Oxidative and excitatory mechanisms of developmental neurotoxicity: transcriptional profiles for chlorpyrifos, diazinon, dieldrin, and divalent nickel in PC12 cells.
ABSTRACT: Oxidative stress and excitotoxicity underlie the developmental neurotoxicity of numerous chemicals.We compared the effects of organophosphates (chlorpyrifos and diazinon), an organo-chlorine (dieldrin), and a metal [divalent nickel (Ni2+)] to determine how these mechanisms contribute to similar or dissimilar neurotoxic outcomes.We used PC12 cells as a model of developing neurons and evaluated transcriptional profiles for genes for oxidative stress responses and glutamate receptors.Chlorpyrifos had a greater effect on oxidative-stress-related genes in differentiating cells compared with the undifferentiated state. Chlorpyrifos and diazinon showed significant concordance in their effects on glutathione-related genes, but they were negatively correlated for effects on catalase and superoxide dismutase isoforms and had no concordance for effects on ionotropic glutamate receptors. Surprisingly, the correlations were stronger between diazinon and dieldrin than between the two organophosphates. The effects of Ni2+ were the least similar for genes related to oxidative stress but had significant concordance with dieldrin for effects on glutamate receptors.Our results point to underlying mechanisms by which different organophosphates produce disparate neurotoxic outcomes despite their shared property as cholinesterase inhibitors. Further, apparently unrelated neurotoxicants may produce similar outcomes because of convergence on oxidative stress and excitotoxicity. The combined use of cell cultures and microarrays points to specific end points that can distinguish similarities and disparities in the effects of diverse developmental neurotoxicants.
Project description:Unrelated developmental neurotoxicants can produce similar neurobehavioral outcomes. We examined whether disparate agents affect neuromodulators that control numerous neurotransmitters and circuits, employing PC12 cells to explore the targeting of neuroactive peptides by organophosphates (chlorpyrifos, diazinon), an organochlorine (dieldrin) and a metal (Ni(2+)); we utilized microarrays to profile gene expression for the peptides and their receptors. Chlorpyrifos evoked robust upregulation of cholecystokinin, corticotropin releasing hormone, galanin, neuropeptide Y, neurotensin, preproenkephalin and tachykinin 1; this involved a critical period at the commencement of neurodifferentiation, since the effects were much less notable in undifferentiated PC12 cells. Diazinon targeted a similar but smaller repertoire of neuropeptide genes and the magnitude of the effects was also generally less. Surprisingly, dieldrin shared many of the same neuropeptide targets as the organophosphates and concordance analysis showed significant overlap among all three pesticides. However, dieldrin had more notable effects on neuropeptide receptors, and overlap between diazinon and dieldrin for the receptors led to a stronger resemblance of these two agents than of chlorpyrifos and dieldrin. Ni(2+) was unique, evoking upregulation of only one of the peptides affected by the other agents, while causing downregulation of several others. Nevertheless, there was still significant concordance between Ni(2+) and either diazinon or dieldrin, reflecting similarities toward the receptors. Our results show that neuropeptides are likely to be a prominent target for the developmental neurotoxicity of organophosphates and other neurotoxicants, and further, that the convergence of disparate agents on the same genes and pathways may contribute to similar neurobehavioral outcomes.
Project description:The developmental neurotoxicity of organophosphates involves mechanisms other than their shared property as cholinesterase inhibitors, among which are excitotoxicity and oxidative stress. We used PC12 cells as a neurodevelopmental model to compare the effects of chlorpyrifos and diazinon on the expression of genes encoding glutamate transporters. Chlorpyrifos had a greater effect in cells undergoing nerve growth factor-induced neurodifferentiation as compared to undifferentiated PC12 cells, with peak sensitivity at the initiation of differentiation, reflecting a global upregulation of all the glutamate transporter genes expressed in this cell line. In differentiating cells, chlorpyrifos had a significantly greater effect than did diazinon and concordance analysis indicated no resemblance in their expression patterns. At the same time, the smaller effects of diazinon were highly concordant with those of an organochlorine pesticide (dieldrin) and a metal (divalent nickel). We also performed similar evaluations for the cystine/glutamate exchanger, which provides protection against oxidative stress by moving cystine into the cell; again, chlorpyrifos had the greatest effect, in this case reducing expression in undifferentiated and differentiating cells. Our results point to excitotoxicity and oxidative stress as major contributors to the noncholinesterase mechanisms that distinguish the neurodevelopmental outcomes between different organophosphates while providing a means whereby apparently unrelated neurotoxicants may produce similar outcomes.
Project description:Cell-signaling cascades are convergent targets for developmental neurotoxicity of otherwise unrelated agents. We compared organophosphates (chlorpyrifos, diazinon), an organochlorine (dieldrin) and a metal (Ni(2+)) for their effects on neuronotypic PC12 cells, assessing gene transcription involved in the cyclic AMP pathway. Each agent was introduced during neurodifferentiation at a concentration of 30 microM for 24 or 72 h and we assessed 69 genes encoding adenylyl cyclase isoforms and regulators, G-protein alpha-and beta,gamma-subunits, protein kinase A subtypes and the phosphodiesterase family. We found strong concordance among the four agents across all the gene families, with the strongest relationships for the G-proteins, followed by adenylyl cyclase, and lesser concordance for protein kinase A and phosphodiesterase. Superimposed on this pattern, chlorpyrifos and diazinon were surprisingly the least alike, whereas there was strong concordance of dieldrin and Ni(2+) with each other and with each individual organophosphate. Further, the effects of chlorpyrifos differed substantially depending on whether cells were undifferentiated or differentiating. To resolve the disparities between chlorpyrifos and diazinon, we performed analyses in rat brain regions after in vivo neonatal exposures; unlike the in vitro results, there was strong concordance. Our results show that unrelated developmental neurotoxicants can nevertheless produce similar outcomes by targeting cell signaling pathways involved in neurodifferentiation during a critical developmental period of vulnerability. Nevertheless, a full evaluation of concordance between different toxicants requires evaluations of in vitro systems that detect direct effects, as well as in vivo systems that allow for more complex interactions that converge on the same pathway.
Project description:Epidemiologic studies support a connection between organophosphate pesticide exposures and subsequent risk of Parkinson's disease (PD). We used differentiating, neuronotypic PC12 cells to compare organophosphates (chlorpyrifos, diazinon), an organochlorine (dieldrin) and a metal (Ni(2+)) for their effects on the transcription of PD-related genes. Both of the organophosphates elicited significant changes in gene expression but with differing patterns: chlorpyrifos evoked both up- and downregulation whereas diazinon elicited overall reductions in expression. Dieldrin was without effect but Ni(2+) produced a pattern resembling that of diazinon. We then exposed neonatal rats to chlorpyrifos or diazinon for the first 4 days after birth and examined the expression of PD-related genes in the brainstem and forebrain. Chlorpyrifos had no significant effect whereas diazinon produced significant increases and decreases in expression of the same PD genes that were targeted in vitro. Our results provide some of the first evidence for a mechanistic relationship between developmental organophosphate exposure and the genes known to confer PD risk in humans; but they also point to disparities between different organophosphates that reinforce the concept that their neurotoxic actions do not rest solely on their shared property as cholinesterase inhibitors. The parallel effects of diazinon and Ni(2+) also show how otherwise unrelated developmental neurotoxicants can nevertheless produce similar outcomes by converging on common molecular pathways, further suggesting a need to examine metals such as Ni(2+) as potential contributors to PD risk.
Project description:Organophosphate pesticides are developmental neurotoxicants. We gave diazinon via osmotic minipumps implanted into dams prior to conception, with exposure continued into the second postnatal week, at doses (0.5 or 1?mg/kg/day) that did not produce detectable brain cholinesterase inhibition. We evaluated the impact on acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT) systems in brain regions from adolescence through full adulthood. Diazinon produced deficits in presynaptic ACh activity with regional and sex selectivity: cerebrocortical regions and the hippocampus were affected to a greater extent than were the striatum, midbrain or brainstem, and females were more sensitive than males. Diazinon also reduced nicotinic ACh receptors and 5HT1A receptors, with the same regional and sex preferences. These patterns were similar to those of diazinon given in a much more restricted period (postnatal day 1-4) but were of greater magnitude and consistency; this suggests that the brain is vulnerable to diazinon over a wide developmental window. Diazinon's effects differed from those of the related organophosphate, chlorpyrifos, with regard to regional and sex selectivity, and more importantly, to the effects on receptors: chlorpyrifos upregulates nicotinic ACh receptors and 5HT receptors, effects that compensate for the presynaptic ACh deficits. Diazinon can thus be expected to have worse neurodevelopmental outcomes than chlorpyrifos. Further, the disparities between diazinon and chlorpyrifos indicate the problems of predicting the developmental neurotoxicity of organophosphates based on a single compound, and emphasize the inadequacy of cholinesterase inhibition as an index of safety.
Project description:Organophosphates affect mammalian brain development through a variety of mechanisms beyond their shared property of cholinesterase inhibition. We used microarrays to characterize similarities and differences in transcriptional responses to chlorpyrifos and diazinon, assessing defined gene groupings for the pathways known to be associated with the mechanisms and/or outcomes of chlorpyrifos-induced developmental neurotoxicity. We exposed neonatal rats to daily doses of chlorpyrifos (1mg/kg) or diazinon (1 or 2mg/kg) on postnatal days 1-4 and evaluated gene expression profiles in brainstem and forebrain on day 5; these doses produce little or no cholinesterase inhibition. We evaluated pathways for general neural cell development, cell signaling, cytotoxicity and neurotransmitter systems, and identified significant differences for >60% of 252 genes. Chlorpyrifos elicited major transcriptional changes in genes involved in neural cell growth, development of glia and myelin, transcriptional factors involved in neural cell differentiation, cAMP-related cell signaling, apoptosis, oxidative stress, excitotoxicity, and development of neurotransmitter synthesis, storage and receptors for acetylcholine, serotonin, norepinephrine and dopamine. Diazinon had similar effects on many of the same processes but also showed major differences from chlorpyrifos. Our results buttress the idea that different organophosphates target multiple pathways involved in neural cell development but also that they deviate in key aspects that may contribute to disparate neurodevelopmental outcomes. Equally important, these pathways are compromised at exposures that are unrelated to biologically significant cholinesterase inhibition and its associated signs of systemic toxicity. The approach used here demonstrates how planned comparisons with microarrays can be used to screen for developmental neurotoxicity.
Project description:Developmental organophosphate exposure reduces the numbers of neural cells, contributing to neurobehavioral deficits. We administered chlorpyrifos or diazinon to newborn rats on postnatal days 1-4, in doses straddling the threshold for barely-detectable cholinesterase inhibition, and evaluated gene expression in the cell cycle and apoptosis pathways on postnatal day 5. Both organophosphates evoked transcriptional changes in 20-25% of the genes in each category; chlorpyrifos and diazinon targeted the same genes, with similar magnitudes of change, as evidenced by high concordance. Furthermore, the same effects were obtained with doses above or below the threshold for cholinesterase inhibition, indicating a mechanism unrelated to anticholinesterase actions. We then evaluated the effects of chlorpyrifos in undifferentiated and differentiating PC12 cells and found even greater targeting of cell cycle and apoptosis genes, affecting up to 40% of all genes in the pathways. Notably, the genes affected in undifferentiated cells were not concordant with those in differentiating cells, pointing to dissimilar outcomes dependent on developmental stage. The in vitro model successfully identified 60-70% of the genes affected by chlorpyrifos in vivo, indicating that the effects are exerted directly on developing neural cells. Our results show that organophosphates target the genes regulating the cell cycle and apoptosis in the developing brain and in neuronotypic cells in culture, with the pattern of vulnerability dependent on the specific stage of development. Equally important, these effects do not reflect actions on cholinesterase and operate at exposures below the threshold for any detectable inhibition of this enzyme.
Project description:Human, animal and cell models support a role for pesticides in the etiology of Parkinson disease. Susceptibility to pesticides may be modified by genetic variants of xenobiotic enzymes, such as paraoxonase, that play a role in metabolizing some organophosphates.We examined associations between Parkinson disease and the organophosphates diazinon, chlorpyrifos, and parathion, and the influence of a functional polymorphism at position 55 in the coding region of the PON1 gene (PON1-55). From 1 January 2001 through 1 January 2008, we recruited 351 incident cases and 363 controls from 3 rural California counties in a population-based case-control study. Participants provided a DNA sample, and residential exposure to organophosphates was determined from pesticide usage reports and a geographic information system (GIS) approach. We assessed the main effects of both genes and pesticides in unconditional logistic regression analyses, and evaluated the effect of carrying a PON1-55 MM variant on estimates of effects for diazinon, chlorpyrifos, and parathion exposures.Carriers of the variant MM PON1-55 genotype exposed to organophosphates exhibited a greater than 2-fold increase in Parkinson disease risk compared with persons who had the wildtype or heterozygous genotype and no exposure (for diazinon, odds ratio = 2.2 [95% confidence interval = 1.1-4.5]; for chlorpyrifos, 2.6 [1.3-5.4]). The effect estimate for chlorpyrifos, was more pronounced in younger-onset cases and controls (<or=60 years) (5.3 [1.7-16]). No increase in risk was noted for parathion.The increase in risk we observed among PON1-55 variant carriers for specific organophosphates metabolized by PON1 underscores the importance of considering susceptibility factors when studying environmental exposures in Parkinson disease.
Project description:Neurotrophic factors control neural cell differentiation and assembly of neural circuits. We previously showed that organophosphate pesticides differentially regulate members of the fibroblast growth factor (fgf) gene family. We administered chlorpyrifos and diazinon to neonatal rats on postnatal days 1-4 at doses devoid of systemic toxicity or growth impairment, and spanning the threshold for barely-detectable cholinesterase inhibition. We evaluated the impact on gene families for different classes of neurotrophic factors. Using microarrays, we examined the regional expression of mRNAs encoding the neurotrophins (ntfs), brain-derived neurotrophic factor (bdnf), nerve growth factor (ngf), the wnt and fzd gene families and the corresponding receptors. Chlorpyrifos and diazinon both had widespread effects on the fgf, ntf, wnt and fzd families but much less on the bdnf and ngf groups. However, the two organophosphates showed disparate effects on a number of key neurotrophic factors. To determine if the actions were mediated directly on differentiating neurons, we tested chlorpyrifos in PC12 cells, an in vitro model of neural cell development. Effects in PC12 cells mirrored many of those for members of the fgf, ntf and wnt families, as well as the receptors for the ntfs, especially during early differentiation, the stage known to be most susceptible to disruption by organophosphates. Our results suggest that actions on neurotrophic factors provide a mechanism for the developmental neurotoxicity of low doses of organophosphates, and, since effects on expression of the affected genes differed with test agent, may help explain regional disparities in effects and critical periods of vulnerability.
Project description:The fibroblast growth factor (FGF) superfamily of neurotrophic factors plays critical roles in neural cell development, brain assembly, and recovery from neuronal injury.We administered two organophosphate pesticides, chlorpyrifos and diazinon, to neonatal rats on postnatal days 1-4, using doses below the threshold for systemic toxicity or growth impairment, and spanning the threshold for barely detectable cholinesterase inhibition: 1 mg/kg/day chlorpyrifos and 1 or 2 mg/kg/day diazinon.Using microarrays, we then examined the regional expression of mRNAs encoding the FGFs and their receptors (FGFRs) in the forebrain and brain stem.Chlorpyrifos and diazinon both markedly suppressed fgf20 expression in the forebrain and fgf2 in the brain stem, while elevating brain stem fgfr4 and evoking a small deficit in brain stem fgf22. However, they differed in that the effects on fgf2 and fgfr4 were significantly larger for diazinon, and the two agents also showed dissimilar, smaller effects on fgf11, fgf14, and fgfr1.The fact that there are similarities but also notable disparities in the responses to chlorpyrifos and diazinon, and that robust effects were seen even at doses that do not inhibit cholinesterase, supports the idea that organophosphates differ in their propensity to elicit developmental neurotoxicity, unrelated to their anticholinesterase activity. Effects on neurotrophic factors provide a mechanistic link between organophosphate injury to developing neurons and the eventual, adverse neurodevelopmental outcomes.