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(Circulation. 1996;93:552-557.)
© 1996 American Heart Association, Inc.

Articles

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

Arthur Penn, PhD; Carroll A. Snyder, PhD

From the Nelson Institute of Environmental Medicine, New York University Medical Center, New York, NY.

Correspondence to Arthur Penn, PhD, Nelson Institute of Environmental Medicine, New York University Medical Center, Long Meadow Road, Tuxedo, NY 10987.

	Abstract

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Background Our 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.

Methods and Results Cockerels 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.

Conclusions (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.

Key Words: arteriosclerosis • butadiene • sidestream smoke

	Introduction

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Cigarette smoking is a major contributor to morbidity and mortality in North America and Western Europe and is becoming a growing health problem in many other parts of the world. In addition to the well-known risks posed to smokers by inhalation of mainstream smoke, there is growing evidence of risks posed to the health of nonsmokers due to involuntary ("passive") inhalation of ETS. The latter is composed of aged and diluted sidestream smoke (85%) and exhaled mainstream smoke (15%). Estimates have been made that passive smoking increases the coronary death rate among US never-smokers by 20% to 70% and that >60 000 deaths yearly from ischemic heart disease are associated with exposure to ETS.¹ Recent in vivo studies from this laboratory have demonstrated that inhalation exposure to ETS is sufficient to promote arteriosclerotic plaque development. Steady-state 6-hour exposures to sidestream smoke for 16 weeks produced significant increases in plaque sizes in cockerels.^{2 3} In addition, the level of carbon monoxide—a surrogate for ETS—in bars in which heavy smoking was taking place equaled or exceeded the carbon monoxide levels in the exposure chambers of cockerels exposed to the steady state sidestream smoke from one cigarette.³ This suggested strongly that plaque-promoting levels of ETS can be encountered routinely by people in smoke-filled environments.

Experiments have demonstrated that exposure to carcinogens present in cigarette smoke profoundly affects the origin and development of both lung cancer and heart disease. Many of these studies have concentrated on individual PAH carcinogens, eg, benzo(a)pyrene, present in the particulate (tar) fraction of cigarette smoke. These compounds are metabolized via enzymes of the cytochrome P-450 system to mutagenic and carcinogenic forms. Previously, we demonstrated that weekly injections of PAH carcinogens, including benzo(a)pyrene and DMBA, at subtumorigenic doses, accelerate arteriosclerotic plaque development in the abdominal aortas of cockerels without causing any increases in plaque numbers.^{4 5 6} We found that a DMBA dosage as low as 5 mg/kg weekly was sufficient to elicit a statistically significant increase in plaque size compared with control plaques.⁴ Noncarcinogenic PAHs, eg, anthracene, were ineffective.

PAH carcinogens are metabolized by arteries of rabbits, chickens, and pigeons^{7 8 9 10} and by cultured human fetal arterial smooth muscle cells.¹¹ A recent study demonstrated that benzo(a)pyrene is metabolized by rat aortas and suggested that this may mimic events occurring in people exposed to cigarette smoke.¹² However, concerns about dose have led to the question of whether PAH carcinogens are responsible for the plaque-promoting activity of cigarette smoke in vivo. For example, even when these agents were administered to cockerels at subtumorigenic doses, the concentrations were still orders of magnitude higher than those at which these agents are found individually in the tar fraction.¹³ In the experiments reported here, we asked whether the in vivo plaque-promoting effects of ETS can be attributed to components other than PAH carcinogens. Cockerels were exposed to one of two prominent ETS components: NNK, a tar fraction component of ETS, or 1,3 butadiene, a vapor phase component of ETS.

The tobacco-specific (particulate fraction) N-nitrosamine, NNK, is a potent carcinogen in rats when administered at high doses.¹⁴ To the best of our knowledge, there are no data that implicate NNK in the development of arteriosclerotic heart disease.

Butadiene (MW=54.09) is a colorless, mildly pungent gas, poorly soluble in water, that is widely used in the synthetic rubber industry. Butadiene is also a component of automotive exhaust and of the vapor phase of ETS, 400 µg per cigarette.¹⁵ The United States produces approximately one fourth of the world's total industrial butadiene output of more than 5x10⁶ metric tons per year. Butadiene was identified as a hazardous air pollutant in the 1990 Clean Air Act amendments.¹⁶ For butadiene, the TLV-the maximum concentration of an airborne contaminant to which a worker can be exposed-is 10 ppm. Butadiene has been reported to be carcinogenic in mice at levels as low as 6.25 ppm¹⁷ and in rats at high levels (1000 to 8000 ppm).¹⁸ Direct data demonstrating that butadiene is a human carcinogen are largely lacking. Butadiene is metabolized via cytochrome P-450 2E1.¹⁹ Most published investigations have failed to identify statistically significant increases in specific disease-associated mortality in workers due to butadiene exposures. One exception is arteriosclerotic heart disease among black men in the butadiene rubber industry. The standardized mortality ratio for black production workers is 1.48 and for black maintenance workers is 1.76.²⁰ There are no animal studies that implicate butadiene in arteriosclerotic plaque development.

The results presented here show clearly that butadiene, at only twice the TLV, stimulates plaque development to the same extent as a moderately high dose of ETS. NNK at a similar relative dose is without effect on plaque development. The results cast further doubt on the atherogenicity of cigarette tar components in vivo and point to vapor phase components as likely plaque-promoting agents in ETS.

	Methods

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Animals
White leghorn cockerels (Avian Services) were adapted to a 12-hour light/dark cycle, and their health and behavior were monitored constantly. Before each study began, the cockerels were distributed randomly into treated and control groups. American Association for Laboratory Animal Care guidelines were followed throughout for housing and care.

Exposures
NNK
Twenty white leghorn cockerels received eight biweekly intramuscular injections (10 mg/kg) of NNK (Chemsyn) dissolved in DMSO (Sigma) starting at 6 weeks of age, according to protocols described previously.^{4 5 6} Ten control cockerels received biweekly injections of DMSO.

Butadiene
Simultaneous inhalation exposures were carried out in stainless steel 1.3-m³ dynamic exposure chambers.²¹ Eight 15-week-old cockerels in each of four chambers were exposed (6 hours per day, 5 days per week, 16 weeks) to a steady state concentration of 20 ppm butadiene (>99.0% purity, Matheson Gases). The butadiene was mixed continually with filtered HEPA, which entered the chambers at a rate of 300 L/min (ie, about 14 air changes per hour). Eight age-matched control cockerels in an adjacent chamber were exposed to the same filtered HEPA without the butadiene. Butadiene concentrations were measured hourly during the daily exposures with a MIRAN 1A infrared gas analyzer (Foxboro Analytical). The analytical wavelength was 11.1 µ, and all readings were compared with a previously prepared calibration curve.

Plaque Analysis
After the animals were euthanatized with Nembutol, at 22 weeks of age in the NNK study and at 31 to 32 weeks of age in the butadiene study, the distal 50 mm of each abdominal aorta was fixed and processed as described previously.^{2 3 4} Plaque location, frequency, and size (plaque index equals plaque cross-sectional area divided by artery wall inner circumference multiplied by 100) were determined for all animals. A PC-based morphometry system using the Bioquant System IV software program described previously^{2 3} was used to generate plaque index data in the NNK study. A Macintosh-based morphometry system using the NIH BioImage 1.55 software package was used to generate the plaque index data in the butadiene study. The same individual coded and processed the tissues for both studies. Two people determined plaque index values in a double-blinded fashion (a different person for each study).

Data Analysis
As reported in all our previous studies, the measured plaque sizes fit log-normal distributions.^{2 3 4 5 6} The logarithms of exposed and control plaque index values were arranged according to increasing value and plotted on log-probability coordinates. Linear regression lines were calculated by least squares analysis and drawn for each data set. ANCOVA was used to test for differences between the two regression lines.²²

	Results

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In the NNK study, plaques were present in all cockerels in both the NNK and control groups. In the butadiene study, microscopic plaques were present in 31 of 32 butadiene-exposed roosters and in all 8 air controls. (All plaques, including those whose plaque index values were too small to be measured reliably, are included in this accounting.) Plaque location along the artery wall was similar in treated and control birds in both studies. The majority of the plaque-containing segments in both groups in each study were found in the most distal 25 to 30 mm of the abdominal aorta. In addition, there were no significant differences between groups in each study in the numbers of plaques per aorta or in the numbers of plaque-containing segments per aorta (Table). In the NNK study there were 7.9 versus 6.8 plaque-containing segments per cockerel (P>.2; DMSO versus NNK). In the butadiene study there were 7.1 versus 6.3 plaque-containing segments per rooster (P>.1; air versus butadiene). These results are consistent with data we have published previously on cockerels exposed to ETS by inhalation^{2 3} or to PAH carcinogens by injection.^{4 5 6} That is, these agents produce neither changes in the location of plaques nor increases in their numbers.

View this table: [in this window] [in a new window]   Table 1. Number of Plaques and Plaque-Containing Segments per Cockerel

The plaque size results with NNK were clear and unexpected. NNK was totally ineffective at augmenting plaque development. Mean plaque index values exceeded median values for both NNK-injected cockerels and DMSO-injected controls (Fig 1). Both skewed data sets fit log-normal distributions. This was consistent with our previous findings^{2 3 4 5 6} (also see butadiene results below). The logarithms of 76 control (DMSO) and 126 NNK-exposed samples were ranked by increasing value and plotted on log-probability coordinates (Fig 2). For each regression line, the linear correlation coefficients (R) exceeded .985. The two regression lines were tested for differences by ANCOVA. The lines had the same slope (0.45), and there were no significant differences in plaque sizes in the two groups (F=0.38; P<.537). These results show that repeated injections with a carcinogen that is prominent in the tar fraction of ETS have no effect on development of arteriosclerotic plaques.

View larger version (17K): [in this window] [in a new window]   Figure 1. Comparison of mean (____) and median (----) plaque index (PI) values per segment for NNK-treated (A) and DMSO-treated (B) cockerels. Note that mean values exceed corresponding medians in 17 of 20 cases.

View larger version (20K): [in this window] [in a new window]   Figure 2. Log-normal distribution of plaque sizes. Plaque indexes (PI) are plotted against probit units (lower abscissa) and cumulative percent of plaque-containing segments (upper abscissa) on log-probability coordinates. Linear regression lines calculated via least squares analysis were drawn for each data set. Seventy-six plaque index values are represented on the upper line (DMSO) and 126 on the lower line (NNK). A probit value of 5 corresponds to the geometric mean of the log-normal distribution. The two lines have nearly identical slopes, and there are no significant differences between plaque sizes in the two groups.

In contrast, butadiene exposures had a striking effect on plaque size. When plaque index values were determined on a segment-by-segment basis, the values were larger for butadiene-exposed animals (Fig 3A) than for the corresponding air controls (Fig 3B). In Fig 4, the logarithms of 46 air control plaque index values and the 210 butadiene plaque index values are plotted on log-probability coordinates, as described above. The 95% confidence limits for the slope of each regression line are also presented. The R values for each line were >.98. There is no overlap anywhere between these two regression lines, which have different slopes (0.49 versus 0.61). The difference in plaque size between the butadiene and air regression groups is highly significant (F=57.24; P<.0001). The mean plaque index of the butadiene group (ie, the median value before log-normal transformation) is 50% greater than the mean plaque index of the air controls. These results are both qualitatively and quantitatively similar to those obtained in our initial ETS (5 cigarette) study.² Thus, daily exposure of young outbred animals to butadiene at a level only 2 times higher than the TLV is sufficient to markedly accelerate arteriosclerotic plaque development.

View larger version (16K): [in this window] [in a new window]   Figure 3. Comparison of mean and median plaque index (PI) values per segment for butadiene (BD)-exposed (A) and air control (B) cockerels.

View larger version (26K): [in this window] [in a new window]   Figure 4. Comparison of log-normal distribution of plaque sizes in butadiene-exposed (upper line) and air control (lower line) cockerels. The 95% confidence limits around each line are indicated by the dashed lines. There are 46 control plaque index (PI) values and 210 butadiene plaque index values. The lines have different slopes, and the differences in plaque size between the two groups are highly significant (see text).

	Discussion

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Studies of the effects of cigarette smoke or its components on morbidity and mortality have emphasized the roles of these agents in carcinogenesis, especially lung cancer. There has been considerably less attention paid to the effects of these agents on cardiovascular disease even though (1) the correlation between cigarette smoking and heart disease is nearly as strong as that between cigarette smoking and lung cancer and (2) each year the number of deaths from heart disease far exceeds the number of deaths from lung cancer. Largely in response to the suggestion that arteriosclerotic plaques can be viewed as benign smooth muscle cell tumors of the artery wall,²³ a number of laboratories, including our own, investigated the role of known PAH carcinogens in the initiation/development of arteriosclerotic plaques.^{4 5 6 14 15 16 17 24 25} The striking effectiveness of these agents on plaque development, albeit at relatively high doses, combined with the presence of PAH carcinogens in cigarette smoke (especially in the tar fraction of sidestream smoke) and the strong epidemiological correlation between smoking and heart disease, led us to investigate first whether exposure to sidestream tobacco smoke would accelerate plaque development. Our studies showed that it does.^{2 3} Six hours of daily exposure to the steady state sidestream smoke from even one filtered moderate tar cigarette was sufficient to accelerate plaque development significantly. We had reported earlier that daily exposure to relatively low levels of carcinogen-rich mainstream smoke (steady state smoke from 2 cigarettes, 2 hours per day, 80 days) also resulted in a significant increase in plaque size.²⁶ In the experiments described here we asked whether individual, non-PAH cigarette smoke carcinogens play a role in plaque development.

Two sets of results are presented. First, we demonstrate that a prominent particulate fraction ETS component, NNK, has no effect on plaque development. NNK is a tobacco-specific nitrosamine, which at high doses is a potent carcinogen in rodents.^{14 27 28 29} In vitro findings have strongly implied that NNK behaves similarly in humans.^{30 31} However, in all these studies, the effective doses of NNK were far higher than the actual doses supplied during the combustion of cigarettes. The dose selected for the experiments described here, 5 mg/kg, is in the range described for optimal formation of the promutagenic O-6 methylguanine adduct in rat lungs.³² The injection protocols used in this study for NNK closely matched those we used previously to demonstrate that some PAH carcinogens, including benzo(a)pyrene, which, like NNK and butadiene is a component of cigarette smoke, can markedly accelerate plaque development.^{4 5 6} Significant acceleration of plaque development by benzo(a)pyrene was evident when as few as 6 treated and 6 control animals were compared.⁶ In the experiments described here, 20 NNK-treated and 10 control cockerels were examined. Thus, NNK, tested in a proven protocol for the promotion of arteriosclerosis, does not accelerate plaque development.

Second, in contrast to the results from the NNK study, those from the butadiene study demonstrate that this vapor phase component of ETS accelerates arteriosclerotic plaque development. Here, butadiene effectively promoted plaque development at a concentration only twice the TLV. This represents the first time that a single cigarette smoke component has been shown to directly affect the development of heart disease at doses that are within an order of magnitude of those found in cigarette smoke. Butadiene is far more concentrated in sidestream smoke than either benzo(a)pyrene or NNK. In ETS from 1R4F cigarettes, the model moderate tar- and nicotine-filtered cigarette used in our previous studies,^{2 3} the butadiene concentration is 1000 times that of NNK and 2500 times that of benzo(a)pyrene.¹³

At best, butadiene has proven to be a very weak carcinogen. The 20-ppm dose of butadiene effective here as a promoter of plaque development in cockerels stands in strong contrast to the butadiene doses of 1000 ppm and higher that are necessary to induce tumor formation in rats.¹⁷ Evidence from occupational studies that butadiene may be a human carcinogen is very limited. There is one published epidemiological study linking deaths of black male workers from arteriosclerotic heart disease to chronic occupational exposure to butadiene.²⁰ For black male production workers in styrene-butadiene manufacturing plants in the United States from 1943 to 1982, the standardized mortality ratio for deaths from arteriosclerotic heart disease was 1.47. For white male production workers in the same facilities, the standardized mortality ratio was 0.91. Among black men in maintenance jobs, the standardized mortality ratio for arteriosclerotic heart disease deaths jumped to 1.76, nearly twice the value for white maintenance workers in the same facilities. The results presented here provide direct experimental support for these epidemiological observations.

There is also a recent report that a subset of the black population exhibits polymorphisms in the cytochrome P-450 1A1 gene (CYP1A1) and that these may be associated with an increased susceptibility to lung cancer.³³ CYP1A1 metabolizes PAH carcinogens, including some in ETS. CYP2E1 metabolizes butadiene in human and rodent livers.¹⁹ There are no reports that we know of in which (1) genetic polymorphisms in human CYP2E1 have been investigated or (2) the enzyme has been identified in human arteries. However, the existence of genetic polymorphisms in at least one member of the cytochrome P-450 family, in a population with an increased standardized mortality ratio for arteriosclerotic heart disease that is also exposed occupationally to butadiene, suggests that the cockerel model system can be exploited to understand the mechanism(s) underlying this butadiene effect.

The cockerel/rooster model is one of the few sensitive and discriminating animal models for studying nondietary environmental contributions to the development of arteriosclerosis. Among cigarette smoke constituents tested previously, benzo(a)pyrene, like butadiene, accelerates plaque development,¹³ whereas carbon monoxide, even at relatively high levels (200 ppm) is without effect.³⁴ We recently published two sets of studies on acceleration of plaque development resulting from exposure to sidestream cigarette smoke.^{2 3} In the first of these, in which eight cockerels per chamber were exposed to steady state sidestream smoke from 5 cigarettes, the steady state exposure chamber butadiene levels were calculated to be 0.7 ppm. Assuming 50% retention of inhaled sidestream smoke during each 6-hour exposure period, the butadiene body burden to each cockerel at the end of each exposure was 105 µg/kg. In contrast, the body burden after a 6-hour exposure to 20 ppm butadiene is 28 times higher.

Although butadiene and NNK are each metabolized at different rates and by different components of the cytochrome P-450 system, a comparison of the carcinogenicity, plaque-promoting potential, and concentration in ETS of these compounds can be instructive. In the studies reported here, 80 days of inhalation exposure to butadiene resulted in a total dose of 240 mg/kg. In carcinogenesis studies with female mice, the lowest effective dose of butadiene was 6.25 ppm, 6 hours per day, 5 days per week, for 2 years.¹⁷ For a 25-g mouse, this translates to a total butadiene dose of 500 mg (ie, on a milligram per kilogram basis, 80 times greater than the cockerel dose). In rats, the lowest effective carcinogenic dose of butadiene is 1000 ppm, 6 hours per day, 5 days per week, for 2 years.¹⁷ For a 250-g rat, this translates to a total butadiene dose of 75 g (ie, on a milligram per kilogram basis, 1000 times greater than the cockerel dose). In carcinogenesis studies with F344 rats, a total dose of 34 mg/kg NNK over a 2-year period produced lung tumors in 20% of the animals.³⁵ A total dose of 136 mg/kg over the same time period yielded lung tumors in 90% of the animals. In the NNK studies reported here with cockerels, the total dose over a 16-week period was intermediate (80 mg/kg) between those two doses, yet NNK had no effect on plaque number or size.

The lack of a plaque-promoting effect of the tar component, NNK, is consistent with results from plaque promotion studies with whole tar extract. Weekly injections (25 mg/kg) of acetone–dry ice extracts of cigarette tar into cockerels were ineffective at accelerating plaque development (Penn et al, manuscript in preparation). Since butadiene is found in such high concentration in ETS relative to NNK and benzo(a)pyrene, it is probably one of the compounds most responsible for the arteriosclerotic plaque–promoting potential of ETS. However, literally thousands of compounds have been identified in the gas and particulate fractions of cigarette smoke, both sidestream and mainstream.³⁶ Thus, other compounds besides butadiene must contribute to the plaque-promoting qualities of ETS. Our NNK and tar injection studies strongly suggest that the bulk of the plaque-promoting factors in ETS may be in the vapor phase.

In the experiments reported here, butadiene exposure levels, while higher than those present in sidestream smoke from one cigarette, were still only twice the occupational TLV. This, combined with the elevated standardized mortality ratio for death from arteriosclerotic heart disease among black production and maintenance workers in the butadiene rubber industry, strongly suggests that the TLV for butadiene may have to be lowered.

	Selected Abbreviations and Acronyms

 

DMBA = 7,12 dimethylbenz(a)anthracene DMSO = dimethylsulfoxide ETS = environmental tobacco smoke HEPA = high-efficiency particulate air NNK = N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone PAH = polynuclear aromatic hydrocarbon TLV = threshold limit value

	Acknowledgments

 
This work was supported in part by a National Institute of Environmental Health Sciences Center Grant (ES 00260). We thank Ken Magar, Michael Barbieri, and Jim Currie for excellent technical assistance and Dr Lung-Chi Chen for advice and help with the statistical analyses.

Received June 8, 1995; revision received August 21, 1995; accepted September 25, 1995.

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