Liver microsomal metabolism of N-methylcarbazole: A probe for induction, inhibition, and species differences

N-Methylcarbazole is converted to four major metabolites by rabbit liver microsomal preparations: N-hydroxymethylcarbazole, 1-hydroxy-N-methylcarbazole, 2-hydroxy-N-methylcarbazole, and 3-hydroxy-N-methylcarbazole. Small amounts of carbazole, presumably formed by the decomposition of N-hydroxymethylcarbazole were also formed. An assay procedure using high pressure liquid chromatography to separate and quantitate the metabolites was developed. The formation of the four major hydroxylated products requires NADPH or NADH. Formation of the hydroxylated products is inhibited by carbon monoxide, SKF-525A or 5,6-benzoflavone to similar extents. Metyrapone inhibited the formation of the 2-hydroxy-N-methylcarbazole to a greater extent than any of the others. 7,8-Benzoflavone, which had little effect on the formation of N-hydroxymethylcarbazole, inhibited the formation of 3-hydroxy-N-methylcarbazole by 30% and the formation of 2-hydroxy-N-methylcarbazole by 60% while stimulating the formation of 1-hydroxy-N-methylcarbazole. The liver microsomal metabolism of N-methylcarbazole by rabbits was compared with that of rats and mice. The major metabolite formed by rabbit liver microsomes is N-hydroxymethylcarbazole with slightly smaller amounts of the 3-hydroxy-N-methylcarbazole being formed. In rats, the major metabolite is the 1-hydroxy-N-methylcarbazole with N-methylcarbazole being formed in slightly lesser amounts. Metabolism of N-methylcarbazole by mice results in the formation of the 3-hydroxy- and N-hydroxymethylcarbazole as the major metabolites in essentially equal amounts. Relatively small amounts of 2-hydroxy-N-methylcarbazole are formed by microsomes from rabbits, rats, or mice. Phenobarbital treatment of rabbits preferentially induces the formation of 2-hydroxy-N-methylcarbazole by 20-fold with lesser induction of the rates of formation of the 1-hydroxy-, 3-hydroxy-, and N-hydroxy- metabolites of N-methylcarbazole (2-, 4-, and 4-fold respectively). Although treatment of the rabbits with 3-methylcholanthrene did not alter the rates of formation of 2-hydroxy- or 3-hydroxy-N-methylcarbazole, the rates of formation of 1-hydroxy- and N-hydroxymethylcarbazole were decreased significantly. In the rat, phenobarbital induction caused a 2-fold increase in the formation of 3-hydroxy-N-methylcarbazole and a 1.5-fold increase in the rate of formation of N-hydroxymethylcarbazole. Steady-state kinetic studies on the metabolism of N-methylcarbazole by liver microsomes from rabbits treated with phenobarbital, 3-methylcholanthrene, or 5,6-benzoflavone provided kinetic evidence for the involvement of different forms of cytochrome P-450 in the formation of each of the four major metabolites. These results suggest that N-methylcarbazole may be an excellent substrate for investigating the multiple forms of cytochrome P-450 in crude microsomal preparations as well as for investigating the substrate specificities and kinetic properties of the purified forms of cytochrome P-450.