The Degradation, Metabolism and EndocrineDisrupting Effects of Triadimefon Enantiomers in Eremiasargus

爬行动物自石炭纪开始出现以来，迄今已有三亿年历史。作为生态系统的 重要组成部分，其种类仅次于鸟类而排在陆地脊椎动物第二位。近年来，随着 外源污染物的持续入侵，爬行动物的生境面临着严重威胁。国际上进行化学品 评估和登记使用的一系列模式生物中却没有爬行动物，而以鸟类代替爬行动物 的生态毒理学评价存在着严重的片面性。因此，本研究选择了广泛存在于我国 的小型蜥蜴—丽斑麻蜥作为研究对象，将其暴露于三唑酮（ Triadimefon，TF） 的两个手性对映体，分别进行了暴露实验，分析了三唑酮手性对映体在蜥蜴不 同组织内的降解、代谢以及代谢产物的生成情况，评价了三唑酮对丽斑麻蜥的 性腺轴、甲状腺轴的内分泌干扰效应。 研究表明，将 50 mg/kgbw三唑酮手性对映（S-(+)-TF和 R-(-)-TF）分别暴 露于丽斑麻蜥后，S-(+)-TF和 R-(-)-TF在蜥蜴的肝脏、脑、肾脏和血液中均可 检测到，并且在 12 h内达到最大浓度。研究表明，三唑酮在各个组织内的降解 速率相对较慢，168 h后，各个组织内均有少量三唑酮对映体的残留，只有血液 在 72h时间后没有检测到。肝脏和脑组织中检测到的 TF浓度相对较高，说明 这两个组织是三唑酮暴露的靶器官。同时通过分析代谢产物三唑醇的生成情况， 发现两暴露组中代谢产物的生成比例差异性很大，R-(-)-TF暴露组中的主要代 谢产物是 TN-B1，而 S-(+)-TF中是 TN-A2，这一结果论证了三唑酮代谢过程中 的立体选择性。 在对三唑酮代谢途径的研究中，通过设计两种不同代谢途径（P450酶和羟 基类固醇脱氢酶途径）中关键作用酶的简并引物并进行定量 PCR实验，发现肝 脏和脑中的 cyp1a, cyp3a基因均有明显的上调趋势，且 cyp3a最为明显。这一 现象说明在肝脏和脑中均有 P450酶参与到三唑酮的代谢过程中来，并且由于 P450酶的高表达可能会造成视黄酸代谢的活跃，增加肝脏肿瘤发生的几率。在 对 hsd11β的研究中发现，肝脏中也出现了上调趋势，而在脑中 hsd11β基因的表 达量基本没有变化，这一差异性说明脑中没有羟基类固醇脱氢酶参与 TF的代 谢，这也可以解释三唑酮在各个组织内降解的差异性的原因。 在三唑酮对丽斑麻蜥下丘脑 -脑垂体-性腺轴（HPG）的内分泌干扰效应研 究中，将三唑酮手性对映体短期（21天）暴露于丽斑麻蜥，对血液中的性激素 含量进行了测定，发现高浓度组血液中的睾酮含量明显增加，而雌二醇的含量 却低于对照组。造成这样现象的原因可能是由于 cyp19基因的表达受到抑制。 在对性腺轴敏感基因的定量 PCR实验中，发现肝脏和性腺中的 cyp19基因显著 下调，cyp17和 hsd17β表现出显著的上调现象。此外，erα和 Ar基因出现了雌 雄性别上的差异性，对雄性的影响表现为上调，而对雌性的基本没有影响。以 上的实验结果可以看出，三唑酮通过影响性腺轴的敏感基因的表达对蜥蜴的性 腺系统产生内分泌干扰效应。 经过三唑酮的短期暴露，测定甲状腺系统的敏感基因的相对表达量发现， tra和 trβ基因在肝脏中的表达量明显上调，且雄性对 tra较为敏感。ttr基因作 为甲状腺干扰的生物标志物，在三唑酮的暴露下显著上调，说明蜥蜴体内甲状 腺激素与转运蛋白的结合能力有所提高。肝脏中的脱碘酶基因 d1的表达量下降， 而 d2的表达量却显著上调，结合已有的研究推测三唑酮的暴露可能对蜥蜴产生 了甲减作用，但是这种作用在脑中相对较弱。 综上所述，三唑酮手性对映体对丽斑麻蜥不同组织内的降解代谢具有一定 的差异性，同时短期的三唑酮暴露对蜥蜴的性腺和甲状腺系统均造成了不同程 度的损伤，这为评价手性农药的安全性提供了可靠的依据。Reptiles, with an enormous population, played an irreplaceable role in the ecosystem. Recently, anthropogenic pollutants have been identified as one of the major contributing factors in the global decline of reptiles, while there are less of researches on it. Though there are many model animals used in the risk assessment and chemicals registration, reptiles are not involved in these animals. In this study, we exposured enantioseparated triadimefon (TF) to Eremias argus, a kind of lizard widely distributed in China. The absorption, distribution of triadimefon and the formation of triadimenol were analyzed at different sampling times. The metabolic pathways were demonstrated through relative gene expression using quantitative real-time PCR reaction. At the same time, we evaluated the disrupting effects of triadimefon on the gonad or thyroid system of lizards. In order to understand the toxicity mechanism of triadimefon in reptiles, the current study administrated S-(+)-TF or R-(-)-TF triadimefon (50 mg/kgbw) to Eremias argus respectively. During the experiment time, triadimefon was quickly peaked to the maximum concentration within 12 hours in liver, brain, kidney, and plasma, while eliminated slowly. After 168 h, there was still slight amount of triadimefon residued in all organs expceted blood. Liver and brain possessed relatively high concentration of TF which may verify these two organs are the target organs for TF exposure. The mainly metabolite in R-(-)-TF and S-(+)-TF treated groups were TN-B1 and TN-A2, respectively showed the selective metabolism to other species. The related gene expression of cyp1a1, cyp3a1 and hsd11β mRNA levels in lizards showed different metabolic pathways in the liver and brain. The up-regulation of cyp1a1, cyp3a1 in liver and brain indicated that P450 enzymes made mainly contribution to the metabolism of triadimefon accompanying with the side-effect of enrichment the metabolite in retinoic acid which may cause tumorigenesis. The differentiation of hsd11β mRNA level in liver and brain implied the dissimilarity of triadimefon in degradation and elimination, so as to the formation of triadimenol. These analyses may give an explanation the different metabolism of triadimefon. In the study of the endocrine disrupting effect of triadimefon on gonad system, the lizards orally exposed to TF and its enantiomers for 21 days. After exposure, the plasma testosterone level in the 100 mg/kgbw group was elevated, while the oestradiol level was reduced. This phenomenon may be caused by the transformation of cyp19, which may inhibit the conversion of testosterone to oestradiol and affect sexual behavior. Furthermore, the cyp19 mRNA level in liver and gonad of the TF2 and TF group (100 mg/kgbw) were significantly down-regulated, while the cyp17 and hsd17β mRNA levels were up-regulated. The expression of erα and Ar mRNA levels were up-regulated in males but not in females, which may indicate that TF has sex differences on these two genes. As seen from the above results, TF and its enantiomers may have endocrine-disrupting effects on lizards (E. argus) by acting sensitively on sex steroid hormones and steroidogenic-related genes. After analysing the related gene expression of thyroid system, we found the exposure of triadimefon might also affect the thyroid. The expression of trα and trβ in liver and brain were up-regulated，especially in males. The gene of ttr, as a biomarker of thyroid, was significantly up-regulated under the exposure of triadimefon and this phenomenon indicated the binding capacity of thyroid hormone and transporter had been improved. D1 expression in liver was decreased, whereas the expression of D2 was significantly evaluated which might imply triadimefon had hypothyroidism effect on lizards. In summary, the degradation and metabolism of triadimefon in different tissues of lizards are both dissimilarity. The short term exposure of triadimefon caused endocrine disrupting effect on gonad and thyroid system and this study provided reliable data on the safety evaluation of triadimefon