Kinetic studies on toluene metabolism in ethanol- and phenobarbital-induced rat liver microsomes in vitro

Abstract

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 o- and p-cresol to as high as 5.5% and 8.0%, respectively, following the increase in toluene concentration. There were two different isozymes with different Km involved in the side-chain hydroxylation of toluene in microsomes from control and ethanol-treated rats. One had a low Km value (0.13-0.17 mM) and could be greatly induced with ethanol treatment. The other was a high Km isozyme (0.60-0.87 mM). PB-induced isozyme showed a similar Km value to that of the high Km 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-Km type had a similar value (0.15 mM) to the low isozyme of BA formation, but the high Km 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 Km (2.11 mM) to that of the high Km isozyme of p-cresol.(ABSTRACT TRUNCATED AT 250 WORDS)

Toluene metabolism in isolated rat hepatocytes: effects of in vivo pretreatment with acetone and phenobarbital

“…In spiked samples the cresols eluted close to benzoic acid, and they might therefore have been determined as such. However, Wang and Nakajima (1991 ) showed in microsomes that no or only small amounts of o-and p-cresol were formed at toluene concentrations used in the present study.…”

“…Microsomal preparations have so far been used to study the oxidation of toluene to its first metabolite, benzyl alcohol (Sato et al 1980;Sato and Nakajima 1985;Wang and Nakajima 1991;Nakajima et al 1991). In the present study the metabolic conversion of toluene through three consecutive steps to its main excretion product, hippuric acid, was investigated in intact hepatocytes.…”

“…Toluene oxidation is increased in microsomal preparations after increase of cytochrome P-450s by long term ethanol and phenobarbital treatment (Ikeda and Ohtsuji 1971;Sato et al 1980;Wang and Nakajima 1991). Decreased toluene oxidation is observed both in vivo and in vitro in the presence of ethanol (Sato et al 1981;Waldron et al 1983;Wall6n et al 1984Wall6n et al , 1985Takahashi et al 1987).…”

The Metabolic Rate Constants and Specific Activity of Human and Rat Hepatic Cytochrome P-450 2E1 Toward Toluene and Chloroform

“…Differences in rates of BA formation between CYP2E1 expressed in MSP isolated from virus expression systems and CYP2E1 contained in samples of human MSP may be explained, in part, by differences in test systems employed, the effect of a lower substrate concentration employed in the Nakajima et al (1997) studies (5 v. 10 mM), and the consistent relationship between reductases and CYP enzymes in our authentic MSP samples. Using rat liver MSP, Wang and Nakajima (1991) demonstrated kinetic constants for BA formation from toluene in control and ethanol-treated (CYP2E1-induced) male Wistar rats. Their results indicated the contribution of two separate enzymes, and that the lowaffinity form was the same form whose activity was induced by ethanol treatment.…”

“…Toluene is a groundwater contaminant whose conversion to benzyl alcohol (BA) is catalyzed by CYP2E1 at low concentrations (Wang & Nakajima, 1991). Liver and kidney toxicity (weight changes) have been used to quantify human health risk to toluene following oral exposure (NTP, 1989).…”