Human CYP2A6, CYP2B6, AND CYP2E1 Atropselectively Metabolize Polychlorinated Biphenyls to Hydroxylated Metabolites

Abstract

Exposure to chiral polychlorinated biphenyls (PCBs) has been associated with neurodevelopmental disorders. Their hydroxylated metabolites (OH-PCBs) are also potentially toxic to the developing human brain; however, the formation of OH-PCBs by human cytochrome P450 (P450) isoforms is poorly investigated. To address this knowledge gap, we investigated the atropselective biotransformation of 2,2',3,4',6-pentachlorobiphenyl (PCB 91), 2,2',3,5',6-pentachlorobiphenyl (PCB 95), 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132), and 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) by different human P450 isoforms. In silico predictions with ADMET Predictor and MetaDrug software suggested a role of CYP1A2, CYP2A6, CYP2B6, CYP2E1, and CYP3A4 in the metabolism of chiral PCBs. Metabolism studies with recombinant human enzymes demonstrated that CYP2A6 and CYP2B6 oxidized PCB 91 and PCB 132 in the meta position and that CYP2A6 oxidized PCB 95 and PCB 136 in the para position. CYP2B6 played only a minor role in the metabolism of PCB 95 and PCB 136 and formed meta-hydroxylated metabolites. Traces of para-hydroxylated PCB metabolites were detected in incubations with CYP2E1. No hydroxylated metabolites were present in incubations with CYP1A2 or CYP3A4. Atropselective analysis revealed P450 isoform-dependent and congener-specific atropselective enrichment of OH-PCB metabolites. These findings suggest that CYP2A6 and CYP2B6 play an important role in the oxidation of neurotoxic PCBs to chiral OH-PCBs in humans.

Figure 1.

Simplified metabolism scheme showing the chemical structures and abbreviations of metabolites of PCB 91, PCB 95, PCB 132 and PCB 136 identified in incubations with human P450 enzymes. For the chemical names of the different PCB metabolites, see the Supporting Information.

Figure 2.. In silico predictions and in vitro incubations show that chiral PCBs are metabolized to hydroxylated metabolites by human CYP2A6, CYP2B6, and CYP2E1.

ADMET Predictor and Metadrug software packages were initially used to predict human P450 isoforms involved in the metabolism of chiral PCBs. Metabolism studies with recombinant human P450 enzymes were performed to assess if the P450 isoforms identified in silico indeed form OH-PCBs. In vitro metabolism studies were carried out using the following incubation conditions: 50 µM PCB; 5-minute incubation at 37 ºC; 10 pmol/mL P450 content; see the Experimental Section for additional details.^, Data are presented as mean ± standard deviation, n = 3. ^a ✓ indicates that the OH-PCB metabolite was predicted to be formed. ^b In vitro metabolite formation rate (+) below 80 fmol/pmol P450/min, (++) between 80 and 180 fmol/pmol P450/min, (+++) between 180 and 420 fmol/pmol P450/min and (++++) above 420 fmol/pmol P450/min. ^c Experimental data for incubation with CYP2E1 for 60 min are shown. ^d Peak corresponding to this metabolite was below the limit of detection (LOD).

Figure 3.. Chiral PCB 91, PCB 95, PCB 132 and PCB 136 are oxidized in a congener-specific manner by human CYP2A6 and CYP2B6 to OH-PCBs.

Representative GC-TOF chromatograms of OH-PCB metabolites (as methylated derivatives) in extracts from incubations of CYP2A6 with (A1) PCB 91; (A2) PCB 95; (A3) PCB 132; and (A4) PCB 136; and in extracts from incubations of CYP2B6 with (B1) PCB 91 and (B2) PCB 132. Incubation conditions were as follow: 50 μM PCB; 60 minutes, 37 ºC; and 10 pmol/mL P450. See the Supporting Information for the corresponding mass spectra. The metabolites were separated on DB5-ms column; see the Experimental Section above for additional details. ^a No mass spectra were obtained due to low analyte levels. However, the peak was identified by matching the retention time with the authentic standard. ^b Accurate mass not determined due to background carbon interference. RS, peak corresponding to the recovery standard.

Figure 4.. Formation rates of PCB metabolites (analyzed as methylated derivatives) reveal that (A) PCB 91; (B), PCB 95, (C) PCB 132 and (D) PCB 136 are metabolized to OH-PCBs by human CYP2A6, CYP2B6, and CYP2E1.

Metabolism studies were performed using the following incubation conditions: 50 µM PCB; 5-minute incubation at 37 ºC; 10 pmol/mL P450 content; NADPH regenerating system. ^c Incubation with CYP2E1 for 60 min. ^d Data are presented as mean ± standard deviation, n = 3.

Figure 5.. OH-PCB metabolites are formed atropselectively from (A) PCB 91, (B) PCB 95, (C) PCB 132 and (D) PCB 136 in incubations with recombinant human CYP2A6 (dark red), CYP2B6 (orange) and CYP2E1 (light green) enzymes.

No metabolites were detected in experiments with CYP1A2 and CYP3A4. Open circles (○), diamonds (◇) and squares (□) indicate 1,2-shift, meta- and para-substituted metabolites, respectively. Data are expressed as mean ± SD, n = 3; error bars are typically hidden behind the symbols. Metabolism studies were performed using the following incubation conditions: 50 μM PCB; 60 min incubation at 37 ºC; 10 pmol/mL P450 content; and ~1 mM NADPH regenerating system. Metabolites were separated on BDM (5–91, 4–91, 3–103 and 4’−95); GTA (3–100, 3’−140) or CD columns (5’−132, 3–150, 5– 136 and 4–136). EF value of 5–95 and 4–95 could not be calculated due to co-elution of E₁-5–95 and E₁-4–95.