Metabolism of clivorine in female rat liver microsomes

The metabolism of clivorine, an othonecine-type hepatotoxic pyrrolizidine alkaloid, was investigated in female rat liver microsomes. The major in vitro metabolites of clivorine found in the incubation mixture are two non-pyrrolic metabolites (M₁ and M₂) and the formation of hepatotoxic pyrrolic metabolites from clivorine is only the minor in vitro metabolic pathway of clivorine in female rats. It suggests that the major in vitro metabolic pathway of clivorine in female rats is different from that in male rats. Selective CYP450 inhibitors pilocarpine (Pil, CYP2A1), diethyldithiocarbamate (DDC, CYP2E1), sulfaphenazole (Sul, CYP2C) and ketoconazole (Ket, CYP3A) had no significant effects on the formation of M₁ and M₂. Selective flavin-containing monooxygenase (FMO) inhibitor methimazole could inhibit the formation of M₂, but had no inhibitory effect on the formation of M₁ and the formation of M₁ was NADPH independent. The above results suggests that the hydrolase and FMO in microsomes are involved in the formation of M₁ and M₂ respectively. On the other hand, Selective CYP450 inhibitors Pil, DDC, Sul had no significant effects on the formation of pyrrolic metabolites from clivorine, but selective CYP3A inhibitor Ket could significantly inhibited the formation of pyrrolic metabolites of clivorine whereas clivorine was not metabolized by recombinant rat CYP2C12 and CYP2E1, but could be metabolized by recombinant mt CYP3A1 and CYP3A2 to produce its corresponding hepatotoxic pyrrolic metabolites. The above results indicate that mt CYP3A1 and CYP3A2 are the primary CYP450 involved in the formation of hepatotoxic pyrrolic metabolites. The difference in clivorine-induced toxicity can be partly attributed to this metabolic difference in sex.