细胞色素P450酶在氰戊菊酯所致肝脏和睾丸毒效应中的作用研究

发布时间：2018-07-24 16:33

【摘要】：目的：通过氰戊菊酯雄性SD大鼠染毒模型，从整体动物水平探讨了CYP450酶在氰戊菊酯所致肝脏和睾丸毒效应中的作用，为氰戊菊酯农药毒性及机制研究提供新的线索。方法：将50只大鼠随机分为1个溶剂对照组和4个氰戊菊酯染毒组(0.00625、0.125、2.5、30mg/kg.bw)，每组10只，连续灌胃染毒8周。染毒结束后，对其各个脏器的脏器系数、血清生化（ALT、TP、BUN、CHOL）以及血浆T-AOC、GSH、MDA等一般毒性指标进行评价，采用放射免疫法检测大鼠血清T_3、T_4、TSH、T、E_2、FSH、LH等激素水平，CASA仪分析氰戊菊酯对大鼠精子运动能力和精子数量的影响，采用GC-MS检测大鼠血和尿液中氰戊菊酯的残留量，荧光定量PCR分析氰戊菊酯对CYP2C11和CYP3A1/23的诱导效应。对肝脏进行组织病理学检查，提取0.125mg/kg染毒组和溶剂对照组的肝脏RNA，每组3只，逆转录为cDNA后进行基因表达谱分析，筛选差异基因，验证所诱导的CYP450酶相关基因，，对差异基因进行GO分类和Pathway分析，并采用荧光定量PCR对钙离子通路相关基因进行验证。采用钙离子探针Fluo-3AM标记BRL细胞内钙离子，观察氰戊菊酯对细胞内钙离子浓度的影响，并用荧光探针DCFH-DA检测细胞内ROS的生成，验证Pathway分析。构建地塞米松诱导CYP450酶的氰戊菊酯染毒大鼠模型，对CYP2C11及CYP3A1/23与氰戊菊酯所致的睾丸毒性进行关联性验证。结果： (1)现有剂量的氰戊菊酯未对大鼠的生长造成明显影响，但高剂量组(30mg/kg)大鼠肝脏脏器系数显著增高。各染毒组血清的BUN含量均显著高于对照组，血浆T-AOC、GSH的含量在2.5mg/kg和30mg/kg组显著下降，而MDA的含量在各染毒组均显著高于对照组。 (2)随着染毒剂量的增加，血液和尿液中的氰戊菊酯的浓度也逐步增加，且相对于血液而言，尿液中氰戊菊酯浓度较低。 (3)氰戊菊酯显著诱导了CYP2C11和CYP3A1/23的mRNA表达，且对CYP2C11的诱导效应比CYP3A1/23更强。 (4)肝脏病理显示高剂量组出现明显的肝脂肪变性，2.5mg/kg染毒组肝脏细胞出现轻微水肿，而0.00625和0.125mg/kg染毒组未见明显病理改变。基因芯片质检及荧光定量PCR验证结果显示芯片数据可靠性较好,芯片结果发现CYP2C11和CYP3A1表达上调。 (5)经Pathway Studio分析发现氰戊菊酯诱导了细胞内Ca~(2+)过载，通过Ca~(2+)信号通路和MAPK信号通路，引起肝脏氧化应激损伤，糖类、谷胱甘肽脂质代谢紊乱,进而导致肝损伤。 (6)分别采用10μM和20μM的氰戊菊酯24h染毒大鼠肝细胞株BRL后，细胞内的钙离子浓度呈剂量依赖性升高。当加入细胞内钙离子螯合剂BAPTA联合处理后，细胞内钙离子浓度与氰戊菊酯单独处理相比显著降低，而当活性氧抑制剂CAT和氰戊菊酯处理后，与氰戊菊酯单独处理组相比，细胞荧光强度没有明显下降。同时氰戊菊酯染毒BRL细胞发现，细胞内ROS显著升高。 (7)血清激素检测发现，T3浓度从0.125mg/kg剂量组开始显著升高，血清睾酮水平呈现剂量依赖性升高，在2.5mg/kg剂量下就出现统计学差异，染毒组其它血清激素与对照组相比没有明显区别。各染毒组精子的运动能力与对照组相比差异不明显，精子数量呈剂量依赖关系的减少，2.5和30mg/kg剂量下呈现统计学差异。 (8)地塞米松预处理发现，其显著增强了氰戊菊酯诱导CYP2C11蛋白的表达，同时血清睾酮的浓度和精子计数显著下降。随CYP2C11表达的升高，睾丸的病理损伤加重，表现为管腔内生精上皮层数减少，细胞排列紊乱，空泡化严重。结论： (1)氰戊菊酯引起了大鼠抗氧化能力的降低，可能与其诱导了CYP2C11和CYP3A1/23表达的升高，加快了对氰戊菊酯的代谢，产生过多的氧自由基有关。 (2)氰戊菊酯引发了肝脏的氧化应激反应和肝细胞内钙离子浓度增加，激活了钙离子信号通路，促使了肝细胞损伤，造成糖类以及脂类的代谢紊乱。 (3)氰戊菊酯破坏了生殖内分泌激素的平衡，造成了精子数量的减少。结合地塞米松诱导模型，发现其生殖毒性可能与CYP2C11诱导表达所致的活性代谢产物的增加有关。
[Abstract]:Objective: To explore the role of CYP450 enzyme in the toxic effect of fenvalerate in liver and testis from the whole animal level through the exposure model of fenvalerate male SD rat, and provide a new clue for the study of the toxicity and mechanism of fenvalerate pesticide.
Methods: 50 rats were randomly divided into 1 solvent control groups and 4 fenvalerate group (0.00625,0.125,2.5,30mg/kg.bw). Each group had 10 rats in each group for 8 weeks. After the treatment, the organ coefficient, serum biochemical (ALT, TP, BUN, CHOL) as well as the plasma T-AOC, GSH, MDA and other general toxicity indexes were evaluated and radioactivity was used. The serum levels of T_3, T_4, TSH, T, E_2, FSH, LH and other hormones were detected by immunoassay. The effects of fenvalerate on sperm motility and sperm quantity in rats were analyzed by CASA instrument. The residues of fenvalerate in blood and urine of rats were detected by GC-MS, and the induction effect of Fenvalerate on CYP2C11 and CYP3A1/23 was analyzed by fluorescence quantitative PCR. The liver was organized. Pathological examination, the liver RNA in the 0.125mg/kg group and the solvent control group was extracted from each group of 3. After cDNA, the gene expression profiles were analyzed, the differential genes were screened, the induced CYP450 related genes were verified, the differential genes were classified by GO and Pathway analysis, and the fluorescence quantitative PCR was used to test the calcium pathway related genes. The effect of fenvalerate on intracellular calcium concentration was observed with calcium ion probe Fluo-3AM, and the effect of fenvalerate on intracellular calcium concentration was observed. The formation of intracellular ROS was detected by fluorescence probe DCFH-DA, and Pathway analysis was verified. The rat model of fenvalerate induced by dexamethasone induced CYP450 enzyme was constructed, and CYP2C11 and CYP3A1/23 and fenvalerate were caused by the induced CYP450 enzyme. The toxicity of the testis was verified by association.
Result:
(1) the current dose of fenvalerate did not significantly affect the growth of rats, but the liver organ coefficient of the high dose group (30mg/kg) increased significantly. The content of BUN in the serum of each group was significantly higher than that of the control group. The content of T-AOC and GSH in the plasma was significantly decreased in the 2.5mg/kg and 30mg/kg groups, while the content of MDA in each group was significantly higher than that of the control group. Group.
(2) The concentration of fenvalerate in blood and urine increased gradually with the increase of the dose of fenvalerate, and the concentration of fenvalerate in urine was lower than that in blood.
(3) Fenvalerate significantly induced the mRNA expression of CYP2C11 and CYP3A1/23, and the induction effect on CYP2C11 was stronger than that on CYP3A1/23.
(4) liver pathology showed obvious hepatic steatosis in the high dose group. The liver cells in the 2.5mg/kg group showed slight edema, but there was no obvious pathological changes in 0.00625 and 0.125mg/kg. The chip data and fluorescence quantitative PCR showed that the reliability of the chip data was better, and the expression of CYP2C11 and CYP3A1 was up regulated by the results of the chip.
(5) the results of Pathway Studio analysis showed that fenvalerate induced Ca~ (2+) overload in cells, induced oxidative stress in the liver through Ca~ (2+) signaling pathway and MAPK signaling pathway, and the metabolic disorder of carbohydrates and glutathione lipid, which led to liver damage.
(6) the intracellular calcium concentration increased in a dose-dependent manner after 10 and 20 micron M and 20 micron of fenvalerate 24h in rat liver cell line. When combined with intracellular calcium ion chelating agent BAPTA, the intracellular calcium concentration was significantly lower than that of fenvalerate alone, while the active oxygen inhibitor CAT and fenvalerate were compared. The fluorescence intensity of BRL cells treated with fenvalerate did not decrease significantly compared with that treated with fenvalerate alone.
(7) serum hormone detection showed that the concentration of T3 increased significantly from the 0.125mg/kg dose group, the serum testosterone level showed a dose-dependent increase, and there was a statistical difference at the dose of 2.5mg/kg. The other serum hormones in the infected group were not significantly different from those of the control group. The number of spermatozoa decreased in a dose-dependent manner, with a statistical difference between 2.5 and 30 mg/kg.
(8) dexamethasone preconditioning significantly enhanced the expression of CYP2C11 protein induced by fenvalerate, while the concentration of serum testosterone and the sperm count decreased significantly. With the increase of CYP2C11 expression, the pathological damage of the testicles was aggravated, which showed that the number of endotheli was reduced, the cell arrangement was disorderly, and the vacuolization was serious.
Conclusion:
(1) fenvalerate causes the decrease of antioxidant capacity in rats, which may induce the increase of CYP2C11 and CYP3A1/23 expression, accelerate the metabolism of fenvalerate and produce excessive oxygen free radicals.
(2) fenvalerate triggered oxidative stress in the liver and increased calcium concentration in the liver cells, activating the calcium signal pathway, causing liver cell damage and causing metabolic disorders of carbohydrates and lipids.
(3) fenvalerate disrupted the balance of reproductive endocrine hormones and resulted in a decrease in the number of sperm. Combined with the dexamethasone induced model, it was found that its reproductive toxicity may be related to the increase of active metabolites induced by CYP2C11 induced expression.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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