HUMAN HEPATIC ALCOHOL-DEHYDROGENASE AND HUMAN ERYTHROCYTE CATALASE DONOT METABOLIZE THE CYTOCHROME P-4502E1 SUBSTRATE, CHLORZOXAZONE

Studies of cytochrome P-4502E1 (CYP2E1)-mediated oxidation of ethanol have been hampered by the lack of a suitable probe for in vivo human s tudies. Chlorzoxazone, a prescribed skeletal muscle relaxant, is metab olized to 6-hydroxychlorzoxazone by CYP2E1 and has been advocated as a specific probe of this enzyme on the basis of microsomal studies. The applications of this probe may include delineating the contribution o f CYP2E1 to in vivo human ethanol metabolism. However, the activity of nonmicrosomal enzymes in metabolizing chlorzoxazone is unknown. Alcoh ol dehydrogenase (ADH), predominantly a hepatic cytosolic enzyme, may be more important than CYP2E1 in the oxidation of ethanol to acetaldeh yde. The contribution of catalase in the in vivo oxidation of ethanol to acetaldehyde is controversial. To determine if either of these enzy mes metabolizes chlorzoxazone and whether ethanol oxidation by either enzyme is inhibited by chlorzoxazone or its metabolite, multiple in vi tro studies were performed. ADH enzyme kinetics were performed with hu man recombinant beta(1) beta(1) and beta(3) beta(3) ADH with ethanol a nd chlorzoxazone (0.5 to 2.5 mM). Neither ADH isoenzyme exhibited NAD( +)-dependent oxidation of chlorzoxazone, but displayed Michaelis-Mente n kinetics for ethanol with K-m values of 89 mu M and 34 mM, for beta( 1) beta(1) and beta(3) beta(3), respectively. Typical in vivo concentr ations of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone, di d not alter beta(1) beta(1) or beta(3) beta(3) ADH-mediated oxidation of ethanol to acetaldehyde. Studies of human hepatic nonmicrosomal enz yme activity were expanded to include all nonmicrosomal NAD(+)-depende nt hepatic enzymes by starch gel electrophoresis assessment. Human hep atic enzymatic activity in the presence of chlorzoxazone was similar t o that observed in the control sample (no added substrate), suggesting a lack of metabolism by NAD(+)-dependent enzymes. Similarly, human er ythrocyte catalase, in the presence of a hydrogen peroxide generating system, did not metabolize chlorzoxazone. Furthermore, neither chlorzo xazone nor 6-hydroxychlorzoxazone altered the catalase-induced formati on of acetaldehyde from ethanol. These data are consistent with chlorz oxazone as a specific probe of CYP2E1 that may be useful to alcohol re searchers.