In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides
In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides
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DOI：
10.1016/0300-483x(95)03156-a
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发表时间：
1995-12-15
期刊：
影响因子：
4.5
通讯作者：
Stohs, SJ
中科院分区：
文献类型：
--
作者：
Bagchi, D;Bagchi, M;Stohs, SJ
活性氧可能与各种农药的毒性有关，因此，我们研究了结构不同的多卤代环烃（PCH），如异狄氏剂和氯霉素，氯化乙酰胺除草剂（CAH），如甲草胺，和有机磷农药（OPS），如毒死蜱和倍硫磷，对肝和脑脂质过氧化和DNA单链断裂（SSB）的产生，这两个指标的氧化应激和氧化组织损伤。所选农药经口施用。在0 h和21 h以0.25 LD（50）剂量对雌性Sprague-Dawley大鼠进行两次给药，并在24 h处死。在一组平行的实验中，我们已经确定了这些农药对DNA-SSB和增强的乳酸脱氢酶（LDH）从培养的神经活性PC-12细胞的体外效应。这些农药的活性氧物种的体外生产也进行了评估，通过确定增强的化学发光反应的肝和脑匀浆。用异狄氏剂、毒死蜱、甲草胺、毒死蜱和倍硫磷处理大鼠后，观察到肝脏脂质过氧化作用分别增加2.8、3.0、4.2、4.3和4.8倍，而在这些相同剂量下，脂质过氧化作用分别增加2.4、2.1、3.6、4.6和5.3倍，在脑匀浆中观察到。用异狄氏剂、氯丹、甲草胺、毒死蜱和倍硫磷分别处理大鼠后，观察到肝脏DNA-SSB增加4.4、3.9、1.6、3.0和3.5倍，而在这些相同剂量下，观察到脑核DNA-SSB分别增加1.9、1.7、2.2、1.4、1.4倍。肝和脑组织分别与1 nmol/ml的五种农药体外孵育后，化学发光在孵育后4-7分钟内出现最大增加，并持续10分钟以上。毒死蜱和倍硫磷的含量分别增加，而脑匀浆中的含量分别增加了1.7倍、1.8倍、2.0倍、3.4倍和3.7倍。在用异狄氏剂、毒死蜱、甲草胺、毒死蜱和倍硫磷处理的动物的肝匀浆中，观察到化学发光反应分别增加2.2、2.3、2.9、2.9和3.4倍，而在脑匀浆中观察到化学发光反应分别增加1.8、2.0、3.2、2.9和2.4倍。将培养的神经活性PC-12细胞与农药一起孵育，并检查释放到培养基中的乳酸脱氢酶（LDH）作为细胞损伤和细胞毒性的指标。在100 nM农药浓度下观察到培养的PC-12细胞释放的LDH最大。在PC-12细胞与异狄氏剂、毒死蜱、甲草胺、毒死蜱和倍硫磷孵育后，观察到LDH渗漏分别增加2.3、2.5、2.8、3.1和3.4倍。用100 nM浓度的这些相同农药孵育培养的PC-12细胞后，DNA-SSB分别增加2.5、2.2、2.1、2.4和2.5倍，结果清楚地表明，这些不同类别的农药诱导活性氧的产生和氧化组织损伤，这可能有助于这些异生物素活性氧在许多毒物和病理条件下可作为程序性细胞死亡（凋亡）的共同介质。
Reactive oxygen species may be involved in the toxicity of various pesticides and we have, therefore, examined the in vivo effects of structurally dissimilar polyhalogenated cyclic hydrocarbons (PCH), such as endrin and chlordane, chlorinated acetamide herbicides (CAH), such as alachlor, and organophosphate pesticides (OPS), such as chlorpyrifos and fenthion, on the production of hepatic and brain lipid peroxidation and DNA-single strand breaks (SSB), two indices of oxidative stress and oxidative tissue damage. The selected pesticides were administered p.o. to female Sprague-Dawley rats in two 0.25 LD(50) doses at 0 h and 21 h and killed at 24 h. In a parallel set of experiments, we have determined the in vitro effects of these pesticides on the DNA-SSB and enhanced lactate dehydrogenase leakage (LDH) from neuroactive PC-12 cells in culture. In vitro production of reactive oxygen species by these pesticides was also assessed by determining the enhanced chemiluminescence responses of hepatic and brain homogenates. Following treatment of rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, increases of 2.8-, 3.0-, 4.2-, 4.3- and 4.8-fold were observed in hepatic lipid peroxidation, respectively, while at these same doses, increases in lipid peroxidation of 2.4-, 2.1-, 3.6-, 4.6- and 5.3-fold, respectively, were observed in brain homogenates. Increases of 4.4-, 3.9-, 1.6-, 3.0- and 3.5-fold were observed in hepatic DNA-SSB following treatment of the rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while at these same doses, increases of 1.9-, 1.7-, 2.2-, 1.4-, 1.4-fold, respectively, were observed in brain nuclear DNA-SSB. Following in vitro incubation of hepatic and brain tissues with 1 nmol/ml of each of the five pesticides, maximum increases in chemiluminescence occurred within 4-7 min of incubation and persisted for over 10 min. Increases of 3.0-, 2.7-, 3.6-, 4.9- and 4.4-fold were observed in chemiluminescence following in vitro incubation of the liver homogenates with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.7-, 1.8-, 2.0-, 3.4- and 3.7-fold, respectively, were observed in the brain homogenates. Increases of 2.2-, 2.3-, 2.9-, 2.9- and 3.4-fold were observed in the chemiluminescence responses in the liver homogenates of the animals treated with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.8-, 2.0-, 3.2-, 2.9- and 2.4-fold, respectively, were observed in the brain homogenates. Cultured neuroactive PC-12 cells were incubated with the pesticides and the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular damage and cytotoxicity was examined. Maximal release of LDH from cultured PC-12 cells was observed at 100 nM concentrations of the pesticides. Increases of 2.3-, 2.5-, 2.8-, 3.1 and 3.4-fold were observed in LDH leakage following incubation of the PC-12 cells with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively. Following incubation of the cultured PC-12 cells with 100 nM concentrations of these same pesticides, increases in DNA-SSB of 2.5-, 2.2-, 2.1-, 2.4- and 2.5-fold, respectively, were observed.The results clearly demonstrate that these different classes of pesticides induce production of reactive oxygen species and oxidative tissue damage which may contribute to the toxic manifestations of these xenobiotics. Reactive oxygen species may serve as common mediators of programmed cell death (apoptosis) in response to many toxicants and pathological conditions.