L-Menthol Attenuates Acetaminophen-Induced Acute Liver Injury Associated with Reduced Oxidative Stress and Ferroptosis-Related Changes.

Acetaminophen (APAP) overdose is a major cause of drug-induced liver injury and remains a widely used model of xenobiotic-induced hepatotoxicity. Oxidative stress, mitochondrial dysfunction, and ferroptosis are key events in APAP-mediated liver damage. In this study, we investigated whether L-menthol pretreatment protects against APAP-induced acute liver injury and explored the underlying mechanisms in vivo and in vitro. Male C57BL/6 mice were pretreated with L-menthol (100 mg/kg/day) for 7 days before APAP challenge (300 mg/kg). L-menthol markedly attenuated hepatic necrosis, inflammatory infiltration, and hepatocyte injury, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, suppressed IL-1β, IL-6, and TNF-α production, restored hepatic glutathione and superoxide dismutase levels, and decreased malondialdehyde accumulation. Transcriptomic analysis revealed significant enrichment of differentially expressed genes in reactive oxygen species- and ferroptosis-related pathways. In APAP-challenged HepG2 cells, L-menthol improved cell viability, preserved mitochondrial ultrastructure, reduced ferrous iron accumulation, was associated with upregulation of Keap1/Nrf2/HO-1/NQO1 pathway-related proteins, and restored GPX4 expression. Collectively, these findings indicate that L-menthol pretreatment attenuates APAP-induced hepatotoxicity, possibly through enhancement of antioxidant defenses and attenuation of ferroptosis-associated changes, supporting its potential as a preventive hepatoprotective small molecule against xenobiotic-induced liver injury.