Probing the Role of CYP2 Enzymes in the Atropselective Metabolism of Polychlorinated Biphenyls Using Liver Microsomes from Transgenic Mouse Models

Abstract

Chiral polychlorinated biphenyls (PCB) are environmentally relevant developmental neurotoxicants. Because their hydroxylated metabolites (OH-PCBs) are also neurotoxic, it is necessary to determine how PCB metabolism affects the developing brain, for example, in mouse models. Because the cytochrome P450 isoforms involved in the metabolism of chiral PCBs remain unexplored, we investigated the metabolism of PCB 91 (2,2',3,4',6-pentachlorobiphenyl), PCB 95 (2,2',3,5',6-pentachlorobiphenyl), PCB 132 (2,2',3,3',4,6'-hexachlorobiphenyl), and PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) using liver microsomes from male and female Cyp2a(4/5)bgs-null, Cyp2f2-null, and wild-type mice. Microsomes, pooled by sex, were incubated with 50 μM PCB for 30 min, and the levels and enantiomeric fractions of the OH-PCBs were determined gas chromatographically. All four PCB congeners appear to be atropselectively metabolized by CYP2A(4/5)BGS and CYP2F2 enzymes in a congener- and sex-dependent manner. The OH-PCB metabolite profiles of PCB 91 and PCB 132, PCB congeners with one para-chlorine substituent, differed between null and wild-type mice. No differences in the metabolite profiles were observed for PCB 95 and PCB 136, PCB congeners without a para-chlorine group. These findings suggest that Cyp2a(4/5)bgs-null and Cyp2f2-null mice can be used to study how a loss of a specific metabolic function (e.g., deletion of Cyp2a(4/5)bgs or Cyp2f2) affects the toxicity of chiral PCB congeners.

Figure 1

Mouse models used in this study. Cyp2f2- and Cyp2a(4/5)bgs-null mice were generated by deleting the indicated segments from the Cyp2f2–2s1 cluster on mouse cytochrome 7, as described previously.⁻ The organization of this gene cluster was published previously.

Figure 2

Simplified metabolism scheme showing the structures and abbreviations of mono- and dihydroxylated metabolites of PCB 91, PCB 95, PCB 132, and PCB 136 observed in metabolism studies with mouse liver microsomes. R₁–R₄ are H if not indicated otherwise.

Figure 3

Differences in the total OH-PCB (ΣOH-PCB) metabolite profiles are due to genotype-dependent differences in the levels of individual PCB metabolites in experiments with pooled liver microsomes from (A) male and (B) female mice. The data were expressed as ng/mg protein. Metabolite levels were compared using one-way ANOVA.

Figure 4

A heatmap-type comparison of the metabolite profiles formed from PCB 91 in incubations with pooled liver microsomes from (A) male and (B) female Cyp2a(4/5)bgs-null, Cyp2f2-null, and the corresponding wild-type mice reveals differences in the hydroxylated PCB metabolite levels across genotypes. These differences in the metabolite profiles are due to genotype-dependent differences in the levels of individual PCB 91 metabolites in experiments with pooled liver microsomes from (C) male and (D) female mice; significant changes are noted by p-values in the figures. The data are expressed as ng/mg protein and, in panels A and B, are visualized using the heatmap function implemented by Metabolanalyst. Metabolite levels were compared using one-way ANOVA. 3-100, 2,2′,4,4′,6-pentachlorobiphenyl-3-ol; 5-91, 2,2′,3,4′,6-pentachlorobiphenyl-5-ol; and 4-91, 2,2′,3,4′,6-pentachlorobiphenyl-4-ol.

Figure 5

A heatmap-type comparison of the metabolite profiles formed from PCB 95 in incubations with pooled liver microsomes from (A) male and (B) female Cyp2a(4/5)bgs-null, Cyp2f2-null, and the corresponding wild-type mice reveals differences in the hydroxylated PCB metabolite levels across genotypes. These differences in the metabolite profiles are due to genotype-dependent differences in the levels of individual PCB 95 metabolites in experiments with pooled liver microsomes from (C) male and (D) female mice; significant changes are noted by p-values in the figures. The data are expressed as ng/mg protein and, in panels A and B, are visualized using the heatmap function implemented by Metabolanalyst. Metabolite levels were compared using one-way ANOVA. 3-103, 2,2′,4,5′,6-pentachlorobiphenyl-3-ol; 5-95, 2,2′,3,5′,6-pentachlorobiphenyl-5-ol; 4′-95, 2,2′,3,5′,6-pentachlorobiphenyl-4-ol; and 4-95, 2,2′,3,5′,6-pentachlorobiphenyl-4-ol.

Figure 6

A heatmap-type comparison of the metabolite profiles formed from PCB 132 in incubations with pooled liver microsomes from (A) male and (B) female Cyp2a(4/5)bgs-null, Cyp2f2-null, and the corresponding wild-type mice reveals differences in the hydroxylated PCB metabolite levels across genotypes. These differences in the metabolite profiles are due to genotype-dependent differences in the levels of individual PCB 132 metabolites in experiments with pooled liver microsomes from (C) male and (D) female mice; significant changes are noted by p-values in the figures. The data are expressed as ng/mg protein and, in panels A and B, are visualized using the heatmap function implemented by Metabolanalyst. Metabolite levels were compared using one-way ANOVA. 3′–140, 2,2′,3,4,4′,6′-hexachlorobiphenyl-3-ol; 5′–132, 2,2′,3,3′,4,6′-hexachlorobiphenyl-5′-ol; and 4′–132, 2,2′,3,3′,4,6′-hexachlorobiphenyl-4′-ol.

Figure 7

A heatmap-type comparison of the metabolite profiles formed from PCB 136 in incubations with pooled liver microsomes from (A) male and (B) female Cyp2a(4/5)bgs-null, Cyp2f2-null, and the corresponding wild-type mice reveals differences in the hydroxylated PCB metabolite levels across genotypes. These differences in the metabolite profiles are due to genotype-dependent differences in the levels of individual PCB 136 metabolites in experiments with pooled liver microsomes from (C) male and (D) female mice; significant changes are noted by p-values in the figures. The data are expressed as ng/mg protein and, in panels A and B, are visualized using the heatmap function implemented by Metabolanalyst. Metabolite levels were compared using one-way ANOVA. 3–150, 2,2′,3′,4,6,6′-hexachlorobiphenyl-3-ol; 5–136, 2,2′,3,3′,6,6′-hexachlorobiphenyl-5-ol; and 4–136, 2,2′,3,3′,6,6′-hexachlorobiphenyl-4-ol.