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1、Organphosphorus Compounds-Induced Neurotoxicity,Mohamed B.Abou-Donia,Ph.D.Department of Pharmacology and Cancer BiologyDuke University Medical Center Durham,North Carolina,USAdoniaduke.edu919-684-2221,Organophosphorus compounds,Used in medicine,industry,agriculture and as warfare agents.Have a wide
2、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 Compounds,Cholinergic Neurotoxicity2.Organophosphorus Ester-Induced Neurotoxicity(OPIDN)3.Organophosphorus Ester-Induced Chr
3、onic Neurotoxicity(OPICN),1.Cholinergic Neurotoxicity,Inhibition of Acetylcholinesterase(AChE),an enzyme essential for life,ORGANOPHOSPHATE,Manifestations of Organophosphate Poisoning,Optic SystemPupil ConstrictionBlurred VisionLacrimation,Respiratory SystemBronchospasmBronchial SecretionPulmonary E
4、demaTightness of ChestWheezingCoughDifficulty Breathing,Gastrointestinal TractSalivationNauseaCrampsAbdominal PainVomitingDiarrheaFecal Incontinence,Urinary-GenitalUrinary IncontinenceImpotenceUterus Contraction,BrainHeadacheDizzinessVertigoAnxietyApathyConfusionAnorexiaInsomniaLethargyFatigueInabil
5、ity to ConcentrateMemory ImpairmentConvulsionComa,Cardiovascular SystemTachycardiaIncreased Blood Pressure,MusculatureWeaknessTremorFasciculationsTwitchingCrampsIncreased Sweating,Treatment of Cholinergic Toxicity,1.2-PAM(2-pyridine aldoxime methiodide)hydrolyzes phosphorylated enzyme thus accelerat
6、ing the regeneration of active AChE;should be administered rapidly within 10 to 15 minutes of exposure,before AChE aging.2.Atropine,an antagonist of muscarinic ACh receptor(AChR),3.Shielding of AChE,Organophosphorus nerve agents,such as sarin act by irreversibly inhibiting AChE in the peripheral and
7、 central nervous systems.2.Pyridostigmine Bromide(PB)is administered to protect against toxicity.PB is 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 inhibitio
8、n by the nerve gas.4.Enzyme activity is restored following spontaneous decarbamyalation of the AChE.Result Free enzyme and near-normal neuromuscular an autonomic functions.,4.Bioscavengers,Butyrylcholinesterase(BChE)is a naturally occurring enzyme in blood.2.Its blood concentration is 2 mg/liter.BCh
9、E has no known function;however,it functions as the first line of defense against poisoning with organphosphorus compounds.It acts as a bioscavenger,like a sponge to absorb and degrade organphosphorus compounds(e.g.,nerve agents and insecticides).,Recombinant BChE(rBCHE),Recombinant human BChE(rBChE
10、)is being developed under the trade name Protexia as a pre-and post-exposure therapy for organphosphorus compound poisoning.Protexia is a pegylated rBChE,that is formed by conjugation of the rBChE with polyethylene glycol in order to:Decrease rBChE immunogenicityIncrease rBChE stability Increase cir
11、culating serum of rBChE3.A limited human study of Protexia has started.,2.OPIDN,OPIDN 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.
12、5.Inhibition of neurotoxicity target esterase(NTE).,Tri-ortho-cresyl phosphate,Synthetic pathways of TOCP,TCPs,Uses of Tricresyl Phosphates TCPs,Antiwear and additive in synthetic lubricants.Flame retardantPlasticizer,Neurotoxity of TCPs,TOCP is a weak inhibitor of AChEIt is a potent producer of OPI
13、DNOther 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 OPIDN,Year Country Incidence Cases France Creosote 59 USA Contamin
14、ated 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 3 Britain Contaminated Cottonseed Oil 171943-1947 Germany Used as cooking oil 10-20 Swi
15、tzerland Contaminated food 73 Switzerland Contaminated Olive Oil 80 South Africa Contaminated Water 11 Morocco Used as cooking Oil 10,000 India Contaminated Cooking Oil 58 Rumania Contaminated Alcohol 12 Fiji Islands Contaminated Flower Morocco Shoe Glue Exposure 401977-1978 Sri Lanka Contaminated S
16、esame Oil 231988 India Contaminated Cooking Oil 2,Neurological dysfunction of OPIDN,Latent period:Days to weeksProgressive phase:Symmetric cramping,numbness and tingling in feet and legs,bilateral dragging of toes(foot-drop),flaccid paralysis.3.Stationary Phase4.Improvement Phase:Results from regene
17、ration of PNS;CNS damage becomes unmasked as spasticity and exaggerated knee jerk.5.Prognosis:Depends on severity of initial symptoms,Total Paralysis below knees with toe drop.,Factors involved in the Development of OPIDN,Chemical StructureAnimal Species:Humans are most sensitive Individual differen
18、cesAnimal AgeDose or Concentration at Neurotoxicity Site:a.Exposure doseb.Frequency of exposure c.Duration of exposured.Route of Exposuree.Other chemical exposure f.Stress,Factors involved in the Development of OPIDN,Metabolic Activation:TOCP is activated to saligenin cyclic-o-tolyl phosphate.Phosph
19、orothioate insecticides are activated to phosphatesCombined exposure to chemicals that increase activity of CYP 450 enhances TOCP neurotoxicity.,Activation,Factors Involved in the Development of OPIDN,Route Of Exposure:Organophosphorus compounds have more access to the nervous system and neurotoxici
20、ty target through inhalation and skin penetration than the gastrointestinal tract.Inhalation is the most effective route of entry,preceded only by intravenous injection.,Factors involved in the Development of OPIDN,3.OPICN,Organophosphorus ester-Induced Chronic Neurotoxicity(OPICN)1.Is a neurodegene
21、rative 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.Damage is greater in the CNS than PNS.4.Neuronal cell death is seen in various brain areas including cerebral
22、 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 cause neuronal cell death or oxidative stress.7.Because CNS injury predominates,improvement is slow and complete
23、recovery is unlikely.,OPICN in the Literature,OPICN has been referred to AS:“Chronic neurobehavioral effects”“Chronic organophosphate-induced neuro-psychiatric disorder(COPIND)”“Psychiatric sequelae of chronic exposure”“Psychological and neurological alterations”“CNS system effects of chronic exposu
24、re”“Neuropsychological abnormalities”“Long-term effects”“Neurobehavioral effects”“Chronic nervous effects of acute organophosphate poisoning”“Chorea and psychiatric changes”“Delayed neurologic behavioral effects of long-term exposure”“Central cholinergic involvement in behavioral hyperactivity”,Orga
25、nophosphorus 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 years after exposure and is distinct from both cholinergic and OPIDN affects.,Characteristics of OPICN1.Neurologi
26、cal alterations,Headache,drowsiness,dizziness,anxiety,increased tension,apathy,restlessness,labile emotions,anorexia,insomnia,bad dreams,weakness,lethargy,fatigue,inability to concentrate,cognitive and memory deficits,depression,social isolation,neurological deficits,irritability,confusion,reduced m
27、otor coordination,and tremors.(Not every patient has all of these symptoms),Characteristics of OPICN2.Neuropathological Changes,A large toxic dose of organophosphates produced necrotic neuronal cell death in the following regions of experimental animals:cerebral and piriform cortices,basal ganglia,t
28、halamus,septum,hypothalamus,hippocampus,corticospinal trac and cerebellum.3.The lesions did not resemble those present in hypoxia or OPIDN.,Characteristics of OPICN2.Neuropathological Changes,Exposure to Ops caused delayed apoptotic neuronal cell death in the following regions:Motor cortexHippocampu
29、sCerebellum andCervical Spinal cord,Human cases of OPICN,1.Three years and nine months after the Tokyo attack,some victims complained of chronic decline of memory(Nishiwaki et al,2001).Three years after the Matsumoto attack,some victims complained of fatigue,shoulder stiffness,weakness,blurred visio
30、n(Nakajima et al.,1999)3.Others complained of insomnia,had bad dreams,husky voice,slight fever,and palpitation.,Neuronal cell Death Consequences,Significant death of cerebral cortex neurons results in muscular weakness and loss of strength.A loss of significant amount of hippocampal neurons leads to
31、 progressive loss of memory and results in learning disabilities.3.Loss of Purkinje cells in the cerebellum may cause:a.Delays in initiating and terminating movements.b.Terminal tremor at the end of the movement.c.Disorders in the spatial coordination of hand and finger muscle.,Specific Aims,This st
32、udy was designed to investigate the long-term,chronic effects following a single dose of sarin that does not produce clinical signs in male Sprague-Dawley rats.,Experimentals,1.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
33、)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 signsb.Neurobehavioral performancec.Brain AChE and plasma BChE activityd.Integrity of the blood brain barriere.Neuropathological changes in the brain,Clinical Signs,24 Hours,7 days,one month
34、,one year after Treatment1.All animals looked and behaved similar to controls.2.Brain AChE and Plasma BChE activities remained normal3.Blood brain barrier was intact4.M2 ACh muscarinic receptor ligand binding was increased in brainstem after one year.,Neurobehavioral Performance,Sensorimotor functio
35、ns were assessed using the following tests:1.Beam walking and beam score 2.Inclined Plane3.Forepaw grip timeThe 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 Year,Histological assessment
36、s demonstrated neuronal cell death in:1.Motor cortex2.Hippocampus3.Cerebellum and4.Cervical Spinal cord,Sarin-Induced Apoptosis,Apoptosis was confirmed using:1.Apoptosis-specific stain TUNNL.2.Neuronal nitric oxide synthase(NOS)immunohistostaining.,Mechanisms of Neuronal Cell Death in OPICN:High lev
37、el,Cholinergic PathwaysOrganophosphatesAChE InhibitionAChE RiseMuscarinic Receptor ActivationGlutamate Receptor ActivationNMDA Receptor ActivationCa2+ReleaseCa2+Entry into Neuronal CellsNecrotic Neuronal Cell Death,Mechanisms of Neuronal Cell Death in OPICN:Low Level,Oxidative StressOrganophosphates
38、Oxidative StressReactive Oxygen Species(ROS)+NO Peroxynitrile(ONOO.)DNA Protein 8-Hydroxy-2-deoxy-guanosine 3-Nitrotyrosine Apoptotic Cell Death,Involvement of CaMKII in Neuronal Cell Death,Chemicals in Cabin Air,Cabin contained the following chemicals:Vapors of lubricating oils and hydraulic fluids
39、(TCPs)2.Insecticides.,Components of Some Engine Lubricating Oils and Hydraulic Fluids,Product Components(wt%)Engine lubricating oilsMobil jet oil254 Tricresyl phosphate(TCP,1-5%)Mobil jet oil I Tricresyl phosphate,(N-Phenyl-1-naphthalamine(1-5%)Hydraulic fluids Skydrol 5 Triisobutyl phosphate,Triphe
40、nyl 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 Tributyl phosphate,Dibutyl phenyl phosphate(Solutia Inc)E
41、poxy modified alkyl ester HyJet IV-A+Tributyl phosphate(79%)(Chevron)Cyclic aliphatic epoxide(2.9%),Additives(21%),Pesticide Sprays,A pesticide spray consists of:a pesticide,a propellant,and solvents.Aerosol spray:2%d-phenothrin(immediate)and 2%permethrin(residual).Application:10 g/ft3.Action:Pyreth
42、roids act by slowing the opening of neuronal sodium channels,resulting in hyper-excitability of the nerves,and subsequent tremors,ataxia,and paralysis.,Permethrin,Phenothrin(1R)-trans-isomer,d-Phenothrin(a mixture of isomers),Pyrethroid,United States Eliminated Disinsection Practice in 1979,In 1979,
43、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 passengers and crew.,Symptoms Related to Cabin AirRespiratory,IrritationPain(eyes,nose,sinu
44、ses,throat)Difficulty breathingBreathing discomfortPain in chestCoughingDry,stuffy nose,Symptoms Related to Cabin AirNeurological and Neurobehavioral,Headache Altered visionDizziness Inco-ordinationDisorientation Loss of balanceConfusionSlurred speechLightheadednessParesthesiasWeakness Impaired memo
45、ryFatigue Inability to concentrateTrouble countingCognitive problems,Symptoms Related to Cabin AirOther Symptoms,Dry skinRapid Heart Rate and palpitationsReproductive effectsLung functions effectsAcute infectionsImmunosuppressionHair loss,Products of O3/Alkene Reactions Identified indoors,Hydroxyl r
46、adicalHydro-peroxy and alkyl-peroxy radicalsHydrogen peroxidesOrganic hydro-peroxides OzonidesFormaldehydeOther volatile aldehydes and ketonesProduct-containing hydroxyl,carbonyl,and/or carboxyl groups(Many of these chemicals cause oxidative stress),Neurobehavioral Effects of TCP isomers,Male Spragu
47、e-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.Control:(vehicle,1 ml/kg/day)2.TOCP 3.TMCP 4.TPCP 5.TCP 6.TOCP+TMCP 7.TOCP+TPCP 8.TMCP+TPCP 9.TOCP+
48、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),Incline Plane(2),GRIP TIME,PURPOSE:To assess forepaw grip strength PROCEDURE:Have the rats grip a 5-mm diameter wood
49、dowelTime 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),Neuronal Cell Death in Brain,Pesticide Sprays,A pesticide spray consists of:a pesticide,a propellant,and solvents.Aerosol spray:
50、2%d-phenothrin(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.,Conclusions,Symptoms of cabin crew are consistent with exposur
