Mechanistic Insight into Difference in Hepatotoxicity of Methyleugenol and Safrole

Methyleugenol (MTL) and safrole (SFL) are hepatotoxic flavorants with distinct IARC classifications (Group 2A versus 2B), and yet the structural reason for this differential toxicity is unclear. Results showed that MTL was more hepatotoxic than SFL, owing to its more efficient metabolic activation. This was evidenced by greater glutathione (GSH) depletion, GSH conjugate formation, and protein adduction. CYP3A4 was identified as the key enzyme mediating this difference. Molecular simulations demonstrated that although SFL binds CYP3A4 with higher affinity, its rigid 3,4-methylenedioxy group displaces the allylic moiety from heme iron, resulting in suboptimal catalytic positioning. In contrast, MTL's 3-methoxy substituent enables stable orientation near the catalytic site, facilitating reactive metabolite formation. Thus, minor structural differences critically dictate the CYP3A4-mediated metabolic activation and subsequent hepatotoxic risk of allylbenzenes. This work provides a mechanistic basis for understanding structure-toxicity relationships and improving the safety assessment of flavor compounds in spices.