Abstract
Cytochrome P450 and carboxylesterase enzymes have been implicated in the metabolism of the carcinogen ethyl carbamate (EC). In this study, we have used a murine liver microsomal system to investigate the relative contributions of P450 and carboxylesterase isozymes to hepatic metabolism of EC. N-Nitrosodimethylamine (NDMA) demethylation and p-nitrophenyl acetate (PNA) hydrolysis were used as catalytic markers of CYP2E1 and carboxylesterase enzymes, respectively. Incubation of liver microsomes with EC (1 mM) produced slight but significant decreases in NDMA demethylation and PNA hydrolysis activities. Incubation of microsomes with paraoxon (PAX), a general carboxylesterase inhibitor, or phenylmethylsulfonyl fluoride (PMSF), a specific inhibitor of hydrolase A, produced decreases of 85 and 45%, respectively, in carboxylesterase activities; neither of the inhibitors elicited alterations in levels of NDMA demethylation. Reaction of microsomes with either PAX or PMSF and then with EC exacerbated the reduction (285%) of NDMA demethylation, and this loss corresponded to decreases in immunodetectable CYP2E1 content. The reduction in PNA hydrolysis activity induced by PAX, PMSF, or EC correlated with decreased immunodetectable hydrolase A in liver microsomes; however, reaction with PAX and not PMSF or EC resulted in loss of immunoreactivity for hydrolase B. These data correlated with levels of covalent binding of [ethyl-14C]EC to liver microsomes, which were 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 CYP2E1 and hydrolase A in liver microsomes of mice.
Footnotes
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Send reprint requests to: Dr. P.-G. Forkert, Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada, K7L 3N6.
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This research was supported by Grant MT-11706 from the Medical Research Council of Canada (P.-G.F.) and Grant RO1-CA73220–01 from the National Cancer Institute, National Institutes of Health (P.-G.F.).
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↵2 The antibodies for the carboxylesterases were raised against rat liver microsomal hydrolase A and hydrolase B. For ease of description, the murine liver microsomal proteins recognized by these antibodies are designated with the same nomenclature.
- Abbreviations used are::
- EC
- ethyl carbamate
- DMSO
- dimethylsulfoxide
- Mab
- monoclonal antibody
- NDMA
- N-nitrosodimethylamine
- PAX
- paraoxon
- PMSF
- phenylmethylsulfonyl fluoride
- PNA
- p-nitrophenyl acetate
- SDS
- sodium dodecyl sulfate
- VC
- vinyl carbamate
- CYP or P450
- cytochrome P450
- Received July 3, 1997.
- Accepted September 11, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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