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Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse and rat liver microsomes
1Chemical Industry Institute of Toxicology, Research Triangle Park NC, USA
2Pennsylvania State University College of Medicine, Hershey PA, USA
3To whom correspondence should be addressed
1,3-Butadiene is carcinogenic in B6C3F1 mice and Sprague- Dawley rats, and has been classified as a probable human carcinogen. The genetic basis for butadiene carcinogenicity is likely mediated by its metabolite, 1,2:3,4-diepoxybutane (BDE). Oxidation of butadiene to 1,2-epoxy-3-butene (BMO) and further activation to BDE is catalysed by cytochrome P450 (CYP) isozymes. The production of BMO from butadiene is mediated by CYP2E1 and, at high butadiene concentrations, by CYP2A6. The purpose of the present study was to identify which human CYP isozymes have the ability to oxidize BMO to BDE, and to determine the extent to which this reaction occurs in B6C3F1 mouse, Sprague-Dawley rat, and human liver microsomes. Of the human cDNA-expressed CYP isozymes tested, only CYP2E1 formed detectable concentrations of BDE at 80 µ.M BMO. CYP2E1 and CYP3A4 were active at 5.0 mM BMO. Interindividual and interspecies variation in the Initial rate of oxidation of 80 µM BMO to BDE was determined using 10 samples of human liver microsomes and single pooled samples from rats and mice. Those experiments revealed a 60-fold variation in activity among 10 human liver samples (range: 0.005–0.324 nmol/mg protein/mm). Rates of BMO oxidation for mouse and rat liver microsomes were 0.473 and 0.166 nmol/mg protein/mm, respectively. Apparent kinetic constants for the oxidation of BMO to BDE by four human microsomal preparations, and pooled samples from mice and rats were estimated from detailed investigations of BMO oxidation at various BMO substrate concentrations. Apparent Km for the human liver samples ranged from 0.304–0.880 mM, and Vmax values ranged from 0.38 to 1.2 nmol/mg protein/mim The apparent values of Km and Vmax for mouse liver microsomes were 0.141±0.007 mM (mean±SE) and 1.303±0.141 nmol/mg protein/ min, respectively. For rat liver microsomes, apparent Km and V were 0.145±0.036 mM and 0.408±0.031 mnol/mg protein/mm, respectively. Measured rates of BDE formation correlated well with CYP2E1 protein concentrations in the human microsome samples. These results implicate human CYP2E1 as a hepatic isoform responsible for the oxidation of BMO to BDE at low concentrations of BMO. Moreover, our in vitro results reveal that microsomes prepared from human, rat and mouse liver possess the ability to form BDE from BMO. Previous in vitro results suggest that following exposure to butadiene more BMO would probably be present in mice than in rats or humans. Thus, in mice more BMO would be available for activation to BDE. This may explain in part the high sensitivity of this mouse strain to butadlene-induced cancer.
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