Reproductive Toxicity Of BPA On Male Zebrafish And Its Epigenetic Mechanism

In recent years,the effects of the environmental endocrine disrupting chemicals (EDCs) on the health of humans and wildlife have been a growing concern among researchers and policy makers. Bisphenol A (BPA) is a kind of phenolic substances which belongs to EDCs and is mainly used as the plasticizer and softener in plastic products.In recent years,BPA have become one of the most common global environmental pollutants with their hazards on human health being widely concerned.To investigate the impact of BPA exposure on development and reproduction, zebrafish were exposure to 0.5mg/L、1.5mg/L BPA or 0.01% solvent control from 20 dpf to 100 dpf.We found that the gonadal-somatic index (GSI) of the male group exposed to 1.5mg/L BPA is 6.24‰±0.71‰,which reduced significantly when comparing with control group and 0.5mg/L BPA group.In 27dpf (one week after exposure)、34dpf(two weeks after exposure)、41 dpf (three weeks after exposure)、100dph, RT-PCR were used to detected VTG mRNA expression,and found that VTG mRNA expression started after three weeks exposure to BPA1.5mg/L,VTG mRNA were also expressed in both BPA treated male zebrafish.Histopathologic examination of testis showed that the development of testis inl.5mg/L BPA group was inhibited significantly,manifested as the co-existence of abnormal seminiferous tubules, reduced sperm count and increased the cell-free zone.Breeding studies with the normal female fishes revealed significant reproductive dysfunctions in BPA treated groups.Female zebrafish produced fewer eggs、the egg viability at 12 hpf were lower、the egg malformation rate at 72 hpf were higher than that of the control.Gonadal abnormalities in male zebrafish were caused after BPA exposured,that wound lead to birth defects in offspring. Previous studies on this issue is not related to epigenetics.We found that 5-methyl cytosine was significantly expressed in mature sperm in BPA1.5mg/L exposured group,indicating the increased level of genome-wide methylation.Compared to the normal zebrafish, the dnmt1、dnmt6、dnmt7、dnmt8 mRNA expression level in BPA 0.5mg/L group are:1.755±0.735 2.143±0.365、4.147±1.366、1.603±0.143,the dnmt1、dnmt6、dnmt7、dnmt8 mRNA expression level in BPA1.5mg/L group are:3.101±0.653、5.036±0.477、3.703±0.19、1.861±0.167,statistically significant differences to the control group (P<0.05) Immunohistochemistry,Western blot results showed that:H3K9me3 significantly increased in BPA treated group,H3K4me3、H3K27me3、H3K36 me3 did not change.VTG as biomarker of zebrafish exposured to EDCs is widely recognized, Privious experiments also confirmed this point (VTG1 mRNA were expressioned in juvenile and adult male after BPA exposure).whether DNA methylation is involved in this process is not known.In order to prove this point,5 months old male zebrafish were exposured to 4mg/L BPA for 30 days,4 months old male zebrafish and female were exposure to 0.01% solvent control for for 30 days,too.Compared to the normal male liver the VTG 1 mRNA expression level in BPA4mg/L male liver is 1761.08±782.59, the demethylation rate of six CpG sites is twice more than.It confirmed that DNA methylation involved in BPA regulating the expression of VTG1.