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Extramural Research

Final Report: Developmental Neurotoxicity in Offspring Induced by Combined Maternal Exposure of Rats to Nicotine and Chlorpyrifos

EPA Grant Number: R829399
Title: Developmental Neurotoxicity in Offspring Induced by Combined Maternal Exposure of Rats to Nicotine and Chlorpyrifos
Investigators: Abou-Donia, Mohamed B.
Institution: Duke University Medical Center
EPA Project Officer: Laessig, Susan A.
Project Period: October 1, 2001 through September 30, 2004
Project Amount: $750,000
RFA: Children's Vulnerability to Toxic Substances in the Environment (2001)
Research Category: Children's Health , Health Effects , Human Health

Description:

Objective:

The objective of this research project was to test the hypothesis that combined exposure to nicotine and chlorpyrifos (CPF) during the critical periods of development of cholinergic pathways in the central nervous system disrupts the structural organization of the cholinergic system and interferes with the cholinergic transmission, resulting in neurologic deficits such as impairments in learning and memory performance.

Summary/Accomplishments (Outputs/Outcomes):

The following has been accomplished during the 3-year funding period.

Year 1

The neurotoxic effects in the offspring on postnatal day (PND) 30 were studied following maternal exposure to nicotine and CPF, alone and in combination. Female Sprague-Dawley rats (300-350 gm) with known pregnancy dates were treated daily with nicotine (1mg/kg, s.c., in normal saline) or CPF (0.1 mg/kg, dermal, in ethanol) or a combination of nicotine and CPF for the gestational days (GD) 4-20. Control animals were treated with saline and ethanol. Following parturition, the body weight and number of litters were recorded. There was no significant difference between the litter size or body weight of the offspring on PNDs 2, 9, and 16 between the control and treated groups. Following completion of the lactational period (PND 23), the animals were weighed according to sex. Male offspring from the mothers treated with nicotine alone gained significantly less weight by PND 30 compared to the control. On PND 7, there was a significant increase in the brain acetylcholinesterase (AChE) activity in the pups from the nicotine and CPF group, whereas plasma butyrylcholinesterase (BChE) activity showed a significant increase (~167 and 176% of control) in the pups from the mothers treated with either CPF alone or in combination with nicotine, respectively. There was no change in the ligand binding for muscarinic or nicotinic acetylcholine receptors in the whole brains of PND 7 day offspring from any of the treatment groups. On PND 30, male offspring showed a significant increase in the AChE activity in the brainstem (~134-148 % of control) and cerebellum (~299-345 % of control) in all the treated groups. Females on PND 30 showed a significant increase in the AChE activity in the brainstem of the CPF-alone group and in the cerebellum of the pups from the combination of nicotine and CPF. There was no change in the plasma BChE activity of male or female pups on PND 30. Histopathological evaluation by H&E staining did not show any gross pathological abnormalities. A significant increase in the immunostaining for glial fibrillary acidic protein (GFAP) was observed in the cortex, the CA1 and CA3 subfields of hippocampus, and dentate gyrus on PND 30 in the pups from nicotine- and CPF-treated mothers. These data suggest that maternal exposure during the entire gestational period with low doses of nicotine and CPF, alone and in combination, does not lead to any significant observable developmental abnormalities in the offspring by PND 30.

Year 2

During this period, the neurotoxic effects in the offspring on PND 60 were studied following maternal exposure to nicotine and CPF, alone and in combination. Female Sprague-Dawley rats (300-350 gm) with known pregnancy dates were treated daily with nicotine (1mg/kg, s.c., in normal saline) or CPF (0.1 mg/kg, dermal, in ethanol) or a combination of nicotine and CPF from GD 4-20. Control animals were treated with saline and ethanol. On PND 60, the offspring were evaluated for cholinergic changes and pathological effects. Plasma BChE activity in the female offspring from CPF-treated mothers showed a significant increase (~183% of control). Male offspring from mothers treated with either CPF or nicotine alone showed a significant increase in the AChE activity in the brainstem, whereas female offspring from mothers treated with either nicotine or a combination of nicotine and CPF showed a significant increase (~134 and 126 % of control, respectively) in AChE activity in the brainstem. No significant changes were observed in the ligand binding densities for a4 b2 and a7 nicotinic acetylcholine receptors in the cortex. Histopathological evaluation using cresyl violet staining showed a significant decrease in surviving Purkinje neurons in the cerebellum of the offspring from nicotine-treated mothers. An increase in GFAP immunostaining was observed in the cerebellum of the offspring from the mothers treated with nicotine. The results of these studies suggest that maternal exposure to real-life levels of nicotine and/or CPF causes differential regulation of brainstem AChE activity. Nicotine also caused a decrease in the surviving neurons and an increased expression of GFAP in cerebellum of the offspring on PND 60.

Year 3

During the last and final year, the neurochemical, neurobehavioral, and neuropathological studies were carried out on PND 90 offspring following maternal exposure to nicotine and CPF, alone and in combination. Timed pregnant Sprague-Dawley rats (300-350g) were treated daily with nicotine (3.3mg/kg, in bacteriostatic water via subcutaneous implantation of mini-osmotic pump), CPF (1.0 mg/kg, dermal, ethanol) or a combination of nicotine and CPF on GD 4-20. Control animals were treated with bacteriostatic water via subcutaneous implantation of mini-osmotic pump and dermal application of ethanol. Offspring on PND 90 were evaluated for neurobehavioral performance, changes in the ligand binding for a7 and a4 b2 nicotinic acetylcholine receptors, and neuropathological alterations in the cerebellum. Beam-walk time, incline plane, and forepaw grip strength showed significant impairments in both male and female offspring from mothers treated with nicotine and CPF, alone or in combination. Male offspring showed greater deficits in behavioral performance than the female offspring. Female offspring from mothers treated with a combination of nicotine and CPF showed a significant increase in plasma BChE activity. Brain regional AChE activity showed differential changes in male and female offspring. Brainstem and cerebellum of female offspring from mothers treated with nicotine or CPF, alone or in combination, showed a significant increase, whereas brainstem of male offspring from mothers treated with nicotine alone or a combination of nicotine and CPF showed a significant increase in AChE. Histopathological evaluations using cresyl violet staining showed a significant decrease in surviving Purkinje neurons in the cerebellum. An increase in GFAP immuno-staining was observed in cerebellum white matter as well as granular cell layer of cerebellum. These data suggest that maternal exposure to nicotine and CPF, alone and in combination, produces neurobehavioral deficits in male and female offspring, a decrease in the surviving neurons, and an increased expression of GFAP in the cerebellum in the offspring at PND 90.

Conclusions:

There is an increasing concern about developmental neurotoxicity following exposure to insecticides and other neurotoxins, such as nicotine. Both CPF and nicotine are developmental neurotoxic agents that primarily affect the cholinergic pathways. Maternal smoking could produce an added toxic burden to the health of the children born to smoking mothers because of concurrent exposure to CPF and nicotine during fetal development. In the present study, we evaluated the neurotoxicity in the offspring at PND 90 following maternal exposure to nicotine and CPF, alone and in combination, during the gestation days. The doses and route of exposure to both chemicals were selected to approximate real-life scenarios. The nicotine dose selected in our studies in rats produces plasma levels of nicotine similar to moderate smoking (~0.5-1 packs/day) by human smokers. CPF exposure was through dermal application at 1 mg/kg, which lies below the threshold to produce any fetal abnormalities. The findings of the present study show that maternal exposure to nicotine, alone or in combination with CPF, produces deficits in beam-walk time and forepaw grip in adult male offspring, whereas the female offspring showed significant deficits in beam-walk time following maternal exposure to nicotine alone. There was a significant increase in AChE activity in brainstem following exposure to nicotine, alone and in combination with CPF, in both male and female offspring. Neuropathological alterations in adult male and female offspring are characterized by a significant decrease in the number of surviving Purkinje neurons and an increase in expression of GFAP in the cerebellum, granular cell layer, and white matter following maternal treatment with nicotine and CPF, alone and in combination. These results suggest that maternal exposure to real-life levels of nicotine and/or CPF produce neurobehavioral deficits and differential regulation of brainstem AChE activity in adult offspring. Maternal exposure to these chemicals also caused a decrease in the surviving neurons in certain brain regions. These changes may be important in assessing long-term neurological adverse health effects in human populations.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 9 publications 3 publications in selected types All 3 journal articles

Type Citation Project Document Sources
Journal Article Abdel-Rahman A, Dechkovskaia A, Mehta-Simmons H, Guan X, Khan W, Abou-Donia M. Increased expression of glial fibrillary acidic protein in cerebellum and hippocampus: differential effects on neonatal brain regional acetylcholinesterase following maternal exposure to combined chlorpyrifos and nicotine. Journal of Toxicology and Environmental Health, Part A 2003;66(21):2047-2066. R829399 (2003)
R829399 (2004)
R829399 (Final)
  • Abstract from PubMed
  • Journal Article Abdel-Rahman A, Dechkovskaia AM, Mehta-Simmons H, Sutton JM, Guan X, Khan WA, Abou-Donia MB. Maternal exposure to nicotine and chlorpyrifos, alone and in combination leads to persistently elevated expression of glial fibrilary acidic protein in the cerebellum of the offspring at late puberty. Archives of Toxicology 2004;78(8):467-476. R829399 (2004)
    R829399 (Final)
  • Abstract from PubMed
  • Journal Article Abou-Donia MB, Abdel-Rahman A, Goldstein LB, Dechkovskaia AM, Shah DU, Bullman SL, Khan WA. Sensorimotor deficits and increased brain nicotinic acetylcholine receptors following exposure to chlorpyrifos and/or nicotine in rats. Archives of Toxicology 2003;77(8):452-458. R829399 (2002)
    R829399 (2003)
    R829399 (2004)
    R829399 (Final)
  • Abstract from PubMed
  • Full-text: SpringerLink Full Text
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  • Supplemental Keywords:

    risk assessment, indoor, infants, neuropathological, vulnerable, pregnancy, chlorpyrifos, nicotine, acetylcholinesterase, nicotinic acetylcholine receptor, combined exposure, smoking, glial fibrillary acidic protein, cerebellum,, RFA, Health, Scientific Discipline, Toxicology, Health Risk Assessment, Susceptibility/Sensitive Population/Genetic Susceptibility, Children's Health, genetic susceptability, sensitive populations, infants, vulnerability, developmental neurotoxicity, chlorpyrifos, children, neurotoxicity, assessment of exposure, indoor air, cigarette smoke, Nicotine, pregnancy, tobacco smoke, toxics, maternal exposure, developmental toxicants

    Progress and Final Reports:
    Original Abstract
    2002 Progress Report
    2003 Progress Report
    2004 Progress Report

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