氧化应激在氰戊菊酯、辛硫磷卵巢毒性中的作用
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摘要
第一部分 氰戊菊酯、辛硫磷对大鼠卵巢功能影响的整体实验研究
研究氰戊菊酯(Fen)、辛硫磷(Pho)对大鼠卵巢内分泌干扰作用及抗氧化系统功能的影响。应用Fen、Pho对雌性SD大鼠进行经口染毒，剂量分别为1.91、9.55、31.80mg／kg和5.88、29.4、98mg／kg，对照组给予等量色拉油。阴道脱落细胞涂片法观察动情周期变化。连续染毒30d后处死，分离卵巢、子宫、脑、肝、脾、肾、肾上腺等脏器，计算脏器系数；分光光度法测定大鼠卵巢组织和血清中的超氧化物歧化酶(SOD)、谷胱甘肽硫转移酶(GST)的活性以及丙二醛(MDA)、谷胱甘肽(GSH)的含量；放射免疫法测定血清中雌二醇
南京医科大学硕士学位论文
(E:)和孕酮(P;)的水平;提取卵巢组织的DNA，进行琼脂糖凝
胶电泳，观察DNA完整性;免疫组化SP法观察bc人2蛋白在卵巢组
织中的表达;运用光镜和电镜观察卵巢组织和细胞超微结构的变化。
结果显示:Fen各染毒组大鼠的动情前期和动情后期缩短，与对
照组相比，在1.91mg瓜g和31.80mg/kg组具有显著性差异;动情期和
动情间期则延长，31.80mg/kg组的动情间期持续时间明显比对照组
短;各组动情周期异常率均高于对照组，在31 .80m眺g组差异具有
显著性。各染毒组大鼠的卵巢、子宫重量均降低，在31.80m眺g组
具有显著性差异;卵巢SOD、GST和GSH水平均增高，其中各剂量
组GST活力均显著增强，MDA含量降低;血清中SOD、GST水平
降低，MDA含量升高;血清中EZ含量显著高于对照组，P4含量则逐
渐下降，在9.55mg/kg和31 .80m眺g组与对照组相比差异具有显著性;
卵巢bcl一2蛋白表达增强，在9.55m留kg组差异具有显著性。Pho各
染毒组大鼠的各动情周期未见明显变化，但动情周期异常率均高于对
照组，在98mg瓜g组差异具有显著性;体重增长均低于对照组，其中
98mg/kg组与对照组相比差异具有显著性;各染毒组大鼠的卵巢、子
宫重量均显著降低;卵巢SOD、GST和GSH水平均增高，MDA含
量降低，血清中SOD、GST水平显著降低，MDA含量升高;各剂量
组E:含量降低，P;含量升高，但与对照组相比差异均无显著性;卵
巢bcl一2蛋白表达增强，在5.88mg瓜g组显著具有显著性。Fen、Pho
各剂量组的大鼠卵巢组织DNA完整、无凋亡样断裂。光镜下观察，
Fen、Pho各组闭锁卵泡增多，但与对照组相比差异均无显著性。电
南京医科大学硕万}学位论文
镜下观察:在Fen 31 .80mg/kg组可见卵巢黄体细胞部分内质网肿胀;
线粒体呈空泡状，峪消失;核仁边聚。Pho 29.4m留kg组卵巢黄体细
胞胞浆内脂滴增大、
质不均，峪消失。
以上结果提示:
增多;
在31.
核膜不完整;线粒体扩张，出现空泡，基
80mg/kg剂量下，Fen对大鼠的动情周期和
激素分泌有明显的干扰作用，而Pho的作用不明显。在1.91、9.55、
31.somg/kg剂量和5.88、29.4和98mg/kg剂量下，Fen、pho均能使
卵巢的抗氧化能力代偿性增强，Pho的作用更为明显，但尚未对卵巢
的抗氧化系统功能造成损伤。
关健词:氰戊菊醋;辛硫磷;大鼠;卵巢;动情周期;bcl-2
第二部分氛戊菊醋、辛硫磷对大鼠卵粱颖拉细胞线拉体功能的影响
研究Fen、Pho对卵巢颗粒细胞线粒体功能的影响，为探讨其内
分泌干扰作用机制提供依据。原代培养大鼠卵巢颗粒细胞(rGCS)，
分别用Feno、5、25、1 25协mol/L或PhoO、5、25、1 25林mol/L处
理细胞24h。MTT试验检测线粒体酶活力;二乙酞二氯氢化荧光素
(DCFH一DA)负载，流式细胞术检测细胞内ROS水平;碘布七丙唆(PI)
和罗丹明123(Rh123)双标记，流式细胞术检测细胞活力和线粒体
膜电位(MMP);生物发光法检测细胞内ATP水平。
结果显示:Fen引起细胞内ROS水平升高，25和1 25月mol几与
对照组相比差异具有显著性;PI的荧光强度下降，细胞活力降低，25
南京医科人学硕士学位论文
似mol几和125尽mol几组与对照组相比差异具有显著性;细胞损伤率
逐渐升高，与对照组比较，25、125拼mol/L组差异具有显著性意义;
反映线粒体酶活性的MTT OD值显著升高，5、25、1 25尽mol几组与
对照组相比，差异均具有显著性;反映线粒体膜电位的Rh123荧光
强度比对照组明显升高;细胞ATP水平呈明显的下降趋势，各剂量
组与对照组相比差异均具有显著性。Pho染毒后，细胞内ROS产生
增加，其中5林mol几组明显高于对照组;PI荧光强度升高，在125
月mol几组与对照组相比差异具有显著性。细胞损伤率逐渐升高，与
对照组相比，125林mol几组差异具有显著性。MTT OD值升高，但
各剂量组与对照组相比，差异均未见显著性。Rh123荧光强度上升，
但与对照组相比，各剂量组差异均未见显著性。与对照组相比，各剂
量组的细胞ATP水平随染毒剂量降低，但差异均未见显著性。
以上结果提示:Fen、 Pho能引起大鼠卵巢颗粒细胞的损伤，表
现为细胞损伤率增加，ATP水平下降。但Fen并非通过影响线粒体功
能而损伤细胞，Fen使细胞活力增强，促进线粒体功能，使线粒体膜
电位增加，线粒体酶活性增强;Pho对线粒体功能作用不明显。
Part I In Vivo Study on the Effects of Fenvalerate and Phoxim on
the Function of Rat Ovary
Objective To investigate the effects of fenvalerate (Fen) and Pho (Pho) on the ovarian antioxidant function.
Methods Fen and Pho were administrated ig to the adult female Sprague Dawley rats at dose of 1.91, 9.55, 31.80mg/kg and 5.88, 29.4, 98 mg/kg respectively for one month. Vaginal smears of rats were performed to determine estrous cycle. Some organs such as ovary, uterus, brain, liver,
spleen, kidney and adrenal were dissected and organ coefficients were calculated. The activities of superoxide dismutase (SOD) and glutathione-s-transferase (GST), and the levels of malondialdehyde (MDA) and glutathione (GSH) in the ovary and serum were measured by photometry. The estradiol (E) and progesterone (P) in the serum were determined by radioimmunoassay (RIA). Total DNA was extracted from ovary and the DNA fragmentation was valued by agarose gel electrophoresis. The expression of bcl-2 protein in the ovary was observed with SP immunohistochemical method. The morphological changes and ultrastructure of ovary were examined by the light and electronic microscopy.
Results In the Fen group, the rate of estrous cylce abnormality significantly increased and the gain of body weight was obviously lower in 31.80mg/kg group than that of control. Proestrus and metestrus prolonged significantly in 1.91mg/kg and 31.80mg/kg group than that of the control and the duration of estrus and diestrus was reduced. The relative organ weights (ovary, uterus) were declined in a dose dependent manner, which was markedly reduced at the dose of 31.80mg/kg. Fen increased the levels of SOD and GSH in ovary and decreased the content of MDA. The levels of GST declined obviously. On the contrary, the SOD and GST activities in the serum of exposed rats were reduced while the content of MDA was enhanced. The synthesis of E was stimulated
-6-
significantly by Fen in serum at doses of 9.55 and 31.80mg/kg while the level of P was declined obviously. The expression of bcl-2 protein in ovary was up-regulated by Fen in 9.55mg/kg group. Pho increased the rate of estrous cycle abnormality significantly and reduced the gain of body weight obviously in 98mg/kg group than that of control. No any obvious change was observed in the duration of each estrous cycle in the exposed groups. Meanwhile, the ovary and uterus weights were declined in a dose dependent manner. Pho increased the levels of SOD, GST and GSH in ovary and decreased the content of MDA. On the contrary, the SOD and GST activities in the serum of exposed rats had been markedly reduced than that of the control while the contents of MDA was enhanced. No any significant effect of E and P was observed in Pho Treated group. The expression of bcl-2 protein in ovary was increased in low dose group. The morphological changes under the light and electronic microscopy examination indicated that the amount of atrestic follicle in the Fen and Pho exposed groups was increased, but there is no obvious alteration in atrestic follicle of treated group as compared with the control. The ultrastructure changes of ovary under the electronic microscopy indicated that parts of endoplasmic reticulum expanded, mitochondria vacuolized and cristae lost in the Fen group. Nucleolus showed margin assemblling. The morphological alterations in Pho group showed the increase and enlargement of the lipid dropletand the loss of
-7-
integrity of the nuclear membrane. Mictochondrial swelled and cristae
disappeared, and the matrix showed asymmetry.
Conclusion The results suggest that Fen at the doses of 1.91, 9.55, and 31.80mg/kg could significantly disrupt the estrous cycle and secretion of hormone, while Pho at the doses of 5.88, 29.4, 98 mg/kg had not obvious effect as Fen did. Fen and Pho could induce the compensatory increase of antioxidant capacity and the action of Pho was more notable, yet they could not result in a significant damage to the antioxidant system of rat ovary.
Key words fenvalerate; phoxim; rat; ovary; estrous
研究氰戊菊酯(Fen)、辛硫磷(Pho)对大鼠卵巢内分泌干扰作用及抗氧化系统功能的影响。应用Fen、Pho对雌性SD大鼠进行经口染毒，剂量分别为1.91、9.55、31.80mg／kg和5.88、29.4、98mg／kg，对照组给予等量色拉油。阴道脱落细胞涂片法观察动情周期变化。连续染毒30d后处死，分离卵巢、子宫、脑、肝、脾、肾、肾上腺等脏器，计算脏器系数；分光光度法测定大鼠卵巢组织和血清中的超氧化物歧化酶(SOD)、谷胱甘肽硫转移酶(GST)的活性以及丙二醛(MDA)、谷胱甘肽(GSH)的含量；放射免疫法测定血清中雌二醇
南京医科大学硕士学位论文
(E:)和孕酮(P;)的水平;提取卵巢组织的DNA，进行琼脂糖凝
胶电泳，观察DNA完整性;免疫组化SP法观察bc人2蛋白在卵巢组
织中的表达;运用光镜和电镜观察卵巢组织和细胞超微结构的变化。
结果显示:Fen各染毒组大鼠的动情前期和动情后期缩短，与对
照组相比，在1.91mg瓜g和31.80mg/kg组具有显著性差异;动情期和
动情间期则延长，31.80mg/kg组的动情间期持续时间明显比对照组
短;各组动情周期异常率均高于对照组，在31 .80m眺g组差异具有
显著性。各染毒组大鼠的卵巢、子宫重量均降低，在31.80m眺g组
具有显著性差异;卵巢SOD、GST和GSH水平均增高，其中各剂量
组GST活力均显著增强，MDA含量降低;血清中SOD、GST水平
降低，MDA含量升高;血清中EZ含量显著高于对照组，P4含量则逐
渐下降，在9.55mg/kg和31 .80m眺g组与对照组相比差异具有显著性;
卵巢bcl一2蛋白表达增强，在9.55m留kg组差异具有显著性。Pho各
染毒组大鼠的各动情周期未见明显变化，但动情周期异常率均高于对
照组，在98mg瓜g组差异具有显著性;体重增长均低于对照组，其中
98mg/kg组与对照组相比差异具有显著性;各染毒组大鼠的卵巢、子
宫重量均显著降低;卵巢SOD、GST和GSH水平均增高，MDA含
量降低，血清中SOD、GST水平显著降低，MDA含量升高;各剂量
组E:含量降低，P;含量升高，但与对照组相比差异均无显著性;卵
巢bcl一2蛋白表达增强，在5.88mg瓜g组显著具有显著性。Fen、Pho
各剂量组的大鼠卵巢组织DNA完整、无凋亡样断裂。光镜下观察，
Fen、Pho各组闭锁卵泡增多，但与对照组相比差异均无显著性。电
南京医科大学硕万}学位论文
镜下观察:在Fen 31 .80mg/kg组可见卵巢黄体细胞部分内质网肿胀;
线粒体呈空泡状，峪消失;核仁边聚。Pho 29.4m留kg组卵巢黄体细
胞胞浆内脂滴增大、
质不均，峪消失。
以上结果提示:
增多;
在31.
核膜不完整;线粒体扩张，出现空泡，基
80mg/kg剂量下，Fen对大鼠的动情周期和
激素分泌有明显的干扰作用，而Pho的作用不明显。在1.91、9.55、
31.somg/kg剂量和5.88、29.4和98mg/kg剂量下，Fen、pho均能使
卵巢的抗氧化能力代偿性增强，Pho的作用更为明显，但尚未对卵巢
的抗氧化系统功能造成损伤。
关健词:氰戊菊醋;辛硫磷;大鼠;卵巢;动情周期;bcl-2
第二部分氛戊菊醋、辛硫磷对大鼠卵粱颖拉细胞线拉体功能的影响
研究Fen、Pho对卵巢颗粒细胞线粒体功能的影响，为探讨其内
分泌干扰作用机制提供依据。原代培养大鼠卵巢颗粒细胞(rGCS)，
分别用Feno、5、25、1 25协mol/L或PhoO、5、25、1 25林mol/L处
理细胞24h。MTT试验检测线粒体酶活力;二乙酞二氯氢化荧光素
(DCFH一DA)负载，流式细胞术检测细胞内ROS水平;碘布七丙唆(PI)
和罗丹明123(Rh123)双标记，流式细胞术检测细胞活力和线粒体
膜电位(MMP);生物发光法检测细胞内ATP水平。
结果显示:Fen引起细胞内ROS水平升高，25和1 25月mol几与
对照组相比差异具有显著性;PI的荧光强度下降，细胞活力降低，25
南京医科人学硕士学位论文
似mol几和125尽mol几组与对照组相比差异具有显著性;细胞损伤率
逐渐升高，与对照组比较，25、125拼mol/L组差异具有显著性意义;
反映线粒体酶活性的MTT OD值显著升高，5、25、1 25尽mol几组与
对照组相比，差异均具有显著性;反映线粒体膜电位的Rh123荧光
强度比对照组明显升高;细胞ATP水平呈明显的下降趋势，各剂量
组与对照组相比差异均具有显著性。Pho染毒后，细胞内ROS产生
增加，其中5林mol几组明显高于对照组;PI荧光强度升高，在125
月mol几组与对照组相比差异具有显著性。细胞损伤率逐渐升高，与
对照组相比，125林mol几组差异具有显著性。MTT OD值升高，但
各剂量组与对照组相比，差异均未见显著性。Rh123荧光强度上升，
但与对照组相比，各剂量组差异均未见显著性。与对照组相比，各剂
量组的细胞ATP水平随染毒剂量降低，但差异均未见显著性。
以上结果提示:Fen、 Pho能引起大鼠卵巢颗粒细胞的损伤，表
现为细胞损伤率增加，ATP水平下降。但Fen并非通过影响线粒体功
能而损伤细胞，Fen使细胞活力增强，促进线粒体功能，使线粒体膜
电位增加，线粒体酶活性增强;Pho对线粒体功能作用不明显。
Part I In Vivo Study on the Effects of Fenvalerate and Phoxim on
the Function of Rat Ovary
Objective To investigate the effects of fenvalerate (Fen) and Pho (Pho) on the ovarian antioxidant function.
Methods Fen and Pho were administrated ig to the adult female Sprague Dawley rats at dose of 1.91, 9.55, 31.80mg/kg and 5.88, 29.4, 98 mg/kg respectively for one month. Vaginal smears of rats were performed to determine estrous cycle. Some organs such as ovary, uterus, brain, liver,
spleen, kidney and adrenal were dissected and organ coefficients were calculated. The activities of superoxide dismutase (SOD) and glutathione-s-transferase (GST), and the levels of malondialdehyde (MDA) and glutathione (GSH) in the ovary and serum were measured by photometry. The estradiol (E) and progesterone (P) in the serum were determined by radioimmunoassay (RIA). Total DNA was extracted from ovary and the DNA fragmentation was valued by agarose gel electrophoresis. The expression of bcl-2 protein in the ovary was observed with SP immunohistochemical method. The morphological changes and ultrastructure of ovary were examined by the light and electronic microscopy.
Results In the Fen group, the rate of estrous cylce abnormality significantly increased and the gain of body weight was obviously lower in 31.80mg/kg group than that of control. Proestrus and metestrus prolonged significantly in 1.91mg/kg and 31.80mg/kg group than that of the control and the duration of estrus and diestrus was reduced. The relative organ weights (ovary, uterus) were declined in a dose dependent manner, which was markedly reduced at the dose of 31.80mg/kg. Fen increased the levels of SOD and GSH in ovary and decreased the content of MDA. The levels of GST declined obviously. On the contrary, the SOD and GST activities in the serum of exposed rats were reduced while the content of MDA was enhanced. The synthesis of E was stimulated
-6-
significantly by Fen in serum at doses of 9.55 and 31.80mg/kg while the level of P was declined obviously. The expression of bcl-2 protein in ovary was up-regulated by Fen in 9.55mg/kg group. Pho increased the rate of estrous cycle abnormality significantly and reduced the gain of body weight obviously in 98mg/kg group than that of control. No any obvious change was observed in the duration of each estrous cycle in the exposed groups. Meanwhile, the ovary and uterus weights were declined in a dose dependent manner. Pho increased the levels of SOD, GST and GSH in ovary and decreased the content of MDA. On the contrary, the SOD and GST activities in the serum of exposed rats had been markedly reduced than that of the control while the contents of MDA was enhanced. No any significant effect of E and P was observed in Pho Treated group. The expression of bcl-2 protein in ovary was increased in low dose group. The morphological changes under the light and electronic microscopy examination indicated that the amount of atrestic follicle in the Fen and Pho exposed groups was increased, but there is no obvious alteration in atrestic follicle of treated group as compared with the control. The ultrastructure changes of ovary under the electronic microscopy indicated that parts of endoplasmic reticulum expanded, mitochondria vacuolized and cristae lost in the Fen group. Nucleolus showed margin assemblling. The morphological alterations in Pho group showed the increase and enlargement of the lipid dropletand the loss of
-7-
integrity of the nuclear membrane. Mictochondrial swelled and cristae
disappeared, and the matrix showed asymmetry.
Conclusion The results suggest that Fen at the doses of 1.91, 9.55, and 31.80mg/kg could significantly disrupt the estrous cycle and secretion of hormone, while Pho at the doses of 5.88, 29.4, 98 mg/kg had not obvious effect as Fen did. Fen and Pho could induce the compensatory increase of antioxidant capacity and the action of Pho was more notable, yet they could not result in a significant damage to the antioxidant system of rat ovary.
Key words fenvalerate; phoxim; rat; ovary; estrous
引文
1. WHO: Environmental Health Criteria 95: Fenvaerate. Whorld Health Organization, Geneva 1990.
2. Data Sheets on Pesticidies, No. 31, Phoxim. WHO/FAO, 1990.
3. Kelly SA, Havrilla CM, Brady TC, et al. Oxidative stress in toxicology:established mammalian and emerging piscine model systems. Environ Health Perspect, 1998, 106(7): 375-384.
4. Findel T, Holbrook NJ. Oxidants, oxidative stress and the bilolgy of aging. Nature, 2000, 408: 239-247.
5．任春兰，高坚瑞，股若元等．氰戊菊酯与甲苯对小鼠脑细胞氧化性损伤作用的研究．卫生研究，1995，24(1)：12-14．
6．易宗春，徐汉宫，蔡红梅等．拟除虫菊酯对大鼠脑组织及肝脏生物转化酶的影响．中华劳动卫生职业病杂志，2000，18(4)：216-219．
7．牛玉杰，石年，吴爱国等．溴氰菊酯对大鼠中枢神经系统一氧化氮合成酶活性的影响．卫生毒理学杂志，1997，11(2)：75-77．
8. Giray B,Grubay, Hincal F. Cypermethrin-induced oxidative stress in rat brain and liver is prevented by vitamin E or allopurinol. ToxicoI Lett,2001, 118(3): 139-146.
9. Kale M, Rathore N, John S, Bhatnagar D. Lipid peroxidative damage on pyrethroid exposure and alterations in antioxidant status in rat erythrocytes: a possible involvement of reactive oxygen species. Toxicol Lett, 1999, 105(3):197-205.
10. Gupta A, Agarwal R, Shukla GS. Functional impairment of
blood-brain barrier following pesticide exposure during early development in rats. Hum Exp Toxicol, 1999, 18(3): 174-179.
11. Rao RP, Kaliwal BB. Monocrotophos Induced dysfunction on estrous cycle and follicular development in mic [J]. Ind Health, 2002, 40:237-244.
12. Dhondup P, Kaliwal BB. Inhibition of ovarian compensatory hypertrophy by the administration of methyl parathion in hemicastrated albino rats. Eprod Toxicol, 1997.11(1): 77-84.
13. Atef M, Youssef SA, Ramadan A, et al. Influence of Phoxim on testicular and seminal vesicle organs,testosterone and cholinesterase level and its tissue residues in male rats [J]. Dtsth Tierarztl Wochemschr, 1995,102: 301-305.
14．詹宁育，王沭沂，王心如．辛硫磷对大鼠精子生产量和精子运动能力的影响．卫生研究，2000，29(1)：4-6．
15．詹宁育，王心如，王沭沂，等．辛硫磷与氰戊菊酯对大鼠睾丸的联合毒性作用．中华劳动卫生职业病杂志，2001，19(4)：261-264．
16. Vandana SP, Hong Huang, Bdulla KS. Role of reactive oxygen metabolites in organophosphate-bidrin-induced renal tubular cytotoxicity.JAm Soc Nephrol, 1999, 10: 1746-1752.
17．何军山，卢新华．有机磷农药中毒及其解救对过氧化脂质、肾功能的影响．梆州医学高等专科学校校报，2002，4(1)：6-8．
18．方伟钧，李赵梅，杨建新等．急性有机磷农药口服中毒与自由基变化关系的初步探讨．中华急诊医学杂志，2002，11(4)：232-235．
19．张博，李光明，汤伯基，等．超氧化物歧化酶、过氧化脂质和谷胱
甘肽过氧化物酶变化与急性有机磷农药中毒的关系．标记免疫分析与临床，1996，3(1)：17-19．
20. Wen XD, Han MS, Yi S, et al. Microcystic Cyanobacteria Causes Mitochondrial Membrane Potential Alteration and Reactive Oxygen Species Formation in Primary Cultured Rat Hepatocytes. Environ Health Perspect, 1998, 106(7): 409-413.
21. Murdoch WJ. Inhibition by oestradiol of oxidative stress-induced apoptosis in pig ovarian tissues. Journal of Reproduction and Fertility,1998, 114: 127-130.
22. Seino T, Saito H, Kaneko T, et al. Eight-hydroxy-2'-deoxyguanosine in granulose cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program.Fertility and Sterility,2002, 77(6): 1184-1190.
23．曾晓，马彦，佟颖等．杀虫剂对家蝇线粒体膜脂流动性影响的研究．中国媒介生物学及控制杂志，1998，9(6)：429-435．
24. Gassner B, Wuthrich A, S choltysik G, Solioz M. The pyrethroids permethrin and cyhalothrin are potent inhibitors of the mitochondrial complex I . J Pharmacol Exp Ther, 1997, 281(2): 855-860.
25. Spetale MR, Morisoli LS, Rodriguez Garay EA. The effect of organophosphorus compounds on respiration by rat liver mitochondria.Farmaco Sci, 1977, 32(2): 116-132.
26. Sambrook J, Fritsch EF, ManiatisT著．金冬雁，黎孟枫，等译．分子克隆实验指南．第2版．北京：科学出版社，1998：463-469．
27．孙存普，张建中，段绍瑾．自由基生物学导论．合肥：中国科学技
术大学出版社，1999：150-156．
28．杨洁，高飞，易静．活性氧与细胞凋亡的研究进展．国外医学肿瘤学分册，2002，29(4)：248-251．
29．李莉，周兰，陈文缘．Bcl-2与细胞凋亡．国外医学临床生物化学与检验学分册，1999，20(6)：272-274．
30. Alexander RR, Sabine S, Karl MS, et al. A microplate assay for the detection of oxidative products using 2',7'-dichloroflurescin-diacetate. J Immunol Methods, 1985, 82: 253-261.
31．李楠，王凤翔，周春喜．荧光探针应用技术．北京：军事医学科学出版社，1998：62-65，84．
32. Enrenberg B, Montana V, Wei MD, et al. Membrane potential can be determined in individual cells from the Nernstian distribution of cationic dyes. Biophys J. 1998, 53: 785-794.
33. Rabn CA, Bombick DW, Doolittle DJ. Assessment of mitochondrial membrane potential as an indicator of cytotoxicity. Fundam Appl Toxicol, 1991,16: 435-440.
34. Mosmann T. Rapid colorimetric assay for cellular grouth and survival: application to prolification and cytotoxicity assay. J Immunol Methods, 1983, 65(1-2): 55-63.
35．陈国南，张帆．化学发光与生物发光．福州：福建科学技术出版社，1998：6-7，158-162．
36．聂松青．生物发光分析及其应用．生理科学进展，1984，15(3)：232-237．
37．戴定威，吴圣楣，李敏等．生化发光法测定体外培养小肠上皮细
胞内微量ATR．临床检验杂志，1997，15(5)：279-280．
38. Stanley PE, Williams SG. Use of the liquid scintillation spectrometer for determining adenosin triphosphate by the luciferase enzyme. Anal Biochem, 1969, 29(3): 381-92.
39. John DJ, Virginia FK, Samuel CS. Increased ATPand Creatine Phosphate Turnover in Pphagocytosing Mouse Peritoneal Macrophages. J Biol Chem, 1979, 254: 9558-9564.
40．张西蘅．生物化学．北京：北京医科大学，中国协和医科大学联合出版社，1994，207-226．
41. Donald N. Metabolic Minimap: Mitochondrial ATP Foramation. Biochem Mol Bio Edu, 2002, 30(1): 3-5.
42. Malaviya M, Husain R, Seth PK, et al. Perinatal effects of two pyrethroid insecticides on brain neurotransmitter function in the neonatal rat. Vet Hum Txoicol, 1993,35(2): 119-122.
43. Xiao C, He FS, Li OS, et al. Potental biochemical mechanisms of neuromuscular junction transmissiondysfunciton induced by organophosphorus insecticides. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 2003, 21(3): 191-193.
44．曾晓．杀虫剂对家蝇线粒体ATP酶活性影响的研究．中国媒介生物学及控制杂志，1995，4：
45．何俊，陈建峰，刘茹等．氰戊菊酯对卵巢细胞／组织钙稳态和激素水平的影响．中华预防医学杂志，2004，1：
46. Richer C, Schweizer M, Cossarizza A. Control of apoptosis by the cellular ATP level. FEB S Letter, 1996, 378: 107-110.
2. Data Sheets on Pesticidies, No. 31, Phoxim. WHO/FAO, 1990.
3. Kelly SA, Havrilla CM, Brady TC, et al. Oxidative stress in toxicology:established mammalian and emerging piscine model systems. Environ Health Perspect, 1998, 106(7): 375-384.
4. Findel T, Holbrook NJ. Oxidants, oxidative stress and the bilolgy of aging. Nature, 2000, 408: 239-247.
5．任春兰，高坚瑞，股若元等．氰戊菊酯与甲苯对小鼠脑细胞氧化性损伤作用的研究．卫生研究，1995，24(1)：12-14．
6．易宗春，徐汉宫，蔡红梅等．拟除虫菊酯对大鼠脑组织及肝脏生物转化酶的影响．中华劳动卫生职业病杂志，2000，18(4)：216-219．
7．牛玉杰，石年，吴爱国等．溴氰菊酯对大鼠中枢神经系统一氧化氮合成酶活性的影响．卫生毒理学杂志，1997，11(2)：75-77．
8. Giray B,Grubay, Hincal F. Cypermethrin-induced oxidative stress in rat brain and liver is prevented by vitamin E or allopurinol. ToxicoI Lett,2001, 118(3): 139-146.
9. Kale M, Rathore N, John S, Bhatnagar D. Lipid peroxidative damage on pyrethroid exposure and alterations in antioxidant status in rat erythrocytes: a possible involvement of reactive oxygen species. Toxicol Lett, 1999, 105(3):197-205.
10. Gupta A, Agarwal R, Shukla GS. Functional impairment of
blood-brain barrier following pesticide exposure during early development in rats. Hum Exp Toxicol, 1999, 18(3): 174-179.
11. Rao RP, Kaliwal BB. Monocrotophos Induced dysfunction on estrous cycle and follicular development in mic [J]. Ind Health, 2002, 40:237-244.
12. Dhondup P, Kaliwal BB. Inhibition of ovarian compensatory hypertrophy by the administration of methyl parathion in hemicastrated albino rats. Eprod Toxicol, 1997.11(1): 77-84.
13. Atef M, Youssef SA, Ramadan A, et al. Influence of Phoxim on testicular and seminal vesicle organs,testosterone and cholinesterase level and its tissue residues in male rats [J]. Dtsth Tierarztl Wochemschr, 1995,102: 301-305.
14．詹宁育，王沭沂，王心如．辛硫磷对大鼠精子生产量和精子运动能力的影响．卫生研究，2000，29(1)：4-6．
15．詹宁育，王心如，王沭沂，等．辛硫磷与氰戊菊酯对大鼠睾丸的联合毒性作用．中华劳动卫生职业病杂志，2001，19(4)：261-264．
16. Vandana SP, Hong Huang, Bdulla KS. Role of reactive oxygen metabolites in organophosphate-bidrin-induced renal tubular cytotoxicity.JAm Soc Nephrol, 1999, 10: 1746-1752.
17．何军山，卢新华．有机磷农药中毒及其解救对过氧化脂质、肾功能的影响．梆州医学高等专科学校校报，2002，4(1)：6-8．
18．方伟钧，李赵梅，杨建新等．急性有机磷农药口服中毒与自由基变化关系的初步探讨．中华急诊医学杂志，2002，11(4)：232-235．
19．张博，李光明，汤伯基，等．超氧化物歧化酶、过氧化脂质和谷胱
甘肽过氧化物酶变化与急性有机磷农药中毒的关系．标记免疫分析与临床，1996，3(1)：17-19．
20. Wen XD, Han MS, Yi S, et al. Microcystic Cyanobacteria Causes Mitochondrial Membrane Potential Alteration and Reactive Oxygen Species Formation in Primary Cultured Rat Hepatocytes. Environ Health Perspect, 1998, 106(7): 409-413.
21. Murdoch WJ. Inhibition by oestradiol of oxidative stress-induced apoptosis in pig ovarian tissues. Journal of Reproduction and Fertility,1998, 114: 127-130.
22. Seino T, Saito H, Kaneko T, et al. Eight-hydroxy-2'-deoxyguanosine in granulose cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program.Fertility and Sterility,2002, 77(6): 1184-1190.
23．曾晓，马彦，佟颖等．杀虫剂对家蝇线粒体膜脂流动性影响的研究．中国媒介生物学及控制杂志，1998，9(6)：429-435．
24. Gassner B, Wuthrich A, S choltysik G, Solioz M. The pyrethroids permethrin and cyhalothrin are potent inhibitors of the mitochondrial complex I . J Pharmacol Exp Ther, 1997, 281(2): 855-860.
25. Spetale MR, Morisoli LS, Rodriguez Garay EA. The effect of organophosphorus compounds on respiration by rat liver mitochondria.Farmaco Sci, 1977, 32(2): 116-132.
26. Sambrook J, Fritsch EF, ManiatisT著．金冬雁，黎孟枫，等译．分子克隆实验指南．第2版．北京：科学出版社，1998：463-469．
27．孙存普，张建中，段绍瑾．自由基生物学导论．合肥：中国科学技
术大学出版社，1999：150-156．
28．杨洁，高飞，易静．活性氧与细胞凋亡的研究进展．国外医学肿瘤学分册，2002，29(4)：248-251．
29．李莉，周兰，陈文缘．Bcl-2与细胞凋亡．国外医学临床生物化学与检验学分册，1999，20(6)：272-274．
30. Alexander RR, Sabine S, Karl MS, et al. A microplate assay for the detection of oxidative products using 2',7'-dichloroflurescin-diacetate. J Immunol Methods, 1985, 82: 253-261.
31．李楠，王凤翔，周春喜．荧光探针应用技术．北京：军事医学科学出版社，1998：62-65，84．
32. Enrenberg B, Montana V, Wei MD, et al. Membrane potential can be determined in individual cells from the Nernstian distribution of cationic dyes. Biophys J. 1998, 53: 785-794.
33. Rabn CA, Bombick DW, Doolittle DJ. Assessment of mitochondrial membrane potential as an indicator of cytotoxicity. Fundam Appl Toxicol, 1991,16: 435-440.
34. Mosmann T. Rapid colorimetric assay for cellular grouth and survival: application to prolification and cytotoxicity assay. J Immunol Methods, 1983, 65(1-2): 55-63.
35．陈国南，张帆．化学发光与生物发光．福州：福建科学技术出版社，1998：6-7，158-162．
36．聂松青．生物发光分析及其应用．生理科学进展，1984，15(3)：232-237．
37．戴定威，吴圣楣，李敏等．生化发光法测定体外培养小肠上皮细
胞内微量ATR．临床检验杂志，1997，15(5)：279-280．
38. Stanley PE, Williams SG. Use of the liquid scintillation spectrometer for determining adenosin triphosphate by the luciferase enzyme. Anal Biochem, 1969, 29(3): 381-92.
39. John DJ, Virginia FK, Samuel CS. Increased ATPand Creatine Phosphate Turnover in Pphagocytosing Mouse Peritoneal Macrophages. J Biol Chem, 1979, 254: 9558-9564.
40．张西蘅．生物化学．北京：北京医科大学，中国协和医科大学联合出版社，1994，207-226．
41. Donald N. Metabolic Minimap: Mitochondrial ATP Foramation. Biochem Mol Bio Edu, 2002, 30(1): 3-5.
42. Malaviya M, Husain R, Seth PK, et al. Perinatal effects of two pyrethroid insecticides on brain neurotransmitter function in the neonatal rat. Vet Hum Txoicol, 1993,35(2): 119-122.
43. Xiao C, He FS, Li OS, et al. Potental biochemical mechanisms of neuromuscular junction transmissiondysfunciton induced by organophosphorus insecticides. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 2003, 21(3): 191-193.
44．曾晓．杀虫剂对家蝇线粒体ATP酶活性影响的研究．中国媒介生物学及控制杂志，1995，4：
45．何俊，陈建峰，刘茹等．氰戊菊酯对卵巢细胞／组织钙稳态和激素水平的影响．中华预防医学杂志，2004，1：
46. Richer C, Schweizer M, Cossarizza A. Control of apoptosis by the cellular ATP level. FEB S Letter, 1996, 378: 107-110.
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