Characterization of the Metabolic Pathways of 4-Chlorobiphenyl (PCB3) in HepG2 Cells Using the Metabolite Profiles of Its Hydroxylated Metabolites

[Image: see text] The characterization of the metabolism of lower chlorinated PCB, such as 4-chlorobiphenyl (PCB3), is challenging because of the complex metabolite mixtures formed in vitro and in vivo. We performed parallel metabolism studies with PCB3 and its hydroxylated metabolites to characterize the metabolism of PCB3 in HepG2 cells using nontarget high-resolution mass spectrometry (Nt-HRMS). Briefly, HepG2 cells were exposed for 24 h to 10 μM PCB3 or its seven hydroxylated metabolites in DMSO or DMSO alone. Six classes of metabolites were identified with Nt-HRMS in the culture medium exposed to PCB3, including monosubstituted metabolites at the 3′-, 4′-, 3-, and 4- (1,2-shift product) positions and disubstituted metabolites at the 3′,4′-position. 3′,4′-Di-OH-3 (4′-chloro-3,4-dihydroxybiphenyl), which can be oxidized to a reactive and toxic PCB3 quinone, was a central metabolite that was rapidly methylated. The resulting hydroxylated-methoxylated metabolites underwent further sulfation and, to a lesser extent, glucuronidation. Metabolomic analyses revealed an altered tryptophan metabolism in HepG2 cells following PCB3 exposure. Some PCB3 metabolites were associated with alterations of endogenous metabolic pathways, including amino acid metabolism, vitamin A (retinol) metabolism, and bile acid biosynthesis. In-depth studies are needed to investigate the toxicities of PCB3 metabolites, especially the 3′,4′-di-OH-3 derivatives identified in this study.