摘要
丁草胺属于酰胺类除草剂,它通过抑制杂草体内蛋白质合成而起到除草作用。斑马鱼具有产卵量多、前期胚胎透明、繁殖快、易于饲养等优点,成为继小鼠、果蝇、线虫之后又一新型模式生物。本论文采用鱼类活体(In vivo)实验方法,以丁草胺原药(我国应用最多的三个除草剂之一)为供试药剂,从下丘脑-垂体-甲状腺轴、下丘脑-垂体-性腺轴和氧化损伤等角度,研究丁草胺对斑马鱼内分泌干扰效应,并探讨其可能的作用机制。
丁草胺对斑马鱼成鱼的急性毒性结果显示：丁草胺对斑马鱼成鱼96h的LC50值是0.49mg/L,根据农业部《农药安全评价准则》农药对鱼类毒性等级的划定标准,表明丁草胺对斑马鱼成鱼的毒性为高毒。采用GC—MS\MS对丁草胺原药中的杂质进行了鉴定,结果表明,96%丁草胺原药中主要杂质是二丁氧基甲烷和(2-氯-N-(2,6-二乙基苯基)乙酰胺。我们测定了斑马鱼整个生命周期中的甲状腺激素变化动态,结果表明斑马鱼的胚胎在28±1℃条件下,受精后48～72h内孵化,授精后0-3天胚胎孵化前T4和T3的含量没有显著变化；幼鱼体内T3含量在授精后第10天达到最高峰；幼鱼体内T4含量在授精后第21天达到最高峰。在斑马鱼生长后期,甲状腺激素会出现一个低平台期,本研究为将来选用斑马鱼进行污染物的内分泌干扰研究奠定了基础。
从下丘脑-垂体-甲状腺轴(HPT)调控角度出发,研究了丁草胺对斑马鱼幼鱼和成鱼体长、体重和生长因子等形态学指标的影响；运用酶联免疫吸附分析(ELISA)检测丁草胺对斑马鱼成鱼血浆和幼鱼组织中甲状腺激素水平的影响；采用实时荧光定量PCR技术检测丁草胺对斑马鱼幼鱼甲状腺轴相关基因表达的影响。结果表明(1)丁草胺染毒30天后,25μg/L和100μg/L剂量可以导致斑马鱼幼鱼的生长因子(CF)增加,但是不同浓度丁草胺处理组中成鱼的生长因子与溶剂对照组相比,差异不显著；(2)丁草胺可以提高斑马鱼成鱼血浆中和幼鱼组织中甲状腺激素四碘甲状腺原氨酸(T4)和三碘甲状腺原氨酸(T3)水平；(3)丁草胺可以使斑马鱼幼鱼促甲状腺激素释放激素(CRH)、促甲状腺激素(TSH)、转运蛋白(TTR)、甲状腺激素受体(TRα)、脱碘酶(Deio1)和纳/碘同向转运体(Slc5a5)等基因的表达量上调,葡萄糖醛酸转移酶基因(UGT1ab)基因的表达量下调,甲状腺过氧化物酶(TPO)、甲状腺球蛋白(TG)、脱碘酶(Deio2)和甲状腺激素受体(TRβ)的表达量没有变化。结果显示丁草胺对斑马鱼成鱼和幼鱼都具有一定的甲状腺干扰作用。
从下丘脑-垂体-性腺轴(HPG)调控角度出发,研究丁草胺对斑马鱼成鱼产卵及后代的影响；运用酶联免疫吸附分析(ELISA)检测丁草胺对斑马鱼成鱼血浆中性激素睾酮、雌二醇和卵黄蛋白原水平；采用实时荧光定量PCR技术检测丁草胺对斑马鱼幼鱼性腺轴的相关基因表达的影响,包括雌激素受体(ERα、ERβ1和ERβ2)、卵黄蛋白原(VTGI和VTG II)和芳香化酶(CYP19)等基因。结果显示丁草胺对斑马鱼的性腺轴具有一定的干扰作用,主要表现在(1)丁草胺可以降低斑马鱼雌鱼的产卵次数和产卵量；(2)丁草胺可以降低雄鱼的性腺指数(GSI),但是对成鱼的肝腺指数(LSI)没有影响；(3)丁草胺可以降低斑马鱼雌鱼血浆中的睾酮(T)和雌二醇(E2)水平,提高成鱼血浆中卵黄蛋白原(VTG)水平；(4)丁草胺可以使斑马鱼幼鱼卵黄蛋白原VTG II表达量上调,但是对幼鱼雌激素受体(ERα、ERβ1和ERβ2)和芳香化酶CYP19a的表达量没有影响。
以雌性成鱼为受试对象,研究丁草胺对斑马鱼肝脏抗氧化防御系统的影响。肝脏是主要的生物富集器官,故被大量地用于外源物质毒理研究。结果显示丁草胺对斑马鱼具有氧化损伤作用。主要表现在丁草胺可以降低斑马鱼雌鱼肝脏中超氧化物歧化酶(SOD)和谷胱甘肽转移酶(GST)的酶活性以及谷胱甘肽(GSH)含量,对肝脏过氧化氢酶(CAT)活性没有影响。
Butachlor is a chloracetamide herbicide, which is one of the top three herbicides for controlling weeds in rice fields in China. Zebrafish (Danio rerio) is a model organism widely used for evaluating reproductive toxicity and endocrine-disrupting effects of chemicals due to its characters of small size, ease of culture, short life cycle and prolific egg production rate with high rate of fertilization and hatching. The present study evaluated the effects of butachlor on endocrine system of zebrafish were evaluated and the potential mechanisms.
The result of acute toxicity test on adult zebrafish showed that the LC50value of96%butuchlor within96h was0.49mg/L, indicating that butachlor was highly toxic to zabrafish. The normal levels of thyroxine (T4) and3,5,3'-triiodothyronine (T3) during the development in zebrafish were also measured. The result showed that whole-body content of both T4and T3remained stable during the pre-hatch period (0-3d). After hatching, both T3and T4significantly increased, with peaking at10days post-fertilization (dpf) and21dpf, respectively, and then thyroid hormones subsequently declined in later development. This work has provided the baseline data for future studies of the thyroid system in zebrafish.
The potential effects of butachlor on hypothalamus-pituitary-thyroid (HPT) axis of zebrafish were assessed in the present study. The parameters to be measured include body weight, length, and the contents of T4and T3. In order to assess the potential mechanisms of thyroid disruption of butachlor, the expressions of related genes in HPT axis were determined. The result demonstrated that butachlor could increase the condition factor of zebrafish in the early life stage after30d's exposure, while no difference of condition factor of zebrafish was observed between the control group and treated group in adult stage; T4and T3levels in zebrafish were increased after exposure to butachlor in both the early life stage and adult stage. After the exposure of butachlor, the mRNA levels of the genes of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSH), transthyretin (TTR), deiodinase (Deiol) and thyroid hormone receptor (TRa) and Slc5a5were significantly upregulated in zebrafish of the early life stage, while the mRNA level of uridinediphosphate-glucuronosyl transferase (UGTlab) was significantly downregulated. No significant differences were observed in expressions of the genes thyroid peroxidase (TPO), globulin (TG), D2and TRβ. In summay, the hypothalamus-pituitary-thyroid (HPT) axis of zebrafish was affected by butachlor.
The effects of butachlor on hypothalamus-pituitary-gonad (HPG) axis of zebrafish were also evaluated. The fecundity was assessed during the exposure period in a short-term assay. At the end of the exposure period, various endpoints such as gonadosomatic index (GSI), liver somatic index (LSI), and the levels of plasma sex steroids (T and E2) and vitellogenin (VTG) of zebrafish were examined. In order to assess the potential mechanisms of reproductive disruption of butachlor, the expressions of related genes in hypothalamus-pituitary-gonad (HPG) axis were determined in the early life stage of zebrafish. Our result showed that hypothalamus-pituitary-gonad (HPG) axis of zebrafish was affected by butachlor:(1) The number of eggs and number of spawning events per female were significately reduced after exposure to butachlor;(2) The gonadosomatic index (GSI) in males was decrased, while no affect on liver somatic index (LSI) was observed in both females and males;(3) The levels of T and E2in plasma were significately decreased in females, and the levels of vitellogenin (VTG) in males were significately increased.(4) There was no significant alteration in expression of vitellogenin (VTGI), estrogen receptor (ERα, ERβ1, ERβ2) and aromatase (CYP19a) in the early life stage after the exposure of butachlor, whereas the transcription of VTG II gene was significantly upregulated.
Addtionally, the liver of zebrafish, which is the organ responsible for detoxifying exogenous substance, was used to evaluate the effect of butachlor on antioxidant system of the fish. Our results showed that either the activitie of hepatic SOD and GST, or the contents of GSH in female zebrafish were significantly decreased, while the activity of CAT was not altered.
引文
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