Unrelated developmental neurotoxicants elicit similar transcriptional profiles for effects on neurotrophic factors and their receptors in an in vitro model

Diverse developmental neurotoxicants can often produce similar functional and behavioral outcomes. We examined an organophosphate pesticide (diazinon), an organochlorine pesticide (dieldrin) and a metal (Ni2+) for effects on the expression of neurotrophic factors and their receptors and modulators in differentiating PC12 cells, an in vitro model of neuronal development. Each agent was introduced at 30 M for 24 or 72 h, treatments devoid of cytotoxicity. Using microarrays, we examined the mRNAs encoding members of the fibroblast growth factor (fgf) family, the neurotrophins (ntfs), brain-derived neurotrophic factor (bdnf), nerve growth factor (ngf), the wnt and fzd gene families, and the receptors and modulators for each class. All three agents evoked highly concordant patterns of effects on genes encoding the fgf family, whereas the correlations were poor for the group comprising bdnf, ngf and their respective receptors. For wnt, fzd and their receptors/modulators, the relationships between diazinon and dieldrin were highly concordant, whereas the effect of Ni2+ was less similar, albeit still significantly correlated with the others. Our results show that otherwise disparate developmental neurotoxicants converge on common sets of neurotrophic pathways known to control neuronal differentiation, likely contributing to similarities in functional outcomes. Further, cell culture models can provide a useful initial screen to identify members of a given class of compounds that may be greater or lesser risks for developmental neurotoxicity, or to provide an indication of agents in different classes that might produce similar effect

Targeting of neurotrophic factors, their receptors, and signaling pathways in the developmental neurotoxicity of organophosphates in vivo and in vitro

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 vulnerabilit

Determining the optimal size of small molecule mixtures for high throughput NMR screening

High-throughput screening (HTS) using NMR spectroscopy has become a common component of the drug discovery effort and is widely used throughout the pharmaceutical industry. NMR provides additional information about the nature of small molecule-protein interactions compared to traditional HTS methods. In order to achieve comparable efficiency, small molecules are often screened as mixtures in NMR-based assays. Nevertheless, an analysis of the efficiency of mixtures and a corresponding determination of the optimum mixture size (OMS) that minimizes the amount of material and instrumentation time required for an NMR screen has been lacking. A model for calculating OMS based on the application of the hypergeometric distribution function to determine the probability of a \u27hit\u27 for various mixture sizes and hit rates is presented. An alternative method for the deconvolution of large screening mixtures is also discussed. These methods have been applied in a high-throughput NMR screening assay using a small, directed library