METABOLISM OF ETHYL CARBAMATE BY PULMONARY CYTOCHROME-P450 AND CARBOXYLESTERASE ISOZYMES - INVOLVEMENT OF CYP2E1 AND HYDROLASE-A

The lung is highly susceptible to ethyl carbamate (EC)-induced tumorig enesis. Our goal in this study was to investigate the in vitro isozyme -selective metabolism of EC in lung microsomes by cytochrome P450 and carboxylesterase enzymes, Our results showed that incubations with EC produced significant reduction in p-nitrophenol (PNP) hydroxylation an d N-nitrosodimethylamine (NDMA) demethylation; there were no alteratio ns in 7-pentoxyresorufin- and 7-ethoxyresorufin O-dealkylase activitie s, Reaction of microsomes with an inhibitory CYP2E1 antibody and subse quent reaction with EC abolished the EC-induced diminution in NDMA dem ethylase activity, Carboxylesterase activity, as assessed by hydrolysi s of p-nitrophenyl acetate, was significantly decreased in microsomes incubated with EC. Reactions with EC in conjunction with the carboxyle sterase inhibitors, paraoxon (PAX) or phenylmethylsulfonyl fluoride (P MSF), abolished the EC-induced decrease in carboxylesterase activity; PAX is a broad-spectrum carboxylesterase inhibitor, whereas PMSF is a specific inhibitor of hydrolase A, a carboxylesterase isozyme. Incubat ions of EC in combination with either PAX or PMSF exacerbated the EC-i nduced reduction in PNP hydroxylase and NDMA demethylase activities, A lterations in immunodetectable CYP2E1 protein levels were not apparent in microsomes incubated with EC alone, but the amounts were decreased in reactions with EC in conjunction with either PAX or PMSF. Immunobl otting with antibodies for the carboxylesterase isozymes, hydrolase A and B, revealed loss of immunodetectable hydrolase A in microsomes inc ubated with EC, PAX, or PMSF, However, immunodetectable hydrolase B wa s only decreased in microsomes reacted with PAX but not with PMSF or E C. These findings correlated with our covalent binding data, which sho wed that levels of binding of [C-14-ethyl]EC to lung microsomes were s ignificantly higher in incubations conducted in conjunction with PAX o r PMSF, compared with control levels. Antibody inhibition of the CYP2E 1 enzyme significantly reduced the extent of binding. Our results demo nstrated that EC metabolism in lung microsomes, as estimated from magn itudes of covalent binding, is mediated by the P450 isozyme CYP2E1 and the carboxylesterase isozyme hydrolase A. (C) 1997 Academic Press.