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.

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.

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.

Uranyl acetate-induced sensorimotor deficit and increased nitric oxide generation in the central nervous system in rats.

We investigated the effects of uranyl acetate on sensorimotor behavior, generation of nitric oxide and the central cholinergic system of rats. Male Sprague-Dawley rats were treated with intramuscular injection of 0.1 and 1 mg/kg uranyl acetate in water, daily for 7 days. Control animals received equivalent amount of water. The treatment was stopped after the seventh injection because the animals in the 1-mg/kg group appeared lethargic. The animals were maintained for an additional observation period of 30 days. The study was initiated as a dose-finding study that covered doses of 10 and 100 mg/kg, as well. However, all the animals in the 100-mg/kg treatment group died after the third and fourth injections, and all animals given 10 mg/kg died after the fifth and sixth injections. On Day 30 following the cessation of treatment, the sensorimotor functions of the animals in the 0.1- and 1-mg/kg treatment groups were evaluated using a battery of tests that included measurements of postural reflexes, limb placing, orientation to vibrissae touch, grip time, beam walking and inclined plane performance. The animals were sacrificed the same day and the cerebral cortex, brainstem, cerebellum and midbrain were dissected. The levels of nitric oxide as marker for increased oxidative stress, and the integrity of the cholinergic system as reflected in acetylcholinesterase (AChE) activity and m2 muscarinic acetylcholine receptors ligand binding, were determined. The data from behavioral observations show that there was a dose-related deficit at the 0.1- and 1-mg/kg treatment groups for inclined plane performance. Both doses reduced grip time, but there was no significant difference between the two doses. Similarly, both beam-walk score and beam-walk time were impaired at both doses as compared with the controls. A significant increase in nitric oxide was seen at 0.1 mg/kg dose in cortex and midbrain, whereas brainstem and cerebellum showed an insignificant decrease at both the doses. Similarly, there was no significant change in nitric oxide levels in kidneys and liver of the treated animals as compared with the controls. There was a significant increase in AChE activity in the cortex of the animals treated with 1 mg/kg uranyl acetate, but not in other brain regions. Ligand binding densities for the m2 muscarinic receptor did not show any change. These results show that low-dose, multiple exposure to uranyl acetate caused prolonged neurobehavioral deficits after the initial exposure has ceased.

Sensorimotor deficit and cholinergic changes following coexposure with pyridostigmine bromide and sarin in rats.

A myriad of neurological symptoms including muscle and joint pain, ataxia, chronic fatigue, headache, and difficulty in concentration have been reported by Persian Gulf War (PGW) veterans. A large number of these veterans were prophylactically treated with pyridostigmine bromide (PB) and possibly exposed to sarin. In the present study we investigated the effects of PB and sarin, alone and in combination, on sensorimotor performance and the central cholinergic system of rats. Male Sprague-Dawley rats were treated with PB (1.3 mg/kg, 15 daily doses, oral) and sarin (50, 75, 90, and 100 microg/kg, single im dose on day 15), alone and in combination. The animals were evaluated for postural reflexes, limb placing, orienting to vibrissae touch, incline plane performance, beam-walk time, and forepaw grip time 7 and 15 days following treatment with sarin. Treatment with either PB or sarin alone resulted in significant sensorimotor impairments. Coexposure to sarin and PB resulted in significant sensorimotor deficits that worsened over time. By 15 days following sarin treatment, plasma butyrylcholinesterase (BChE) activity returned to normal levels in the animals treated with sarin alone, whereas in the animals exposed to PB or PB plus sarin, there was an increase in the enzyme activity. Cortical acetylcholinesterase (AChE) activity remained inhibited in the animals treated with sarin alone and in combination with PB. Muscarinic acetylcholine receptor (m2 mAChR) ligand binding with [(3)H]AFDX-384 in cortex and brain stem showed significant increases (approximately 120-130% of control) following coexposure to PB and sarin at higher doses. To evaluate the potential of PB for augmentation or inhibition of the toxicity induced by acute sarin exposure, the animals were exposed to either 10 or 100 microg/kg sarin (single im injection) with or without pretreatment with PB, and sacrificed 3 h after treatment with sarin. Pretreatment with PB offered slight protection in the plasma as well as brain regional enzyme activities. Pretreatment with PB did not have any effect on sarin-inhibited brain regional AChE activity following treatment with 100 microg/kg sarin. These results show that prophylactic treatment with PB offers some degree of protection in peripheral cholinesterase. Furthermore, these results show that treatment with either sarin or PB alone resulted in sensorimotor impairments, while coexposure to high doses of sarin with PB caused an exacerbated deficit.