Grantee Research Project Results
Final Report: Pulmonary Metabolism of Benzene
EPA Grant Number: R826191Title: Pulmonary Metabolism of Benzene
Investigators: Carlson, Gary P.
Institution: Purdue University
EPA Project Officer: Aja, Hayley
Project Period: October 20, 1997 through October 19, 2000
Project Amount: $389,120
RFA: Exploratory Research - Human Health (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
Benzene is associated with leukemia due to the action of its metabolites on the bone marrow. The hypothesis tested was that the lung makes a significant contribution to the bioactivation of benzene, either directly inhaled or reaching the lungs via the circulation. The first objective was to compare benzene metabolism by human lung and liver microsomes with that by microsomes from experimental animals. The second objective was to examine the metabolism of benzene that is inhaled and that which is systemically delivered, using the isolated perfused rabbit or rodent lung (IPRL) to approximate an in vivo situation as closely as possible. The third objective was to identify the pulmonary cell types responsible for benzene metabolism.Summary/Accomplishments (Outputs/Outcomes):
Studies on the hepatic and microsomal metabolism of benzene in the rat, mouse, rabbit, and human indicate that the lung as well as the liver can biotransform benzene in all four species. In comparing species, the rat is most similar to the human in oxidative pulmonary microsomal metabolism of benzene to phenol and hydroquinone but not catechol, which was not found to be formed in human microsomes, at all benzene concentrations studied ranging from 24 mM to 1,000 mM. In oxidative hepatic microsomal benzene metabolism, the rat is most similar to the human at the lower concentrations, 24 M and 200 M. At the higher benzene concentrations, 700 M and 1,000 M, the mouse is most similar to the human in phenol formation. Thus, overall, the rat is most similar to the human in oxidative benzene metabolism at the lower environmental levels in both liver and lung. In all species, the cytochrome P450 enzyme system responsible for benzene metabolism approached saturation in hepatic microsomes but not in pulmonary microsomes. The hydroquinone to phenol ratio decreased in the hepatic microsomes for all species, suggesting that hepatic microsomes have a greater affinity for benzene than for phenol. The opposite effect occurred in pulmonary microsomes from all species except for rabbit. This confirms the findings of a number of researchers as to the competitive interactions of benzene and phenol. Considering that the lung is the initial site of contact for inhaled benzene, receives 100 percent of the cardiac output, and has been found to be the site of tumor formation in animals exposed to benzene, these studies strengthen the argument that the lung plays an important role in benzene metabolism and, therefore, toxicity. Our results also indicate the importance of considering relevant benzene concentrations when measuring metabolic rates, since qualitative as well as quantitative differences were observed between the high and low substrate concentrations.Studies were conducted with specific chemical inhibitors to determine which cytochromes P450 are the most important in the metabolism of benzene. These inhibitors were 300 mM diethyldithiocarbamate (DDTC) as an inhibitor of CYP2E1, 1 mM -methylbenzylamino-benzotriazole (MBA) as an inhibitor of CYP2B, and 5 mM 5-phenyl-1-pentyne (5P1P) as an inhibitor of CYP2F. CYP2E1 was found to be the cytochrome P450 isozyme most responsible for hepatic microsomal benzene metabolism in mouse, human, and rat. Results of pulmonary microsomal experiments indicated that members of the CYP2F subfamily as well as CYP2E1 are involved especially at the lower, environmentally relevant benzene concentration studied. Isozymes of the CYP2B subfamily were not notably involved at the benzene concentrations studied. This is not surprising because other researchers found CYP2B1 to be capable of metabolizing benzene to an extent comparable to CYP2E1 only at concentrations much higher than that used in our studies (24 mM and 1,000 mM).
Confirmation of the importance of CYP2E1 in the liver and lung was shown in studies in which CYP2E1 knockout mice were used, and activities were compared with those of wild-type mice. The results showed that CYP2E1 is the most important isozyme in the liver, accounting for 96 percent of the total hydroxylated metabolite formation. However, in the lung CYP2E1 was responsible for only 45 percent of the formation of total hydroxylated metabolite. When chemical inhibitors of CYP2E1 and CYP2F2 were used to further examine the contributions of these isozymes to benzene metabolism, the results confirmed the finding that while CYP2E1 is the most important isozyme in the liver, CYP2F2 and CYP2E1 are both significantly involved in the lung.
Species and tissue comparisons also were made of phenol metabolism using microsomes from the lungs and livers of non-Swiss albino, CYP2E1 knockout, and wild-type mice. Cytochrome P450 isozymes involved were again evaluated utilizing chemical inhibitors of CYP2E1, CYP2B, and CYP2F2. Based on a comparison of the knockout and wild-type mice, CYP2E1 was found to be responsible for only approximately 50 percent of 20 M phenol metabolism in the liver. This suggests another isozyme(s) is involved in hepatic phenol metabolism. In pulmonary microsomes, both CYP2E1 and CYP2F2 were significantly involved.
To evaluate the metabolism of benzene using a system that more clearly mimics the in vivo situation, the isolated perfused lung preparation was utilized. Lungs from the rabbit, rat, and mouse were employed with exposure via the pulmonary vasculature. In all three species, the metabolism of benzene delivered via the pulmonary circulation was shown to occur with phenol being the primary metabolite. Phenylsulfate also was detected in perfusate from rabbits and mice but at low levels. Hydroquinone and phenol were identified in tissue. Benzene metabolism was concentration dependent in mice because when three concentrations (55 M, 120 M, and 200 M) were used, greater metabolism was observed at the highest concentration as compared to the lower two. To evaluate the ability of the lung to metabolize inhaled benzene, the isolated perfused mouse lung was exposed to benzene via the trachea (maximum of 175 ppm). Benzene was metabolized as it crossed from the air space into the perfusate. These results demonstrate that the lung can metabolize benzene in an in vivo simulation when exposed via the pulmonary vasculature or via inhalation.
Experiments also were conducted to examine the types of cells in the lung that are associated with the metabolism of benzene. Clara and type II pneumocytes were isolated from mouse lung. The use of chemical inhibitors was included to again examine the importance of CYP2E1 and CYP2F2. When Clara cells were isolated alone from NSA mice, they were able to metabolize benzene. As in the microsomal studies, both DDTC and 5P1P had inhibitory effects. Hydroquinone was detected in several samples but not consistently. When both Clara cells and type II cells were isolated by centrifugal elutriation and benzene metabolism was measured, the data were not of sufficient quality to draw conclusions regarding the activity of the type II pneumocytes.
Journal Articles on this Report : 6 Displayed | Download in RIS Format
Other project views: | All 14 publications | 6 publications in selected types | All 6 journal articles |
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Carlson GP, Mantick NA, Powley MP. Metabolism of styrene by human lung and liver. Journal of Toxicology and Environmental Health 2000;59(8):591-595. |
R826191 (Final) |
not available |
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Powley MW, Carlson GP. Species comparison of hepatic and pulmonary metabolism of benzene. Toxicology, Volume 139, Issue 3, 6 December 1999, Pages 207-217. |
R826191 (Final) |
not available |
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Powley MW, Carlson GP. Cytochromes P450 involved with benzene metabolism in hepatic and pulmonary microsomes. Journal of Biochemical and Molecular Toxicology 2000;14(6):303-309. |
R826191 (Final) |
not available |
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Powley MW, Carlson GP. Hepatic and pulmonary microsomal benzene metabolism in CYP2E1 knockout mice. Toxicology 2001, Volume: 169, Number: 3 (DEC 28), Page: 187-194. |
R826191 (Final) |
not available |
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Powley MW, Carlson GP. Cytochrome P450 isozymes involved in the metabolism of phenol, a benzene metabolite. Toxicology Letters 2001, Volume: 125, Number: 1-3 (DEC 15), Page: 117-123. |
R826191 (Final) |
not available |
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Powley MW, Carlson GP. Benzene metabolism by the isolated perfused lung. Inhalation Toxicology 2002;14(6):569-584 |
R826191 (Final) |
not available |
Supplemental Keywords:
air, health effects, solvents, metabolism., Health, Scientific Discipline, Toxics, Toxicology, Environmental Chemistry, Health Risk Assessment, HAPS, Risk Assessments, Biochemistry, bone marrow, detection, animal model, benzene, pulmonary vasculature, inhalation, human exposure, pulmonary metabolism, Benzene (including benzene from gasoline), leukemia, bioaccumulation, biomarker, carcinogenic, human health riskProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.