| Agricultural advances have improved the lifestyle of humankind. However, these advances have brought about new problems that affect the environment and future generations. The trace has the potential to influence the organism, interfer with endocrine function, cause hormone disorders and reproductive toxicity. Therefore, endocrine disruption caused by the pesticides have become a major problem. Here, this thesis used zebrafish as model to investigate the estrogen activity of triadimefon, atrazine and azocyclotin, and the effects on reproduction, sex steroid hormones, and transcription of the genes belonging to the hypothalamic-pituitary-gonad (HPG) axis for azocyclotin were investigated. The whole investigation included:1. We investigated estrogen activity of pesticides based on biomarker, such as hepatic vitellogenin (vtgl), estrogenic receptor a (era) gene, and estradiol level (E2) in female zebrafish. Adult zebrafish were exposed triadimefon, atrazine and azocyclotin for 21 days. Exposure to azocyclotin (0.45 ug/L) significantly reduced whole-body E2 concentration in females. Furthermore, vtgl and era were downregulated in females, suggesting azocyclotin has anti-estrogenic activity.2. Adult zebrafish was exposed to azocyclotin (0.09,0.45 μg/L) for 21 days according to OECD 229. This study intended to introduce the negative effect of azocyclotin on the reproductive capability and the gonads maturation of zebrafish. The average number eggs per female was significantly decreased in azocyclotin-exposed fish; Ovarian development was inhibited at the two concentrations, while azocyclotin caused no significant change in GSI of the male zebrafish. Exposure to 0.09 and 0.45 μg/L resulted in significantly more POs and AOs. The ovaries at the 0.45 ug/L group were characterized by the lack of LMOs and instead had higher proportions of CAOs. In the males, a significant increase in the percentage of spermatocytes (Sc) was observed in the both groups. However, the percentage of spermatozoa (Sz) significantly reduced after exposure to 0.09 and 0.45 μg/L. These results indicated that azocyclotin exposure could adversely affect normal ovulation and induce reproductive toxicity. In addition, the high concentration of azocyclotin measured in the gonads may also contribute to impaired ovulation. Azocyclotin was also detected in Fl eggs following parental exposure, indicating maternal transfer of this compound. There appeared to be a concentration-related decrease in malformation rate after exposure, indicating that maternal exposure to azocyclotin caused developmental toxicity in their offspring.3. Up to 13 different steroid hormones were quantified in gonads using UPLC-MS/MS. A more detailed analysis was performed to determine how steroid profiles in the liver tissue differed between the treatments. Exposed of zebrafish to azocyclotin significantly increased androgens and decreased estrogens levels, resulted in lesser luteinizing hormone release, decreased 17- hydroxyprogesterone level responsible for triggering gamete maturation, and inhibited excretion of stress-induced hormones, such as cortisol and cortisone. Our study showed that LTQ profiling of extracts of liver tissue resulted in the detection of significant differences in methoxyestrogens, profiles between the groups, which might accelerate metabolism of E2 and in turn reduce E2 level.4. The effects on mRNA expression of the genes belonging to the hypothalamic- pituitary-gonad (HPG) axis were investigated. The results included:(1) The present observation provide evidence that pituitary gonadotropins lhβ were down-regulated by exposure to azocyclotin and FAD in zebrafish. FAD treatment was shown to lead to decrease in the transcript level of cyp19a1b in brain of zebrafish.(2) In the gonad, the transcriptional levels of 3β-hsd and 17β-hsd3 were generally up-regulated in both sexes exposed to 0.45 μg/L, while the transcriptional levels of cyp17 and 17β-hsdl were significantly up-regulated. In this study, exposure of zebrafish to azocyclotin caused a significant inhibition of cypl9ala expression in testes of males. The mRNA level of cypl9ala in the ovaries was generally inhibited at lower doses. However, the elevation of cyp19a1a was observed at the high dose of azocyclotin.(3) A concentration-specific decrease in era expression was observed at all azocyclotin and FAD concentrations. The effects of 0.45 μg/L and 100 μg/L on down-regulation of vtgl and vtg2 expression in livers of females and males were observed. In livers, similar transcriptional patterns were observed in azocyclotin-exposed females and males. There were remarkable up-regulation of cyp1a1, comt, ugt1a and gstpl in the 0.09 and 0.45 μg/L groups, while remarkable reduction of ahr2 mRNA levels in females. In the liver, expressions of comt and gstplwere significantly up-regulated by exposed to 100 μg/L. In females, significant up-regulation of cyplal ugtla and ahr2 were observed in the 100 μg/L treatment group. But all were not significantly changed in the males.5. Principle component analysis (PCA) showed that sex hormone levels and fecundity were related to the mRNA level of several genes in the HPG and liver axis. PC1 was highly influenced by variables such as fshβ, lhβ, cypl9a,17/3-hsd3, vtgl, ugtla and gstpl and was significantly correlated with the concentrations of T, E2, E1, AE, P4,17P4 and fecundity in female fish. In males, PC1 was significantly correlated with the concentration of 11-KT and E2.In conclusion, the impaired balance of sex steroids in adult zebrafish may lead to adverse effects on fertility such as disrupted maturation of oocytes and spermatozoa in response to azocyclotin exposure. We further discovered that azocyclotin causes sex- and tissue-specific changed in gene expression of steroidogenesis along the HPG axis, it was postulated that azocyclotin exerted its activity in zebrafish by:(1) Azocyclotin may directly act on er to inhibit vtg gene transcription in the liver, the observed downregulation of vtg in zebrafish could be regarded as a consequence of lower E2 concentration.(2) A statistically significant up-regulation of 3/3-hsd mRNAs would result in increase in the levels of P4, and the observed transcriptional changes of cypl 7 and 17β-hsd3 genes would lead to alterations of androgens. With this in mind, a parallel reduction of era and vtg expression in response to azocyclotin exposure might indicate anti-estrogenic effects of azocyclotin.(3) It is possible that azocyclotin was acting as an anti-estrogenic agent by affecting the metabolic processing of E2 by inducing the liver-mediated biotransformation pathway. It is conceivable that the induction of hepatic ugtla and gstpl perhaps mainly was due to the production of reactive oxygen species and lipid peroxidation, indicating that azocyclotin might accelerate metabolism of steroid hormones.(4) The results in the present study suggested that marked down-regulation of lhβ gene expression, decreased levels of E2 might be reasons for azocyclotin-mediated inhibition of ovarian and testicular development in zebrafish. On the basis of the results from histological examination, may cause reproductive toxicity in zebrafish via a down-regulation of estrogen. |
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