Co-exposure to pyridostigmine bromide, DEET, and/or permethrin causes sensorimotor deficit and alterations in brain acetylcholinesterase activity

Military personnel deployed in the Persian Gulf War (PGW) were exposed to a combination of chemicals, including pyridostigmine bromide (PB), DEET, and permethrin. We investigated the dose-response effects of these chemicals, alone or in combination, on the sensorimotor performance and cholinergic system of male Sprague-Dawley rats. Animals were treated with a daily dermal dose of DEET and/or permethrin for 60 days and/or PB (gavage) during the last 15 days. Neurobehavioral performance was assessed on day 60 following the beginning of the treatment with DEET and permethrin. The rats were sacrificed 24 h after the last treatment for biochemical evaluations. PB alone, or in combination with DEET, or DEET and permethrin resulted in deficits in beam-walk score and longer beam-walk times compared to controls. PB alone, or in combination with DEET, permethrin, or DEET and permethrin caused impairment in incline plane performance and forepaw grip strength. PB alone at all doses slightly inhibited plasma butyrylcholinesterase activity, whereas combination of PB with DEET or permethrin increased its activity. Brainstem acetylcholinesterase (AChE) activity significantly increased following treatment with combinations of either DEET or permethrin at all doses, whereas the cerebellum showed a significant increase in AChE activity following treatment with a combination of PB/DEET/permethrin. Co-exposure to PB, DEET, and permethrin resulted in significant inhibition in AChE in midbrain. PB alone or in combination with DEET and permethrin at all doses increased ligand binding for m2 muscarinic acetylcholine receptor in the cortex. In addition, PB and DEET together or a combination of PB, DEET, and permethrin significantly increased ligand binding for nicotinic acetylcholine receptor. These results suggest that exposure to various doses of PB, alone and in combination with DEET and permethrin, leads to sensorimotor deficits and differential alterations of the cholinergic system in the CNS.     

Neurological deficits induced by malathion, DEET, and permethrin, alone or in combination in adult rats

Malathion (O,O-dimethyl-S-[1,2-carbethoxyethyl]phosphorodithionate), DEET (N,N-diethyl-m-toluamide), and permethrin [(+/-)-cis/trans-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane carboxylic acid (3-phenoxyphenyl) methyl ester] are commonly used pesticides. To determine the effects of the dermal application of these chemicals, alone or in combination, the sensorimotor behavior, central cholinergic system, and histopathological alterations were studied in adult male Sprague-Dawley rats following a daily dermal dose of 44.4 mg/kg malathion, 40 mg/kg DEET, and 0.13 mg/kg permethrin, alone and in combination for 30 d. Neurobehavioral evaluations of sensorimotor functions included beam-walking score, beam walk time, inclined plane, and grip response assessments. Twenty-four hours after the last treatment with each chemical alone or in combination all behavioral measures were impaired. The combination of DEET and permethrin, malathion and permethrin, or the three chemicals together resulted in greater impairments in inclined performance than permethrin alone. Only animals treated with a combination of DEET and malathion or with DEET and permethrin exhibited significant increases in plasma butyrlcholinesterase (BChE) activity. Treatment with DEET or permethrin alone, malathion and permethrin, or DEET and permethrin produced significant increases in cortical acetylcholinesterase (AChE) activity. Combinations of malathion and permethrin or of DEET and permethrin produced significant decreases in midbrain AChE activity. Animals treated with DEET alone exhibited a significant increase in cortical m2 muscarinic ACh receptor binding. Quantification of neuron density in the dentate gyrus, CA1 and CA3 subfields of the hippocampus, midbrain, brainstem, and cerebellum revealed significant reductions in the density of surviving neurons with various treatments. These results suggest that exposure to real-life doses of malathion, DEET, and permethrin, alone or in combination, produce no overt signs of neurotoxicity but induce significant neurobehavioral deficits and neuronal degeneration in brain.     

Increased 8-hydroxy-2'-deoxyguanosine, a biomarker of oxidative DNA damage in rat urine following a single dermal dose of DEET (N, N-diethyl-m-toluamide), and permethrin, alone and in combination

Levels of the biomarker of DNA oxidative damage 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat urine following dermal exposure to DEET (N,N-diethyl-m-toluamide) and permethrin, alone and in combination have been determined. A group of five rats for each time point were treated with a single dermal dose of 400 mg/kg of DEET, 1.3 mg/kg of permethrin or their combination. Urine samples were collected 2,4,8,16,24,48, and 72 h following application. Control urine samples of rats treated with ethanol were also collected at the same time intervals. Solid phase extraction coupled with high performance liquid chromatography (HPLC) with UV detection at 254 nm was used for determination of 2'-deoxyguanosine, and (8-OHdG). The limits of detection (LOD) were 0.5 ng of both 2'-deoxyguanosine and 8-OHdG. Their average percentage recoveries from urine samples were between 70-85%. A single dermal dose of DEET or in combination with permethrin significantly induced levels of (8-OHdG) that are excreted in the urine over the time course of the study compared to control urine samples. Permethrin did not cause significant increase in the amount of 8-OHdG in the urine. Levels of 8-OHdG in urine excreted at 24 h were 1009+/-342, 1701+/-321, 1140+/-316, and 1897+/-231 ng following treatment with ethanol, DEET, permethrin, and DEET+permethrin, respectively. The results indicate that dermal administration of DEET could generate free radical species hence cause DNA oxidative damage in rats.     

Induction of urinary excretion of 3-nitrotyrosine, a marker of oxidative stress, following administration of pyridostigmine bromide, DEET (N,N-diethyl-m-toluamide) and permethrin, alone and in combination in rats

In this study, we determined levels of 3-nitrotyrosine in rat urine following administration of a single oral dose of 13 mg/kg pyridostigmine bromide (PB) (3-dimethylaminocarbonyloxy-N-methylpyridinum bromide), a single dermal dose of 400 mg/kg N,N-diethyl-m-toluamide (DEET) and a single dermal dose of 1.3 mg/kg permethrin, alone and in combination. Urine samples were collected from five treated and five control rats at 4, 8, 16, 24, 48, and 72 h following dosing. Solid-phase extraction coupled with high-performance liquid chromatography with ultraviolet detection at 274 nm was used for the determination of tyrosine and 3-nitrotyrosine. A single oral dose of PB and a single dermal dose of DEET or their combination significantly (P<0.05) increased levels of 3-nitrotyrosine starting 24 h after dosing compared with control urine samples. The maximum increase of 3-nitroytyrosine was detected 48 h after combined administration of PB and DEET. The ratio of 3-nitrotyrosine to tyrosine in urine excreted 48 h after dosing was 0.19+/-0.04, 0.20+/-0.05, 0.28+/-0.03, 0.32+/-0.04, 0.19+/-0.05, 0.42+/-0.04, 0.27+/-0.03, 0.36+/-0.04, and 0.48+/-0.04 following administration of water, ethanol, PB, DEET, permethrin, PB+DEET, PB+permethrin, DEET+permethrin, and PB+DEET+permethrin, respectively. The results indicate that an oral dose of PB and a dermal administration of DEET, alone and in combination, could generate free radical species, and thus increase levels of 3-nitrotyrosine in rat urine. Induction of 3-nitrotyrosine, a marker of oxidative stress, following exposure to these compounds could be significant in understanding the proposed enhanced toxicity following combined exposure to these compounds.     

Locomotor and sensorimotor performance deficit in rats following exposure to pyridostigmine bromide, DEET, and permethrin, alone and in combination.

Since their return from Persian Gulf War (PGW), many veterans have complained of symptoms including muscle and joint pain, ataxia, chronic fatigue, headache, and difficulty with concentration. The causes of the symptoms remain unknown. Because these veterans were exposed to a combination of chemicals including pyridostigmine bromide (PB), DEET, and permethrin, we investigated the effects of these agents, alone and in combination, on the sensorimotor behavior and central cholinergic system of rats. Male Sprague-Dawley rats (200-250 gm) were treated with DEET (40 mg/kg, dermal) or permethrin (0.13 mg/kg, dermal), alone and in combination with PB (1.3 mg/kg, oral, last 15 days only), for 45 days. Sensorimotor ability was assessed by a battery of behavioral tests that included beam-walk score, beam-walk time, incline plane performance, and forepaw grip on days 30 and 45 following the treatment. On day 45 the animals were sacrificed, and plasma and CNS cholinesterase, and brain choline acetyl transferase, muscarinic and nicotinic acetylcholine receptors were evaluated. Animals treated with PB, alone or in combination with DEET and permethrin, showed a significant deficit in beam-walk score as well as beam-walk time as compared with controls. Treatment with either DEET or permethrin, alone or in combination with each other, did not have a significant effect on beam-walk score. All chemicals, alone or in combination, resulted in a significant impairment in incline plane testing on days 30 and 45 following treatment. Treatment with PB, DEET, or permethrin alone did not have any inhibitory effect on plasma or brain cholinesterase activities, except that PB alone caused moderate inhibition in midbrain acetylcholinesterase (AChE) activity. Treatment with permethrin alone caused significant increase in cortical and cerebellar AChE activity. A combination of DEET and permethrin or PB and DEET led to significant decrease in AChE activity in brainstem and midbrain and brainstem, respectively. A significant decrease in brainstem AChE activity was observed following combined exposure to PB and permethrin. Coexposure with PB, DEET, and permethrin resulted in significant inhibition in AChE in brainstem and midbrain. No effect was observed on choline acetyl transferase activity in brainstem or cortex, except combined exposure to PB, DEET, and permethrin caused a slight but significant increase in cortical choline acetyltransferase activity. Treatment with PB, DEET, and permethrin alone caused a significant increase in ligand binding for m2 muscarinic acetylcholine receptor (mAChR) in the cortex. Coexposure to PB, DEET, and permethrin did not have any effect over that of PB-induced increase in ligand binding. There was no significant change in ligand binding for nicotinic acetylcholine receptor (nAChR) associated with treatment with the chemical alone; a combination of PB and DEET or coexposure with PB, DEET, and permethrin caused a significant increase in nAChR ligand binding in the cortex. Thus, these results suggest that exposure to physiologically relevant doses of PB, DEET, and permethrin, alone or in combination, leads to neurobehavioral deficits and region-specific alterations in AChE and acetylcholine receptors.     

DEET (N,N-diethyl-m-toluamide) alone and in combination with permethrin increased urinary excretion of 6beta-hydroxycortisol in rats, a marker of hepatic CYP3A induction.

In this study, the ratio of 6beta-hydroxycortisol (6beta-OHF) to free cortisol (F) was determined in urine following a single dermal dose of 400 mg/kg of DEET (N,N-diethyl-m-toluamide), and 1.3 mg/kg of permethrin, alone and in combination, in rats. Urine samples were collected at 2, 4, 8, 16, 24, 48, and 72 h after application. Recoveries of 6beta-OHF and cortisol (F) from control urine samples were between 75 and 85%, with limits of detection at 30 and 10 ng/ml for cortisol and 6beta-OHF, respectively. A single dermal dose of DEET alone and in combination with permethrin significantly increased urinary excretion of 6beta-hydroxycortisol 24 h after dosing. Permethrin did not significantly alter the urinary excretion of 6beta-hydroxycortisol. These results indicate that DEET, alone and in combination with permethrin, increased urinary excretion of 6beta-OHF in rats following a single dermal dose application.     

Combined exposure to DEET (N,N-diethyl-m-toluamide) and permethrin-induced release of rat brain mitochondrial cytochrome c.

The release of cytochrome c from the mitochondrial intermembrane space can induce apoptosis. The levels of mitochondrial cytochrome c in rat brain following a single dermal dose of 400 mg/kg of DEET, and of 1.3 mg/kg of permethrin, alone or in combination were determined. Rats were sacrificed at a time interval of 0.5, 1, 2, 4, 8, 16, 24, 48, or 72 h after dosing. Brain mitochondria were isolated and the levels of cytochrome c were measured using reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Average percentage recovery of cytochrome c spiked with control rat brain mitochondria was 83.2 +/- 8.9%. Limits of detection and quantitation were 1 and 5 ng, respectively. The results showed that a single dermal dose of a combination of DEET and permethrin significantly increased the release of brain mitochondrial cytochrome c starting 24 h after treatment. DEET and permethrin alone did not affect the release of cytochrome c. The results indicate that combined exposure to DEET and permethrin might induce the apoptotic processes in rat brain as seen by the release of cytochrome c.     

Stress and combined exposure to low doses of pyridostigmine bromide, DEET, and permethrin produce neurochemical and neuropathological alterations in cerebral cortex, hippocampus, and cerebellum

Exposure to a combination of stress and low doses of the chemicals pyridostigmine bromide (PB), DEET, and permethrin in adult rats, a model of Gulf War exposure, produces blood-brain barrier (BBB) disruption and neuronal cell death in the cingulate cortex, dentate gyrus, thalamus, and hypothalamus. In this study, neuropathological alterations in other areas of the brain where no apparent BBB disruption was observed was studied following such exposure. Animals exposed to both stress and chemical exhibited decreased brain acetylcholinesterase (AChE) activity in the midbrain, brainstem, and cerebellum and decreased m2 muscarinic acetylcholine (ACh) receptor ligand binding in the midbrain and cerebellum. These alterations were associated with significant neuronal cell death, reduced microtubule-associated protein (MAP-2) expression, and increased glial fibrillary acidic protein (GFAP) expression in the cerebral cortex and the hippocampal subfields CA1 and CA3. In the cerebellum, the neurochemical alterations were associated with Purkinje cell loss and increased GFAP immunoreactivity in the white matter. However, animals subjected to either stress or chemicals alone did not show any of these changes in comparison to vehicle-treated controls. Collectively, these results suggest that prolonged exposure to a combination of stress and the chemicals PB, DEET, and permethrin can produce significant damage to the cerebral cortex, hippocampus, and cerebellum, even in the absence of apparent BBB damage. As these areas of the brain are respectively important for the maintenance of motor and sensory functions, learning and memory, and gait and coordination of movements, such alterations could lead to many physiological, pharmacological, and behavioral abnormalities, particularly motor deficits and learning and memory dysfunction.     

In vitro metabolism and interactions of pyridostigmine bromide, N,N-diethyl-m-toluamide, and permethrin in human plasma and liver microsomal enzymes

1. The in vitro human plasma activity and liver microsomal metabolism of pyridostigmine bromide (PB), a prophylactic treatment against organophosphate nerve agent attack, N,N-diethyl-m-toluamide (DEET), an insect repellent, and permethrin, a pyrethroid insecticide, either alone or in combination were investigated. 2. The three chemicals disappeared from plasma in the following order: permethrin > PB > DEET. The combined incubation of DEET with either permethrin or PB had no effect on permethrin or PB. Binary incubation with permethrin decreased the metabolism of PB and its disappearance from plasma and binary incubation with PB decreased the metabolism of permethrin and its clearance from plasma. Incubation with PB and/or permethrin shortened the DEET terminal half-life in plasma. These agents behaved similarly when studied in liver microsomal assays. The combined incubation of DEET with PB or permethrin (alone or in combination) diminished DEET metabolism in microsomal systems. 3. The present study evidences that PB and permethrin are metabolized by both human plasma and liver microsomal enzymes and that DEET is mainly metabolized by liver oxidase enzymes. Combined exposure to test chemicals increases their neurotoxicity by impeding the body's ability to eliminate them because of the competition for detoxifying enzymes.     

Pyridostigmine bromide modulates the dermal disposition of [14C]permethrin

The cause of the Gulf War Syndrome may be related to soldiers being exposed to insecticides (e.g., permethrin (P)), insect repellents (e.g., N,N-diethyl-m-toluamide (DEET)), an organophosphate nerve agent simulant (e.g., diisopropyl fluorpohosphate (DFP)), and/or prophylactic treatment (e.g., pyridostigmine bromide (PB)) against potential nerve gas attacks. The purpose of this study was to assess the dermal disposition of [14C]permethrin in ethanol or ethanol:water (3:2) in the isolated perfused porcine skin flap (IPPSF) model with simultaneous dermal exposure to DEET or DFP. These IPPSFs were also simultaneously perfused arterially with or without PB, DFP, or DFP + PB. The results indicated that DFP + PB significantly increased [14C]permethrin absorption compared to controls (1.06% dose vs 0.14% dose). PB significantly increased [14C]permethrin disposition in the stratum corneum (SC) in aqueous mixtures only (9.40 vs 3.35% dose), while topical DEET or topical DFP reduced [14C]permethrin levels in the SC especially in nonaqueous mixtures. PB also significantly enhanced [14C]permethrin penetration into all skin tissues and perfusate in aqueous mixtures, while DEET reversed this effect. PB appeared to influence [14C]permethrin disposition in flowthrough diffusion cells, suggesting that the mechanism of this interaction may be associated predominantly with epidermal permeability, although muscarinic effects in the vasculature in IPPSFs should not be ruled out and requires further investigation. These experiments suggest that intraarterial perfusion of PB and/or DFP and topical application of DFP or DEET can alter the disposition of [14C]permethrin in skin and possibly its bioavailability in soldiers simultaneously exposed to these chemicals.     

In vitro metabolism and interactions of pyridostigmine bromide,N,N-diethyl-m-toluamide,and permethrin in human plasma and liver microsomal enzymes

1.?The in vitro human plasma activity and liver microsomal metabolism of pyridostigmine bromide (PB), a prophylactic treatment against organophosphate nerve agent attack, N,N-diethyl-m-toluamide (DEET), an insect repellent, and permethrin, a pyrethroid insecticide, either alone or in combination were investigated.

2.?The three chemicals disappeared from plasma in the following order: permethrin > PB > DEET. The combined incubation of DEET with either permethrin or PB had no effect on permethrin or PB. Binary incubation with permethrin decreased the metabolism of PB and its disappearance from plasma and binary incubation with PB decreased the metabolism of permethrin and its clearance from plasma. Incubation with PB and/or permethrin shortened the DEET terminal half-life in plasma. These agents behaved similarly when studied in liver microsomal assays. The combined incubation of DEET with PB or permethrin (alone or in combination) diminished DEET metabolism in microsomal systems.

3.?The present study evidences that PB and permethrin are metabolized by both human plasma and liver microsomal enzymes and that DEET is mainly metabolized by liver oxidase enzymes. Combined exposure to test chemicals increases their neurotoxicity by impeding the body's ability to eliminate them because of the competition for detoxifying enzymes.     

The effects of pyridostigmine bromide, permethrin, and DEET alone, or in combination, on fixed-ratio and fixed-interval behavior in male and female rats.

Concurrent exposure to pyridostigmine bromide (PB), permethrin (PERM) and/or N,N-diethyl-m-toluamide (DEET) may have contributed to the development of a syndrome that appears to have afflicted military personnel who served during the Gulf War. The present experiment sought to evaluate the behavioral effects of these compounds alone, or in various combinations, in male and female rats. Subjects were exposed to a multiple fixed-ratio (FR) 50, fixed-interval (FI) 2-min schedule of reinforcement. PB dose-dependently decreased FR and FI response rates. FR responding was disrupted by lower doses and there were no differences between the sexes. PERM vehicle administration decreased response rates maintained by both schedules of reinforcement; this was offset by an increase in response rate after the administration of the intermediate dose of PERM. The highest dose of PERM decreased both FR and FI response rates. FR rates in male rats were more disrupted than those in female rats. Only the highest dose of DEET decreased FR and FI response rates in male and female rats. FR rates were more disrupted in female rats than in male rats. Synergistic effects were only observed when FI response rates decreased in male rats upon exposure to half the low dose of PB with half the low dose of PERM or half the low dose of PB with half the low dose of DEET. The results of this experiment thus show that small doses of PB, PERM and DEET disrupt well-established, schedule-controlled behavior in male and female rats in a schedule- and gender-dependent manner; schedule-dependent and gender-dependent synergistic effects were also observed. The mechanism by which the compounds exert these behavioral effects remains to be determined.     

Testicular germ-cell apoptosis in stressed rats following combined exposure to pyridostigmine bromide,N,N-diethyl m-toluamide (DEET), and permethrin

This study reports and characterizes the testicular apoptosis following daily exposure of male Sprague-Dawley rats to subchronic combined doses of pyridostigmine bromide (PB, 1.3 mg/kg/d in water, oral), a drug used for treatment of myasthenia gravis and prophylactic treatment against nerve agents during the Persian Gulf War; the insect repellent N,N-diethyl m-toluamide (DEET, 40 mg/kg/d in ethanol, dermal); and the insecticide permethrin (0.13 mg/kg in ethanol, dermal), with and without stress for 28 d. Combined exposure to these chemicals was implicated in the development of illnesses including genitourinary disorders among many veterans of the Persian Gulf War. Previous studies from this laboratory have shown that exposure to combination of these chemicals produced greater toxicity compared to single components. Exposure to stress alone did not cause any significant histopathological alterations in the testes. Administration of combination of these chemicals induced apoptosis in rat testicular germ cells, Sertoli cells, and Leydig cells, as well as in the endothelial lining of the blood vessels. Testicular damage was significantly augmented when the animals were further exposed to a combination of chemicals and stress. Histopathological examination of testicular tissue sections showed that apoptosis was confined to the basal germ cells and spermatocytes, indicating suppression of spermatogenesis. Increased apoptosis of testicular cells coincided, in timing and localization, with increased expression of the apoptosis-promoting proteins Bax and p53. Furthermore, significant increase of 3-nitrotyrosine immunostaining in the testis revealed oxidative and/or nitrosation induction of cell death. In conclusion, combined exposure to real-life doses of test compounds caused germ-cell apoptosis that was significantly enhanced by stress.     

Binding of pyridostigmine bromide,N,N-diethyl-m-toluamide and permethrin,alone and in combinations,to human serum albumin

In this study we examined the interaction of the anti-nerve agent drug pyridostigmine bromide (PB, 3,3-dimethylaminocarbonyloxy- N-methylpyridiniyum bromide), the insect repellent DEET ( N, N-diethyl- m-toluamide), and the insecticide permethrin [3-(2,2-dichloroethyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methyl ester] in binding to human serum albumin (HSA). Concentrations between 500 ng/ml and 10 microg/ml PB, DEET and permethrin, alone or in combination, were incubated with HSA at 37 degrees C for 60 min. Concentrations of PB, DEET and permethrin were determined using high performance liquid chromatography (HPLC). The results showed that 81.2+/-4.2%, and 84.6+/-2.5% of the initial concentration of PB was bound to HSA when incubated alone or in combination with DEET or permethrin, respectively. DEET and permethrin did not significantly interact with HSA after 1 h of incubation. Incubation of combinations of two or three compounds did not significantly alter the binding pattern of any of the compounds with HSA. These results showed that PB is highly bound to albumin protein, while the competition between PB, DEET and permethrin on binding sites of HSA as a possible site of interaction following combined administration in vivo is not likely.     

Functional impairment of blood-brain barrier following pesticide exposure during early development in rats

1. The effect of certain pesticides on the functional integrity of the developing blood-brain barrier (BBB) was studied following single and repeated exposure, and after subsequent withdrawal in rats. 2. Ten-day-old rat pups exposed orally to quinalphos (QP, organophosphate), cypermethrin (CM, pyrethroid) and lindane (LD, organochlorine) at a dose of 1/50th of LD50, showed a significant increase in the brain uptake index (BUI) for a micromolecular tracer, sodium fluorescein (SF), by 97, 37 and 72%, respectively, after 2 h. Residual increases in the BUI were found even after 3 days of the single treatment of QP (28%) and LD (23%). 3. Repeated exposure for 8 days (postnatal days (PND) 10-17) with QP, CM and LD increased the BBB permeability by 130, 80 and 50%, respectively. Recovery from these changes was complete in QP and LD-treated animals after 13 days (PND 18-30) of withdrawal. However, CM showed persistent effects that were normalized only after 43 days (PND 18-60) of withdrawal. 4. A single dose reduced to 1/100th of LD50 also increased BUI in 10-day-old rat pups following QP (20%) and CM (28%) exposure at 2 h. 5. An age-dependent effect of these pesticides was evident from the study showing higher magnitude of BUI changes in 10-day-old rats as compared to that in 15-day-old rats. Furthermore, adult rats did not show any effect on BBB permeability even at a higher dose (1/25th of LD50) of these pesticides given alone or in combination with piperonyl butoxide (600 mg/kg, i.p.) for 3 consecutive days. 6. This study showed that developing BBB is highly vulnerable to single or repeated exposure of certain pesticides. The observed persistent effects during brain development even after withdrawal of the treatment may produce some neurological dysfunction at later life as well.     

In utero exposure to nicotine and chlorpyrifos alone, and in combination produces persistent sensorimotor deficits and Purkinje neuron loss in the cerebellum of adult offspring rats

This study was carried out to investigate the effect of in utero exposure to the cholinotoxicants, nicotine and chlorpyrifos, alone or in combination on neurobehavioral alterations and neuronal morphology latter in adult age. In the present study, 90 days old (corresponding to a human adult age) male and female offspring rats were evaluated for neurobehavioral, and neuropathological alterations following maternal, gestational exposure to nicotine and chlorpyrifos (O,O-diethyl-O-3,5,6-trichloro-2-pyridinyl phosphorothioate), alone and in combination. Female Sprague-Dawley rats (300–350 g) with timed-pregnancy were treated with nicotine (3.3 mg/kg/day, in bacteriostatic water via s.c. implantation of mini osmotic pump), chlorpyrifos (1.0 mg/kg, daily, dermal, in 75% ethanol, 1.0 ml/kg) or a combination of both chemicals, on gestational days (GD) 4–20. Control animals received bacteriostatic water via s.c. implantation of mini osmotic pump and dermal application of 70% ethanol. The offspring at postnatal day (PND) 90 were evaluated for neurobehavioral performance, changes in the activity of plasma butyrylcholinesterase (BChE) and acetylcholinesterase (AChE), and neuropathological alterations in the brain. Neurobehavioral evaluations included beam-walk score, beam-walk time, incline plane performance and forepaw grip time. Male and female offspring from mothers treated with nicotine and CPF, alone or in combination showed impairments in the performance of neurobehavioral tests, indicating sensorimotor deficits. Female offspring from mothers treated with a combination of nicotine and chlorpyrifos showed significant increase in plasma BChE activity. Brain regional AChE activity showed differential increases in male and female offspring. Brainstem and cerebellum of female offspring from mothers treated with nicotine or chlorpyrifos, alone or in combination showed increased AChE activity, whereas brainstem of male offspring from mothers treated with nicotine alone or a combination of nicotine and chlorpyrifos showed increase in AChE activity. Also, male offspring exposed in utero to nicotine exhibited increased AChE activity. Histopathological evaluations using cresyl violet staining showed a decrease in surviving Purkinje neurons in the cerebellum in offspring of all treatments groups. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer (GCL) of cerebellum following all exposures. These results indicate that in utero exposure to nicotine and chlorpyrifos, alone and in combination produced significant sensorimotor deficits in male and female offspring, differential increase in brain AChE activity, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum in adult offspring rats at a corresponding human adult age. Collectively, this study demonstrates that maternal exposure to environmental neurotoxic chemicals, i.e., nicotine and chlorpyrifos leads to developmental abnormalities in the offspring that persist latter into adulthood.     

Effects of single and repeated exposure to biocidal active substances on the barrier function of the skin in vitro

The dermal route of exposure is important in worker exposure to biocidal products. Many biocidal active substances which are used on a daily basis may decrease the barrier function of the skin to a larger extent than current risk assessment practice addresses, due to possible skin effects of repeated exposure. The influence of repeated and single exposure to representative biocidal active substances on the skin barrier was investigated in vitro. The biocidal active substances selected were alkyldimethylbenzylammonium chloride (ADBAC), boric acid, deltamethrin, dimethyldidecylammonium chloride (DDAC), formaldehyde, permethrin, piperonyl butoxide, sodium bromide, and tebuconazole. Of these nine compounds, only the quaternary ammonium chlorides ADBAC and DDAC had a clear and consistent influence on skin permeability of the marker compounds tritiated water and [(14)C]propoxur. For these compounds, repeated exposure increased skin permeability more than single exposure. At high concentrations the difference between single and repeated exposure was quantitatively significant: repeated exposure to 300 mg/L ADBAC increased skin permeability two to threefold in comparison to single exposure. Therefore, single and repeated exposure to specific biocidal products may significantly increase skin permeability, especially when used undiluted.     

Sensorimotor deficits and increased brain nicotinic acetylcholine receptors following exposure to chlorpyrifos and/or nicotine in rats

Despite well-known adverse effects associated with cigarette smoking, approximately 20% of the US population continues to smoke and many more are exposed to environmental tobacco smoke. Many of the same individuals are also exposed to environmental neurotoxic chemicals such as the organophosphorus insecticide chlorpyrifos. In the present study, the effects of exposure to low doses of nicotine and chlorpyrifos alone and in combination, were studied on the central cholinergic system and sensorimotor performance in rats. Male Sprague-Dawley rats (250-300 g) were treated with nicotine (1 mg/kg s.c., in normal saline), chlorpyrifos (0.1 mg/kg dermally, in 0.1 ml 70% ethanol), or a combination of both, daily for 30 days. Control rats were treated with saline and dermally with ethanol. Sensorimotor behavior was evaluated 24 h following the last dose using a battery of tests. There was a significant deficit in incline plane performance, beam-walk score and beam-walk time following exposure to each chemical, alone or in combination. The deficit in incline plane performance was greater when the two chemicals were given in combination than with either compound alone. Biochemical analysis showed a decrease in cerebellar and an increase in midbrain acetylcholinesterase (AChE) activity following combined exposure. Exposure to nicotine alone resulted in a significant increase in AChE activity in brainstem and midbrain, whereas there was no significant change after exposure to chlorpyrifos, alone. A significant increase in ligand binding to nicotinic acetylcholine receptors (nAChR) was observed in brainstem and cortex following exposure to nicotine or chlorpyrifos. This was further augmented with combined exposure, which caused a modest but significant increase in m2 muscarinic acetylcholine receptors (m2-mAChR) ligand binding in the cortex. These data suggest that exposure to either nicotine or chlorpyrifos or a combination of the two may impair neurobehavioral performance and affect the central nervous system cholinergic pathways.