辛硫磷对大鼠肝脏毒性的研究
摘要
为探讨低浓度辛硫磷农药对肝脏的慢性毒性作用及其机理,本研究通过观察大鼠肝脏病理组织与超微结构改变及检测血浆、肝脏ChE、抗氧化物酶和脂质过氧化指标,研究了辛硫磷农药慢性灌胃染毒条件下对大鼠肝脏的氧化损伤作用;并进行了原代培养大鼠肝细胞的染毒试验,通过扫描电镜、荧光显微镜下观察,Annexin-V-FITC和PI双标记流式细胞仪测定凋亡率,双氢罗丹明标记细胞、流式细胞仪测定细胞内活性氧(ROS)等方法,探讨了辛硫磷对大鼠肝脏的毒性作用、致细胞凋亡作用以及ROS在辛硫磷诱导细胞凋亡中的作用。
结果表明,辛硫磷慢性染毒,大鼠血浆和肝脏中ChE活性均显著降低,但没有出现明显的临床中毒症状,推测中毒症状的出现不仅与血浆ChE活性降低程度有关,更主要与ChE降低速度有关。大鼠肝脏的病变程度随着染毒剂量的增加和染毒时间的延长有加重趋势。30μmol/kg剂量染毒15d,血浆和肝脏SOD、GSH-Px活性及MDA含量均无明显变化,这也进一步说明辛硫磷是一种短效型低毒杀虫剂对人畜的毒性较低。但是随着染毒时间的延长,染毒30d后,肝脏抗氧化酶活性升高和MDA浓度升高,血浆抗氧化酶活性降低,是否表明肝脏的抗氧化能力代偿性增强,有待进一步探讨。300μmol/kg剂量的辛硫磷染毒15d,血浆SOD、GSH-Px活性降低,MDA含量明显上升,但肝脏SOD、GSH-Px活性升高。持续染毒30d时,血浆和肝脏SOD、GSH-Px活性均明显下降,MDA含量极显著升高(P﹤0.01),表明辛硫磷农药持续染毒后,不仅可引起大鼠机体氧化应激,而且可以通过诱导肝细胞发生脂质过氧化造成肝脏结构的损伤。
本试验中30μmol/L辛硫磷对体外培养的肝细胞持续染毒24h,扫描电镜下以及吖啶橙染色后荧光显微镜下都观察到凋亡细胞,证明了低浓度的辛硫磷可以诱导肝细胞凋亡。不同剂量的辛硫磷对原代培养的肝细胞染毒,肝细胞染毒12h,100μmol/L剂量组死亡率极显著增加(P<0.01)、300μmol/L剂量组死亡率达到3.01%。染毒24h,随着染毒剂量的增大,细胞死亡率呈明显的上升趋势,差异均极显著(P<0.01),300μmol/L剂量组死亡率已高达14.28%。说明辛硫磷农药对肝细胞具有直接的毒性作用,而且细胞死亡率随染毒剂量增大和染毒时间延长呈明显的剂量-效应关系,表明辛硫磷作用剂量的增加或作用时间的延长,均可增加对细胞的毒性损伤。本试验测得各染毒组大鼠肝细胞随染毒剂量的增大和染毒时间延长,细胞凋亡率增大,与对照组比较,30μmol/L染毒12h凋亡率显著增加(P<0.05),100μmol/L剂量组凋亡率极显著增加(P<0.01),并且300μmol/L剂量组细胞凋亡率达到最大3.93%;染毒24h后,与对照组相比,各染毒组细胞凋亡率极显著增加(300μmol/L剂量组除外),100μmol/L剂量组细胞凋亡率达到最大7.53%。说明低浓度的辛硫磷农药可以诱导肝实质细胞凋亡。本研究发现随着辛硫磷浓度的升高,肝细胞内ROS水平逐渐升高,细胞凋亡率与细胞内ROS水平呈正相关。提示在辛硫磷农药致大鼠肝细胞凋亡过程中ROS发挥重要作用。
To investigate the effects and mechanism of low concentration phoxim on the liver of rat, in this paper we studied the toxicity and oxidative damage on testes under the exposure of phoxim especially the phoxim of chronic intragastric administration. The microstructure and ultra-microstructure changes of liver were examined and the changes of ChE、antioxidase and production of lipid peroxidation were detected. In addition, we also exposed phoxim to primary cultures of rat hepatocytes. To explore the induction of phoxim on the apoptosis、necrosis of hepatocytes and the effect of ROS on the induction of apoptosis. Apoptotic cells were observed by scanning electron microscope(SEM) and fluorescence microscope, apoptotic rates were determined in flow cytometry (FCM) by Annexin-V、PI double labeling and intra-cellular ROS levels by dihydrorhodamine 123.
The research results showed that the activity of ChE excessively decreased in plasma and liver under the chronic phoxim exposure, but no clinical symptom obviously was observed. The activity of ChE in high dose group decreased to 22% and 75% in plasma and liver respectively compared to the control. It can be concluded that appearance of toxicity clinical symptom was not only with the degree of the ChE reduction but mainly with the speed of reduction. The activity of SOD and GSH-Px both decreased in plasma and the content of MDA increased in plasma and liver along with the exposed time extended. Histopathological changes showed fatty degeneration in liver cells of rats. It was concluded that rats exposed to phoxim successively could induce oxidative stress and lesions of structure in the liver.
Different dose of phoxim was exposed to primary cultured hepatocytes of rat, It was found that phoxim could damage the hepatocytes directly. The rates of cell apoptosis and death were obviously enhanced and the change was dose-time-dependent. The results showed that intracellular Reactive oxygen species (ROS) levels were advanced with the dose of phoxim. The apoptotic rate was directly correlated with the level of intracellular ROS. It suggested that ROS plays an important role in phoxim inducing apoptosis of hepatocytes.
结果表明,辛硫磷慢性染毒,大鼠血浆和肝脏中ChE活性均显著降低,但没有出现明显的临床中毒症状,推测中毒症状的出现不仅与血浆ChE活性降低程度有关,更主要与ChE降低速度有关。大鼠肝脏的病变程度随着染毒剂量的增加和染毒时间的延长有加重趋势。30μmol/kg剂量染毒15d,血浆和肝脏SOD、GSH-Px活性及MDA含量均无明显变化,这也进一步说明辛硫磷是一种短效型低毒杀虫剂对人畜的毒性较低。但是随着染毒时间的延长,染毒30d后,肝脏抗氧化酶活性升高和MDA浓度升高,血浆抗氧化酶活性降低,是否表明肝脏的抗氧化能力代偿性增强,有待进一步探讨。300μmol/kg剂量的辛硫磷染毒15d,血浆SOD、GSH-Px活性降低,MDA含量明显上升,但肝脏SOD、GSH-Px活性升高。持续染毒30d时,血浆和肝脏SOD、GSH-Px活性均明显下降,MDA含量极显著升高(P﹤0.01),表明辛硫磷农药持续染毒后,不仅可引起大鼠机体氧化应激,而且可以通过诱导肝细胞发生脂质过氧化造成肝脏结构的损伤。
本试验中30μmol/L辛硫磷对体外培养的肝细胞持续染毒24h,扫描电镜下以及吖啶橙染色后荧光显微镜下都观察到凋亡细胞,证明了低浓度的辛硫磷可以诱导肝细胞凋亡。不同剂量的辛硫磷对原代培养的肝细胞染毒,肝细胞染毒12h,100μmol/L剂量组死亡率极显著增加(P<0.01)、300μmol/L剂量组死亡率达到3.01%。染毒24h,随着染毒剂量的增大,细胞死亡率呈明显的上升趋势,差异均极显著(P<0.01),300μmol/L剂量组死亡率已高达14.28%。说明辛硫磷农药对肝细胞具有直接的毒性作用,而且细胞死亡率随染毒剂量增大和染毒时间延长呈明显的剂量-效应关系,表明辛硫磷作用剂量的增加或作用时间的延长,均可增加对细胞的毒性损伤。本试验测得各染毒组大鼠肝细胞随染毒剂量的增大和染毒时间延长,细胞凋亡率增大,与对照组比较,30μmol/L染毒12h凋亡率显著增加(P<0.05),100μmol/L剂量组凋亡率极显著增加(P<0.01),并且300μmol/L剂量组细胞凋亡率达到最大3.93%;染毒24h后,与对照组相比,各染毒组细胞凋亡率极显著增加(300μmol/L剂量组除外),100μmol/L剂量组细胞凋亡率达到最大7.53%。说明低浓度的辛硫磷农药可以诱导肝实质细胞凋亡。本研究发现随着辛硫磷浓度的升高,肝细胞内ROS水平逐渐升高,细胞凋亡率与细胞内ROS水平呈正相关。提示在辛硫磷农药致大鼠肝细胞凋亡过程中ROS发挥重要作用。
To investigate the effects and mechanism of low concentration phoxim on the liver of rat, in this paper we studied the toxicity and oxidative damage on testes under the exposure of phoxim especially the phoxim of chronic intragastric administration. The microstructure and ultra-microstructure changes of liver were examined and the changes of ChE、antioxidase and production of lipid peroxidation were detected. In addition, we also exposed phoxim to primary cultures of rat hepatocytes. To explore the induction of phoxim on the apoptosis、necrosis of hepatocytes and the effect of ROS on the induction of apoptosis. Apoptotic cells were observed by scanning electron microscope(SEM) and fluorescence microscope, apoptotic rates were determined in flow cytometry (FCM) by Annexin-V、PI double labeling and intra-cellular ROS levels by dihydrorhodamine 123.
The research results showed that the activity of ChE excessively decreased in plasma and liver under the chronic phoxim exposure, but no clinical symptom obviously was observed. The activity of ChE in high dose group decreased to 22% and 75% in plasma and liver respectively compared to the control. It can be concluded that appearance of toxicity clinical symptom was not only with the degree of the ChE reduction but mainly with the speed of reduction. The activity of SOD and GSH-Px both decreased in plasma and the content of MDA increased in plasma and liver along with the exposed time extended. Histopathological changes showed fatty degeneration in liver cells of rats. It was concluded that rats exposed to phoxim successively could induce oxidative stress and lesions of structure in the liver.
Different dose of phoxim was exposed to primary cultured hepatocytes of rat, It was found that phoxim could damage the hepatocytes directly. The rates of cell apoptosis and death were obviously enhanced and the change was dose-time-dependent. The results showed that intracellular Reactive oxygen species (ROS) levels were advanced with the dose of phoxim. The apoptotic rate was directly correlated with the level of intracellular ROS. It suggested that ROS plays an important role in phoxim inducing apoptosis of hepatocytes.
引文
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