【作者】 王慧；

【导师】 田华； 汝少国；

【作者基本信息】 中国海洋大学 ， 生态学， 2013， 硕士

【摘要】 久效磷农药已被证实具有潜在的环境雌激素活性，可导致雄性金鱼、孔雀鱼体内雄激素睾酮(testosterone, T)水平降低、雌激素17β-雌二醇(17β-estradiol, E2)水平升高。鱼类性激素的合成涉及一系列的酶促反应，久效磷农药对性激素合成途径中间环节的干扰可能是导致鱼类雄雌激素比例失衡的原因。本文采用0.01mg/L、0.10mg/L和1.00mg/L久效磷农药暴露雄性金鱼21天，首先检测性激素合成底物胆固醇含量变化情况，其次在克隆金鱼多种类固醇生成酶cDNA片段的基础上检测久效磷暴露对其mRNA表达水平的影响，最后检测久效磷暴露条件下性激素合成途径中间产物和效应激素的含量，并分析类固醇生成酶基因表达变化与性激素含量异常的相关关系，以期从性激素合成与转化的角度阐明久效磷农药导致雄性鱼类雄雌激素比例失衡的机制。本文研究结果表明：(1)0.01mg/L、0.10mg/L和1.00mg/L久效磷农药暴露雄性金鱼21天，采用全自动生化分析仪测定雄性金鱼血浆中总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、甘油三酯的含量。结果表明：0.10mg/L久效磷农药可使雄鱼血浆中高密度脂蛋白胆固醇含量显著降低，可能会导致运输到精巢组织的性激素合成底物含量降低，从而导致T合成受到抑制。(2)0.01mg/L、0.10mg/L和1.00mg/L久效磷农药暴露雄性金鱼21天，采用实时定量PCR(real-time PCR)检测精巢中类固醇激素合成急性调节蛋白(steroidogenic acute regulatory protein, StAR)及胆固醇侧链裂解酶(cholesterol sidechain cleavage enzyme, P450scc)、3β-羟类固醇脱氢酶(3β-hydroxysteroiddehydrogenase,3β-HSD)、17α羟化酶(cytochrome P45017alpha-hydroxylase,17,20-lyase, P450c17)等类固醇生成酶mRNA的表达水平，采用酶联免疫分析方法(enzyme-linked immunosorbent assay, ELISA)测定血浆中孕烯醇酮、孕酮、17α-羟基孕烯醇酮、17α-羟基孕酮、脱氢表雄酮、雄烯二酮、雌酮、11β-羟基睾酮等性激素合成中间产物的含量。结果表明：久效磷农药抑制雄性金鱼精巢中StAR mRNA的表达水平，可能会导致转运到线粒体内膜的胆固醇含量减少；久效磷农药抑制P450scc的表达，从而降低血浆中孕烯醇酮的含量；久效磷农药降低3β-HSD、P450c17mRNA的表达水平，导致雄烯二酮含量减少；由此推测久效磷农药对上述类固醇生成途径的抑制作用，是造成雄性鱼类体内雄激素T含量降低的另一机制。(3)0.01mg/L、0.10mg/L和1.00mg/L久效磷农药暴露雄性金鱼21天，采用real-time PCR检测精巢中20β-羟类固醇脱氢酶(20β-hydroxysteroid dehydrogenase,20β-HSD)、芳香化酶(cytochrome P450aromatase, P450arom)、17β-羟类固醇脱氢酶I(17β-hydroxysteroid dehydrogenase type I,17β-HSD1)、11β-羟化酶(11β-hydroxylase, P45011β)、11β-羟类固醇脱氢酶II(11β-hydroxysteroiddehydrogenase type II,11β-HSD2) mRNA表达水平，采用ELISA法检测血浆中17α,20β-双羟孕酮、11-酮基睾酮(11-ketotestosterone,11-KT)等效应激素的含量。结果表明：久效磷农药提高P450arom、17β-HSD1mRNA表达水平，推测久效磷农药对这两种类固醇生成酶基因表达的促进作用，是造成雄性鱼类体内雌激素E2含量升高的机制，且P450arom表达的升高可能进一步造成T含量的降低；久效磷农药促进20β-HSD mRNA表达，导致17α,20β-双羟孕酮含量升高；久效磷农药虽然引起P45011β、11β-HSD2mRNA表达水平的降低，但是本文结果表明其对11-KT含量无显著影响。综上所述，本文从胆固醇含量、StAR和类固醇生成酶mRNA的表达量以及性激素合成途径中涉及的中间产物及效应激素的含量三个方面，探讨了久效磷农药对雄性金鱼性激素合成与转化的影响机制，研究表明久效磷农药造成雄性金鱼血浆中雄雌激素比例失衡主要是由于：(1)降低血浆中高密度脂蛋白胆固醇含量，抑制雄激素合成相关酶类mRNA表达，从而降低雄激素(主要为T)的合成；(2)促进雌激素合成相关酶类mRNA表达，进而提高雌激素(主要为E2)的合成。本文系统研究了结构与内源性激素并不相似的久效磷农药发挥环境雌激素效应的机制，以期为非典型环境雌激素(不能直接与激素受体相结合)的作用靶点研究提供一定的借鉴。更多还原

【Abstract】 Our previous studies have demonstrated that monocrotophos (MCP) haspotential estrogenic properties, since it increased17β-estradiol (E2) levels anddecreased testosterone (T) contents in male goldfish (Carassius auratus) and guppy(Poecilia reticulata). Sex hormone biosynthesis involves a series of reactionscatalyzed by several steroidogenic enzymes, thus disruption on the process ofsteroidogenesis may be responsible for the imbalance of androgen and estrogencaused by MCP pesticide. In this study, following a21-day exposure to0.01mg/L,0.10mg/L, and1.00mg/L MCP in male goldfish, changes of cholesterol (thesubstrate for sex hormones synthesis) contents were investigated, and then the effectsof MCP exposure on mRNA levels of steroidogenic acute regulatory protein (StAR)and steroidogenic enzymes were studied based on cloning cDNA fragments of severalsteroidogenic enzymes. Finally, the contents of intermediate products and effectorhormones involved in the sex steroid biosynthesis pathway were detected after MCPexposure, to establish the correlation of steroidogenic enzyme expression and sexhormone levels. The aim of this study is to elucidate the mechanisms causingimbalance of sex steroids by MCP pesticide in male fish from the perspective of sexhormone synthesis and transformation. The results showed that:(1) After a21-day exposure to0.01mg/L,0.10mg/L and1.00mg/L MCP,concentration of total cholesterol, high density lipoproteins cholesterol (HDL-C), lowdensity lipoprotein cholesterol, and triglyceride in plasma of male goldfish were eachdetected using automatic biochemistry analyzer. MCP decreased the HDL-C levels inplasma, which may lead to a decline of the substrate of sex hormones took into thetestis and thus inhibit T synthesis.(2) After a21-day exposure to0.01mg/L,0.10mg/L and1.00mg/L MCP,real-time PCR was used to detect mRNA levels of StAR and several steroidogenicenzymes, including cholesterol side chain cleavage enzyme (P450scc),3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P45017alpha-hydroxylase(P450c17), and ELISA was adopted to determine levels of intermediate products involved in the steroid biosynthesis pathway, including pregnenolone, progesterone,17α-hydroxypregnenolone,17α-hydroxyprogesterone, dehydroepiandrosterone,androstenedione, estrone, and11β-hydroxy testosterone. MCP inhibited StAR mRNAexpression levels in testis of male goldfish, which may result in reducedintra-mitochondrial cholesterol pools; MCP decreased the abundance of P450scctranscripts, leading to a decline of pregnenolone; MCP reduced3β-HSD and P450c17mRNA expression levels, which resulted in a decline of androstenedione; thus it wasdeduced that the inhibition of MCP pesticide on the above mentioned steroidogenesispaths might be another reason that T in male fish was decreased.(3) After a21-day exposure to0.01mg/L,0.10mg/L and1.00mg/L MCP,real-time PCR was used to quantify mRNA levels of20β-hydroxysteroiddehydrogenase (20β-HSD), cytochrome P450aromatase (P450arom),17β-hydroxysteroid dehydrogenase type I (17β-HSD1),11β-hydroxylase (P45011β),and11β-hydroxysteroid dehydrogenase type II (11β-HSD2), and ELISA was adoptedto test levels of effector hormones involved in the sex steroid biosynthesis pathway,including17α,20β-dihydroxy-4-pregnen-3-one and11-ketotestosterone (11-KT). MCPraised P450arom and17β-HSD1mRNA levels, thus it was suggested that theenhancement of MCP pesticide on gene expression of these two steroidogenicenzymes contributed to the increase of E2, and the increase of P450arom expressionmight strengthen the decrease of androgen T; MCP increased20β-HSD mRNAexpression levels in testis of male goldfish, leading to an increase of17α,20β-dihydroxy-4-pregnen-3-one; however, although MCP decreased mRNAexpression of P45011βand11β-HSD2,11-KT contents were not affected according tothis study.In conclusion, the action mechanisms of monocrotophos pesticide on sexhormone synthesis and transformation in the male goldfish were discussed byevaluating the levels of cholesterol contents, mRNA levels of StAR and steroidogenicenzymes, and contents of intermediate products and effector hormones involved in thesteroid biosynthesis pathway. The results showed that MCP caused the imbalance ofestrogen and androgen mainly via (a) decreasing HDL-C concentrations in the plasma,inhibiting mRNA expression of steroidogenic enzymes related to androgen synthesis,and consequently reducing the androgen (mainly T) levels;(b) promoting mRNAexpression of steroidogenic enzymes related to estrogen synthesis, and then increasingestrogen (mainly E2) levels. Through systematic investigation on action machnisms of MCP pesticide which does not have a similar structure to E2, the present study canprovide a reference for a better understanding of the acting targets of non-typicalenvironmental estrogens.更多还原