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

Abstract

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. Indepth studies are needed to investigate the toxicities of PCB3 metabolites, especially the 3′,4′-di-OH-3 derivatives identified in this study.

Assessment of Polychlorinated Biphenyls and Their Hydroxylated Metabolites in Postmortem Human Brain Samples: Age and Brain Region Differences

“…Dichlorinated OH-PCBs were detected in five samples, and tri- and tetrachlorinated OH-PCBs were observed in all 18 samples analyzed. As described earlier, OH-PCBs were identified based on their accurate mass and isotope pattern. , For example, a dichlorinated OH-PCB was detected in the Nt-LCMS analysis in BA19 from a 1-day-old female donor (Figure ; donor 2017-3, see Table S1). In the GC–MS/MS analysis, we observed a peak corresponding to 4′-9+4–14 (as methylated derivatives), dichlorinated OH-PCBs, in the same brain region from this donor.…”

“… 33 , 34 Several studies suggest that PCB congeners are preferentially oxidized to meta- and para-hydroxylated metabolites by human cytochrome P450 enzymes. 35 − 38 Some OH-PCB congeners are selectively retained in human blood 39 and are present in human populations . 40 − 43 Other, mostly lower chlorinated, PCBs are readily eliminated, either as free OH-PCBs or OH-PCB conjugates.…”

“…PCBs are oxidized by cytochrome P450 enzymes to hydroxylated PCBs (OH-PCBs) in humans. , Several studies suggest that PCB congeners are preferentially oxidized to meta- and para-hydroxylated metabolites by human cytochrome P450 enzymes. − Some OH-PCB congeners are selectively retained in human blood and are present in human populations . − Other, mostly lower chlorinated, PCBs are readily eliminated, either as free OH-PCBs or OH-PCB conjugates. − Levels of OH-PCBs in human blood and placenta have received considerable attention, and epidemiological studies implicate OH-PCBs in adverse health outcomes, including neurotoxic outcomes . For example, maternal exposure to OH-PCBs during pregnancy may increase neonatal T4 levels .…”

PCB Sulfates in Serum from Mothers and Children in Urban and Rural U.S. Communities

“… 34 Another example is the identification of monohydroxylated, sulfate, and glucuronide metabolites in HepG2 cells exposed to PCB 3. 35 …”

Metabolism of 3-Chlorobiphenyl (PCB 2) in a Human-Relevant Cell Line: Evidence of Dechlorinated Metabolites

“…29,30 Importantly, putative dechlorinated metabolites (class 4) were detected, suggesting for the first time that some LC-PCBs may undergo dechlorination reactions in humans. Similar to previous studies, 29,30 we did not detect PCB 2 quinone metabolites or HepG2 cells were exposed for 24 h to PCB 2 [high concentration (10 μM) or low concentration (3.6 nM)] as described in the Experimental Section; metabolites were extracted from the cell culture medium by QuEChERS extraction, and extracts were analyzed by LC-Orbitrap MS. Most of these PCB 2 metabolites were also detected in LC-QTof analyses of the same extracts (Table S1).…”