Developmental Neurotoxicity Of Chlorpyrifos Research Articles

Toxicogenomic profiling in maternal and fetal rodent brains following gestational exposure to chlorpyrifos

Considering the wide variety of effects that have been reported to occur in the developmental neurotoxicity of chlorpyrifos (CP) and the lack of consensus on their dependence of brain acetylcholinesterase (AChE) activity inhibition, we applied microarray technology to explore dose-dependent alterations in transcriptional response in the fetal and maternal C57BL/6 mouse brain after daily gestational exposure (days 6 to 17) to CP (2, 4, 10, 12 or 15 mg/kg, sc). We identified significantly altered genes across doses and assessed for overrepresentation of Gene Ontology (GO) biological processes and KEGG pathways. We further clustered genes based on their expression profiles across doses and repeated the GO/pathways analysis for each cluster. The dose–effect relationship of CP on gene expression, both at the gene and pathway levels was non-monotonic and not necessarily related to brain AChE inhibition. The largest impact was observed in the 10 mg/kg dose group which was also the LOAEL for brain AChE inhibition. In the maternal brain, lower doses (4 mg/kg) influenced GO categories and pathways such as cell adhesion, behavior, lipid metabolism, long-term potentiation, nervous system development, neurogenesis, synaptic transmission. In the fetal brain, lower doses (2 and/or 4 mg/kg) significantly altered cell division, translation, transmission of nerve impulse, chromatin modification, long-term potentiation. In addition, some genes involved in nervous system development and signaling were shown to be specifically influenced by these lower CP doses. Our approach was sensitive and reflected the diversity of responses known to be disrupted by CP and highlighted possible additional consequences of CP neurotoxicity, such as disturbance of the ubiquitin proteasome system.

Ameliorating the Developmental Neurotoxicity of Chlorpyrifos: A Mechanisms-Based Approach in PC12 Cells

BackgroundOrganophosphate developmental neurotoxicity involves multiple mechanisms converging on neural cell replication and differentiation.ObjectivesWe evaluated mechanisms contributing to the adverse effects of chlorpyrifos (CPF) on DNA synthesis, cell number and size, and cell signaling mediated by adenylyl cyclase (AC) in PC12 cells, a neuronotypic cell line that recapitulates the essential features of developing mammalian neurons.ResultsIn undifferentiated cells, cholinergic receptor antagonists had little or no protective effect against the antimitotic actions of CPF; however, when nerve growth factor was used to evoke differentiation, the antagonists showed partial protection against deficits in cell loss and alteration in cell size elicited by CPF, but were ineffective in preventing the deterioration of AC signaling. Nicotine, which stimulates nicotinic acetylcholine receptors but also possesses a mixture of prooxidant/antioxidant activity, had adverse effects by itself but also protected undifferentiated cells from the actions of CPF and had mixed additive/protective effects on cell number in differentiating cells. The antioxidant vitamin E also protected both undifferentiated and differentiating cells from many of the adverse effects of CPF but worsened the impact on AC signaling. Theophylline, which prevents the breakdown of cyclic AMP, was the only agent that restored AC signaling to normal or supranormal levels but did so at further cost to cell replication.ConclusionsOur results show definitive contributions of cholinergic hyperstimulation, oxidative stress, and interference with AC signaling in the developmental neurotoxicity of CPF and point to the potential use of this information to design treatments to ameliorate these adverse effects.

Organophosphates and Outdoor Air Concentrations

Organophosphates and Outdoor Air Concentrations

Critical periods for the role of oxidative stress in the developmental neurotoxicity of chlorpyrifos and terbutaline, alone or in combination

The developmental neurotoxicity of chlorpyrifos (CPF) involves mechanisms other than inhibition of cholinesterase. In the current study, we examined the ability of CPF to evoke lipid peroxidation in the developing brain of fetal and neonatal rats. CPF given to pregnant rats on gestational days 17–20 or to neonatal rats on postnatal days 1–4, failed to elicit increases in thiobarbituric acid-reactive species (TBARS) in brain regions even when the dose was raised above the threshold for systemic toxicity and hepatic damage. In contrast, CPF administration during the second postnatal week, the peak period of neuronal cell differentiation and synaptogenesis, did evoke significant increases in TBARS even at a dose devoid of systemic toxicity. Terbutaline, which is chemically unrelated to CPF and which stimulates neuronal cell metabolism through direct actions on β-adrenoceptors, also elicited oxidative damage in the developing brain with greater sensitivity in the second postnatal week. These results indicate that diverse compounds can exert convergent effects on brain development through their shared potential to elicit oxidative stress, and that the net outcome is dependent upon specific developmental stages in which metabolic demand is especially high. Furthermore, given the common use of terbutaline in the therapy of preterm labor, and the nearly ubiquitous exposure of the human population to organophosphorus pesticides, the combined oxidative burden of exposure to both agents may contribute to the worsened neurodevelopmental outcomes noted in animal models of such dual exposures.

Chlorpyrifos exposure during neurulation: cholinergic synaptic dysfunction and cellular alterations in brain regions at adolescence and adulthood

The developmental neurotoxicity of chlorpyrifos (CPF) involves multiple mechanisms, thus rendering the immature brain susceptible to adverse effects over a wide window of vulnerability. Earlier work indicated that CPF exposure at the neural tube stage elicits apoptosis and disrupts mitotic patterns in the brain primordium but that rapid recovery ensues before birth. In the current study, we assessed whether defects in cholinergic synaptic activity emerge later in development. CPF was given to pregnant rats on gestational days 9–12, using regimens devoid of overt maternal or fetal toxicity. We then examined subsequent development of acetylcholine systems and compared the effects to those on general biomarkers of cell development. Choline acetyltransferase (ChAT), a constitutive marker for cholinergic nerve terminals, was increased in the hippocampus and striatum in adolescence and adulthood. In contrast, hemicholinium-3 (HC-3) binding to the presynaptic choline transporter, an index of nerve impulse activity, was markedly subnormal. Furthermore, m 2-muscarinic cholinergic receptor binding was significantly reduced, instead of showing the expected compensatory upregulation for reduced neural input. CPF also elicited delayed-onset alterations in biomarkers of cell packing density, cell number, cell size and neuritic projections, involving brain regions both with and without reductions in indices of cholinergic activity. In combination with earlier results, the current findings indicate that the developing brain, and especially the hippocampus, is adversely affected by CPF regardless of whether exposure occurs early or late in brain development, and that defects emerge in adolescence or adulthood even in situations where normative values are initially restored in the immediate post-exposure period.

Developmental neurotoxicity elicited by gestational exposure to chlorpyrifos: when is adenylyl cyclase a target?

The developmental neurotoxicity of chlorpyrifos (CPF) involves mechanisms over and above cholinesterase inhibition. In the present study, we evaluated the effects of gestational CPF exposure on the adenylyl cyclase (AC) signaling cascade, which regulates the production of cyclic AMP, a major controller of cell replication and differentiation. In addition to basal AC activity, we assessed the AC response to direct enzymatic stimulants [forskolin, manganese (Mn(2+))]; the response to isoproterenol, which activates signaling through beta-adrenoceptors (betaARs); and the concentration of betaAR binding sites. CPF administered to pregnant rats on gestational days (GD) 9-12 elicited little or no change in any components of AC activity or betaARs. However, shifting the treatment window to GD17-20 produced regionally selective augmentation of AC activity. In the brainstem, the response to forskolin or Mn(2+) was markedly stimulated by doses at or below the threshold for observable toxicity of CPF or for inhibition of fetal brain cholinesterase, whereas comparable effects were seen in the forebrain only at higher doses. In addition, low doses of CPF reduced betaAR binding without impairing receptor-mediated stimulation of AC. These results indicate that signal transduction through the AC cascade is a target for CPF during a discrete developmental period in late gestation, an effect that is likely to contribute to the noncholinergic component of CPF's developmental neurotoxicity.

Serotonergic systems targeted by developmental exposure to chlorpyrifos: effects during different critical periods.

During brain development, serotonin (5HT) provides essential neurotrophic signals. In the present study, we evaluated whether the developmental neurotoxicity of chlorpyrifos (CPF) involves effects on 5HT signaling, as a potential mechanism underlying noncholinergic neuroteratogenic events. We evaluated four different treatment windows ranging from the neural tube stage [gestational days (GD) 9-12] and the late gestational period (GD17-20) through postnatal phases of terminal neuronal differentiation and synaptogenesis [postnatal days (PN) 1-4, PN11-14]. Exposure to CPF on GD9-12 elicited initial suppression, immediately followed by rebound elevation, of 5HT1A and 5HT2 receptors as well as the 5HT transporter, all at doses below the threshold for cholinergic hyperstimulation and the resultant systemic toxicity. In contrast, with GD17-20 exposure, the initial effect was augmentation of all three components by low doses of CPF. Sensitivity of these effects declined substantially when exposure was shifted to the postnatal period. We also identified major alterations in 5HT-mediated responses, assessed for the adenylyl cyclase signaling cascade. Although GD9-12 exposure had only minor effects, treatment on GD17-20 elicited supersensitivity to both stimulatory and inhibitory responses mediated by 5HT. Our results indicate that CPF affects 5HT receptors, the presynaptic 5HT transporter, and 5HT-mediated signal transduction during a discrete critical gestational window. These effects are likely to contribute to the noncholinergic component of CPF's developmental neurotoxicity.

Developmental neurotoxicity of chlorpyrifos: what is the vulnerable period?

Previously, we found that exposure of neonatal rats to chlorpyrifos (CPF) produced brain cell damage and loss, with resultant abnormalities of synaptic development. We used the same biomarkers to examine prenatal CPF treatment so as to define the critical period of vulnerability. One group of pregnant rats received CPF (subcutaneous injections in dimethyl sulfoxide vehicle) on gestational days (GD) 17-20, a peak period of neurogenesis; a second group was treated on GD9-12, the period of neural tube formation. In the GD17-20 group, the threshold for a reduction in maternal weight gain was 5 mg/kg/day; at or below that dose, there was no evidence (GD21) of general fetotoxicity as assessed by the number of fetuses or fetal body and tissue weights. Above the threshold, there was brain sparing (reduced body weight with an increase in brain/body weight ratio) and a targeting of the liver (reduced liver/body weight). Indices of cell packing density (DNA per gram of tissue) and cell number (DNA content) similarly showed effects only on the liver; however, there were significant changes in the protein/DNA ratio, an index of cell size, in fetal brain regions at doses as low as 1 mg/kg, below the threshold for inhibition of fetal brain cholinesterase (2 mg/kg). Indices of cholinergic synaptic development showed significant CPF-induced defects but only at doses above the threshold for cholinesterase inhibition. With earlier CPF treatment (GD9-12), there was no evidence of general fetotoxicity or alterations of brain cell development at doses up to the threshold for maternal toxicity (5 mg/kg), assessed on GD17 and GD21; however, augmentation of cholinergic synaptic markers was detected at doses as low as 1 mg/kg. Compared with previous work on postnatal CPF exposure, the effects seen here required doses closer to the threshold for fetal weight loss; this implies a lower vulnerability in the fetal compared with the neonatal brain. Although delayed neurotoxic effects of prenatal CPF may emerge subsequently in development, our results are consistent with the preferential targeting of late developmental events such as gliogenesis, axonogenesis, and synaptogenesis.

Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos?

The increasing use of chlorpyrifos (CPF) has elicited concern about neurotoxic effects on the fetus and neonate. CPF targets a number of events specific to brain development, over and above the ability of its active metabolite, CPF oxon, to inhibit cholinesterase. We used PC12 cells, a model system which displays many of the neurodevelopmental effects of CPF, in order to examine whether oxidative stress underlies the direct effects of CPF on development. Production of reactive oxygen species (ROS) was measured with a fluorescent intracellular dye. When PC12 cell suspensions were treated acutely with CPF for 10 min, ROS generation was increased in a concentration-dependent manner; CPF oxon was much less effective than the native compound. CPF also increased the ROS production in response to an acute sodium nitroprusside challenge, indicating sensitization of the cells to other oxidant stressors. Next, PC12 cells were grown in an undifferentiated state in the presence of CPF or CPF oxon for extended time periods, under conditions in which CPF inhibits mitosis, and the cells were then washed and ROS production measured. Neither compound elicited a significant change in ROS production. Finally, differentiation was initiated with nerve growth factor and the cells were exposed continuously to CPF or CPF oxon over a 72 h period; under these conditions, CPF inhibits neurite outgrowth. When the cells were washed and evaluated for ROS production, no significant differences were seen. These results indicate that CPF, but not CPF oxon, has the ability to elicit acute increases in ROS production. However, the effect disappears immediately once CPF exposure is terminated, possibly reflecting cellular defense mechanisms that lessen the impact of oxidant injury.

Developmental neurotoxicity of chlorpyrifos in vivo and in vitro: effects on nuclear transcription factors involved in cell replication and differentiation

Chlorpyrifos is a widely used organophosphate insecticide that is a suspected developmental neurotoxin. Although chlorpyrifos exerts some effects through cholinesterase inhibition, recent studies suggest additional, direct actions on developing cells. We assessed the effects of chlorpyrifos on nuclear transcription factors involved in cell replication and differentiation using in vitro and in vivo models. HeLa nuclear protein extracts were incubated with the labeled consensus oligonucleotides for AP-1 and Sp1 transcription factors in the presence and absence of chlorpyrifos. In concentrations previously shown to affect cell development, chlorpyrifos reduced AP-1, but not Sp1 DNA-binding activity. Next, chlorpyrifos was incubated with PC12 cells either during cell replication or after initiation of differentiation with NGF. Chlorpyrifos evoked stage-specific interference with the expression of the transcription factors: Sp1 was reduced in replicating and differentiating cells, whereas AP-1 was affected only during differentiation. Finally, neonatal rats were given apparently subtoxic doses of chlorpyrifos either on postnatal days 1–4 or 11–14 and the effects were evaluated in the forebrain (an early-developing, cholinergic target region) and cerebellum (late-developing region, poor in cholinergic innervation). Again, chlorpyrifos evoked stage-specific changes in transcription factor expression and binding activity, with greater effects on Sp1 during active neurogenesis, and effects on AP-1 during differentiation. The changes were present in both forebrain and cerebellum and were gender-specific. These results indicate that chlorpyrifos interferes with brain development, in part by multiple alterations in the activity of transcription factors involved in the basic machinery of cell replication and differentiation. Noncholinergic actions of chlorpyrifos that are unique to brain development reinforce the need to examine endpoints other than cholinesterase inhibition.

Developmental neurotoxicity of chlorpyrifos: delayed targeting of DNA synthesis after repeated administration

Despite the fact that they recover more rapidly from cholinesterase inhibition than do adults, developing animals are more sensitive to delayed neurotoxicity caused by chlorpyrifos exposure. Previous studies indicate that acute, high dose chlorpyrifos exposure of developing rats interferes with synthesis of brain macromolecules, dependent upon a critical maturational stage and upon regional disparities in cholinergic innervation. In order to determine whether chronic, lower level chlorpyrifos exposure targets similar developmental events, rats were treated daily on postnatal days 1–4, using a dose (1 mg/kg, s.c.) that caused no mortality or weight deficits and that produces minimal cholinesterase inhibition. At the end of the treatment period, we examined macromolecule synthesis in three brain regions possessing disparate maturational profiles and cholinergic innervation: the brainstem, which undergoes its primary phase of neurogenesis prenatally and develops prominent cholinergic innervation, the forebrain, which develops somewhat later but also becomes cholinergically enriched, and the cerebellum, which undergoes neurogenesis postnatally and remains poor in cholinergic innervation. Four h after the last chlorpyrifos treatment, no effects were seen for DNA, RNA or protein synthesis. However, on postnatal day 5 (24 h after the last treatment), robust deficits in DNA synthesis were observed in brainstem and forebrain, with lesser effects on the cerebellum. Although the brain regional selectivity is compatible either with differences in critical maturational phases or with targeting of cholinergically-enriched brain regions, we found no significant effects in the heart, despite the fact that it is also receives cholinergic innervation. Effects on DNA synthesis were not evident 4 h after the last dose, but then appeared after 24 h. As the 4-h point is 28 h after the third dose, this suggests that a cumulative threshold needs to be exceeded in order for the delayed neurotoxicity to appear. At the point at which DNA synthesis was inhibited in brainstem and forebrain, no effects were seen for RNA or protein synthesis, indicating selectivity for macromolecule synthesis associated with cell replication. These data indicate that otherwise subtoxic, chronic exposures to chlorpyrifos nevertheless target DNA synthesis, and by inference, cell replication, in selective brain cell populations, early events that are likely contributors to the deficits in cell number that appear several days later.