CHLORZOXAZONE METABOLISM BY WINTER FLOUNDER LIVER-MICROSOMES - EVIDENCE FOR EXISTENCE OF A CYP2E1-LIKE ISOFORM IN TELEOSTS

Winter flounder (Pleuronectes americanus) liver microsomes metabolized chlorzoxazone (CZX), a CYP2E1 (cytochrome P450 2E1)- specific substra te, to a single product, 6-OH CZX, The liver microsomal fraction had t he highest level of activity, which varied among fish, ranging from 60 to 220 pmol/mg/min. CZX metabolism was linearly related to microsomal protein and dependent on NADPH. The optimal pH range for this activit y was from 7.2 to 8.0, Maximal activity was found at 18-22 degrees C ( 154 +/- 62 pmol/mg/min) but was detectable at all temperatures tested. CZX 6-hydroxylation displayed first-order kinetics as shown by Line-w eaver-Burk plots. The average apparent K-m, 280 mu M, was higher than that reported for human liver microsomes but similar to the K-m determ ined for human CYP2E1 expressed in vaccinia virus. V-max values ranged from 65 to 141 pmoles/mg/min. The CYP2E1-specific inhibitors, diethyl dithiocarbamate (DDC) and aniline, significantly reduced 6-OH CZX prod uction by 80 and 35%, respectively, 7,8-Benzoflavone and ethoxyresoruf in, both CYP1A-specific inhibitors, did not significantly inhibit CZX metabolism nor did trioleandomycin (TAO) a CYP3A-specific inhibitor. D iethyldithiocarbamate (DDC), at 50 mu M, had no significant effect on winter flounder liver microsomal ethoxyresorufin-O-deethylase activity (EROD), a CYP1A-specific activity. 7,8-Benzoflavone, at 10 mu M, inhi bited EROD activity by 60%. This is the first report of CYP2E1 activit y in teleost liver microsomes, (C) 1998 Academic Press.