BHT and Its Metabolites Disrupt β2-Adrenergic Signaling: A Biotransformation-Dependent Cardiotoxicity Mechanism in Zebrafish

Butylated hydroxytoluene (BHT) is a widely used synthetic phenolic antioxidant, ubiquitously detected in aquatic environments. While traditionally considered safe based on in vitro data, BHT adversely affects aquatic organisms at environmentally relevant concentrations. Here, we demonstrate that BHT-induced cardiotoxicity is primarily mediated by its metabolites through disruption of β2-adrenergic receptor (β2AR) signaling. In zebrafish larvae exposed to 0.01-1 μM BHT for 6 days, metabolites BHT-CHO, BHT-COOH, and BHT-Q accumulated prominently, inducing cardiac morphological and histological alterations. Transcriptomic analysis highlighted pronounced neuro-cardiovascular network disruption, with significant suppression of adrenergic signaling in cardiomyocytes at 1 μM. Molecular docking and biolayer interferometry identified β2AR as a critical target for BHT metabolites, and functional rescue with a β-adrenoceptor agonist confirmed pathway dependency. However, paradoxical activation of β2AR-cAMP/PKA with concomitant suppression of downstream PKA-mediated phosphorylation resulting from competitive BHT-Q binding to the PKA catalytic subunit, indicates a functional uncoupling effect. This disrupts intracellular Ca2+ homeostasis in cardiomyocytes and drives cardiomyocyte dysfunction. Our findings reveal an unconventional toxicological paradigm, wherein environmental pollutants exploit kinase ATP pockets to corrupt signaling fidelity rather than simply inhibit or activate receptors, highlighting the necessity of metabolic activation assessment in environmental risk evaluation.