Covalently Active Metabolites of Bisphenol A Analogs by Mass Spectrometry Diagnostic Ions: Possible Mechanisms of Their Toxicity

Bisphenol A analogs (BPs), used as BPA alternatives, have drawn great concerns due to their potential adverse effects. Studies have shown that reactive metabolites (RMs) formed in vitro and in vivo could covalently bind to nucleophilic macromolecules to elicit toxicity. However, the bioactivation potential of BPs and their capacity to covalently modify amino acid residues within proteins have been poorly characterized. Thus, this study systematically characterized the metabolic activation of eight BPs and their reactivity toward cysteine. Using <i>N</i>-acetylcysteine (NAC) as a trapping agent to capture RMs, we developed a novel nontargeted fragment screening strategy for cysteine adduct identification and mechanistic exploration. Integrating calculated electron affinity results, mechanistic analyses revealed a common activation pathway across multiple BPs involving oxidation, ipso-addition, and ipso-substitution. Also, the abundances of cysteine adducts correlated with metabolic rates of individual BPs, underscoring structure-reactivity relationships. These results provided critical mechanistic insight into BPs bioactivation, implicating their potential toxicity risk and supporting environmental risk evaluation.