PCB126 induced toxic actions on liver energy metabolism is mediated by AhR in rats

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in the regulation of biological responses to more planar aromatic hydrocarbons, like TCDD. We previously described the sequence of events following exposure of male rats to a dioxin-like polychlorinated biphenyl (PCB) congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), that binds avidly to the AhR and causes various types of toxicity including metabolic syndrome, fatty liver, and disruption of energy homeostasis. The purpose of this study was, to investigate the role of AhR to mediate those toxic manifestations following sub-acute exposure to PCB126 and to examine possible sex differences in effects. For this goal, we created an AhR knockout (AhR-KO) model using CRISPR/Cas9. Comparison was made to the wild type (WT) male and female Holtzman Sprague Dawley rats. Rats were injected with a single IP dose of corn oil vehicle or 5 μmol/kg PCB126 in corn oil and necropsied after 28 days. PCB126 caused significant weight loss, reduced relative thymus weights, and increased relative liver weights in WT male and female rats, but not in AhR-KO rats. Similarly, significant pathologic changes were visible which included necrosis and regeneration in female rats, micro- and macro-vesicular hepatocellular vacuolation in males, and a paucity of glycogen in livers of both sexes in WT rats only. Hypoglycemia and lower IGF1, and reduced serum non-esterified fatty acids (NEFAs) were found in serum of both sexes of WT rats, low serum cholesterol levels only in the females, and no changes in AhR-KO rats. The expression of genes encoding enzymes related to xenobiotic metabolism (e.g. CYP1A1), gluconeogenesis, glycogenolysis, and fatty acid oxidation were unaffected in the AhR-KO rats following PCB126 exposure as opposed to WT rats where expression was significantly upregulated (PPARα, females only) or downregulated suggesting a disrupted energy homeostasis. Interestingly, Acox2, Hmgcs, G6Pase and Pc were affected in both sexes, the gluconeogenesis and glucose transporter genes Pck1, Glut2, Sds, and Crem only in male WT-PCB rats. These results show the essential role of the AhR in glycogenolysis, gluconeogenesis, and fatty acid oxidation, i.e. in the regulation of energy production and homeostasis, but also demonstrate a significant difference in the effects of PCB126 in males verses females, suggesting higher vulnerability of glucose homeostasis in males and more changes in fatty acid/lipid homeostasis in females. These differences in effects, which may apply to more/all AhR agonists, should be further analyzed to identify health risks to specific groups of highly exposed human populations.

Keywords: AhR Knockout rats; Energy homeostasis; Fatty acid oxidation; Gene expression; Glucose homeostasis; Liver toxicity; PCB126; Sex differences.

Fig-1. PCB126 caused AhR– and sex-dependent pathologies in livers of WT rats (H&E Staining).

Microvesicular and macrovesicular hepatocellular cytoplasmic vacuolation in male WT rats and hepatocellular necrosis and mitotic figures (regeneration) in female WT rats were observed after PCB126 exposure. No significant histologic changes were identified in AhR KO rats or WT rats treated with vehicle alone (n=5 to 7 per group; n=3 for male WT-PCB126). (Thin arrows: microvesicular hepatocellular vacuolation; Thick arrows: periportal inflammation; Arrowhead: mitotic figure)

Fig-2. PCB126 significantly reduced serum glucose and the liver transcript level of Insulin-like growth factor 1 (IGF-1) of WT male and female rats, not in AhR-KO rats.

Serum glucose level for male (A) and female rats (B), relative liver transcript level of IGF-1 in male (C) and female rats (D) reported as mean ± SE. Statistical differences between WT control and treatment groups, and AhR-KO control and treatment groups were analyzed by Two-way ANOVA (*P <0.05; n=5 to 7 per group; n=3 for male WT-PCB126 for serum glucose level; and n= 3 to 5 for gene expression).

Fig-3:. Low liver glycogen levels in WT-PCB126 rats and reduced glycogenolysis pathway gene.

PAS staining showed low glycogen in the liver of WT rats after PCB126 exposure (A). Liver transcript level of Glycogen Phosphorylase (Pygl) in male (B) and female rats (C) reported as mean ± SE. Statistical differences between WT control and treatment groups, and AhR-KO control and treatment groups were analyzed by Two-way ANOVA (*P <0.05; n=5 to 7 per group; n=3 for male WT-PCB126 for histology; and n= 3 to 5 for gene expression).

Fig-4. PCB126 downregulated the transcript level of genes related to gluconeogenesis and glucose transporter in male liver, less so in female liver.

Relative transcript level of Pck1 for male (A) and female rats (B), of G6Pase for male (C) and female rats (D), of GLUT2 for male (E) and female rats (F) reported as mean ± SE. Statistical differences between WT control and treatment groups, and AhR-KO control and treatment groups were analyzed by Two-way ANOVA (*P <0.05; n= 3 to 5 for each group).

Fig-5:. Some liver gluconeogenesis gene transcript levels were reduced by PCB126 in WT rats.

Relative transcript level of Pc in male (A) and female (B), of Sds in male (C) and female (D), and of Ldh-A in male (E) and female (F) rat livers reported as mean ± SE. Statistical differences between WT control and treatment groups, and AhR-KO control and treatment groups were analyzed by Two-way ANOVA (*P <0.05; n= 3 to 5 for each group).

Fig-6:. Among tested coactivators of gluconeogenesis only Crem was downregulated by PCB126 and only in male rat livers.

Relative liver transcript level of Creb1 in male (A) and female rats (B), of Icer in male (C) and female rats (D), of Pgc1-α in male (E) and female rats (F), and of Crem in male (G) and female rat (H) reported as mean ± SE. Statistical differences between WT control and treatment groups, and AhR-KO control and treatment groups were analyzed by Two-way ANOVA (*P <0.05; n= 3 to 5 for each group).

Fig-7. PCB126 mediated AhR activation caused decreased serum glucose levels and lower transcript levels of genes involved in liver gluconeogenesis and glucose transport, particularly in male rats.

Fig-8. PCB126 exposed WT rats had lower transcript levels of genes involved in β-oxidation but female rats had increased PPARα levels.

Relative transcript level of Pparα in male (A) and female rats (B), of Acox1 in male (C) and female rats (D), and of Hmgcs2 in male (E) and female rats (F) reported as mean ± SE. Statistical differences between WT control and treatment groups, and AhR-KO control and treatment groups were analyzed by Two-way ANOVA (*P <0.05; n= 3 to 5 for each group).

Fig-9. PCB126 reduced serum non-esterified fatty acids (NEFAs) and cholesterol in WT rats, not in AhR-KO rats.

Serum NEFAs level of male (A) and female rats (B), serum cholesterol level of male (C) and female rats (D), reported as mean ± SE. Statistical differences between WT control and treatment groups, and AhR-KO control and treatment groups were analyzed by Two-way ANOVA (*P <0.05; n= 3 to 7 for each group).

Fig-10. PCB126 significantly imbalanced the energy homeostasis in rats, which depended on AhR activation.

The scheme depicts the effects of PCB126 on gluconeogenesis, glycogenolysis, and fatty acid oxidation in the liver resulting in an imbalance in energy homeostasis after 28 days exposure that is mediated by AhR activation. Sex differences in gene regulation suggest a larger role of ß-oxidation in female and gluconeogenesis in male rats (DNL-De Novo Lipogenesis; TG- Triglyceride; NEFA- non-esterified fatty acid).