Inactivation of cytochrome P-450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes by vinyl carbamate in murine pulmonary microsomes

We tested the hypothesis that vinyl carbamate (VC) is metabolized in vitro by cytochrome P-450 and carboxylesterase enzymes in murine lung. Incubation s with VC and an NADPH-generating system produced a 50% decrease in N-nitro sodimethylamine (NDMA) demethylation and a corresponding loss in the amount s of immunodetectable CYP2E1, Preincubation of microsomes with a CYP2E1 inh ibitory antibody or the CYP2E1-selective inhibitor diallyl sulfone (DASO(2) ) inhibited demethylase activity; no alterations were detected upon subsequ ent exposure to VC, Carboxylesterase-mediated hydrolysis of p-nitrophenyl a cetate was reduced by 22% in microsomes incubated with VC, Decreased carbox ylesterase activity also was detected in microsomes incubated with phenylme thylsulfonyl fluoride (PMSF), an inhibitor of hydrolase A, a carboxylestera se isozyme. No change in enzyme activity was detected when microsomes were subsequently incubated with VC, The loss in carboxylesterase activity corre lated with decreased immunodetectable hydrolase A in microsomes incubated w ith VC, PMSF, or PMSF and VC, The reduction in VC-induced NDMA demethylase activity was increased to 85% of the control in microsomes previously incub ated with PMSF, and this corresponded with a marked decrease in CYP2E1 immu noreactivity in the immunoblots. Covalent binding of VC to proteins was det ected in microsomes incubated with VC and an NADPH-generating system. Bindi ng was inhibited in microsomes preincubated with either an inhibitory CYP2E 1 antibody or DASO(2). In contrast, binding levels were augmented in micros omes preincubated with PMSF, These data supported VC metabolism by CYP2E1 a nd hydrolase A in murine lung microsomes and is consistent with involvement of CYP2E1 and hydrolase A in the activation and detoxication of VC, respec tively.