Dog liver microsomal P450 enzyme-mediated toluene biotransformation

1. We studied toluene metabolism in dog liver microsomes and the major metabolite was benzyl alcohol with o- and p-cresol as minor metabolites. 2. The enzyme kinetics of toluene biotransformation were examined by means of Lineweaver-Burk analyses. The Michaelis-Menten values differed among the three pathways, the order being; Km, o-cresol > p-cresol > benzyl alcohol; Vmax, benzyl alcohol > o-cresol > p-cresol; and Cl(int), benzyl alcohol > p-cresol > o-cresol. 3. The formation of benzyl alcohol, o- and p-cresol from toluene was substantially inhibited by the P4502E inhibitors such as DDC (diethyldithiocarbamate) and 4-methylpyrazole in all pathways, with IC50's in the range of 0.02-0.59 mM. The P4502B inhibitors, metyrapone and secobarbital also inhibited benzyl alcohol and p-cresol formation, whereas o-cresol was not inhibited by these latter compounds. 4. Anti-rat P4502E1 antibodies inhibited benzyl alcohol, o- and p-cresol formation from 26 to 30% 0.2 ml serum/mg microsomal protein. Furthermore, anti-rat P4502B1/2 antibody inhibited benzyl alcohol and p-cresol formation (47 and 44% respectively), but not that of o-cresol. Anti-rat P4502C11/6 antibody also inhibited benzyl alcohol and p-cresol formation 31 and 24% respectively in a similar manner to that by the anti-rat P4502B1/2 antibody. 5. These results suggested that the P4502B, 2C and 2E isozymes in dog liver contribute to the formation of benzyl alcohol and p-cresol from toluene, and 2E isozyme preferentially contributes to the formation of o-cresol.