Hepatic Lipid Accumulation and Nrf2 Expression following Perinatal and Peripubertal Exposure to Bisphenol A in a Mouse Model of Nonalcoholic Liver Disease

Abstract

Background: Exposure to chemicals during critical windows of development may re-program liver for increased risk of nonalcoholic fatty liver disease (NAFLD). Bisphenol A (BPA), a plastics component, has been described to impart adverse effects during gestational and lactational exposure. Our work has pointed to nuclear factor E2-related factor 2 (Nrf2) being a modulator of hepatic lipid accumulation in models of NAFLD.

Objectives: To determine if chemical exposure can prime liver for steatosis via modulation of NRF2 and epigenetic mechanisms.

Methods: Utilizing BPA as a model exposure, pregnant CD-1 mice were administered 25μg/kg/day BPA via osmotic minipumps from gestational day 8 through postnatal day (PND)16. The offspring were weaned on PND21 and exposed to same dose of BPA via their drinking water through PND35. Tissues were collected from pups at week 5 (W5), and their littermates at week 39 (W39).

Results: BPA increased hepatic lipid content concomitant with increased Nrf2 and pro-lipogenic enzyme expression at W5 and W39 in female offspring. BPA exposure increased Nrf2 binding to a putative antioxidant response element consensus sequence in the sterol regulatory-element binding protein-1c (Srebp-1c) promoter. Known Nrf2 activators increased SREBP-1C promoter reporter activity in HepG2 cells. Methylated DNA immunoprecipitation-PCR and pyrosequencing revealed that developmental BPA exposure induced hypomethylation of the Nrf2 and Srebp-1c promoters in livers of W5 mice, which was more prominent in W39 mice than in others.

Conclusion: Exposure to a xenobiotic during early development induced persistent fat accumulation via hypomethylation of lipogenic genes. Moreover, increased Nrf2 recruitment to the Srebp-1c promoter in livers of BPA-exposed mice was observed. Overall, the underlying mechanisms described a broader impact beyond BPA exposure and can be applied to understand other models of NAFLD. https://doi.org/10.1289/EHP664.

Figure 1.

Effects of PNPP BPA exposure on body weight and hepatic lipid accumulation in female CD-1 mice. A) Body weight and liver weight. Asterisk (*) represents significant difference in weights between BPA treated and vehicle treated animals of same age ($p\le 0.05$). B) Hepatic triglyceride quantification. C) Oil Red O staining of lipids in the liver tissue. Representative images are displayed in $200\mathrm{X}$ magnification. D) Quantification of oil red staining density from all the samples.

Figure 2.

Effects of PNPP BPA exposure on mRNA expression of lipogenic targets in livers of female CD-1 mice (A.W5; B. W39). (*) represents significant difference in weights between BPA treated and vehicle treated animals of same age ($p\le 0.05$).

Figure 3.

Effects of PNPP BPA exposure on protein expression of lipogenic transcription factors and enzymes in livers of female CD-1 mice. (A: W5 blots, B: quantification of W5 blots, C: W39 blots, D: quantification of W39 blots). Nuclear proteins were detected for expression using specific antibodies for peroxisome proliferator activated receptor gamma ($\text{Ppar}-\mathrm{\gamma }$), sterol regulatory element binding protein-1c (Srebp-1c), and phosphorylated Srebp-1c. Fatty acid synthase (Fas), acetyl-CoA carboxylase (Acc) and phosphorylated Acc were quantified from total protein lysates by Western blot using specific antibodies. The mean blot intensity is presented as percent expression. (*) represents significant difference in weights between BPA-treated and vehicle-treated animals of same age ($p\le 0.05$).

Figure 4.

Effects of PNPP BPA exposure on Nrf2 signaling in livers of female CD-1 mice. A) Nrf2 and its target gene glutamate cysteine ligase (Gclc) expression in livers of W5 animals. Messenger RNA expression was quantified using real time polymerase chain reaction (RT-PCR). B) Protein expression of nuclear Nrf2 and Gclc by Western blot for W5 animals and blot intensity quantification. C) Nrf2 and Gclc mRNA expression in W39 animals. D) Protein expression of nuclear Nrf2 and Gclc in W39 animals and blot intensity quantification. E & F) Glutathione (GSH) content in W5 and W39 animals. (*) represents significant difference in weights between BPA treated and vehicle treated animals of same age ($p\le 0.05$).

Figure 5.

Effect of PNPP BPA exposure on Srebp-1c, Fas, and Nrf2 promoter methylation. CpG sites in promoter sequences of Srebp-1c, Fas, and Nrf2 (A, B, and C, respectively) up to $2\text{kb}$ upstream of translational start site were analyzed to check methylation effects of PNPP BPA. Results are plotted as fold enrichment.

Figure 6.

Nrf2 recruitment to the Srebp-1c promoter by chromatin immunoprecipitation (ChIP). A) Schematic of Nrf2 recruitment on promoter of Srebp-1c. B) End-point PCR. C) Real-time PCR amplification by using primers as enlisted in supplementary Table S3, which covers putative ARE consensus sequence on promoter of Srebp-1c. Results are plotted as fold enrichment in comparison with negative control.

Figure 7.

NRF2 mediated transactivation of human SREBP-1C in vitro. A) NRF2 expression plasmid were transiently co-transfected with the SREBP-1C promoter luciferase reporter constructs ($-1.5\text{kb}$) or pGL3 basic, in HepG2 cells. Luciferase activity was measured as relative firefly/renilla luciferase and was recorded as relative light units data are presented as mean fold change $\pm \text{SEM}$. B) SREBP-1C promoter luciferase reporter constructs or pGL3 basic were transiently transfected into HepG2 cell. After 24 hrs of transfection, HepG2 cell were treated with the oleanolic acid ($50\text{uM}$) and sulforaphane ($10\mathrm{\mu }\mathrm{M}$) along with DMSO (0.05%) for 12hrs. Luciferase activity was measured using a commercial kit.