ISOZYME-SELECTIVE METABOLISM OF ETHYL CARBAMATE BY CYTOCHROME-P450 (CYP2E1) AND CARBOXYLESTERASE (HYDROLASE-A) ENZYMES IN MURINE LIVER-MICROSOMES

Cytochrome P450 and carboxylesterase enzymes have been implicated in t he metabolism of the carcinogen ethyl carbamate (EC). In this study, w e have used a murine liver microsomal system to investigate the relati ve contributions of P450 and carboxylesterase isozymes to hepatic meta bolism of EC. N-Nitrosodimethylamine (NDMA) demethylation and p-nitrop henyl acetate (PNA) hydrolysis were used as catalytic markers of CYP2E 1 and carboxylesterase enzymes, respectively, Incubation of liver micr osomes with EC (1 mM) produced slight but significant decreases in NDM A demethylation and PNA hydrolysis activities, Incubation of microsome s with paraoxon (PAX), a general carboxylesterase inhibitor, or phenyl methylsulfonyl fluoride (PMSF), a specific inhibitor of hydrolase A, p roduced decreases of 85 and 45%, respectively, in carboxylesterase act ivities; neither of the inhibitors elicited alterations in levels of N DMA demethylation, Reaction of microsomes with either PAX or PMSF and then with EC exacerbated the reduction (285%) of NDMA demethylation, a nd this lass corresponded to decreases in immunodetectable CYP2E1 cont ent, The reduction in PNA hydrolysis activity induced by PAX, PMSF, or EC correlated with decreased immunodetectable hydrolase A in liver mi crosomes; however, reaction with PAX and not PMSF or EC resulted in lo ss of immunoreactivity for hydrolase B, These data correlated with lev els of covalent binding of [ethyl-C-14]EC to liver microsomes, which w ere significantly elevated in incubations conducted with PAX or PMSF. Antibody inhibition of the CYP2E1 enzyme significantly reduced levels of binding to microsomal proteins, compared with control levels, These results are consistent with the premise that EC is metabolized by CYP 2E1 and hydrolase A in liver microsomes of mice.