【作者】 阮国洪；

【导师】 周志俊； 金泰廙； 常秀丽；

【作者基本信息】 复旦大学 ， 卫生毒理学， 2006， 博士

【摘要】 乐果(Dimethoate)是中国最常用的有机磷农药(OPs)之一。有机磷急性中毒救治是一个重大医学课题，国家十五攻关将迟发性神经病变和中间肌无力综合症治疗列为重要攻关内容。有机磷中毒的主要原因是乙酰胆碱酯酶(AChE)的磷酸化，其活性的丧失造成乙酰胆碱(ACh)在神经突触处的过量积聚，进而乙酰胆碱受体(M样和N样)过度兴奋，引起一系列中毒症状。虽然针对M胆碱受体(阿托品)、乙酰胆碱酯酶(肟类复能剂)和对症处理，在有机磷农药中毒救治中起了重要作用，也积累了丰富的经验，但中毒的病死率仍处于相当高的水平，一些问题急需研究，例如有机磷中毒的有些临床症状震颤、癫痫涉及非胆碱能机制多巴胺(DA)神经递质的异常变化，因而关注有机磷农药对单胺类神经递质的影响具有重要意义。 与有机磷中毒有关的单胺类神经递质具有一系列重要生理功能。例如，去甲肾上腺素(NE)增加具有升压和升高体温作用，又具有降压和心率减慢作用，还能增加食欲等，NE减少会增加痛感等。肾上腺素(E)主要是升压作用。多巴胺(DA)与帕金森症(Parkinson disease，PD)和精神分裂症有关，DA神经系统还参与调节脑垂体激素的分泌、心脑血管功能、中枢性催吐、胃肠道的蠕动、眼内压调节和视网膜信息的传递等。NE和5-羟色胺(5-HT)递质系统还与阿尔采默型老年痴呆(Alzheimer’s disease)有关。此外，5-HT与多方面的生理功能有关，包括睡眠、行为和记忆、下丘脑-垂体前叶功能、疼痛感受、体温调节、摄食和心血管功能调节。 由于单胺类神经递质具有一系列重要生理功能，因此研究乐果对单胺类神经递质的影响，有助于进一步探讨乐果的神经毒性机制，对于改善乐果中毒病人的临床治疗具有重要意义。而目前国内外在此方面只有对有机磷战争毒剂索曼(soman)的研究报告，发现其可以影响中枢神经系统其它神经递质，如儿茶酚胺的变化以及相应受体变化。虽有研究提示，有机磷酸酯可以影响单胺类神经递质，但这些变化的规律并不完全清楚。 因此，本文针对乐果影响单胺类神经递质影响作用机制进行了深入研究。首先建立递质检测方法。采用ODS柱，通过对流动相的组成、配比和pH进行实验研究，在不加离子对试剂条件下发现柠檬酸三钠、磷酸氢二钠和甲醇为最佳组合，流速1.0ml／min，建立一种快速准确测定大鼠血清及脑组织中单胺类神经递质去甲肾上腺素(NE)、肾上腺素(E)、多巴胺(DA)、5-羟色胺(5-HT)含量和多巴胺-β-羟化酶(DβH)活性的高效液相色谱电化学检测方法。又通过对流动相的组成优化实验研究，发现柠檬酸三钠、磷酸氢二钠和乙腈为最佳组合，建立一种快速准确测定大鼠脑片及其培养液中多巴胺及其代谢物的高效液相色谱电化学检测方法。通过线性试验、回收试验、精密度试验以及样品的测定，验证了方法的可行性。 观察乐果染毒对大鼠大脑分区单胺类神经递质去甲肾上腺素(NE)、肾上腺素(E)、多巴胺(DA)和5—羟色胺(5-HT)浓度变化的影响，对乐果中毒机制作进一步的探讨。雄性SD大鼠随机分为对照组(生理盐水)、低(38.9mg／kg)、中(83.7mg／kg)、高(180mg／kg)四个剂量组，腹腔注射染毒，给药后0.5h、2h、8h、24h断头处死并分离脑组织(大脑皮层、纹状体、海马、脑干和小脑五部分)。用正丁醇、正庚烷、高氯酸、三氯甲烷处理脑组织样品，用高效液相色谱电化学检测法检测。结果发现：不同时程的染毒组(低、中、高剂量组)大脑DA及其代谢物3，4二羟苯乙酸(DOPAC)、5-HT、NE和E的浓度与对照组相比均有显著性差异(P＜0.05)。变化规律非常复杂，在不同时程、不同剂量呈现不同方向的变化。DA及其代谢物DOPAC在纹状体分别增加了27—122％、89—538％；DA在其它分区变化没有显著性差异，DOPAC在大脑皮层有的剂量和时程增加了50—72％、有的剂量和时程减少了31—51％；在海马没有显著性差异；在脑干有的剂量和时程增加了4—102％，有的剂量和时程减少了10—16％；在小脑有的剂量和时程增加了6—39％，有的剂量和时程减少了6—23％。5-HT的浓度在纹状体减少了4—15％；在大脑皮层增加了34—169％；在海马有的剂量和时程增加了33—62％，有的剂量和时程减少了28—58％；在脑干增加了11—172％；在小脑有的剂量和时程增加了13—328％，有的剂量和时程减少了15—40％。NE的浓度在纹状体减少了18—70％；在大脑皮层有的剂量和时程增加了2—8％，有的剂量和时程减少了5—11％；在海马有的剂量和时程增加了7％，有的剂量和时程减少了8—24％；在脑干有的剂量和时程增加了3—20％、有的剂量和时程减少了8—16％；在小脑减少了4—23％。E的浓度在纹状体减少了7—45％；在大脑皮层减少了14—37％；在海马减少了33—56％；在脑干有的剂量和时程增加了4—8％、有的剂量和时程减少了7—33％；在小脑减少了4—19％。DA、DOPAC、NE、E和5-HT的浓度在不同时程的低、中、高剂量组间均有显著性差异(P＜0.05)。DA、DOPAC和5-HT浓度有随染毒剂量和时程的增加而增加的趋势，相反NE和E浓度有随染毒剂量和时程的增加而减少的趋势，存在剂量时程效应关系。因此乐果的中毒存在非胆碱能机制，即单胺类能机制，在临床上单胺类神经递质异常变化所引起的中毒症状应给予必要的关注。 研究发现：乐果对血清单胺类DA、DOPAC、5-HT、NE、E浓度有影响，不同时程的染毒组(低、中、高剂量组)血清DA及其代谢物DOPAC和5-HT的浓度比对照组分别增加了8.42—296％、13—67％和3.2—27％。不同时程的染毒组血清NE和E的浓度比对照组分别减少了9.58—55.4％和11—32.6％。DA、DOPAC、NE、E和5-HT的浓度在不同时程的低、中、高剂量组间均有显著性差异(P＜0.05)。DA、DOPAC和5-HT浓度有随染毒剂量和时程的增加而增加的趋势，相反NE和E浓度有随染毒剂量和时程的增加而减少的趋势。 通过体外和体内乐果染毒对大鼠大脑纹状体和血清DβH活性的影响试验，发现乐果对大鼠大脑纹状体和血清DβH活性有抑制效应，与对照组相比，染毒组纹状体和血清DβH活性下降6—59％。体外试验有剂量反应关系(r=-0.8912)，体内试验有剂量效应(r=-0.9967)和时程效应(r=-0.9134)。乐果对大鼠体内DβH活性的抑制作用，会造成去甲肾上腺素与肾上腺素合成障碍，体内去甲肾上腺素与肾上腺素减少或缺失，同时体内多巴胺增多，导致血压调节紊乱，血管扩张，血压下降，必然引起一系列的病理反应。 通过体外和体内乐果对大鼠大脑纹状体和血清MAO酶活性的影响试验，发现乐果对大鼠大脑纹状体和血清MAO酶活性没有抑制作用。 体外离体脑片染毒实验证实：乐果对单胺类神经递质DA及其代谢物DOPAC、高香草酸(HVA)有影响。与对照组相比，染毒剂量组黑质脑片及其培养液，纹状体脑片及其培养液DA、DOPAC和HVA浓度分别增加了36—174％、9—266％和18—546％，染毒时程组分别增加了8—260％、3—302％和41—696％。乐果对黑质脑片及其培养液，纹状体脑片及其培养液DA、DOPAC和HVA浓度有影响，并随染毒剂量和时程增加而增加，有显著性剂量效应和时程效应(p＜0.05)。 总之，通过乐果染毒大鼠，发现大脑分区和血清中单胺类神经递质DA及其代谢物DOPAC和神经递质5-HT浓度有随染毒剂量和时程的增加而增加的趋势，相反NE和E浓度有随染毒剂量和时程的增加而减少的趋势，存在剂量时程效应关系。推测乐果对单胺类神经递质的影响机制是通过抑制多巴胺-β-羟化酶活性来实现毒性效应。通过体外和体内乐果对大鼠大脑纹状体和血清DβH活性和MAO酶活性的影响试验，发现乐果对大鼠大脑纹状体和血清DβH活性有抑制效应，体外试验有剂量反应关系，体内试验有剂量效应和时程效应；乐果对大鼠大脑纹状体和血清MAO酶活性没有抑制作用，直接证明乐果对单胺类神经递质的影响机制。乐果对黑质脑片及其培养液，纹状体脑片及其培养液DA、DOPAC和HVA浓度有影响，并随染毒剂量和时程增加而增加，有显著性剂量效应和时程效应，乐果染毒脑片实验结果进一步证实乐果对单胺类神经递质的影响机制。因此，乐果对单胺类神经递质影响机制主要是通过对大鼠体内DβH活性的抑制而发挥作用的。以上结果提示乐果通过抑制DβH活性来改变体内单胺类神经递质的水平，而单胺类神经递质的异常变化与一系列精神疾病、情感障碍和运动失衡有关，有机磷农药乐果又能影响单胺类神经递质的异常变化和抑制DβH活性，因此长期接触有机磷农药可能与帕金森症、精神分裂症和阿尔采默型老年痴呆等精神和神经疾病有关，有关这方面的相互作用尚有待于进一步的深入研究。更多还原

【Abstract】 Dimethoatc (O,O-dimcthyl 5-mcthyl carbamoylmethyl phosphorodithioate, DM) is one of organophosphorus insecticides with anticholinesterase activity, used in agriculture to treat fruit trees, olive trees, vegetable plants, ornamental plants, tobacco, cotton, and other crops. It is the most widely used as a pesticide in China. DM is often a cause of accidental or suicidal poisoning. Deliberate self-harm by ingestion of organophosphorus insecticides is a common health problem all over the world, most in the developing country. Once it has entered the body, it undergoes metabolic activation by oxidative desulfuration to omethoate .The poisoning results in an initial life-threatening cholinergic crisis and several intermediate and late neurological and psychiatric manifestations.Symptoms of acute exposure to organophosphale or cholinesterase-inhibiting compounds may include the following: numbness, tingling sensations, incoordination, headache, dizziness, tremor, nausea, abdominal cramps, sweating, blurred vision, difficulty breathing or respiratory depression, and slow heartbeat. Very high doses may result in unconsciousness, incontinence, and convulsions or fatality. Persons with respiratory ailments, recent exposure to cholincsterase inhibitors, impaired cholinesterase production, or liver malfunction may be at increased risk from exposure to DM. Mutagenic effects due to dimcthoate exposure were seen in mice. An increase in malignant tumors was reported in rats given oral doses of 5. 15 or 30 mg/kg/day dimcthoate for over a year. The toxicity of the DM was extensively studied. However, many problems, e.g., the effect of DM on the monoamines ncurotransmittcrs in the rat brain and the mechanisms, are still unsolved and became the object of this study. This report summarizes our recent findings regarding the changes of neurotransmittcr and their biosynthesis metabolic enzyme that occur in discrete brain regions and serum. The results are discussed in relation to the mechanisms controlling the nerve agent DM-induccd seizures and ncuropathology.A rapid, reliable and simple method detecting monoamine ncurotransmitters in rat serum and brain tissue by high performance liquid chromatography with electrochemical detector was developed. An ODS column was selected as separation column at room temperature, and the mobile phase (pH4.50) consisted of .0.02M trisodium citrate and 0.05M disodium phosphate.containing 15 %mcthanol(volumc ratio)in distilled water. The mobile phase was pumped at a flow rale of 1.0ml/min and the oxidation potential was set at +0.65V. Dihydroxybenzylaminc was used as the internal standtard for quantification. Four compounds. epinephrine(E),dopamine(DA) and 5-hydroxytryptaminc(5-I(?)T) in the brain tissue(wholc cerebral cortex, hippocampus, slriatum. midbrain. cerebellum) and serum were simultaneouslyseparated and determined under the above conditions. RSD% of the retention times and peak areas of standard sample was in the range 0.29-0.67 and 0.09-0.36.The recoveries of all analytes were over 85%.The detection limits of 0.083ng/ml for NE,0.51ng/ml for E,0.046 ng/ml for DA and 0.078 ng/ml for 5-HT were achieved with standard solutions. The linearity is over the range 1.0-100 ng/ml for NE, 5.0-80 ng/ml for E, 1.0-80 ng/ml for DA and 1.0-60 ng/ml for 5-HT. The assay has been applied successfully to measure simultaneously cortex and serum concentrations of these four monoamincs in rats.The alterations of monoaminc levels in the rat brain and serum were determined after the Dimelhoale(DM) induced intoxication, including the alterations of (?)pincphinc(NE), cpinephinc(E). serotonin(5-HT), dopamine(DA) and its metabolite (3.4-hydroxypheny(?)aceticacid, DOPAC) level. Groups of rats were treated with saline and 38.9. 83.7 and 180 mg/kg DM respectively and were decapitated at different lime course from 0.5 to 24 hours after the administration. The monoamines ncurotransmitters were determined by reverse-phase high-performance liquid chroniatography with electrochemical detection. The concentrations of DA(8-296% of control). DOPAC(13-67% of control) and 5-HT(3-27% of control) increased according with the DM dose and the exposure time, while the levels of NE(10-55 % of control) and E.(11-33% of control) contents decreased at the same time. These findings indicate that DM-induced toxic effects can alter the monoamine levels after the different dose and time exposure in the rat brain and scrum suggesting that monoaminergic mechanism is involved in the DM intoxication.To testify monoaminergic mechanism, the change of enzyme dopamine β-hydroxylase (Dβ(?)) activity in the rat striatum and scrum was determined after the dimethoate (DM) induced intoxication. The 3 administration groups received DM (38.90mg/kg, 83.70mg/kg. 180.0mg/kg, respectively) and killed at different time-course(0.5,2.8,24h respectively )after DM administration. The DβH activity is determined by reverse-phase high-performance liquid chromatography with electrochemical detection. Levels of DβH activity (6-59% of control) decreased with the increase of DM dose and changed with different time-course. Level change of DβH activity in dose-effect showed significant linear relationship (correlation r= -0.9967) at 2h time-course in the rat striatum and serum. These findings indicated that DM-induced toxic effects can decline DβH activity level in different exposure dose and at different treated time-course, suggesting that different toxic effect mechanisms arc involvedThe activity of monoaminc oxidase (MAO) in the rat striatum and scrum was also determined after the dimethoate (DM) induced intoxication. No significant changes of MAO activity level were observed with,the increase of DM dose and changed with different lime-course. These results indicated that DM-induced toxic effects can not change MAO activity level in different exposure dose and at different treated time-course, suggesting that toxic effect mechanisms dimethoate induced intoxication arc not involved in the inhibition of MAO.These changes in monoaminergic systems include a significant increase in the turnover rate of DA and marked release and depletion of NE. and E ensued, the inhibition of enzyme dopamine β-hydroxylase (DβH) activity or the reduction in DβH, not the inhibition of monoamine oxidasc (MAO). Some tests are designed to determine the activity of DβH. The findings testify that a slow inhibition of DβH activity or the reduction in DβH content result in concurrent elevation of DA and decrease in levels of NE and E.A rapid, reliable and simple method detecting dopamine (DA) and two metabolites of DA. 3. 4-hydroxyphcnylaccticacid(DOPAC) and homovanillic acid (HVA) in the rat nigrostriatal slice by high performance liquid chromatography with electrochemical detector was also developed. An ODS column was selected as separation column at room temperature, and the mobile phase (pH4.50)consisted of 0.02M trisodium citrate and 0.05M disodium phosphatc.containing 10 % acetonitril(volumc ratio)in distilled water. The mobile phase was pumped at a flow rate of 1.0ml/min and the oxidation potential was set at +0.85V. RSD% of the retention times and peak areas of standard samples was in the range 0.34-0.47, 0.61-0.78 and 0.41-0.58. The recoveries of all analytes were over 85%.The detection limits of 0.045ng/ml for DA.0.18ng/ml for DOPAC.0.36 ng/ml for HVA were achieved with standard solutions. The linearity is over the range 1.0-100 ng/ml for DA. 5.0-100 ng/ml for DOPAC and 5.0 100 ng/ml forHVA .The assay has been applied successfully to measure simultaneously concentrations of these dopamine (DA) and two metabolites of DA. DOPAC and HVA in the rat nigrostriatal sliceThe alterations of dopamine (DA) and two metabolites of DA. DOPAC and HVA in the rat nigrostriatal slice were determined after the dimcthoate (DM) induced intoxication. Groups of slices were treated with saline and 3.89. 8.37 and 18 mg/ml DM respectively at 2hr time cousc, and were treated with 18 mg/ml at different time course from 0.5 to 24 hours. The levels of DA, DOPAC and HVA were determined by reverse-phase high-performance liquid chromatography with electrochemical detection. The concentrations of DA (36-260% of control). DOPAC (9-302% of control) and HVA (18-696% of control) increased according with the DM dose and the exposure time. These findings further demonstrate that DM-induced toxic effects can alter the levels of DA. DOPAC and HVA after the different dose and time exposure in the rat nigrostriatal slice, suggesting that monoaminergic mechanism is involved in the DM intoxication.In conclusion, the dimcthoate is not only a cholinesterase-inhibiting compound but also a dopamine-β-hydroxylase inhibitor. These findings prove the monoaminergic mechanism in the DM intoxication.更多还原