Metabolism of ethyl carbamate by pulmonary cytochrome P450 and carboxylesterase isozymes: involvement of CYP2E1 and hydrolase A

Abstract

The lung is highly susceptible to ethyl carbamate (EC)-induced tumorigenesis. 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 and N-nitrosodimethylamine (NDMA) demethylation; there were no alterations in 7-pentoxyresorufin- and 7-ethoxyresorufin O-dealkylase activities. Reaction of microsomes with an inhibitory CYP2E1 antibody and subsequent reaction with EC abolished the EC-induced diminution in NDMA demethylase activity. Carboxylesterase activity, as assessed by hydrolysis of p-nitrophenyl acetate, was significantly decreased in microsomes incubated with EC. Reactions with EC in conjunction with the carboxylesterase inhibitors, paraoxon (PAX) or phenylmethylsulfonyl fluoride (PMSF), 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. Incubations of EC in combination with either PAX or PMSF exacerbated the EC-induced reduction in PNP hydroxylase and NDMA demethylase activities. Alterations 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. Immunoblotting with antibodies for the carboxylesterase isozymes, hydrolase A and B, revealed loss of immunodetectable hydrolase A in microsomes incubated with EC, PAX, or PMSF. However, immunodetectable hydrolase B was only decreased in microsomes reacted with PAX but not with PMSF or EC. These findings correlated with our covalent binding data, which showed that levels of binding of [14C-ethyl]EC to lung microsomes were significantly higher in incubations conducted in conjunction with PAX or PMSF, compared with control levels. Antibody inhibition of the CYP2E1 enzyme significantly reduced the extent of binding. Our results demonstrated that EC metabolism in lung microsomes, as estimated from magnitudes of covalent binding, is mediated by the P450 isozyme CYP2E1 and the carboxylesterase isozyme hydrolase A.