Integrated analysis of detoxification pathways and hepatotoxicity of butylated hydroxytoluene (BHT) in the Manila clam (Ruditapes philippinarum)

Synthetic phenolic antioxidants (SPAs) are emerging contaminants (ECs) of growing concern in marine ecosystems, yet their detoxification and hepatotoxicity in marine invertebrates remain largely unknown. Here, we integrated transcriptomic profiling, in silico prediction, and experimental validation to characterize the detoxification of butylated hydroxytoluene (BHT) in Ruditapes philippinarum and to elucidate its hepatotoxicity. BHT metabolism was transcriptionally regulated by the aryl hydrocarbon receptor ( AHR ) and hormone receptor 96 ( HR96 ) pathways. Key enzymes included Phase I cytochrome P450 isoforms ( CYP1A1, CYP1A2, CYP3A4 ) and the Phase II enzyme UDP-glucuronosyltransferase ( UGT ). Metabolite profiling revealed the transformation products in the order: BHT-CHO > BHT-Q > BHT-OH > BHT-COOH, and a cascade detoxification pathway was subsequently proposed. BHT exposure impaired antioxidant defenses, induced oxidative stress, and caused macromolecular damage. Molecular dynamics simulations revealed a compromised DPPC bilayer integrity, suggesting structural membrane damage. In parallel, histopathological analysis exhibited characteristic hepatic lesions and cytoplasmic vacuolization, accompanied by significant increases in inflammatory cytokines ( TNF , IL16 ) and transaminase levels (AST, ALT). Together, these results delineate a metabolism–oxidative stress–hepatotoxicity cascade for BHT in bivalves, providing novel mechanistic insights into SPA-induced toxicity. Our findings highlight the ecological risks posed by persistent SPAs in marine environments and underscore their potential to compromise the health of sentinel bivalves, supporting their inclusion in environmental monitoring and regulatory frameworks. • Transcriptomics and in silico prediction identified BHT detoxification genes. • AHR and HR96 were revealed as central regulators of BHT metabolism. • CYP450 enzymes mediated Phase I, while UGT dominated Phase II conjugation. • BHT caused hepatotoxicity via membrane damage, oxidative stress, and inflammation. • This is the 1st mechanistic evidence of BHT hepatotoxicity in marine invertebrates.