辛硫磷对大鼠肝脏毒性的研究

发布时间：2018-08-23 19:16

【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㩳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(P0.01)、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(P0.01),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(P0.05),100μmol/L剂量组凋亡率极显著增加(P0.01),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。
[Abstract]:To investigate the chronic toxicity of phoxim pesticide at low concentration on liver and its mechanism, the oxidative damage of rat liver was studied by observing the pathological and ultrastructural changes of liver and detecting the indexes of plasma, liver ChE, antioxidant enzyme and lipid peroxidation. The toxicity of Phoxim on primary cultured rat hepatocytes was studied by scanning electron microscopy, fluorescence microscopy, Annexin-V-FITC and PI double-labeled flow cytometry, dihydrorhodamine-labeled cells and reactive oxygen species (ROS) assay. The toxicity of Phoxim on rat liver and its effect on cell apoptosis were investigated. Apoptosis and the role of ROS in phoxim induced apoptosis.
The results showed that the ChE activity in plasma and liver of rats was significantly decreased after chronic exposure to phoxim, but no obvious clinical toxic symptoms were observed. It was speculated that the occurrence of toxic symptoms was not only related to the degree of decrease of plasma ChE activity, but also to the rate of decrease of plasma ChE activity. There was no significant change in SOD, GSH-Px activity and MDA content in plasma and liver after 15 days of exposure at a dose of 30 micromol/kg, which further indicated that phoxim was a short-acting and low-toxic insecticide with low toxicity to human and livestock. However, with the prolongation of exposure time, liver antioxidant enzyme activity and MDA concentration increased 30 days after exposure. The activity of SOD, GSH-Px and MDA in plasma and liver decreased significantly after 15 days of Phoxim exposure, but the activity of SOD and GSH-Px in liver increased significantly. The activity of SOD, GSH-Px in plasma and liver decreased significantly after 30 days of continuous exposure. The content of phoxim increased significantly (P?0.01), indicating that phoxim could not only induce oxidative stress in rats, but also damage the liver structure by inducing lipid peroxidation in hepatocytes.
Apoptotic cells were observed under scanning electron microscopy and fluorescence microscopy after staining with acridine orange. It was proved that low concentration of phoxim could induce apoptosis of hepatocytes. Different doses of phoxim were given to primary cultured hepatocytes and the hepatocytes were exposed to phoxim for 12 hours and 100 micromol/L for 12 hours. The mortality of L-dose group was significantly increased (P 0.01), and that of 300 micromol/L group was 3.01%. The cell mortality increased significantly with the increase of dose in 24 hours (P 0.01). The mortality of 300 micromol/L group was up to 14.28%. The cell death rate showed a dose-effect relationship with the increase of dose and duration of exposure, indicating that the increase of dose or duration of phoxim could increase the toxic damage to cells. Compared with the control group, the apoptosis rate increased significantly at 12 h (P 0.05), and significantly at 100 micromol/L (P 0.01), and the apoptosis rate reached the maximum of 3.93% in the 300 micromol/L dose group; after 24 h, the apoptosis rate of each exposure group was significantly increased (except 300 micromol/L dose group), and fine in the 100 micromol/L dose group. The apoptosis rate was 7.53%. It indicated that low concentration phoxim pesticide could induce apoptosis of hepatic parenchyma cells. With the increase of Phoxim concentration, the level of ROS in hepatocytes increased gradually, and the apoptosis rate was positively correlated with the level of ROS in hepatocytes. Effect.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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