Dihydralazine is known to induce immunoallergic
hepatitis. Since anti-liver microsome (anti-LM)
autoantibodies found in the serum of the patients react with P450 1A2, it is suggested that
dihydralazine is biotransformed into a reactive metabolite, which covalently binds to
cytochrome P450 1A2 and triggers an immunological response as a neoantigen. We investigated inactivation of
P450 enzymes, including P450 1A2, during the metabolism of
dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and a target of
dihydralazine. Human liver microsomes or microsomes from lymphoblastoid cells expressing
P450 enzymes were preincubated with
dihydralazine in the presence of
NADPH, followed by an assay of several
monooxygenase activities. Preincubation of human liver microsomes with
dihydralazine in the presence of
NADPH resulted in decreases in
phenacetin O-deethylase activity (an
indicator of P450 1A2 activity) and
testosterone 6beta-hydroxylase activity (P450 3A4), but not in
diclofenac 4'-hydroxylase activity (P450 2C9), an indication of inactivation of P450s 1A2 and 3A4 during the
dihydralazine metabolism. The inactivation of both of the P450s followed pseudo-first-order kinetics and was saturable with increasing
dihydralazine concentrations. Similar time-dependent decreases in the activities were obtained in the case for use in microsomes expressing P450 1A2 and P450 3A4 instead of the human liver microsomes. The data presented here demonstrated that
dihydralazine was metabolically activated not only by P450 1A2 but also by P450 3A4, and the chemically reactive metabolite bound to and inactivated the
enzyme themselves, suggesting that
dihydralazine is a mechanism-based inactivator of P450s 1A2 and 3A4. The data support the postulated covalent binding of a reactive metabolite of
dihydralazine to P450 1A2 as a step in the formation of anti-LM
antibodies in
dihydralazine hepatitis, but it is not the unique factor for determining the specificity of the
autoantibodies.