The Effects And Regulatory Mechanism Of BPA On Lipid Metabolism In Rare Minnow Gobiocypris Rarus

As its ubiquitous existence in the environment and extensive application in human life,bisphenol A(BPA),a representative endocrine disrupting compounds(EDCs),has raised considerable concerns of environmental remediation and human health.Increasingly,researches have also detected BPA contamination in various aquatic environments.Therefore,its detrimental effects have also drawn scientific and public concern about the healthy and sustainable development of aquatic organisms,especially fish.The previous studies on BPA toxicology in fish mainly focused on reproductive toxicity and detrimental effect on steroidogenesis in response to BPA.But to date,the underlying regulation metabolisms of BPA on lipid metabolism has not been elucidated.The present study firstly evaluated the effects and potential mechanism of waterborne BPA exposure on lipid metabolism.The effects of BPA on lipid metabolism and its regulatory mechanism in rare minnow(G.rarus)were studied in this research.The adult G.rarus were exposed at 15 μg/L BPA for 3-,4-and 6-week.Firstly,triglyceride content in liver and serum were quantified by GPO-PAP,and the activity of ACC,FASN,GPAT and CPT1 were analysed by ELISA.Then,the mRNA expression of acaca,acacb,fasn,gpat1,cpt1α and srebp-1c in liver were detected by qRT-PCR.Besides,the 5’ flanking sequences of acaca,fasn and cpt1α were cloned with Genome Walker and sterol responsive element(SRE)and CpG loci were predicted in them.Additionally,the expression of SREBP-1 protein was detected by Western Blot.Finally,the interaction between SREBP-1 and acaca,fasn and cpt1α were detected by CHIP method,and the methylation status of acaca,fasn and cpt1α were detected by BSP method.The main results were showed as follow:1.In the present study,15 μg/L BPA exposure for 4 weeks caused significant decrease of serum triglyceride contents but a non-significant effect in liver in female G.rarus,which suggests that BPA might affect triglyceride transportation from liver to blood in the females.The increasing tendency of triglyceride contents in liver after BPA exposure for 3 weeks,which is consistent with the changes in body weight and HSI.What’s more,The significantly increasing of triglyceride contents in liver after BPA exposure for 3 and 6 weeks revealed that BPA exposure could suppress triglyceride accumulation to some extent in males.2.In male G.rarus after 15 μg/L BPA exposure for 4 weeks,the activity of CPT1 was significantly increased compared to the control.The increased CPT1 activity suggests that BPA exposure might increase the capacity for fatty acid β-oxidation.While the increased enzyme activities for ACC,FASN and GPAT may indicate an increase in lipid synthesis.Taking account of the trend to increase in liver and serum triglyceride contents in our study,we concluded that BPA could promote both lipid synthesis and fatty acid β-oxidation in males,however its potential stronger effect on lipid synthesis might result in the rising trend of triglyceride contents.In the present study,BPA exposure caused respective significant increase and decrease of CPT1 activity in liver of the males and females,which suggests that the effect of BPA on fatty acid β-oxidation pathway might be gender-dependent in G.rarus.3.BPA caused respective significant decrease and increase of the precursor and nuclear forms of SREBP-1 proteins and mRNA expressionin the females after 3-and 6-week exposure.which is consistent with the changes in mRNA expression of acaca and fasn.In males,the precursor andmature forms of SREBP-1 proteins and mRNA leves were decreased,which is consistent with the changes in mRNA expression of acaca and fasn and not consistent with cpt1α mRNA changes.4.Our study displayed that SREBP-1 could bind to the SRE fragments of fasn,acaca and cpt1α.In females,BPA exposure for 3 weeks significantly inhibited the combination of SREBP-1 and SRE in acaca and fasn 5’ flanking sequence,whereas BPA exposure for 6 weeks extremely significantly promoted SREBP-1 to bind to the SREs of them,which is consistent with the changes of their mRNA expression.We concluded that SREBP-1 could regulate the expression of acaca and fasn by binding to the SRE site directly as a response to the effects of BPA.In males,BPA exposure for 3 weeks significantly inhibited the combination of SREBP-1 and SRE in acaca and fasn 5’ flanking sequence,whereas BPA exposure for 6 weeks promoted SREBP-1 to bind to the SREs of them.For cpt1α,BPA exposure for 3 weeks significantly promoted the binding of SREBP-1 and its SRE site,whereas 6 weeks treatment had not caused significant effect in it.The mRNA expression of these three genes is consistent with the binding of SREBP-1 and SRE in the corresponding gene except that the mRNA expression of acaca was abnormal after 6 weeks’ exposure.Thus,the study indicated that BPA could regulate the mRNA expressions of acaca,fasn and cpt1α by inducing the interaction of SRE sites and SREBP-1 in male lives.5.In the liver tissues of the females,methylation levels of cpt1 a in-445 and-433 CpG sites were significantly increased after BPA exposure for 3 weeks.However,methylation levels of cpt1 a in-638 and-433 CpG sites were decreased after 6 weeks BPA exposure.The present study firstly evaluated the effects and potential mechanism of waterborne BPA exposure on lipid metabolism in G.rarus.This will contribute to understand the in vivo effects of BPA on lipid metabolism,and thus provide some novel insights on BPA effects in fish.