Glutathione conjugation and protein adduction derived from oxidative debromination of benzbromarone in mice

Benzbromarone (BBR), a uricosuric agent, has been known to induce hepatoxicity, and its toxicity has a close relation to P450s-mediated metabolic activation. An oxidative debromination metabolite of BBR has been reported in microsomal incubations. The present study attempted to define the oxidative debromination pathway of BBR in vivo. One urinary mercapturic acid (M1) and one biliary GSH conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice after solid phase extraction. M1 and M2 shared the same chromatographic behavior and mass spectral identities as those detected in NAC/GSH- and BBR-fortified microsomal incubations. The structure of M1 was verified characterized by chemical synthesis, along with mass spectrometry analysis. In addition, hepatic protein modification that occurs at cysteine cysteine-based protein modification residues (M93) was observed in mice given BBR. The observed protein adduction reached its peak 4 h after administration and elicited a dose-dependent manner. A GSH conjugate derived from oxidative debromination of BBR was detected in liver of mice treated with BBR, and the formation of the GSH conjugate apparently took place earlier than the protein adduction. In summary, our in vivo work provided strong evidence for the proposed oxidative debromination pathway of BBR, which facilitates the understanding of the mechanisms of BBR-induced hepatotoxicity. SIGNIFICANCE STATEMENT The present study investigated the oxidative debromination pathway of benzbromarone (BBR) in vivo. One urinary mercapturic acid (M1) and one biliary glutathione GSH conjugate (M2) derived from the oxidative debromination metabolite were detected in BBR-treated mice. M1 and M2 were also observed in microsomal incubations. The structure of M1 was characterized by chemical synthesis, followed by mass spectrometry analyseswas verified characterized by chemical synthesis. Cysteine-based protein More importantly, protein adduction derived from oxidative debromination of BBR (M93) was observed in mice given BBR, and the observed protein adduction occurred in dose- and time-dependent manners. The success in detection of GSH conjugate, urinary NAC conjugate, and hepatic protein adduction in mice given BBR provided solid evidence for in vivo oxidative debromination of BBR. The in vivo studies allowed us to better understand the metabolic activation of BBR.