Carroll A. Snyder

The inhalation toxicology of benzene: Incidence of hematopoietic neoplasms and hematotoxicity in AKRJ and C57BL6J mice

Abstract AKR J mice and C57BL 6J mice were given lifetime exposures to 100 and 300 ppm benzene, respectively. Peripheral blood cell counts were obtained biweekly throughout the exposures. Anemia and lymphocytopenia were produced in benzene-exposed AKR mice. Twenty percent of the exposed AKR mice developed bone marrow hypoplasia, compared to 2% for the controls. The benzene exposures did not alter the incidence or induction time of the viral-induced lymphomas commonly seen in AKR mice. In C57BL mice, exposure to benzene produced anemia, lymphocytopenia, and neutrophilia accompanied by a left shift. Thirteen (33%) of the exposed C57BL mice developed bone marrow hyperplasia and in four of these mice, hyperplasia was essentially limited to granulopoietic elements. None of the control C57BL mice developed bone marrow hyperplasia. In benzene-exposed C57BL mice there was a significant increase in the incidence of hematopoietic neoplasms including six cases (15%) of thymic lymphoma. Although two control mice (5%) died with lymphoma neither of these tumors involved the thymus. Thymic lymphoma is rare in C57BL mice but can be produced by ionizing radiation and chemical carcinogens.

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.

DIFFERENTIAL DNA-PROTEIN CROSSLINKING IN LYMPHOCYTES AND LIVER FOLLOWING CHRONIC DRINKING WATER EXPOSURE OF RATS TO POTASSIUM CHROMATE

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. CONCLUSION (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.

Acute and chronic dose/response effects of inhaled benzene on multipotential hematopoietic stem (CFU-S) and granulocyte/macrophage progenitor (GM-CFU-C) cells in CD-1 mice

Carcinogenic chromium (VI) compounds are persistent environmental contaminants with potential for human exposure through drinking water. One lesion associated with chromium (VI) exposure is the formation of DNA-protein crosslinks (DPC). In an attempt to develop markers of chromium exposure, the formation of DPC in lymphocytes was investigated. Fisher 344 rats were exposed to K2CrO4 in their drinking water for 3 and 6 weeks at concentrations of 100 and 200 ppm chromium. No DPC could be detected in isolated splenic lymphocytes using the alkaline elution technique or by using a polyclonal antibody to chromate-induced DPC. However, increased complexing of proteins with DNA was demonstrated in liver following 3 weeks of exposure at both 100 and 200 ppm chromium. Intraperitoneal administration of potassium chromate did not induce detectable DPC in lymphocytes; however, an increased association of proteins with isolated DNA was detected in the liver. DPC were also induced in isolated splenic lymphocytes following a 2-hr exposure in vitro to 100 microM K2CrO4 in a salts-glucose medium. Although chromium was detected in blood, liver, and kidney, blood levels were comparatively much lower. A comparison of chromium levels required to induce DPC in lymphocytes in vitro and the amount absorbed orally suggests that the white blood cell chromium levels following oral exposure may be too low to induce measurable DNA-protein crosslinks in lymphocytes.

Repeated exposure of C57B1 mice to inhaled benzene at 10 ppm markedly depressed erythropoietic colony formation

Abstract Many blood dyscrasias are associated with hematopoietic stem cell anomalies. In order to investigate the interaction of inhaled benzene with hematopoietic stem cells, marrow and spleen cells from male CD-1 mice were assayed for CFU-S by the spleen colony method, and for GM-CFU-C by an in vitro agar technique following benzene exposure using a number of regimes. Specifically, these consisted of a 6 hr/day × 5 days exposure to either 1.1, 9.9, 103, 306, 603, 1276, 2416, or 4862 ppm (Experiment 1); a 6 hr/day × 5 days/week × 50 days exposure to 9.6 ppm (Experiment 2); and a 6 hr/day × 5 days/week × 26 weeks exposure to 302 ppm (Experiment 3). In Experiment 1 femoral and splenic cellularites were significantly reduced at concentrations ≥ 103 ppm. Marrow concentration of GM-CFU-C was equivalent to or greater than control values at all levels, however, splenic GM-CFU-C concentration was decreased at 103 ppm and above. Femoral and splenic CFU-S and GM-CFU-C per organ were depressed at 103 ppm and above. No change in colony/cluster ratio was observed. Since changes in stem cell number were detected at 100 ppm, Experiment 2 was designed to compare the effects of a 10-ppm exposure delivered over 50 days with the 100-ppm exposure delivered over 5 days. In Experiment 2, no detectable changes were observed in bone marrow, but splenic cellularity and the number and concentration of splenic CFU-S were elevated vs matched control. Experiment 3 repeated a regime that produced two cases of myeloid leukemia in CD-1 mice and a marked depression was observed in marrow and spleen cellularity. The concentration and number of marrow and spleen CFU-S and marrow GM-CFU-C were also depressed. The number of splenic GM-CFU-C were also reduced, however, splenic GM-CFU-C concentration was increased relative to control. Splenic colony/cluster ratio was also significantly reduced in this experiment. These data demonstrate that 5-day inhalation exposure to benzene concentrations 10 times the current TLV depresses marrow and splenic CFU-S and GM-CFU-C with splenic cells showing greater sensitivity. Stem cell depletion seems, therefore, to be involved in the pathogenesis of benzene-induced hematotoxicity.

Acute and chronic dose/response effect of benzene inhalation on the peripheral blood, bone marrow, and spleen cells of CD-1 male mice

Exposure of C57Bl mice to 10 ppm benzene (the current occupational exposure limit) for 6 h/day, 5 days/week causes a progressive depression in the in vitro colony forming ability of one of the erythroid progenitor cells, the colony-forming unit-erythroid (CFU-E). Colony growth of cells from exposed mice was only 5% of control colony growth after 178 days of exposure. Burst-forming-cell growth was depressed to 55% of control growth after 66 days but returned to control growth values at 178 days. In addition, benzene-exposed mice exhibited depressions in the numbers of splenic nucleated red cells and in the numbers of circulating red cells and lymphocytes. These results suggest that low-level exposure to benzene may be hematotoxic.

The carcinogenicity of discontinuous inhaled benzene exposures in CD-1 and C57Bl/6 mice

Abstract Inhaled benzene hematotoxicity to recognize stem cell progeny was studied in male CD-1 mice exposed for 6 hr/day × 5 days to one of the following mean benzene concentrations: 1.1, 9.9, 103, 306, 603, 1276, 2416, and 4862 ppm. Additional groups of mice were exposed for 6 hr/day × 5 days/week × 10 weeks to 9.6 ppm, or 6 hr/day × 5 days/week × 26 weeks to 302 ppm. Following the 5-day exposures, granulocytopenia and lymphocytopenia were observed at levels ≥ 103 ppm with no change in WBC differential. RBC counts were depressed only at the two highest exposure levels while hematocrits were variably affected and showed no clear dose/response effect. Marrow and splenic cellularities were reduced at all levels ≥ 103 ppm. Marrow lymphocytes, splenic lymphocytes, and marrow granulocytes were reduced in accordance with the reduction in total cellularity, however, splenic granulocytes and spleen weights were depressed at almost all exposure levels. Nucleated RBCs in the marrow and spleen were depressed at almost all levels ≥ 103 ppm. Exposure for 50 days to 9.6 ppm benzene, a total dose equivalent to that delivered over 5 days at 103 ppm, induced no detectable changes in the peripheral blood or bone marrow, however, increases in splenic weight and cellularity were observed. Twenty-six weeks of exposure to 302 ppm benzene resulted in lymphocytopenia and anemia. Marrow cellularity was reduced to 32% of control and was due primarily to a reduction in lymphocytes and granulocytes. Spleen cellularity and weight were reduced to 17 and 67% of control, respectively. Decreased spleen cellularity was due primarily to a reduction in lymphocytes to 5% of control. These extended exposures to 302 ppm benzene resulted in atypical cell morphology in the peripheral blood, bone marrow, and spleen.

Mice Exposed in Utero to 20 ppm Benzene Exhibit Altered Numbers of Recognizable Hematopoietic Cells Up to Seven Weeks after Exposure

Groups of male C57Bl and CD-1 mice were exposed to benzene via inhalation using two different exposure protocols. One protocol consisted of repetitive week-long exposures to 300 ppm benzene (6 h/d×5 d/wk) interrupted by 2 weeks of non-exposure. The exposure pattern (1 week of exposure followed by 2 weeks of non-exposure) was continued until the death of the last exposed animal. The second protocol consisted of exposures to 1200 ppm benzene (6 h/d×5 d/wk) for 10 weeks. Exposures were then terminated and the animals allowed to live out their lives. For each protocol, appropriate age-matched control mice received comparable exposures to filtered, conditioned air. The discontinuous exposure patterns mimic the patterns of exposure often encountered in the workplace and, in addition, prolong the survival of exposed animals so as to maximize potential tumorigenic responses. Both exposure protocols were markedly hematotoxic to both mouse strains as measured by peripheral blood counts. Both strains of mice responded to the intermittent 300 ppm benzene exposures with elevated incidences of malignant tumors. Particularly noteworthy was a 35% incidence of zymbal gland tumors in the C57Bl mice. In contrast, only the CD−1 mice responded to the 1200 ppm benzene exposures delivered over 10 weeks with elevated tumor incidences. A 46% incidence of lung adenoma was particularly striking in these mice. Neither of the benzene exposure protocols induced elevated incidences of leukemia/lymphoma in either strain. These studies demonstrate that discontinuous exposures to benzene are tumorigenic and that a lifetime exposure to benzene, even if delivered at a lower concentration and in an intermittent exposure pattern, is more tumorigenic than a short-term exposure to benzene.