【摘要】：目的：通過氰戊菊酯雄性SD大鼠染毒模型，從整體動(dòng)物水平探討了CYP450酶在氰戊菊酯所致肝臟和睪丸毒效應(yīng)中的作用，為氰戊菊酯農(nóng)藥毒性及機(jī)制研究提供新的線索。 方法：將50只大鼠隨機(jī)分為1個(gè)溶劑對(duì)照組和4個(gè)氰戊菊酯染毒組(0.00625、0.125、2.5、30mg/kg.bw)，每組10只，連續(xù)灌胃染毒8周。染毒結(jié)束后，對(duì)其各個(gè)臟器的臟器系數(shù)、血清生化（ALT、TP、BUN、CHOL）以及血漿T-AOC、GSH、MDA等一般毒性指標(biāo)進(jìn)行評(píng)價(jià)，采用放射免疫法檢測大鼠血清T_3、T_4、TSH、T、E_2、FSH、LH等激素水平，CASA儀分析氰戊菊酯對(duì)大鼠精子運(yùn)動(dòng)能力和精子數(shù)量的影響，采用GC-MS檢測大鼠血和尿液中氰戊菊酯的殘留量，熒光定量PCR分析氰戊菊酯對(duì)CYP2C11和CYP3A1/23的誘導(dǎo)效應(yīng)。對(duì)肝臟進(jìn)行組織病理學(xué)檢查，提取0.125mg/kg染毒組和溶劑對(duì)照組的肝臟RNA，每組3只，逆轉(zhuǎn)錄為cDNA后進(jìn)行基因表達(dá)譜分析，篩選差異基因，驗(yàn)證所誘導(dǎo)的CYP450酶相關(guān)基因，，對(duì)差異基因進(jìn)行GO分類和Pathway分析，并采用熒光定量PCR對(duì)鈣離子通路相關(guān)基因進(jìn)行驗(yàn)證。采用鈣離子探針Fluo-3AM標(biāo)記BRL細(xì)胞內(nèi)鈣離子，觀察氰戊菊酯對(duì)細(xì)胞內(nèi)鈣離子濃度的影響，并用熒光探針DCFH-DA檢測細(xì)胞內(nèi)ROS的生成，驗(yàn)證Pathway分析。構(gòu)建地塞米松誘導(dǎo)CYP450酶的氰戊菊酯染毒大鼠模型，對(duì)CYP2C11及CYP3A1/23與氰戊菊酯所致的睪丸毒性進(jìn)行關(guān)聯(lián)性驗(yàn)證。 結(jié)果： (1)現(xiàn)有劑量的氰戊菊酯未對(duì)大鼠的生長造成明顯影響，但高劑量組(30mg/kg)大鼠肝臟臟器系數(shù)顯著增高。各染毒組血清的BUN含量均顯著高于對(duì)照組，血漿T-AOC、GSH的含量在2.5mg/kg和30mg/kg組顯著下降，而MDA的含量在各染毒組均顯著高于對(duì)照組。 (2)隨著染毒劑量的增加，血液和尿液中的氰戊菊酯的濃度也逐步增加，且相對(duì)于血液而言，尿液中氰戊菊酯濃度較低。 (3)氰戊菊酯顯著誘導(dǎo)了CYP2C11和CYP3A1/23的mRNA表達(dá)，且對(duì)CYP2C11的誘導(dǎo)效應(yīng)比CYP3A1/23更強(qiáng)。 (4)肝臟病理顯示高劑量組出現(xiàn)明顯的肝脂肪變性，2.5mg/kg染毒組肝臟細(xì)胞出現(xiàn)輕微水腫，而0.00625和0.125mg/kg染毒組未見明顯病理改變�；蛐酒|(zhì)檢及熒光定量PCR驗(yàn)證結(jié)果顯示芯片數(shù)據(jù)可靠性較好,芯片結(jié)果發(fā)現(xiàn)CYP2C11和CYP3A1表達(dá)上調(diào)。 (5)經(jīng)Pathway Studio分析發(fā)現(xiàn)氰戊菊酯誘導(dǎo)了細(xì)胞內(nèi)Ca~(2+)過載，通過Ca~(2+)信號(hào)通路和MAPK信號(hào)通路，引起肝臟氧化應(yīng)激損傷，糖類、谷胱甘肽脂質(zhì)代謝紊亂,進(jìn)而導(dǎo)致肝損傷。 (6)分別采用10μM和20μM的氰戊菊酯24h染毒大鼠肝細(xì)胞株BRL后，細(xì)胞內(nèi)的鈣離子濃度呈劑量依賴性升高。當(dāng)加入細(xì)胞內(nèi)鈣離子螯合劑BAPTA聯(lián)合處理后，細(xì)胞內(nèi)鈣離子濃度與氰戊菊酯單獨(dú)處理相比顯著降低，而當(dāng)活性氧抑制劑CAT和氰戊菊酯處理后，與氰戊菊酯單獨(dú)處理組相比，細(xì)胞熒光強(qiáng)度沒有明顯下降。同時(shí)氰戊菊酯染毒BRL細(xì)胞發(fā)現(xiàn)，細(xì)胞內(nèi)ROS顯著升高。 (7)血清激素檢測發(fā)現(xiàn)，T3濃度從0.125mg/kg劑量組開始顯著升高，血清睪酮水平呈現(xiàn)劑量依賴性升高，在2.5mg/kg劑量下就出現(xiàn)統(tǒng)計(jì)學(xué)差異，染毒組其它血清激素與對(duì)照組相比沒有明顯區(qū)別。各染毒組精子的運(yùn)動(dòng)能力與對(duì)照組相比差異不明顯，精子數(shù)量呈劑量依賴關(guān)系的減少，2.5和30mg/kg劑量下呈現(xiàn)統(tǒng)計(jì)學(xué)差異。 (8)地塞米松預(yù)處理發(fā)現(xiàn)，其顯著增強(qiáng)了氰戊菊酯誘導(dǎo)CYP2C11蛋白的表達(dá)，同時(shí)血清睪酮的濃度和精子計(jì)數(shù)顯著下降。隨CYP2C11表達(dá)的升高，睪丸的病理損傷加重，表現(xiàn)為管腔內(nèi)生精上皮層數(shù)減少，細(xì)胞排列紊亂，空泡化嚴(yán)重。 結(jié)論： (1)氰戊菊酯引起了大鼠抗氧化能力的降低，可能與其誘導(dǎo)了CYP2C11和CYP3A1/23表達(dá)的升高，加快了對(duì)氰戊菊酯的代謝，產(chǎn)生過多的氧自由基有關(guān)。 (2)氰戊菊酯引發(fā)了肝臟的氧化應(yīng)激反應(yīng)和肝細(xì)胞內(nèi)鈣離子濃度增加，激活了鈣離子信號(hào)通路，促使了肝細(xì)胞損傷，造成糖類以及脂類的代謝紊亂。 (3)氰戊菊酯破壞了生殖內(nèi)分泌激素的平衡，造成了精子數(shù)量的減少。結(jié)合地塞米松誘導(dǎo)模型，發(fā)現(xiàn)其生殖毒性可能與CYP2C11誘導(dǎo)表達(dá)所致的活性代謝產(chǎn)物的增加有關(guān)。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

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