The Mechanisms Of Hepatic Lipid Accumulation Induced By Long-Term Bisphenol A Exposure In Mouse Liver

Accumulating evidence suggests a role of bisphenol A (BPA) in metabolic diseases. However, the underlying mechanism is still unclear. Using a mouse BPA exposure model, we investigated the effects of long-term BPA exposure on lipid metabolism of male mice and the underlying mechanisms. The male mice exposed to BPA (0.5μg BPA /kg/day, a human relevant dose) for 10 months exhibited significantly hepatic accumulation of triglycerides and cholesterol. The liver cells from the BPA-exposed mice showed significantly increased expression levels of the genes related to fatty acid and cholesterol synthesis, and significantly reduced expression levels of the genes related to fatty acid oxidation. These liver cells also showed increased expression levels of the transcription factors, Srebf1 and Srebf2, which may upregulate the genes related to fatty acid and cholesterol synthesis, and decreased methylation levels in the promoters of Srebfl and Srebf2 genes. The expression levels of DNA methyltransferases were decreased, and could partially explain the hypomethylaiton of Srebfl and Srebf1. Knockdown DNA methyltransferases in Hepal-6 cell line led to demethylation and increased expression levels of Srebfl and Srebf1. BPA administration down-regulated the expression of DNMTs in Hepa 1-6. Our results suggest that long-term BPA exposure could induce hepatic lipid accumulation, which may be due to the epigenetic reprogramming of the genes involved in lipid metabolism, such as the alterations of DNA methylation patterns.Part Ⅰ Establishment of Mouse Model of Long-term BPA Exposure and the Effects of BPA on Lipid MetabolismObjectives:To investigate the effects of long-term human relevant BPA exposure on lipid metabolism.Materials and Methods:From the first day of childbirth and throughout lactation, mother mice were randomly assigned to two groups. Mothers had free access to drinking water that contained BPA at O(control) or 2.5ug/L (estimated 0.5μg BPA/kg body weight, a human relevant dose). Their male offspring were administered BPA accordingly after weaning. Drinking water of both groups contained 0.1% vehicle (ethanol) by volume. Part of male offspring from each group were randomly selected and then weighted and sacrificed after fasting overnight at the age of 8 weeks. The rest mice were killed at the age of ten months. Fasting blood glucose levels were tested using an Accu-check compact glucometer. Hepatic content of triglyceride (TG) and total cholesterol were measured using TG and total cholesterol assay kit respectively. Serum levels of TG, total cholesterol, low-and high-density lipoprotein (LDL, HDL) cholesterol, glutamic pyruvic transaminase (ALT) and glutamic oxalacetic transaminase (AST) were assayed on a biochemical analyzer, TBA120FR. Serum insulin was determined with the mouse insulin ELISA kit. Oil-Red-O staining and H&E staining were used to detect the lipid accumulation of liver.Conclusions:Long-term BPA exposure induced obesity, mediated the homeostasis of glucose and lipid metabolism, and, induced hepatic TG and cholesterol accumulation in BPA-exposed male mice of middle age.Part II The Expression of Genes Related to Hepatic Lipid Metabolism in Livers from Mice of Long-term BPA ExposureObjectives:To explore the effects of long-term BPA exposure on the expression profile of genes related to lipid metabolism in mouse liver and to investigate the mechanism underlying the abnormal lipid metabolism induced by BPA.Materials and Methods:Total RNA was isolated with Trizol reagent from mouse livers of both ages (8 weeks and 10 months). cDNA was synthesized using RT reagent Kit. The amplification of the genes of interest was performed using real-time quantitative PCR on a 7900 Real-Time PCR System using SYBR-Green Premix Ex Taq.Results:In 10-month male mice, the liver cells from the BPA-exposed group showed significantly increased expression levels of the genes related to fatty acid and cholesterol synthesis, and significantly reduced expression levels of the genes related to fatty acid oxidation, and, triglyceride and cholesterol transportation. These liver cells also showed increased expression levels of the transcription factors, Srebfl and Srebf2.The mean level of Fasn in BPA exposure mice was almost 2 times of that in control mice, and, the levels of Scdl, Hmgcr and Sqle in BPA group were 4 times of those in control group.Conclusions:The mechanisms potentially underlying the aberrant hepatic lipid metabolism and associated dyslipidemia induced by BPA were increased fatty acid and cholesterol synthesis, impaired triglyceride and cholesterol transportation, and, reduced fatty acid oxidation. The activation of fatty acid and cholesterol synthesis may be of the most importance. Increased expression levels of the transcription factors, Srebfl and Srebf2, could partially explain the increased expression of genes related to fatty acid and cholesterol synthesis.PartⅢ The Alterations of the DNA Methylation Patterns of Genes Involved in Lipid Metabolism Contributed to the BPA-Modulated Expression Profile of These GenesObjectives:To investigate the role of DNA methylation in the BPA-modulated expression profile of genes related to lipid metabolism.Materials and Methods:DNA, total RNA and protein was isolated from mouse livers of both ages (8 weeks and 10 months). MassArray EpiTYPER quantitative DNA methylation analysis was applied to determine the methylation levels of unit or each CpG site in the promoters of selected genes related to lipid metabolism. The mRNA levels of DNA methyltransferases (Dnmtl, Dnmt3a and Dnm3b) was measured using real-time quantitative PCR and the protein levels were tested with western blot. We knockdown DNMTs with siRNA in Hepal-6 mouse hepatocyte cell line and study the expression of genes involved in lipid synthesis with real-time PCR and measured the DNA methylation levels of genes involved in lipid synthesis with MassArray EpiTYPER quantitative DNA methylation analysis. We treated Hepal-6 cell with BPA and measured the expression of DNMTs with real-time PCR.Results:Overall methylation levels were significantly decreased in the promoters of Srebf2 and Hmgcr in 8-week BPA mice, and, promoters of Srebfl, Srebf2, Fasn and Hmgcr in 10-month BPA mice compared with control group. Methylation levels of single CpG sites from the four genes were also statistically decreased in BPA-exposed mice of both ages. In 8-week mice, the mRNA and protein levels of Dnmtl and Dnmt3a were significantly reduced in BPA group compared with control. In 10-month mice, BPA group showed reduced mRNA and protein levels of Dnmt3b. DNA methyltransferase knockdown led to demethylation and promoted expression of lipogenesis genes in hepal-6 mouse hepatocyte cell line. BPA administration downregulated the expression of DNMTs in hepal-6 cell line.Conclusions:The hypomethylation of the promoters of Srebfl, Srebf2, Fasn and Hmgcr from BPA-exposed mice is very likely to contribute to the increased transcription of the four genes. The decreased expression of DNA methyltransferases induced by BPA led to the hypomethylaiton of the genes related to lipid synthesis.