Comparative developmental neurotoxicity of organophosphates in vivo: Transcriptional responses of pathways for brain cell development, cell signaling, cytotoxicity and neurotransmitter systems

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Abstract

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 (1 mg/kg) or diazinon (1 or 2 mg/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.

Introduction

Despite their widespread use [17], organophosphate insecticides are a major concern for human health because of their propensity to damage the developing brain at exposures below the threshold for signs of systemic intoxication [55], [56], [62], [72], [73], [86], [87], [108]. Although it was originally thought that these agents act solely through inhibition of cholinesterase and consequent cholinergic hyperstimulation, it is now evident that there are multiple mechanisms that contribute to neurodevelopmental abnormalities [10], [17], [42], [73], [74], [77], [112]. Accordingly, whereas all the organophosphates share cholinesterase as a target, they are likely to differ to a greater or lesser extent in their effects unrelated to that particular mechanism. Indeed, chlorpyrifos (CPF), the best-studied agent, affects brain development through diverse targets such as oxidative stress, cell signaling cascades, expression and function of nuclear transcription factors, and neuronal-glial cell interactions [42], [73], [86], [87], [89], mechanisms that may be shared in varying degrees by other organophosphates [1], [67], [73], [75], [86], [87], [91], [110].

We recently compared the thresholds for cholinesterase inhibition, systemic toxicity and several developmental neurotoxicity endpoints for CPF, diazinon (DZN) and parathion [50], [91], [97] and found distinct disparities in both the sensitivity of various pathways involved in cell differentiation as well as in outcomes related to abnormal brain development. These findings point to the need to screen the various organophosphates for similarities and differences in their targeting of the key pathways that contribute to their ultimate neurodevelopmental consequences. Profiling of gene transcription responses in these pathways represents a potentially valuable and informative approach to identification of common and disparate mechanisms of neural damage. To date, this strategy has been applied with only a handful of genes at a time and yet has yielded some promising results, including demonstrations of effects on the expression of factors involved in neural growth, glial cell development, myelination, apoptosis, muscarinic acetylcholine receptors (mAChRs) and acetylcholinesterase splice variants that typify neural damage and repair [13], [27], [50]. With gene microarray techniques, cell culture systems of tumor lines expressing neurohumoral characteristics further indicate a broader range of potential target pathways [61], [63]. In the current study, we cast a broader net for in vivo effects of organophosphates on brain development by using microarrays to conduct planned comparisons of families of genes based on the known pathway targets of CPF, both for mechanisms of brain cell damage and the types of neurons ultimately affected [42], [87], [88], [89]. In essence, we used CPF as a method of validation of the microarray results because the phenotypic outcomes of CPF treatment are already established. We then compared the results for CPF with those for DZN, for which far less is known, in order to emphasize points of similarity and difference that may enable the prediction of disparities in the ultimate neurodevelopmental outcomes of these two organophosphates. We concentrated on doses that evoke no cholinesterase inhibition or barely detectable inhibition, too low to elicit any signs of cholinergic hyperstimulation [97], [100]. For our evaluations of gene transcription, we chose specific pathways involved in: (a) neural cell growth and neurite formation; (b) transcription factors and cell signaling cascades that mediate neural cell differentiation; (c) cytotoxic events including oxidative stress, apoptosis and expression of ionotropic glutamate receptors (iGluRs); (d) neurotransmitter pathways known to be especially targeted by CPF. The latter include acetylcholine (ACh) and the monoamines, serotonin (5HT), dopamine and norepinephrine; in addition, we compared effects on metabotropic glutamate receptors (mGluRs), which are not involved in excitotoxicity, to those on the iGluRs. Our assessments were conducted in two brain regions that differ both in anatomical attributes as well as in maturational timetables [81]. The brainstem develops earliest of the regions and contains many of the cell bodies for the neural pathways targeted by CPF, whereas the forebrain develops later and contains a high concentration of the nerve terminal zones to which the cells originating in the brainstem project.

Section snippets

Animal treatments

All experiments were carried out in accordance with federal and state guidelines and with prior approval of the Duke University Institutional Animal Care and Use Committee; all animals were treated humanely and with due care for alleviation of distress. Timed-pregnant Sprague–Dawley rats (Charles River, Raleigh, NC) were housed in breeding cages, with a 12 h light–dark cycle and free access to food and water. On the day of birth, all pups were randomized and redistributed to the dams with a

Results

Our planned comparisons approach included 252 of the genes that passed quality control filters. The global ANOVA indicated significant treatment effects that were restricted to specific genes and regions: p < 0.0001 for interactions of treatment × gene and treatment × region × gene, with both regions individually showing significant (p < 0.0001) interactions of treatment × gene. This global test justified the examination of values for individual genes in each region. In addition, looking at each gene

Strategic issues and limitations

In the current study, we used planned comparisons of defined groupings of genes defining the pathways known to be associated with the mechanisms and/or outcomes of CPF-induced developmental neurotoxicity, rather than relying on a genome-wide approach. The disadvantage of this strategy is that it does not identify completely novel or unsuspected targets. On the other hand, there are distinct benefits that outweigh this liability. First, inclusion of all 42,000 transcripts generates over 2000

Conflicts of interest

The authors state that they have no conflicts of interest.

Acknowledgments

Research was supported by NIH ES10356. The authors thank Drs. Elwood Linney and Seth Kullman for assistance with technical aspects and manuscript preparation.

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