Organphosphorus Compounds-Induced Neurotoxicity

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Organphosphorus Compounds-Induced Neurotoxicity

• Mohamed B. Abou-Donia, Ph.D.
• Department of Pharmacology and Cancer Biology
• Duke University Medical Center
• Durham, North Carolina, USA
• donia_at_duke.edu
• 919-684-2221

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Organophosphorus compounds

• Used in medicine, industry, agriculture and as
  warfare agents.
• Have a 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.

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Actions of Organophosphorus Compounds

• Cholinergic Neurotoxicity
• 2. Organophosphorus Ester-Induced Neurotoxicity
  (OPIDN)
• 3. Organophosphorus Ester-Induced Chronic
  Neurotoxicity (OPICN)

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1. Cholinergic Neurotoxicity

• Inhibition of
• Acetylcholinesterase (AChE), an enzyme
  essential for life

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ORGANOPHOSPHATE
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Manifestations of Organophosphate Poisoning
Optic System Pupil Constriction Blurred
Vision Lacrimation
Brain Headache Dizziness Vertigo Anxiety Apathy Co
nfusion Anorexia Insomnia Lethargy Fatigue Inabili
ty to Concentrate Memory Impairment Convulsion Com
a
Respiratory System Bronchospasm Bronchial
Secretion Pulmonary Edema Tightness of
Chest Wheezing Cough Difficulty Breathing
Gastrointestinal Tract Salivation Nausea Cramps Ab
dominal Pain Vomiting Diarrhea Fecal Incontinence
Cardiovascular System Tachycardia Increased Blood
Pressure
Musculature Weakness Tremor Fasciculations Twitchi
ng Cramps Increased Sweating
Urinary - Genital Urinary Incontinence Impotence U
terus Contraction
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Treatment of Cholinergic Toxicity

• 1. 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 AChE aging.
• 2. Atropine, an antagonist of muscarinic ACh
  receptor (AChR)

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3. Shielding of AChE

• Organophosphorus 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 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 AChE.
• Result
• Free enzyme and near-normal neuromuscular an
  autonomic functions.

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4. Bioscavengers

• Butyrylcholinesterase (BChE) is a naturally
  occurring enzyme in blood.
• 2. Its blood concentration is 2 mg/liter.
• BChE 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).

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Recombinant BChE (rBCHE)

• Recombinant human BChE (rBChE) 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 immunogenicity
• Increase rBChE stability
• Increase circulating serum of rBChE
• 3. A limited human study of Protexia has
  started.

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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.
• 5. Inhibition of neurotoxicity target esterase
  (NTE).

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Synthetic pathways of TOCP
Tri-ortho-cresyl phosphate
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TCPs
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Uses of Tricresyl Phosphates TCPs

1. Antiwear and additive in synthetic lubricants.
2. Flame retardant
3. Plasticizer

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Neurotoxity of TCPs

1. TOCP is a weak inhibitor of AChE
2. It is a potent producer of OPIDN
3. Other isomers have not been thoroughly tested for
   OPIDN

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Isomers of Tri-cresyl Phosphate (TCP)

• There are 10 possible TCP structures
• Isomers
  OPIDN
• o,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 -
  

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Chronology of TOCP-Induced OPIDN

• Year Country
  Incidence Cases
• France
  Creosote
  59
• USA Contaminated Ginger
  Extract Approx. 50,000
• 1925-1934 France, Germany, Apiol
  Abortfacient
  200-500
• Switzerland
• 1937 South Africa
  Contaminated Cooking Oil 600
• 1940 Switzerland
  Contaminated Cooking oil
  80
• 1942 Britain
  Manufacturing
  3
• Britain Contaminated
  Cottonseed Oil 17
• 1943-1947 Germany Used as cooking oil
  10-20
• Switzerland
  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 40
• 1977-1978 Sri Lanka
  Contaminated Sesame Oil 23

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Neurological dysfunction of OPIDN

• Latent period Days to weeks
• Progressive phase Symmetric cramping,
• numbness and tingling in feet and legs,
  bilateral dragging of toes (foot-drop), flaccid
  paralysis.
• 3. Stationary Phase
• 4. Improvement Phase Results from regeneration
  of PNS CNS damage becomes unmasked as spasticity
  and exaggerated knee jerk.
• 5. Prognosis Depends on severity of initial
  symptoms

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Total Paralysis below knees with toe drop.
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Factors involved in the Development of OPIDN

• Chemical Structure
• Animal Species Humans are most sensitive
• Individual differences
• Animal Age
• Dose or Concentration at Neurotoxicity Site
• a. Exposure dose
• b. Frequency of exposure
• c. Duration of exposure
• d. Route of Exposure
• e. Other chemical exposure
• f. Stress

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Factors involved in the Development of OPIDN

• Metabolic Activation
• TOCP is activated to saligenin cyclic-o-tolyl
  phosphate.
• Phosphorothioate insecticides are activated to
  phosphates
• Combined exposure to chemicals that increase
  activity of CYP 450 enhances TOCP neurotoxicity.

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Activation
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Factors Involved in the Development of OPIDN

• Route 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,
  preceded only by intravenous injection.

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Factors involved in the Development of OPIDN
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3. 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. Damage 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 cause neuronal
  cell death or oxidative stress.
• 7. Because CNS injury predominates, improvement
  is slow and complete recovery is unlikely.

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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 exposure
• 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

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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 years after
  exposure and is distinct from both cholinergic
  and OPIDN affects.

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Characteristics of OPICN1. Neurological
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 motor
  coordination, and tremors.
• (Not every patient has all of these symptoms)

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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, thalamus, septum, hypothalamus,
  hippocampus, corticospinal trac and cerebellum.
• 3. The lesions did not resemble those present in
  hypoxia or OPIDN.

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Characteristics of OPICN2. Neuropathological
Changes

• Exposure to Ops caused delayed apoptotic neuronal
  cell death in the following regions
• Motor cortex
• Hippocampus
• Cerebellum and
• Cervical Spinal cord

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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 vision (Nakajima et
  al., 1999)
• 3. Others complained of insomnia, had bad
  dreams, husky voice, slight fever, and
  palpitation.

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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 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.

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Specific Aims

• This study 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.

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Experimentals

• 1. Groups of 15 animals were treated with a
  single intramuscular injection of sarin (LD50
  100 µg/kg)
• a) 1.0 µg/kg (0.01 x LD50) or
• b) 10.0 µg/kg (0.1x LD50)
• 2. The following parameters were studied at 24 h,
  7 days, one month,
• and one year.
• a. Clinical signs
• b. Neurobehavioral performance
• c. Brain AChE and plasma BChE activity
• d. Integrity of the blood brain barrier
• e. Neuropathological changes in the brain

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Clinical Signs

• 24 Hours, 7 days, one month, one year after
  Treatment
• 1. All animals looked and behaved similar to
  controls.
• 2. Brain AChE and Plasma BChE activities remained
  normal
• 3. Blood brain barrier was intact
• 4. M2 ACh muscarinic receptor ligand binding was
  increased in brainstem after one year.

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Neurobehavioral Performance

• Sensorimotor functions were assessed using the
  following tests
• 1. Beam walking and beam score
• 2. Inclined Plane
• 3. Forepaw grip time
• The results showed sensorimotor deficits 3 months
  after treatment that were exacerbated by the end
  of the year after treatment.

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0,01 and 0.1 x LD50 Sarin after one Year

• Histological assessments demonstrated neuronal
  cell death in
• 1. Motor cortex
• 2. Hippocampus
• 3. Cerebellum and
• 4. Cervical Spinal cord

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Sarin-Induced Apoptosis

• Apoptosis was confirmed using
• 1. Apoptosis-specific stain TUNNL.
• 2. Neuronal nitric oxide synthase (NOS)
  immunohistostaining.

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Mechanisms of Neuronal Cell Death in OPICN High
level

• Cholinergic Pathways
• Organophosphates
• AChE Inhibition
• AChE Rise
• Muscarinic Receptor Activation
• Glutamate Receptor Activation
• NMDA Receptor Activation
• Ca2 Release
• Ca2 Entry into Neuronal Cells
• Necrotic Neuronal Cell Death

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Mechanisms of Neuronal Cell Death in OPICN Low
Level

• Oxidative Stress
• Organophosphates
• Oxidative Stress
• Reactive Oxygen Species (ROS)
• NO
• 
  Peroxynitrile (ONOO.)
  
• DNA
  Protein
• 8-Hydroxy-2-deoxy-guanosine
  3-Nitrotyrosine
• 
  Apoptotic Cell Death

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Involvement of CaMKII in Neuronal Cell Death
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Chemicals in Cabin Air

• Cabin contained the following chemicals
• Vapors of lubricating oils and hydraulic fluids
  (TCPs)
• 2. Insecticides.

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Components of Some Engine Lubricating Oils and
Hydraulic Fluids

• Product
  Components (wt)
• Engine lubricating oils
• Mobil 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, 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 Tributyl
  phosphate, Dibutyl phenyl phosphate
  
• (Solutia Inc) Epoxy
  modified alkyl ester
• 
  
• HyJet IV-A Tributyl
  phosphate (79)
• (Chevron) Cyclic aliphatic epoxide
  (lt2.9) , Additives (lt21)

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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 Pyrethroids act by slowing the opening of
  neuronal sodium channels, resulting in hyper-
  excitability of the nerves, and subsequent
  tremors, ataxia, and paralysis.

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Permethrin
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Phenothrin ( 1R)-trans-isomer
d-Phenothrin ( a mixture of isomers)
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United 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 passengers
  and crew.

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Symptoms Related to Cabin AirRespiratory

• Irritation
• Pain (eyes, nose, sinuses, throat)
• Difficulty breathing
• Breathing discomfort
• Pain in chest
• Coughing
• Dry, stuffy nose

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Symptoms Related to Cabin AirNeurological and
Neurobehavioral

• Headache Altered vision
• Dizziness Inco-ordination
• Disorientation Loss of balance
• Confusion Slurred speech
• Lightheadedness Paresthesias
• Weakness Impaired memory
• Fatigue Inability to concentrate
• Trouble counting Cognitive problems

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Symptoms Related to Cabin AirOther Symptoms

• Dry skin
• Rapid Heart Rate and palpitations
• Reproductive effects
• Lung functions effects
• Acute infections
• Immunosuppression
• Hair loss

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Products of O3/Alkene Reactions Identified
indoors

• Hydroxyl radical
• Hydro-peroxy and alkyl-peroxy radicals
• Hydrogen peroxides
• Organic hydro-peroxides
• Ozonides
• Formaldehyde
• Other volatile aldehydes and ketones
• Product-containing hydroxyl, carbonyl, and/or
  carboxyl groups
• (Many of these chemicals cause oxidative stress)

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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. Control (vehicle, 1 ml/kg/day)
• 2. TOCP
• 3. TMCP
• 4. TPCP
• 5. TCP
• 6. TOCP TMCP
• 7. TOCP TPCP
• 8. TMCP TPCP
• 9. TOCPTMCP TPCP
• Twenty hours after the last dose, the rats were
  subjected to behavioral evaluations following
  which they were sacrificed.

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Beam-Walk Time (1)
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Beam-Walk Time (2)
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Incline Plane (1)
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Incline Plane (2)
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GRIP TIME

• PURPOSE To assess forepaw grip strength
• PROCEDURE
• Have the rats grip a 5-mm diameter wood dowel
• Time to release grip is recorded in seconds.
  

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Grip Time (1)
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Grip Time (2)
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AChE Activity in the different brain regions (1)
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AChE Activity in the different brain regions (2)
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Neuronal Cell Death in Brain
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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 Pyrethroids act by slowing the opening of
  neuronal sodium channels, resulting in hyper-
  excitability of the nerves, and subsequent
  tremors, ataxia, and paralysis.

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Conclusions

• Symptoms of cabin crew are consistent with
  exposure to concurrent exposure to chemicals
  including tri-cresyl phosphate isomers.