Evaluation of Aroclor 1260 exposure in a mouse model of diet-induced obesity and non-alcoholic fatty liver disease

Abstract

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with non-alcoholic fatty liver disease (NAFLD) in epidemiologic studies. The purpose of this study was to evaluate the hepatic effects of a PCB mixture, Aroclor 1260, whose composition mimics human bioaccumulation patterns, in a mouse model of diet-induced obesity (DIO). Male C57Bl/6J mice were fed control diet or 42% high fat diet (HFD) and exposed to Aroclor 1260 (20mg/kg or 200mg/kg in corn oil) for 12weeks. A glucose tolerance test was performed; plasma/tissues were obtained at necropsy for measurements of adipocytokine levels, histology, and gene expression. Aroclor 1260 exposure was associated with decreased body fat in HFD-fed mice but had no effect on blood glucose/lipid levels. Paradoxically, Aroclor 1260+HFD co-exposed mice demonstrated increased hepatic inflammatory foci at both doses while the degree of steatosis did not change. Serum cytokines, ALT levels and hepatic expression of IL-6 and TNFα were increased only at 20mg/kg, suggesting an inhibition of pro-inflammatory cytokine production at the 200mg/kg exposure. Aroclor 1260 induced hepatic expression of cytochrome P450s including Cyp3a11 (Pregnane-Xenobiotic Receptor target) and Cyp2b10 (constitutive androstane receptor target) but Cyp2b10 inducibility was diminished with HFD-feeding. Cyp1a2 (aryl hydrocarbon Receptor target) was induced only at 200mg/kg. In summary, Aroclor 1260 worsened hepatic and systemic inflammation in DIO. The results indicated a bimodal response of PCB-diet interactions in the context of inflammation which could potentially be explained by xenobiotic receptor activation. Thus, PCB exposure may be a relevant "second hit" in the transformation of steatosis to steatohepatitis.

Keywords: AhR; Aroclor 1260; CAR; NAFLD; PCBs; PXR.

Fig. 1

Effects of Aroclor 1260 exposure on body weight and visceral adiposity. (A). Increase in body weight with time for C57BL/6 mice (n = 10) fed with a 42% milk fat diet (vs. CD). Body weight measurements were taken from Week 1 to Week 12 (12 weeks). (B). The % increase in body weight gain with time was calculated and the body weight at Week 1 was taken as 100%. (C). The % fat composition was measured using a Lunar PIXImus densitometer and Aroclor 1260 (20 and 200 mg/kg) exposure lowered % fat composition in HFD-fed mice vs. HFD + vehicle group. (D). Epididymal weight (EW) to body weight (BW) ratio was calculated for all groups of mice. (E). Epididymal adipose tissue was stained with H&E. (F). Adipocyte size (μm²) was measured and average cell size of >100 cells for each group was calculated. Values are mean ± SEM, p < 0.05, a—Δ due to HFD. CD—control diet, HFD— high fat diet, Ar—Aroclor 1260.

Fig. 2

Aroclor 1260 exposure caused steatohepatitis in HFD-fed mice. (A). H&E staining of hepatic sections established the occurrence of centrilobular hepatocellular hypertrophy, karyomegaly, and multinucleate (arrow head) hepatocytes in the CD + Aroclor 1260 (200 mg/kg) group [A]. HFD consumption resulted in variable, centrilobular, microvesicular lipidosis [B–D], while centrilobular hepatocellular hypertrophy was observed in HFD + Aroclor 1260 (20 mg/kg) mice [C]. HFD-fed mice exposed to Aroclor 1260 (200 mg/kg) exhibited occasional, small areas of necrosis and inflammation (steatohepatitis) [D], characterized by neutrophils (arrow head) and pyknotic debris (arrow). (B). CAE staining demonstrated neutrophil infiltration in the HFD + Aroclor 1260 (20 and 200 mg/kg) groups. (C). Serum ALT and (D) AST levels (U/L) were measured (n = 10) using the Piccolo Xpress chemical analyzer. Values are mean ± SEM, p < 0.05, a—Δ due to HFD, b—Δ due to Aroclor 1260 exposure at 20 mg/kg. CD—control diet, HFD—high fat diet, Ar—Aroclor 1260, PV—portal vein, CV—central vein.

Fig. 3

Low dose Aroclor 1260 increased serum cytokines in HFD-fed mice. Serum (A) IL-6 (pg/mL), (B) tPAI-1 (pg/mL), (C) leptin (pg/mL) and (D) resistin (pg/mL) levels were measured using the Luminex IS 100 system (n = 10). Values are mean ± SEM, p < 0.05, a—Δ due to HFD, b—Δ due to Aroclor 1260 exposure at 20 mg/kg, c—Δ due to Aroclor 1260 exposure at 200 mg/kg. CD—control diet, HFD—high fat diet, Ar—Aroclor 1260.

Fig. 4

HFD increased insulin resistance, and this was unaffected by Aroclor 1260 co-exposure. (A). HOMA-IR was calculated from fasting blood glucose and insulin levels for all six groups of animals (n = 10). (B). Glucose tolerance test was performed, and blood glucose levels were measured for mice (n = 10) fed with CD or HFD, with or without Aroclor 1260 co-exposure. (C). AUC was calculated, and the HFD groups showed higher AUC levels than the CD groups. (D). Fasting blood glucose levels (mg/dL) were measured, and the CD-fed mice co-exposed to Aroclor 1260 showed lower levels vs. CD + vehicle. Values are mean ± SEM, p < 0.05, a—Δ due to HFD, b—Δ due to Aroclor 1260 exposure at 20 mg/kg, c—Δ due to Aroclor 1260 exposure at 200 mg/kg. CD—control diet, HFD—high fat diet, Ar—Aroclor 1260.

Fig. 5

Effects of Aroclor 1260 exposure on genes involved in lipid metabolism. Real-time PCR experiments showed the changes in hepatic mRNA expressions caused by Aroclor 1260 exposure for (A) FAS, (B) SCD1, (C) SREBP-1c and (D) CD36. Values are mean ± SEM, p < 0.05, a—Δ due to HFD, b—Δ due to Aroclor 1260 exposure at 20 mg/kg, c—Δ due to Aroclor 1260 exposure at 200 mg/kg. CD—control diet, HFD—high fat diet, Ar—Aroclor 1260.

Fig. 6

Effects of Aroclor 1260 exposure on TLR-4 target genes. Real-time PCR experiments showed the changes in hepatic mRNA expressions caused by Aroclor 1260 exposure for (A) TNFα, (B) IL-6, (C) MCP2 and (D) MIP1α. Values are mean ± SEM, p < 0.05, a—Δ due to HFD, b—Δ due to Aroclor 1260 exposure at 20 mg/kg. CD—control diet, HFD— high fat diet, Ar—Aroclor 1260.

Fig. 7

Aroclor 1260 exposure altered hepatic expression of CAR, PXR and AhR target genes. Real-time PCR experiments showed the changes in hepatic mRNA expressions caused by Aroclor 1260 exposure for (A) Cyp2b10 (CAR target gene), (B) Cyp3a11 (PXR target gene), (C) Cyp1a2 (AhR target gene) and (D) PEPCK-1 (an indirect target of CAR and PXR). Values are mean ± SEM, p < 0.05, a—Δ due to HFD, b—Δ due to Aroclor 1260 exposure at 20 mg/kg, c—Δ due to Aroclor 1260 exposure at 200 mg/kg. CD—control diet, HFD—high fat diet, Ar—Aroclor 1260.