* Departments of Pharmacology and Cancer Biology and
Medicine (Neurology), Duke University Medical Center; and
VA Medical Center, Durham, North Carolina 27710
Received August 23, 2000; accepted November 27, 2000
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ABSTRACT |
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Key Words: Persian Gulf War; sensorimotor; pyridostigmine bromide; DEET; permethrin; combined exposure; CNS.
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INTRODUCTION |
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PB is a quaternary dimethyl carbamate used as a treatment for myasthenia gravis at a higher dose range than what was given to PGW veterans (Breyer-Pfaff et al., 1985, 1990
). PB reversibly inhibits 3040% of the AChE in the peripheral nervous system, thus limiting irreversible inhibition of the enzyme by nerve agents (Blick et al., 1991
). AChE activity is restored following spontaneous decarbamylation resulting in near-normal neuromuscular and autonomic functions (Blick et al., 1991
). Toxic symptoms associated with PB overdose results from overstimulation of nicotinic and muscarinic receptors in the peripheral nervous system, resulting in exaggerated cholinergic effects such as muscle fasciculations, cramps, weakness, muscle twitching, tremor, respiratory difficulty, gastrointestinal tract disturbances, and paralysis (Abou-Donia et al., 1996
; McCain et al., 1997
). With severe intoxication, death may occur because of asphyxia. Central nervous system effects of PB are not expected unless BBB permeability is compromised. The positive charge on the quaternary pridinyl nitrogen prevents PB from crossing the intact BBB (Birtley et al., 1966
).
The insect repellent N,N-diethyl-m-toluamide (DEET) and the insecticide pyrethroid permethrin 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl) methyl ester have been used extensively by humans since their introduction. DEET is commonly used as a repellant against mosquitoes, flies, ticks, and other insects (McConnel et al., 1986; Robbins and Cherniack, 1986
). However, extensive and repeated topical DEET applications can cause human poisoning, including death (Edwards and Johnson, 1987
; Gryboski et al., 1961
; Roland et al., 1985
). The symptoms associated with DEET poisoning include tremors, restlessness, difficulty with speech, seizures, impairment of cognitive function, and coma (McConnel et al., 1986
). DEET efficiently crosses the dermal barrier and may localize to dermal fat deposits (Blomquist and Thorsell, 1977
: Snodgrass et al., 1982
). Although the exact mechanisms of DEET toxicity are not known, extremely high levels of DEET exposure cause demyelination and spongiform myelinopathy in the rat (Verschoyle et al., 1992
).
Permethrin is a type I synthetic pyrethroid insecticide that exists in four different stereoisomers (Casida et al., 1983). It provides insecticidal activity for several weeks following a single application and is used in a variety of public buildings, industrial premises, and private dwellings to control fleas, flies, mites, and cockroaches. Permethrin intoxication results as a consequence of modification of sodium channels, leading to prolonged depolarization and repetitive discharges in presynaptic nerve fibers after a single stimulus (Narahashi, 1985
). This repetitive nerve action is associated with tremor, hyperactivity, ataxia, convulsions, and in some cases to paralysis. Permethrin is detoxified by ester hydrolysis in the blood and most tissues.
We previously reported that concurrent exposure to relatively large doses of PB, DEET, and permethrin in hens resulted in neurotoxic effects greater than those produced by exposure to the single components (Abou-Donia et al., 1996). In the present study we have extended these observations to include doses similar to levels of human exposure. We evaluated whether PB would enhance the neurotoxic effects caused by low-dose, combined exposure to DEET and permethrin. These results suggest that treatment with PB, DEET, and permethrin, alone or in combination, causes a significant impairment in sensorimotor abilities and region-specific effects on brain AChE and mAChR.
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MATERIALS AND METHODS |
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Animals.
Male Sprague-Dawley rats weighing 225250 g were obtained from Zivic-Miller Laboratories, Allison Park, PA, and housed at Duke University Medical Center vivarium. The animals were randomly assigned to control and treatment groups and housed at 2123°C with a 12-h light/dark cycle. They were supplied with food and water ad libitum. The rats were allowed to adjust to their environment for a week before starting the treatment. Animal care was in accordance with institutional guidelines.
Treatment.
The dosages of chemicals used were as follows: PB (1.3 mg/kg/day in water, oral), DEET (40 mg/kg/day in 70% ethanol, dermal), and permethrin (0.13 mg/kg/day in 70% ethanol, dermal). These doses of PB, DEET, and permethrin correspond to real-life exposure to military personnel during the PGW. Oral doses were given by gavage, while dermal applications were applied to the back of the neck on a 1-in2 area preshaved with electric clippers. Groups with five animals each were treated as described below. Animals were sacrificed 24 h after treatment with the last dose.
Behavioral Studies.
A battery of standardized tests was employed on days 30 and 45 following the treatment. These behavioral tests were designed to measure sensorimotor reflexes, motor strength, and coordinated gait (Bederson et al., 1986; Goldstein, 1993
; Markgraf et al., 1992
). All behavioral testing was performed by a trained observer blind to the treatment status of the animal and was carried out in a soundproof room with subdued lighting (less than 10.76 lumens/m2, ambient light). Rats were handled for 2 min daily for 5 days during the week prior to behavioral testing.
Postural Reflexes
Description.
Rats were held gently by the tail, one meter above the floor, and observed for forelimb extension. Normal rats extend both forelimbs. Consistent flexion of the forelimb is an abnormal response. Rats with consistent forelimb flexion are then further assessed by being placed on a large sheet of plastic-coated paper that can be gripped with the forepaws. With the tail held, gentle lateral pressure was applied behind the shoulder of the rat until the forelimb slid several inches. The maneuver was repeated five times in each direction. Normal rats resist lateral pressure by gripping the coated paper.
Scoring.
Grade 0: rats without evidence of consistent forelimb flexion when held above the floor; grade 1: rats with consistent forelimb flexion; grade 2: otherwise grade 1 rats that do not resist lateral pressure on at least three of five trials in either direction.
Limb Placing
Description.
Visual, tactile, and proprioceptive forelimb placing responses were examined. For visual placing, rats were held in the hands of the examiner 10 cm above the tabletop, with forelimbs hanging free. The rats were then slowly tilted toward the table. Intact rats reach toward the table with both forepaws. For tactile placing, the dorsal and then lateral portions of the forepaws were touched to the table edge. Intact rats immediately place the paw on the surface of the table. Proprioceptive placing was tested by pushing the forepaw onto the table edge. Care was taken to avoid the vibrissae touching the table.
Scoring.
For each test: grade 0, the placing response is immediate; grade 1, the placing response is slow or delayed; grade 2, the placing response does not occur within 2 s.
Orienting to Vibrissae Touch
Description.
The rat was placed atop an inverted polycarbonate cage and allowed 1 min for habituation. Its vibrissae were then touched with a cotton-tipped swab.
Scoring.
Grade 0: rat orients to the side of the probe on at least two of three trials from each side; grade 1, rat fails to orient on at least two of three trials on either side.
Grip Time.
Forepaw grip time of the rats was assessed by having them hang from a 5 mm diameter wood dowel gripped with both forepaws. Time to release their grip was recorded in seconds.
Beam-Walking and Beam Score
Description.
The testing apparatus was a 2.5 x 122 cm wooden beam elevated 75.5 cm above the floor with wooden supports. A 20 x 25 x 24 cm goal box with a 9.5 cm opening is located at one end of the beam. A switch-activated source of bright light (75 watt Tungsten bulb) and white noise (41 dB at 8000 Hz, 58 dB at 4000 Hz, 56 dB at 2000 Hz, 56 dB at 1000 Hz, 58 dB at 500 Hz, and 52 dB at 250 Hz SPL at the center of the frequency at each octave band) were located at the start end of the beam and served as avoidance stimuli. The rats were first trained with a series of three approximate trials. Rats are readily trained to perform the beam-walking task (Goldstein, 1993).
Scoring.
Both the latency until the animal's nose entered the goal box (up to 90 s) and the use of the hind paw to aid locomotion were recorded. Beam-walking ability was measured with a seven-point scoring system scale as previously described (Goldstein, 1993): 1, the rat is unable to place the affected hindpaw on the horizontal surface of the beam; 2, the rat places the hindpaw on the horizontal surface of the beam and maintains balance for at least 5 s; 3, the rat traverses the beam while dragging the affected hindpaw; 4, the rat traverses the beam and at least once places the affected hindpaw on the horizontal surface of the beam; 5, the rat crosses the beam and places the affected hindlimb on the horizontal surface of the beam to aid less than half its steps; 6, the rat uses the affected hindpaw to aid more than half its steps; and 7, the rat traverses the beam with no more than two footslips. In addition, the latency until the animal's nose enters the goal box (up to 90 s) is recorded for the final trial. Rats that fell off the beam were assigned latencies of 90 s.
Incline Plane
Description.
The rats were placed on a flat plane in the horizontal position, with the head facing the side of the board to be raised according to the method described by Yonemori et al. (1998). The board was slowly rotated to the vertical position. Two trials were performed for each testing session.
Scoring.
The angle that the rat began to slip downward was recorded. The results of two trials were averaged at each time point.
Statistical Analysis.
For continuous data (beam-walk time, beam-walk score, grip time, and incline plane), groups were compared by two-way ANOVA, with repeated measures as appropriate. The significance of post hoc pairwise comparison was determined with Fisher's LSD tests. For nonparametric data (postural reflexes, limb placing, and vibrassal touch), comparisons across treatment groups were made with the Kruskal-Wallis test. If the Kruskal-Wallis test indicated a significant difference among the groups, Dunn's procedure would be applied to the ranks of the data to determine the significance of post hoc, pairwise comparisons.
Assays
Acetylcholinesterase and butyrylcholinesterase assays.
Brain acetylcholinesterase (AChE) and plasma cholinesterase (BChE) activities were measured by the Ellman assay (Ellman et al., 1961). For AChE assays, dissected brain regions were homogenized in Ellman buffer and centrifuged for 5 min at 5000 x g; the resulting supernatant was used for AChE analysis. AChE activity was measured using acetylthiocholine as substrate in a Molecular Devices UV Max Kinetic Microplate Reader at 412 nm. 5,5'-Dithio-bis-2-nitrobenzoic acid (DTNB) was used as the color reagent as described by Abou-Donia et al. (1996). Protein concentrations in tissue samples and plasma were determined by the method of Smith et al. (1985).
Choline acetyl transferase.
Choline acetyl transferase activity in brain was determined using methods by Fonnum (1975).
Muscarinic acetylcholine receptor binding.
For the assay of the ligand binding for m2 mAChR, the tissue was homogenized in 10 mM phosphate buffer, pH 7.4, and centrifuged at 40,000 x g for 10 min. The membranes were suspended in the same buffer at a protein concentration of 1.252.5 mg/ml as described by Huff et al. (1994), and the ligand binding was carried out according to Slotkin et al. (1999). The m2 mAChR binding was carried out by using m2 mAChR-specific ligand, [3H]AF-DX 384 at room temperature for 60 min. Results are presented as specific receptor binding (dpm)/mg protein (percent of control).
Nicotinic acetylcholine receptor binding.
[3H]Cytisine was used as specific ligand for nAChR according to the method described by Slotkin et al. (1999). An aliquot of membrane preparation containing 200 µg protein was used to carry out the incubation with 1 nM [3H]cytisine at 4°C for 75 min. Results are presented as specific receptor binding (dpm)/mg protein (percent of control).
Statistics.
For biochemical assays, treatment groups were compared to control groups by two-way unpaired t-test using Prism GraphPadTM software, and results were plotted using Excel graphics for Macintosh.
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RESULTS |
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Effect of DEET, Permethrin or PB, Alone or in Combination, on Sensorimotor Function
A battery of behavioral tests was carried out to assess the sensorimotor function. We focused on sensorimotor ability because the majority of PGW veterans' complaints related to muscle and joint pain, fatigue, disorientation, and ataxia. Animals were tested on days 30 and 45 from the beginning of the treatment. Although statistical analyses were performed on the actual data, for the sake of comparability, the data obtained from beam-walk score, beam-walk time, incline plane, and grip time are presented as a percent of control. In the figures, error bars reflect SEM based on the raw data and recalculated to reflect percent of control. Figure 1 represents the measurements carried out on day 30, and Figure 2
represents the activity measured on day 45 following the beginning of the treatment with DEET and permethrin.
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For beam-walking time, two-way repeated measures ANOVA showed a significant effect of treatment group (ANOVA F7,70 = 8.4, p = 0.006) but no treatment group x time interaction (ANOVA F1,7 = 0.7, p = 0.65). Given alone, pyridostigmine, but not DEET or permethrin, differed significantly from control (Fisher LSD, p = 0.0002). There was not a significant difference between rats given PB and those given DEET, permethrin, and PB (Fisher LSD, p = 0.20).
For incline plane performance (Figs. 1 and 2), two-way repeated measures ANOVA showed a significant effect of treatment group (ANOVA F7,70 = 38.1, p < 0.0001) and a significant treatment group x time interaction (ANOVA F1,7 = 4.5, p = 0.004). All drugs given alone differed significantly from control (Fisher LSD, p < 0.0001). There was no significant difference between rats given PB and those given DEET, permethrin, and PB (Fisher LSD, p > 0.9).
Finally, for forepaw grip time (Figs. 1 and 2), two-way repeated measures ANOVA showed a significant effect of treatment group (ANOVA F7,70 = 44.5, p = 0.001) and a significant treatment group x time interaction (ANOVA F1,7 = 4.9, p = 0.001). All drugs given alone differed significantly from control (Fisher LSD, p < 0.0001). There was no significant difference between rats given PB and those given DEET, permethrin, and PB (Fisher LSD, p = 0.06).
A similar decreasing but insignificant trend in horizontal and vertical movement was observed in the animals treated with the combination of PB, DEET, and permethrin (data not shown).
In summary, each drug given alone had a significant behavioral effect, which tended to become more evident over time. There also was no significant difference on any parameter between rats given PB alone and those given DEET, permethrin, and PB. Most significant deficits were observed in animals given PB or a combination of PB with other chemicals.
Effect of PB, DEET, and Permethrin, Alone or in Combination, on Plasma and Brain Cholinesterase Activities
Plasma BChE and AChE activities in cortex, brainstem, midbrain, and cerebellum from the animals treated with PB, DEET, and permethrin, alone or in combination, were assayed. Data on the effects of single-chemical treatment are presented in Figure 3 (top panel). Treatment with PB, alone or in combination with DEET and permethrin, caused slight but insignificant inhibition (
96% of controls) of plasma BChE activity. Treatment with DEET, alone or in combination with PB or permethrin, caused a variable but insignificant increase (
125140% of control) in BChE activity. Treatment with permethrin, either alone or in combination, did not have any effect on BChE activity.
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Data on combination of two chemicals are presented in Figure 3 (middle panel). A combination of PB and DEET treatment resulted in significant AChE inhibition in the brainstem and midbrain (p < 0.001 and 0.01, respectively). Combined exposure with PB and permethrin resulted in significant inhibition (
67% of control, p < 0.04) in brainstem AChE activity, whereas cortex activity remained significantly increased. Combined exposure with DEET and permethrin resulted in significant inhibition (p < 0.001) in brainstem AChE activity, whereas other regions did not show any change in the activity that was different than individual chemical alone. This was consistent with our previous studies (Abou-Donia et al., 1996
).
Data presented in Figure 3 (bottom panel) indicate that brainstem and midbrain AChE activity was significantly inhibited when animals were exposed to the combination of PB, DEET, and permethrin (
6065% of control, p < 0.001 and 0.04, respectively). The magnitude of inhibition is similar to that observed when animals were exposed to a combination of PB and DEET, suggesting that PB under these treatment conditions has the potential to inhibit the brainstem and midbrain AChE activity.
Effect of PB, DEET, and Permethrin, Alone or in Combination, on Brain Choline Acetyl Transferase (ChAT) Activity
Choline acetyl transferase (ChAT) catalyzes the final step in the biosynthesis of acetylcholine by facilitating the irreversible transfer of acetyl groups of acetylCoA to choline. In view of the changes induced by PB in CNS on AChE activity, and because PB-induced inhibition of AChE is reversible and short-lived (Watts and Wilkinson, 1977), we argued that there may exist alternative mechanisms of acetylcholine buildup. Therefore, we studied the effects of treatment with PB, DEET, and permethrin, alone or in combination, on ChAT activity in brainstem and cortex. In the CNS, the majority of ChAT activity is localized in brainstem and cortex (Wu and Hersh, 1994
). Data in Figure 4
represent the enzyme activity in the cortex and brainstem. Treatment with PB and permethrin alone did not have any significant effect on brainstem or cortex enzyme activities, whereas DEET treatment caused a significant increase in the enzyme activity in the cortex (p < 0.001). Combined exposure of PB and permethrin caused a significant increase (p < 0.001) in ChAT activity in the cortex. However, coexposure with PB, DEET, and permethrin did not result in any significant change in enzyme activity in either region.
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DISCUSSION |
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The anatomical and molecular bases of the behavioral effects observed in the present study are complex. Different lesion studies have shown that severe sensorimotor impairment occurs in the animals with lesions of anteromedial and caudal forelimb cortex (Barth et al., 1990). Similarly, studies with bilateral large lesions in the rat somatic sensorimotor cortex have shown impairment in limb-placing response. Additionally, it has also been suggested that limb placing is a function of corticospinal tract (Hicks and D'Amato, 1975
). Thus, it is possible that treatment with PB, DEET, and permethrin, alone or in combination, could affect these innervations as well as innervations in other brain regions, and as a consequence, sensorimotor deficit may occur after prolonged exposure.
Beam-walking performance is an integrated form of behavior necessitating pertinent levels of consciousness, memory, sensorimotor, and cortical functions mediated by cortical area, and it has been suggested that an injury to cortex is reflected by a deficit in beam-walk task. A role for norepinephrine (NE) has strongly been proposed in the deficit caused by cortical injury (Boyeson et al., 1992; Goldstein, 1995
). It has been suggested that NE facilitates the recovery from locomotor deficit by alleviating injury-induced decrease and turnover of NE levels in the cortex (Boyeson and Feeney, 1990
; Kikuchi et al., 2000
). NE originates from the locus coeruleus and is widely distributed in the CNS, including cerebral cortex, hippocampus, cerebellum, and spinal cord. Therefore, it is possible that treatment with PB, DEET, and permethrin, alone and in combination, might regulate the noradrenergic and other catecholaminergic pathway in these animals. However, in view of the complexity of the behavioral outcome, it is also possible that these deficits are the result of exposure of these chemicals on multiple regions in the brain
PB has been used to protect against organophosphate nerve agent poisoning. It provides protection by shielding the peripheral AChE by reversibly binding to it. Because PB is a positively charged quaternary ion, it does not cross the BBB under ordinary circumstances. Thus, the toxic effects of PB are thought to be mediated through peripheral ACh nicotinic and muscarinic receptors (Albuquerque et al., 1997). Indeed, Chaney et al. (1999) found that PB-induced seizures in the mouse were mediated via peripheral nervous system (PNS) muscarinic and nicotinic receptors. However, other studies also suggest that PB toxicity is mediated through CNS ACh receptors as well as through the PNS (Servatius et al., 1998
). Our results indicate that low-dose PB treatment for 15 days inhibited midbrain AChE activity, whereas plasma BChE activity showed little inhibition. It is possible that PB entry into the CNS and the consequent inhibition of AChE in the CNS may enhance the toxic potential of neurotoxic agents. It is not certain yet how PB could affect the CNS. It is possible that continuous treatment PB, alone or in combination with DEET and permethrin, could affect the BBB permeability, thus allowing PB to enter the CNS. Other studies also demonstrate that treatment with chemicals that cause inhibition of AChE lead to m2 mAChR up-regulation (Majocha and Baldessarini, 1984
; Witt-Enderby et al., 1995
).
Some PGW veterans were exposed to a combination of pesticides and insecticides such as DEET and permethrin. In addition, they were exposed to PB because they were allowed to ingest twenty-one 30-mg tablets of PB. DEET is highly permeable to the skin and has been studied (Baynes et al., 1997; Selim et al., 1995
) for its metabolism and toxicity. Permethrin has been used to impregnate the clothing of military personnel as protection against pestiferous and vector insects (Taplin and Meinking, 1990
). It is possible that combined exposure to these chemicals would result in differential effects than exposure to single chemicals. Indeed our biochemical data show this phenomenon. Our data on cholinesterase suggest that DEET and permethrin alone do not inhibit the AChE activity in the CNS or plasma, whereas a combination with PB resulted in significant inhibition in brainstem and midbrain activity. These data are consistent with our previous studies in chickens (Abou-Donia et al., 1996
). An intriguing finding in our study is that treatment with permethrin alone caused a significant increase in cortical and cerebellar AChE activity, whereas DEET treatment alone caused a significant increase in brainstem AChE activity. The combination treatment led to a significant inhibition of AChE activity in brainstem, suggesting that brainstem may be the most susceptible to combined exposure. This inhibition may be mediated by PB, which might gain entry in the CNS following coexposure with DEET and permethrin. Treatment with DEET or permethrin caused an increase in AChE activity that may be due to an increase in AChE protein levels. Although not universally accepted, an increase in AChE protein may reflect an increased axonal repair and synaptic modeling, as has been shown recently (Bigbee et al., 2000
; Guizzetti et al., 1996
; Sternfeld et al., 1998
). Therefore, it is possible DEET and permethrin treatment alone may cause subtle changes that are reflected in increased synaptic modeling and repair. The behavioral data on single chemicals substantiate this notion. Coexposure with PB, DEET, and permethrin together caused a significant inhibition in brainstem and midbrain AChE activity, suggesting that treatment with three chemicals together could lead to added neurotoxic effects.
Cholinergic system in the CNS plays an important role in learning and memory (Lena and Changeux, 1998; Levey et al., 1995
). We studied the receptor ligand binding for m2AChR and nicotinic AChR in the cortex. Based on our data, it appears that increased receptor ligand binding density for both of the receptors in the cortex in response to treatment with PB, DEET, and permethrin, alone or in combination, may be a compensatory mechanism for a reduced ability of these receptors to bind their respective ligands. It is known that treatment with muscarinic antagonists induces receptor up-regulation (Ben-Barak and Dudai, 1980
; Coccini et al., 2000
; Majocha and Baldessarini, 1984
; Smiley et al., 1998
). Wang et al. (1996) reported the regulation of muscarinic receptor by repeated treatment with nicotine. The up-regulation of cortex m2AChR may be related to an increase in the AChE levels in the cortex of the animals treated with DEET or permethrin. Increased AChE activity in the cortex suggests that ACh levels are depleted. It is possible that subsequent receptor up-regulation is a response to reduced neurotransmitter levels. Increased ligand binding for m2 muscarinic receptor results in the inhibition of adenylate cyclase activity through a pertussis toxin-sensitive G-protein, resulting in an inhibitory postsynaptic response (Brann et al., 1993
; Wess, 1996
). The inhibitory nature of m2 receptor may have regulatory response on (GABA)ergic system in the cortex. It is known that cholinergic input in certain brain regions tonically inhibits (GABA)ergic system and is inhibitory to vasomotor glutamergic neurons. Thus, an increase in m2AChR in response to treatments with PB, DEET, and permethrin, alone or in combination, may regulate the glutamergic pathway leading to a decreased motor response. Also, it is well accepted that most of the toxic effects of pyrethroid insecticides are mediated through the modification of axonal Na+ channels (Narahashi, 1996
). Moreover, there is additional evidence that some of the toxic effects of pyrethroids are mediated by the interaction with GABA receptor-ionophore complex (Crofton and Reiter, 1987
; Gammon and Sander, 1985
; Lawrence et al., 1985
). However, no clear association between the modification of Na+ channels and development of sensorimotor deficit has yet been established.
In summary, our results suggest that exposure to physiologically relevant doses of PB, DEET, and permethrin, alone or in combination, lead to sensorimotor deficits and alteration in the cholinergic system in rats. These results further suggest that exposure with these chemicals, alone or in combination, may have played a role in the development of long-term health consequences associated with the PGW veterans. The contribution of cholinergic changes to the behavioral deficit following treatment with these chemicals is not clear at the moment, as these changes may involve a combination of mechanisms related to central and peripheral or neuromuscular system. In a recent study, Nostrandt et al., (1997) observed an insignificant correlation between changes in muscarinic receptor and AChE in the CNS following treatment with chlorpyrifos, which is a more potent cholinotoxic than the chemicals we used in the current studies. However, the possibility remains that the behavioral impairment observed in our studies may also have been a consequence of other generalized abnormalities such as deficit in cognition and motivation because of the changes in cholinergic system. Further studies are in progress to evaluate the histopathological correlates of these behavioral changes.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: VA Medical Center, Durham, North Carolina 27710.
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