A Preliminary Study On The Hepatotoxicity And Mechanisms Of 17α-ethinylestradiol (EE2) Exposure On The Marine Medaka Oryzias Melastigma

The typical environmental endocrine disruptor 17α-Ethinylestradiol(EE2)is widely present in water environment.It has been reported to have reproductive toxicity and immunotoxicity,but the hepatotoxicity of EE2 has been rarely reported.Understanding the cytotoxicity of EE2 to fish and its potential toxicity mechanism is conducive to fully understand the harmfulness of estrogen-based pollutants.In this study,EE2 was selected as the research subject and the marine model animal,marine medaka(Oryzias melastigma)was used as the experimental material.Through water exposure experiments,combined with cell biology and molecular biology techniques,the effects of exposure to low concentrations of EE2 on embryo development were explored,and the endoplasmic reticulum stress,lipid metabolism and apoptosis in marine medaka liver cells under different concentrations of EE2 were investigated.The obtained results are as follows:(1)The effect of low concentration EE2 on the embryo development of medaka was analyzed.The fertilized eggs of the medaka were exposed to the low concentration EE2(50 ng/L)until the embryos hatched.The incubation time and mortality analysis of medaka embryos showed that exposure to low concentrations of EE2(50 ng/L)increased the hatching rate of medaka embryos,but the incubation time was significantly prolonged.(2)The effects of different concentrations of EE2 exposure on the endoplasmic reticulum stress and immune-related genes of marine medaka liver tissue were studied.Three-month-old male marine medaka were exposed to different concentrations of EE2(0.1 μg/L,1 μg/L and 10 μg/L)for 24 h and 72 h,and liver samples were collected for real-time quantitative PCR(qPCR)analysis.The results showed that ER stress-related genes,including PERK,eIF2a,ATF4,CHOP and IRE1,were significantly induced.The expression of apoptosis suppressors gene Bcl2 was up-regulated,the expression of pro-apoptosis gene BIRC2 was inhibited,and the expression of apoptosis executive genes caspase9 and caspase3 were downregulated.The expression of HLA gene,which helps T cells recognize antigens;Lyn,which regulates B lymphocyte differentiation;and C7 and CFB genes,which are components of the complement system,were significantly inhibited.These results suggest that exposure to EE2 affects the expression of endoplasmic reticulum stress and immune-related functional genes in medaka.(3)The effects of EE2 exposure on lipid metabolism medaka hepatocytes were analyzed.Three-month-old male and female medaka were exposed to different concentrations of EE2(0.1 μg/L,0.5 μg/L and 1 μg/L),and the changes of total cholesterol in the liver were detected at 12,24,48 and 72 h after exposure.The results showed that the total cholesterol content of male medaka liver significantly increased in the 0.5 μg/L and 1 μg/L EE2 groups after 48 h of exposure to EE2.At 72 h,the total cholesterol content of the 0.5 μg/L EE2 group continued to increase,but there was no significant difference between the total cholesterol content of the 1 pg/L EE2 group and the control group.The total cholesterol content in the liver of female medake was significantly increased only at 24 h after EE2 exposure in the 1 μg/L EE2 experimental group.when the Oryzias latipes liver cell line(DIT29 cells)was exposed to different concentrations of EE2(50 ng/L,500 ng/L and 5000 ng/L)for 24 h,the lipid content of the cells was significantly increased in both 500 ng/L and 5000 ng/L treatment groups.Exposure of 50 ng/L EE2 to DIT29 cells for 24 h significantly reduced the total cholesterol content in the cells.The expression levels of genes related to lipid metabolism in DIT29 cells were analyzed after exposure to different concentrations of EE2(50 ng/L,500 ng/L,and 5000 ng/L).It was found that SREBF1,which controls triglyceride synthesis;SREBF2,which regulates cholesterol metabolism;fatty acid synthase gene FASN;and PLIN2,which is related to long chain fatty acid transport,had no significant changes at 24 h and 48 h after EE2 exposure.These results suggested that EE2 exposure caused the disturbance of lipid metabolism in the liver cells of medaka.(4)The effect of EE2 exposure on DIT29 cells apoptosis was analyzed.DIT29 cells were exposed to 5 ng/L,50 ng/L,500 ng/L,5000 ng/L and 50000 ng/L EE2 for 24 h,and there was no significant changes in cell viability.However,after exposure to 50 ng/L and 50000 ng/L EE2 for 24 h,the enzyme activity of Caspase3/7 was significantly inhibited,and the inhibitiory effect was positively correlated with the concentration.The enzyme activity of Caspase3/7 was still inhibited after adding hydrogen peroxide to the exposed system.The Annexin V-FITC/PI assay showed that exposure to EE2 at concentrations of 50 ng/L,500 ng/L and 5000 ng/L for 24 h and 48 h significantly inhibited the apoptosis of DIT29 cells.There was no significant difference in the proliferation-related RXRA gene of DIT29 cells after EE2 exposure,while the pro-apoptotic gene SPHK2 was significantly up-regulated.These results indicate that EE2 exposure significantly inhibits the apoptotic pathway of DIT29 cells.In summary,by studying the embryonic developmental toxicity and hepatotoxicity effect of EE2 and its mechanism,it was found that EE2 can delay embryonic development and significantly affect the process of medaka liver cell endoplasmic reticulum stress,lipid metabolism and cell apoptosis.