Effects And Regulatory Mechanisms Of Copper And Oxytetracycline On Hepatic Lipid Metabolism In Grass Carp Ctenopharyngodon Idella

Copper(Cu)is an essential trace element for living organisms.In husbandry,Cu(Cu²⁺)was usually added into feed and accordingly entered the environment.In aquaculture,Cu distributed in the water along with the use of disinfection and insecticide.Herein,adaptive Cu can be obtained from food and aquacultural environment,which can be further accumulated in the liver.However,once Cu is highly existed in water,Cu will affect normal physiological and metabolic function to inhibit fish growth,threats the aquaculture industry.Antibiotics,which was used to alleviate infection for bacterial and fungus,was commonly used in aquaculture as well.Owing to the long-term existence of antibiotics in the environment,the antibiotics,which is hard to degradation and easy to migration,accumulates and causes adverse effects on bacterial resistance,further threats aquaculture industry.At present,there are increasing studies focused on the threaten and side effects of single pollutant in aquaculture.However,only few reports focused on the side effects and regulatory mechanisms induced by the co-existence of multiple pollutants in aquaculture.Herein,based on the regulatory mechanism of lipogenesis in grass carp(Ctenopharyngodon idella),we studied the mechanisms of Cu-induced lipid metabolism,and further illustrated the regulation on lipid metabolism in the co-existence of Cu and oxytetracycline(OTC)environment in fish.1.Structure and functional analysis of SREBP1,ACC?,FAS and SCD1 promoters in grass carpThe SREBP1 transcription factor is one of major regulators of lipid metabolism.The present study determined the transcriptional regulation of SREBP1 and its target genes(including acc?,fas and scd1)in grass carp.We cloned and characterized the 1998 bp,2043 bp,1632 bp and 1889 bp sequences of srebp1,acc?,scd1,and fas promoters,respectively.A cluster of putative binding sites of transcription factors,such as SP1,YY1,NF-Y,SREs and E-box element,were predicted on their promoter regions.By overexpressing nuclear SREBP1(n SREBP1),we found n SREBP1 overexpression reduced srebp1 promoter activity,increased scd1 and fas promoter activity,but did not influence acc?promoter activity.The site mutation and EMSA analysis indicated that srebp1,fas and scd1 promoters,but not acc?promoter,possessed SREBP1 binding sites.Based on the results that n SREBP1 overexpression in CIK cells,we illustrated the regulatory mechanism on lipogenesis,which SREBP1 regulated the expression of lipogenesis through a self-regulated mechanism for maintaining the balance in lipid metabolism in fish.2.Effects of waterborne copper on lipid deposition and DNA methylation in liver of grass carpDNA methylation is important in regulating physiological processes by influencing gene transcription.Thus,we conducted an 8-wks experiment of waterborne copper exposure on juvenile grass carp to study the mechanisms of DNA methylation on lipid metabolism induced by Cu²⁺in the liver of grass carp.Here,we found that Cu exposure increased lipid content and induced the DNA hypermethylation at the whole genome level.Cu induced oxidative stress,increased reactive oxygen species(ROS)production,activated endoplasmic reticulum stress and damaged mitochondrial function,and accordingly increased lipid deposition.Cu incubation resulted in hypermethylation of glucose-regulated protein 78(grp78)and peroxisome proliferator-activated receptor gamma coactivator-1?(pgc1?)promoter regions.Moreover,CCAAT/Enhancer binding protein?(C/EBP?)could be bound to the methylated sequence of grp78,whereas C/EBP?could not be bound to the methylated sequence of grp78,influenced grp78expression and further induced ER stress,resulted in the increased lipogenesis;in contrast,DNA methylation of pgc1?blocked the specific protein 1(SP1)binding on the promoter of pgc1?,interfered mitochondrial function,resulted in the inhibition of lipolysis.Altogether,Cu²⁺induced generation of ROS,altered changes of DNA methylation on genome,and finally resulted in lipid deposition in liver through interrupting the balance of lipogenesis and lipolysis.3.Effects of waterborne oxytetracycline and copper on lipid deposition in liver of grass carpOxytetracycline(OTC)and Cu²⁺are both prevalent contaminations in water.We conducted an experiment of waterborne Cu,OTC and Cu+OTC complex exposure on grass carp for 6-wks,and then illustrated the mechanisms of lipid metabolisms in the liver.Here,we discovered that OTC and Cu interacted to inhibit the growth performance of grass carp.We found that OTC and Cu induced oxidative stress,activated antioxidant response,aggravated lipid deposition through NRF2-SREPB1-mediated lipogenesis in the liver of grass carp.In addition,Cu caused severed lipid peroxidation and mitochondrial dysfunction,resulted in the deficiency of lipid utilization.Consequently,the synergistic effects of antioxidant response and mitochondrial dysfunction,resulted in the lipid deposition under OTC and Cu co-exposure,which hindered the growth of grass carp.The present study indicated the side-effects of the Cu+OTC complex were greater than the effects of OTC and Cu alone,enlightened our understanding on the risk of multiple pollutants assessment.The current study is a fundamental research for illustrating the molecular mechanisms of the nutrition metabolisms under the threat of co-existence of multiple pollutants in the aquaculture environment,which can further promote guidance to build a healthy aquaculture.