Roles of Hepatic Cytochrome P450 Enzymes in 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) Metabolism and N -Methyl Protoporphyrin IX Formation

Abstract 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) is a porphyrinogenic compound that induces hepatic formation of N-methyl protoporphyrin IX (NmePPIX), a potent endogenous inhibitor of ferrochelatase (FECH). Inhibition of FECH by NmePPIX blocks the conversion of protoporphyrin IX (PPIX) into heme, resulting in PPIX accumulation and PPIX-mediated cholestatic liver injury. Although cytochrome P450 (CYP)-mediated transfer of a methyl group from DDC has long been proposed as the mechanism underlying DDC-induced NmePPIX formation, the specific CYP enzymes responsible for DDC metabolism and NmePPIX formation remain poorly characterized. Here, a metabolomics-based approach identified six CYP-dependent DDC metabolites (M1–M6) in the incubation with mouse liver S9 fractions. Notably, no demethylated DDC metabolite was detected. We further demonstrated that CYP3A contributes to four metabolic pathways of DDC, including a previously unrecognized pathway involving deamination and intramolecular cyclization. Given the important role of CYP3A in DDC metabolism and its high hepatic expression, we investigated whether CYP3A contributes to NmePPIX formation. Our in vivo study found that Cyp3a deficiency had no effect on DDC-induced NmePPIX formation, PPIX accumulation, or PPIX-mediated liver injury. Collectively, these findings provide the first comprehensive characterization of CYP-dependent DDC metabolism and demonstrate that CYP3A is not essential for DDC-induced NmePPIX formation, indicating that the major CYP-dependent metabolic pathways of DDC are mechanistically distinct from the pathway leading to NmePPIX formation.