5
Animal Studies
Studies of laboratory animals and other nonhuman systems are essential to understanding possible health effects when experimental research in humans is not ethically or practically possible (NRC, 1991). Animal studies can be used to characterize absorption, distribution, metabolism, elimination, and excretion of chemicals, and they may examine acute (short-term) or chronic (long-term) exposures. Such studies permit a potentially toxic agent to be introduced under controlled conditions (with respect to dose, duration, and route of exposure) to probe effects on many body systems.
Although, animal models are not always ideal replicates of human conditions, there are sufficient similarities between human and animal responses to many toxicants such that animal models can be used to examine mechanistic hypotheses, that is, how the toxic agent exerts its deleterious effects at the cellular and molecular levels. Mechanism-of-action (or mechanistic) studies encompass a range of laboratory approaches with whole animals and with in-vitro systems using tissues or cells from humans or animals. Animal studies can be a valuable complement to human studies of genetic susceptibility or other biomarkers and they can facilitate the study of chemical mixtures and their potential interactions. If the animal models are successful they may be used to evaluate potential therapeutic strategies and interventions.
Early volumes in the Gulf War and Health series described animal toxicology studies that focused on the association between exposure to a specific toxicant (e.g., sarin, solvents, combustion products, depleted uranium [DU]) and the health outcomes that may result from that exposure. Among the exposures that have been assessed in animal models are vaccines, pyridostigmine bromide (PB), DU, pesticides, sarin, and stress; the exposures have been assessed individually and as mixtures. Volumes 4 and 8 of the Gulf War and Health series were concerned only with associations between being deployed to the Persian Gulf region during the war and the prevalence of health outcomes in deployed vs nondeployed veterans; the two volumes did not examine specific toxicant exposures and thus animal studies were not considered in them.
Based on the premise that different populations of Gulf War veterans inevitably experienced multiple and variable chemical exposures, in this chapter, the committee focuses its review on animal toxicology studies that used multiple exposures which were generally considered to be relevant to those exposures
that veterans might have experienced in theater. Thus, studies examining single chemical exposures are not considered here. Because such a review has not been previously conducted by a Gulf War and Health committee, no publication date limits were applied to the literature search for animal studies (see Chapter 2).
ANIMAL MODELS OF GULF WAR ILLNESS
A substantial body of research has been conducted in animal models to determine whether a relationship exists between chemical exposures experienced by veterans during the Gulf War and manifestations of Gulf War illness and other health conditions. Animal models have been used to determine whether the symptoms observed in veterans with Gulf War illness can be reproduced in animals by chemical and vaccine exposures, and if so, what mechanistic underpinnings could contribute to the observed effects. However, it is difficult to design animal models to study Gulf War illness because the types and extent of exposures are unknown and the symptoms of Gulf War illness are diverse.
Neuropathological studies have examined hypotheses that Gulf War–related symptoms are based on the ability of toxicants to increase permeability of the blood–brain barrier, particularly under stressful conditions, and thereby alter brain function, mainly metabolism and histopathology, to produce the symptoms of Gulf War illness. Neurobehavioral approaches have been used to examine whether exposed animals have symptoms that mimic the memory and motor function symptoms of veterans, and whether the symptoms could reflect changes in brain metabolism or histopathology. In addition, some animal studies have examined potential effects of Gulf War toxicants on the reproductive system, the musculoskeletal system, and the immune system as veterans have complained of adverse health effects related to those systems.
There are some challenges in the interpretation of animal study data for the purposes of elucidating Gulf War illness. For example, with the exception of fatigue, symptoms such as headache, and muscle and joint pain, as reported by veterans are difficult to study with standard tests in animals (OTA, 1990). In carrying out its charge, the committee used animal studies to determine whether they provided support for health findings from epidemiologic studies in Gulf War veterans.
This chapter describes studies using the multiple exposures that were reported by veterans during the Gulf War and the effects of those exposures on the brain, and the reproductive, musculoskeletal, and immune systems, as well as behavior and pain. Table 5-1 at the end of this chapter provides a brief overview of the species, exposures, and main results for each study described in the following sections.
BLOOD–BRAIN BARRIER PERMEABILITY, BRAIN HISTOPATHOLOGY,
AND BRAIN METABOLISM AND FUNCTION
Blood–Brain Barrier Integrity
Several investigators have hypothesized that alterations in the blood–brain barrier may be caused by exposure to cholinesterase inhibitors such as the insecticides used to treat the military uniforms, the nerve agent prophylactic PB, and possibly sarin, and that such alterations could be enhanced by the stress of deployment (Abdel-Rahman et al., 2002, 2004; Amourette et al., 2009; Friedman et al., 1996; Grauer et al., 2000; Kant et al., 2001; Lallement et al., 1998; Shaikh and Pope, 2003; Shaikh et al., 2003; Sinton et al., 2000; Song et al., 2002, 2004; Tian et al., 2002). Normally the quaternary ammonium structure of PB reduces its ability to enter the brain, precluding its effects on the central nervous system (CNS). Friedman et al. (1996) found that exposing mice to PB accompanied by an inescapable forced swim
stress protocol reduced the dose of PB required to produce a 50% inhibition in brain acetylcholinesterase activity to less than one-hundredth of the usual dose, thus supporting the proposed hypotheses. However, numerous subsequent studies using different species (e.g., rats, mice, and guinea pigs) and multiple different stress paradigms have not replicated those initial findings. Rats exposed to pole climbing avoidance stress plus tritiated PB to assess its blood–brain barrier permeability, showed no radioactivity in brain micropunches and cryosections (Amourette et al., 2009). The authors concluded that although PB induced effects on the CNS (based on earlier studies), these effects did not seem to be mediated by a central passage of PB linked to increased permeability of the blood brain barrier caused by stress. Shaikh and Pope (2003) and Shaikh et al. (2003) also found no evidence that stress (forced running) in combination with paraoxon exposure (an organophospshate insecticide that inhibits acetylcholinesterase) enhanced PB uptake into brain in rats. Song et al. (2002, 2004) reported that restraint stress failed to affect uptake of PB into the brain; treatment with paraoxon did compromise the blood–brain barrier, but only in young rats. Tian et al. (2002) reported that neither forced running nor forced swimming had any effect on PB toxicity, PB uptake into brain, or PB-induced brain cholinesterase inhibition in rats. Kant et al. (2001) reported that rats exposed to PB and given foot shock stress did not show enhanced stress effects on performance or in levels of stress hormones, nor did stress enhance the passage of PB into the brain. Furthermore, in three other studies, stress actually mitigated or even precluded uptake of PB into the brains of mice, guinea pigs, and rats (Grauer et al., 2000; Lallement et al., 1998; Sinton et al., 2000).
In contrast, Abdel-Rahman et al. (2002) combined 28 days of restraint stress in rats with dermal exposure to PB, DEET (N,N-diethyl-meta-toluamide), or permethrin, either individually or as a mixture of the three chemicals. The combination of chemicals, but not any chemical or stress alone, disrupted blood–brain barrier integrity and produced neuronal death in the cingulate cortex, dentate gyrus, thalamus, and hypothalamus, but not in other regions of the brain. In a later study by this group (Abdel-Rahman et al., 2004), rats exposed to both stress and the chemicals exhibited decreased brain acetylcholinesterase in the midbrain, brainstem, and cerebellum and decreased m2 muscarinic acetylcholine receptor ligand binding in the midbrain and the cerebellum, areas of the brain where no disruption of the blood–brain barrier was observed. The authors suggested that combined stress and the three specific chemicals can damage the cerebral cortex, hippocampus, and cerebellum even in the absence of apparent blood–brain barrier damage.
Brain Metabolism and Histopathology
Multiple aspects of brain metabolism and associated histopathology have also been studied. Scremin et al. (2005) sought to determine if the exposure of veterans to PB as a protective agent would enhance the response to sarin, and lead to delayed behavioral dysfunction, that is, whether induced neurobehavioral dysfunction outlasted effects on inhibition of cholinesterase. Sprague Dawley rats were exposed to low (subsymptomatic) doses of sarin, with or without PB, for 3 weeks with observations of neurobehavioral symptoms as well as brain metabolism, cerebral blood flow, glucose utilization, heart rate, and locomotor activity carried out at 2, 4, and 16 weeks after exposure. Early changes observed at 2 and 4 weeks postexposure included increased regional cerebral blood flow, a finding consistent with known vascular effects of cholinergic agonists. The results indicate that PB protected against some changes seen in the rats exposed to sarin alone. No symptoms, including any changes in regional cerebral blood flood and glucose metabolism, were present in the animals at 16 weeks postexposure. Thus, these studies do not support the hypothesis that delayed symptoms observed in Gulf War illness could be caused by exposure to PB.
A potential synergism of these chemical exposures was subsequently tested by Buchholz et al. (1997), using lower doses in adult Sprague Dawley rats. CNS uptake of C14-labeled permethrin in rats that had received a very high dose of PB (7.75 mg/kg) for 10 days was decreased by 30%, while blood levels of permethrin were not altered by PB treatment. The authors concluded that these results were inconsistent with the hypothesized synergy of such compounds in Gulf War illness. Kant et al. (2001) exposed rats to PB alone or in combination with stress (foot shock) and observed that PB decreased blood acetylcholinesterase by half, but had no effect on cortical brain acetylcholinesterase. PB did not further modify blood corticosterone levels elevated by stress. Thus, PB did not exacerbate the effects of stress on either performance measures or on levels of stress hormones.
A mouse model of Gulf War illness was used to study effects on phospholipid metabolism following coadministration of 2mg/kg PB and 200 mg/kg permethrin intraperitoneal for 10 days followed by up to a 150-day observation period (Abdullah et al., 2011). The doses used were equal to the LD50 (lethal dose for 50% of sample cells) for both compounds: the permethrin dose calculated as the highest expected dose of permethrin expected for Gulf War veterans. No effects were observed at 8 days postexposure, but chronic effects, including cognitive impairment and anxiety, as well as increased astrogliosis in the cortex but not the hippocampus, were observed at 150 days; there was no evidence of microgliosis or neuronal loss. Exploratory brain proteomic analysis found alterations in proteins associated with lipid metabolism and molecular transport in the brain, along with endocrine and immune system metabolic changes. In conjunction with the observations of cortical astrogliosis, the findings suggest a persistent and residual adverse effect on the immune system.
In further experiments by the same group, mice exposed to 1.3 mg/kg/day PB orally, plus 0.13 mg/kg/day permethrin and 40 mg/kg/day DEET dermally, and restraint stress for 28 days (Abdullah et al., 2012) were reported to exhibit sensorimotor deficits, anxiety, and increased astrocytosis in the cerebral cortex, but no histopathological consequences in the hippocampus, nor any increase in microgliosis. Brain phosphatidyl choline (PC) and sphingomyelin content were increased, with the increase in PC containing monounsaturated fatty acids being higher than PC with saturated and polyunsaturated fatty acids. The authors interpreted the lipid changes as suggestive of alterations in peroxisomal pathways and stearoyl-CoA desaturase activity that might relate to neurobehavioral and neuropathological symptoms following exposure to Gulf War agents. In further study, it was hypothesized that PB and permethrin would modulate the concentration of PC and sphingomyelin, which are reservoirs required for acetylcholine synthesis, and that this modulation would persist at the chronic (150 days) time point (Abdullah et al., 2013). PC and sphingomyelin were elevated in brains of exposed mice at 150 days after a 10-day exposure. Lysoplatelet-activating factors, which are products of PC, were decreased. Catalase expression (a marker for peroxisomes) was increased in exposed mice. The authors state that these results are similarly suggestive of peroxisomal and lysosomal dysfunction in the mouse brains at chronic time points postexposure.
Husain and Somani (2004) examined exposure to sarin, PB, and PB plus sarin, each with or without exercise, in mice. Exercise was carried out daily for 10 weeks on a treadmill. Daily sarin and PB dosing was administered during the fifth and sixth week only. Biochemical effects were only assayed at 1 day following the 10-week exposures. The exposures, regardless of exercise, significantly lowered butyrylcholinesterase, acetylcholinesterase, and neurotoxic esterase in plasma, platelets, and nerves, as well as the spinal cord, striatum, and cortex. While the authors concluded that exercise enhanced the adverse effects of Gulf War chemical exposures in mice, the effects observed were minimal and not likely to have biological significance.
DEET (40 mg/kg) or permethrin (0.13 mg/kg) or the combination in 70% ethanol was applied daily to adult male rats for 60 days (Abdel-Rahman et al., 2001). All three conditions produced diffuse neuronal cell death in the cerebral cortex, hippocampus, and cerebellum.
Exposure to chlorpyrifos alone or to a combination of PB, permethrin, and chlorpyrifos for 10 days in adult mice both produced pathological changes in hippocampal, cortical, and amygdalar morphometry (Ojo et al., 2014), including an impairment of synaptic integrity in the hippocampus and altered neuronal differentiation in the dentate gyrus at 3 days postexposure. Both exposures also increased astrocytic glial fibrillary acidic protein immunoreactivity in the piriform cortex, motor cortex, and basolateral amygdala and were accompanied by an increase in acetylcholine levels in the brain. The study purported to show early changes due to Gulf War agents, but it did not examine the persistence of these effects.
The hypothesis that stress-induced corticosteroids would prime the CNS to produce a robust proinflammatory response to neurotoxicants and lead to systemic inflammation was tested in male mice. In a 17-day exposure period, the mice were treated with PB/DEET (14 days), corticosterone (7–17 days), and 1 day (day 15) of di-isopropyl fluorophosphate (DFP), a sarin surrogate (O’Callaghan et al., 2015). The level of corticosterone used is known to be immunosuppressive; the weights of the thymus and spleens of all mice receiving it were reduced 20%. DFP caused neuroinflammation throughout the brain in a manner similar to that caused by sarin, with increased expression of multiple proinflammatory mediators. Corticosterone caused a marked exacerbation of the neuroinflammatory effect of DFP. The combination of PB and DEET did not enhance the DFP-induced neuroinflammation with or without corticosterone. There was no effect of DFP, with or without corticosterone, on neurodegeneration, astrogliosis, or microglial activation in the frontal cortex, hippocampus, striatum, or hypothalamus.
Brain Function
PB, permethrin, and DEET exposures combined with restraint stress in rats for 4 weeks reduced numbers of parvalbumin-expressing GABAergic interneurons in some portions of the hippocampus (Megahed et al., 2014). Rats also showed diminished hippocampal neurogenesis at 3 months postexposure. Unfortunately, the ability to apply this information to human health is limited by the use of only 6 rats per group and a relatively high dose of PB (1.3 mg/kg/day). The hippocampus is critical to executive functions, thus this study suggests that, at relatively high doses, PB exposure in the presence of stress could induce delayed neurotoxicity
Other Brain Effects
Williams et al. (2006) used marmosets to study the effect of vaccination or PB on sleep activity and brain electroencephalography. The investigators were blinded as to treatment group. There were no long-term changes in brain electrical activity or sleep architecture that could be attributed to the treatments with vaccines or with PB.
Summary of Brain Effects
Inconsistent results on the permeability of the blood–brain barrier and associated effects to certain combinations of toxic exposures, but not others, give weak support for the hypothesis that stress-induced damage to the blood–brain barrier is a cause for symptoms of Gulf War illness. Three studies showed increased permeability (Abdel-Rahman et al., 2002, 2004; Freidman et al., 1996), but 10 studies found no such effect (Amourette et al., 2009; Grauer et al., 2000; Kant et al., 2001; Lallement et al., 1998; Shaikh
and Pope, 2003; Shaikh et al., 2003; Sinton et al., 2000; Song et al., 2002, 2004; Tian et al., 2002). One animal study suggested that stress-induced elevations in cortisone can exacerbate the neuroinflammatory effects of acetylcholinesterase inhibitors (O’Callaghan et al., 2015). Furthermore, altered phospholipid metabolism, which may be associated with adverse effects on the immune system and peroxisomal and lysosomal function, might persist long after exposure to chemicals associated with Gulf War illness (Abdullah et al., 2011, 2012, 2013). Loss of parvalbumin-expressing GABAergic interneurons could be associated with cognitive deficits according to one study (Megahed et al., 2014).
BEHAVIORAL EFFECTS
Veterans with Gulf War illness have reported cognitive or executive dysfunctions, in particular, deficits in memory, and the illness has also been associated with increases in mood disorders, including anxiety and depression. Relevant to these conditions, experimental animal models have examined changes in acquisition (learning) behavior, in short-term memory, and behavioral characteristics, albeit with the use of widely varying approaches. Several studies have evaluated the effect of Gulf War–related toxicants and aspects of motor function likely based on numerous reports of fatigue in Gulf War illness.
Executive Function
Learning
In general, only two different paradigms of learning have been examined in response to Gulf War illness–related toxicants. Early studies examined the acquisition of lever press responding of rats employing what was effectively a differential reinforcement of low rate (0 second or 16 second) schedule of reward following either repeated administration of PB alone or in combination with permethrin (van Haaren et al., 1999, 2000). The authors found that PB slowed acquisition of this behavior, an effect that was not produced by permethrin alone, nor did permethrin enhance the effects of PB. There are several difficulties with the interpretation of these studies, as PB effects appeared only in a fraction of the exposed group and the results are inconsistent and marginal. Furthermore, the behavioral paradigm was dependent upon the animals making contact with the lever after an autoshaping paradigm. Such effects are highly dependent on the animal’s basal activity levels and thus not necessarily reflective of deficient learning per se; the basal activity levels may have been influenced by the drug. In addition, no information is presented on the extent to which these deficits may have reflected motor, motivational, or sickness-related effects.
More recent studies have used other stressors, such as a water maze or climbing a pole to avoid a shock, to measure learning changes in response to Gulf War–related toxicants. One study combined PB with stress induced by pole climbing avoidance (Lamproglou et al., 2009); other studies have combined PB, permethrin, DEET, and stress to mirror reported Gulf War exposures (Abdullah et al., 2012; Parihar et al., 2013). While all researchers report that the toxicants produced deficits in learning, alternative interpretations cannot be ruled out for any of the studies. Combined PB and stress (climbing a pole to escape or avoid intermittent shock delivery) resulted in a longer latency (i.e., slower learning) to find the platform in the water maze. However, in these studies, the animals also had reduced body weights, which could have affected their swimming response (swimming is a highly effortful motor response, and the stress activity requires significant physical endurance). In addition in some cases, the statistical analysis failed to incorporate a repeated measures component of testing that makes the day-by-day comparisons across groups questionable; moreover, of the 8 days of testing, only 1 day shows a significant effect
(Abdullah et al., 2012). Furthermore, PB can cause hypothermia in rodents, an effect that by itself has been shown to impair water maze learning (e.g., Iivonen et al., 2003).
Of the three studies that have examined combined effects of PB, permethrin, and DEET with stress (immobilization restraint stress) on water-maze learning in rats, Abdullah et al. (2012) appears to show suggestive beneficial effects of these treatments, at least in males, in the form of shorter path lengths to the submerged platform. Exposed animals also had potential motor deficits, manifest as shorter latencies to fall from a rotating rod (although this could have been a learned escape response). Using this same combination of toxicants, chronic restraint stress exacerbated the effects of the chemicals on water-maze learning (Parihar et al., 2013). The effects, however, were inconsistent, and the absence of a group that received stress alone makes it difficult to conclude such synergism exists. Data on body weights across time (supplementary data) show increased body weights in the chemical-alone group relative to other groups indicating systemic effects of chemical exposure that may have affected motivation to perform and learning capability.
Thus, the ability of Gulf War–related toxicants to produce changes in learning has yet to be fully established, given that other potential motor, motivational, or physical factors that might affect learning cannot be ruled out.
Memory
Surprisingly, despite reported complaints from Gulf War veterans, there have been few studies to assess explicit memory deficits. Kant et al. (2001) examined the combined effects of PB and stress on delayed alternation performance, a behavioral paradigm that required rats to alternate responses between two response devices, with an imposed delay between such response opportunities. No effects of PB or its enhancement by stress were found, although the delay used in the study (1 second) was likely too short to tap working memory; longer delays were not examined.
Following combined exposures to PB, DEET, and permethrin alone or in conjunction with restraint stress, no deficits were reported in novel object recognition memory, suggesting no deficits in short-term memory (Parihar et al., 2013). This task first familiarizes animals with two objects in an enclosed environment, after which the animal is removed from the environment for some designated period and one object replaced by a novel object. The time spent with the novel object is typically greater, as the subject “remembers” the other object. However, it was clear in this study that total exploration behavior was severely impaired in the group stressed with a combination of PB, DEET, and permethrin, suggesting potential motor or motivational impairments. These impairments could affect the extent to which this group was actually familiarized with the two objects in the initial testing session, and this would ultimately compromise the study’s ability to measure memory.
A subsequent study by the same group using the same exposures did find a deficit in the novel object recognition index in treated animals (Hattiangady et al., 2014). However, as in the earlier study, the extent of contact with the novel objects and the exploration levels were significantly lower in the treated groups, findings that could suggest inadequate learning or familiarity with the objects in this group to begin with, which by itself would produce the type of deficits in novel object recognition memory that were seen. The authors did, however, observe deficits in another measure of short-term memory (i.e., place recognition in the absence of such deficits in total exploration levels). Thus, findings were inconsistent across these studies, making it unclear which toxicants impair memory functions per se and which ones might play a supporting role.
Potential memory deficits in mice treated for 10 days with PB and permethrin were reported using a maze, as were neuropathological changes in the brain (Zakirova et al., 2015). Symptoms were observed
in the short term (11–18 days) and in the long term (5 months) postexposure. No cognitive deficits were observed at the short-term point, but by 5 months memory deficits had emerged as had increased astrogliosis and reduced staining of synaptophysin in the hippocampus and cerebral cortex. The doses were 0.7 mg/kg/day for PB and 200 mg/kg/day for permethrin, but the authors did not attempt to compare them to what would be expected in veterans. Unfortunately, no information on body weight of the animals in response to the treatments was provided.
Considered overall, information on memory-related deficits in response to Gulf War exposures is limited and inconsistent, and requires further data to assess potential confounding effects of deficits in motor or sensory functions, and systemic toxicity. For example, few studies provided information on body weight which might confound observed effects.
Anxiety and Mood-Related Behaviors
Parihar et al. (2013) examined anxiety-like behavior with a widely used approach—the elevated plus maze. Number of entries into open arms and time spent in open arms was reduced in rats exposed to a combination of PB, DEET, and permethrin, and further reduced when the exposure was combined with restraint stress. These findings are consistent with interpretations of anxiety-like behavior as rats spent more time in the closed areas of the maze.
Several other investigators have measured behavior obtained in an open field, where increased behavior in the periphery of the field compared with behavior in the center of the open field is considered to be an index of anxiety-like behavior. Results have been inconsistent. Hoy et al. (2000b) found increases rather than decreases in time spent by male rats in the central area of the open field in response to PB plus DEET, and to permethrin plus DEET in the immediate 24 hours after a 7-day exposure; a greater time in the center arena was seen in female rats in response to PB plus permethrin following a 7-day exposure. Similarly, mice exposed to PB plus permethrin for 10 days showed a reduction in time spent in the perimeter (i.e., greater time in the center) at day 15 postexposure; however, they spent more time in the perimeter at day 30 postexposure in a study by Abdullah et al. (2011).
Two studies have examined combinations of two or more Gulf War chemicals. Combined exposures of both male and female mice to PB, permethrin, DEET, and restraint stress resulted in more time spent in the perimeter in the 15-minute open field test (Abdullah et al., 2012); however, the differences tend to be about 5 seconds or less, raising questions about their biological significance. Conversely, Hattiangady et al. (2014) examined the effect of the same exposure combination in a similar test in rats and found no effects on time spent in the central zone.
A potentially interesting approach was taken by Servatius and Beck (2005), who examined the possibility that nonspecific symptoms reported in Gulf War illness could reflect classical conditioning effects generated during military service. This hypothesis stipulates that conditioning of symptom effects of toxicant-related exposures could result from environmental stimuli such as sounds or visual cues that would then become conditioned stimuli. Then when these or very similar sounds or visual cues occurred later, the cues could evoke the symptomatology associated with the chemical toxicants. Specifically, the study demonstrated that treatment with PB led to potentiated auditory startle responses occurring in the presence of odors associated with exposures to the stressor (PB), suggesting that such odors could function as conditioned stimuli eliciting PB-related effects. The study found that in rats olfactory stimuli are more significant than visual stimuli; visual stimuli were less or not effective.
Avoidance and escape responses have been examined in two studies. In this paradigm, a cue is presented that signals pending onset of an aversive stimulus (frequently a shock); a designated response after the cue but prior to the onset of the shock is an avoidance response, whereas an escape is defined
as a behavioral response occurring in the presence of the shock. Using this approach, Lamproglou et al. (2009) offered rats an opportunity to either avoid or escape shock presentation by climbing a pole. The number of pole climbing trials declined notably across sessions in the PB plus stress groups relative to the stress only group, suggesting more efficient or beneficial avoidance behavior in the former group. This is because climbing the pole prior to the sound initiating the trials actually decreased the numbers of trials (and hence potential shock exposures). No effects of PB, sarin, or the combination on passive avoidance responding were found in rats over a 16-week observation period (Scremin et al., 2003).
Animal models of depression appear limited to a study by Hattiangady et al. (2014) who found rats exposed to a combination of PB, permethrin, and DEET had a reduction in voluntary wheel running and significantly greater latency to eating following a 24-hour fast. Those findings are reminiscent of anhedonia, and as noted by the authors, consistent with decreased motivational levels and depression-like behavior.
As with learning and memory, the studies to date that have examined animal models of anxiety or mood-related disorders do not support any conclusions about the relationship between these disorders and any specific Gulf War-related toxicants.
Motor Function
Most animal studies have evaluated motor function in terms of locomotor activity (ambulation, rearing) and aspects of motor or sensorimotor function.
Locomotor Activity
Locomotor activity is generally assessed in an open arena where levels of ambulation (horizontal movement or distance) and hind-limb rearing-related behaviors are measured. Hoy et al. (2000a,b) examined the effects of PB, permethrin, and DEET, alone and in combinations. Following acute exposures (Hoy et al., 2000a), reductions in locomotor activity in male rats were seen in response to PB and permethrin combined and for DEET and permethrin combined. Following 7 days of such exposures, both male and female rats had reduced locomotor speed in response to PB and DEET (Hoy et al., 2000b). An increase in activity levels in males given DEET and permethrin was observed, but no effects were found in response to either chemical alone. Another study of combined PB and permethrin reported a very slight increase in time spent in the perimeter of the open field coupled with a reduction in total distance traveled (i.e., lowered locomotor activity levels) at 30 days postexposure (Abdullah et al., 2011). However, no significant effects on locomotor activity were found after exposure to PB for 7 days with or without stress in another study (Dubovicky et al., 2007). Hattiangady et al. (2014) showed that combined exposures to PB, permethrin, DEET, and stress produced no effects on open field behavior, but markedly reduced levels of voluntary wheel running behavior (a self-reinforcing behavior in rodents) were seen.
Two studies examined the effects of sarin and stress. Sustained measurement of activity beginning 2 days prior to exposure through 1 month postexposure to sarin and mild heat stress found no changes in activity levels (Conn et al., 2002). Conversely, a study of sarin alone or in combination with shaking stress, found a decrease in several measures of locomotor activity (Mach et al., 2008).
Some studies suggest that various Gulf War toxicants could reduce locomotor activity, which may be related to the fatigue reported by veterans with Gulf War illness. However, it is difficult to ascertain what the behavioral mechanism(s) of locomotor alterations are, given potential confounding (e.g., environmental distractions) and possible systemic toxicant-related sickness behavior which could also reduce locomotor activity levels. This may be more evident when measured during or immediately after
toxicant exposures, although such effects have not been reported and body weight changes in response to chemical exposures in these studies are seldom reported. Such interpretations are less likely to confound delayed measures of behavioral effects. Furthermore, slight differences in the size of the open field and the specific operational definitions of the behaviors being measured can translate into substantial differences in outcomes even when the same toxicant administration protocols are used.
Motor and Sensorimotor Function
Aspects of fine motor and sensorimotor function following exposure to Gulf War toxicants were measured in rats using beam walk, inclined plane, forepaw grip, postural reflexes, limb placing, and orienting to vibrissae touch (Abou-Donia et al., 2001, 2002, 2004). Following exposures to PB, permethrin, and DEET, alone or in combination, for up to 45 days, a complex profile of effects led the authors to suggest that PB alone might increase beam walk latency (time to cross a beam) during the treatment period (Abou-Donia, 2001). A subsequent study administered DEET, or DEET and permethrin, for 60 days with PB for the last 15 days (Abou-Donia et al., 2004). PB alone or in combination with DEET or DEET and permethrin increased beam walking time, impaired incline plane performance, and reduced grip strength. However, these exposures also resulted in reduced body weights that could alter these motor functions as well. Exposure to PB and sarin produced deficits similar to those described above, providing some evidence that combined PB and sarin enhanced motor function deficits (Abou-Donia et al., 2002). It is important to note, however, that the exposures in the last study included high doses of sarin with its associated systemic toxicity, which could also lead to motor deficits.
Based on the available studies, it is not clear that lower levels of Gulf War toxicants that may mirror Gulf War veteran exposures alter fine motor functions in the absence of more overt toxicity.
Summary of Behavioral Effects
The committee finds that it is difficult to arrive at any generalities regarding the behavioral studies discussed in this section for the following reasons:
- The studies frequently do not use the same chemicals, doses, or dosing regimens or the same measurement protocols for effects (e.g., timing).
- The studies often use different behavioral paradigms to measure the specific behavioral activities.
- The relevance of the administered doses to humans is often not evident or the doses are quite high compared with suspected human exposures.
- Interpretation of study findings are often difficult given the lack of important data, such as changes in body weights or signs of motivational deficits, which might influence the animal’s ability to perform behavioral tests.
- The potential for toxicant-induced confounders, including sickness behavior and hypothermia, which also affect behavior, are frequently not reported and not considered in the data interpretation.
Given these issues, it is not possible to draw reliable conclusions about the effects of Gulf War toxicants on animal behavior as measured by learning, memory, mood, or motor function.
ORGAN SYSTEM EFFECTS
In addition to brain and behavioral effects, investigators have also reported on other potential effects of Gulf War exposures, including testicular changes, musculoskeletal problems, immune effects, and pain response. Because Gulf War illness has many manifestations and symptoms may include joint and muscle pain, infections, and headaches, among others, the committee also reviewed the few studies that focused on other organ system effects. Testicular changes, as a reproductive system effect, are included because they have been a concern to veterans.
Reproductive System
One study assessing sperm and testicular changes in animals exposed to toxicants meant to mirror Gulf War exposures was identified. Male rats were exposed to PB, DEET, and permethrin with and without restraint stress for 28 days (Abou-Donia et al., 2003). Stress alone did not influence these outcomes. Testes of animals exposed to the chemical combination showed histologic abnormalities including arrested spermatogenesis and seminiferous tubule degeneration, effects enhanced under conditions of stress. The authors further investigated the causal mechanism for the observed toxicity and identified apoptosis due to the increased expression of two apoptosis-promoting proteins in testicular tissue. While this study could suggest that exposures such as those experienced by Gulf War veterans may contribute to sperm abnormalities and male infertility, the findings should be viewed with caution, as the animals in the chemical plus stress group also gained less weight, indicating poorer health overall, which compromises the validity of the findings.
Musculoskeletal System
Studies of Gulf War exposures on musculoskeletal function or injury have been carried out in mice and in marmosets. PB and vaccine exposures such as those experienced by Gulf War veterans had no effects on muscle function in marmosets (Stevens et al., 2006). Three 10-week studies in male mice investigated the skeletal muscle effects of combined exposures to PB and exercise stress (Jagannathan et al., 2001; Somani et al., 2000) and PB and/or sarin and exercise stress (Husain and Somani, 2004). Animals were exercised daily, and treatment groups received PB and/or sarin at weeks 5 and 6; doses of PB and sarin were 1.2 mg/kg PB and 0.01 mg/kg sarin (or 1/20th of the LD50). PB with exercise stress, and PB and sarin with exercise stress reduced respiratory exchange ratios during and for several weeks after treatment; the authors suggest that these changes in muscle respiration with and following exercise may enhance oxidative muscle injury. No data was provided on body weight or other indicators of systemic toxicity. The studies in mice suggest a potential mechanism for muscle injury following combined exposures to chemicals and stress designed to mirror those exposures reported by Gulf War veterans.
Immune System
Studies of Gulf War exposures on immune function have been carried out in mice and in marmosets. Little evidence of impaired immune response was observed in marmosets given PB in combination with vaccines (Griffiths et al., 2006; Hornby et al., 2006). Most immune parameters examined were unaffected in female mice exposed to DEET, PB, or JP-8 (jet fuel), singly or in combination (Peden-Adam et al., 2001). All exposures suppressed immunoglobulin-M responses, but this was not exacerbated with mixture exposure. The highest mixture dose increased CD4+ T helper cells, decreased the CD4/CD8 ratio
in spleen, and suppressed delayed-type hypersensitivity. In animals treated with melanoma tumor cells and Listeria monocytogenes, susceptibility was not affected (Peden-Adam et al., 2001). As mentioned earlier, lipid metabolism studies in mice exposed to combined PB and permethrin over 10 days suggested an adverse effect on the immune system that lasted long after the exposure (Abdullah et al., 2011).
Pain Response
Nutter et al. (2015) reported a study in which 30 rats were exposed to permethrin, chlorpyrifos, and PB, over 30 or 60 days. The dosages were calculated on the basis of what Gulf War veterans might have experienced and are described as an intensified anticholinesterase exposure protocol relative to earlier studies by this group that did not influence pain behavior (Nutter and Cooper, 2014; Nutter et al., 2013). The exposures did not statistically affect body weight in comparison with controls. Assessment of pain behavior relied on right hind-limb withdrawal in response to force application as well as activity levels (15-minute test periods in an open field). The study reported increases in resting time in the 30-day treated group for approximately 8 weeks postexposure, and the 60-day treated group exhibited increased resting time and reduced movement for 12 weeks postexposure, which the authors interpret as consistent with a delayed myalgia in rats and which was seen in conjunction with a decline in activity of muscle nociceptor channel activity and protein expression.
CONCLUSIONS
Animal studies have suggested physiological alterations in response to the toxicant exposures that Gulf War veterans might have experienced while deployed. However, animal studies have typically examined isolated symptoms of Gulf War illness rather than the symptom clusters that are reported by veterans. The lack of information on the actual exposures experienced by veterans during the Gulf War has resulted in a multiplicity of animal study designs that provide inconsistent results.
Animal studies have not been successful in suggesting a mechanism by which deployment exposures during the Gulf War might lead to Gulf War illness or its many symptoms.
The committee concludes that although the existence of an animal model would be advantageous for identifying and evaluating treatment strategies for Gulf War illness, it cautions that developing such an animal model is not possible given researchers’ inability to realistically determine the exposures associated with Gulf War deployment, let alone the frequency, duration, or dose of those exposures, or the effect of multiple exposures.
TABLE 5-1 Animal Studies on Gulf War Illness and Other Organ System Effects
| Study | Exposure | Result |
|---|---|---|
| Blood–Brain Barrier Integrity | ||
| Abdel-Rahman et al., 2002 Rats | DEET + PB + permethrin + stress Exposed dermally for 28 days to 11.3 mg/kg/d PB, 40 mg/kg/d DEET, and 0.13 mg/kg/d permethrin individually or as a mixture of the three chemicals, with or without restraint stress |
Evidence of increased blood–brain barrier permeability with exposures to mixture of three chemicals plus stress, but not to any one chemical with or without stress; only affected certain regions: cingulate cortex, dentate gyrus, thalamus, and hypothalamus |
| Abdel-Rahman et al., 2004 Rats | DEET + PB + permethrin + stress Expose dermally for 28 days to 11.3 mg/kg/d PB, 40 mg/kg/d DEET, and 0.3 mg/kg/d permethrin individually or as a mixture of the three chemicals, with and without restraint stress |
Stress plus combined exposure to the three chemicals resulted in decreased brain AChE in certain areas of the brain (midbrain, brainstem, cerebellum) and decreased m2 muscarinic acetylcholine receptor binding (midbrain and cerebellum), areas where no disruption of the blood–brain barrier was observed |
| Amourette et al., 2009 Rats | PB + stress Two 5-day periods of daily exposure to 1.5 mg/kg 3H-PB with and without stress of pole climbing avoidance |
No evidence of increased blood–brain barrier permeability to PB with stress |
| Friedman et al., 1996 Mice | PB + stress Daily ip injections of 0.5 or 1.0 mg/kg of PB with or without stress of two 4-min forced swim sessions |
Evidence of increased blood–brain barrier permeability to PB with stress |
| Grauer et al., 2000 Mice | PB + stress Stress by two 4-min forced swim sessions or 5 min with feet in ice water and then treated with saline, 0.4 mg/kg PB or 0.2 mg/kg physostigmine im or ip |
Evidence of decreased blood–brain barrier permeability to PB with stress |
| Kant et al., 2001 Rats | PB + stress Treated with 200 μl of saline, 25 mg/ml PB, or 20 mg/ml physostigmine, administered by osmotic minipump, with and without avoidance/escape stress, or yoked stress |
No evidence of increased blood–brain barrier permeability to PB with stress |
| Lallement et al., 1998 Guinea pigs | PB + stress Exposure to saline or 0.2 mg/kg PB with or without stress: low (24.3–25.9°C for 2 hrs); medium (38.4–39.6°C for 2 hrs); or high (42.6°C for 2 hrs) |
Evidence of decreased blood–brain barrier permeability in response to PB with stress |
| Shaikh and Pope, 2003 Rats | PB + paraoxon 1: Control, vehicle 2: 10 mg/kg PB 3: 30 mg/kg PB 4: 0.1 mg/kg paraoxon 5: 10 mg/kg PB + 0.1 mg/kg paraoxon 6: 30 mg/kg PB + 0.1 mg/kg paraoxon |
No evidence of increased blood–brain barrier permeability to PB with paraoxon |
| Study | Exposure | Result |
|---|---|---|
| Shaikh et al., 2003 Rats | PB + stress + paraoxon 1: Control: DMSO im and saline 2: 10 mg/kg PB 3: 30 mg/kg PB 4: 0.1 mg/kg paraoxon 5: 0.1 mg/kg paraoxon + 10 mg/kg PB 6: 0.1 mg/kg paraoxon + 30 mg/kg PB 60 min forced running after treatment |
No evidence of increased blood–brain barrier permeability to PB with stress or paraoxon |
| Sinton et al., 2000 Rats | PB + stress Restraint stress or forced swimming (or both), or heat stress, followed by PB (0.5, 1, 2, 3, or 5 mg/kg ip); No stress + PB or physostigmine (0.1, 0.2, 0.5, 1, or 2 mg/kg) or saline |
Evidence of decreased blood–brain barrier permeability to PB with stress |
| Song et al., 2002 Rats | PB + stress 1: Controls: saline and no stress 2: 30 mg/kg PB, no stress 3: stress protocol only 4: stress protocol with PB Stress protocols-A: restraint tube (90 min) then PB; B: PB then restraint tube (60 min); C: restraint tube (3 hr), then PB, then restraint tube (60 min) |
No evidence of increased blood–brain barrier permeability to PB with stress |
| Song et al., 2004 Rats | PB + paraoxon Treatment: 30 mg/kg PB by gavage 50 min before 100 μg/kg paraoxon im |
Paraoxon increased the number of leaky capillaries in the brain of young rats but not older rats |
| Tian et al., 2002 Rats | PB + stress 1: Controls, no stress or PB 2: no stress, 30 mg/kg PB 3: stress and saline 4: stress and 30 mg/kg PB Stress: forced run on a treadmill after, before, or before and after PB treatment; or forced swimming before or after PB treatment |
No evidence of increased PB toxicity or increased blood–brain barrier permeability to PB with stress |
| Brain Metabolism and Histopathology | ||
| Abdel-Rahman et al., 2001 Rats | DEET + permethrin All conditions applied dermally 1: Control: 70% ethanol 2: 40 mg/kg DEET in ethanol 3: 0.13 mg/kg permethrin in ethanol 4: DEET + permethrin in ethanol 7 d per week for 60 d |
Evidence of diffuse neuronal cell death in cerebral cortex, hippocampus, and cerebellum for all three exposures |
| Study | Exposure | Result |
|---|---|---|
| Abdullah et al., 2011 Mice | PB + permethrin Single dose of 2 mg/kg PB and 200 mg/kg permethrin in DMSO, or DMSO only, ip for 10 d followed by 115-d observation period | Cognitive impairment and anxiety observed at 115 days as well as astrogliosis in the cortex. No effects at 8 d. No neuronal loss or microgliosis. Changes in proteins associated with lipid metabolism and molecular transport and changes in endocrine and immune associated proteins |
| Abdullah et al., 2012 Mice | DEET + PB + permethrin + stress Control: water, and topical 70% ethanol Treatment: 1.3 mg/kg PB in water; 0.13 mg/kg permethrin and 40 mg/kg DEET in 70% ethanol dermally; and 5min of restraint stress; 28 days 42-d observation period | Evidence of astrocytosis in the cerebral cortex and increased lipids (phosphatidyl choline and sphingomyelin) in the brain |
| Abdullah et al., 2013 Mice | PB + permethrin Single dose of 2 mg/kg PB and/or 200 mg/kg permethrin in DMSO, or DMSO only, ip for 10 days. 150-d observation period | Evidence of increased phosphatidyl choline and sphingomyelin in the brain and other changes (decreased lysoplatelet activating factors and increased catalase expression) indicating peroxisomal and lysosomal dysfunction |
| Buchholz et al., 1997 Rats | PB + permethrin 1: PB 7.75 mg/kg in food 2: PB +14C-permethrin 4.75 μg/kg ip 3: Controls, plain food for 10 days |
No evidence of harmful synergistic effect of PB and permethrin on central nervous system |
| Husain and Somani, 2004 Mice | PB + sarin + stress 1: Controls 2: 0.01 mg/kg/d sarin sc for weeks 5 and 6 3: exercise on treadmill daily for 10 weeks with increasing speed and time 4: sarin + exercise 5: 1.2 mg/kg/d PB orally for weeks 5 and 6 6: PB + exercise 7: PB + sarin 8: PB + sarin + exercise Animals sacrificed 24 hrs after last treatment |
Little evidence that exercise increased the effects of PB or sarin on butyrcholinesterase, AChE, neurotoxic esterase in plasma or brain |
| Kant et al., 2001 Rats | PB + stress Treated with 200 μl of saline, 25 mg/ml PB, or 20 mg/ml physostigmine administered by osmotic minipump at a rate of 1.5 or 1.2 mg/kg/d, with and without avoidance or escape stress, or yoked stress | No evidence that PB exacerbated the effects of stress. PB decreased blood AChE by half, but had no effect on cortical brain AChE and no effect on blood corticosterone levels elevated by stress |
| Study | Exposure | Result |
|---|---|---|
| O’Callaghan et al., 2015 Mice | DEET + PB + corticosterone + DFP 1: PB 3 mg/kg/d sc + DEET 30 mg/kg/d sc for 14 days, 300 mg/L cortisone in drinking water on days 8-15, and DFP on day 15 2: Controls, saline sc |
Evidence that cortisone exacerbated the neuroinflammatory effect of DFP |
| Ojo et al., 2014 Mice | PB + permethrin + chlorpyrifos 1: 5 mg/kg/d chlorpyrifos ip 2: chlorpyrifos + 0.7 mg/kg/d PB + 200 mg/kg/d permethrin, ip 3: control, DMSO ip 10 days |
Evidence of pathological changes in hippocampal, cortical, and amygdalar morphometry in both exposure groups at 3 days post exposure, but these early changes did not persist |
| Scremin et al., 2005 Rats | PB + sarin 1: control, tap water and saline injection 2: 80 mg/L PB in drinking water and saline sc 3: tap water and 62.5 μg/kg sarin sc 3 times/week 4: PB in water and sarin sc For 3 weeks; sacrificed at 2, 4, 16 weeks after treatment |
Evidence of effects on brain metabolism at 2 and 4 weeks (increased cerebral blood flow, glucose utilization but not at 16 weeks |
| Brain Function | ||
| Megahead et al., 2014 Rats | PB + permethrin + DEET + stress controls; treatment: 1.3 mg/kg/d PB in water by oral gavage, 40 mg/kg/d DEET and 0.13 mg/kg/d permethrin dermally, and 5 min/d restraint stress for 4 weeks. 3-month observation period | Evidence of reduced numbers of parvalbumin and neuropeptide Y expressing interneurons in some parts of hippocampus and diminished hippocampal neurogenesis |
| Other Brain Effects | ||
| Williams et al., 2006 Marmosets | PB + vaccines 1: vaccinated with 20% of human dose or vehicle in period 1 (first 51 days); 2: 500 μg/kg/d PB by miniosmotic pump or sterile saline 21 months |
No evidence of long-term effects of vaccination or PB on brain electrical activity or sleep architecture |
| Executive Function | ||
| Abdullah et al., 2012 Mice | DEET + PB + permethrin + stress Control: water, and topical 70% ethanol Treatment: 1.3 mg/kg PB in water; 0.13 mg/kg permethrin and 40 mg/kg DEET dermally; and 5 min of restraint stress; 28 days. 42-d observation period | Evidence of possible beneficial effects of PB in water maze performance but deficits on a rotating rod |
| Hattiangady et al., 2014 Rats | DEET + PB + peermethrin + stress Treatment group: 40 mg/kg/d DEET dermally + 0.13 mg/kg/d permethrin dermally + 1.3 mg/kg/d PB by gavage + 5 min/d restraint stress; for 4 weeks Testing conducted 3 months after exposure | Evidence that treatment impaired place recognition memory and caused novel object recognition memory dysfunction |
| Kant et al., 2001 Rats | PB + physostigmine + stress Treated with 200 μl of saline, 25 mg/ml PB, or 20 mg/ml physostigmine administered by osmotic minipump; with and without avoidance or escape stress, or yoked stress | No evidence of effect of PB or physostigmine and stress on working memory |
| Study | Exposure | Result |
|---|---|---|
| Lamproglou et al., 2009 Rats | PB + stress 1: 1.5 mg/kg/d PB in water 2: stress (pole climbing avoidance days 1–12) 3: stress + PB (given 30 min before stress, treatment daily on days 1–5 and 8–12) Testing on days 15–199; sacrifice at day 12 or 199 |
Evidence of long-term behavioral effects (aggressiveness, impulsiveness, learning dysfunction) |
| Parihar et al., 2013 Rats | DEET + PB + permethrin + stress 1: control vehicle and handling daily 2: 40 mg/kg/d DEET and 0.13 mg/kg/d permethrin dermally, and 1.3 mg/kg/d PB by gavage 3: DEET + PB + permethrin + stress (restraint stress 5 min/d) 4: control group 4 weeks |
Evidence of increased depressive and anxiety-like behavior and spatial learning and memory dysfunction; and effects of stress on mood and cognitive dysfunction; no evidence of effect on novel object recognition memory, possible effect on exploration behavior in rats exposed to chemicals + stress |
| van Haaren et al., 2000 Rats | PB + permethrin 1: controls (water and vehicle) 2: 1.5 mg/kg/d PB for 7 d by gavage 3: permethrin (0, 15, or 60 mg/kg) by gavage before session in an operant chamber 4: PB + permethrin |
No evidence of behavioral effects of permethrin; evidence of delayed response acquisition in rats treated with PB |
| Zakirova et al., 2015 Mice | PB + permethrin Treatment: 0.7 mg/kg/d PB and 200 mg/kg/d permethrin ip for 10 days. Testing at 18-d and 5-months postexposure | Evidence of long-term effects on cognition (memory deficits) and neuropathology associated with PB and permethrin |
| Anxiety and Mood-Related Behaviors | ||
| Abdullah et al., 2011 Mice | PB + permethrin Single dose of 2 mg/kg PB and/or 200 mg/kg Per in DMSO, or DMSO only, ip for 10 d 115-d observation period | Evidence of decreased anxiety-like behavior in rats exposed to PB and permethrin at 15 days postexposure, but increased anxiety-like behavior at 30 days |
| Abdullah et al., 2012 Mice | DEET + PB + permethrin + stress Control: water, and topical 70% ethanol Treatment: 1.3 mg/kg PB in water; 0.13 mg/kg permethrin and 40 mg/kg DEET dermally; and 5 min of restraint stress; 28 days 42-d observation period | Evidence of increased anxiety-like behavior |
| Hattiangady et al., 2014 Rats | DEET + PB + permethrin + stress Treatment group: 40 mg/kg/d DEET dermally + 0.13 mg/kg/d permethrin dermally + 1.3 mg/kg/d PB by gavage + 5 min/d restraint stress For 4 weeks; testing conducted 3 months after exposure | No evidence of effect on anxiety-like behavior; evidence for depression-type behavior |
| Study | Exposure | Result |
|---|---|---|
| Hoy et al., 2000b Rats | DEET + PB + permethrin 1: 7.5 mg/kg PB by gavage 2: 200 mg/kg DEET by gavage 3: 60 mg/kg permethrin ip 4: 3.75 mg/kg PB + 30 mg/kg permethrin 5: 3.75 mg/kg PB + 100 mg/kg DEET 6: 2.5 mg/kg PB + 20 mg/kg permethrin + 67 mg/kg DEET 7: 100 mg/kg DEET + 30 mg/kg permethrin Daily for 7 days; testing conducted 24 hr after last treatment |
Evidence of reduced anxiety-like behavior in male rats exposed to PB + DEET and permethrin + DEET and female rats exposed to PB + permethrin |
| Lamproglou et al., 2009 Rats | PB + stress 1: 1.5 mg/kg/d PB in water 2: stress (pole climbing avoidance days 1–12) 3: stress + PB (given 30 min before stress, treatment daily on days 1–5 and 8–12) Testing on days 15–199 |
Evidence of beneficial avoidance behavior |
| Parihar et al., 2013 Rats | DEET + PB + permethrin + stress 1: control vehicle and handling daily 2: 40 mg/kg/d DEET and 0.13 mg/kg/d permethrin dermally, and 1.3 mg/kg/d PB by gavage; 3: DEET + PB + permethrin + stress (restraint stress 5 min/d) 4: control group 4 weeks |
Evidence of increased anxiety-like behavior in rats exposed to PB, DEET, permethrin, and stress |
| Scremin et al., 2003 Rats | PB + sarin 1: PB 80 mg/L in drinking water 2: sarin 62.5 μg/kg 3 times/week sc 3: PB + sarin 3 weeks; assessed at 2, 4, or 16 weeks after treatment |
No evidence of effects of PB or sarin on avoidance behavior at 16 weeks |
| Servatius and Beck, 2005 Rats | PB + neostigmine + interleukin PB: 0.1 or 1.0 mg/kg ip Neostigmine: 0.016 mg/kg ip Interleukin: 1 or 3μg/kg ip Testing on days 2 and 15 | Evidence that PB potentiated auditory startle responses in the presence of odors associated with stressors |
| Motor Function | ||
| Abdullah et al., 2011 Mice | PB + permethrin Single dose of 2 mg/kg PB and/or 200 mg/kg permethrin in DMSO, or DMSO only, ip for 10 d, 115-d observation period | Evidence of reduction in locomotor activity 30 days postexposure |
| Abou-Donia et al., 2001 Rats | DEET + PB + permethrin 1: 1.3 mg/kg PB for days 30-45 2: 40 mg/kg/d DEET dermally 3: 0.13 mg/kg/d permethrin dermally 4: DEET + permethrin 5: DEET + PB 6: permethrin + PB 7: DEET + permethrin +PB 45 days |
Evidence that PB increased beam walk latency, but not other measures of motor function |
| Study | Exposure | Result |
|---|---|---|
| Abou-Donia et al., 2002 Rats | PB + sarin 1: 1.3 mg/kg/d PB for 15 days 2: 50, 75, 90, or 100 μg/kg sarin on day 15 3: PB + sarin |
Evidence of impaired motor function |
| Abou-Donia et al., 2004 Rats | DEET + PB + permethrin 1: controls, 70% ethanol dermally and water by gavage 2: PB (0.13, 1.3, or 13 mg/kg/d) for days 46 to 60 3: PB + DEET (DEET at 4, 40, or 400 mg/kg/d for 60 days dermally) 4: PB + permethrin (permethrin at 0.013, 0.13 or 1.3 mg/kg/d for 60 days) 5: PB + DEET + permethrin |
Evidence that PB, PB + DEET, and DEET + Per impaired motor function (beam walk time, incline plane performance, and grip strength) |
| Conn et al., 2002 Rats | Stress (heat) + sarin stress 25°C or 32°C for 1h/day for 1, 5, or 10 days sarin 0, 0.2, 0.4 mg/m3 by inhalation Assessed for 1 month postexposure | No evidence of effect on activity levels |
| Dubovicky et al., 2007 Mice | PB + stress (chronic shaker) PB 10 mg/kg/day sc for 7 d Assessed on days 2 and 7 during stress and days 7, 14, 21, and 28 after treatment. Separate group of mice tested in open field on day 1, 3, and 6 during stress and PB | No evidence of effect of PB on locomotor activity |
| Hattiangady et al., 2014 Rats | DEET + PB + permethrin + stress Treatment group: 40 mg/kg/d DEET dermally + 0.13 mg/kg/d permethrin dermally + 1.3 mg/kg/d PB by gavage + 5 min/d restraint stress For 4 weeks; testing conducted 3 months postexposure | Evidence of reduced locomotor activity in one test (voluntary wheel running) but no effect on other behaviors (open field test) |
| Hoy et al., 2000a Rats | DEET + PB + permethrin 1: 10 mg/kg PB by gavage 2: 50, 200, or 500 mg/kg DEET by gavage 3: 15, 30, or 60 mg/kg permethrin ip 4: 100 mg/kg DEET + 5 mg/kg PB 5: 15 mg/kg permethrin + 5 mg/kg PB 6: 100 mg/kg DEET + 15 mg/kg permethrin Testing conducted 30 min after last treatment |
Evidence of reduced locomotor speed in male rats exposed to PB + permethrin and DEET + permethrin |
| Hoy et al., 2000b Rats | DEET + PB + permethrin 1: 7.5 mg/kg PB by gavage 2: 200 mg/kg DEET by gavage 3: 60 mg/kg permethrin ip 4: 3.75 mg/kg PB + 30 mg/kg permethrin 5: 3.75 mg/kg PB + 100 mg/kg DEET 6: 2.5 mg/kg PB + 20 mg/kg permethrin + 67 mg/kg DEET 7: 100 mg/kg DEET + 30 mg/kg permethrin Daily for 7 days; testing conducted 24 hrs after last treatment |
Evidence of reduced locomotor speed with PB + DEET |
| Mach et al., 2008 Mice | Stress + sarin Shaker stress 90 min/d for 7 days 6.4 μg/kg/d sarin on days 4-6 Tests conducted on days 5 and 7, and 21 days after exposure | Evidence of decreased locomotor activity |
| Study | Exposure | Result |
|---|---|---|
| Reproductive System | ||
| Abou-Donia et al., 2003 Rats | PB + permethrin + DEET + stress 1: 1.3 mg/kg/d PB in water 2: 0.13 mg/kg/d permethrin dermally, and 40 mg/kg/d DEET dermally 3: PB + permethrin + DEET + restraint stress 28 days |
Evidence of sperm and testicular changes |
| Musculoskeletal System | ||
| Husain and Somani, 2004 Mice | PB + sarin + stress 1: 0.01 mg/kg/d sarin sc for weeks 5 and 6 2: exercise on treadmill daily for 10 weeks 3: sarin + exercise 4: 1.2 mg/kg/d PB orally for weeks 5 and 6 5: PB + exercise 6: PB + sarin 7: PB + sarin + exercise Animals sacrificed 24 hrs after last treatment |
Evidence of effects on skeletal muscle (changes in muscle respiration) |
| Jagannathan et al., 2001 Mice | PB + stress 1: sedentary control 2: 1.2 mg/kg PB orally for weeks 5 and 6 3: PB + stress exercise daily for 10 weeks Sacrificed 24 hrs after last treatment |
Evidence of effects on skeletal muscle (changes in muscle respiration) |
| Somani et al., 2000 Mice | PB + stress 1: sedentary control 2: stress exercise daily for 10 weeks 3: PB 1.2 mg/kg orally for weeks 5 and 6 4: PB + stress Sacrificed 24 hrs after last treatment |
Evidence of effects on skeletal muscle (changes in muscle respiration) |
| Stevens et al., 2006 Marmosets | PB + vaccines; 1: 500 μg/kg/d PB days 15–44 2: vaccines at 20% of human dose days 0-51 3: vaccines + PB Animals sacrificed 18 months after first vaccinations |
No evidence of effects on muscle function |
| Immune System | ||
| Abdullah et al., 2011 Mice | PB + permethrin Single dose of 2 mg/kg PB and/or 200 mg/kg permethrin in DMSO, or DMSO only, ip for 10 d, 115-d observation period | Evidence of long-term immune effects |
| Griffiths et al., 2006 Marmosets | PB + vaccines 1: 10 vaccines at 20% of human dose scheduled the same as service members, and 500 μg/kg/d PB between days 15 and 44 2: vaccines and saline |
Little evidence of immune system impairment |
| Hornby et al., 2006 Marmosets | PB + vaccines 1: 500 μg/kg/d PB on days 15 and 44 2: vaccines at 20% of human dose between days 0-51 3: vaccines + PB |
Little evidence of immune system impairment |
| Study | Exposure | Result |
|---|---|---|
| Peden-Adam et al., 2001 Mice | DEET + PB + JP-8 Treatment: high or low dose (single or combined mixture daily) 15.5 or 31 mg/kg DEET sc, 2 or 5 mg/kg PB orally, and 500 or 1,000 mg/kg JP-8 by gavage 14 days | Little evidence of effects on immune function except for depression of plaque forming cells at both low and high dose combined treatments and a depression of delayed hypersensitivity response after the combined high dose treatment |
| Pain Response | ||
| Nutter and Cooper, 2014 Rats | PB + permethrin Treatment: 2.6 mg/kg/d permethrin dermally, 120 mg/kg chlorpyrifos sc every 14 days; 13 mg/kg/d PB by gavage on days 1–14 30 or 60 days; animals sacrificed 8 and 12 weeks postexposure | Evidence of changes in pain behavior (prolonged rest period and decreased muscle press withdrawal) noted in treated animals during treatment but indices returned to normal after cessation of treatment |
| Nutter et al., 2013 Rats | PB + permethrin Treatment: 2.6 mg/kg/d permethrin dermally; 120 mg/kg chlorpyrifos sc every 14 days; 13 mg/kg/d PB by gavage on days 1–14 30 or 60 days; animals sacrificed 8 and 12 weeks postexposure | No evidence of effects on pain behavior or activity measures |
NOTE: AChE = acetylcholinesterase; DEET = N,N-diethyl-meta-toluamide; DMSO = dimethyl sulfoxide; im = intramuscular; ip = intraperitoneal; JP-8 = jet propellant 8; PB = pyridostigmine bromide; sc = subcutaneous.
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