Study On The Novel Determinations Of Paraquat And Postmortem Redistribution, Diffusion In Mouse Models

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 C₁₂H₁₄N₂·2Cl 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 serumObjective: 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 was0.02⁵.0μg/ml, and the linear equation of the method was A=36977c－517, R²=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 detectionObjective: 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^-51.0×10^-3mol/L, and the linear equation of the method was H=5.5921c+0.2537,R²=0.9957. The detection limit was3.3×10^-6mol/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 modelsObjective:（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 of8LD₅₀. 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 received8LD₅₀parquat 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 received4LD₅₀doseparaquat 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 received2LD₅₀and8LD₅₀dose 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 by8LD₅₀paraquat 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）2LD₅₀,4LD₅₀and8LD₅₀threedoses with stable temperature20℃and72hours diffusion time were designedto study the influence of dose on postmortem diffusion;（7） Three temperaturelevels of-20℃,4℃and20℃with8LD₅₀dose and72hours diffusion timewere used to study the storage temperature on postmortem diffusion.Summary:（1） The postmortem distribution, decomposition kinetics anddiffusion models of paraquat (8LD₅₀/4LD₅₀) 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 by8LD₅₀paraquat 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-ECDObjective:（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:40⁴5g; 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 optimization①Blood 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 of0.5⁴0ng/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 of10¹000ng/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 experiments①Catecholamines 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 by8LD₅₀paraquat 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,4LD₅₀PQ 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.