Maternal exposure to nicotine and chlorpyrifos,alone and in combination,leads to persistently elevated expression of glial fibrillary acidic protein in the cerebellum of the offspring in late puberty

We previously showed that maternal exposure to nicotine, alone or in combination with chlorpyrifos, caused an increase in glial fibrillary acidic protein (GFAP) immunostaining in the CA1 subfield of hippocampus and cerebellum in postnatal day (PND) 30 offspring. In the present study, PND 60 offspring were evaluated for histopathological and cholinergic effects following maternal exposure to nicotine and chlorpyrifos, alone and in combination. Timed-pregnant Sprague-Dawley rats (300–350 g) were treated daily with nicotine (1 mg/kg, s.c., in normal saline) or chlorpyrifos (0.1 mg/kg, dermal, in ethanol) or a combination of nicotine and chlorpyrifos from gestational days (GD) 4 to 20. Control animals were treated with saline and ethanol. On PND 60, the offspring were evaluated for cholinergic changes and pathological effects. Plasma butyrylcholinesterase (BChE) activity in the female offspring from chlorpyrifos treated mothers showed a significant increase (~183% of control). Male offspring from mothers treated with either chlorpyrifos or nicotine alone showed a significant increase in the acetylcholinesterase (AChE) activity in the brainstem while female offspring from mothers treated with either nicotine or a combination of nicotine and chlorpyrifos 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 42 and 7 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 in cerebellar white matter was observed in the offspring from the mothers treated with nicotine. These results suggest that maternal exposure to real-life levels of nicotine and/or chlorpyrifos causes differential regulation of brainstem AChE activity. Also, nicotine caused a decrease in the surviving neurons and an increased expression of GFAP in cerebellar white matter of the offspring on PND 60. These changes can lead to long-term neurological adverse health effects later in life.     

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.     

Maternal exposure of rats to nicotine via infusion during gestation produces neurobehavioral deficits and elevated expression of glial fibrillary acidic protein in the cerebellum and CA1 subfield in the offspring at puberty

Maternal smoking during pregnancy is known to be a significant contributor to developmental neurological health problems in the offspring. In animal studies, nicotine treatment via injection during gestation has been shown to produce episodic hypoxia in the developing fetus. Nicotine delivery via mini osmotic pump, while avoiding effects due to hypoxia-ischemia, it also provides a steady level of nicotine in the plasma. In the present study timed-pregnant Sprague-Dawley rats (300-350 g) were treated with nicotine (3.3 mg/kg, in bacteriostatic water via s.c. implantation of mini osmotic pump) from gestational days (GD) 4-20. Control animals were treated with bacteriostatic water via s.c. implantation of mini osmotic pump. Offspring on postnatal day (PND) 30 and 60, were evaluated for changes in the ligand binding for various types of nicotinic acetylcholine receptors and neuropathological alterations. Neurobehavioral evaluations for sensorimotor functions, beam-walk score, beam-walk time, incline plane and grip time response were carried out on PND 60 offspring. Beam-walk time and forepaw grip time showed significant impairments in both male and female offspring. Ligand binding densities for [3H]epibatidine, [3H]cytisine and [3H]alpha-bungarotoxin did not show any significant changes in nicotinic acetylcholine receptors subtypes in the cortex at PND 30 and 60. Histopathological evaluation using cresyl violet staining showed significant decrease in surviving Purkinje neurons in the cerebellum and a decrease in surviving neurons in the CA1 subfield of hippocampus on PND 30 and 60. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer of cerebellum and the CA1 subfield of hippocampus on PND 30 and 60 of both male and female offspring. These results indicate that maternal exposure to nicotine produces significant neurobehavioral deficits, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum and CA1 subfield of hippocampus of the offspring on PND 30 and 60. The results show that although 60-day-old male and female rat offspring of mothers exposed to nicotine during gestation did not differ from control in body weight gain or nicotinic acetylcholine receptors ligand binding, they exhibited significant sensorimotor deficits that were consistent with the neuropathological alterations seen in the brain. These neurobehavioral and pathological deficits indicate that maternal nicotine exposure may produce long-term adverse health effects in the offspring.     

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.     

Imidacloprid induces neurobehavioral deficits and increases expression of glial fibrillary acidic protein in the motor cortex and hippocampus in offspring rats following in utero exposure

Imidacloprid, a neonicotinoid, is one of the fastest growing insecticides in use worldwide because of its selectivity for insects. The potential for neurotoxicity following in utero exposure to imidacloprid is not known. Timed pregnant Sprague-Dawley rats (300-350 g) on d 9 of gestation were treated with a single intraperitoneal injection (i.p.) of imidacloprid (337 mg/kg, 0.75 x LD50, in corn oil). Control rats were treated with corn oil. On postnatal day (PND) 30, all male and female offspring were evaluated for (a) acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity, (b) ligand binding for nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (m2 mAChR), (c) sensorimotor performance (inclined plane, beam-walking, and forepaw grip), and (d) pathological alterations in the brain (using cresyl violet and glial fibrillary acidic protein [GFAP] immunostaining). The offspring of treated mothers exhibited significant sensorimotor impairments at PND 30 during behavioral assessments. These changes were associated with increased AChE activity in the midbrain, cortex and brainstem (125-145% increase) and in plasma (125% increase). Ligand binding densities for [3H]cytosine for alpha4beta2 type nAchR did not show any significant change, whereas [3H]AFDX 384, a ligand for m2mAChR, was significantly increased in the cortex of offspring (120-155% increase) of imidacloprid-treated mothers. Histopathological evaluation using cresyl violet staining did not show any alteration in surviving neurons in various brain regions. On the other hand, there was a rise in GFAP immunostaining in motor cortex layer III, CA1, CA3, and the dentate gyrus subfield of the hippocampus of offspring of imidacloprid-treated mothers. The results indicate that gestational exposure to a single large, nonlethal, dose of imidacloprid produces significant neurobehavioral deficits and an increased expression of GFAP in several brain regions of the offspring on PND 30, corresponding to a human early adolescent age. These changes may have long-term adverse health effects in the offspring.     

Effects of maternal intravenous nicotine administration on locomotor behavior in pre-weanling rats

Maternal tobacco use is associated with adverse developmental outcomes in offspring, including hyperactivity. Animal studies attempting to model this phenomenon have primarily used continuous s.c. nicotine infusion as the method of nicotine administration, which does not model the intermittent bolus delivery of nicotine associated with smoking in humans. The purpose of the present experiment was to examine the locomotor activity of pre-weanling offspring of pregnant rats exposed to an i.v. nicotine dosing protocol that approximates the pattern of nicotine exposure in moderate to heavy smokers. Pregnant rats were administered an i.v. bolus of 0.03 mg/kg nicotine (N=13) or saline (N=10) every 14 min for 16 h/day, resulting in a total daily dose of 2 mg/kg (base), from gestational day 4 to delivery. Pups from each litter were tested for spontaneous locomotor activity on postnatal days (PND) 19-21 and nicotine-induced locomotor activity on PND 22. Mean birth weight was significantly lower in nicotine-exposed pups compared to controls, but body weights were equivalent between groups by the time of behavioral testing. Mean total distance traveled, vertical counts, and stereotypy counts were lower on PND 19 in nicotine-exposed pups compared to controls, but only the difference in mean stereotypy counts was statistically significant. Within-session analysis revealed that both distance traveled and stereotypy were significantly decreased in nicotine-exposed pups in the first 5 min of the session on PND 19. Total time spent in the center of the field was also lower in nicotine-exposed pups. Nicotine-induced increases in activity on PND 22 did not differ according to gestational exposure. These findings demonstrate that prenatal nicotine exposure in a model that mimics the pattern of nicotine exposure from cigarette smoking in humans results in offspring that exhibit low birth weight and hypoactivity in a novel environment.     

Inhibition of cholinesterase enzymes following a single dermal dose of chlorpyrifos and methyl parathion, alone and in combination, in pregnant rats

Pregnant Sprague-Dawley rats (14-18 d of gestation) were treated with either a single dermal subclinical dose of 30 mg/kg (15% of dermal LD50) chlorpyrifos (O,O-diethyl-O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate) or a single dermal subclinical dose of 10 mg/kg (15% of dermal LD50) methyl parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate) or the two in combination. Chlorpyrifos inhibited maternal and fetal brain acetylcholinesterase (AChE) activity within 24 h of dosing, (48% and 67% of control activity, respectively). Following application of methyl parathion, peak inhibition of maternal and fetal brain AChE activity occurred at 48 h and 24 h after dosing (17% and 48% of control activity, respectively). A combination of chlorpyrifos and methyl parathion produced peak inhibition of maternal and fetal brain AChE activity at 24 h postdosing (35% and 73% of control activity, respectively). Maternal and fetal brain AChE activity recovered to various degrees of percentage of control 96 h after dosing. Application of methyl parathion or chlorpyrifos alone or in combination significantly inhibited maternal plasma butyrylcholinesterase (BuChE) activity. No significant inhibition of fetal plasma BuChE activity was detected. Peak inhibition of maternal liver BuChE occurred 24 h after application of methyl parathion or chlorpyrifos alone or in combination (64%, 80%, and 61% of control activity, respectively). Significant inhibition of placental AChE occurred within 24 h after application of methyl parathion or chlorpyrifos alone or in combination. The results suggest that methyl parathion and chlorpyrifos, alone or in combination, were rapidly distributed in maternal and fetal tissues, resulting in rapid inhibition of cholinesterase enzyme activities. The lower inhibitory effect of the combination could be due to competition between chlorpyrifos and methyl parathion for cytochrome P-450 enzymes, resulting in inhibition of the formation of the potent cholinesterase inhibitor oxon forms. The faster recovery of fetal plasma BuChE is attributed to the de novo synthesis of cholinesterase by fetal tissues compared to maternal tissues.     

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.     

Neonatal ethanol and nicotine exposure causes locomotor activity changes in preweanling animals

Sprague-Dawley rats were used to investigate the effects of neonatal ethanol (ETOH) and nicotine (NIC) exposure on activity levels in preweanling offspring. Male and female pups received daily oral intubations of ethanol ((ETOH) 5 g/kg/day), nicotine ((NIC) 12 mg/kg/day), ethanol and nicotine ((ETOH+NIC) 5 g/kg/day+12 mg/kg/day) or isocaloric maltose (control) on either postnatal days (PND) 1-7 or PND 8-14. A non-treated control group was also included. Peak blood ethanol concentrations (BECs) measured in a separate subset of animals ranged from 167 and 344 mg/dl depending upon neonatal treatment and period of exposure. Subjects were tested in an open field apparatus on PND 19-21. Animals exposed to ETOH or ETOH+NIC on PND 1-7 were hyperactive relative to the other treatment groups. In contrast, animals exposed to NIC or ETOH+NIC during PND 8-14 were hypoactive relative to other treatment groups. Males appeared more sensitive than females on measures of anxiety (distance traveled in the center of the open field) but this also varied dependent on neonatal treatment and period of exposure. These findings suggest that the third trimester is a critical period for ETOH and NIC effects on offspring activity although the pattern of effects on activity are different depending on when drug exposure occurred during the neonatal period.     

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.     

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.     

Developmental neurotoxicity of organophosphorous pesticides: fetal and neonatal exposure to chlorpyrifos alters sex-specific behaviors at adulthood in mice.

Developmental exposure to the organophosphorous insecticide chlorpyrifos (CPF) induces long-term effects on brain and behavior in laboratory rodents. We evaluated in adult mice the behavioral effects of either fetal and/or neonatal CPF exposure at doses not inhibiting fetal and neonatal brain cholinesterase. CPF (3 or 6 mg/kg) was given by oral treatment to pregnant females on gestational days 15-18 and offspring were treated sc (1 or 3 mg/kg) on postnatal days (PNDs) 11-14. Serum and brain acetylcholinesterase (AChE) activity was evaluated at birth and 24 h from termination of postnatal treatments. On PND 70, male mice were assessed for spontaneous motor activity in an open-field test and in a socioagonistic encounter with an unfamiliar conspecific. Virgin females underwent a maternal induction test following presentation of foster pups. Both sexes were subjected to a plus-maze test to evaluate exploration and anxiety levels. Gestational and postnatal CPF exposure (higher doses) affected motor activity in the open field and enhanced synergically agonistic behavior. Postnatal CPF exposure increased maternal responsiveness toward pups in females. Mice of both sexes exposed to postnatal CPF showed reduced anxiety response in the plus-maze, an effect greater in females. Altogether, developmental exposure to CPF at doses that do not cause brain AChE inhibition induces long-term alterations in sex-specific behavior patterns of the mouse species. Late neonatal exposure on PNDs 11-14 was the most effective in causing behavioral changes. These findings support the hypothesis that developmental CPF may represent a risk factor for increased vulnerability to neurodevelopmental disorders in humans.     

Tissue manganese concentrations in lactating rats and their offspring following combined in utero and lactation exposure to inhaled manganese sulfate.

There is little information regarding the tissue distribution of manganese in neonates following inhalation. This study determined tissue manganese concentrations in lactating CD rats and their offspring following manganese sulfate (MnSO4) aerosol inhalation. Except for the period of parturition, dams and their offspring were exposed to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m3) for 6 h/day, 7 days/week starting 28 days prior to breeding through postnatal day (PND) 18. Despite increased manganese concentrations in several maternal tissues, MnSO4 inhalation exposure did not affect body weight gain, terminal (PND 18) body weight, or organ weights in the dams. Exposure to MnSO4 at 1 mg Mn/m3 resulted in decreased pup body weights on PND 19 and decreased brain weights in some PND 14 to PND 45 pups. Exposure to MnSO4 at > or =0.05 mg Mn/m3 was associated with increased stomach content, blood, liver, and skull cap manganese concentrations in PND 1 pups, increased brain, lung, and femur manganese concentrations in PND 14 pups, and elevated olfactory bulb, cerebellum, and striatum manganese concentrations in PND 19 pups. When compared to controls, MnSO4 exposure to > or =0.5 mg Mn/m3 increased liver and blood manganese concentrations in PND 14 pups and increased liver, pancreas, and femur manganese concentrations in PND 19 pups. Manganese concentrations returned to control values in all offspring tissues by PND 45 +/- 1. Our data demonstrate that neonatal tissue manganese concentrations observed following MnSO4 inhalation are dependent on the MnSO4 exposure concentration and the age of the animal.     

The effects of perinatal exposure to nicotine on plasma LH levels in prepubertal rats

Adult female rats were chronically treated with nicotine administered via the drinking water during pregnancy and/or lactation. The approximate doses of nicotine consumed per day were 2.4 mg/kg and 4.5 mg/kg of body weight. The pups were weaned at 20 days of age. The pups were killed by decapitation on postnatal days 20, 30, or 40 and plasma from heparinized trunk blood was assayed for luteinizing hormone (LH). At 30 days of age untreated male and female offspring had the highest levels of plasma LH compared to 20 and 40 days of age. This level was not affected by any subsequent dose or treatment. Prepubertal females exposed to nicotine during pregnancy failed to exhibit the pattern of LH levels seen in control animals, whereas those exposed during lactation or throughout the perinatal period showed a distinctive pattern of plasma LH. Chronic exposure of female offspring to the low dose of nicotine during lactation tended to increase plasma LH levels at 20 and 40 days. Female offspring exposed to nicotine during pregnancy or to the low dose during lactation showed significant deficits in body weight at 40 days of age which appeared to correlate with a delay in vaginal opening. The results suggest that perinatal exposure to maternally administered nicotine may disrupt normal patterns of LH release in the offspring of both sexes and alter sexual development in female offspring.     

Pubertal exposure to Bisphenol A increases anxiety-like behavior and decreases acetylcholinesterase activity of hippocampus in adult male mice

The negative effects of Bisphenol A (BPA) on neurodevelopment and behaviors have been well established. Acetylcholinesterase (AChE) is a regulatory enzyme which is involved in anxiety-like behavior. This study investigated behavioral phenotypes and AChE activity in male mice following BPA exposure during puberty. On postnatal day (PND) 35, male mice were exposed to 50 mg BPA/kg diet per day for a period of 35 days. On PND71, a behavioral assay was performed using the elevated plus maze (EPM) and the light/dark test. In addition, AChE activity was measured in the prefrontal cortex, hypothalamus, cerebellum and hippocampus. Results from our behavioral phenotyping indicated that anxiety-like behavior was increased in mice exposed to BPA. AChE activity was significantly decreased in the hippocampus of mice with BPA compared to control mice, whereas no difference was found in the prefrontal cortex, hypothalamus and cerebellum. Our findings showed that pubertal BPA exposure increased anxiety-like behavior, which may be associated with decreased AChE activity of the hippocampus in adult male mice. Further studies are necessary to investigate the cholinergic signaling of the hippocampus in PBE induced anxiety-like behaviors.     

Neurobehavioral assessment of rats exposed to low doses of PCB126 and methyl mercury during development

Epidemiological and laboratory studies have suggested that polychlorinated biphenyls (PCBs) and methyl mercury (MeHg) may have additive or synergistic effects on CNS function. Aim of this study was to characterize the effects of exposure to low levels of MeHg (0.5mg/kgday in drinking water) and PCB126 (100ng/kgday in food), alone and in combination, on neurobehavioral development in Wistar rats. Dams were treated from gestational day 7 to post-natal day (PND) 21. Animals were tested for developmental landmarks and reflexes (PND1-21), attention deficits (PND40), locomotor activity (PND30, 110), spatial learning (PND75), coordination and balance (PND90), object discrimination (PND80), anxiety (PND100), and conditioned learning (PND110). Parameters related to pregnancy, sex ratio at birth, and physical development (at weaning) did not differ among groups, though PCB126 decreased number of pups at birth. A slight delay in negative geotaxis was found in female rats in all treatment groups. No significant effects were seen in attention, coordination and balance, object discrimination, and spatial and conditioned learning. Increased motor activity was present in PCB126-treated male and in MeHg+PCB-treated female rats in the elevated plus maze test, and in PCB126-treated male rats in the open field test (PND110). The results do not support the hypothesis that co-exposure to MeHg and PCB126 results in additive or synergistic effects. This finding is in agreement with more recent in vitro and in vivo studies.     

Neurobehavioral toxicity study of dibutyl phthalate on rats following in utero and lactational exposure

To investigate the neurobehavioral effects of dibutyl phthalate (DBP), an important endocrine disruptor known for reproductive toxicity, on rodent offspring following in utero and lactational exposure, pregnant Wistar rats were treated with DBP (0, 0.037, 0.111, 0.333 and 1% in the diet) from gestation day (GD) 6 to postnatal day (PND) 28, and selected developmental and neurobehavioral parameters of the offspring were measured. There were no significant effects of DBP on body weight gain of the dams during GD 6–20 or on the pups' ages of pinna detachment, incisor eruption or eye opening. Exposure to 1% DBP prolonged gestation period, decreased body weight in both male and female pups, depressed surface righting (PND 7) in male pups, shortened forepaw grip time (PND 10), enhanced spatial learning and reference memory (PND 35) in male pups. Exposure to 0.037% DBP also shortened forepaw grip time (PND 10), but inhibited spatial learning and reference memory in male pups. Sex × treatment effects were found in forepaw grip time (PND 10), spatial learning and reference memory, and the male pups appeared to be more susceptible than the females. However, all levels of DBP exposure did not significantly alter surface righting (PND 4), air righting (PND 16), negative geotaxis (PND 4 or 7), cliff avoidance (PND 7) or open field behavior (PND 28) in either sex. Overall, the dose level of DBP in the present study produced a few adverse effects on the neurobehavioral parameters, and it may alter cognitive abilities of the male rodent. Copyright © 2009 John Wiley & Sons, Ltd.     

Developmental toxicity of toluene in male rats: effects on semen quality, testis morphology, and apoptotic neurodegeneration

In one study, pregnant Wistar rats were exposed to 1200 ppm toluene by inhalation 6 h a day from gestational day (GD) 7 to postnatal day (PND) 18. Sperm analysis was performed in the adult male offspring at PND 110 by using computer-assisted sperm analysis. Toluene did not affect the semen quality of exposed rats. In another study, pregnant rats were exposed to 1800 ppm from GD 7 to GD 20, and the male offspring were killed at PND 11, 21 or 90. Paired testes weight, histopathology and immunoexpression of vimentin in Sertoli cells were used as markers of testis toxicity. In the brain, the number of apoptotic cells in the hippocampus and cerebellum were counted after visualisation by means of the TUNEL assay. Mean body weight in pups of exposed dams was lower than in pups from control litters. This decrease was still statistically significant at PND 11, but at PND 21 and 90 the body weight of toluene-exposed males tended to approach that of the controls. Absolute and relative testes weights were reduced in all three age groups, although not to a statistically significant degree. Histopathological examinations of the testis and immuno-expression of vimentin did not reveal any differences between toluene-exposed animals and control animals. In the hippocampus, almost no apoptosis was observed in any age group, and there were no differences in apoptotic neurodegeneration between male rats exposed to 1800 ppm and control animals at PND 11, 21 or 90. Generally, a marked increase in number of apoptotic cells was observed in cerebellar granule cells at PND 21 compared with the other age groups. Toluene induced a statistically significant increase in the number of apoptotic cells in the cerebellar granule layer at PND 21. The mean was increased from 37 in the control group to 71 in the toluene-exposed group. Thus, the granular cell layer in cerebellum is a highly relevant tissue with which to study toluene-induced apoptosis, because of the continuous migration of neurons and high frequency of neuronal apoptosis during the weaning period. In summary, it is concluded, that neither pre- and postnatal exposure to 1200 ppm toluene nor prenatal exposure to 1800 ppm induced significant effects on the reproductive parameters investigated. However, prenatal exposure to 1800 ppm toluene did increase neuronal apoptosis in the cerebellum of weaned male rats, possibly by delaying postnatal migration of granule cells to their final destination, or by toluene-induced retardation of generalised fetal growth.     

Long-term effects of postnatal aluminium exposure on acetylcholinesterase activity and biogenic amine neurotransmitters in rat brain

The long-term effects of early postnatal exposure to aluminium on acetyl choline esterase (AChE) activity and on biogenic amines were studied in different brain regions. The subjects were eight days old male Wistar rat pups. They were grouped into normal control and aluminium exposed groups. For aluminium exposure, the pups were gastric intubated with aluminium chloride (40 mg/Kg body weight) for two weeks. Control rats were given equal volumes of distilled water. After the treatment, they were rehabilitated for forty days. On the sixtieth day, the rats from both the groups were sacrificed and AChE activity, levels of dopamine, noradrenaline and serotonin were estimated in the cerebral cortex, hippocampus, septum, brainstem and striatum. In the aluminium exposed group: the AChE activity was significantly decreased in the hippocampus, septum, striatum and brainstem; serotonin levels were reduced by 20% in the cortex, hippocampus, septum and striatum; in brain stem, the serotonin level was decreased by 40%. A 60% reduction in noradrenaline levels was observed in the striatum whereas it was reduced by 25% in other regions except in hippocampus. Though dopamine levels were not altered in the cortex, septum and brainstem, they were reduced by 40% in the striatum. The study documents the long-term consequences of exposure to aluminium during the developmental periods.     

Effects of repeated oral postnatal exposure to chlorpyrifos on open-field behavior in juvenile rats.

Organophosphorus (OP) insecticides have the potential to cause behavioral effects in children. This study was designed to determine if repeated oral exposure of preweanling rats to chlorpyrifos would produce behavioral changes at both pre- and postweanling ages. Treatment occurred every second day beginning on post-natal day (PND) 1, and continued through PND 21. The rats received one of the following regimens: a low-dosage (3 mg/kg) from PND 1-21; a medium dosage (mg/kg from PND 1-5, and then 6 mg/kg from PND 7-21; or a high-dosage schedule of 3 mg/kg on PND 1-5, then 6 mg/kg from PND 7-13, and 12 mg/kg from PND 15-21. There were no differences in body weights among the control-, low-, and medium-dosage groups but the high-dosage group had significantly lower body weights on PND 13-21. An open field was used to measure locomotor activity on PND 10, 12, 14, 16, 18, 20, 25, and 30. There were no differences in locomotor activity levels or treatment effects between males and females. On PND 10, 12, 14, 16, 18, and 20 there was no effect on locomotor activity with any dosage. On days 25 and 30, locomotor activity was significantly decreased with the medium- and high-dosage groups. Brain cholinesterase (ChE) inhibition was about 25-38% on PND 25 and 14-34% on PND 30. On PND 25 but not 30, lung and diaphragm ChE and serum butyrylcholinesterase (BChE), with the high-dosage animals, and heart ChE with the medium- and high-dosage groups were significantly inhibited. There was no significant inhibition of skeletal muscle ChE or serum acetylcholinesterase (AChE) on PND 25 and 30. These data suggest that early postnatal chlorpyrifos exposures will depress locomotor activity in juvenile rats, with the effects most pronounced after brain ChE activity has substantially recovered.