Neonatal Chlorpyrifos Exposure Research Articles

Persistent alterations in seizure susceptibility, drug responsiveness and comorbidities associated with chemical kindling after neonatal exposure to an organophosphate

Developmental exposure to organophosphates (OPs), at doses that do not cause cholinergic crisis, induces profound and lasting alterations in different neurotransmitter systems, which contribute to several behavioral outcomes. The present work examines whether neonatal exposure to low dose of chlorpyrifos (CPF), a widely used OP insecticide, alters the general excitability of the adult brain, its responsiveness to drugs with antiepileptic properties, the process of chemical kindling and the kindling-induced behavioral outcomes. Neonatal rats were exposed to daily doses of CPF (1 mg/kg) or dimethyl sulfoxide (DMSO, vehicle) on postnatal days (PND) 1–4. On PND 60, a subgroup of animals from both CPF and DMSO groups were injected with additive doses of pentylenetetrazole (PTZ) to evaluate the latency time to the first seizure, the threshold of PTZ-induced convulsion, and to determine the anticonvulsive action of phenobarbital (20 mg/kg), ethosuximide (100 mg/kg) and scopolamine (0.6 mg/kg) when used as pretreatment. Rats in the other subgroups were kindled by repeated intraperitoneal injections of an initially subconvulsive dose of PTZ (37.5 mg/kg) at 48-h intervals for 4 weeks. Kindled rats were then subjected to radial arm maze, sweet taste preference and forced swim test. Neonatal exposure to CPF shortened the latency time to the first seizure after pretreatment with scopolamine in female rats and decreased the threshold for PTZ-induced clonic convulsions after phenobarbital pretreatment in male rats. Neonatal CPF exposure also decreased the rate of kindling progression in female rats during early stages of PTZ kindling. On the other hand, CPF exposure sex-selectively reduced the number of working memory errors after kindling only in male rats. Drug challenge with MK-801 induced more impairment in the working memory of female kindled rats, indicating more dependence of working memory on NMDA receptor activity in these animals. Female kindled rats from CPF exposed group also showed longer time of immobility in forced swim test, showing an increase in the depressive-like behavior. This difference was also observed in the second session of forced swim test, after treating with fluoxetine, a selective inhibitor of serotonin reuptake. The recent finding, together with lack of difference in the sweet taste preference, suggests that mechanism beyond the reduction of serotonergic activity underlie the increased depressive-like behavior in this animals. To our knowledge, this is the first report describing the potential contribution of developmental exposure to an OP in susceptibility to antiepileptic drug resistance and alteration of seizure-induced behavioral deficits.

Neonatal exposure to chlorpyrifos affects maternal responses and maternal aggression of female mice in adulthood

CD-1 mice were exposed to the organophosphate pesticide chlorpyrifos (CPF) throughout postnatal days (PND) 11–14 at the subtoxic dose of 3 mg/kg. At adolescent age, females and males underwent a sociability test in which level of sociability and social preference were measured. At adulthood only females' behavior was analyzed. Maternal behavior of CPF-exposed females was assessed on postpartum day 1 after removal of the pups for 1 h, while anxiety levels were measured in a 5 min dark–light test on postpartum day 2. Nest defense response to an unfamiliar male intruder was assessed on postpartum day 7. In addition, from birth to postpartum day 7 a detailed analysis of nest building activity was carried out. Neonatal CPF exposure does not interfere with social behavior and social preferences at adolescence, whereas at adulthood it induces significant behavioral alterations in lactating females. Motivation to build and defend the nest was decreased in CPF females that were also less anxious than controls in the dark–light paradigm. These results confirm that developmental exposure to CPF induces long-lasting alterations in selected sexual-dimorphic responses of the adult social repertoire, and suggest that early exposure to CPF might interfere with hypothalamic neuroendocrine mechanisms regulating social responses.

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.

The alterations in CNS serotonergic mechanisms caused by neonatal chlorpyrifos exposure are permanent

Fetal or neonatal exposure to chlorpyrifos (CPF) or related organophosphate pesticides leads to abnormalities of brain cell development, synaptic function, and behavior. Recent studies in rats indicate profound effects on serotonin (5HT) systems that originate during CPF exposure and that are still present at 2 months posttreatment in the young adult. To determine if these changes are permanent, we administered 1 mg/kg of CPF daily to neonatal rats on postnatal days 1–4, a regimen devoid of systemic toxicity, and examined 5HT synaptic markers at 5 months of age: radioligand binding to 5HT 1A and 5HT 2 receptors and to the 5HT transporter. There were global elevations in all three synaptic proteins, with pronounced sex selectivity (effects on males > females) and a regional hierarchy of effects, viz. striatum > midbrain ≈ brainstem > cerebral cortex. Because there is a normal sex disparity for 5HT synaptic proteins, with females having higher values than males, the increase caused by CPF exposure in males completely eliminated this difference. Our findings at 5 months of age replicate those seen in young adulthood and strongly suggest that the effects of neonatal CPF exposure on 5HT systems are permanent.

Developmental Exposure of Rats to Chlorpyrifos Elicits Sex-Selective Hyperlipidemia and Hyperinsulinemia in Adulthood

Developmental exposure to chlorpyrifos alters cell signaling both in the brain and in peripheral tissues, affecting the responses to a variety of neurotransmitters and hormones. We administered 1 mg/kg/day chlorpyrifos to rats on postnatal days 1–4, a regimen below the threshold for systemic toxicity. When tested in adulthood, chlorpyrifos-exposed animals displayed elevations in plasma cholesterol and triglycerides, without underlying alterations in nonesterified free fatty acids and glycerol. This effect was restricted to males. Similarly, in the postprandial state, male rats showed hyperinsulinemia in the face of normal circulating glucose levels but demonstrated appropriate reduction of circulating insulin concentrations after fasting. These outcomes and sex selectivity resemble earlier findings at the cellular level, which identified hepatic hyperresponsiveness to gluconeogenic inputs from β-adrenoceptors or glucagon receptors. Our results thus indicate that apparently subtoxic neonatal chlorpyrifos exposure, devoid of effects on viability or growth but within the parameters of human fetal or neonatal exposures, produce a metabolic pattern for plasma lipids and insulin that resembles the major adult risk factors for atherosclerosis and type 2 diabetes mellitus.

Alterations in Central Nervous System Serotonergic and Dopaminergic Synaptic Activity in Adulthood after Prenatal or Neonatal Chlorpyrifos Exposure

Exposure to chlorpyrifos (CPF) alters neuronal development of serotonin (5HT) and dopamine systems, and we recently found long-term alterations in behaviors related to 5HT function. To characterize the synaptic mechanisms underlying these effects, we exposed developing rats to CPF regimens below the threshold for systemic toxicity, in three treatment windows: gestational days (GD) 17–20, postnatal days (PN) 1–4, or PN11–14. In early adulthood (PN60), we assessed basal neurotransmitter content and synaptic activity (turnover) in brain regions containing the major 5HT and dopamine projections. CPF exposure on GD17–20 or PN1–4 evoked long-term increases in 5HT turnover across multiple regions; the effects were not secondary to changes in neurotransmitter content, which was unaffected or even decreased. When the treatment window was shifted to PN11–14, there were no long-term effects. Dopamine turnover also showed significant increases after CPF exposure on GD17–20, but only when the dose was raised above the threshold for overt toxicity; however, hippocampal dopamine content was profoundly subnormal after exposures below or above the acute, toxic threshold, suggesting outright neurotoxicity. These results indicate that, in a critical developmental period, apparently nontoxic exposures to CPF produce lasting activation of 5HT systems in association with 5HT-associated behavioral anomalies.

Quantitative morphological assessment reveals neuronal and glial deficits in hippocampus after a brief subtoxic exposure to chlorpyrifos in neonatal rats

Neurochemical and behavioral studies indicate that the widely used organophosphorus insecticide, chlorpyrifos (CPF), evokes neurobehavioral teratogenicity with a wide window of vulnerability, ranging from embryonic life through postnatal development. Few studies have detailed morphological damage that corresponds to the operational deficits. We administered 5 mg/kg of CPF sc daily on postnatal days (PN) 11–14, a regimen that is devoid of systemic toxicity, but that elicits long-term cognitive impairment and disruption of cholinergic, catecholaminergic, and serotonergic synaptic function. On PN15 and 20, we conducted quantitative morphologic examinations of neurons and glia in CA1, CA3, and dentate gyrus regions of the hippocampus. Although hippocampal morphology after CPF exposure was normal on gross observation, morphometric analysis revealed a significant overall reduction in the total number of neurons and glia. Superimposed on this basic effect, CPF elicited a delayed-onset increase in the neuron/glia ratio that emerged by PN20, connoting selective gliotoxicity. The alterations in cell numbers were accompanied by significant perikaryal swelling and by enhanced development of astrocytic processes. Layer thickness also showed delayed-onset effects of CPF, with thinning of the CA1 and CA3 layers and enlargement of the dentate gyrus. Our results indicate that there are subtle morphological changes in the juvenile rat brain after neonatal CPF exposure that are detectable only with quantitative analysis and that correlate with regional and cell-specific targets identified earlier in neurochemical studies. The simultaneous targeting of neurons and glia by CPF is likely to play an important role in its developmental neurotoxicant effects.

Morphologic effects of subtoxic neonatal chlorpyrifos exposure in developing rat brain: regionally selective alterations in neurons and glia

The widely used organophosphate insecticide, chlorpyrifos (CPF), elicits neurobehavioral teratogenesis with exposure windows ranging from the embryonic neural tube stage through postnatal development. To explore the morphologic changes occurring in late-stage exposure, newborn rats were given 5 mg/kg of CPF s.c. daily on postnatal days (PN) 11–14, a regimen that is devoid of systemic toxicity, but that elicits long-term cognitive impairment. On PN15 and 20, we examined the septal nucleus, striatum and somatosensory cortex. Across all three regions, CPF elicited a significant decrease in the number of glial cells. Superimposed on this basic pattern, there were region-specific alterations in the number and type of neurons, and neuronal perikaryal dimensions. In the septal nucleus, the CPF group exhibited an increase in the number of neurons on PN20, representing a delay in the normal maturational decline; there was a parallel decrease in the glial/neuronal ratio. In the striatum, the number of neurons per unit area was reduced in the CPF group, accompanied by perikaryal hypertrophy, as evidenced by an increase in the average neuronal cell diameter. In the somatosensory cortex, the distribution of cell sizes indicated a decrease in the proportion of small, nonpyramidal cells. Thus, there are subtle morphological changes in the juvenile rat brain after neonatal CPF exposure that are detectable with quantitative analysis and that correlate with later emergence of behavioral alterations. Furthermore, the current findings support the hypothesis that CPF interferes with gliogenesis, a relatively late event in brain development; accordingly, the vulnerable period for adverse effects of CPF is likely to extend into childhood or adolescence.

Behavioral alterations in adolescent and adult rats caused by a brief subtoxic exposure to chlorpyrifos during neurulation

The widely used organophosphate insecticide, chlorpyrifos (CPF), elicits neurobehavioral abnormalities after apparently subtoxic neonatal exposures. In the current study, we administered 1 or 5 mg/kg/day of CPF to pregnant rats on gestational days 9–12, the embryonic phase spanning formation and closure of the neural tube. Although there were no effects on growth or viability, offspring showed behavioral abnormalities when tested in adolescence and adulthood. In the CPF-exposed groups, locomotor hyperactivity was noted in early T-maze trials, and in the elevated plus-maze; alterations in the rate of habituation were also identified. Learning and memory were adversely affected, as assessed using the 16-arm radial maze. Although all CPF-exposed animals eventually learned the task, reference and working memory were impaired in the early training sessions. After training, rats in the CPF group did not show the characteristic amnestic effect of scopolamine, a muscarinic acetylcholine antagonist, suggesting that, unlike the situation in the control group, muscarinic pathways were not used to solve the maze. These results indicate that apparently subtoxic CPF exposure during neurulation adversely affects brain development, leading to behavioral anomalies that selectively include impairment of cholinergic circuits used in learning and memory. The resemblance of these findings to those of late gestational or neonatal CPF exposure indicates a prolonged window of vulnerability of brain development to CPF.

Functional alterations in CNS catecholamine systems in adolescence and adulthood after neonatal chlorpyrifos exposure

Chlorpyrifos (CPF), one of the most widely used pesticides, is a neurobehavioral teratogen in animals. We administered CPF to neonatal rats on postnatal days (PN) 1–4 (1 mg/kg) or PN11–14 (5 mg/kg), regimens devoid of overt systemic toxicity. We then examined the impact on catecholaminergic systems in adolescence (PN30) and adulthood (PN60), assessing basal neurotransmitter content and transmitter utilization rates (turnover) in brain regions comprising the major noradrenergic and dopaminergic projections. Although CPF had only sporadic effects on basal norepinephrine and dopamine content, it profoundly suppressed norepinephrine turnover across multiple regions, indicative of net reductions in presynaptic activity. Dopamine turnover showed less consistent effects, with subnormal turnover in some regions and activation in others. We also evaluated whether CPF exposure altered the ability of catecholamine systems to respond to acute cholinergic stimulation, elicited by administration of a single challenge dose of nicotine. In the normal brain, nicotine increases the utilization of norepinephrine and dopamine. With only a few exceptions, animals receiving neonatal CPF exposure showed lasting desensitization of the nicotine response; not only was the activation by nicotine blunted in the CPF group, but in some regions the nicotine response was reversed, eliciting a reduction in transmitter turnover. These results indicate that neonatal CPF exposure produces widespread deficiencies in catecholaminergic synaptic function that persist into adulthood, and that are best revealed by dynamic measures of synaptic activity and responsiveness, as opposed to static markers like basal transmitter levels. The effects seen here are likely to contribute to alterations in behavioral performance that persist or emerge long after the termination of CPF exposure.

Alterations in serotonin transporter expression in brain regions of rats exposed neonatally to chlorpyrifos

Chlorpyrifos (CPF), one of the most widely-used organophosphate pesticides, is a suspected neuroteratogen. We administered CPF to neonatal rats on postnatal days (PN) 1–4 (1 mg/kg) or PN11–14 (5 mg/kg), treatments devoid of overt toxicity. At the end of the treatment period (PN5 and 15, respectively) and 5–7 days later, we then examined the effects on paroxetine (PXT) binding to the presynaptic 5HT high-affinity transporter, a marker for serotonin (5HT) projections. In males, we found a persistent decrease in PXT binding across the two different treatment regimens, with deficits apparent in a brain region containing 5HT terminal fields (forebrain) as well as in a region containing 5HT cell bodies (brainstem). In contrast, females given the early treatment regimen (PN1–4) showed deficits in the brainstem but transient elevations in the forebrain; the later treatment regimen (PN11–14) had no significant effect on PXT binding in females. These data are consistent with earlier work showing brainstem cell injury resulting from neonatal CPF exposure, and indicate specific damage to 5HT neurons, with a consequent loss of transporter expression in both terminal fields and perikarya. In females, the damage may be temporarily offset by initial trophic effects in the terminal region, consequent to the cholinergic stimulation evoked by cholinesterase inhibition via the active metabolite, CPF oxon. The gender-selective effects on 5HT systems are likely to contribute to similar gender dimorphism in behavioral performance. Because the CPF effects involve 5HT, a neurotransmitter intimately involved in the control of mood, we suggest the need to evaluate behaviors that typify animal models of depression.

Persistent cholinergic presynaptic deficits after neonatal chlorpyrifos exposure

The commonly-used organophosphate insecticide, chlorpyrifos (CPF), impairs brain cell development, axonogenesis and synaptogenesis. In the current study, we administered CPF to neonatal rats on postnatal (PN) days 1–4 (1 mg/kg) or PN11–14 (5 mg/kg), treatments that were devoid of overt toxicity. We then examined two cholinergic synaptic markers, choline acetyltransferase activity (ChAT) and [ 3H]hemicholinium-3 binding (HC-3) in the hippocampus, midbrain, striatum, brainstem and cerebral cortex in the juvenile (PN30) and young adult (PN60). Across all brain regions, CPF exposure evoked significant reductions in both markers, with larger effects on HC-3 binding, which is responsive to neuronal impulse activity, than on ChAT, a constitutive marker. Superimposed on the deficits, there were gender-selective effects and distinct regional disparities in the critical exposure period for vulnerability. In the hippocampus, either the early or late treatment regimen evoked decreases in ChAT but the early regimen elicited a much larger decrease in HC-3; effects persisted into adulthood. In the midbrain, CPF administration on PN1–4 elicited deficits similar to those seen in the hippocampus; however, exposure on PN11–14 elicited changes preferentially in females. Gender selectivity was also apparent in the striatum, in this case reflecting deficits in females after CPF treatment on PN1–4. In contrast, the effects of CPF on the brainstem were relatively more robust in males; effects in the cerebral cortex were less notable than in other regions. These results indicate that neonatal CPF exposure produces widespread deficiencies in cholinergic synaptic function that persist into adulthood. The effects are likely to contribute to gender-selective alterations in behavioral performance that persist or emerge long after the termination of exposure and well after the restoration of cholinesterase activity.

Neonatal chlorpyrifos exposure targets multiple proteins governing the hepatic adenylyl cyclase signaling cascade: implications for neurotoxicity

Chlorpyrifos has been hypothesized to interact with receptors and transduction proteins involved in the production of cyclic AMP, contributing to adverse effects on cell replication and differentiation. We studied the effects of neonatal chlorpyrifos exposure on hepatic adenylyl cyclase (AC) activity, as the liver accumulates the highest concentrations of chlorpyrifos and is the site for generation of its active metabolite, chlorpyrifos oxon. Newborn rats were given 1 mg/kg of chlorpyrifos s.c. on PN1-4. On PN5, 24 h after the last dose, AC catalytic activity was induced as assessed by the response to the direct AC stimulant, Mn 2+. In contrast, AC activation dependent upon interaction of the enzyme with G-proteins (forskolin) did not show any enhancement, suggesting impairment of G-protein function. This conclusion was confirmed by impaired responsiveness to fluoride, which directly activates G-proteins. In addition, the response of AC to hormonal signals was altered in a receptor-selective manner, with an enhanced response to glucagon but not to the β-adrenoceptor agonist, isoproterenol. The effects of chlorpyrifos on AC signaling displayed a critical developmental period of vulnerability, as treatment of older rats (PN11-14) failed to cause substantial induction of AC or interference with G-protein signaling, although it did still enhance the glucagon response. In all cases, the effects of chlorpyrifos disappeared within a few days of discontinuing treatment. These results stand in contrast to the delayed deterioration of AC signaling seen in the brain after the same chlorpyrifos treatment. The temporal and organ selectivity of chlorpyrifos’ effects on the AC cascade suggest that disruption of membrane signaling occurs consequent to selective effects on cell development, rather than representing a direct interaction between chlorpyrifos and signaling proteins.

Neonatal chlorpyrifos exposure alters synaptic development and neuronal activity in cholinergic and catecholaminergic pathways

After routine home application of chlorpyrifos (CPF), infant and child exposures can exceed acceptable levels. We treated neonatal rats daily on postnatal days (PN) 1–4 (1 mg/kg) or days 11–14 (5 mg/kg), treatments that evoked no overt signs of toxicity. Effects on the development of cholinergic neuronal function were assessed using choline acetyltransferase (ChAT) activity and hemicholinium-3 (HC-3) binding as indices of synaptic proliferation and synaptic activity, respectively. In the forebrain, early CPF treatment caused a decrease in ChAT without affecting HC-3 binding; late treatment decreased HC-3 binding without affecting ChAT. In the brainstem, early treatment had no effect on either parameter but late treatment decreased both ChAT and HC-3 binding. Effects of CPF were not limited to development of cholinergic synapses but also involved catecholamine pathways. For norepinephrine or dopamine, either early or late CPF treatment evoked an increase in synaptic activity (transmitter turnover). The cerebellum, a region with sparse cholinergic innervation, was affected the most. Effects on catecholamine systems were unrelated to the magnitude or temporal pattern of cholinesterase inhibition. Our results suggest that CPF exposure during the postnatal period of synaptogenesis elicits widespread disruption of cholinergic and catecholaminergic pathways. As this is the period in which patterns of synaptic responsiveness is programmed by neural input, the period of developmental vulnerability to CPF is likely to extend into childhood.