Metabolomics Investigation On 2,3,7,8-tetrachlorodibenzofuran Induced Non-alcoholic Fatty Liver Disease In Mice

Objectives: 2,3,7,8-tetrachlorodibenzofuran is one of typical dioxin-like persistent organic pollutants produced from municipal waste combustion.Previous studies showed that TCDF exerted toxic effects on human healthy and could induce fatty liver or obesity.In this study,long-term treatment with low doses of TCDF was performed orally on mice.We used metabolomics combined with molecular biology method to investigate the effects of TCDF exposure on lipid homeostasis in mice.Methods: 8-week-old male C57BL/6J mice were divided into three weight-matched group(Control,Low TCDF and High TCDF).Mice in group of Low TCDF and High TCDF were treated with the final dose of TCDF 0.25 and 1.0 μg/kg body weight,respectively.At the end of 14 weeks post-dose,mice were sacrificed and samples including liver and blood were collected.We measured the level of triglyceride(TG)in liver and detected the pathological change of liver tissue.Metabolomic techniques based on 1H NMR,ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry(UHPLC-Qq Q-MS),and gas chromatography-flame ionization detector(GC-FID)was used to study lipid metabolic consequences of mice.The effect of TCDF on the m RNA and protein expression level of key enzymes involved in metabolic pathways was obtained from QPCR and Western Blot method.Results: Ahr-Cyp1a1 pathway was activated by TCDF exposure.Compared with the control group,mice exposed to TCDF shown a significant increase in body weight and hepatic TG.A significant increase in the ratio of liver to body weight after relatively higher dose of TCDF treatment in mice was observed.Photomicrograph of H&E-stained liver sections of the TCDF treated mice indicated the lipid droplets.Metabolomics revealed that TCDF exposure cause significant up-regulation of hepatic hepatic fatty acids levels.Marked increases in gene expression of Acaca,Fasn,Scd1,Fabp4,Cd36,Dgat1,Pparγ,Srebp1 c,encoding the proteins involved in biosynthesis,transport and transformation of fatty acid,were observed in TCDF-treated mice.Quantitative results showed that levels of some phospholipids including PC,LPC,PE,LPE,SM were decreased after exposure to TCDF.The level of TMAO,betaine,sarcosine,creatinine were significantly decreased in the liver and serum of mice exposured to TCDF,and the expression of enzymes involved in CDP-choline biosynthetic pathway was increased.Targeted ceramides analysis revealed that TCDF exposure induced significant elevation in the levels of ceramides in the liver of mice.Consistently,marked increases in gene expression of Sptlc1,Sptlc2,Kdsr,Cers2,Cers4,Cers5,Cers6,Degs1,Degs2,Smpd1,Smpd2,Smpd4,Neu1,Glb1,Gba1,encoding enzymes associated with three ceramide biosynthetic pathways was observed in the liver of mice exposure to TCDF.And significant increased in gene expression of XBP1、ATF6、Eif2α、Gadd34、ATF4、Chop、Srebp2,encoding enzymes involved in endoplasmic reticulum stress was shown in TCDF-treated mice.The results of Western blot showed that the expression of ATF4、XBP1 protein was increased.Conclusions: TCDF induced hepatic steatosis in C57BL/6J mice by up-regulating the synthesis and uptake of fatty acids in the liver and promoting the biosynthesis of TG.Lipoprotein is indispensable in the output of TG from liver.As the excessive consumption of phospholipid and choline,lipoprotein synthesis was imped.TG accumulated in the liver and hepatic steatosis was being aggravated.Excessive synthesis of lipotoxic ceramides as well as endoplasmic reticulum stress and inflammation occurred in the liver,which ultimately promoted the occurrence of nonalcoholic fatty liver disease in mice.