William R. Mundy

Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth.

In vitro models may be useful for the rapid toxicological screening of large numbers of chemicals for their potential to produce toxicity. Such screening could facilitate prioritization of resources needed for in vivo toxicity testing towards those chemicals most likely to result in adverse health effects. Cell cultures derived from nervous system tissue have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and function, and have been used to understand the mechanism of action of neurotoxic chemicals. Recently, it has been suggested that in vitro models could be used to screen for chemical effects on critical cellular events of neurodevelopment, including differentiation and neurite growth. This review examines the use of neuronal cell cultures as an in vitro model of neurite outgrowth. Examples of the cell culture systems that are commonly used to examine the effects of chemicals on neurite outgrowth are provided, along with a description of the methods used to quantify this neurodevelopmental process in vitro. Issues relating to the relevance of the methods and models currently used to assess neurite outgrowth are discussed in the context of hazard identification and chemical screening. To demonstrate the utility of in vitro models of neurite outgrowth for the evaluation of large numbers of chemicals, efforts should be made to: (1) develop a set of reference chemicals that can be used as positive and negative controls for comparing neurite outgrowth between model systems, (2) focus on cell cultures of human origin, with emphasis on the emerging area of neural progenitor cells, and (3) use high-throughput methods to quantify endpoints of neurite outgrowth.

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny.

Because the Developmental Neurotoxicity Testing Guidelines require large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemical or morphological, and do not assess function of neuronal networks. In this study, microelectrode arrays (MEAs) were used to determine if chemical-induced changes in function could be detected by assessing the development of spontaneous network activity. MEAs record individual action potential spikes as well as groups of spikes (bursts) in neuronal networks, and activity can be assessed repeatedly over days in vitro (DIV). Primary cultures of rat cortical neurons were prepared on MEAs and spontaneous activity was assessed on DIV 2, 6, 9, 13, and 20 to determine the in vitro developmental profile of spontaneous spiking and bursting in cortical networks. In addition, 5 μM of the protein kinase C inhibitor bisindolylmaleamide-1 (Bis-1) was added to MEAs (n = 9–18) on DIV 5 to determine if changes in spontaneous activity could be detected in response to inhibition of neurite outgrowth. A clear profile of in vitro activity development occurred in control MEAs, with the number of active channels increasing from 0/MEA on DIV 2 to 37 ± 5/MEA by DIV 13; the rate of increase was most rapid between DIV 6 and 9, and activity declined by DIV 20. A similar pattern was observed for the number of bursting channels, as well as the total number of bursts. Bis-1 decreased the number of active channels/MEA and the number of bursting channels/MEA. Burst characteristics, such as burst duration and the number of spikes in a burst, were unchanged by Bis-1. These results demonstrate that MEAs can be used to assess the development of functional neuronal networks in vitro, as well as chemical-induced dysfunction.

Organophosphorus compounds preferentially affect second messenger systems coupled to M2/M4 receptors in rat frontal cortex

Recent reports indicate that organophosphate insecticides, in addition to inhibiting acetylcholinesterase activity, can bind directly at a subset of muscarinic receptors, which also bind cis-methyldioxolane with high affinity. Muscarinic receptors are known to act through at least two second messenger systems, either the stimulation of phosphoinositide turnover (mediated through the M1 and M3 receptor subtypes) or the inhibition of cAMP formation (mediated through the M2 and M4 receptor subtypes). We have investigated the action of the active forms of parathion, malathion, and chlorpyrifos (paraoxon, malaoxon, and chlorpyrifos oxon, respectively) on these second messenger systems in cortical slices from adult male Long-Evans rats. Paraoxon, malaoxon, and chlorpyrifos oxon (10(-8) to 10(-4) M) inhibited forskolin-stimulated cAMP formation in a concentration-dependent manner. The effect on cAMP formation was blocked by the muscarinic antagonist atropine (10 microM). These results suggest that paraoxon, malaoxon, and chlorpyrifos oxon can act as agonists at the M2 and/or M4 subset of muscarinic receptors. In addition, chlorpyrifos may have another site of action. In contrast, none of the organophosphates had any effect on basal or carbachol-stimulated phosphoinositide hydrolysis. The differential activity on these two second messenger systems make it unlikely that the observed effects on cAMP formation are due to increases in endogenous acetylcholine resulting from inhibition of acetylcholinesterase.

CAN THE MECHANISMS OF ALUMINUM NEUROTOXICITY BE INTEGRATED INTO A UNIFIED SCHEME

Regardless of the host, the route of administration, or the speciation, aluminum is a potent neurotoxicant. In the young adult or developmentally mature host, the neuronal response to Al exposure can be dichotomized on morphological grounds. In one, intraneuronal neurofilamentous aggregates are formed, whereas in the other, significant neurochemical and neurophysiological perturbations are induced without neurofilamentous aggregate formation. Evidence is presented that the induction of neurofilamentous aggregates is a consequence of alterations in the posttranslational processing of neurofilament (NF), particularly with regard to phosphorylation state. Although Al has been reported to impact on gene expression, this does not appear to be critical to the induction of cytoskeletal pathology. In hosts responding to Al exposure without the induction of cytoskeletal pathology, impairments in glucose utilization, agonist-stimulated inositol phosphate accumulation, free radical-mediated cytotoxicity, lipid peroxidation, reduced cholinergic function, and altered protein phosphorylation have been described. The extent to which these neurochemical modifications correlate with the induction of a characteristic neurobehavioral state is unknown. In addition to these paradigms, Al is toxic in the immediate postnatal interval. Whether unique mechanisms of toxicity are involved during development remains to be determined. In this article, the mechanisms of Al neurotoxicity are reviewed and recommendations are put forth with regard to future research. Primary among these is the determination of the molecular site of Al toxicity, and whether this is based on Al substitution for divalent metals in a number of biological processes. Encompassed within this is the need to further understand the genesis of host- and developmental-specific responses.

Methylmercury decreases NGF-induced TrkA autophosphorylation and neurite outgrowth in PC12 cells

Neurotrophin signaling through Trk receptors is important for differentiation and survival in the developing nervous system. The present study examined the effects of CH(3)Hg on (125)I-nerve growth factor (NGF) binding to the TrkA receptor, NGF-induced activation of the TrkA receptor, and neurite outgrowth in an in vitro model of differentiation using PC12 cells. Whole-cell binding assays using (125)I-NGF revealed a single binding site with a K(d) of approximately 1 nM. Methylmercury (CH(3)Hg) at 30 nM (EC(50) for neurite outgrowth inhibition) did not affect NGF binding to TrkA. TrkA autophosphorylation was measured by immunoblotting with a phospho-specific antibody. TrkA autophosphorylation peaked between 2.5 and 5 min of exposure and then decreased but was still detectable at 60 min. Concurrent exposure to CH(3)Hg and NGF for 2.5 min resulted in a concentration-dependent decrease in TrkA autophosphorylation, which was significant at 100 nM CH(3)Hg. To determine whether the observed inhibition of TrkA was sufficient to alter cell differentiation, NGF-stimulated neurite outgrowth was examined in PC12 cells after exposure to 30 nM CH(3)Hg, a concentration that inhibited TrkA autophosphorylation by approximately 50%. For comparison, a separate group of PC12 cells were exposed to a concentration of the selective Trk inhibitor K252a (30 nM), which had been shown to produce significant inhibition of TrkA autophosphorylation. Twenty-four hour exposure to either CH(3)Hg or K252a reduced neurite outgrowth to a similar degree. Our results suggest that CH(3)Hg may inhibit differentiation of PC12 cells by interfering with NGF-stimulated TrkA autophosphorylation.

Neurotoxicity of environmental chemicals and their mechanism of action

Despite a ban on their manufacture in 1977, polychlorinated biphenyls (PCBs) are still found in significant quantities in the environment. Developmental exposure to PCBs and related compounds has been reported to be neurotoxic in human and animals. Research in our laboratory has focused on the possible site(s) and mechanism(s) of PCB-induced developmental neurotoxicity. Recent experiments with rats found that developmental exposure to Aroclor-1254 (ARC) affects the acquisition of a lever press response and produces long-term changes in calcium buffering and protein kinase C (PKC) activity in the brain. In vitro studies in our laboratory have found that ARC increases [3H]phorbol ester binding, an indirect measure of PKC translocation, and inhibits calcium buffering in microsomes and mitochondria. Other experiments indicate that PCB congeners with chlorine substitutions at ortho- or low lateral substitutions are active in vitro, while non-ortho-substituted congeners are less active or inactive. Other research suggests that the lack of coplanarity of the PCB molecule is related to in vitro activity of PCB congeners. These studies indicate that in vivo developmental exposure to PCBs alters behavior and second messenger systems during adulthood, while in vitro experiments indicate that nervous system activity is related to ortho-substituted congeners that tend to be non-coplanar in configuration. Our results are consistent with the hypothesis that developmental neurotoxicity of ARC is due, in part, to the presence of ortho-substituted PCB congeners.

Use of high content image analysis to detect chemical-induced changes in synaptogenesis in vitro.

Synaptogenesis is a critical process in nervous system development whereby neurons establish specialized contact sites which facilitate neurotransmission. Early life exposure to chemicals can result in persistent deficits in nervous system function at later life stages. These effects are often the result of abnormal development of synapses. Given the large number of chemicals in commerce with unknown potential to result in developmental neurotoxicity (DNT), the need exists for assays that can efficiently characterize and quantify chemical effects on brain development including synaptogenesis. The present study describes the application of automated high content image analysis (HCA) technology for examining synapse formation in rodent primary mixed cortical cultures. During the first 15 days in vitro (DIV) cortical neurons developed a network of polarized neurites (i.e., axons and dendrites) and expression of the pre-synaptic protein synapsin increased over time. The localization of punctate synapsin protein in close apposition to dendrites also increased, indicating an increase in synapse formation. Results demonstrated that: (1) punctate synapsin protein with a spatial orientation consistent with synaptic contact sites could be selectively measured, (2) the critical period for synaptogenesis in cortical cultures was consistent with previous reports, (3) chemicals known to inhibit synapse formation decreased automated measurements of synapse number and (4) parallel evaluation of neuron density, dendrite length and synapse number could distinguish frank cytotoxicity from specific effects on synapse formation or neuronal morphology. Collectively, these data demonstrate that automated image analysis can be used to efficiently assess synapse formation in primary cultures and that the resultant data is comparable to results obtained using lower throughput methods.

Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2 cells using automated high-content image analysis.

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and immortalized human and non-human clonal cell lines have been used to investigate the molecular mechanisms controlling neurite outgrowth and examine chemical effects on this process. The present study characterizes the molecular phenotype of hN2 human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24h post-plating hN2 cells express a number of protein markers indicative of a neuronal phenotype, including: nestin, beta(III)-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite outgrowth in hN2 cells proceeded rapidly, with a majority of cells extending one to three neurites by 48h in culture. In addition, concentration-dependent decreases in neurite outgrowth and ATP-content were observed following treatment of hN2 cells with either bisindolylmaleimide I, U0126, lithium chloride, sodium orthovanadate and brefeldin A, all of which have previously been shown to inhibit neurite outgrowth in primary rodent neural cultures. Overall, the molecular phenotype, rate of neurite outgrowth and sensitivity of hN2 cells to neurite outgrowth inhibitors were comparable to other in vitro models previously characterized in the literature. hN2 cells provide a model in which to investigate chemical effects on neurite outgrowth in a non-transformed human-derived cells and provide an alternative to the use of primary rodent neural cultures or immortalized clonal cell lines.

Advancing the science of developmental neurotoxicity (DNT): Testing for better safety evaluation

test Guidelines OPPtS 8706300 on DNt (US ePA, 1998) and in 2007 the Organization for economic Cooperation and Development (OeCD) endorsed a new OeCD DNt test Guideline 426 (OeCD, 2007). these guidelines are largely based on animal studies and are used as higher tiered, triggered tests based on structure activity relationships or evidence of neurotoxicity in standard adult, developmental, or reproduction studies (Makris et al., 2009). experts at the conference stated that these in vivo tests are unsuitable for screening large numbers of chemicals for many reasons including low throughput, high cost, and questions regarding reliability. there was also consensus that new, reliable, and efficient screening and assessment tools are needed for better identification, prioritization, and evaluation of chemicals with the potential to induce developmental neurotoxicity. the information obtained from these screening studies will likely also help to refine animal tests and to inform epidemiological studies.