who can complete this by 11:30pm tonight?

Acute and long-term consequences of exposure to organophosphate nerve agents in humans

Taiza H. Figueiredo, James P. Apland¥, Maria F. M. Braga, and Ann M. Marini*,§

*Department of Neurology, Department of Anatomy, Physiology and Genetics; Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

¥Neuroscience Program, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, United States

Summary

Nerve agents are organophosphate (OP) compounds and among the most powerful poisons known

to man. A terrorist attack on civilian or military populations causing mass casualties is a real

threat. The OP nerve agents include soman, sarin, cyclosarin, tabun and VX. The major

mechanism of acute toxicity is the irreversible inhibition of acetylcholinesterase (AChE). AChE

inhibition results in the accumulation of excessive acetylcholine levels in synapses leading to

progression of toxic signs including hypersecretions, tremors, status epilepticus, respiratory distress and death. Miosis and rhinorrhea are the most common clinical findings in those

individuals acutely exposed to OP nerve agents. Prolonged seizures are responsible for the

neuropathology. The brain region that shows the most severe damage is the amygdala followed by

the piriform cortex, hippocampus, cortex, thalamus, and caudate/putamen. Current medical

countermeasures are only modestly effective in attenuating the seizures and neuropathology.

Anticonvulsants such as benzodiazepines decrease seizure activity and improve outcome but their

efficacy depends upon the administration time post-exposure to the nerve agent. Administration of

benzodiazepines may increase the risk for seizure recurrence. Recent studies document long-term

neurologic and behavior deficits while technological advances demonstrate structural brain

changes on magnetic resonance imaging.

Keywords

organophosphate nerve agents; acetylcholinesterase; human; acute effects; long-term effects

§Address correspondence to: Ann M. Marini, Ph.D., M.D., Department of Neurology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814, [email protected], Phone number: 301-295-9686.

Disclosure of Conflicts of Interest The authors have no conflicts of interest.

Ethical Publication We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

HHS Public Access Author manuscript Epilepsia. Author manuscript; available in PMC 2019 October 01.

Published in final edited form as: Epilepsia. 2018 October ; 59(Suppl 2): 92–99. doi:10.1111/epi.14500.

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Introduction

Background

Organophosphate (OP) G-series nerve agents were synthesized in the 1930s by German

scientists.1 An accidental spill in a laboratory where synthetic production of nerve agents

was being conducted highlighted the extreme toxicity of these compounds.2 These

compounds quickly and efficiently penetrate the human body via the skin, inhalation, and

through the bloodstream. OP nerve agent use during war is particularly advantageous given

that they cause mass casualties by inducing status epilepticus and incapacitating organ systems in the body that may result in death. These compounds were deployed during the

Iraq-Iran war3 and on Kurdish people in Northern Iraq.4 In 2013, sarin was dropped in

Damascus killing over 1400 Syrians,5,6 including 426 children;5,7 sarin gas was suspected to

have been deployed again outside of Damascus in 2016 (http://www.telegraph.co.uk/news/

2016/05/17/assads-forces-have-used-sarin-nerve-gas-for-the-first-time-since/).

The recent death of a high profile figure by VX, a V-series nerve agent that exerts higher

toxicity in comparison with some other G-series nerve agents possibly due to the inability of

phosphorylphosphatases to break the phosphorous-sulfur bond in the bloodstream,8 at an

international airport (http://www.bbc.com/news/world-asia-39096172) demonstrates facile

concealment, accessibility, mobility and immediate deployment of OP nerve agents. Similar

but more widespread human intoxication occurred in June of 1994 when sarin gas was

surreptitiously released at midnight while people slept in the city of Matsumoto, Japan and

again in March 1995 when sarin gas was deployed in the Tokyo subway where thousands of

individuals were intoxicated and nineteen died.9 These events illustrate the versatility and

destructive capability of chemical warfare agents i.e., killing one person versus incurring

mass casualties.

Acetylcholinesterase

Nerve agents have the ability to irreversibly inhibit the enzyme AChE in central and

peripheral nervous system synapses. In the mammalian brain, AChE, located in membranes

of postsynaptic neurons10, exists mostly as a four subunit enzyme of 70 kDa. The catalytic

subunits are linked together by disulfide bonds to the hydrophobic subunit P. Subunit P is

required for localization at the cell surface11. The acetylcholinesterase gene is transcribed by

alternative splicing at the carboxyl terminal with peptide sequences of R (readthrough), H

(hydrophobic). or T (tail) catalytic subunit isoforms that determine post-translational

processing, quaternary associations and anchoring12. The soluble and cell membrane-

localized acetylcholinesterase forms are generated from the AChER form. AChEH is a

glycosylphosphatidylinositol-anchored dimer that is primarily expressed in red blood cells

and liver. Perhaps the most interesting, diverse and dynamic isoform is the AChET form

because it produces monomers, dimers and the collagen-and hydrophobic-tailed forms and

soluble forms13. The “T’ peptide directs the assembly of tetramers of AChE14 and the

product of this specific transcript is the synaptic form of AChE that is expressed

predominately in the CNS and muscle tissue15. Inducers of the readthrough AChE transcript

include stress i.e. forced swim, continued use and toxic concentrations of AChE inhibitors

and inflammation16–19. The molecular diversity of AChE leading to three isoforms serves

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the functional range and location of this enzyme as soluble monomers, dimers, and tetramers

that are anchored in membranes, amphiphilic dimers, hydrophobic- and collagen-tailed

tetramers13.

Mechanism of Action

Exposure to nerve agents results in rapid absorption of agent into the bloodstream from

every route, including percutaneous, inhalation and oral administration8,20. Nerve agents

selectively target and irreversibly inhibit acetylcholinesterase (AChE), the enzyme that

breaks down the excitatory neurotransmitter acetylcholine. In the central nervous system,

acetylcholinesterase inhibition results in the overactivation of muscarinic receptors leading

to the initiation of status epilepticus whereas other types of toxic signs are observed in the peripheral nervous system (Figure 1). Miosis and rhinorrhea are the most common clinical

signs of exposure to OP nerve agents.9,21,22 Excessive synaptic acetylcholine levels cause a

massive release of glutamate which in turn, sustains and maintains status epilepticus23,24

resulting in hypoxic-ischemic neuronal cell death via N-methyl-D-aspartate (NMDA)

receptor-mediated excitotoxicity.23, 25–28

Status epilepticus is a serious complication of nerve agent exposure and is defined as a prolonged seizure or continuous seizures lasting more than five minutes without regaining

consciousness.29 It is well-established that prolonged seizures cause the neuropathology. 30–32 Neurodegeneration occurs most frequently in the amygdala, followed by the piriform

cortex, hippocampus, cerebral cortex, thalamus, and caudate/putamen.30,33, 34–37

Some characteristics of OP nerve agents

Sarin belongs to the same class of G-series OP nerve agents as tabun, soman and cyclosarin.

The OP nerve agents are volatile liquids and persist for a short time in the environment.38

Nerve agents are clear, and odorless liquids at room temperature. The vapor pressures of the

nerve agents (2.9 mm Hg (sarin), 0.4 mm Hg (soman), 0.07 mm Hg (tabun) and 0.044 mm

Hg (cyclosarin)) are high and the lethal vapor risk follows in descending order with sarin

exhibiting the highest lethal vapor risk39. The higher the vapor pressure, the higher the

volatility of the organophosphate nerve agent at any given temperature. The vapor/aerosol

state enters the body through the respiratory tract and eyes, and the liquid state enters the

body through eyes, skin and mouth. While it has been reported that nerve agents that exhibit

low volatility do not cause miosis as an initial symptom40, the Center for Disease Control,

the Agency for Toxic Substances and Disease Registry (ATSDR) and the National Institutes

of Health all list miosis and other symptoms including diarrhea on their websites as

symptoms following exposure to organophosphate nerve agents regardless of route of

administration (https://www.atsdr.cdc.gov/MMG/MMG.asp?id=523&tid=93; https://

emergency.cdc.gov/agent/nerve/tsd.asp; https://chemm.nlm.nih.gov/nerveagents.htm).

Clinical examination of any patient exposed to an organophosphate nerve agent regardless of

route of administration (oral, systemic, dermal, inhalation) should include evaluation of all

signs of acetylcholinesterase inhibition within the sympathetic and parasympathetic system.

In particular, evaluation of miosis is rapid and easy to recognize during the examination. A

thorough examination of individuals thought or confirmed to be exposed to organophosphate

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nerve agents will minimize oversight of even an uncommon symptom that may otherwise

result in respiratory failure and death.

Evaluation after OP nerve agent exposure

Exposure to OP nerve agents requires immediate evaluation and treatment because vapor

and systemic exposure in particular can result in the rapid development of symptoms leading

to death. It is extremely important for personnel caring for individuals exposed to OP nerve

agents to keep in mind that these agents can emanate from clothing. Liquid droplets from an

OP nerve agent that are absorbed by clothing can cross contaminate the skin of personnel

that in turn can result in local signs initially such as sweating and fasciculations but more

progressive signs of toxicity will occur as the nerve agent is absorbed and carried

systemically throughout the body leading to generalized signs of toxicity including status epilepticus, defecation, miosis, bronchospasm, bronchorrhea, paralysis and respiratory failure40. If dermal exposure is suspected, multiple clinical examinations may be important

in detecting delayed systemic signs and symptoms. Dermal exposure via touching

contaminated clothing can result in devastating consequences if the contaminated skin like

the fingers rub the eyes as ocular contact results in rapid local and systemic toxic effects40.

A highly organized and coordinated effort must be in place after dissemination of an OP

nerve agent. Tents with showers should be set up in the field and victims must be

decontaminated with copious amounts of water. A basic outline of the exposure levels,

clinical signs and symptoms and treatments is shown in Table 1.

At the hospital, a detailed history and examination of individuals suspected of OP nerve

agent exposure need to be performed as the rapid development of symptoms could lead to

death depending upon the specific agent, route of exposure, and the amount of agent and

time of exposure. Taking advantage of easily recognized clinical signs such as sweating,

fasciculations and miosis helps to arrive at the correct diagnosis and provide urgent and life-

saving treatment.

It is incumbent upon emergency department staff to be properly prepared if victims

suspected of OP nerve agent exposure are expected to arrive at the hospital. Medical staff

were not wearing the proper equipment in the emergency department while triaging sarin

victims after the subway attack in Tokyo and were exposed to sarin vapor leading to signs

and symptoms of exposure9. All personnel involved with the care of nerve agent victims

need to wear personal protective equipment, and air supplied respirators. Butyl rubber

gloves and aprons are protective against dermal exposure. This equipment is required until

the patient is decontaminated; air purifying respirators and double latex gloves are not

protective41. In addition to protecting staff and caring for victims urgently, it should be

emphasized that chemical weapons inflict mass casualties. The deployment and

dissemination of OP nerve agents cause chaos in the field as well as in the emergency

department due to the overwhelming number of critically ill patients that need urgent

treatment. If a significant amount of an OP nerve agent is deployed in a small but crowded

area, one can expect that first responders will not be able to administer life-saving

countermeasures within minutes of the deployment. Under these circumstances, it is not

unreasonable to predict that status epilepticus in some if not many victims may continue for

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up to an hour before currently approved medical countermeasures can be administered to

these victims.

In summary, cognizance of the possibility of OP nerve agent exposure requires an organized,

carefully crafted and coordinated plan involving local hazmat teams, police, fire and experts

in the field as well as a network of medical centers where well-trained and equipped staff are

prepared to further decontaminate and urgently treat the expected large number of victims.

Treatment

Standard-of-care treatment for OP nerve agent acute exposure includes atropine, a

muscarinic antagonist, pralidoxime (2-PAM), an oxime that regenerates acetycholinesterase

activity in those molecules that are not aged, and diazepam, a benzodiazepine to stop/

attenuate seizures.8,25 Repeated administration of atropine five minutes after the first

injection may be necessary to reduce secretions, difficulty breathing and improve

ventilation. It is critical to thoroughly wash the eyes for 5–10 minutes to limit eye injury

after exposure to liquid OP nerve agents; decontamination of eyes is of no use when

individuals are exposed to the vapors of OP nerve agents41. Inhibition of AChE activity by

OP nerve agents is initially reversible but over time the covalent bond between the active site

and the OP nerve agent stabilizes by removal of an alkyl group, a process called aging42, and

results in irreversible AChE inhibition.

Current medical countermeasures are only modestly effective when given post-exposure in a

mass casualty situation because it is anticipated that large crowds or a crowded areas of

limited space would delay administration of life-saving medical countermeasures. Thus, it

will take some time for first responders to identify survivors that need urgent treatment with

medical countermeasures while victims are being decontaminated. The use of

benzodiazepines is relatively ineffective when given thirty minutes or longer after OP nerve

agent exposure and may increase seizure recurrence.43–45 Given the expected number of

victims and the time it will take for first responders to identify and treat critically ill victims,

it is not inconceivable that victims may be in status epilepticus for at least 30 minutes prior to administration of currently approved medical countermeasures.

Long-term effects after exposure to OP nerve agents

Years after exposure to sarin, victims of the Tokyo subway attack presented with significant

declines in psychomotor and memory functions,46,47 signifying long-term cognitive

impairment. An active duty soldier exposed to low-dose sarin while deployed to Iraq six

years prior to evaluation exhibited poor informational processing speed, difficulties with

speed-related bilateral manual motor coordination, poor attention, reduced memory and

recall.48 In a recent article, 344 adults who were children at the time they were exposed to

mustard gas and sarin in the Kurdish city of Halabja and surrounding areas deployed by

Iraqi forces in 1988, participated in a study investigating the long-term effects of chemical

warfare agents. Eighteen participants were excluded from the study after their forms were

lost in transit. Participants exposed to chemical warfare agents were children (10 years or

younger) at the time of chemical warfare exposure. The mean age of the group at the time of

exposure was 4.9 years and the mean age of subjects when they were examined was 20

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years. There were 202 females and 142 males in the study. Only four participants received

emergency treatment within 24 hours of exposures; the remainder received treatment

between 24 hours and more than one week after chemical warfare exposure. There was no

mention whether anyone received currently approved medical countermeasures. Current or

previous smokers, anyone who had a medical condition that affected organs relevant to the

study or had a history of occupational dust exposure were excluded from the study.

Documentation of both acute and long-term effects of chemical warfare agents was

conducted in the subjects fourteen to twenty-two years after exposure. Among the maladies

at the time of examination by investigators, seventy-four percent of subjects had

neurological symptoms. Twenty percent of subjects had convulsions while ninety percent

exhibited signs of anxiety/restlessness/muscular cramps. Additional clinical neurologic signs

that were found in the subjects included ataxia (31%), paralysis (4%), fasciculations (7%),

confusion (38%), dysarthria (22%), headaches (43%) and coma (38%)49.

People exposed to OP pesticides also exhibit long-term effects. Sheep farmers exposed to

low-level OP pesticides demonstrated significant cognitive impairment on working, verbal

and visual memory testing, response speed, fine motor control, mental flexibility and

strategy making. In contrast, other cognitive domains such as visuospatial, verbal abilities

and verbal reasoning were not impaired. Two different control groups were used in this

study to ensure that there was no selection bias.50 Depression is a major neuropsychiatric

disorder found in individuals exposed to either intoxicating or low-dose OP nerve agents,

deployed veterans exposed to nerve agents,51,52 individuals of the terrorist attack in the

Tokyo subway53 as well as those exposed to OP pesticides,50, 54–58 particularly women.59

Post-traumatic stress disorder (PTSD), an anxiety disorder, was reported in human studies

following exposure to OP nerve agents despite administration of standard-of-care drugs to

promote survival and stop the nerve agent-induced seizures.60,61 Anxiety in the absence of

PTSD has also been found in individuals exposed to OP pesticides.50,57,62

The long-term effects of OP nerve agent exposure in humans are similar to results reported

in rodents exposed to OP nerve agents demonstrating neuropathological changes in several

brain regions following soman exposure63,64. Neuronal damage continues for days and

weeks after OP exposure in rodents despite lifesaving treatment with an oxime, atropine and

diazepam.65–67 Learning and memory deficits and depressive-like behavior are also

associated with soman-induced brain damage.64–69 In summary, long-term neuropsychiatric

deficits have been reported in humans exposed to either low or intoxicating levels of OP

nerve agents. A recent report in children exposed to chemical warfare agents suggests that

exposure to chemical warfare agents can lead to long-term neurological and

neuropsychiatric deficits. Animal models of OP nerve agents replicate many of the adult

human cognitive deficits.

Structural brain changes on magnetic resonance imaging after OP nerve agent exposure

Structural brain alterations were observed in the amygdala and left anterior cingulate cortex

in those individuals exposed to sarin in the Tokyo subway attack carrying a diagnosis of

post-traumatic stress disorder (PTSD).60,70,71 On brain magnetic resonance imaging (MRI),

there was a significant reduction in the amygdalar volume on both sides of the brain; a

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negative correlation existed between the left amygdalar volume and PTSD and the left

anterior cingulate cortex was also smaller.60 Exposure to an intoxicating dose of sarin as

occurred during the Tokyo subway attack resulted in regional reductions in gray and white

matter; the significant decrease in the white matter volume in the left temporal stem near the

insula correlated with the serum cholinesterase levels and the severity of the somatic

complaints.71 A significant reduction in regional gray matter volume was found in the right

insula, temporal cortices and left hippocampus in comparison with controls.71 Structural

brain changes occurred in veterans exposed to low-dose sarin and cyclosarin. A significant

reduction was found in total gray and white matter volume72,73. Significant reductions in the

CA2 and CA3/dentate gyrus subfields of the hippocampus were reported in individuals

exposed to low-dose chemical warfare agents74 and remodeling of the white matter is

suspected in the temporal stem, corona radiata, superior/inferior cingulum, internal and

external capsule, inferior and superior fronto-occipital fasciculus and nine superficial white

matter areas located between the cortex and deep white matter.75 Taken together, these

results underscore that low-level as well as intoxicating levels of OP nerve agents can result

in structural gray and white matter alterations in the brain long after exposure to OP nerve

agents.

Acknowledgments

Our research has been supported by the CounterACT Program, National Institutes of Health, Office of the Director and the National Institute of Neurologic Disorders and Stroke [Grant Number 5U01NS058162-07 to MFB].

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Key Point Box

• Organophosphate (OP) nerve agents are deadly poisons that inflict mass casualties in human populations.

• Irreversible inhibition of acetylcholinesterase activity by OP nerve agents leads to accumulation of acetylcholine in synapses and hyperstimulation of

muscarinic and nicotinic receptors in the central and peripheral nervous

systems.

• Hyperstimulation of muscarinic receptors in brain results in status epilepticus

• A plethora of clinical manifestations in the central and peripheral nervous system results from exposure to OP nerve agents; miosis and rhinorrhea are

the most common clinical signs of OP nerve agent exposure.

• Long-term effects occur after low or intoxicating levels of exposure to OP nerve agents.

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Figure 1. Overview of the mechanism of action and clinical manifestations of the acute exposure to organophosphate nerve agents Exposure to organophosphate (OP) nerve agents leads to the irreversible inhibition of

acetylcholinesterase (AChE) resulting in the accumulation of the excitatory neurotransmitter

acetylcholine (ACH) in synapses and hyperstimulation of muscarinic and nicotinic

acetylcholine receptors in the central and peripheral nervous system. The major clinical

manifestation in the central nervous system after exposure to OP nerve agents is

overactivation of muscarinic receptors resulting in status epilepticus. Prolonged status epilepticus results in hypoxic-ischemic neuronal cell death via an N-methyl-D-aspartate (NMDA) receptor-mediated mechanism. Clinical manifestations in the peripheral nervous

system depend upon the type of acetylcholine receptors expressed in the particular organ.

Miosis and rhinorrhea are the most common clinical findings of exposure to OP nerve agents

whereas excessive bronchial secretions and respiratory depression lead to death.

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Table 1

Preparations and Actions in the field after deployment of an OP nerve agent

Guidelines for First responders

Field Equipment and Actions

Exposure Levels

Clinical symptoms and signs

Treatment

Personal Protective Equipment, air supplied respirators, butyl rubber gloves and aprons.

HAZMAT teams, experts in chemical warfare agents, rapid decontamination of individuals exposed to organophosphate nerve agents.

Very high Patient is unconscious, in status epilepticus, having breathing difficulties, muscle paralysis, cardiac dysfunction

Diazepam, 2-PAM and atropine, oxygen mask, intravenous access, watch for deterioration

First responders should

have ATNAA* autoinjector kits used by the military as this is the most efficacious way to administer life-saving medical countermeasures

If showers are available, all patients need to be divested of their clothes and washed down rapidly and extensively to minimize further absorption, dermal and ocular contact of vapors and liquid

Moderate with caution Patient is recovering from exposure to organophosphate nerve agent. Patient may need to remain if dermal contact is suspected or confirmed in anticipation of delayed symptoms and signs

2-PAM, atropine and diazepam should be on stand-by

Ensure open airway, breathing and circulation. Start intravenous fluids and monitor heart rate, blood pressure, evidence of seizures. Have 2-PAM and atropine close by in case patient suddenly deteriorates

Run water over eyes extensively to reduce eye injury

Minimal Patient has a few minor symptoms i.e. miosis, secretions

No treatment necessary

Report to hospital approximate number of seriously affected patients.

Showers for victims, personal protective equipment for Emergency personnel

Extremely High Patient is in prolonged cardiopulmonary arrest

Assisted ventilation, CPR, 2-PAM, atropine and diazepam if seizing

* ATNAA is defined as Antidote treatment-nerve agent autoinjector

Epilepsia. Author manuscript; available in PMC 2019 October 01.