6PPD metabolic activation by CYP3A initiates cytotoxicity in mouse primary hepatocytes

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is a widely used rubber antioxidant and an emerging environmental pollutant, yet its hepatotoxic potential and the underlying role of metabolic activation remain poorly understood. Here, we integrated metabolite profiling, liver microsomal incubations, chemical oxidation synthesis, and mouse primary hepatocytes and animal exposure to examine the metabolic and toxicological outcomes of 6PPD. A total of 54 metabolites were identified in mice, including phase I oxidation products and phase II conjugates. Importantly, the reactive metabolite p-benzoquinone diimide (6PPD-QDI) was detected and shown to form conjugates with biological thiols. In microsomal incubations containing glutathione (GSH), N-acetylcysteine (NAC), or cysteine conjugates, 6PPD produced the corresponding conjugates, supporting the generation of 6PPD-QDI. The conjugate formation was NADPH-dependent and substantially suppressed by the CYP3A inhibitor ketoconazole, indicating that CYP3A mediates the bioactivation of 6PPD. In vivo, identical GSH and NAC conjugates were detected in bile and urine of exposed mice, and hepatic proteins contained dose-dependent protein adduction at cysteine residues. In primary hepatocytes, 6PPD induced concentration- and time-dependent cytotoxicity associated with GSH depletion and protein adduction, which was mitigated by CYP3A inhibition or thiol supplementation. Notably, CYP3A4-overexpressing HepG2 cells exhibited enhanced susceptibility to cytotoxicity and protein adduction compared to wild-type cells.