Effect And Mechanism Of DEHP On Lipid Metabolism In The Liver Of Zebrafish

Di(2-ethylhexyl)phthalate(DEHP),a widely used plasticizer,can be easily released into the environment.Currently,DEHP can be detected in China’s surfacewaters,which increases the risk of exposure to aquatic organisms.DEHP and its metabolites have been detected in liver and muscle of fish.The liver is one of the target organs of DEHP and is also an important organ for lipid metabolism.However,the study of DEHP on lipid metabolism in fish liver has been rarely reported.In this study,adult zebrafish(Danio rerio)were exposed to DEHP(0,0.02,0.2,2and 20 μg/L)for 4 weeks.And then,the content of lipid in liver was detected by biochemical analysis.It was found that 2 μg/L DEHP is the lower concentration which exhibiting a significant change on fat content of liver.At this concentration,we examined the DEHP content in fish by high performance liquid chromatography,the structure by tissue sectioning,the activity of lipid-metabolism enzyme by enzyme-linked immunoassay,and the expression of genes by real-time fluorescence quantification.Subsequently,the primary hepatocytes from zebrafish were treated with 0.4 mg/L DEHP for 24 h.Fatostatin,an inhibitor of sterol-regulatory element binding protein(SREBP-1)which belongs to transcription factor related to lipid metabolism,was added or transient transfection with SREBP-1 to verify whether DEHP affected hepatic lipid metabolism through SREBP-1.The results were as follows:1.DEHP accumulated in zebrafish.The content of DEHP in females was 3.7times higher than that in males in 2 μg/L DEHP treatment group.2.DEHP caused lipid accumulation in zebrafish liver.DEHP significantly increased the body weight,visceromatic index and hepatic index in female,the content of triglyceride(TG)in plasma,the content of TG and lipid droplets in liver,while DEHP had no effect on male fish.3.DEHP promoted lipid synthesis process in zebrafish liver.(1)In female zebrafish,DEHP enhanced the activity of fatty acid synthase(FAS)and acetyl-coenzyme A carboxylase 1(ACC1)in the liver.DEHP increased the expression of genes associated with lipid synthesis,including fas,acc,scd1,elvol6,dgat1 and acs in female liver.DEHP had no effect on the activity of carnitine palmotoyltransferase(CPT Ⅰ)in female liver.Genes related to lipid catabolism were decreased by DEHP treatment in female liver,including cpt1 and acadm.DEHP increased the expression of cd36 and fabp1 genes in female liver,while decreased the expression of fatp2 gene.DEHP increased the protein level of SREBP-1 in female liver.DEHP also increased the expression of gene related to lipid-metabolism transcription factors,including srebp-1 and pparγ.(2)In male zebrafish,DEHP had no effect on the activities of FAS and ACC1 in liver.But it decreased the activity of CPT Ⅰ in liver.In addition,DEHP increased the expression of lpl,cpt1 a and cpt1 b genes.DEHP increased the expression of fatp2 gene,while it had no effect on the protein expression level of SREBP-1 and the expression of srebp-1 and pparγ genes in liver.4.DEHP affected the structure and function of liver in zebrafish.DEHP increased the round vacuoles in the liver of female.In addition,DEHP increased the content of glutathione activity in plasma of female.DEHP upregulated the expression ratio of bax/bcl2 genes and the expression of caspase3 gene in female,while it did not have such effects on male.5.DEHP caused lipid accumulation in zebrafish liver by SREBP-1.DEHP increased the content of TG and TC,the protein level of SREBP-1,the expression of srebp-1 and its target genes(fas,acc and scd)in primary hepatocytes.Fatostatin recovered the effect of DEHP-induced fat accumulation in hepatocytes.Fatostatin inhibited the protein expression of SREBP-1 and the expression of srebp-1 and its target genes(fas,acc and scd)induced by DEHP.The result from knockdown of SREBP-1 by RNAi in primary hepatocytes is similar with Fatostatin treatment.To sum up,DEHP damaged the liver of zebrafish.DEHP affected the balance of lipid transport,synthesis and catabolism.In addition,DEHP can increase the expression of genes and the activity of enzymes related to lipid-synthesis by activing SREBP-1,and then induces hepatic fat accumulation in zebrafish.In conclusion,this study investigated the effect and mechanism of DEHP on hepatic lipid metabolism in zebrafish,providing a new evidence for an in-depth understanding of the toxic effects of phthalates on fish.