Toxicity Of 17-beta-estradiol For Zebrafish

Many kinds of chemical compounds in the environment have estrogen-like activities. These compounds can disturb the normal function of endocrine system in organisms. Therefore, they are harmful to organisms and their offsprings. These compounds are called environmental estrogens. Among them, 17β-estradiol(E2) is the dominant estrogen.In this study, zebrafish was used as experiment material for the research of toxicology mechanism of E2. Methods included quantitative real-time PCR(qRT-PCR), RNA-Seq and iTRAQ.First, the concentration gradient of E2 solutions, 0, 10 ng/L, 1, 2, 2.5, 3, 4, 5 and 6 mg/L was prepared by using 100% ethanol as cosolvent. Then zebrafish embryos were treated by these E2 solutions. The effects of E2 on development, morphology and hatchability of zebrafish embryos were observed by microscope. Results showed that malformation was observed after E2 treatment, including spinal curvature and pericardialites. In addition, E2 could also delay hatching time of embryos. In summary, E2 could increase malformation rate and mortality and delay hatching time with a dose-dependent manner.qRT-PCR was used to detect genes suggested to be involved in hatch(zhe1) transcription in zebrafish embryos in order to find molecular mechanism of delay of hatching time caused by E2. The result illustrated that high concentration of E2 could delay the time which was needed by the expression level of zhe1 to reach peak. Then zebrafish embryos treated by 2 mg/L E2 for 9 d and larval, juvenile(21 dpf), adult female(6 months old) and adult male(6 months old) zebrafish treated by 10 ng/L E2 for 9 d were collected, respectively. After treatment and collection, the expression patterns of genes suggested to be responsible for sexual differentiation(brca2, sox9 a, sox9 b, dmrt1 and cyp19a1a) in each group were detected by qRT-PCR. The results indicated that E2 could up-regulate the expression levels of the female-predominant genes(brac2, sox9b), down-regulate the expression levels of sectional male-predominant genes(sox9a) and had different effects on sex hormone conversion pathway(cyp19a1a) in different developmental stages of zebrafish.Larval zebrafish treated by 0 and 10 ng/L E2 for 9 days were also collected for the analysis of RNA-Seq and isobaric tags for relative and absolute quantitation(iTRAQ). RNA-Seq analysis revealed 82 up-regulated genes and 236 down-regulated genes in E2 treatment group. The main toxicity of E2 for zebrafish was cleared by the analyses of GO and KEGG, including slowing down metabolism(like reducing efficiency of transcription and translation, inhibiting angiogenesis, proliferation and apoptosis), inhibiting bile acids and steroid hormone synthesis, accelerating the hydrolysis of ATP(because of the up-regulation expression of genes encoding ATPase and slowing down of purine metabolism) and inducing the activation of self-defense mechanism(increasing the barrier effect). iTRAQ analyses revealed E2 could inhibit proliferation and increase the barrier effect, which partly consisted with the result of RNA-Seq.In this study, toxicology of different concentrations of E2 for different developmental stages of zebrafish is researched on molecular, transcriptome and proteinsome levels. Results can be used as a reference for further study of the toxicology of environmental estrogens and sets of their emission standard.