MONOCLONAL ANTIBODY-DIRECTED CHARACTERIZATION OF CYTOCHROME-P450 ISOZYMES RESPONSIBLE FOR TOLUENE METABOLISM IN RAT-LIVER

Monoclonal antibodies (MAbs) were used to study the contribution of cytochromes P450IA1/IA2, P450IIB1/IIB2, P450IIC11/IIC6 and P450IIE1 to toluene side-chain (benzyl alcohol, BA, formation) and ring (o- and p-cresol formation) oxidation in liver microsomes from fed, one-day fasted, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. All rats were fed synthetic liquid diets. MAb 1-7-1 against P450IA1/IA2 inhibited markedly o-cresol formation and slightly p-cresol formation but not BA formation only in microsomes from MC-treated rats. MAbs 2-66-3, 4-7-1 and 4-29-5 against P450IIB1/IIB2 strongly inhibited BA, o-cresol and p-cresol formation only in PB-induced microsomes. MAb 1-68-11 against P450IIC11/IIC6 inhibited BA formation at high toluene concentration in the following order: fed > fasted > ethanol = MC > PB, and ethanol greater-than-or-equal-to fed = fasted > MC > PB on the basis of the percentage and net amount inhibition, respectively. MAb 1-91-3 against P450IIE1 inhibited BA formation at low toluene concentration, but not at high concentration, in the following order: ethanol > fasted = fed > MC, and ethanol > fasted > fed > MC on the basis of percentage and net inhibition, respectively. MAbs 1-68-11 and 1-91-3 also inhibited p-cresol formation at high and low toluene concentrations, respectively. These results indicate that (i) both P450IIE1 and P450II11/IIC6 are constitutive isozymes mainly responsible for the formation of BA and p-cresol from toluene as low- and high-K(m) isozymes, respectively; (ii) P450IIE1, but not P450IIC11/IIC6, is induced by one-day fasting and ethanol treatment; (iii) both P450IIE1 and P450IIC11/IIC6 are decreased by PB and MC treatments; (iv) P450IIE1 is inhibited by high concentration of toluene; (v) P450IIB1/IIB2 can contribute to the formation of BA, o- and p-cresol from toluene, while P450IAI/IA2 preferentially contributes to the formation of o-cresol.