Behavioral Toxicity of Nerve Agents

CHAPTER

33

Behavioral Toxicity of Nerve Agents JIRI KASSA, JIRI BAJGAR, KAMIL KUCA, AND DANIEL JUN

I. INTRODUCTION

II. THE METHODS USED TO EVALUATE BEHAVIORAL EFFECTS OF NERVE AGENTS

Behavioral changes in humans exposed to highly toxic organophosphorus compounds, called nerve agents, have been discussed in numerous reports. The incidence of behavioral effects is higher in individuals who have been severely exposed to nerve agents, but they may occur in individuals who have received a small exposure and have no or minimal physical signs and symptoms. The behavioral effects usually start within a few hours and last from several days to several weeks or months. The most frequent symptoms include feelings of uneasiness, tenseness, and fatigue. Exposed individuals may be forgetful and generally display impaired memory and learning, poor comprehension, decreased ability to communicate, or occasional mild confusion. There are a few reports describing behavioral changes in subjects accidentally exposed to nerve agents. They reported sleep disturbance, mood changes, fatigue, jitteriness or tenseness, an inability to read with comprehension, difficulties with thinking and expression, forgetfulness, a feeling of being mentally slowed, depression, irritability, giddiness, poor performance in arithmetic tests, minor difficulties in orientation, and frightening dreams. It was observed that the complex of central nervous system (CNS) symptoms may not fully develop until 24 h after exposure. In addition, no correlations between the presence or severity of symptoms and the degree of acetylcholinesterase inhibition were seen. Most of the effects of exposure disappear within 3 days. It was concluded that not only severe but also mild intoxication of nerve agents may cause behavioral and psychological disturbances. In general, the behavioral effects have not been permanent but have lasted from weeks to several months, or possibly several years. Long-term behavioral effects after poisoning with nerve agents or organophosphorus insecticides have been reported (Karczmar, 1984; Levin and Rodnitzki, 1976; Sidell, 1974). These reports are based on clinical observations, which are occasionally supported by psychological studies.

Handbook of Toxicology of Chemical Warfare Agents

A. Functional Observatory Battery The functional observatory battery (FOB) is a noninvasive and relatively sensitive type of neurobehavioral examination of 40 sensory, motor, and automatic nervous functions. Some of them are scored (Table 33.1), and the others are measured in absolute units (Frantik and Hornychova, 1995; Slechta, 1989). The first evaluation is made when nerve agent-exposed or control rats are in the home cage. The observer evaluates each animal’s posture, palpebral closure and gait, and the presence or absence of convulsions is noted. Each rat is then removed from the home cage and briefly held in the hand. The presence or absence of spontaneous vocalization, piloerection, and other fur and skin abnormalities as well as irritability is noted. Lacrimation and salivation are also observed. Other signs such as exophthalmus, crustiness around the eyes, or emaciation are recorded too. The rats are then placed on a flat surface which serves as an open field. A timer is started for 3 min during which the frequency of rearing responses is recorded. At the same time, gait characteristics are noted and ranked, and activity, tremor, convulsions, and abnormal posture are evaluated. At the end of the third minute, the number of fecal boluses and urine pools on the absorbent pad is registered. Then, a reflex test that consists of recording each rat’s response to the frontal approach of the blunt end of a pen, a touch of the pen to the posterior flank, and an auditory click stimulus is used. The responsiveness to a pinch on the tail and the ability of pupils to constrict in response to light are then assessed. These measurements are followed by a test for the aerial righting reflex, then by the measurements of forelimb and hindlimb grip strength, body weight, rectal temperature, and finally hindlimb landing foot splay. The whole battery of tests requires approximately 6–8 min per rat. Motor activity data are collected using an apparatus for testing

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Scored values only Marker

L2

L1

0

Posture

1

2

3

4

Sitting or standing Passive Very easy

Rearing

Asleep

Flattened

Normal Easy

Flight Difficult

Hypertonia Slight

Rigidity Severe

Defense Moderately difficult Fasciculations Crusta

Open

Slightly

Half-way

Normal No Normal None None Normal

Exo Yes Pale Slight Slight Repetitive

Erythema Severe Severe Nonrhythmic

Cyanosis

Tonic movements

Normal

Gait

Normal

Contraction of extensors Ataxia

Catch difficulty Ease of handling Muscular tonus Lacrimation

Atonia Hypotonia Normal None

Palpebral closure Endoexophthalmus Piloerection Skin abnormalities Salivation Nose secretion Clonic movements

Endo

Opisthotonus Overcompensation of hindlimb movement

Colored Mild tremors

Colored crusta Completely shut

Pigmented

Severe tremors Emprosthotonus Explosive jumps Forelimbs are Feet point extended outwards from body

5 Lying on side Escape

6

7

Crouched Head over bobbing Aggression

Ptosis

Cold

Injury

Myoclonic

Clonic

Tonic convulsions Walks Hunched on tiptoes body

Body is flattened against surface

SECTION III $ Target Organ Toxicity

TABLE 33.1. Functional observational battery (FOB)

Gait score

Normal

Slightly impaired

Totally impaired Totally impaired Normal

Mobility score

Normal

Slightly impaired

Arousal (level of unprovoked activity) Tension Stereotypy Bizarre behavior

Very low Partial (ears) Head weaving Head

Stupor Body weaving Body

Grooming Self-mutilation

Approach response

No reaction

Normal

Slow reaction

Touch response

No reaction

Normal

Slow reaction

Click response

No reaction

Normal

Slow reaction

Tail-pinch response

No reaction

Normal

Slow reaction

Circling Abnormal movements Energetic reaction Energetic reaction Energetic reaction Energetic reaction

Mydriasis Normal reaction Normal

Slightly uncoordinated Lands on side

Pupil size Pupil response Righting reflex

None None None

Miosis

Normal No reaction

Lands on back

Enhanced

Permanent

Others Others Exaggerated reaction Exaggerated reaction Exaggerated reaction Exaggerated reaction

CHAPTER 33 $ Behavioral Toxicity of Nerve Agents

Sporadic

Somewhat impaired Somewhat impaired Reduced

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SECTION III $ Target Organ Toxicity

a spontaneous motor activity of laboratory animals. The animals are placed for a short period (10 min) in the measuring cage and their movement (total horizontal activity, stereotypical activity, rearing, jumping, scratching, and total vertical activity) is recorded.

B. Performance on the RAM Task RAM sessions are conducted using an eight-arm commercially available radial maze measuring 137.2 cm in diameter. The center of the maze is a plastic octagon hub measuring 26.67 cm across, with a Plexiglass lid and wire grid floor. A Plexiglass arm with a wire mesh floor is attached to each of the eight sides of the hub. The entrance to each arm contains a motorized guillotine door allowing access to and from the hub. Each arm’s runway contains two floor-mounted switches, which are depressed by the weight of the rat when present in the proximal and distal portion of the runway, respectively. The terminal portion of each arm contains a food dispenser, for delivering food pellets, connected to a trough that is outfitted with the photoemitter/ detector unit that can detect access by the rats. Experiments are controlled and monitored using a commercial hardware interface and a microcomputer using the L2T2S software control system (Coulbourn Instruments). For the RAM task, four of eight arms are ‘‘baited’’. That is, a single food pellet is available upon a nose-poke into the food trough at the terminal portion of four arms. Each rat is randomly assigned a maze configuration of four baited arms from 37 possible configurations which excludes more than two consecutive baited arms. Thus, the same configuration of baited arms is used for a particular rat for each of the sessions, but different configurations can be used for different rats. Sessions begin with the rat placed in the center hub compartment and the doors to the eight arms are raised. The rat is then free to explore the maze to obtain the food rewards available from the four baited arms. The session is terminated when a rat obtains all four food rewards or 15 min have elapsed. If a rat does not complete the maze within 15 min, a completion time of 15 min is assigned and errors are analyzed. Failure to complete the maze, however, is infrequent and only occurs during the initial few sessions on the maze. No familiarity training with the maze is conducted prior to the first session. The major dependent variables characterizing performance on the RAM task are the time to complete the maze and the number of errors made. Errors are designated as occurring when a rat chooses an unbaited arm (reference memory error) or when a rat returns to a baited arm after obtaining the food reward (working memory error) (Genovese et al., 2006).

C. Acoustic Startle Response and Pre-pulse Inhibition The animals are tested for acoustic startle response (ASR) and pre-pulse inhibition (PPI) in the SM100 Startle Monitor

system. The system is usually programmed for six types of white-noise burst stimulus trials: no stimulus (background, 60 dB), pre-pulse (70 dB), pulse (100 dB and 120 dB), prepulse plus pulse (70 dB þ 100 dB and 70 dB þ 120 dB). Each trial type is presented ten times in ten blocks. Stimuli are presented in random order to avoid order effects and habituation. The inter-trial interval can vary from 9 to 16 s. All animals are regularly handled before individual tests in order to minimize handling-related stress. Animals are pair matched according to baseline values into the experimental groups using the average of the response to 100 dB and 120 dB. The tested animals are restrained loosely in holders that are placed on a sensing plate transforming movements of the body (jerks) into an analog signal through an interface. Finally, the percentage pre-pulse inhibition measures are calculated as the difference between the pulse alone and multiplied by 100. Percentage scores are typically used to minimize the effect of individual variation of startle amplitude on pre-pulse inhibition (Mach et al., 2008).

D. Performance on Y-Maze Cognitive functioning can be tested using a Y-maze with averse motivation by a strong electric footshock, evaluating learning and spatial memory (Koupilova et al., 1995). The Y-maze is a fully automated apparatus used for the study of behavior of laboratory rats. It is a plastic box consisting of a square start area (285  480 mm) separated by a Plexiglass sliding door from two trapezoid, black and white arms – choice area (140  324 mm). The grid-floor in the start and choice area is electrifiable. The animal (usually rat) is placed on the start area and after 48 s electric footshocks (60 V, 50 Hz, duration 0.5 s) are applied at 5 s intervals. The rats try to avoid the shock by escaping to one of two arms. In the case of a rat moving to the wrong (dark) arm, the rat fails to avoid further footshock. The animals are taught spatial discrimination with the preference of the black or white arm in the Y-maze. The latency to enter the correct arm is measured and the number of wrong entries is counted. Before exposure to nerve agent, the rats are trained to avoid footshock by moving to the correct (white) arm in the Y-maze. It usually takes 4 weeks of training to reach the criterion which was 80% or more correct averse behavior (moving to the correct arm) within less than 1.5 s. During the training, ten sessions (two trials/session) per week lasting 4 min are realized. The exposure starts the day after the animals reached this criterion. The latency time to enter the correct arm by nerve agent-exposed rats and the number of entry errors are compared to the values obtained from the control rats exposed to the pure air instead of nerve agent.

E. Performance on T-Maze Cognitive functioning can be also tested using a T-maze, consisting of five segments, a starting and a goal compartment

CHAPTER 33 $ Behavioral Toxicity of Nerve Agents

to evaluate learning, spatial memory and spatial orientation (Koupilova and Herink, 1995). The rats are trained, with the food reward, to run through the maze in less than 10 s without entering the side arm. The time necessary to reach the goal box is recorded. Before exposure to nerve agent, the rats are trained to reach the goal box as soon as possible by moving to the correct segment in the T-maze. It usually takes 4–6 weeks of training to reach the criterion which was 80% or more correct behavior. The exposure starts the day after the animals reached this criterion. The time of reaching the goal box by nerve agent-exposed rats is compared to the values obtained from the same rats immediately before nerve agent exposure and from control rats exposed to pure air instead of nerve agent.

or 4, 20 cm off the pool wall. The platform is sunk 2 cm below water surface, so it is not visible from the rats’ view owing to the water mirror effect. The yellow rectangle (30 cm  40 cm) is fixed on the pool wall, immediately close to the platform, as the spatial conditional cue (Robinson et al., 2004). Its place is variable according to the platform. Another dark rectangle is randomly fixed on the pool wall in different compartments (without platform) as the negative conditional cue. Round the pool, there are several stable extramaze cues in the room that the rat could use to navigate the maze (Morris, 1984). However, the impact of extramaze cues is not significant due to high maze walls.

III. LONG-TERM BEHAVIORAL EFFECTS OF ACUTE HIGH-LEVEL EXPOSURE TO NERVE AGENTS

F. Performance on Morris Water Maze The water maze is often used for the evaluation of effects of various compounds on memory functions, i.e. memory formation, consolidation and retrieval effects due to its advantages and broad utilization. The Morris water maze (WM) is a widely used measurement of visuospatial learning that has been demonstrated to have high validity in identifying cognitive effects of various brain lesions and the effects of drugs used to treat cognitive deficits (Morris, 1984; Myhrer, 2003). Special motivation such as food and water deprivation is not required for the WM performance. The effect of odor cue is eliminated in the WM. In addition, rats are forced to swim in the WM. They cannot choose whether or not to move, so failure to respond is not a confound (Shukitt-Hale et al., 2004). The place learning version with submerged platform can be used for working memory tests (Myhrer, 2003). The WM can be used to measure spatial learning and memory in the case of the evaluation of cognitive impairment in rats because of mentioned advantages. The rats perform cognitive tasks that require spatial learning and memory – the ability to acquire a cognitive representation of location in space and the ability to effectively navigate the environment in the WM (Shukitt-Hale et al., 2004). Memory alterations appear to occur mostly in secondary memory systems and are reflected in the storage of newly acquired information (Bartus et al., 1989; Joseph, 1992). It is thought that hippocampus mediates allocentric spatial navigation (i.e. place learning) and prefrontal cortex is critical to acquiring the rules that govern performance in particular tasks (i.e. procedural knowledge), while the dorsomedial striatum mediates egocentric spatial orientation (i.e. response and cue learning) (McDonald and White, 1994; Oliviera et al., 1997). The water maze consists of a black circular pool (180 cm diameter  80 cm high) filled to a depth of 25 cm with water of room temperature (Raveh et al., 2002). The pool is imaginarily divided into four same compartments numbered 1–4 clockwise. The black antireflective circular escape platform (15 cm diameter) is placed into compartment no. 1

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Much of the data regarding long-term neurological sequelae to exposures to cholinesterase inhibitors in humans have been gathered following accidental exposures to organophosphorus (OP) compounds (pesticides as well as nerve agents). Nevertheless, the extrapolation from these exposures to prediction of effects from nerve agent is difficult because: 





the cholinergic crisis caused by acute, severe intoxication with the OP pesticides is generally much longer than that caused by OP nerve agents OP pesticides-induced delayed peripheral neuropathy can be caused by nerve agents only at doses many times greater than the LD50 (Davis et al., 1960) a delayed manifestation of OP poisoning has not been described after administration of nerve agents to animals or in the instances of nerve agent poisoning in humans (Sidell, 1997).

There have been descriptions of the acute effects in humans that follow high-dose exposure (LD50) to nerve agents soman, sarin, and VX (Inoue, 1995; Nakajima et al., 1997; Nozaki et al., 1995; Sidell, 1974). The similar cluster of behavioral symptoms (anxiety, psychomotor depression, intellectual impairment, and sleep disturbance) was observed in the immediate period following resolution of the acute signs of intoxication and then slowly faded with time, sometimes taking months to be fully resolved. The CNS symptoms noted following short-term exposure of humans to diisopropyl fluorophosphate (DFP) were excessive dreaming, insomnia, jitteriness and restlessness, increased tension, emotional lability, subjective tremulousness, nightmares, giddiness, drowsiness, and mental confusion. CNS symptoms were correlated with the depression of red blood cell acetylcholinesterase (AChE; EC 3.1.1.7) to 70 and 60% of original activity and they disappeared within 1 to 4 days (Grob et al., 1947). It was also noted that more severely exposed individuals and those with multiple exposures tended to display persistent symptoms that included forgetfulness, irritability, and confused

486

SECTION III $ Target Organ Toxicity

thinking, although the duration of these persistent symptoms was never clearly defined (Holmes and Gaon, 1956). These CNS symptoms are virtually identical to those that have been reported to occur following high-level exposure to nerve agents. It was shown in the study of human sarin poisoning that sarin-induced behavioral effects were virtually identical to those reported for DFP. These effects coincided with the depression of plasma ChE and red blood cell AChE activity to approximately 60 and 50% of original activity (Grob and Harvey, 1958). The behavioral symptoms such as anxiety, psychomotor depression, a general intellectual impairment consisting of difficulties in concentration and retention, and sleep impairment generally involving insomnia due to excessive dreaming were also described during human poisoning with nerve agent VX (Bowers et al., 1976). The exposure to high doses of OP compounds including nerve agents has been demonstrated to result in severe brain neuropathology that involves not only neuronal degeneration and necrosis of various brain regions (Lemercier et al., 1983; McLeod et al., 1982; Petras, 1981) but also persistent severe alteration in behavior and cognitive incapacitation especially impairments of learning and memory (Bushnell et al., 1991; McDonald et al., 1988). The most significant injury caused by OP poisoning is neuronal degeneration of the hippocampus that is associated with spatial learning and memory. Therefore, impairment of cognitive functions, especially incapacitation of learning and memory, belongs to the most frequent central signs of acute OP poisoning (Marrs, 1993; McDonald et al., 1988). In addition, the adverse effects of OP compounds on cognition functions, such as learning and memory, may persist for quite some time after termination of toxicant exposure. The results from several studies have demonstrated the presence of OP compounds-induced learning impairments several days after the classic signs of OP toxicity have subsided (Buccafusco et al., 1990; Bushnell et al., 1991; McDonald et al., 1988). Behavioral effects are typically evident before the occurrence of physical symptoms. These effects were associated with whole blood ChE inhibitions of >60%. Several studies of the long-term effects of the sarin exposure victims from Japan have been published. Exposure to nerve agents in humans was found to produce effects that include cognitive deficits and memory loss (Hatta et al., 1996; Hood, 2001; Okudera, 2002). Eighteen victims of the Tokyo subway incident were evaluated at 6 to 8 months after exposure (Yokoyama et al., 1998). Sarin-exposed individuals scored significantly lower than controls on a digit symbol substitution test, and scored significantly higher than controls on a general health questionnaire (GHQ, psychiatric symptoms) and a profile of mood states (POMS, fatigue). The elevated scores on the GHQ and POMS were positively related to the increased PTSD (post-traumatic stress disorder) scores and were considered to be due to PTSD (Yokoyama et al., 1998). There have been two brief reports of severely poisoned nerve agent victims (one sarin

and one VX) in Japan who experienced retrograde amnesia, possibly due to prolonged periods of seizures and/or hypoxia (Hatta et al., 1996; Nozaki et al., 1995). Symptoms related to sarin exposure in Japan still exist 1–3 years after the incident and include fatigue, asthenia, shoulder stiffness, and blurred vision (Abu-Qare and Abou-Donia, 2002). The existence of long-term behavioral effects following acute exposure to high doses of nerve agents was many times verified with the help of laboratory experiments on animals. There are numerous studies in animals showing that survivors of high-level OP exposure can experience subtle but significant long-term neurological and neuropsychological outcomes that are detectable months or even years following the recovery from acute poisoning (Brown and Kelley, 1998). Exposure of animals to nerve agents was shown to produce neurotoxicity in the CNS areas associated with cognition and memory functions (Koplovitz et al., 1992; Petras, 1994). There are a few studies that revealed changes in the brain following sublethal nerve agent exposure that involve not only the cholinergic system but also the glutamatergic system (Lallement et al., 1992; McDonough and Shih, 1997). Excitotoxic injury caused by increased levels of glutamate has repeatedly been shown to cause cognitive dysfunction (O’Dell et al., 2000). Therefore, the disruption of cognitive functions, especially spatial and working memory, seems to be the most frequent and the most observable behavioral effect of nerve agent poisoning. The studies show changes in the brain following sublethal nerve agent exposure that lead to memory and attention deficits that normally involve the hippocampus (Hatta et al., 1996; Miyaki et al., 2005; Nishikawi et al., 2001). The role of hippocampus in complex visuospatial learning and memory has been well established. The high concentration of NMDA and AMPA glutamate receptors, which play a key role in hippocampal-mediated learning and memory, also makes the hippocampus highly vulnerable to glutamate-induced excitotoxic injury from nerve agent poisoning (Filliat et al., 2007; Lallement et al., 1992; Shih et al., 1990). Following a high-dose exposure (above 0.5 LD50) seizures are a prominent sign of nerve agent intoxication and these prolonged seizures can produce neural lesions (McDonough and Shih, 1997). Thus, neurological and behavioral deficits are predictable long-term effects following exposure to such doses of nerve agents. Animals exposed to high (convulsive) doses of nerve agent can develop spontaneous seizures, and display hyperactive and aggressive behavior and profound deficits in learning and/or performance of a variety of behavioral tasks. Animal studies have demonstrated deficits in acquisition of several types of operant tasks, performance of serial probe recognition task, maze learning, and passive avoidance learning following acute poisoning with nerve agents (McDonough et al., 1986; Modrow and Jaax, 1989; Raffaele et al., 1987). The inhalation exposure to high-level sarin induced in rats impaired memory processes seen at 1 month post-exposure

CHAPTER 33 $ Behavioral Toxicity of Nerve Agents

with no recovery of cognitive function during the 6 month follow-up period. In the open field, sarin-exposed rats showed a significant increase in overall activity with no habituation over days. In a working memory paradigm in the water maze, the same rats showed impaired working and reference memory processes with no recovery. These data suggest long lasting impairment of brain functions in surviving rats following a single sarin exposure. Animals that seem to fully recover from the exposure, and even animals that initially show no toxicity signs, develop some adverse neurobehavioral changes with time (Grauer et al., 2008). These findings are in accord with reports on long-term behavioral impairment following exposure to OP pesticides used in agriculture (Wesseling et al., 2002). Similarly, longterm follow-up of victims of the sarin attacks in Japan demonstrated neurological as well as emotional and cognitive changes up to 7 years post-exposure (Miyaki et al., 2005; Ohbu et al., 1997; Yokoyama et al., 1998). Generally, according to high-dose exposure studies, animals exposed to nerve agents that exhibit seizures that are not promptly controlled develop brain damage and subsequent neurobehavioral problems. Animals that do not develop seizures or those that are rapidly and effectively treated with drugs that stop the seizures suffer no brain lesion and display no long-term neurobehavioral deficits.

IV. CHRONIC BEHAVIORAL EFFECTS OF SINGLE OR REPEATED LOW-LEVEL EXPOSURE TO NERVE AGENTS Anticholinesterase compounds such as nerve agents can alter behavioral functions even after small subtoxic doses. There are very few data on human exposures. Based on the data describing the signs and symptoms in accidentally exposed humans, some long-term health effects, including behavioral effects of repeated subclinical exposures to OP compounds, were observed (Wesseling et al., 2002). When the workers were exposed to small amounts of nerve agents they showed mild toxic signs of exposure including CNS effects such as insomnia, excessive dreaming, restlessness, drowsiness, and weakness (Craig and Freeman, 1953). It was shown that psychological symptoms are probably more common than usually recognized and may persist in more subtle forms for much longer (days, weeks) than physical symptoms (Sidell and Hurst, 1997). Recently, a dose–response association was found between low-dose exposure to sarin and cyclosarin inhalation during the 1991 Gulf War and impaired neurobehavioral functioning as well as subtle CNS pathology as revealed by MRI study (Heaton et al., 2007; Proctor et al., 2006). It is interesting that functional impairments were detected even in people who initially developed only mild or no signs of sarin or cyclosarin toxicity. These data correspond to the published epidemiological studies showing alterations in cognitive functions, impaired memory, and concentrations

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in humans after chronic low-dosage occupational exposure to OP insecticides (Parro´n et al., 1996; Stephens et al., 1995). Increased reported forgetfulness and difficulties in thinking, exposure-related increases in work-related tension, sleep disturbance, restlessness, and nervousness have been documented among sheep farmers exposed to OP pesticides (Beach et al., 1996; Stephens et al. 1995). Based on the experimental animal data, the progression of signs, their neuropharmacological basis, and toxic consequence elicited from acute high-dose exposures have been well characterized (McDonough and Shih, 1993; Shih et al., 2003). However, much less is known about the long-term effects of repeated low-dose nerve agent exposure. Several comprehensive reviews on the long-term health effects of exposure to low-level nerve agent exposure have been published (Moore 1998; Romano et al., 2001). It is known that a significant, clinically manifested AChE inhibition in the central nervous system leading to the neuronal degeneration of some brain regions including the hippocampus, associated with spatial learning and memory, is not necessary for clinically manifested cognitive impairments. This fact corresponds with earlier published data about neurological and neurophysiological outcomes detectable months or even years following recovery from acute OP poisoning (Savage et al., 1988; Yokoyama et al., 1998). It is very difficult to find the real reason for the memory impairments in the case of low-level nerve agent exposure. Recently, a temporal relationship has been demonstrated between OP-induced impairment in performance of a spatial memory task and the protracted decrease in the expression of cholinergic receptors in specific brain regions (including the hippocampus) following the asymptomatic exposure to OP compounds (Stone et al., 2000). Nerve agent-induced impairment of cognitive functions is probably caused by subsequent desensitization and internalization of cholinergic receptors as a reaction of nerve agent-exposed organisms on hyperstimulation of cholinergic receptors, especially in parts of the brain with a high density of cholinergic synapses such as the hippocampus (McDonald et al., 1988; Stone et al., 2000). This means that a decrease in the number of cholinergic receptors in the hippocampus following low-level exposure to OPs without significant AChE inhibition could cause memory impairments. In the available literature on repeated low-dose exposure to nerve agents, soman is the nerve agent studied most often. Mice, rats, guinea pigs, and primates were used to investigate repeated low-dose soman exposure. The effects of repeated soman exposures ranged from performance decrements on a well-learned compensatory tracking task (Blick et al., 1994b) to development of attention deficits (Gause et al., 1985) and hyperreactive responses to handling (Shih et al., 1990). Unlike soman, the amount of literature regarding the effects of repeated low-level exposure to sarin is rather sparse and sometimes conflicting. Rhesus monkeys exposed to low levels of intramuscular sarin showed no signs of adverse health or long-term

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SECTION III $ Target Organ Toxicity

behavioral effects (Burchfiel et al., 1976). In contrast, it has been observed in rats and mice that intraperitoneal injections of subtoxic doses of sarin or soman decreased locomotor activity, altered behavior on the plus-maze and elevated horizontal bridge tests (Baille et al., 2001; Nieminen et al., 1990; Sirkka et al., 1990). It was also shown that repeated low-level sarin inhalation in rats at clinically asymptomatic doses was disruptive to neurophysiological function and caused long-term memory impairments (Kassa et al., 2001a, b). The results of the study related to the measurement of sarin-induced alteration of behavioral and neurophysiological functions at 3 months following low-level sarin inhalation exposure of rats showed a significant alteration of mobile activity and gait characterized by ataxia and an increase in stereotypical behavior. These signs were observed in rats repeatedly exposed to sarin at clinically asymptomatic doses or singly exposed to sarin at doses causing mild muscarinic signs of exposure. These animals had awkward hindlimbs and their mobility was markedly diminished (Kassa et al., 2001d). Spatial discrimination in the Y-maze was also altered in rats exposed to low levels of sarin. While spatial orientation of rats singly exposed to clinically asymptomatic doses of sarin was significantly influenced for a short time only (1 or 2 h following exposure), the rats repeatedly exposed to clinically asymptomatic doses of sarin showed a decrease in Y-maze performance for a relatively long time (until the third week following the exposure) (Kassa et al., 2004). The significant impairment of spatial memory of rats exposed to clinically asymptomatic concentrations of sarin was also observed when cognitive functions were evaluated with the help of T-maze performance. Rats exposed to low-level sarin showed a significant decrease in T-maze performance for a short time (until the first day following the exposure). In addition, the effects of low-level sarin inhalation exposure were dose dependent. When the rats were exposed to lowlevel sarin causing moderate signs of poisoning, their time of passage through the maze was more lengthened at 1 and 2 h following the inhalation exposure compared to the rats exposed to clinically asymptomatic levels of sarin (Kassa et al., 2001c). A single exposure to another nerve agent, cyclosarin, at concentrations that do not produce convulsions or severe clinical signs of toxicity can also produce performance deficits on learned behavioral tasks. However, with repeated exposure, the deficits are not persistent and recovery is complete. In addition, exposure concentrations not producing any evaluated clinical signs of toxicity, other than temporary miosis (in the case of inhalation exposure), do not produce performance deficits on the behavioral tasks (Genovese et al., 2006). Reports in the literature of animal studies show that nerve agents can be administered repeatedly with minimal overt neurobehavioral effects if care is taken in choosing the dose and the time between doses (Sterri et al., 1980, 1981). The

repeated low-level nerve agent exposure made the cognitive impairments longer and higher compared to the single nerve agent exposure. The repeated exposure to low doses of soman can produce small, transient performance decrements only, probably due to the development of a physiological and behavioral tolerance to low levels of ChE activity (Blick et al., 1994a, b). Nevertheless, a progressive and long-lasting inhibition of ChE in CNS following repeated administration of low doses of nerve agent soman was demonstrated (Hartgraves and Murphy, 1992). This study was corroborated by Olson using nerve agent sarin (Olson et al., 2000). Generally, repeated or long-term exposure to low levels of nerve agents can cause neurophysiological and behavioral alterations (Abu-Quare and Abou-Donia, 2002). The rats repeatedly exposed to sarin at doses corresponding to 0.5  LD50 (three times per week, s.c.) showed an increase in acoustic startle and a decrease in distance explored in the open field 2 weeks after sarin exposure. On the other hand, no effect of sarin exposure on passive avoidance was noted at the same time after sarin poisoning. Brain regional AChE was not affected at any time after sarin exposure, but muscarinic receptors were down-regulated in the hippocampus, caudate putamen, and mesencephalon in the sarin group at 2 weeks after sarin exposure. Thus, down-regulation of muscarinic receptors in the hippocampus as a reaction to acetylcholine accumulation at muscarinic receptor sites based on AChE inhibition can be considered a cause of behavior performance deficits, especially disruption of cognitive functions (Scremin et al., 2003). In addition, protracted impairment of cognitive functions in rats exposed repeatedly to low-level organophosphorus compounds may be associated with a decreased rate of AChE recovery in the hippocampus (Prendergast et al., 1997). The results from several studies have demonstrated the presence of OP-induced learning impairments several days after the behavioral signs of OP toxicity have subsided (Bushnell et al., 1991, McDonald et al., 1988). Chronic exposure to OP compounds can also result in specific long-term cognitive deficits even when signs and symptoms of excessive cholinergic activity are not present (Prendergast et al., 1998). Thus, the significant, clinically manifested AChE inhibition in the CNS leading to the neuronal degeneration of some brain regions including the hippocampus is not necessary for the clinically manifested cognitive impairments. This conclusion corresponds with earlier published data about neurological and neurophysiological outcomes detectable months or even years following recovery from acute OP poisoning (Savage et al., 1988; Yokoyama et al., 1998). A current study attempts to show a temporal relationship between OP-induced impairment in performance of a spatial memory task and the protracted decrease in the expression of cholinergic receptors in specific brain regions caused by asymptomatic exposure to an OP compound (Stone et al., 2000). In addition, low-level

CHAPTER 33 $ Behavioral Toxicity of Nerve Agents

OP-induced memory impairment may be associated with a decreased AChE recovery in the hippocampus relative to the cortex. This decreased rate of enzyme recovery may contribute to hippocampal toxicity undelying protracted impairment of working memory and other cognitive functions (Prendergast et al., 1997). Repeated or chronic low-level nerve agent exposure can cause a prolonged inhibition of extracellular AChE leading to a prolonged increase in extracellular acetylcholine (ACh). The prolonged availability of ACh in the synaptic clefts results in feedback inhibition on muscarinic, presynaptic receptors to decrease further ACh release (Russell et al., 1985). The greater ACh release in the nerve agentexposed group may be due to the known down-regulation of muscarinic receptors in response to chronic nerve agent exposure (Churchill et al., 1984). Neurochemical analyses showed that the normal brain neurotransmitter and receptor homeostasis is disrupted even at 10–12 days after 2 weeks of chronic nerve agent exposure at least in the striatum but probably throughout the whole cholinergic system in the brain (Shih et al., 2006).

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noncholinergic outcomes of low-level nerve agent exposure. The long-term behavioral toxicity of nerve agents, especially the alteration of cognitive functions (T-maze, Ymaze, Morris maze test) due to nerve agent-induced delayed toxicity, seems to be connected with the neuropathological damage observed in the hippocampus. Thus, neuropathology of the hippocampus connected with the alteration of cognitive functions can occur after high-level as well as repeated or long-term low-level nerve agent exposure. Neurochemical analysis of repeated or low-level nerve agent exposure provokes the suggestion that the prolonged nerve agent-induced alteration in brain chemistry may be a pharmacological basis for neurobehavioral changes. Thus, it is necessary to follow brain homeostasis during acute as well as chronic nerve agent exposure. Repeated or long-term exposure to low levels of nerve agents can cause neurophysiological and behavioral alterations due to down-regulation of muscarinic receptors in the hippocampus as a reaction to acetylcholine accumulation at muscarinic receptor sites based on AChE inhibition. This phenomenon is considered to be the cause of behavior performance deficits, especially disruption of cognitive functions.

V. CONCLUDING REMARKS AND FUTURE DIRECTION Exposure to high doses of nerve agents has been demonstrated to result in severe brain neuropathology that involves not only neuronal degeneration and necrosis of various brain regions but also persistent severe alterations in behavior and cognitive functions, especially impairment of learning and memory. The most significant injury caused by nerve agent poisoning is neuronal degeneration of the hippocampus which is associated with spatial learning and memory. Therefore, impairment of cognitive functions, especially incapacitation of learning and memory, belongs to the most frequent central signs of acute nerve agent poisoning. In addition, the adverse effects of nerve agents on cognitive functions, such as learning and memory, may persist for a relatively long time following the termination of nerve agent exposure. Behavioral alterations and impairments of cognitive functions were found following acute exposure to nerve agents with the absence of any classic signs of cholinergic toxicity. It was shown based on the experimental results that not only convulsive doses but also clinically asymptomatic doses of nerve agents can cause subtle long-term neurophysiological and neurobehavioral dysfunctions. The neurological and neurophysiological outcomes are detectable months or even years following the recovery from acute poisoning. This probably means that systems other than the cholinergic nervous system can be involved in nerve agent-induced long-term signs of alteration of neurological and neurophysiological functions. Thus, it is necessary in the future to find new markers describing

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