Studies On Poisoning Characteristics Of Organophosphosphorus Toxicant On AChE And Efficacy Of Reactivators Based On Quantitative Determination Of Effect Biomarkers By LC-MS

Organophosphosphorus toxicant is a class of compounds containing phosphorus,and normally divided into nerve agents and pesticides.Effective standard therapy for acute OP poisoning includes administration of atropine as symptomatic therapy(antagonizing muscarinic receptors)and oximes as causal therapy,in which atropine can antagonize the muscarinic receptors and release the poisoning symptoms,but the administrated amount of atropine is difficult to master during the emergency.It often happens to poisoning of atropine due to its overdose in the clinical treatment.It's well known that the enzyme reactivators representing oxime derivatives of quarternary pyridinium compounds are considered as the specific medications which are generally used to the therapy of OP poisoning.However,the exactly clinical effectiveness of oxime reactivators is a matter of controversy in recent years.On one hand,oxime reactivators can recover the activity of intoxicated AChE in nerve or muscular tissues and improve neuromuscular transmission,thus the body functions will gradually restore.On the other hand,some study reports found oximes could also react with OPs and form more potent inhibitors of AChE that are identified as phosphylated oxime during the process of reactivation,which may intoxicate the reactivated AChE once again,even worsen the poisoning of enzyme due to the high reactivity.Up to now,the characterization of OPs poisoning and the evaluation of the effectiveness of antidotes are mainly studied by the determination of AChE activity such as the well-known Ellman assay,which has several limitations due to the low sensitivity and the lack of specificity.The published research demonstrated the assays based on the determination of enzyme activity could not give the accurate results in the case that the enzyme inhibition level was lower than 20%.Furthermore,these assays are also easy to be interfered by BChE or many other chemicals such as cholinesterase inhibitors and some drugs etc.The assays cannot precisely and directly demonstrate the exact statuses(uninhibited,inhibited and aged)of AChE before and after NAs intoxication or reactivator administration.The proteolysis-combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)method was developed in our laboratory for studying the poisoning and remedy of nerve agent through quantitative determination of the specifically adducted peptides of AChE.In comparison with Ellman assay,this method can not only describe the different statuses of the target enzyme during poisoning and therapy,but also accurately characterize the case that the inhibition rate of AChE was less than 1%.In order to solve the problems of OPs poisoning and the shortcomings of existing methods,we carry out an in-depth study.This dissertation is composed of four chapters.The first chapter is the preface.The introduction,toxicological mechanism,analytical methods of biomarkers and the research and development of specific antidotes of organophosphosphorus compounds were systemically described.The purposes and significance,contents and innovations of this study were proposed.In chapter two,with the curtailed use of organochlorine insecticides,organophosphorus compounds became one of the most important pesticides throughout the world,the security problems are increasingly concerned.There are normally dimethoxy or diethoxy convalently linked to phosphorus in the structures of OP pesticides.Based on these features,the specifically adducted peptides were prepared,and then the poisoning characteristics of DDVP,dipterex,monocrotophos paraoxon on AChE and efficacy of reactivators were studied by quantitative determination of effect biomarkers previously established by our group.In chapter three,the reaction product diethylphosphoryl obidoxime(DEP-obidoxime)was first prepared between paraxon and obidoxime in the suitale veronal buffer solution.Then,the poisoning characteristics of phosphylated oxime on AChE and efficacy of reactivators were studied by quantitative determination of the effect biomarkers formed between phosphylated oxime and AChE.The experimental results confirmed that DEP-obidoxime can intoxicate AChE in extremely short time,fortunately,the poisoned AChE by DEP-obidoxime can still be reactivated by typical oximes(pralidoxime,pyramidoxime monohydrate(HI-6),obidoxime).EC50 values of each oxime were determined and indicated that the reactivation effectiveness of the above oximes as the following ranking: Pralidoxime> HI-6> obidoxime.A new founding was first reported that some oxime reactivators can worsen the inhibition or aging levels of GD poisoned AChE in our previous research work.We speculate that the oxime reactivatiors can react with GD and form more potent inhibitors of AChE during the process of reactivation,which may intoxicate the reactivated AChE once again,even worsen the poisoning of enzyme due to the high reactivity.Based on this discovery,we studied the reaction characteristics of GD and reactivatiors by UV and GC in chapter four.,The results showed that GD didn't react with tertiary amine reactivatiors but react significantly with oximes.At the same time,the reaction products PMPA-obidoxime and PMPA-pralidoxime formed between GD and obidoxime or pralidoxime were successfully obtained.