摘要
百草枯又名克芜踪、对草快,化学名是1,1-二甲基-4,4联吡啶阳离子盐,分子式为C_(12)H_(14)N_2·2Cl,分子量为257.2Da,是目前全世界广泛使用的有机杂环类接触性脱叶剂,具有触杀作用和一定内吸作用。纯品为白色结晶,以阳离子形式存在,易溶于水,微溶于乙醇和丙酮,在酸中稳定存在,在碱中易被分解,常用制剂为20%的水溶液。口服中毒死亡率可达90%以上,目前已被20多个国家禁止或者严格限制使用。
百草枯对哺乳动物具有较强的毒性,尤其是人。目前也没有特效药可以解毒,口服中毒死亡率可达90%以上。据报道,每年在亚洲、美洲、欧洲都有大量的百草枯死亡案例出现,已报告的死亡病例达数百例,多是经口误服致死。
百草枯可通过消化道、受损的皮肤等途径进人体,中毒后死亡率高,尤其以肺部最为突出,此外还对全身其他脏器造成严重损伤。大脑作为神经中枢部位,对毒物反应极为敏感。百草枯对哺乳动物脑组织中单胺类神经递质存在的危害,为探讨其神经毒性提供了试验素材。
百草枯可以引起多脏器的损伤,尤其是肺部最为明显。肺部损伤主要表现形式为肺水肿、肺泡出血、炎症细胞侵润、胶原沉积和成纤维细胞增生等。早期患者在中毒几天内死于急性呼吸窘迫综合征和多脏器功能的衰竭。即使早期存活的患者也会因肺间质纤维化在数周内死亡,个别幸存病人也由于肺间质的纤维化其生存质量严重下降。
基于上述原因,本实验研究首先建立了两种检测百草枯的新方法,又将小鼠作为动物模型,研究了百草枯在其体内的死后分布、死后再分布和死后弥散现象,观察了中毒小鼠血液及脑中单胺类神经递质的变化情况。第一部分除草剂百草枯测定方法的建立
(1)毛细管电泳—紫外检测法测定血清中百草枯方法的建立
目的:建立一种准确、简便、快速的方法用于测定百草枯的含量。
方法:将商品除草剂直接稀释后上样;血清中的百草枯则需要C_(18)固相萃取后上样,在256nm紫外波长处测定百草枯的含量。毛细管电泳法采用重力进样,进样高度25cm,进样时间15s,弹性石英毛细管内径75μm,有效分离长度55cm,分离电压12kV,以浓度20mmol/L,pH3.5的NaH_2PO_4缓冲溶液,1.0mmol/L的十二烷基硫酸钠(SDS)作为电泳缓冲液进行分离。
结果:毛细管电泳—紫外检测法测得百草枯线性范围为0.02~5.00μg/ml,线性回归方程A=36977c-517,相关系数0.9984。平行进样7次,其RSD为4.52%,计算所得百草枯的检出限为3.0ng/ml。测得除草剂中百草枯的实际含量是182.1g/L。将百草枯实际样品稀释2×105倍过滤后,加入0.02,1.00,2.50μg/ml的标准品测得PQ的回收率分别为96.7%,90.1%,95.6%,平均回收率为94.1%,平均RSD为2.17%。向血清中分别加入上述PQ的标准品固相萃取后,测得其回收率分别为85.0%,88.0%,82.0%,平均回收率为85.0%,平均RSD为4.79%。
小结:毛细管电泳紫外检测法测定除草剂中百草枯的含量方法准确可靠,结果令人满意。
(2)修饰电极—电化学法测定除草剂中百草枯的含量
目的:研究百草枯在番红花红/金纳米/DNA修饰的玻碳电极的电化学行为,并用于实际样品的测定,从而建立一种准确、简便、快速的方法用于测定百草枯的含量。
方法:(1)修饰电极的制备:将裸玻碳电极置于Al2O3粉末的悬浆之中进行打磨至镜面,取出,依次用1:1的丙酮、1:1的硝酸、二次蒸馏水,各超声清洗5分钟,氮气吹干。(2)条件选择:找出SFR聚合最佳浓度、SFR最佳聚合圈数、金纳米浸泡最佳时间、DNA浸泡最佳时间和缓冲液最佳pH值。(3)在最佳条件下,利用差分脉冲法测定不同浓度百草枯在修饰电极上的线性关系。(4)在2×10-4mol/L百草枯溶液中,考察修饰膜的稳定性及干扰物对其测定时的影响。(5)利用SFR/Au/DNA聚合膜电极对百草枯样品进行了测定,将其稀释至所在范围浓度,在最佳条件下用差分脉冲法进行测定。
结果:(1)条件选择:SFR的最佳浓度为2mmol/L;SFR的最佳聚合圈数为30圈;金纳米修饰时间为8h;DNA修饰时间为4h;缓冲溶液最佳pH为7.0;(2)利用修饰电极,电化学检测法测得百草枯线性范围为1.0×10~(-5)~1.0×10~(-3)mol/L,线性回归方程H=5.5921c+0.2537,相关系数R~2=0.9957。平行测定11次,其RSD为5.84%,计算所得百草枯的检出限为3.3×10~(-6)mol/L。测得除草剂中百草枯的实际含量是183.6g/L。将百草枯实际样品稀释后,加入0.05,0.25,0.50mmol/L的标准品测得PQ的回收率分别为100.0%,97.4%,97.4%,平均回收率为98.3%;平均RSD为4.70%。
小结:发现SFR/Au/DNA聚合膜对于百草枯的还原能够起到明显的电催化作用,该电极已用于实际样品测定,结果令人满意。
第二部分百草枯在小鼠体内的死后分布、死后再分布及死后弥散研究
目的:(1)建立小鼠百草枯死后分布、埋葬尸体中的分解动力学及死后百草枯灌胃死后弥散的动物模型;(2)研究百草枯在小鼠体内的死后分布及埋葬尸体中的分解动力学;(3)研究百草枯在小鼠体内的死后弥散现象。
方法:(1)百草枯在埋葬尸体中的分解动力学研究
①取健康昆明小鼠28只,8LD_(50)剂量百草枯灌胃染毒致死。0天的死后立即解剖取材,其余装入塑料袋,不封口,埋于河北医科大学本部公共卫生学院西面花园中。分别于21、42、63、84、105、126天后挖出,解剖取材,高效液相色谱法检测其中百草枯的含量;②取健康昆明小鼠8只,8LD_(50)剂量百草枯灌胃染毒致死,死后随机分成2组,分别装于木箱和编织袋中,埋于花园中,63天后挖出解剖取材,检测其中百草枯的含量;③取健康昆明小鼠8只,以同样剂量处死后,分别于2012.3.3与2012.5.5埋葬,63天后(即2012.5.5及2012.7.7)挖出;解剖取材,检测百草枯的含量。
(2)百草枯在小鼠体内的死后弥散研究
①将小鼠脊椎脱臼处死,1小时后,4LD_(50)百草枯灌胃,将染毒的小鼠置于常温环境下(20℃),分别于6、12、24、48、72、96h各解剖4只小鼠,取心、肾、脑、肺、右下肢肌肉,血液。将小鼠的肝脏分为四个部分,分别是:(Ⅰ)肝左叶;(Ⅱ)肝中间部分;(Ⅲ)肝右叶;(Ⅳ)肝基底部位。称重,测其百草枯的含量;②将小鼠脊椎脱臼处死,1小时后,4LD_(50)百草枯灌胃,将染毒的小鼠分别置于4℃和-20℃,72h后解剖,取心、肝、肾、脑、肺、右下肢肌肉,血液,称重,测其百草枯的含量;③将小鼠脊椎脱臼处死,1小时后,以2LD_(50)和8LD_(50)百草枯灌胃,将染毒的小鼠置于20℃,72h后解剖动物,取心、肝、肾、脑、肺、右下肢肌肉、血液,称量,测其百草枯的含量。
结果:(1)百草枯在小鼠体内的死后分布:中毒小鼠死亡后体内各个组织都检测出百草枯,其中胃>肠>肾>肝>脾>肺>血液>右下肢肌肉>脑>心;(2)埋葬尸体中百草枯随时间变化:8LD_(50)灌胃致死小鼠埋葬尸体中PQ的含量呈现先上升,后下降的趋势。埋葬42天后尸体心、肝、脾、肺、肾、胃、肠、脑、右下肢肌肉中百草枯含量升至最高,之后缓慢下降;(3)不同埋葬方式研究结果表明埋葬63天塑料袋包装小鼠尸体心、肺、肝、肾、脑、肌肉中百草枯含量平均值均高于编织袋及木箱包装,说明编织袋保存尸体中百草枯分解较快,木箱次之,塑料袋最慢。(4)不同季节埋葬结果显示:冬天埋葬的小鼠尸体心、肺、肝、肾、脑、肌肉中百草枯含量平均值均高于春天及夏天埋葬的小鼠;(5)死后6小时在小鼠体内各脏器检出百草枯,说明百草枯在死后尸体内是可以发生弥散的。肝左叶是百草枯集中的地方,其百草枯浓度/百草枯血液浓度的比值最高,达到7.87±2.32。另外肝脏中四个不同区域的百草枯平均浓度分别是13.57、11.94、3.89和3.36μg/g(肝左叶、肝基底部位、肝中间部位、肝右叶)。将每一只小鼠测定的不同组织中的百草枯浓度标出,并作出各个脏器的线性关系,其中:肝(R~2=0.4565)、肾(R~2=0.3882)、肺(R~2=0.5414)、脑(R~2=0.6846)、心(R~2=0.6600)、右下肢肌肉(R~2=0.4937);(6)不同储存温度结果显示:72h后,20℃储存的小鼠尸体中,心、肺、肝、肾、脑、右下肢肌肉中百草枯含量平均值均高于4℃及-20℃;(7)不同灌胃剂量结果显示:8LD_(50)灌胃剂量的小鼠尸体中心、肺、肝、肾、脑、右下肢肌肉中百草枯含量平均值均高于2LD_(50)和4LD_(50)。
小结:(1)本实验分别采用8LD_(50)和4LD_(50)剂量百草枯灌胃染毒,建立了百草枯的死后分布和死后弥散模型,可应用于百草枯中毒(死)的法医学检验及法医毒物动力学研究。(2)8LD_(50)灌胃致死小鼠埋葬尸体中百草枯的含量呈现先上升,后下降的趋势。埋葬42天后尸体心、肝、脾、肺、肾、胃、肠、脑、右下肢肌肉中百草枯含量升至最高,之后缓慢下降。埋葬126天后,小鼠心、肝、脾、胃、肾、肺、肠、右下肢肌肉仍能检测到百草枯;不同埋葬方式对中毒小鼠体内百草枯的含量有一定影响,编织袋分解较快,木箱次之,塑料袋最慢;埋葬季节对中毒小鼠体内百草枯的含量也有影响,冬天温度低,百草枯分解较慢;夏天温度高,百草枯分解较快。(3)小鼠死后1小时4LD_(50)百草枯灌胃后,6h后可在其各个脏器中检测到百草枯,说明百草枯在小鼠体内可发生死后弥散。从肝脏四个部分可以看出,离胃越近的部分,弥散的浓度越大。这与百草枯本身的理化性质、染毒剂量、尸体保存温度及时间、弥散距离和尸体的姿势有关。(4)在百草枯中毒死亡埋葬尸体法医学鉴定时,应根据埋葬时间、埋葬方式、埋葬季节、服毒剂量等因素对尸体中百草枯分解的影响,结合中毒方式和生前抢救情况,及早进行尸体挖掘和检验,全面取材,大致推断埋葬当时尸体中百草枯的含量范围。除了要提取胃内容物、肺、心血等常见的检材外,还应当采取肾,肝,脾,脑等组织进行定性、定量分析,并充分考虑毒物的剂量、死亡时间、尸体所处的环境、体内微生物等影响因素,结合临床表现、病理报告等综合分析,为百草枯误服案件、中毒死亡案件和死后灌毒案件的甄别提供科学客观的依据。
第三部分单胺类神经递质在百草枯中毒小鼠血、脑组织中的变化研究
目的:(1)建立单胺类神经递质在小鼠血液和脑中的检测方法;(2)研究百草枯中毒小鼠血液和脑中单胺类神经递质含量的变化情况。
方法:(1)条件优化:本研究采用高效液相色谱—库仑阵列电化学检测器(HPLC-ECD)对小鼠血液及脑组织中的单胺类神经递质及其代谢产物的检测方法进行了系统研究。通过对样品的前处理方法的改进,对检测电极电势的选择和流动相条件的优化,确定了小鼠血液及脑组织中神经递质及其代谢产物的含量的检测条件。
(2)动物实验:对照组:健康雄性昆明小鼠,40~45g;中毒组:将健康昆明小鼠以10mg/kg灌胃后,分别于1、3、7、14、21天断头处死,解剖(解剖前注入肝素,以防血液凝固),取心血、脑。小鼠血液前处理用固相萃取;小鼠脑组织样品直接用5%的磺基水杨酸处理,取50μl直接进入HPLC进行检测。
结果:(1)条件优化:
①检测小鼠血液中儿茶酚胺的色谱条件为:流动相缓冲液为20mmol/L柠檬酸三钠,用HCl调pH为3.25,经0.22μm的滤膜过滤后加11%(v/v)的甲醇,应用前经超声脱气,流速为0.5ml/min,柱温保持25℃,电化学检测器电压设置为0.85V。在此条件下,NE、E、DA在0.5~40ng/ml呈现良好的线性关系,检出限依次为0.4、0.4、0.2ng/ml,回收率为89.0%~100.0%,日内及日间RSD均小于6%。
②检测小鼠脑中神经递质的色谱条件为:流动相缓冲液为20mmol/L柠檬酸三钠,HCl调pH4.25,经0.22μm的滤膜过滤后加10%(v/v)的甲醇,应用前经超声脱气,流速为1.0ml/min,柱温保持25℃,电化学检测器电压设置为0.75V。在此条件下,DA、MHPG、5-HT、DOPAC、5-HIAA、HVA在10~1000ng/ml呈现良好的线性关系,其检出限为依次为0.2、0.4、0.1、0.4、0.1、0.3ng/ml,六种神经递质的回收率在86.6%~94.5%,日内及日间RSD均小于5%。
(2)动物实验:
①正常及中毒组小鼠血液中儿茶酚胺类物质的测定:中毒后小鼠的儿茶酚胺含量比正常组的平均值低,到21天后,中毒小鼠血液中的NE、E、DA分别是正常组的53%、73%和40%,达到了极显著差异(P<0.01)。
②正常及中毒组小鼠脑组织中六种单胺类神经递质的测定:中毒后小脑中的神经递质含量比正常组的平均值低,到21天后,中毒小鼠脑中的DA、DOPAC、HVA、MHPG、5-HT、5-HIAA分别是正常组的77%、82%、58%、85%、81%和84%,DA达到了显著差异(P<0.05),其余五组达到了极显著差异(P<0.01)。
小结:(1)本文对所建立的方法均进行了周密的验证,结果显示此方法准确、灵敏,操作简单,方便快捷,适宜生物样品的检测;(2)NE、E、DA、DOPAC、5-HT、5-HIAA、MHPG及HVA都是中枢神经系统内重要的神经递质,在神经信号的传递中发挥重要作用,实验结果表明百草枯可导致小鼠血液中儿茶酚胺及脑组织中单胺类神经递质含量减少,影响神经兴奋的传递,对小鼠神经系统具有一定的毒性作用。
结论:本研究建立了两种检测除草剂百草枯的新方法(CE-UV,ECD),并分别与传统经典高效液相方法做了对比,发现其检测结果准确,检测范围较宽,能满足实际样品的测定需要,可以在法医毒物分析中进行应用。
创建了百草枯急性中毒的小鼠模型,在此基础上对百草枯在小鼠体内的死后分布、死后再分布及死后弥散进行了一系列的研究,发现8LD_(50)灌胃致死小鼠埋葬尸体中百草枯的含量先上升,后下降,在42天达到极值;埋葬126天后,小鼠各个脏器仍能检测到百草枯。小鼠死后1小时4LD_(50)百草枯灌胃后,6h后可在其各个脏器检测到百草枯,说明其在小鼠体内可以发生死后弥散,且离胃越近的部位,弥散的浓度越大。
建立了单胺类神经递质在小鼠血液和脑中的检测方法,并且研究了百草枯中毒前后小鼠血液和脑中单胺类神经递质(NE、E、DA、DOPAC、5-HT、5-HIAA、MHPG及HVA)含量的变化情况,结果显示百草枯可导致小鼠血液中儿茶酚胺及脑组织中单胺类神经递质随着时间的延长而减少(21d),影响神经兴奋的传递,对小鼠神经系统具有一定的毒性。
Paraquat (1,1’-dimethyl-4,4’-bipyridinium) and its dichloride salt arebroad-spectrum contact plant killers and herbage desiccants that wereintroduced commercially during the past50years. It is one of the most widelyused herbicidal chemicals in the world and is now available in more than130countries. Its molecular formula and weight are C_(12)H_(14)N_2·2Cl and257.2Da,respectively. The pure substance is white crystal; it usually exists as cationicform. It can be easily dissolved in water, and slightly dissolved in ethanol andacetone. It is stable in acid solutions, and can easily decompose in alkalinesolutions.20%aqueous solutions are usually used in commercial market.
The biochemical mechanism of paraquat toxicity is due to the cyclicoxidation and reduction in tissues, leading to production of superoxide anionand other free radicals and eventually the highly destructive hydrogenperoxide. The lung is the organ most severely affected in paraquat poisoning,due largely to the preferential accumulation of paraquat in lung alveolar cells.Although many organs are affected by paraquat, death is usually due toprogressive pulmonary fibrosis. At present, there is no completely successfultreatment for paraquat-induced lung toxicity.
Numerous human injuries and deaths have resulted from intentionalingestion of the concentrated commercial product. Most poisonings resultedfrom the ingestion of the21%cation concentrated, which had been decantedand stored in empty beer, soft drink, or lemonade bottles; paraquat is areddish-brown liquid that resembles root beer or cola drinks.
Initial clinical signs depend on the route of exposure. Early symptomsand signs of poisoning by ingestion are burning pain in the mouse, throat,chest, and upper abdomen, due to the corrosive effect of paraquat on themucosal lining. Diarrhea, which is sometimes bloody, can also occur. Giddiness, headache, fever, myalgia, lethargy, and coma are other examples ofCNS and systemic findings. Pancreatitis may cause severe abdominal pain.Proteinuria, hematuria, pyuria, and azotemia reflect renal injury. Oliguriaindicates acute tubular necrosis.
The lung is the primary target organ of paraquat, and pulmonary effectsrepresent the most lethal and least treatable manifestation of toxicity. However,toxicity from inhalation is rare. The primary mechanism is through thegeneration of free radicals with oxidative damage may to lung tissue. Whileacute pulmonary edema and early lung damage may occur within a few hoursof severe acute exposures, the delayed toxic damage of pulmonary fibrosis, theusual cause of death, most commonly occurs7-14days after the ingestion. Inpatients who ingested a very large amount of concentrated solution (20%),some have died more rapidly (within48h) from circulatory failure.
Therefore, firstly in this study, we estebished three methods to detect PQ;secondly, the distribution, redistribution and postmorterm diffusion ofparaquat in mouse models were studied; thirdly, the determination ofmonoamine neurotransmitters in normal/paraquat-induced mouse brains byHPLC-ECD was established.
Part Ⅰ The esteblishments of herbicide paraquat determinations
(1) CE with UV detector in determination of paraquat in serum
Objective: To establish an accurately, simple and fast method todetermine the content of paraquat in serum and herbicide.
Methods: The herbicide contained paraquat was diluted and analyzeddirectly by CE. Firstly the protein in serum was precipitated by chloroformand phenol, and then the PQ in serum can be detected by UV detector set at256nm. The separation and determination condition was as follows:gravitation sampling, the time was15seconds, the height was25cm; runningvoltage:15kV, bouncy silica capillary:75μm id., total length70cm,55cmfrom the inlet to UV detection; electrolyte:20mmol/L monosodium phosphate,1mmol/L lauryl sodium sulfate, pH3.5.
Results: The linear range of PQ was0.02~5.0μg/ml, and the linear equation of the method was A=36977c-517, R~2=0.9984. The detection limitwas3ng/ml. The relative standard deviation (RSD) was4.52%. The recoverywere96.7%,90.1%,95.6%, respectively after diluting PQ production2×105times. The actual content of PQ in the herbicide was182.1g/L. The averageRSD was2.17%. The serum with PQ standard was pre-treated by solid-phaseextraction, and then detected by HPLC. The recovery of standard additionwere85.0%,88.0%,82.0%, respectively, the average RSD was4.79%.
Summary: The proposed method has been satisfactorily applied for thedetermination of herbicide PQ, and the result was accurate and reliable.
(2) The determination of PQ in herbicide by electro-chemical detection
Objective: To establish an accurately, simple and fast method todetermine the content of paraquat in serum and herbicide.
Methods:①The base GCE was polished to a mirror-like surface withalumina slurries down to0.05mm, rinsed after each polishing withdouble-distilled water, and ultrasonicated in HNO3(1:1v/v), acetone (1:1v/v),and double-distilled water in succession. The base GCE was then dried undera nitrogen flow until use;②Optimized condition: the concerntration of SFR,the circles of SFR modified on the electrode, the immersion time of the Au,the immersion time of the DNA, the pH of the buffer;③In the best conditions,the lineary range of PQ was studied by differential pules voltammetry (DPV).④In the best conditions of2×10-4mol/L PQ solution, the stability of themodified electrode was studied.⑤Real samples were detected by themodified elecreode using DPV.
Results:①Optimized condition: the concerntration of SFR was2mmol/L, the circles of SFR modified on the electrode was20, the immersiontime of the Au was8h, the immersion time of the DNA was2h, the pH of thebuffer was7;②In optimum conditions, the hight of the peak wereproportional to the concentration of paraquat in the range of1.0×10~(-5)~1.0×10~(-3)mol/L, and the linear equation of the method was H=5.5921c+0.2537,R~2=0.9957. The detection limit was3.3×10~(-6)mol/L, with a relative standarddeviation (n=11) of5.84%. The PQ in the herbicide marked200g/L was actually183.6g/L, and the recoveries range was97.4%~100.0%, average RSDwas4.70%.
Summary: The proposed method has been satisfactorily applied for thedetermination of herbicide PQ, and the result was accurate and reliable.
Part Ⅱ The studies on the distribution, redistribution and postmortermdiffusion of paraquat in mouse models
Objective:(1) To establish the postmortem distribution model ofparaquat in mouse; to establish the decomposition kinetics model of paraquatin buried cadaver of mouse; and to establish the postmortem diffusion modelof paraquat in mouse;(2) To investigate the postmortem distribution ofparaquat in mouse and the decomposition kinetics of paraquat in buriedcadaver of mouse;(3) To investigate the postmortem diffusion of paraquat inmouse.
Methods:(1) Study on the decomposition kinetics of paraquat in buriedcadavers.
①28mouse were given an intra-gastric administration of PQ with adose of8LD_(50). As soon as they died, the mice were put into plastic unsealedbags, and buried in the garden to the west of our building in Hebei MedicalUniversity. Four of them were dug out, dissected and the specimen werecollected for analysis of PQ at0d,21d,42d,63d,84d,105d and126d after theburying by HPLC-UV;②8mice were divided into2group,4of which wereput into mash bag, and the other4were put into coffins. They were dug out,dissected on the63rdday for analysis, and the data were compared with themice packed with unsealed plastic bags on63rdday;③8mice were received8LD_(50)parquat on Mar/3/2012and May/5/2012, respectively, after63days(May/5/2012and Jul/7/2012) the8mice were dug out, dissected, and the datawere compared with the63rdday from Nov/19/2011to Jan/21/2012.
(2) Study on the postmortem diffusion of paraquat in mice:40mice weresacrificed by cervical dislocation.32of them were received4LD_(50)doseparaquat after1h.4mice were put into a fridge at4℃and-20℃, respectively.After72h, they were dissected for the analysis. The rest24mice were left lying horizontally on their back at room temperature (20℃) for6h,12h,24h,48h,72h and96h. Additionally, the mouse liver was divided into4parts:(Ⅰ):The left lobe of liver,(Ⅱ) The median lobe of liver,(Ⅲ) The right lobeof liver;(Ⅳ) The caudate. Weight and detect PQ;4mice were received2LD_(50)and8LD_(50)dose paraquat after1h, respectively.72h later, they werealso dissected for the analysis.
Results:(1) Distribution: PQ was detected in the tissues of mice,including some important organs. The concentration of PQ was: stomach>intestine> kidney> liver> spleen> lung> blood> right thigh muscle>brain> heart;(2) PQ in mice poisoned by8LD_(50)paraquat firstly increased andthen decreased. The paraquat in brain, heart, kidney, liver, lung, and thighmuscle reached their vertex on the42ndday, after being buried126days; theydecreased to the bottom;(3) Different burial ways showed that after63days,the differences of paraquat concentration in solid tissues among plastic bags,coffins and mash bags. It showed that there were significant concentrationdifferences in brains and hearts (P<0.05);(4) Also the results of burial indifferent seasons showed that PQ concentration in solid tissues (liver, lung andkidney) were significant among winter, spring and summer (P<0.05);(5)Liver had the highest drug concentration of the tissues investigated. There washowever a striking difference in PQ concentration within the liver; the lobeslying closest to the stomach, the left lobe of liver and the caudate lobe, havingthe highest concentrations. Furthermore, the concentration of PQ found in thebrain, heart, kidney, liver, lung and thigh muscle showed a significant rise(P<0.01) with increasing time after death;(6)2LD_(50),4LD_(50)and8LD_(50)threedoses with stable temperature20℃and72hours diffusion time were designedto study the influence of dose on postmortem diffusion;(7) Three temperaturelevels of-20℃,4℃and20℃with8LD_(50)dose and72hours diffusion timewere used to study the storage temperature on postmortem diffusion.
Summary:(1) The postmortem distribution, decomposition kinetics anddiffusion models of paraquat (8LD_(50)/4LD_(50)) in mice have been developed, which can be applied to forensic identification and study on forensictoxicokinetics of decomposition of PQ poisoning death cases;(2) PQ in micepoisoned by8LD_(50)paraquat firstly increased and then decreased. The paraquatin brain, heart, kidney, liver, lung, and thigh muscle reached their vertex onthe42ndday, after being buried126days; they decreased to the bottom;different burial ways showed that after63days, the differences of paraquatconcentration in solid tissues among plastic bags, coffins and mash bags; alsothe results of burial in different seasons showed that PQ concentration in solidtissues (liver, lung and kidney) were significant among winter, spring andsummer;(3) There is a postmortem of PQ in the mice, which probably relatesto its physico-chemical property, dose, poisoning way, poisoning time afterdeath, the storage temperature and time of corpse to preserve, diffusiondistance, corpse posture;(4) When we deal with medico-legal expertiseassociated with paraquat poison, factors that may have impact on paraquatdecomposition should be taken into consideration apart from the poisoningway and rescue situation before death to make a comprehensive judgment ofthe value of the exhumation. Exhumation and examination, sample collection,and toxicology analysis should be done earlier, and given full consideration tothe impact of causal factors according to the phases of decomposition to infera general content range of paraquat when the body is buried. On the otherhand, paraquat postmortem diffusion can occur in the corpses, which mayhave connection with the dose of drug, preserving time, diffusion distance,storage temperature and etc. Therefore, in the forensic identification ofparaquat diffusion and its impact on toxicology analysis results should betaken into consideration. Besides examining the stomach contents, lung andheart blood, other samples like kidney, liver, spleen, and brain tissues shouldalso be extracted for comprehensive qualitative, quantitative analysis,especially in quantitative analysis of the poison in the stomach contents. Fullconsideration should be given to the influential factors such as doses, time ofdeath, environment of the corpse, vivo micro-organisms, etc. All these are combined with clinical manifestations, pathology reports and comprehensiveanalysis to provide scientific and objective basis for paraquat mistaken,poison-death and postmortem poisoning cases.
Part Ⅲ Determination of monoamine neurotransmitters inparaquat-induced mouse brains by HPLC-ECD
Objective:(1) To establish a new method to detect monoamineneurotransmitters in mouse blood and brain;(2) To observe the NTconcentration differences between normal and paraquat-induced mice.
Methods:(1) Chromatography conditions optimization: This study wasresearched NTs in mouse blood and brains by HPLC-ECD. The pre-treatmentsof the bio-samples were improved; the applied potential and mobile phasewere optimized;(2) Animal experiments: CK Group: healthy male KM mice,weight:40~45g; Toxic Group: healthy mice were given10mg/kg PQ,7ofthem were sacrificed at1st,3rd,7th,14th,21stday, respectively. The brains andblood were taken out. The blood pre-treatment was SPE; while5%sulfosalicylic acid was used to pre-treat brains. The supernatant or extractionneeds to be filtrated by a0.45μm filter membrane,50μl of which can beinjected into HPLC for analysis.
Results:(1) Chromatography condition optimization
①Blood conditions: Chromatography was performed at25℃on a250mm×4.6mm column, packed with5μm SinoChrom ODS-BP (Yilite,Dalian, China).The mobile phase consisted of11%v/v0.02M sodiumcitrate in methanol. The pH was adjusted from3.25by hydrochloric acid.The flow rate was0.5ml/min isocratic, and the injection volume was20μl. The applying potential of coulometric detector was set0.85V. Inthis conditions, the limits of detection (LODs, S/N=3) for catecholamineswere0.4,0.4,0.2ng/ml, respectively, with linear range of0.5~40ng/ml.The intra-and inter-day RSD value for the peak area were both <6%(n=7). The recovery was between89.0%and100.0%.
②Brain conditions: Chromatography was performed at25℃ona250mm×4.6mm column, packed with5μm SinoChrom ODS-BP (Yilite, Dalian, China).The mobile phase consisted of10%v/v0.02M sodiumcitrate in methanol. The pH was adjusted from4.25by hydrochloric acid.The flow rate was1.0ml/min isocratic, and the injection volume was20μl. The applying potential of coulometric detector was set0.75V. Inthis conditions, the limits of detection (LODs, s/n=3) for catecholaminesand their metabolites (DA、MHPG、5-HT、DOPAC、5-HIAA、HVA) were0.2、0.4、0.1、0.4、0.1、0.3ng/ml, respectively, with linear range of10~1000ng/ml. The intra-and inter-day RSD value for the peak area wereboth less than5%(n=7). The recovery was between86.6%and94.5%.
(2) Animal experiments
①Catecholamines in blood: The concentration of CAs in allPQ-induced groups was lower than CK group. After21days, CAs (NE, E,DA) concentration in toxic mouse was respectively53%,73%, and40%ofthat of CK group, with a significance level of0.01(P<0.01).
②NTs in brains: The concentration of six NTs in all PQ-induced groupswas lower than CK group. After21days, NTs (DA, DOPAC, HVA, MHPG,5-HT,5-HIAA) concentration in toxic mouse was respectively77%,82%,58%,85%,81%, and84%of that of CK group, with a significance level of0.01, except DA (P<0.05).
Summary:(1) This method is simple, convenient and sensitive, which issuitable for determination of catecholamines and their metabolites in mouseblood and brain;(2) NE, E, DA, DOPAC,5-HT,5-HIAA, MHPG, and HVAare all the important NTs in mammal CNS, which act important roles intransferring neural signals. The results indicated that PQ can lead to anincrease of NTs in mouse blood and brain, have an adverse neurological effect,causes major damage to the nervous system of mammals.
Conclusions: In this study three new methods were established to detectPQ (CE-UV, ECD), and compared with traditional HPLC method, we foundthese new methods were simple, sensitivity accurate, reliable and reproducible.These methods can be employed in forensic toxicology analysis.
To establish the postmortem distribution model of paraquat in mouse and the decomposition kinetics model of paraquat in buried cadaver of mouse; andthe postmortem diffusion model of paraquat in mouse. We found that PQ inmice poisoned by8LD_(50)paraquat firstly increased and then decreased. Theparaquat in brain, heart, kidney, liver, lung, and thigh muscle reached theirvertex on the42nd day, after being buried126days; they decreased to thebottom. The mice were sacrified after1h,4LD_(50)PQ was given. The PQ wasdetected in every tissue after6h. It indicated that there was a postmortem ofPQ in the mice, and the closer to the stomach, the higher the concentrationwas.
To establish a new method to detect monoamine neurotransmitters inmouse blood and brain, and observe the NT concentration differences betweennormal and paraquat-induced mice. We found that HPLC-ECD was simple,convenient and sensitive, which was suitable for determination ofcatecholamines and their metabolites in mouse blood and brain. NE, E, DA,DOPAC,5-HT,5-HIAA, MHPG, and HVA are all the important NTs inmammal CNS, which act an important roles in transferring neural signals. Theresults indicated that PQ can lead to an increase of NTs in mouse blood andbrain, have an adverse neurological effect, causes major damage to thenervous system of mammals.
引文
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