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

1,3-Butadiene is carcinogenic in B6C3F(1) 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 metaboli te, 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 purpo se 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 whic h this reaction occurs in B6C3F(1) mouse, Sprague-Dawley rat, and huma n liver microsomes. Of the human cDNA-expressed CYP isozymes tested, o nly CYP2E1 formed detectable concentrations of BDE at 80 mu M BMO, CYP 2E1 and CYP3A4 were active at 5.0 mM BMO. Interindividual and interspe cies variation in the initial rate of oxidation of 80 mu M BMO to BDE was determined using 10 samples of human liver microsomes and single p ooled samples from rats and mice. Those experiments revealed a 60-fold variation in activity among 10 human liver samples (range: 0.005-0.32 4 nmol/mg protein/min). Rates of BMO oxidation for mouse and rat liver microsomes were 0.473 and 0.166 nmol/mg protein/min, respectively. Ap parent kinetic constants for the oxidation of BMO to BDE by four human microsomal preparations, and pooled samples from mice and rats were e stimated from detailed investigations of BMO oxidation at various BMO substrate concentrations. Apparent K-m for the human liver samples ran ged from 0.304-0.880 mM, and V-max values ranged from 0.38 to 1.2 nmol /mg protein/min. The apparent values of K-m and V-max for mouse liver microsomes were 0.141 +/- 0.007 mM (mean +/- SE) and 1.303 +/- 0.141 n mol/mg protein/ min, respectively. For rat liver microsomes, apparent K-m and V-max were 0.145 +/- 0.036 mM and 0.408 +/- 0.031 nmol/mg prot ein/min, respectively. Measured rates of BDE formation correlated well with CYP2E1 protein concentrations in the human microsome samples. Th ese results implicate human CYP2E1 as a hepatic isoform responsible fo r the oxidation of BMO to BDE at low concentrations of BMO. Moreover, our in vitro results reveal that microsomes prepared from human, rat a nd mouse liver possess the ability to form BDE from BMO. Previous in v itro results suggest that following exposure to butadiene more BMO wou ld probably be present in mice than in rats or humans.