Mitochondrial and Redox Mechanisms of Leflunomide-Induced Hepatotoxicity Following Repeated Exposure

Leflunomide is a disease-modifying antirheumatic drug used for inflammatory arthritis. However, aminotransferase elevations and cases of drug-induced liver injury have been reported during therapy. Although leflunomide or its active metabolite has shown antioxidant or protective effects in acute and extrahepatic models, whether repeated hepatic exposure produces a redox–mitochondrial injury phenotype remains insufficiently defined. This study investigated the effects of repeated oral leflunomide administration on hepatic injury markers, oxidative stress, and mitochondrial function in male BALB/c mice. Animals received leflunomide at ۵, ۱۰, or ۲۰ mg/kg by gavage for ۲۸ days. Plasma ALT, AST, and LDH were measured. Hepatic oxidative status was assessed by reactive oxygen species formation, lipid peroxidation, reduced glutathione content, ferric-reducing antioxidant power, and the activities of superoxide dismutase and catalase. Liver mitochondria were isolated to evaluate dehydrogenase activity, membrane potential, and calcium-triggered swelling. Repeated leflunomide exposure reduced body weight gain at ۱۰ and ۲۰ mg/kg without altering the liver weight index. ALT and AST were increased in treated animals, whereas LDH remained unchanged. Leflunomide also shifted the hepatic redox profile toward oxidative stress, with increased ROS formation and lipid peroxidation, GSH depletion, reduced antioxidant capacity, and lower SOD and CAT activities. These alterations were accompanied by mitochondrial dysfunction, including reduced dehydrogenase activity, loss of mitochondrial membrane potential, and enhanced mitochondrial swelling. These findings indicate that repeated leflunomide exposure induces a hepatic injury phenotype characterized by oxidative stress and mitochondrial impairment. Sustained liver exposure may reveal mitochondrial liabilities not evident in acute or extrahepatic experimental settings.

Keywords:

Leflunomide , Hepatotoxicity , Oxidative stress , Mitochondrial dysfunction , Drug-induced liver injury