1. Urethane is a fermentation by-product abundantly found in alcoholic beverages and foods and significant human exposure to this chemical may occur. Recent National Toxicology program (NTP) studies demonstrated that urethane is a potent animal carcinogen and is classified as """"""""reasonably anticipated to be a human carcinogen."""""""" Contrary to earlier reports, studies in this laboratory demonstrated that CYP2E1 and not esterases is the principal enzyme responsible for urethane epoxidation. Our current hypothesis centers on the premise that urethane metabolism to the epoxide intermediate (vinyl carbamate) via cytochrome P450 2E1 (CYP2E1) is responsible for the formation of DNA adducts and subsequent mutagenesis and carcinogenesis. In order to test this hypothesis, urethane was administered to CYP2E1-null or wild-type mice at 1, 10, or 100 mg/kg/day, 5 days/week for 6 weeks. Significant increase in the mutagenicity of urethane was observed in wild-type mice vs. CYP2E1-null mice. Further, after 7 months recovery (without chemical exposure), animals were sacrificed and evaluated histopathologically. This study revealed that CYPE1-null mice exhibited significantly lower incidence and number of tumors/lung of bronchoalveolar adenomas/carcinomas in the lungs, adenomas in the Harderian glands, and hemangiomas/hemangiosarcomas in the liver and heart. In conclusion, this data directly demonstrated for the first time that CYP2E1 plays an essential role in urethane-induced mutagenicity and carcinogenesis, presumably via epoxidation. 2. Metabolism of aliphatic nitriles is thought to proceeds via conjugation with glutathione or oxidation via cytochrome P450 enzymes to form epoxide intermediates. Recent National Toxicology Program (NTP) reports demonstrated significant variations in the effects of these chemicals and that male rodents are more sensitive to the toxicity/mortality of these chemicals than females. The objectives of this project include the assessment of the enzymatic basis of epoxidation of aliphatic nitriles, the investigation of the structure- metabolism-activity relationships, and the mechanisms of the greater sensitivity of males vs. females. Present results demonstrated that 1) while CYP2E1 is the only enzyme responsible for the epoxidation of acrylonitrile, additional CYPs are involved in the epoxidation of methacrylonitrile, crotononitrile, and allylonitrile; 2) epoxidation is an essential pathway leading to cyanide formation; 3) greater blood cyanide levels were detected in male mice treated with aliphatic nitriles which may explain the greater sensitivity of male vs. female mice to the toxicity/mortality caused by these chemicals; and 4) higher expression of CYP2E1 and EH in male mice may contribute to increased epoxidation of aliphatic nitriles and subsequent metabolism to cyanide.
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