KINETIC-STUDIES ON TOLUENE METABOLISM IN ETHANOL-INDUCED AND PHENOBARBITAL-INDUCED RAT-LIVER MICROSOMES INVITRO

In vitro metabolism of toluene was investigated at substrate concentrations of 0.03-6.25 mM in liver microsomes from control and ethanol- and phenobarbital (PB)-treated rats. Three metabolites, benzylalcohol (BA), o- and p-cresol, were measured by high-performance liquid chromatograph. BA was the main metabolite of toluene, whereas o- and p-cresol contributed only 1.1-1.5% and 1.7-2.8% of total metabolites, respectively, in microsomes from control rats. Ethanol treatment showed little effect on the percentages of three metabolites, but PB increased the percentages of omicron- and rho-cresol to as high as 5.5% and 8.0%, respectively, following the increase in toluene concentration. There were two different isozymes with different K(m) involved in the side-chain hydroxylation of toluene in microsomes from control and ethanol-treated rats. One had a low K(m) value (0.13-0.17 mM) and could be greatly induced with ethanol treatment. The other was a high K(m) isozyme (0.60-0.87 mM). PB-induced isozyme showed a similar K(m) value to that of the high K(m) isozyme existing in microsomes from control and ethanol-treated rats. Two isozymes were involved in the formation of p-cresol in microsomes of control rats; the low-K(m) type had a similar value (0.15 mM) to the low isozyme of BA formation, but the high K(m) isozyme had a larger value (2.04 mM) than the high isozyme of BA. Only one enzyme responsible for o-cresol formation was detected in microsomes of control rats, and had a similar K(m) (2.11 mM) to that of the high K(m) isozyme of p-cresol. The high K(m) and low V(max) values of isozymes of aromatic hydroxylation account for the minor proportion of cresols. Ethanol and PB treatments enhanced the metabolism of toluene, but they differ in that the ethanol-inducible isozyme metabolized toluene at a faster rate at low substrate concentrations, whereas PB did so at high toluene concentrations. The possible involvement of subtypes of cytochrome P450, and the significance of the results in biological monitoring of toluene exposure are discussed.