Identification of neural-relevant toxcast high-throughput assay intended gene targets: Applicability to neurotoxicity and neurotoxicant putative molecular initiating events.

The US EPA's Toxicity Forecaster (ToxCast) is a suite of high-throughput in vitro assays to screen environmental toxicants and predict potential toxicity of uncharacterized chemicals. This work examines the relevance of ToxCast assay intended gene targets to putative molecular initiating events (MIEs) of neurotoxicants. This effort is needed as there is growing interest in the regulatory and scientific communities about developing new approach methodologies (NAMs) to screen large numbers of chemicals for neurotoxicity and developmental neurotoxicity.

Comparison of Acute Effects of Neurotoxic Compounds on Network Activity in Human and Rodent Neural Cultures.

Assessment of neuroactive effects of chemicals in cell-based assays remains challenging as complex functional tissue is required for biologically relevant readouts. Recent in vitro models using rodent primary neural cultures grown on multielectrode arrays allow quantitative measurements of neural network activity suitable for neurotoxicity screening. However, robust systems for testing effects on network function in human neural models are still lacking.

Testing for developmental neurotoxicity using a battery of in vitro assays for key cellular events in neurodevelopment.

Medium- to high-throughput in vitro assays that recapitulate the critical processes of nervous system development have been proposed as a means to facilitate rapid testing and identification of chemicals which may affect brain development. In vivo neurodevelopment is a complex progression of distinct cellular processes. Therefore, batteries of in vitro assays that model and quantify effects on a variety of neurodevelopmental processes have the potential to identify chemicals which may affect brain development at different developmental stages.

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes.

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential.

From the Cover: Developmental Neurotoxicants Disrupt Activity in Cortical Networks on Microelectrode Arrays: Results of Screening 86 Compounds During Neural Network Formation.

Less than 1% of environmental chemicals have been evaluated for developmental neurotoxicity (DNT). Current guideline DNT studies are resource intensive and not amenable to screening large numbers of compounds for hazard. As part of evaluating a battery of more rapid and scalable in vitro assays for DNT hazard, 86 compounds were screened for their ability to alter function during cortical network development.

A multivariate extension of mutual information for growing neural networks.

Recordings of neural network activity in vitro are increasingly being used to assess the development of neural network activity and the effects of drugs, chemicals and disease states on neural network function. The high-content nature of the data derived from such recordings can be used to infer effects of compounds or disease states on a variety of important neural functions, including network synchrony. Historically, synchrony of networks in vitro has been assessed either by determination of correlation coefficients (e.

Editor's Highlight: Evaluation of a Microelectrode Array-Based Assay for Neural Network Ontogeny Using Training Set Chemicals.

Thousands of compounds in the environment have not been characterized for developmental neurotoxicity (DNT) hazard. To address this issue, methods to screen compounds rapidly for DNT hazard evaluation are necessary and are being developed for key neurodevelopmental processes. In order to develop an assay for network formation, this study evaluated effects of a training set of chemicals on network ontogeny by measuring spontaneous electrical activity in neural networks grown on microelectrode arrays (MEAs).

Characterization of Early Cortical Neural Network Development in Multiwell Microelectrode Array Plates.

We examined neural network ontogeny using microelectrode array (MEA) recordings made in multiwell MEA (mwMEA) plates over the first 12 days in vitro (DIV). In primary cortical cultures, action potential spiking activity developed rapidly between DIV 5 and 12. Spiking was sporadic and unorganized at early DIV, and became progressively more organized with time, with bursting parameters, synchrony, and network bursting increasing between DIV 5 and 12.

Expanding the test set: Chemicals with potential to disrupt mammalian brain development.

High-throughput test methods including molecular, cellular, and alternative species-based assays that examine critical events of normal brain development are being developed for detection of developmental neurotoxicants. As new assays are developed, a "training set" of chemicals is used to evaluate the relevance of individual assays for specific endpoints. Different training sets are necessary for each assay that would comprise a developmental neurotoxicity test battery.

Use of alternative assays to identify and prioritize organophosphorus flame retardants for potential developmental and neurotoxicity.

Due to their toxicity and persistence in the environment, brominated flame retardants (BFRs) are being phased out of commercial use, leading to the increased use of alternative chemicals such as the organophosphorus flame retardants (OPFRs). There is, however, limited information on the potential health effects of OPFRs. Due to the structural similarity of the OPFRs to organophosphorus insecticides, there is concern regarding developmental toxicity and neurotoxicity.

A multiplexed assay for determination of neurotoxicant effects on spontaneous network activity and viability from microelectrode arrays.

Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characterize the ability of drugs, chemicals and particles to cause neurotoxicity. While compound effects on spontaneous network activity are easily determined by MEA recordings, compound cytotoxicity is not routinely assessed, particularly within the same network from which recordings are collected. With the advent of higher-throughput 48 and 96 well MEA systems, rapid and simple methods to measure compound effects on cell health are required to facilitate efficient compound screening using MEAs.

Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening.

High-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system.

Ontogeny of biochemical, morphological and functional parameters of synaptogenesis in primary cultures of rat hippocampal and cortical neurons.

Background: Synaptogenesis is a critical neurodevelopmental process whereby pre- and postsynaptic neurons form apposed sites of contact specialized for chemical neurotransmission. Many neurodevelopmental disorders are thought to reflect altered patterns of synaptic connectivity, including imbalances between excitatory and inhibitory synapses. Developing rapid throughput approaches for assessing synaptogenesis will facilitate toxicologic and drug screening studies of neurodevelopmental disorders.

Media formulation influences chemical effects on neuronal growth and morphology.

Screening for developmental neurotoxicity using in vitro, cell-based systems has been proposed as an efficient alternative to performing in vivo studies. One tool currently used for developmental neurotoxicity screening is automated high-content imaging of neuronal morphology. While high-content imaging (HCI) has been demonstrated to be useful in detection of potential developmental neurotoxicants, comparison of results between laboratories or assays can be complicated due to methodological differences.

Improving in vitro to in vivo extrapolation by incorporating toxicokinetic measurements: a case study of lindane-induced neurotoxicity.

Approaches for extrapolating in vitro toxicity testing results for prediction of human in vivo outcomes are needed. The purpose of this case study was to employ in vitro toxicokinetics and PBPK modeling to perform in vitro to in vivo extrapolation (IVIVE) of lindane neurotoxicity. Lindane cell and media concentrations in vitro, together with in vitro concentration-response data for lindane effects on neuronal network firing rates, were compared to in vivo data and model simulations as an exercise in extrapolation for chemical-induced neurotoxicity in rodents and humans.

Current approaches and future role of high content imaging in safety sciences and drug discovery.

High content imaging combines automated microscopy with image analysis approaches to simultaneously quantify multiple phenotypic and/or functional parameters in biological systems. The technology has become an important tool in the fields of safety sciences and drug discovery, because it can be used for mode-of-action identification, determination of hazard potency and the discovery of toxicity targets and biomarkers. In contrast to conventional biochemical endpoints, high content imaging provides insight into the spatial distribution and dynamics of responses in biological systems.

Use of high content image analyses to detect chemical-mediated effects on neurite sub-populations in primary rat cortical neurons.

Traditional developmental neurotoxicity tests performed in vivo are costly, time-consuming and utilize a large number of animals. In order to address these inefficiencies, in vitro models of neuronal development have been used in a first tier screening approach for developmental neurotoxicity hazard identification. One commonly used endpoint for assessing developmental neurotoxicity in vitro is measurement of neurite outgrowth.

Developmental neurotoxicity testing: a path forward.

Great progress has been made over the past 40 years in understanding the hazards of exposure to a small number of developmental neurotoxicants. Lead, polychlorinated biphenyls, and methylmercury are all good examples of science-based approaches to characterizing the hazard to the developing nervous systems from environmental contaminants. However, very little effort has been spent to address the challenge of assessing the potential developmental neurotoxic hazard of the thousands of other chemicals in common commercial use.

Neurotrophic effects of leukemia inhibitory factor on neural cells derived from human embryonic stem cells.

Various growth factor cocktails have been used to proliferate and then differentiate human neural progenitor (NP) cells derived from embryonic stem cells (ESC) for in vitro and in vivo studies. However, the cytokine leukemia inhibitory factor (LIF) has been largely overlooked. Here, we demonstrate that LIF significantly enhanced in vitro survival and promoted differentiation of human ESC-derived NP cells.

Comparison of chemical-induced changes in proliferation and apoptosis in human and mouse neuroprogenitor cells.

There is a need to develop rapid and efficient models to screen chemicals for their potential to cause developmental neurotoxicity. Use of in vitro neuronal models, including human cells, is one approach that allows for timely, cost-effective toxicity screening. The present study compares the sensitivity of human (ReN CX) and mouse (mCNS) neuroprogenitor cell lines to chemicals using a multiplex assay for proliferation and apoptosis, endpoints that are critical for neural development.

In vitro phototoxicity and hazard identification of nano-scale titanium dioxide.

Titanium dioxide nanoparticles (nano-TiO(2)) catalyze reactions under UV radiation and are hypothesized to cause phototoxicity. A human-derived line of retinal pigment epithelial cells (ARPE-19) was treated with six samples of nano-TiO(2) and exposed to UVA radiation. The TiO(2) nanoparticles were independently characterized to have mean primary particle sizes and crystal structures of 22nm anatase/rutile, 25nm anatase, 31nm anatase/rutile, 59nm anatase/rutile, 142nm anatase, and 214nm rutile.

Quantitative assessment of neurite outgrowth in PC12 cells.

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity. In order to identify potential developmental neurotoxicants, assessment of critical neurodevelopmental processes, such as neuronal differentiation and growth has been proposed. PC12 cells have been widely used to study the neurotrophic factor-induced signaling pathways that control differentiation, and as in vitro models to detect the effect of chemicals on neurite outgrowth.

Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth.

There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth.