Perspectives in Pharmacology Animal Models That Best Reproduce the Clinical Manifestations of Human Intoxication with Organophosphorus Compounds

The translational capacity of data generated in preclinical toxicological studies is contingent upon several factors, including the appropriateness of the animal model. The primary objectives of this article are: 1) to analyze the natural history of acute and delayed signs and symptoms that develop following an acute exposure of humans to organophosphorus (OP) compounds, with an emphasis on nerve agents; 2) to identify animal models of the clinical manifestations of human exposure to OPs; and 3) to review the mechanisms that contribute to the immediate and delayed OP neurotoxicity. As discussed in this study, clinical manifestations of an acute exposure of humans to OP compounds can be faithfully reproduced in rodents and nonhuman primates. These manifestations include an acute cholinergic crisis in addition to signs of neurotoxicity that develop long after the OP exposure, particularly chronic neurologic deficits consisting of anxiety-related behavior and cognitive deficits, structural brain damage, and increased slow electroencephalographic frequencies. Because guinea pigs and nonhuman primates, like humans, have low levels of circulating carboxylesterases—the enzymes that metabolize and inactivate OP compounds—they stand out as appropriate animal models for studies of OP intoxication. These are critical points for the development of safe and effective therapeutic interventions against OP poisoning because approval of such therapies by the Food and Drug Administration is likely to rely on the Animal Efficacy Rule, which allows exclusive use of animal data as evidence of the effectiveness of a drug against pathologic conditions that cannot be ethically or feasibly tested in humans. The discovery of the usefulness of organophosphorus (OP) compounds as pesticides in the late 1930s unfortunately led to the identification of some of the most toxic compounds synthetized by humankind, including tabun, soman, sarin, cyclosarin, VR, and VX, which would become collectively known as nerve agents (Tucker, 2006). These nerve agents have been stockpiled and used as weapons of mass destruction in chemical warfare and in terrorist attacks against civilians. The death toll and poor health outcomes of the alleged use of sarin against civilians in Damascus, Syria, as recently as August 2013, and the catastrophic results of the use of tabun or sarin during the Second Sino-Japanese War (1937–1945), the 1980s Iraq-Iran conflict, and the 1990s terrorist attacks in Japan are well documented (Romano and King, 2001; Coupland and Leins, 2005; Tucker, 2006; Dolgin, 2013; Patrick et al., 2013; http://www.un.org/disarmament/content/slideshow/ Secretary_General_Report_of_CW_Investigation.pdf). The fatalities and poor health conditions resulting from acute and/or chronic exposures of humans to OP pesticides have also become a major public health concern. There are estimates that more than 3 million cases of acute OP pesticide This work was supported in part by funds from the National Institutes of Health National Institute of Neurological Disorders and Stroke CounterACT Program [Grant U01-NS059344]; and by federal funds from the Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human Services, under contract with Countervail Corp. [Contract HHSO100201100030C]. The opinions and assertions contained herein are the private views of the coauthors and do not represent the official position or policies of the Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human Services, the CounterACT program, the National Institute of Neurological Disorders and Stroke, the National Institutes of Health, or the US Government. dx.doi.org/10.1124/jpet.114.214932. ABBREVIATIONS: ACh, acetylcholine; AChE, acetylcholinesterase; ADC, apparent diffusion coefficient; AMN, atropine methylnitrate; CNS, central nervous system; EEG, electroencephalogram; FDA, Food and Drug Administration; LCt50, median lethal concentration; MRI, magnetic resonance imaging; OP, organophosphorus; PTSD, post-traumatic stress disorder; T2, spin-spin relaxation time; VEP, visual-evoked potential. 313 at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from poisoning occur per year, with more than 1 million cases being attributed to occupational exposure (Karalliedde and Senanayake, 1989). Just as alarming are the conservative estimates that pesticide self-poisoning worldwide accounts for more than 250,000 deaths per year, which corresponds to about one-third of the world’s suicide cases (Gunnell et al., 2007). Finally, epidemiologic studies have provided compelling evidence that acute or continued exposure to subacute levels of OP pesticides is associated with increased risks of debilitating neurologic disorders in both adults and developing children (Levin and Rodnitzky, 1976; Savage et al., 1988; Dahlgren et al., 2004; Rosas and Eskenazi, 2008; Engel et al., 2011). Acute signs and symptoms of OP poisoning result primarily, although not exclusively, from their common action as irreversible inhibitors of acetylcholinesterase (AChE), the enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh) (Albuquerque et al., 1985). Persistent stimulation of muscarinic receptors by accumulated ACh leads to a muscarinic syndrome characterized by miosis, profuse secretions, bradycardia, bronchoconstriction, hypotension, and diarrhea. Overstimulation of nicotinic receptors triggers tachycardia and skeletal muscle fasciculation, whereas their subsequent desensitization contributes to muscle weakness. The broad range of central nervous system (CNS)–related acute effects includes anxiety, restlessness, confusion, ataxia, tremors, seizures, and central cardiorespiratory paralysis (reviewed in Hurst et al., 2012; see also Yokoyama et al., 1998). Therefore, treatment of acute OP intoxication relies heavily on the use of an oxime to reactivate OP-inhibited AChE, atropine to block the overactivation of muscarinic receptors, and benzodiazepines to reduce the incidence and intensity of OP-induced convulsions and the resulting neuropathology. However, the inadequate outcomes of these treatments have been extensively scrutinized (Buckley et al., 2004). For example, AChE inhibited by some OPs, including the nerve agent soman, ages quickly and cannot be reactivated by clinically available oximes (Kassa, 2002). Some clinical trials have also revealed no clinical benefit of the use of oximes against poisoning with different pesticides, despite the fact that blood AChE was reactivated (Buckley et al., 2011). In addition, as discussed later in this article, recurrent seizures develop even after OP-induced acute seizures are controlled with benzodiazepines (Sekijima et al., 1997). Thus, there is an urgent need to develop new therapeutic strategies to treat and/or prevent the immediate and delayed toxicity of OP compounds. The public health relevance of OP toxicity to humans worldwide, the need for a comprehensive understanding of the AChE-unrelated mechanisms that contribute to the pathologic conditions triggered by exposure of males and females at different ages to OPs, and the urgency to develop efficacious medical countermeasures to treat and/or prevent these conditions underscore the need to identify optimal animal models in which the clinical manifestations of human OP toxicity can be replicated. In the present article, we provide a comprehensive analysis of the natural history of acute and delayed signs and symptoms that develop following exposure of humans to OP compounds, with an emphasis on nerve agents. This analysis, then, is followed by a discussion of how faithfully the clinical manifestations of human exposure to OP compounds can be reproduced in different animal models of OP intoxication and a brief review of mechanisms that contribute to the immediate and delayed OP neurotoxicity. This information placed in perspective lays the groundwork for future research aimed at developing safe and effective therapeutic interventions against the acute and delayed toxic effects induced by OP pesticides and nerve agents. Clinical Manifestations of Human Exposure to OP Compounds: Acute and Long-Term Clinical Signs and Symptoms Studies of humans who had been experimentally exposed to nerve agents under controlled conditions provided some of the first evidence of the direct effects of nerve agents on neurologic functions in humans. For example, Bowers et al. (1964) analyzed the clinical signs presented by 96 young male volunteers on active duty in the Army or the Air Force following their percutaneous exposure to a low dose of an OP compound likely to be VX. Although the volunteers did not develop overt signs of acute toxicity, they presented with a syndrome that was broadly referred to as a “state of altered awareness” and was characterized by difficulty in sustaining attention and slowing of intellectual and motor processes, in addition to subjective feelings of agitation, anxiety, and confusion (Bowers et al., 1964). In general, these clinical manifestations were associated with the inhibition of AChE in red blood cells, and symptomatic recovery was associated with the recovery of AChE activity (Grob and Harvey, 1953; Bowers et al., 1964). Long-term follow-up of these individuals is not available. However, case reports of humans occupationally or intentionally exposed to nerve agents, follow-up studies of the victims of the 1995 terrorist attack with sarin in the Tokyo subway, and case reports and follow-up studies of humans accidentally or intentionally exposed to OP pesticides, summarized below, all support the contention that persistent delayed neurologic deficits develop following an initial exposure to these chemicals. Metcalf and Holmes (1969) reported that workers who had a history of exposure to OP compounds presented years later with attention deficits, memory impairment, and difficulty in maintaining alertness that were accompanied by increased slow electroencephalographic activity in the u range. A subsequent study compared the electroencephalograms (EEGs) recorded from industrial workers years after their confirmed accidental exposure to sarin with those recorded from control subjects (Duffy et al., 1979). In this study, visual inspection of the EEGs revealed that the exposure to sarin was associated with an increase of the slow (d and u) frequencies and a reduction of the a frequency. Spectral analysis of the EEGs also revealed a significant increase of the high-frequency activity in the b range (12–30 Hz) years after the exposure to sarin (Duffy et al., 1979). Follow-up studies of Japanese victims of the sarin attack in Matsumoto also reported that, long after the sarin exposure, victims of the attack presented with significant EEG alterations, some of which were consistent with recurring electrical seizures (Sekijima et al., 1997). One of the victims was a male subject who at the time of the attack presented severe disturbance of consciousness, developed convulsions, and required assisted ventilation. At a 1-year follow-up examination, sharp wave complexes, which are consistent with epileptiform activity (Westmoreland, 1998), were present in the EEG of this victim. Two other victims were women who at the time of the attack presented no convulsions, needed no artificial ventilation, and 314 Pereira et al. at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from had only mild disturbance of consciousness. During the 1-year follow-up examination, bursts of d activity, which often correlate with focal brain lesions (Harmony et al., 1995), were present in their EEGs. Abnormal bursts of d activity could still be detected in the EEG of one of the two female victims during a 2-year follow-up examination (Sekijima et al., 1997). During the 2-year follow-up examination, 14-Hz spikes were also detected in the EEG of a fourth victim, a female who at the time of the attack presented no convulsions and needed no respiratory support (Sekijima et al., 1997). Although the authors suggest that these 14-Hz spikes represented epileptic electroencephalographic changes, caution is warranted given that this patient was 16 years old at the time of the examination and 14-Hz spikes frequently appear in the EEG during adolescence (Klass and Westmoreland, 1985). These case reports clearly indicate that patients who do not develop acute convulsions in response to an acute exposure to nerve agents can, years later, present with abnormal slow d waves that are suggestive of focal brain lesions. They also support the notion that patients who survive a severe case of nerve agent intoxication can years later present with epileptic EEG discharges. It has been suggested that hypoxia resulting from motor convulsions and respiratory distress during the acute phase of OP intoxication contributes to the neurologic deficits that develop and persist long after the acute phase of intoxication subsides (Nozaki et al., 1995; Hatta et al., 1996). For example, Nozaki et al. (1995) reported the case of a patient who presented with amnesia 15 days and 6 months after recovering from convulsions induced by an exposure to VX. Hatta et al. (1996) reported the case of another patient who presented with retrograde amnesia and personality changes after recovering from clonic-tonic generalized convulsions and severe episodes of dyspnea induced by sarin during the terrorist attack in Tokyo. However, case reports and follow-up studies of people who did not experience overt CNS signs of intoxication following an OP exposure and yet developed persistent delayed neurologic deficits, as described below, support the contention that neurologic impairments can develop following an acute exposure to levels of nerve agents or OP pesticides that are not sufficient to induce convulsions and/or hypoxia. The 1995 terrorist attack with sarin in the Tokyo subway is the largest documented exposure of a civilian population to a nerve agent. Approximately 95% of the 111 victims who were admitted to hospitals and diagnosed as moderately or severely intoxicated received the conventional therapeutic interventions for OP poisoning, including atropine to block the overactivation of muscarinic receptors and pralidoxime to reactivate sarin-inhibited AChE; diazepam was also used as needed to treat victims who developed convulsions (Okumura et al., 1996). Even though the treatments effectively countered the acute signs of toxicity, victims of the sarin attack who did not present with episodes of convulsion at the time of the attack presented years later with significant memory decline (Hood, 2001; Nishiwaki et al., 2001). Although it is well recognized that post-traumatic stress disorder (PTSD) associated with the experience of a warfare attack can confound the evaluation of long-term health effects induced by the chemical used in the attack, some studies have provided evidence that PTSD alone cannot explain changes in neurophysiologic functions and disruption of the structural integrity of specific brain regions observed in victims who had been exposed to sarin in the Tokyo subway attack (Murata et al., 1997; Yamasue et al., 2007). For example, 6–8 months after the sarin attack, event-related and visual-evoked potentials (P300 and VEP, respectively) were analyzed in 18 victims of the attack and 18 control subjects (Murata et al., 1997). P300 is an event-related potential associated with decision making, and VEP is an evoked potential that reflects the conduction time from the retina to the visual cortex. In victims of the attack, the latencies of both P300 and VEP were significantly longer than those measured in control subjects. The finding that the longer latencies of those potentials did not correlate with the victims’ high PTSD scores strongly indicated that sarin, rather than PTSD, was the cause of the neurophysiologic alterations measured in those patients (Murata et al., 1997). Likewise, Nishiwaki et al. (2001) reported that first responders who were dispatched to the site of the attack in Tokyo presented 3 years later with memory deficits that were evident in the backward digit span test and were independent of PTSD symptoms. Finally, Yamasue et al. (2007) reported that 5 years after the attack victims presented with significant decrease in gray matter volume in the right insular cortex, the right temporal cortex, and the left hippocampus. In these victims, the volume of the left subinsular white matter was positively correlated with decreased serum cholinesterase levels measured after the incident, but not with the occurrence or severity of PTSD. A case report published in 2010 corroborated the notion that exposure of humans to low levels of sarin can trigger delayed neurocognitive deficits (Loh et al., 2010). This is the report of a US Army sergeant who 8 months after experiencing mild signs of acute toxicity following an exposure to sarin in Iraq presented with attention deficits and impaired motor coordination in the absence of any signs or symptoms of emotional distress or mood disorders. Persisting neurologic symptoms of memory loss, decreased concentration, irritability, and personality changes have also been observed in all members of a family who were accidentally exposed to the OP pesticide diazinon (Dahlgren et al., 2004). A pesticide company mistakenly sprayed the interior of the family’s house with diazinon, and, soon after the exposure, all members of the family presented acute signs and symptoms of OP intoxication, including headaches, nausea, skin irritation, runny nose, and vomiting. They did not present with convulsion. Three months to 3 years later, all family members presented with cognitive deficits and mood disorders. In these cases, the persistent neurologic dysfunctions following the exposure to the OP pesticide clearly developed in the absence of acute convulsions and hypoxia. Population studies have provided additional evidence that signs of emotional distress and memory deficits develop in humans long after their recovery from acute poisoning by cholinesterase inhibitors or from a hypercholinergic syndrome following acute occupational exposure toOP pesticides (Levin andRodnitzky, 1976; Savage et al., 1988; Rosenstock et al., 1991; Steenland et al., 1994; Wesseling et al., 2002; Roldán-Tapia et al., 2005). In summary, a cholinergic crisis defines the acute phase of OP intoxication in humans, even though the prevalence of specific signs and symptoms is OP specific (e.g., Nozaki et al., 1995). In addition, although it is true that subtle differences in the prevalence of delayed signs and symptoms of OP neurotoxicity also exist among individuals exposed to different OP pesticides and nerve agents, it appears that mood disorders, specifically anxiety and depression, and cognitive Acute and Delayed Clinical Manifestations of OP Toxicity 315 at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from deficits, particularly attention deficits and memory impairment, are common persistent neurologic conditions seen in individuals long after their exposure to these chemicals (Metcalf and Holmes, 1969; Levin and Rodnitzky, 1976; Savage et al., 1988; Rosenstock et al., 1991; Steenland et al., 1994; Nozaki et al., 1995; Hatta et al., 1996; Hood, 2001; Nishiwaki et al., 2001; Wesseling et al., 2002; Dahlgren et al., 2004; Roldán-Tapia et al., 2005). In many cases, these delayed neurologic deficits have been observed in the absence of acute convulsions and/or hypoxia induced by the OP compounds. In some cases, these deficits have also been dissociated from the trauma associated with the exposure event and have been correlated with the following: 1) disruption of the structural integrity of different brain structures, including the hippocampus and the cingulate cortex, as seen in magnetic resonance imaging (MRI) studies of victims of the Tokyo subway terrorist attack with sarin (Yamasue et al., 2007); 2) decreased regional cerebral blood flow, particularly in the occipital lobes, as seen in a single-photon emission computed tomography study of subjects long after they had experienced an acute intoxication with OP pesticides (Mittal et al., 2011); and 3) increased slow electroencephalographic activity, as reported in individuals occupationally exposed to sarin (Metcalf and Holmes, 1969). Therefore, at a minimum, translationally relevant animal models should present acute and delayed signs and symptoms of OP toxicity that recapitulate those described in this work and should also predict the effectiveness and safety of medical countermeasures against OP