Chlorzoxazone metabolism by winter flounder liver microsomes: evidence for existence of a CYP2E1-like isoform in teleosts

Abstract

Winter flounder (Pleuronectes americanus) liver microsomes metabolized chlorzoxazone (CZX), a CYP2E1 (cytochrome P450 2E1)- specific substrate, to a single product, 6-OH CZX. The liver microsomal fraction had the 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 activity was from 7.2 to 8.0. Maximal activity was found at 18-22 degreesC (154 +/- 62 pmol/mg/min) but was detectable at all temperatures tested. CZX 6-hydroxylation displayed first-order kinetics as shown by Lineweaver-Burk plots. The average apparent Km, 280 microM, was higher than that reported for human liver microsomes but similar to the Km determined for human CYP2E1 expressed in vaccinia virus. Vmax values ranged from 65 to 141 pmoles/mg/min. The CYP2E1-specific inhibitors, diethyldithiocarbamate (DDC) and aniline, significantly reduced 6-OH CZX production by 80 and 35%, respectively. 7,8-Benzoflavone and ethoxyresorufin, both CYP1A-specific inhibitors, did not significantly inhibit CZX metabolism nor did trioleandomycin (TAO) a CYP3A-specific inhibitor. Diethyldithiocarbamate (DDC), at 50 microM, had no significant effect on winter flounder liver microsomal ethoxyresorufin-O-deethylase activity (EROD), a CYP1A-specific activity. 7,8-Benzoflavone, at 10 microM, inhibited EROD activity by 60%. This is the first report of CYP2E1 activity in teleost liver microsomes.