【作者】 赵岭；

【导师】 马雪梅；

【作者基本信息】 北京工业大学 ， 生物医学工程， 2013， 博士

【摘要】 有机磷农药（Organophosphate pesticides，OPs）是目前使用最为广泛的一种化学合成杀虫剂，它所引起的毒性作用已成为全球关注的公共问题。急性OPs暴露症状明显、便于救护；长期低剂量是人群接触OPs的主要方式，可导致多器官损伤，与多种癌症发生具有相关性并可引起中枢神经发育迟缓、学习、记忆功能下降等。氢分子一直被认为是一种生理惰性气体，随着研究的深入发现它是一种可以选择清除超氧阴离子的新型抗氧化剂，其对OPs毒性的保护作用及分子机制未见报道。本研究针对OPs毒性作用的分子机制和防护方法进行探讨。研究发现，OPs可以引起细胞和生物体产生活性氧，激活EB病毒并对脑、肝组织形成损伤。氢分子可在活性氧产生阶段及时阻断，进而抑制EB病毒激活，缓解脑、肝损伤。这一研究为将氢分子应用于OPs长期低剂量接触危险人群，提供一种简便易行的日常防护方法奠定了基础，同时关于OPs和氢分子作用分子机制的探讨具有重要的科学意义。本论文的主要研究工作如下：1.毒死蜱激活EB病毒及氢分子的抑制作用：EB病毒的人群感染率超过90%，病毒的活性状态与多种肿瘤的发生密切相关。低毒类OPs毒死蜱（Chlorpyrifos，CPF）作用于携带潜伏期EB病毒Raji细胞4h，检测细胞氧化应激相关指标和EB病毒立早期基因BZLF-1表达。荧光探针CM-H2DCFDA检测发现CPF诱导细胞活性氧（Reactive oxygen species，ROS）累积，非酶类抗氧剂GSH含量显著性减低。低浓度CPF引起Raji细胞抗氧化酶SOD、CAT活性升高，高浓度则造成这两个酶活性降低，说明CPF可以诱发细胞氧化应激状态。CPF造成Raji细胞脂质过氧化产物MDA含量显著性升高，DNA断裂水平明显增加；Q-PCR检测发现EB病毒立早期基因BZLF-1表达上调。氢分子可以改善细胞氧化应激状态，保护生物大分子损伤，并且抑制EB病毒BZLF-1表达上调，说明氢分子缓解Raji细胞氧化应激状态，抑制EB病毒激活。以上结果说明有机磷农药可能与EB病毒具有协同致癌作用，氢分子可以在ROS诱发阶段阻断EB病毒的激活和生物大分子损伤，对有机磷农药日常防护有重要意义。2.毒死蜱降低乙酰胆碱酯酶（AChE）活性及氢分子的保护作用。8周龄健康Wistar大鼠每天以1/20LD50毒死蜱灌胃8周建立模型亚毒剂量染毒模型，通过饮用氢水方式补给氢分子。CPF经过细胞色素P450酶CYP2B6亚型的催化转化为毒性很高的CPO，后者可以被对氧磷酶1（PON1）转化成毒性较小的3，5，6-三氯-2-吡啶酚（3,5,6-trichloropyridine-2-phenol，TCP）进行解毒，或者与AChE不可逆的共价结合，使AChE丧失活性，造成乙酰胆碱在作用位点累积从而引发各种中毒症状。实验结果表明，CPF重复染毒造成血清和脑组织AChE活性显著降低，与正常对照组相比分别下降了20.05%和63.48%。氢分子缓解CPF染毒造成的AChE活性降低，提示氢分子除了具有已知的清除羟自由基的功能外，还可能直接作用于有机磷农药的活性代谢产物CPO，这一研究为进一步深入研究氢分子的生物学效应奠定了基础，拓展了氢分子生物学的研究领域。3.毒死蜱引起脑、肝组织损伤及氢分子的保护作用：亚毒剂量CPF引起机体、脑和肝组织氧化应激反应，表现为血清MDA含量升高，SOD和CAT酶活性降低。脑组织MDA含量、SOD活性、CAT活性升高；肝脏MDA含量、SOD活性升高，然而CAT水平降低。海马是脑中与学习记忆功能相关的神经元，浦肯野细胞层是与运动协调相关的神经元，CPF染毒造成海马CA1区、CA3区和浦肯野细胞层神经元胞体减小，胞核皱缩等，说明神经元细胞出现明显损伤；肝脏出现水肿，未见坏死、炎症等损伤；TUNEL染色显示脑组织中神经元细胞出现明显凋亡，而肝脏中未见凋亡细胞，以上实验结果说明CPF染毒造成脑组织的损伤高于肝脏。氢分子缓解氧化应激状态、保护神经元、肝细胞损伤。MnSOD定位于线粒体中，过量表达具有抗凋亡的作用，肝组织中MnSOD显著表达上调，脑组织中则MnSOD没有明显升高，同时脑组织中缺乏CPO解毒酶PON1，使得CPF以毒性很高的CPO形式存在。肝脏由于MnSOD高表达和代谢解毒酶PON1的作用损伤程度较轻。氢分子对损伤有明显的保护作用。4.毒死蜱引起肝脏氧化应激相关基因变化及氢分子的作用。氧化应激信号通路包括ROS代谢相关基因，抗氧化相关基因、氧转运蛋白和其他氧化应激相关基因。PCR芯片技术检测发现CPF染毒造成Fmo2、Sod2、Cygb、Ldha、Gpx、Nox4、Gclc、Gclm、Cat等基因变化。氢分子改善了这些变化。综上所述，有机磷农药可以引起细胞和生物体产生活性氧，激活EB病毒并对脑、肝组织形成损伤。氢分子可在活性氧产生阶段阻断，抑制EB病毒激活，缓解脑、肝损伤。氢分子作为有机磷农药长期低剂量接触危险人群的日常防护措施奠定了一定的基础。更多还原

【Abstract】 Nowadays, organophosphate pesticides (OPs) are the most widely used syntheticchemical compounds, and the toxic effect induced by OPs has become a global issue.Acute OPs exposure causes obvious symptoms, which is easy to rescue. Long-timeexposure with very low doses is the main way to contract OPs for most people, whichcaused various damages, such as neural development delayed, decline in learning andmemory. Moreover, chronic OPs exposure was reported to be associated withtumorigenesis. In the past, hydrogen was considered as the physiological inert gas.With developing of research, hydrogen was determined as a new antioxidant whichselectively scavenger hydroxyl radicals (·OH). Until now, the protective effect andmechanism of hydrogen on OPs toxicity have not been reported. This study wasaimed to explore mechanism and protection of OPs toxicity. Our results showed thatOPs induced reactive oxygen species (ROS) accumulation, reactivated Epstein-Barr(EB) virus, damage brain and liver. Hydrogen inhibited EB reactivation, alleviatedbrain and hepatic damage via scavenger ROS production. Hydrogen can be used as asimple protective method for person who was exposed to OPs for long-time. At thesame time, this study has scientific significance on the mechanism of OPs toxicity andhydrogen effects.The main works of this research is as follow:1. Chlorpyrifos (CPF) reactivated EB virus and the inhibition of hydrogen: EBvirus infected more than90%of the world’s population, and its reactivation isassociated with various tumorgenesis. In this study, In this study, Raji cell line wasexposed to chlorpyrifos (CPF) which is one of OPs with low toxicity. Oxidative stressof Raji cells and EB virus immediately-early gene BZLF-1was detected. CPFexposure caused significantly ROS production and non-enzymatic antioxidant GSHlevel decrease. SOD and CAT activities were increased with low concentration of CPF,while decreased with high CPF concentration. These results showed that CPF inducedoxidative stress of Raji cells. Moreover, MDA level, a biomarker of lipid peroxidation,and DNA breakage were obviously increased after CPF exposure. BZLF-1wasup-regulation, suggesting that EB virus lytic cycle was reactivated. Hydrogenattenuated oxidative stress and EB virus reactivation. The damage of biologicalmolecule induced by OPs was ameliorated. All these results suggested that OPs andEB virus may have synergistic carcinogensis. Hydrogen inhibited EB virusreactivation and biological molecular damage induced by OPs at the stage of ROSproduction. Hydrogen has significance on daily protection of OPs exposure.2. CPF caused decrease in acetyl cholinesterase (AChE) activity and protectiveeffect of hydrogen. Healthy8weeks old Wistar rats were used in this study, and1/20LD50CPF was supplied via gavage daily for8weeks, and drinking hydrogen-richwater. CPF convert into CPO with catalyzing of CYP2B6, a hypotype of P450family.Paraoxon1convert CPO into3,5,6-trichloropyridine-2-phenol (TCP). CPO canirreversibly combine with AChE, t he inhibition of AChE by OPs causesaccumulation of acetycholine at cholinergic synapses, with over-stimulation ofmuscarinic and nicotinic receptors. Our results showed that CPF exposure caused significantly decrease35.5%and62.1%in AChE activates in serum and brain.Hydrogen alleviated AChE activity decrease, suggesting that the protective effect ofhydrogen may be not limited to scavenger OH. Hydrogen may directly impact onCPO. This research expands the field of hydrogen biology.3. CPF caused brain and hepatic damage and protective effect of hydrogen:sub-toxic CPF exposure induced oxidative stress in serum, brain and liver. In serum,MDA level was increased, SOD and CAT activities decreased. In brain and liver,MDA level and SOD activity were significantly increased. CAT activity wasincreased in brain while decreased in liver. Hippocampus neurons are associated withlearning and memory function. Pukenje cell layer are the neurons associated withmotion. CPF intoxication caused nuclear shrinking, losing in cytoplasma in CA1,CA3and Pukenje cell layer. This result showed that CPF exposure caused severedamage in neuron. In liver, edema was observed without necrosis and inflammation.TUNEL staining showed that neuron cells was apoptotic in brain, while there was noobvious apoptosis in liver. All these results suggest CPF intoxication caused moresevere damage in brain compared with liver. Hydrogen ameliorated oxidative stress,and protected neuron and hepatic cell from damage. Overexpression of MnSOD couldinhibite apoptosis. MnSOD gene expression was not significantly increased, andPON1was lack in brain, which made CPF exist as high toxic CPO. In liver, increasein MnSOD expression and PON1make liver lesser damage. Hydrogen showedprotective effect of these injuries.4. CPF causes liver oxidative stress related genes change, and the function ofhydrogen molecules. Oxidative stress signaling pathways including ROS metabolismrelated genes, oxidation resistance related gene, oxygen transport protein and otheroxidative stress related genes. PCR chip results showed CPF exposure regulatedFmo2, Sod2, Cygb, Ldha, Gpx, Nox4, Gclc, Gclm, Cat, etc. hydrogen amelioratedthese regulations to normal levels.In conclusion, OPs can lead to cells and organisms to produce reactive oxygenspecies, activation of EB virus, cause the brain and hepatic tissue damage. Molecularhydrogen can be block in stage of ROS production, inhibition of EB virus activation,relieve brain and hepatic damage. Hydrogen can be as as daily protection measuresfor people exposed with low-dose CPF in long-term.更多还原