【作者】 刘少颖；

【导师】 朱国念；

【作者基本信息】 浙江大学 ， 农药学， 2011， 博士

【摘要】 三唑酮是甾醇脱甲基抑制剂(DMI),对植物体内麦角甾醇合成具有抑制作用。本论文以斑马鱼为受试对象,评价了三唑酮对斑马鱼胚胎-幼鱼阶段暴露产生的胚胎发育毒性。胚胎急性毒性结果表示三唑酮对斑马鱼胚胎毒性为低毒,2μg/mL和4μg/mL暴露后斑马鱼胚胎呈现出致畸效应,主要表现为躯干弯曲、游囊关闭等畸形现象。通过窗口试验及运动行为等综合指标的检测,进一步对致畸机制进行了初步的探讨,推测自主运动减缓从而影响孵化是导致畸形现象产生的可能机制,是否还存在其他方面的潜在机制还需通过其他实验进一步进行研究。另外,通过检测斑马鱼运动行为评价其可能具有的潜在神经毒性效应,主要包括24 hpf的自主运动和120 hpf游动能力,初步证实了三唑酮对斑马鱼胚胎发育具有神经毒性效应,方法具有一定的新颖性。从下丘脑-垂体-甲状腺轴出发探讨三唑酮对斑马鱼内分泌-生殖系统造成的影响。研究了三唑酮对斑马鱼胚胎甲状腺激素T3、T4的影响,进一步从下丘脑-垂体-甲状腺轴角度出发,主要包括促甲状腺激素(TSHβ)、甲状腺激素核受体(TRα和TRβ)、脱碘酶(D1和D2)的基因表达量水平进行了测定,解释其可能存在的干扰机制。结果显示三唑酮对斑马鱼早期胚胎具有一定的甲状腺激素干扰效应。主要表现为诱导促甲状腺激素TSHβ基因表达量升高,从而促进甲状腺激素合成和分泌,导致体内T4水平升高。同时抑制了脱碘酶D1,使得T4到T3之间的脱碘转化受到抑制,导致T3水平降低。T3在体内只有进入靶细胞后,与甲状腺激素核受体TRp结合后才能发挥作用。试验结果显示相应的甲状腺激素受体TRp的基因表达量也受到抑制。T3的生物活性是T4的3-5倍,另外它与甲状腺激素核受体TRp结合后才能发挥作用。因此,三唑酮暴露后降低了斑马鱼体内甲状腺的生物活性。从性腺发育和生殖功能出发探讨三唑酮对斑马鱼内分泌-生殖系统造成的影响。将斑马鱼亲代FO代整个生命周期长时间暴露于三唑酮,为三唑酮的生态毒理学和生态环境风险评价研究提供了一定的科学依据和理论基础。对斑马鱼亲代F0代的内分泌-生殖系统相关的生理生化指标进行了检测。实验结果三唑酮对斑马鱼种群数量、体重体长等生长指标具有一定的抑制作用。另外,通过性腺组织学观察,发现三唑酮对卵巢、精巢的重量没有影响,但是三唑酮处理组雌鱼卵巢处于相对幼稚的阶段,发育不成熟。从性别比例的统计数据可以发现三唑酮处理后,斑马鱼种群出现雄性化的现象,结合Vtg基因表达量也受到抑制,推测三唑酮暴露后斑马鱼种群出现雄性表现型增多的现象主要是由于三唑酮具有芳香酶抑制剂的特性,使其干扰了斑马鱼的性别发育,导致种群中雄性表现型增多的现象。斑马鱼整个生命周期暴露于三唑酮后又进行了配对产卵试验,从整体角度研究了三唑酮对斑马鱼繁殖的影响效应并作了初步的判断,三唑酮处理组雌鱼产卵次数减少,表明抑制了交配行为,导致产卵数量降低。雌鱼产卵降低还与卵巢发育不成熟相关,另一方面,雄鱼的精子密度和精子活力降低等因素也影响了配对产卵试验。另外,本论文通过三唑酮亲代F0整个生命周期期暴露后对子代F1代胚胎的受精率、孵化率和死亡率三个指标的观察,从亲代和子代二世代角度深入研究三唑酮对斑马鱼的内分泌-生殖毒性。结果显示亲代处于0.5μg/mL浓度三唑酮暴露后对子代F1胚胎的受精率显著降低,进一步影响孵化,最终导致死亡。结合雄鱼精子质量的试验,表明亲代雄鱼精子质量显著降低,包括精子活力和精子密度,导致了子代胚胎受精率降低。0.125μg/mL和0.25μg/mL浓度三唑酮亲代F0处理后对子代F1胚胎受精率、孵化率、畸形率未发现任何影响,但其是否具有其他毒性效应有待进一步研究。更多还原

【Abstract】 Triadimefon, a triazole fungicide, has been widely detected in the environment, but few studies have assessed its effect on aquatic organisms. The present study evaluated the effect of triadimefon in embryo-larval stages of zebrafish. Zebrafish embryos exhibited teratogenic evidence of bent spine, uninflated swim bladder and other malformations after being exposed to various triadimefon concentrations (2-4μg/mL) from 6 to 120 h post-fertilization (hpf). Locomotor activities namely spontaneous movement in embryos and swimming activity in larvae were detected to assess the neurotoxicity. This was the first study to detection the neurotoxicity and teratogenic of triadimefon in zebrafish. It seemed that the spontaneous movement was related to the hatching rate and the potential course of the uninflated swim bladder. The results suggested that the need for additional research to identify the mode of triadimefon toxicity.The study evaluated thyroid hormone levels and the expression of related genes in the hypothalamic-pituitary-thyroid (HPT) axis, including thyroid-stimulating hormone (TSHP), deiodinases (D1 and D2) and the thyroid hormone receptor (TRαand TRβ). After triadimefon exposure, increased T4 but decreased T3 were found. It seemed that increased TSHβcould result in increasing T4. The conversion of T4 to T3 occurred by the deiodinases. Decreased T3 could be explained by decreasing deiodinase type I (D1). The action of thyroid hormones was mediated by their binding to nuclear receptors that acted directly on target genes. TRP mRNA levels were significantly down-regulated, possibly as a response to the decreased T3 levels. The overall results indicated that triadimefon exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by triadimefon could occur at several steps in the synthesis, regulation, and action of thyroid hormones.Impact of triadimefon on endocrine reproductive system in zebrafish, including vitellogenin (Vtg) mRNA, mortality rate, growth, sex ratio, gonad histology and sperm parameters was evaluated in this study by using full life cycle test. Zebrafish of 24 hours post fertilization (24 hpf) were exposed to various concentration of triadimefon (0.125,0.25,0.5μg/mL) until 120 days. Increased mortality rate were observed in fish exposed to 0.5μg/mL triadimefon. At 40 dpf, Vtg mRNA expression was decreased in fish exposed to 0.25 and 0.5μg/mL triadimefon. Histological assessment revealed that the female fish in the treated by 0.5μg/mL triadimefon had immature ovaries. The sex ratio had a significantly increased in terms of males. These effects were consistent with aromatase inhibition. Armatase inhibitor could block the balance of estrogens and androgens so as to increase proportion of males in populations.F0 Breeding studies revealed significant reproductive dysfunctions in 0.25 and 0.5μg/mL triadimefon-treated groups. This was a report about fish with reduced reproductive capacity following exposure to triadimefon, had not been published yet. Reproductive success was a very complicated process and it might be influenced by multiple factors. The sperm examination revealed that triadimefon decreased the sperm mobility and sperm concentration, which could partially explain why the triadimefon-treated males were almost infertile. Then some parameters of the offspring F1 were tested, including embryo fertility, mortality, hatching rate and teratogenic effects. It suggested that triadimefon could adversely affect the fertility rate, the hatching and mortality. Decreaed fertily could be explained by decreasing sperm concentration and motility.0.125μg/mL and 0.25μg/mL triadimefon had no significantly influences on the fertility, hatching and mortality. Whether these concentrations had any other toxic effects needed to be illustrated in further study.更多还原