| 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. |
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