Effect Of Bisphenol A On Global DNA Methylation Of Rare Minnow(gobiocypris Rarus)testes And Its Mechanism

Bisphenol A?BPA?is an important chemical raw material and is widely used in food packaging,children's toys and household appliances.Due to the extensive use,BPA is ubiquitous in the environment and poses the threat to human and animal health.The molecular mechanisms of action of BPA are complex.It not only can interfere with the endocrine system homeostasis,but also can cause changes in epigenetic modifications.Previous studies revealed that BPA can result in the disturbance of global DNA methylation levels of gonads in fish.However,the specific mechanism is still unclear.In the present study,Gobiocypris rarus?G.rarus?was used as the experimental animal.The coding sequences?CDs?of target genes were isolated and characterized by homologous cloning method.Adult G.rarus at 5 months dpf was exposed to 1,15 and 225?g L^-1 BPA for7?short-term?and 14?prolonged?days.The levels of methylation of genome DNA,DNA methyltransferases?DNMTs?,DNA demethylases?TETs?and GSH synthesis related enzymes and substrates,and the expressions of target genes in the testes of G.rarus at two time points and three BPA concentrations were measured by enzyme-linked immunosorbent assay,Dot blot,and real-time quantitative PCR?qRT-PCR?.These experiments could contribute to investigating the effects and molecular mechanisms of BPA on the global DNA methylation levels of teleost testes and the relationship between the disturbance of GSH pool and the change of DNA methylation level.Based on these studies,antioxidant N-acetylcysteine?NAC?was added to explore the protective effect of NAC on BPA-induced toxicity.The main findings were as follows:1.The full-length CDs of dnmt1,dnmt3,dnmt4,dnmt6,dnmt7,dnmt8,tet2,tet3,cbsa,cbsb,gnmt and g6pd,and parts of CDs of dnmt5 and tet1 were obtained.qRT-PCR results shown that cbsa and gnmt were mainly expressed in the liver,cbsb was mainly expressed in gonads and brain,dnmt1,dnmt3,dnmt4,dnmt5 and dnmt7 were mainly expressed in the gonads,dnmt6,dnmt8,tet1,tet2 and tet3 were mainly expressed in the brain,and g6pd was mainly expressed in the ovary.Additionally,tet1 was also highly expressed in the testis.2.After short-term?7 days?BPA exposure,no significant difference of global DNA methylation levels in the ovaries of G.rarus was found between treatment and control groups.However,both 15 and 225?g L^-1 BPA resulted in DNA hypermethylation of the testes.The increase of DNA methylation level in 15?g L^-1 BPA group was accompanied by a decrease in TETs level while that of 225?g L^-1 BPA group was accompanied by an increase in DNMTs level.The results indicated that the mechanisms of BPA on DNA methylation of short-term exposure of these two groups were different.The 15?g L^-1 BPA group was related to DNA demethylation progress while 225?g L^-1 BPA group was relevant to DNA methylation progress,respectively.In addition,GSH content and levels of de novo GSH synthesis-related enzymes and substrates were significantly elevated in the 225?g L^-1 BPA group,indicating that the de novo synthesis pathway of GSH was enhanced.Meanwhile,as an additional reaction of de novo GSH production,the DNMTs-mediated DNA methylation process might be promoted.3.After prolonged?14 days?BPA exposure,the global DNA methylation in the ovaries of G.rarus was also unchanged by all concentrations(1,15,and 225?g L^-1).All treatments caused decrease of TETs and 5hmC levels while only 225?g L^-1 BPA resulted in DNA hypermethylation of the testis.The increase in global DNA methylation level of the testis induced by 225?g L^-1 BPA was accompanied by the decrease of TETs and 5hmC levels,indicating that the change of DNA methylation level at this concentration was associated with TETs-mediated DNA demethylation.Meanwhile,the unchanged DNA methylation level and decreased TETs and 5hmC levels of low and medium concentrations suggested that other factors which can affect DNA methylation levels might be also disturbed by BPA exposure.In addition,all treatments resulted in consumption of testicular GSH.And the supplementation of GSH pool would be mainly accomplished through cyclic synthesis for the expressions of cbsa,cbsb,and gclc for de novo synthesis were down-regulated while g6pd for cycle synthesis was up-regulated.Unlike short-term exposure,there was no clear relationship between changes in DNA methylation level and GSH level by prolonged exposure to high concentrations of BPA.4.After NAC addition,the global DNA methylation levels in the testes of G.rarus were lower than that of BPA treatment alone and the level of cysteine was increased while the levels of SAH and HCY were returned to normal,indicating that NAC could directly increase cysteine levels in the testes and attenuate the entry of HCY into the transsulfur pathway for the synthesis of cysteine,and ultimately inhibit BPA-induced DNA hypermethylation.In addition,BPA resulted in oxidative stress in the testis and the scavenging of H₂O₂ was mainly achieved by CAT,indicating that the large amount of GSH synthesized by the testis of G.rarus after BPA exposure might not be used mainly for antioxidation.However,after adding NAC to BPA-exposed groups,the contents of cysteine in the testes were increased and the testes converted to using GPx to eliminate H₂O₂.And this might be related to the increase of cysteine which is the restriction substrate for de novo GSH production by NAC addition.Moreover,the positive response of the antioxidant system in the testes of G.rarus and the antioxidant activity of NAC protected the sperm from oxidative damage.In conclusion,BPA exposure for 7 and 14 days might result in the disturbance of DNMTs-mediated DNA methylation progress or TETs-mediated demethylation process,and eventually lead to DNA hypermethylation in the testes of G.rarus.However,the different concentrations of BPA and exposure time had no significant effect on global DNA methylation levels of the ovaries.The de novo GSH production in the testis during short-term exposure to high concentration of BPA promoted DNA methylation progress.Additionally,the addition of NAC to directly increase the cysteine contents could inhibit the BPA-induced DNA hypermethylation of the testes.Furthermore,the positive response of the testicular antioxidant system and the antioxidant activity of NAC protected the sperm from oxidative damage induced by BPA and/or NAC treatment.