Cytochrome P450 (CYP) super family of enzymes carry out oxidative metabolism of a variety of endogenous and foreign chemicals. Cytochrome P450 2E1 (CYP2E1) is a well conserved CYP and is expressed in liver, kidney, nasal mucosa, brain, lung, and other tissues. CYP2E1 is responsible for the bioactivation of a variety of environmentally important xenobiotics including 1,3-butadiene, acetaldehyde, acetaminophen, aniline, benzene, carbon tetrachloride, trichloroethylene, dichloroethylene, ethylene glycol, vinyl chloride, and nitrosamines. Most of these chemicals undergo oxidative metabolism via the CYP enzymes to form epoxide intermediates. Environmental chemicals that are metabolized to epoxides cause similar pattern of carcinogenicity in animals. It is hypothesized that formation of the epoxide intermediates in vivo is responsible for the toxicity, mutagenicity, and carcinogenicity of most of these chemicals. The objectives of this research include the investigation of the metabolic activation of chemicals to epoxide intermediates and the role of epoxides in chemical-induced toxicity, mutagenicity, and carcinogenicity. In order to achieve these goals, we are using CYP2E1 and epoxide hydrolase knock-out mice to investigate the metabolism of urethane (ethyl carbamate) and acrylonitrile. Urethane is a fermentation by-product abundantly found in alcoholic beverages and foods and significant human exposure to this chemical may occur. Urethane is a animal carcinogen and is classified as """"""""reasonably anticipated to be a human carcinogen."""""""" Early studies indicated that while CYP2E1 is involved, esterases are the primary enzymes responsible for urethane metabolism. Further, urethane metabolism to the epoxide intermediate (vinyl carbamate) via CYP2E1 is thought to be responsible for the formation of DNA adducts and subsequent carcinogenesis. Using CYP2E1 knock out (KO) mice, current studies were undertaken as part of an overall project intended to assess the role of CYP2E1 in the metabolism, toxicity, mutagenicity, and carcinogenicity of urethane, with special emphasis on the role of epoxide intermediate. Comparison of urethane metabolism in wild-type (WT) and KO mice demonstrated that CYP2E1 is the principal enzyme responsible for the metabolism of urethane. However, esterases and CYPs other than CYP2E1 may play a minor role in urethane metabolism. Using CO2 elimination data, a two-compartment model was constructed and the t1/2 of urethane was estimated at 1.75 and 35 hr in WT and KO mice, respectively. This work also showed a great potential for the bioaccumulation of urethane in the absence of CYP2E1-mediated metabolism in KO mice. Whether the bioaccumulation of urethane leads to toxicity remains unknown and is the subject of additional investigation in this laboratory. Acryonitrile (AN) is a heavily produced unsaturated nitrile, which is used in the production of synthetic fibers, plastics, and rubber. AN is a multi-site rodent carcinogen and suspected human carcinogen. Metabolism of AN proceeds via conjugation with glutathione or epoxidation via CYP2E1 to cyanoethylene oxide (CEO). It was hypothesized that CEO metabolism via epoxide hydrolase (EH) is the primary pathway for cyanide formation. Further, it was hypothesized that CEO is responsible for the mutagenicity/carcinogenicity of this chemical. The objectives of this work include the assessment of the enzymatic basis of AN metabolism to CEO and cyanide and the role of metabolism in the toxicity and carcinogenicity of this chemical. Comparison of the metabolism of 13C-AN in WT and KO mice demonstrated that WT mice excreted metabolites derived from CEO and from direct glutathione conjugation with AN. Further, blood cyanide levels increased in the blood of WT mice in a dose-dependent manner. In contrast, no metabolites derived from CEO were found in the urine of AN treated KO mice. Cyanide levels in the blood of KO mice treated with AN were not statistically different from levels found in the blood of vehicle-treated mice. Based on these studies, CYP2E1 is identified as the only enzyme responsible for the epoxidation of AN. Further, this work demonstrated that epoxidation of AN via CYP2E1 is essential for AN metabolism to cyanide and metabolism of CEO via EH may play a major role in the formation of cyanide in mice. Gender differences in the expression of EH may explain the higher cyanide levels and the greater sensitivity of male vs. female mice. These data also indicate that, in the absence of CYP2E1 and the capability to oxidize AN, the internal dose of AN in the KO mice increased to greater than 3 times the levels in WT mice. Further, this work also demonstrated that hemoglobin adducts increased in a dose-dependent manner and were significantly higher in KO mice. Finally, this data suggest that hemoglobin adducts provide a long-term measure of the internal dose of AN and may be used as a marker of humans exposure to this chemical.
