development. When administered after either carcinogen-associated or diet-promoted plaque size increases had occurred, CO elicited no further plaque size increases. Thus, in this animal model, daily exposures to moderately high CO levels were without discernable effect upon arteriosclerotic plaque development, although high COHb levels were attained.

Butadiene inhalation accelerates arteriosclerotic plaque development in cockerels

It has long been recognized that benzene exposure produces disparate toxic responses among different species or even among different strains within the same species. There is ample evidence that species- or strain-dependent differences in metabolic activity correlate with the disparate responses to benzene. However, bone marrow cells (the putative targets of benzene toxicity) may also exhibit species- or strain-dependent differences in susceptibility to the toxic effects of benzene. To investigate this hypothesis, two sets of companion experiments were performed. First, two strains of mice, Swiss Webster (SW) and C57B1/6J (C57), were exposed to 300 ppm benzene via inhalation and the effects of the exposures were determined on bone marrow cellularity and the development of bone marrow CFU-e (Colony Forming Unit-erythroid, an early red cell progenitor). Second, bone marrow cells from the same strains were exposed in vitro to five known benzene metabolites (1,4 benzoquinone, catechol, hydroquinone, muconic acid, and phenol) individually and in binary combinations. Benzene exposure, in vivo, reduced bone marrow cellularity and the development of CFU-e in both strains; however, reductions in both these endpoints were more severe in the SW strain. When bone marrow cells from the two strains were exposed in vitro to the five benzene metabolites individually, benzoquinone, hydroquinone, and catechol reduced the numbers of CFU-e in both strains in dose-dependent responses, phenol weakly reduced the numbers of the C57 CFU-e only and in a non-dose-dependent manner, and muconic acid was without effect on cells from either strain. Only benzoquinone and hydroquinone exhibited differential responses to CFU-e from the two strains and both of these metabolites were more toxic to SW cells than to C57 cells. Six of the ten possible binary mixtures of metabolites were differentially toxic to the CFU-e from the two strains and five of these mixtures were more toxic to SW cells than to C57 cells. Thus, SW mice were more susceptible to the toxic effects of inhaled benzene and their bone marrow cells were more severely affected by in vitro exposure to benzene metabolites. The binary combinations containing phenol produced little or no enhancement of the toxic effects of the non-phenol metabolites. The weak toxic response induced by phenol, whether delivered alone or in binary mixtures, suggests that little metabolism occurred during the 48 h of the in vitro exposures since benzoquinone and hydroquinone, which were clearly toxic when added to the CFU-e culture system, are formed by further metabolic oxidation of phenol. Thus, strain-dependent differential metabolism appeared to play a minimal role in the disparate toxicity observed in the in vitro studies, implying that the diverse responses were due to inherent differences in the susceptibilities of the CFU-e to the toxic action of the benzene metabolites.

Environmental tobacco smoke: Experimental facts and societal issues

1,3-Butadiene (BD), a gas widely used in the rubber industry, is also present in automotive exhaust and in the vapor phase of environmental tobacco smoke (ETS; approximately 400 micrograms/cigarette). The threshold limit value (TLV) for BD which was 10 ppm, has now been reduced to 2 ppm. Extensive investigations of workers have identified very few statistically significant increases in BD-associated cancer mortality. However, two studies have reported increased BD-associated mortality from arteriosclerotic heart disease in black workers in the BD rubber industry. The cockerel is a sensitive animal model for studying effects of environmental agents on arteriosclerosis development. Previous studies showed that inhaled environmental levels of ETS significantly accelerate arteriosclerosis. Surprisingly, the carcinogen-rich tar fraction of ETS was ineffective. The elevated risk of death from arteriosclerotic heart disease in black BD workers and the high BD level in the vapor phase of ETS, raised the question of whether BD would accelerate arteriosclerosis in cockerels. Here, cockerels breathed either 20 ppm BD or filtered air (6 h/day, 80 days). Blinded measurements showed no differences between groups in plaque frequency or location. However, plaque sizes were significantly larger in BD-treated cockerels than in controls--results nearly identical to those reported earlier for ETS-exposed vs. air-exposed cockerels. This indicates that BD may contribute to the atherogenicity of ETS and provides experimental support for the recent reduction in the TLV for BD.

Heat-shock-induced enhanced reactivation of UV-irradiated Herpesvirus

Involuntary exposure to environmental tobacco smoke (ETS) in public or in working places is considered to be a serious risk to human health. This symposium addressed several issues of toxicological interest that are associated with exposure to ETS. Epidemiologic evidence obtained in human studies suggests that passive smoking increases the risk of developing lung cancer in nonsmokers and favors the development of respiratory tract infections in children. Comparatively few data are available from animal studies that provide experimental support of the observations. Exposure of pregnant or neonate rats to cigarette sidestream smoke (SS) affects developmental patterns of drug metabolizing enzymes that may persist up to 90 days. In young roosters, SS accelerates the development of arteriosclerotic plaques. On the other hand, exposure of adult rats for up to 90 days induces only transient signs of damage in the nasal passages, but not in the deep lung, and this only at extremely high concentrations of ETS. So far, experimental toxicology has provided comparatively few data on the correlation between exposure to ETS and adverse health effects. yet, such data are needed, particularly since many conclusions drawn from the epidemiological studies remain open to criticism and questions.

Transforming potential is detectable in arteriosclerotic plaques of young animals.

The objective of this study was to compare the ability of heat shock (HS) with that of another type of cellular stress, UV irradiation, to cause the induction of enhanced viral reactivation, a process that may represent an SOS-type repair process in mammalian cells. Studies performed to evaluate the effect of HS on growth of Vero cells revealed that HS at 45 degrees C for 45 min caused inhibition of cell growth similar to that caused by UV irradiation at 12 J/m2, but this inhibition was not observed at HS treatment for 5-15 min, or at a UV fluence of 2 J/m2. Enhanced reactivation of UV-irradiated Herpesvirus was observed in cells which had been pretreated by HS for greater than 30 min or UV at 12 J/m2. The synthesis of new proteins following HS for 15 and 45 min and UV at 12 J/m2 was examined by [35S]methionine-labeling experiments. The new synthesis of two HS proteins with molecular weights of 46 000 and 78 000 was induced by both levels of HS, but to a much greater extent at the high dose. These proteins were not detected in response to UV irradiation. These results indicate that, like UV irradiation, HS at levels inhibitory to cell growth induced enhanced viral reactivation in Vero cells. The results also suggest that at least two proteins in the HS protein family are not necessary for this response to occur.