Xenobiotic Metabolism

Environmental Health Sciences

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Abstract

Tg.AC and p53+/- genetically altered mice are potential models for chemical carcinogenicity testing by the National Toxicology Program. Some carcinogens are metabolically activated, therefore it is imperative that the altered genome of either mouse model does not change xenobiotic metabolizing capability and capacity inherent in the corresponding FVB/N or C57BL/6 wildt ype. We have demonstrated that urinary metabolite profiles of Tg.AC and p53+/- mice treated with benzene, methacrylonitrile, or ethoxyquin, were similar to that of the corresponding treatment groups of the respective wild type. These three representative substrates undergo biotransformation through several enzymatic pathways, including both phase I and II metabolism. The apparent major oxidative enzyme involved in metabolism of benzene and methacrylonitrile is CYP2E1. Both compounds undergo a measure of glutathione conjugation involving GST. As a follow-up to previous experiments, the present work has characterized relative expression levels of CYP2E1, CYP1A2, and CYP3A in liver microsomal protein and GST in liver cytosol of Tg.AC, p53+/-, FVB/N, and C57BL/6 mice. Western blot analysis indicated that the levels of these representative enzymes were quantitatively similar in liver of each transgenic mouse line and its corresponding wild type strain. CYP2E1 activity, as measured by chlorzoxazone hydroxylation, was also similar between each transgenic mouse line and the respective wild type strain. The present work provides additional evidence that the inherent ability of Tg.AC and p53+/- mice to metabolize xenobiotics would not be a factor in data interpretation of studies using either of these two transgenic mouse lines. Pulegone is a monoterpene found in the essential oil of several mints, including Pennyroyal. These mints are used in flavoring food and beverages. Pennyroyal and Pennyroyal oil have also been used as an abortifacient. The use of Pennyroyal oil, which contains about 85% pulegone, has resulted in serious toxicity and death. Pulegone has been nominated to the NTP for toxicity and carcinogenicity studies. As part of those studies metabolism studies in rats and mice are being conducted by our lab. Fourteen metabolites were isolated by HPLC and characterized by NMR, MS and UV spectroscopy. The metabolites demonstrate that pulegone is metabolized by three major pathways: 1) hydroxylation followed by glucuronidation; 2) reduction to menthone/isomenthone followed by hydroxylation; 3) Michael addition of glutathione. The latter result indicates that pulegone can react directly with tissue nucleophiles. Our distribution studies indicated that there may be bioaccumulation of pulegone-derived material following multiple doses. A multiple dose study was done to study tissue accumulation and metabolic profile changes. Female rats dosed for four consecutive days with 14C-pulegone had higher tissue concentrations of pulegone-derived material (increases of 244, 221, and 272 % in blood, liver and kidney) than single dosed rats substantiating the hypothesis that repeated daily dosing will result in accumulation of pulegone-derived material. Rats were also dosed with three daily non-radioactive doses followed by a single 14C dose. There were shifts in the percentages of several urinary metabolites differing primarily in the site of hydroxylation of the pulegone and in the formation of phenol metabolites. Liver microsomes from multiply dosed rats showed a decrease in cytochrome P450 1A2, as measured by Western blot analysis, but cytochrome P450 2E1, thought to be principally involved in the metabolism of pulegone, was unaffected.