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1、Organphosphorus Compounds-Induced NeurotoxicityMohamed B. Abou-Donia, Ph.D.Department of Pharmacology and Cancer BiologyDuke University Medical Center Durham, North Carolina, USAdoniaduke.edu919-684-2221Organophosphorus compounds Used in medicine, industry, agriculture and as warfare agents. Have a
2、wide range of acute toxicity: a) low acute toxicity chemicals such as tricresyl phosphates (TCPs) b) highly toxic nerve agents such as sarin, soman and tabun.Actions of Organophosphorus Compounds1.Cholinergic Neurotoxicity2. Organophosphorus Ester-Induced Neurotoxicity (OPIDN)3. Organophosphorus Est
3、er-Induced Chronic Neurotoxicity (OPICN)1. Cholinergic NeurotoxicityInhibition of Acetylcholinesterase (AChE), an enzyme essential for lifeORGANOPHOSPHATEXR Organophosphate +dR(a)(b)(d)(c)phe 297phe 295acyl pocket(a)(b)(d)(c)phe 297phe 295acyl pocketPeripheral binding site(a)(b)(d)(c)phe 297phe 295a
4、cyl pocketPeripheral binding siteManifestations of Organophosphate PoisoningOptic SystemPupil ConstrictionBlurred VisionLacrimationRespiratory SystemBronchospasmBronchial SecretionPulmonary EdemaTightness of ChestWheezingCoughDifficulty BreathingGastrointestinal TractSalivationNauseaCrampsAbdominal
5、PainVomitingDiarrheaFecal IncontinenceUrinary - GenitalUrinary IncontinenceImpotenceUterus ContractionBrainHeadacheDizzinessVertigoAnxietyApathyConfusionAnorexiaInsomniaLethargyFatigueInability to ConcentrateMemory ImpairmentConvulsionComaCardiovascular SystemTachycardiaIncreased Blood PressureMuscu
6、latureWeaknessTremorFasciculationsTwitchingCrampsIncreased SweatingTreatment of Cholinergic Toxicity1. 2-PAM (2-pyridine aldoxime methiodide)hydrolyzes phosphorylated enzyme thus accelerating the regeneration of active AChE; should be administered rapidly within 10 to 15 minutes of exposure, before
7、AChE aging.2. Atropine, an antagonist of muscarinic ACh receptor (AChR)3. Shielding of AChEOrganophosphorus nerve agents,such as sarin act by irreversibly inhibiting AChE in the peripheral and central nervous systems. 2. Pyridostigmine Bromide (PB) is administered to protect against toxicity. PB is
8、approved by the FDA for soman. 3. Prophylaxis Principle is that PB acts by shielding AChE in peripheral nervous system to reversibly inhibit 30-40% of the enzyme, protecting it from permanent inhibition by the nerve gas.4. Enzyme activity is restored following spontaneous decarbamyalation of the ACh
9、E.Result Free enzyme and near-normal neuromuscular an autonomic functions.4. Bioscavengers 1.Butyrylcholinesterase (BChE) is a naturally occurring enzyme in blood.2.Its blood concentration is 2 mg/liter.3.BChE has no known function; however, it functions as the first line of defense against poisonin
10、g with organphosphorus compounds.4.It acts as a bioscavenger, like a sponge to absorb and degrade organphosphorus compounds (e.g., nerve agents and insecticides).Recombinant BChE (rBCHE)1.Recombinant human BChE (rBChE) is being developed under the trade name Protexia as a pre- and post-exposure ther
11、apy for organphosphorus compound poisoning.2.Protexia is a pegylated rBChE, that is formed by conjugation of the rBChE with polyethylene glycol in order to:1.Decrease rBChE immunogenicity2.Increase rBChE stability 3.Increase circulating serum of rBChE3. A limited human study of Protexia has started.
12、 2. OPIDNOPIDN is a neurodegenerative disorder:1. A latent period; 6 and 14 days.2. Neuropathological lesions: medulla of the brain, spinal cord, and sciatic nerve.3. Degeneration of the axon and of myelin 4. Species and age sensitivity.5. Inhibition of neurotoxicity target esterase (NTE).+3TOCPPhos
13、phorus oxychlorideTri-ortho-cresyl phosphateSynthetic pathways of TOCPTCPs+ POCL3+ POCL3+ POCL3ortho-cresolmeta-cresolpara-cresolTOCPTMCPTPCP Uses of Tricresyl Phosphates TCPs 1.Antiwear and additive in synthetic lubricants.2.Flame retardant3.Plasticizer Neurotoxity of TCPs1.TOCP is a weak inhibitor
14、 of AChE2.It is a potent producer of OPIDN3.Other isomers have not been thoroughly tested for OPIDN Isomers of Tri-cresyl Phosphate (TCP)There are 10 possible TCP structures:Isomers OPIDNo,o,o + o,o,m; o,o,p +o,m,m,; o,m,p; o,p,p +m,m,m; m,m,p; m,p,p; p,p,p - Chronology of TOCP-Induced OPIDNYear Cou
15、ntry Incidence Cases1899 France Creosote 591930 USA Contaminated Ginger Extract Approx. 50,0001925-1934 France, Germany, Apiol Abortfacient 200-500 Switzerland1937 South Africa Contaminated Cooking Oil 6001940 Switzerland Contaminated Cooking oil 801942 Britain Manufacturing 31945 Britain Contaminat
16、ed Cottonseed Oil 171943-1947 Germany Used as cooking oil 10-201947 Switzerland Contaminated food 731952 Switzerland Contaminated Olive Oil 801955 South Africa Contaminated Water 111959 Morocco Used as cooking Oil 10,0001960 India Contaminated Cooking Oil 581966 Rumania Contaminated Alcohol 121967 F
17、iji Islands Contaminated Flower1973 Morocco Shoe Glue Exposure 401977-1978 Sri Lanka Contaminated Sesame Oil 231988 India Contaminated Cooking Oil 2 Neurological dysfunction of OPIDN1.Latent period: Days to weeks2.Progressive phase: Symmetric cramping,numbness and tingling in feet and legs, bilatera
18、l dragging of toes (foot-drop), flaccid paralysis.3. Stationary Phase4. Improvement Phase: Results from regeneration of PNS; CNS damage becomes unmasked as spasticity and exaggerated knee jerk.5. Prognosis: Depends on severity of initial symptomsTotal Paralysis below knees with toe drop.Factors invo
19、lved in the Development of OPIDN1.Chemical Structure2.Animal Species: Humans are most sensitive 3.Individual differences4.Animal Age5.Dose or Concentration at Neurotoxicity Site: a. Exposure doseb. Frequency of exposure c. Duration of exposured. Route of Exposuree. Other chemical exposure f. StressF
20、actors involved in the Development of OPIDNMetabolic Activation:1.TOCP is activated to saligenin cyclic-o-tolyl phosphate.2.Phosphorothioate insecticides are activated to phosphates3.Combined exposure to chemicals that increase activity of CYP 450 enhances TOCP neurotoxicity.ActivationTOCPSaligenin
21、cyclic-o-tolyl phosphateFactors Involved in the Development of OPIDNRoute Of Exposure:Organophosphorus compounds have more access to the nervous system and neurotoxicity target through inhalation and skin penetration than the gastrointestinal tract.Inhalation is the most effective route of entry, pr
22、eceded only by intravenous injection.Factors involved in the Development of OPIDNCombined Exposure with other chemicalsChlorpyrifosOral LD50 in rats 150 mg/kgCauses OPIDN at lethal dosesPropetamphos (safrotin)Oral LD50 in rats 119 mg/kgDoes not cause OPIDN3. OPICN Organophosphorus ester-Induced Chro
23、nic Neurotoxicity (OPICN)Organophosphorus ester-Induced Chronic Neurotoxicity (OPICN)1. Is a neurodegenerative disorder that results from large toxic or small subclinical doses of Ops.2. Clinical signs, which continue for weeks to years, consist of neurological and neurobehavioral abnormalities.3. D
24、amage is greater in the CNS than PNS.4. Neuronal cell death is seen in various brain areas including cerebral cortex, hippocampal formation and cerebellum.5. Cell death results from early necrosis or delayed apoptosis.6. OPICN is exacerbated by concurrent exposure to stress or other chemicals that c
25、ause neuronal cell death or oxidative stress.7. Because CNS injury predominates, improvement is slow and complete recovery is unlikely.OPICN in the LiteratureOPICN has been referred to AS:“Chronic neurobehavioral effects”“Chronic organophosphate-induced neuro-psychiatric disorder (COPIND)”“Psychiatr
26、ic sequelae of chronic exposure” “Psychological and neurological alterations”“CNS system effects of chronic exposure”“Neuropsychological abnormalities”“Long-term effects”“Neurobehavioral effects”“Chronic nervous effects of acute organophosphate poisoning”“Chorea and psychiatric changes”“Delayed neur
27、ologic behavioral effects of long-term exposure”“Central cholinergic involvement in behavioral hyperactivity” Organophosphorus Ester-Induced Chronic Neurotoxicity (OPICN)Individuals exposed to a single large toxic or small subclinical doses of Ops have developed a chronic neurotoxicity that persists
28、 years after exposure and is distinct from both cholinergic and OPIDN affects. Characteristics of OPICN1. Neurological alterations Headache, drowsiness, dizziness, anxiety, increased tension, apathy, restlessness, labile emotions, anorexia, insomnia, bad dreams, weakness, lethargy, fatigue, inabilit
29、y to concentrate, cognitive and memory deficits, depression, social isolation, neurological deficits, irritability, confusion, reduced motor coordination, and tremors. (Not every patient has all of these symptoms)Characteristics of OPICN2. Neuropathological Changes1.A large toxic dose of organophosp
30、hates produced necrotic neuronal cell death in the following regions of experimental animals: cerebral and piriform cortices, basal ganglia, thalamus, septum, hypothalamus, hippocampus, corticospinal trac and cerebellum.3. The lesions did not resemble those present in hypoxia or OPIDN.Characteristic
31、s of OPICN2. Neuropathological ChangesExposure to Ops caused delayed apoptotic neuronal cell death in the following regions:Motor cortexHippocampusCerebellum andCervical Spinal cordHuman cases of OPICN1. Three years and nine months after the Tokyo attack, some victims complained of chronic decline o
32、f memory (Nishiwaki et al, 2001). 2.Three years after the Matsumoto attack, some victims complained of fatigue, shoulder stiffness, weakness, blurred vision (Nakajima et al., 1999)3. Others complained of insomnia, had bad dreams, husky voice, slight fever, and palpitation. Neuronal cell Death Conseq
33、uences1.Significant death of cerebral cortex neurons results in muscular weakness and loss of strength. 2.A loss of significant amount of hippocampal neurons leads to progressive loss of memory and results in learning disabilities.3. Loss of Purkinje cells in the cerebellum may cause: a. Delays in i
34、nitiating and terminating movements. b. Terminal tremor at the end of the movement. c. Disorders in the spatial coordination of hand and finger muscle.Specific AimsThis study was designed to investigate the long-term, chronic effects following a single dose of sarin that does not produce clinical si
35、gns in male Sprague-Dawley rats. Experimentals1. Groups of 15 animals were treated with a single intramuscular injection of sarin (LD50 = 100 g/kg):a) 1.0 g/kg (0.01 x LD50) or b) 10.0 g/kg (0.1x LD50) 2. The following parameters were studied at 24 h, 7 days, one month,and one year.a. Clinical signs
36、b. Neurobehavioral performancec. Brain AChE and plasma BChE activityd. Integrity of the blood brain barriere. Neuropathological changes in the brainClinical Signs24 Hours, 7 days, one month, one year after Treatment1. All animals looked and behaved similar to controls.2. Brain AChE and Plasma BChE a
37、ctivities remained normal3. Blood brain barrier was intact4. M2 ACh muscarinic receptor ligand binding was increased in brainstem after one year.Neurobehavioral PerformanceSensorimotor functions were assessed using the following tests:1. Beam walking and beam score 2. Inclined Plane3. Forepaw grip t
38、imeThe results showed sensorimotor deficits 3 months after treatment that were exacerbated by the end of the year after treatment. 0,01 and 0.1 x LD50 Sarin after one YearHistological assessments demonstrated neuronal cell death in:1. Motor cortex2. Hippocampus3. Cerebellum and4. Cervical Spinal cor
39、dSarin-Induced ApoptosisApoptosis was confirmed using:1. Apoptosis-specific stain TUNNL.2. Neuronal nitric oxide synthase (NOS) immunohistostaining. Mechanisms of Neuronal Cell Death in OPICN: High level Cholinergic PathwaysOrganophosphatesAChE InhibitionAChE RiseMuscarinic Receptor ActivationGlutam
40、ate Receptor ActivationNMDA Receptor ActivationCa2+ ReleaseCa2+ Entry into Neuronal CellsNecrotic Neuronal Cell Death OPACh accumulation at muscarinic receptorsAChE InhibitionMuscarinic receptors stimulationNeuronal hyper excitationBBBGlutamate overflow+NMDA Receptor activationCholinergic cascade fo
41、llowing Large Toxic dose of Organophosphates (OP)Neuronal cell BodyNa+ and H2OCa2+ Rise Ca2+ levelAberation of cell functionOnset of catabolism enzymeMechanisms of Neuronal Cell Death in OPICN: Low LevelOxidative StressOrganophosphatesOxidative StressReactive Oxygen Species (ROS) + NO Peroxynitrile
42、(ONOO.) DNA Protein 8-Hydroxy-2-deoxy-guanosine 3-Nitrotyrosine Apoptotic Cell DeathInvolvement of CaMKII in Neuronal Cell Death Ca2+ Release CaMKII avtivation ROS Cytochrome C-release Mitochondrial damageCaspase-3 activationCELL DEATH Chemicals in Cabin AirCabin contained the following chemicals:1.
43、Vapors of lubricating oils and hydraulic fluids (TCPs)2. Insecticides.Components of Some Engine Lubricating Oils and Hydraulic FluidsProduct Components (wt%)Engine lubricating oilsMobil jet oil254 Tricresyl phosphate (TCP,1-5%)Mobil jet oil I Tricresyl phosphate, (N-Phenyl-1-naphthalamine (1-5%)Hydr
44、aulic fluids Skydrol 5 Triisobutyl phosphate, Triphenyl phosphate, (Solutia Inc.) Epoxy-modified alkyl ester Skydrol 500B Tributyl Phosphate, Dibutyl phenyl phosphate(Solutia Inc.) Butyl diphenyl phosphate, Epoxy-modified alkyl ester Butyl Di-phenyl Phosphate, 2,6-Di-tert-butyl-p-cresol Skydrol LD-4
45、 Tributyl phosphate, Dibutyl phenyl phosphate (Solutia Inc) Epoxy modified alkyl ester HyJet IV-A + Tributyl phosphate (79%) (Chevron) Cyclic aliphatic epoxide (2.9%) , Additives (21%)Pesticide SpraysA pesticide spray consists of: a pesticide, a propellant, and solvents.Aerosol spray: 2% d-phenothri
46、n (immediate) and 2% permethrin (residual) .Application: 10 g/ ft3. Action: Pyrethroids act by slowing the opening of neuronal sodium channels, resulting in hyper- excitability of the nerves, and subsequent tremors, ataxia, and paralysis.PermethrinPhenothrin ( 1R)-trans-isomerd-Phenothrin ( a mixtur
47、e of isomers)PyrethroidUnited States Eliminated Disinsection Practice in 1979 In 1979, the Centers for Disease Control and Prevention (CDC) concluded that the disinfsection of aircrafts was ineffective in preventing insects from entering a country and that it would pose a potential health risk to pa
48、ssengers and crew. Symptoms Related to Cabin AirRespiratory IrritationPain (eyes, nose, sinuses, throat)Difficulty breathingBreathing discomfortPain in chestCoughingDry, stuffy noseSymptoms Related to Cabin AirNeurological and NeurobehavioralHeadache Altered visionDizziness Inco-ordinationDisorienta
49、tion Loss of balanceConfusionSlurred speechLightheadednessParesthesiasWeakness Impaired memoryFatigue Inability to concentrateTrouble countingCognitive problemsSymptoms Related to Cabin AirOther SymptomsDry skinRapid Heart Rate and palpitationsReproductive effectsLung functions effectsAcute infectio
50、nsImmunosuppressionHair lossProducts of O3/Alkene Reactions Identified indoorsHydroxyl radicalHydro-peroxy and alkyl-peroxy radicalsHydrogen peroxidesOrganic hydro-peroxides OzonidesFormaldehydeOther volatile aldehydes and ketonesProduct-containing hydroxyl, carbonyl, and/or carboxyl groups(Many of
51、these chemicals cause oxidative stress)Neurobehavioral Effects of TCP isomers Male Sprague-Dawley rats (250-300 g) were randomly divided into groups of 10 rats each. A daily dose of 2.5 mg/kg of test compounds was applied on a pre-clipped area on the back of the neck for 30 days as follows: 1. Contr
52、ol: (vehicle, 1 ml/kg/day) 2. TOCP 3. TMCP 4. TPCP 5. TCP 6. TOCP +TMCP 7. TOCP +TPCP 8. TMCP +TPCP 9. TOCP+TMCP +TPCPTwenty hours after the last dose, the rats were subjected to behavioral evaluations following which they were sacrificed. Beam-Walk Time (1)*Beam-Walk Time (2)*Incline Plane (1)*Incl
53、ine Plane (2)*GRIP TIME PURPOSE: To assess forepaw grip strength PROCEDURE: 1.Have the rats grip a 5-mm diameter wood dowel2.Time to release grip is recorded in seconds. Grip Time (1)*Grip Time (2)*AChE Activity in the different brain regions (1)* *AChE Activity in the different brain regions (2)*Ne
54、uronal Cell Death in BrainMOTOR CORTEXLayer 1-3Layer 4-6CONTTOCPTPCPTMCPHistogram showing the density of dying and surviving neurons/mm2Pesticide SpraysA pesticide spray consists of: a pesticide, a propellant, and solvents.Aerosol spray: 2% d-phenothrin (immediate) and 2% permethrin (residual) .Appl
55、ication: 10 g/ ft3. Action: Pyrethroids act by slowing the opening of neuronal sodium channels, resulting in hyper- excitability of the nerves, and subsequent tremors, ataxia, and paralysis.ConclusionsSymptoms of cabin crew are consistent with exposure to concurrent exposure to chemicals including tri-cresyl phosphate isomers.
