【作者】 吴慧明；

【导师】 娄永根； 朱国念；

【作者基本信息】 浙江大学 ， 环境生物学， 2013， 博士

【摘要】 近20年来,农产品的生产越来越依赖于农药、化肥等外源性物质。据统计,我国的农药生产量已超过美国而居世界第一位。由于农药在农林业、工业、学校和住宅区的广泛使用,人们在享受农药所带来利益的同时,承受着其所产生的负面影响。化学农药生产与使用所造成的污染是我国食品安全、生态安全中影响范围最大的有机物污染。论文选择新型杀虫剂螺虫乙酯,进行对雄性SD大鼠的28d和90d灌胃毒性试验,研究农药对大鼠的亚慢性毒性、效应器官及毒性指标；通过分子生物学手段研究效应机制,以期补充螺虫乙酯在毒理学研究上的不足。论文首先进行螺虫乙酯在大鼠体内的分布与代谢研究。大鼠经28d连续口服染毒后处死,采用现代残留检测前处理方法和超高压液相色谱-串接质谱联用仪检测不同组织器官中螺虫乙酯残留量及其主要代谢产物的分布。试验结果显示睾丸中螺虫乙酯的残留量最高为0.025mg/kg,其次肝脏为0.023mg/kg,脂肪和肌肉中最低0.005mg/kg,但睾丸、肝脏、肺、肾、心脏等螺虫乙酯残留量没有明显差异。其主要代谢物enol的残留量肝脏中最高,达0.620mg/kg,其次是肾0.370mg/kg,睾丸中仅为0.083mg/kg,其它组织器官由高至低依次为肾>血浆>肺>心脏>睾丸>脂肪>肌肉。28d灌胃试验结果表明,螺虫乙酯可显著降低大鼠体重和每周取食量,大鼠体重下降和处理浓度存在剂量-效应关系。螺虫乙酯处理浓度为250mg/kg·d时,可显著影响肝脏、肾、睾丸和附睾的脏器系数；相对肝脏和肾脏,睾丸和附睾更易受螺虫乙酯影响,50mg/kg·d的螺虫乙酯灌胃28d即可显著改变二者的脏器系数。染毒持续至90d后,螺虫乙酯对大鼠体重的影响消除,但高剂量组(250mg/kg·d)的肝脏、睾丸脏器系数显著小于对照组。上述结果显示肝和睾丸是螺虫乙酯主要的效应器官。用HE染色法研究28d和90d染毒后,肝脏和睾丸组织形态学以及精子数量、形态的变化。结果说明螺虫乙酯可明显改变大鼠肝脏组织形态,250mg/kg·d染毒28d和90d都发现肝严重变色,肝小窦变窄或消失,肝细胞出现水肿和凋亡；同剂量螺虫乙酯可影响睾丸精小管以及支持细胞的组织形态,睾丸间质细胞出现损坏,生精上皮的层次不明显或消失。10mg/kg·d既可显著减少附睾中的精子数量,精子数量和药剂浓度有剂量效应关系。用ELISA法和全自动生化分析仪检测了染毒后大鼠血清中瘦素、血糖、胰岛素、睾酮及其它衡量肾功能、肝功能、脂质代谢等的生化指标。结果显示,血清中瘦素、血糖和胰岛素的浓度与螺虫乙酯浓度有一定的剂量效应关系。当暴露浓度为250mg/kg·d|时,28d后血清中瘦素的浓度仅为对照的77%,而葡萄糖的浓度则升高了85.6%,胰岛素浓度升高了77.3%；90d后,血清中瘦素的浓度仅为41.1%,葡萄糖和胰岛素浓度分别升高了44.9%和16.6%。数据表明染毒时间越长对瘦素的影响越大,但血糖和胰岛素却有恢复的趋势。睾酮的测定结果显示,28d染毒没有明显改变血清中的睾酮浓度。染毒90d后血清睾酮浓度发生显著变化,并出现剂量-浓度的效应关系。50mg/kg·d和250mg/kg·d染毒后,睾酮浓度显著低于对照组,分别是对照的66.1%和51.7%。各处理组之间血清中血尿素氮(BUN)、肌酐(Creatinine)含量没有显著性差异,说明大鼠肾功能没有受到明显影响。虽然碱性磷酸酯酶(ALP)、谷丙转氨酶(ALT)的浓度没有明显变化,但250mg/kg·d处理组天冬氨酸氨基转移酶(AST)、白蛋白(ALb)、总胆红素(TBIL)含量与对照组有明显差异,说明肝功能受到一定程度损害。试验结果还显示,血清中总胆固醇(CHOL)、甘油三脂(TG)、高密度脂蛋白固醇(HDL-C)的含量没有显著变化,但250mg/kg·d处理组的低密度脂蛋白固醇(LDL-C)浓度却显著提高,是对照组的159.7%,暗示大鼠患动脉粥样硬化等心血管疾病风险增加。荧光定量PCR法测定了28d染毒,螺虫乙酯对肝脏、睾丸中乙酰辅酶A羧化酶(ACC)、AMP活化蛋白激酶a(AMPKa)、脂肪酸合成酶(FAS)、瘦素(leptin)、肉毒碱棕榈酰基转移酶1(CPT-1),以β-actin为内参基因,UNT组的表达量为基础,采用2ΔΔCT法计算分析基因表达差异。试验结果显示：ACC1在肝脏中的表达更易受螺虫乙酯的影响,50mg/kg·d睾丸和肝脏中的ACC1表达量均显著下调,仅为BK组的66%和19%；250mg/kg·d处理组,则分别为57%和22%。ACC2和CPT-I在肝脏的表达量与农药浓度有明显剂量效应关系,50mg/kg·d和250mg/kg·d处理组ACC2在肝脏中的表达量分别是BK组的65.4%和21.4%,显著下调；在睾丸中的表达量分别是BK组的90.3%和66.7%,仅高剂量组出现显著下调。CPT-I在各处理组睾丸的表达量没有显著变化,而在肝脏中的表达受药剂影响显著上调,10mg/kg·d、50mg/kg·d和|250mg/kg·d处理组的基因表达量分别是BK组的1.04倍、11.7倍和37.7倍。250mg/kg·d处理组的ACC1基因表达下调和FAS在肝脏的表达量仅为BK组的2%,表明染毒后大鼠体内脂肪酸氧化加快,而脂肪酸合成受阻得不到补充,导致大鼠体重显著下降。AMPKa是细胞的“能量感应器”,其信号转导涉及三大物质代谢、细胞生长与凋亡等生命基础活动。250mg/kg·d螺虫乙酯染毒后AMPKa1在肝脏中的表达显著下调,仅为UNT组的55.5%,AMPKa2则为31.5%；该基因在肝脏中的显著下调,导致肝脏组织形态发生变化,肝功能受损。在睾丸中表达下调,影响了睾丸间质细胞结构和精小管生精上皮细胞的完整性,导致附睾精子数显著下降,精子畸形率上升；染毒90d后,使血清睾酮含量显著下降。50mg/kg·d和250mg/kg·d染毒处理组大鼠的TNF-α在肝脏中表达量显著下调,可能与LDL-C浓度升高有关。相关试验数据和分析结果可得出如下结论：1)螺虫乙酯及主要代谢产物(螺虫乙酯烯醇)主要分布于大鼠肝脏、肾脏和睾丸器官；2)螺虫乙酯可抑制脂肪合成和大鼠生长发育,其主要效应器官是肝脏和睾丸；3)螺虫乙酯通过抑制ACC的基因表达、影响大鼠机体能量平衡和脂质代谢相关基因的表达、干扰激素平衡等过程产生毒理效应。更多还原

【Abstract】 Since the last20years, agriculture has relied more and more on pesticides and fertilizers. Now, pesticide production in China has exceeded the United States ranked first in the world. Because of pesticides are widely used in farm, forest, industry, schools, people are suffering from pesticide adverse effects while enjoying the benefits of it. Pesticide is the largest organic pollutant that influences food and ecological safety. Experiment processed for studying subchronic toxicity of spirotetramat on male SD rats by28d/90d gavage, then BMI, organs coefficient, Comprehensive Metabolic Panel (CMP) were tested to evaluate the side effects of pesticide on rat. Furthermore, mechanism of toxicity was investigated with molecular biological method. The results of the experiment will be a supplement of the toxicology datum.Firstly, distribution and metabolism of spirotetramat and its metabolite in rat tissue were studied. After repeated dose28d oral administration, the rats were executed. Then, the residues of pesticide and its metabolite in various tissues were detected by ultra performance liquid chromatography equipped with triple quadruple mass spectrometer (UPLC-MS/MS). The results showed that the maximum residue of spirotetramat was in testicle of0.025mg/kg, followed by0.023mg/kg in liver, fat and muscle was smallest for0.005mg/kg respectively. However, there is no significant difference of pesticide concentration in testicle, liver, lung, kidney, heart. Its principal metabolite of the enol residues in liver is the highest for0.620mg/kg, followed by0.370mg/kg in kidney and only0.083mg/kg in testicle. The rank from high to low was liver> kidney> plasma> lung> heart> testicle> fat> muscle.Secondly, the results of the28-day trial indicated that the body weight of the rat and the weekly food intake significantly reduced, and there was a dose-response relationship between weight loss in rats and concentration of spirotetramat. Organ coefficients (OC) of liver, kidney, testicle and epididymis was obviously affected by spirotetramat of250mg/kg.d. Compared with the liver and kidney, testicle and epididymis are more sensitive to spirotetramat, and their OC were evidently changed by spirotetramat of250mg/kg.d repeated dose28-day. However, there were no effects of spirotetramat on weight loss in rats at90-day experiment, the OC of liver and testicle in rat markedly reduced by pesticide of250mg/kg.d. It showed that liver and testicle are the main targets of the attack by the pesticide.Then, HE staining method was used to test the changes of the liver and testicle morphology. Certainly, the sperm amount and deformity rate were examined to assess the reproductive toxicity of spirotetramat. The test results showed the liver tissue morphology perceptibly changed by pesticide, the discoloration, narrowed sinus, hepatocyte edema and apoptosis were found in rat repeated dose28-day or90-day at250mg/kg.d spirotetramat. Synchronously, seminiferous tubule, supporting cells and interstitial cell were injured by pesticide, and structure of seminiferous epithelium was destroyed. The sperm amount and deformity rate in rat epididymis decreased significantly after exposing to pesticide of10mg/kg.d for28days or90days. In addition, there was a dose-response relationship between the sperm count and chemistry levels.Serum leptin, insulin and testosterone contents in rat were tested with ELISA kits, and the comprehensive metabolic panel (CMP) and glucose detected with automatic biochemical analyzer. Results showed that there were dose-response relationships between the serum leptin, glucose, insulin levels and spirotetramat concentration. Exposing to250mg/kg.d spirotetramat, serum leptin concentrations were77%compared to that in control group rat in28-day tests, glucose of185.6%and insulin of177.3%was found in similar experiment. In repeated dose90-day tests, serum leptin levels were decreased by58.9%, but the insulin and glucose in serum were elevated by44.9%and16.6%, respectively. It indicated that the damage extent of leptin related to the exposure time, but blood glucose and insulin levels were on a recovery trend. The testosterone test results displayed there were no significant changes in serum testosterone concentration in28-day trial. In the other experiment (90-day trial), it was radically changed by pesticide of50mg/kg.d and250mg/kg.d, the testosterone levels were reduced by33.9%and48.3%respectively compared to the control group. There were no meaningful differences between the blood urea nitrogen (BUN) and creatinine (Creatinine) contents in the treatment rats; it means the rat kidney function is normal. Although, alkaline phosphatase (ALP), alanine transaminase (ALT) concentration did not .significantly change, but the aspartate transaminase (AST), albumin (Alb) and total bilirubin (TBIL) levels in rat in250mg/kg.d group was significantly different from that of control group. The results indicated the liver injury. However, the serum total cholesterol (CHOL), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) levels were not significantly inflected. The low density lipoprotein cholesterol (LDL-C) level was markedly elevated in250mg/kg.d group. The increased LDL-C level suggested an enhanced risk of suffering from cardiovascular diseases such as atherosclerosis in rats.The gene expressions of ACC, AMPKa, FAS, CPT-I, leptin, β-actin and TNF-α in hepatocyte and testicular cell were detected by real-time quantitative PCR (RT-PCR) for assessing the mechanism of toxicity of spirotetramat. The2-AACT method was used to analysis the data from RT-PCR for evaluating the change in gene expression relative to the untreated control, Where theβ-actin used as keeping house gene and the results of untreated control(UNT group) as calibrator for the method. The experimental results declared gene expression of ACC1was more perceptive in hepatocyte than that in testicular cell. In S50(50mg/kg.d) group, ACC1gene expressions in hepatocyte or testicular cell was significantly down regulated by spirotetramat ranged from66%and19%compared to BK group; and in S250(250mg/kg.d) group, there was about57%and22%respectively. There were obvious dose-response relationships between the amount of gene expression of ACC2or CPT-I in hepatocyte and pesticide levels. In hepatocyte of S10and S250group rats, the ACC2gene expression significantly reduced to65.4%and21.4%of BK group, respectively. It was90.3%and66.7%in testicular cell, simultaneously. There are no noteworthy differences of CPT-Ⅰ expression in testicular cell. On the contrary, CPT-Ⅰ gene was clearly up regulated in hepatocyte. In S10, S50and S250group, the changes in CPT-Ⅰ gene expression were1.04,11.7and37.7times respectively of the BK group. Moreover, the FAS gene in S250group was down regulated to2%of the BK group. Reduced ACC1, ACC2and elevated CPT-Ⅰ gene expression in hepatocyte indicated that the fatty acid oxidation was accelerated, and fatty acid synthesis was blocked. As a result, the body weight lost considerably for lack of fat. AMPKa considered as the cell’s energy sensors, the signal transduction of AMPKa in cells related to metabolism of lipid, carbohydrate and protein, and related to cell growth and apoptosis. In hepatocyte, the AMPKal gene was evidently unhappy regulated to55.5%of UNT group after exposing to250mg/kg.d for28days. The AMPKa2gene was31.5%. Reduced expression of AMPKa in hepatocyte resulted in liver injury. Certainly, the testicle damaged when expression reduced in testicular cells. Reduced sperm amount in rat epididymis and increased sperm deformity rate was found in S250group rats. After administering of250mg/kg.d spirotetramat for90days, serum testosterone levels evidently declined. The sharply dropped TNF-a expression in hepatocyte may be associated with elevated LDL-C in serum.In general, testing datum can be reached the following conclusions:1) Spirotetramat and its metabolite remained mainly in rat liver, kidney and testis.2) Fat biosynthesis and development of rat inhibited by spirotetramat, and liver and testis were primary effector organs.3) Spirotetramat produced toxicology effects through inhibiting ACC gene expression, affecting gene expression about keeping energy balance and normal lipid metabolism, disturbing the hormone balance.更多还原