Translating neurobehavioural endpoints of developmental neurotoxicity tests into in vitro assays and readouts

The developing nervous system is particularly vulnerable to chemical insults. Exposure to chemicals can result in neurobehavioural alterations, and these have been used as sensitive readouts to assess neurotoxicity in animals and man. Deconstructing neurobehaviour into relevant cellular and molecular components may allow for detection of specific neurotoxic effects in cell-based systems, which in turn may allow an easier examination of neurotoxic pathways and modes of actions and eventually inform the regulatory assessment of chemicals with potential developmental neurotoxicity. Here, current developments towards these goals are reviewed. Imaging genetics (CB) provides new insights into the neurobiological correlates of cognitive function that are being used to delineate neurotoxic mechanisms. The gaps between in vivo neurobehaviour and real-time in vitro measurements of neuronal function are being bridged by ex vivo measurements of synaptic plasticity (RW). An example of solvent neurotoxicity demonstrates how an in vivo neurological defect can be linked via the N-methyl-d-aspartate (NMDA)-glutamate receptor as a common target to in vitro readouts (AB). Axonal and dendritic morphology in vitro proved to be good correlates of neuronal connectivity and neurobehaviour in animals exposed to polychlorinated biphenyls and organophosphorus pesticides (PJL). Similarly, chemically induced changes in neuronal morphology affected the formation of neuronal networks on structured surfaces. Such network formation may become an important readout for developmental neurotoxicity in vitro (CvT), especially when combined with human neurons derived from embryonic stem cells (ML). We envision that future in vitro test systems for developmental neurotoxicity will combine the above approaches with exposure information, and we suggest a strategy for test system development and cell-based risk assessment.     

Qualitative and quantitative comparability of human and animal developmental neurotoxicants: a workshop summary

A Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity was held in Williamsburg, Va. on April 11-13, 1989. Based upon data presented at the Workshop, the degree of qualitative and quantitative comparability between data obtained from humans and experimental animals is reviewed for several developmental neurotoxicants (lead, agents of abuse, alcohol, PCBs, phenytoin, methylmercury, and ionizing radiation). Qualitative comparability was considered for the following functional categories: motor development and function, cognitive function, sensory function, motivation/arousal behavior, and social behavior. Quantitative comparability was assessed by comparing administered dose as well as measures of internal dose (e.g., blood levels) for selected agents. Comparability of qualitative changes between humans and rodents was most apparent when comparisons were made on the basis of general categories of behavioral function. These data support the use of animal models in assessing risk for developmental neurotoxicants and provide guidance on the types of functional end points that can be incorporated into a developmental neurotoxicity testing battery. Evidence of quantitative comparability was most apparent when an internal measure of dose (e.g., blood level) was used.     

Structure-activity comparison of organotin species: dibutyltin is a developmental neurotoxicant in vitro and in vivo

Human exposure to the organotins can occur due to their use as polyvinyl chloride heat stabilizers and as marine biocides. The consequences of this exposure for human health are unknown. We initially compared the toxicity of monomethyltin, dimethyltin, and dibutyltin to the known neurotoxicant trimethyltin using an in vitro model of neuronal development in PC12 cells. Dibutyltin, a compound traditionally thought to target the immune system, was the most potent neurotoxicant. Dibutyltin significantly inhibited neurite outgrowth and caused cell death at concentrations approximately 40-fold lower than the lowest toxic concentrations of trimethyltin. Dimethyltin was less potent than trimethyltin and monomethyltin was not toxic at any concentration examined. These results suggested the importance of prioritizing in vivo neurotoxicity testing with dibutyltin. To accomplish this, pregnant rats were dosed orally with low levels of dibutyltin from gestational day 6 through weaning. In response to developmental dibutyltin exposure, the incidence of apoptotic cell death, measured by DNA fragmentation and TUNEL staining, was increased in the neocortex and hippocampus of postnatal day 38 offspring. No effect was observed at other ages examined.     

Application of in vitro neurotoxicity testing for regulatory purposes: Symposium III summary and research needs

Prediction of neurotoxic effects is a key feature in the toxicological profile of many compounds and therefore is required by regulatory testing schemes. Nowadays neurotoxicity assessment required by the OECD and EC test guidelines is based solely on in vivo testing, evaluating mainly effects on neurobehavior and neuropathology, which is expensive, time consuming and unsuitable for screening large number of chemicals. Additionally, such in vivo tests are not always sensitive enough to predict human neurotoxicity and often do not provide information that facilitates regulatory decision-making processes. Incorporation of alternative tests (in vitro testing, computational modelling, QSARs, grouping, read-across, etc.) in screening strategies would speed up the rate at which compound knowledge and mechanistic data are available and the information obtained could be used in the refinement of future in vivo studies to facilitate predictions of neurotoxicity. On 1st June 2007, the European Commission legislation concerning registration, evaluation and authorisation of chemicals (REACH) has entered into force. REACH addresses one of the key issues for chemicals in Europe, the lack of publicly available safety data sheets. It outlines a plan to test approximately 30,000 existing substances. These chemicals are currently produced in volumes greater than 1ton/year and the essential data on the human health and ecotoxicological effects are lacking. It is estimated that approximately 3.9 million test animals (including 2.6 million vertebrates) (Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortnaer S, et al. ECVAM's response to the changing political environment for alternatives: consequences of the European Union chemicals and cosmetics policies. ATLA 2003;31:473-81) would be necessary to fulfill the requirements of REACH if the development and establishment of alternative methods is not accepted by regulatory authorities. In an effort to reduce animal use and testing costs within this tonnage band, the European Commission has advocated the use of alternative approaches. Neurotoxicity testing is not directly addressed within REACH, however when alerts are observed based on organ specific toxicity studies then neurotoxicity assessment has to be performed. This session at the 11th International Neurotoxicology Association Meeting provided a forum to openly discuss and debate the potential of in vitro testing strategies that could be relevant for neurotoxicity evaluation in the context of regulatory requirements. The EU FP6 project A-Cute-Tox was presented as an example of a possible in vitro testing strategy for prediction of human acute systemic toxicity. Other presentations focused on the characterization of the available in vitro models (cell lines and primary culture) and neuronal specific endpoints, with a special emphasis on electrical activity, metabonomics and modulation of vesicular neurotransmitter release as possible neuronal endpoints relevant for in vitro neurotoxicity testing. Finally, it was underlined that in vitro systems (strategies) that have the potential to be applied for neurotoxicity assessment have to be formally validated under standardised conditions that have been recognised by national and international validation bodies.     

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury,lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.     

Integration of epidemiology and animal neurotoxicity data for risk assessment

Most human health risk assessments are based on animal studies that can be conducted under conditions where exposure to multiple doses of a single chemical can be controlled. Data from epidemiology studies also provide valuable information about human exposure and response to pesticides. Human studies have the potential of evaluating neurobehavioral and other outcomes that may be more difficult to evaluate in animals. The human data together with animal data can contribute to a weight-of-evidence analysis in the characterization of human health risks. Epidemiology data do, however, pose challenges with respect to characterizing human health risks. Similarly, animal data at high doses or routes of exposure not typical for humans also pose challenges to dose-response evaluations needed for risk assessments. This paper summarizes some of the presentations given at a symposium held at the Xi'an, China, International Neurotoxicology Conference held in June 2011. This symposium brought together scientists from government, industry and academia to discuss approaches to evaluating and conducting animal and human neurotoxicity studies for risk assessment purposes, using the pesticides paraquat and chlorpyrifos as case studies.     

Guidelines for developmental neurotoxicity and their impact on organophosphate pesticides: a personal view from an academic perspective

The appropriate regulation of drugs, chemicals and environmental contaminants requires the establishment of clear and accepted guidelines for developmental neurotoxicity. Ideally, these guidelines should encompass the ability to assess widely disparate classes of compounds through routine tests, with high throughput and low cost. Increasingly, however, the progress in primary research from academic laboratories deviates from this goal, focusing instead on categorizing novel effects of toxicants, development of new testing paradigms, and extension of techniques into molecular biology. The differing objectives of academic science as opposed to those of regulatory agencies or industry, are driven in part, by the priorities of the agencies that fund primary research. Recent work on organophosphate pesticides (OPs) such as chlorpyrifos (CPF) illustrate this dichotomy. Originally, OPs were thought to affect brain development through their ability to elicit cholinesterase inhibition and consequent cholinergic hyperstimulation. This common mechanism allowed for parallels to be drawn between standard measures of systemic toxicity, gross morphological examinations, and exposure testing utilizing an easily-assessed surrogate endpoint, plasma cholinesterase activity. In the past decade, however, it has become increasingly evident that CPF, and probably other OPs, have direct effects on cellular processes that are unique to brain development, and that these effects are mechanistically unrelated to inhibition of cholinesterase. The identification and pursuit of these mechanisms and their consequences for brain development represent new and exciting scientific findings, while at the same obscuring the ability to sustain a uniform approach to neurotoxicity guidelines or biomarkers of exposure. In the future, a new set of test paradigms, relying on primary work in cell culture, invertebrates, or non-mammalian models, followed by more targeted examinations of specific processes in mammalian models, may unite cutting-edge academic research with the need for establishing flexible guidelines for developmental neurotoxicity.     

Neurotoxicants and central catecholamine systems

The ubiquitous functional roles of brain catecholamines have led to the notion that in vitro neurochemical changes in these systems may predict neurotoxicity. Conversely, others have argued that the appropriate use of neurochemical methods is for testing specific hypotheses that are developed based on observed phenomena. Three studies from this laboratory are presented in support of the latter hypothesis. The first example is with inorganic lead, a major environmental pollutant. The effects of small doses of lead on CNS development have been difficult to quantify or study mechanistically. However, the serendipitous finding that lead exposure during early postnatal development increased lithium-induced polydipsia (LIP) has provided clues that have permitted testing of specific neurochemical hypotheses related to dopamine systems. Conversely, the administration of either trimethyl- or triethyltin to rats during perinatal periods causes profound neurotoxicity. However, although some changes in the neurochemistry of catecholamine systems have been found, these data have provided little insight into either the cause or sequelae of toxicity. Finally, the food color erythrosin (FD & C Red #3) was hypothesized to be a neurotoxicant because it disrupted neurotransmitter uptake in vitro. Our data suggested this was an artifact of the methodology, a position supported by clinical and behavioral studies. These data provide examples of the strengths and weaknesses in neurochemical approaches to neurotoxicity.     

Role of autophagy inhibitors and inducers in modulating the toxicity of trimethyltin in neuronal cell cultures

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Earlier ultrastructural studies indicated the formation of autophagic vacuoles in neurons after TMT intoxication. However, no evaluation has been attempted to determine the role of the autophagic pathway in TMT neurotoxicity. To assess the contribution of autophagy to TMT-induced neuronal cell death, we checked the vulnerability of neuronal cultures to TMT after activation or inhibition of autophagy. Our results show that autophagy inhibitors (3-methyladenine and l-asparagine) greatly enhanced TMT neurotoxicity. Conversely, known activators of autophagy, such as lithium and rapamycin, displayed neuroprotection against this toxic compound. Due to its diverse targets, the action of lithium was complex. When lithium was administered according to a chronic treatment protocol (6?days pretreatment) it was able to rescue both hippocampal and cortical neurons from TMT (or from glutamate toxicity used as reference). This effect was accompanied by an increased phosphorylation of glycogen synthase kinase 3 which is a known target for lithium neuroprotection. If the pre-incubation time was reduced to 2?h (acute treatment protocol), lithium was still able to counteract TMT toxicity in hippocampal but not in cortical neurons. The neuroprotective effect of lithium acutely administered against TMT in hippocampal neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. These data indicate that TMT neurotoxicity can be dramatically modified, at least in vitro, by lithium addition which seems to act through different mechanisms if acutely or chronically administered.     

Toxicological screening for organophosphorus-induced delayed neurotoxicity: complications in toxicity testing

The acute biocidal effects of organophosphorus pesticides are a central feature of modern agricultural chemistry, and also define the concerns of regulatory toxicology. Less well known, but more complex and idiosyncratic, is the potential for some agents to produce a delayed and progressive polyneuropathy--Organophosphorus Induced Delayed Neurotox-icity (OPIDN). On three occasions during the past ten years, the National Institute for Occupational Safety and Health (NIOSH) had been asked to evaluate human delayed neurotoxicity from three commercially available pesticides. These were leptophos, fenthion, and isofenphos. In each case, human disease was either observed or suggested by specialized toxicity testing. The reasons that federally recommended screening measures failed to identify a potential for human neurotoxicity were not accidental, but stem from a systematic approach that focuses on a traditional definition of acute lethal toxicity. The oral single dose study on one species appears to be insufficient for recognizing the delayed neurotoxic hazard of many representatives of this chemical class. The recent addition of a recommended biochemical assay--neurotoxic esterase (NTE)--to federal guidelines potentially improves sensitivity, but it is purely adjunctive and does not amend underlying ambiguities in selecting the dose and route of administration. It is also quite probable that human neurotoxicity may be a potential hazard from exposure to more than the handful of organophosphorus pesticides that have been described in the literature.     

Glucocorticoid exacerbation of gp120 neurotoxicity: role of microglia

Gp120 protein, part of the HIV coat, may be a causative agent in AIDS-Related Dementia (ARD) because of its demonstrated neurotoxicity in vitro and in vivo. There are two possible mechanisms for this toxicity, namely through release of toxins from the microglia or through direct action on neuronal chemokine receptors. In tissue culture, glucocorticoids (GCs), the adrenal steroids released during stress, exacerbate gp120 neurotoxicity. In this paper, we examine the means by which GCs may increase toxicity, focusing on interactions with microglia and glia. Media from microglia treated with gp120 was toxic to neurons but not to glia. The effects of GCs upon the extent of gp120-induced release of toxins by microglia seemed to be dependent on the time of exposure to the hormone. Twenty-four-hour exposure of microglia to GCs decreased the toxicity of gp120-treated microglial conditioned media. In contrast, longer-term GC exposure enhanced neurotoxicity. There also appeared to be a component of gp120 neurotoxicity in hippocampal cultures that was exacerbated by GCs, independent of the amount of microglia present. Thus, GCs appear to act at a number of different sites in the multi-cellular pathway to exacerbate the neurotoxic effects of gp120.     

Regulatory developmental neurotoxicity and human risk assessment.

A number of efforts, in the last 15 years, have been directed at developing protocols to assess the potential developmental neurotoxicity (DN) of test agents. Japan and the United Kingdom have general protocols that describe the behavioral parameters that should be evaluated as part of other types of testing protocols, such as standard developmental and/or reproductive toxicity studies. In 1986, EPA published a proposed separate guideline for the testing of glycol ethers. Since then, this protocol has undergone extensive review and comment by an agency-wide workgroup, participants of a workshop sponsored by EPA and NIDA, EPA's Scientific Advisory Panel, and the public. Comments were taken into consideration and the final DN testing protocol has been published recently by EPA's Office of Pesticide Programs. This protocol provides specific guidance on issues of study design, aspects of CNS function to be evaluated and criteria for selection of testing procedures. It is designed to be a "generic" protocol could be applied to testing of pesticides and other chemicals and that could be modified on a case-by-case basis depending on the data available on a specific agent of concern. With the development of testing protocols for assessing DN, there comes a need for the development of guidance as to how the data should be interpreted and applied toward conducting a risk assessment for extrapolation to humans. Some guidance was developed at the EPA-NIDA Workshop. EPA has published specific risk assessment guidelines in the area of developmental toxicity that include a section on interpretation of data on functional deficits, including DN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Do we really want to REACH out to in vitro?

To comply with international regulations on chemicals, such as REACH (registration, evaluation, and authorization of chemicals), an enormous amount of toxicity testing is required. Traditional tests will fall short, since these strongly rely on in vivo studies, in particular for neurotoxicity. Therefore, a shift to alternative/in vitro toxicity testing is essential, in particular for neurotoxicity testing. However, the use of in vitro models and in vitro endpoints appears far from well accepted. This brief personal view highlights some of the concerns regarding in vitro research, e.g. using clonal cell lines such as PC12 cells and SH-SY5Y cells, to illustrate that many of these concerns may not be justified. A better characterization of specific in vitro models as well as a better understanding of the motive for using these in vitro models for neurotoxicity testing in the scientific community is necessary. The future of neurotoxicity testing will involve an increased use of in vitro experiments that are carefully designed with respect to compatibility of the exposure paradigm, the in vitro model and the chosen endpoint(s).     

Application of micro-electrode arrays (MEAs) as an emerging technology for developmental neurotoxicity: evaluation of domoic acid-induced effects in primary cultures of rat cortical neurons

Due to lack of knowledge only a few industrial chemicals have been identified as developmental neurotoxicants. Current developmental neurotoxicity (DNT) guidelines (OECD and EPA) are based entirely on in vivo studies that are both time consuming and costly. Consequently, there is a high demand to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. One of the most promising tools for neurotoxicity assessment is the measurement of neuronal electrical activity using micro-electrode arrays (MEAs) that provides a functional and neuronal specific endpoint that until now has been used mainly to detect acute neurotoxicity. Here, electrical activity measurements were evaluated to be a suitable endpoint for the detection of potential developmental neurotoxicants. Initially, primary cortical neurons grown on MEA chips were characterized for different cell markers over time, using immunocytochemistry. Our results show that primary cortical neurons could be a promising in vitro model for DNT testing since some of the most critical neurodevelopment processes such as progenitor cell commitment, proliferation and differentiation of astrocytes and maturation of neurons are present. To evaluate if electrical activity could be a suitable endpoint to detect chemicals with DNT effects, our model was exposed to domoic acid (DomA), a potential developmental neurotoxicant for up to 4 weeks. Long-term exposure to a low concentration (50nM) of DomA increased the basal spontaneous electrical activity as measured by spike and burst rates. Moreover, the effect induced by the GABA(A) receptor antagonist bicuculline was significantly lower in the DomA treated cultures than in the untreated ones. The MEA measurements indicate that chronic exposure to DomA changed the spontaneous electrical activity leading to the possible neuronal mal functioning. The obtained results suggest that the MEAs could be a useful tool to identify compounds with DNT potential.     

Neurotoxicology risk assessment guidelines: developmental neurotoxicology

EPA's Neurotoxicity Risk Assessment Guidelines were recently published in final form in the Federal Register (1998). This document was developed over a period of nearly ten years and is intended to establish operating principles used in the evaluation of data for neurotoxicity risk assessment. The guidelines contain a number of assumptions and definitions of key concepts, as well as guidance as to the evaluation of various behavioral and structural changes produced by chemical exposure in humans and animals. With regard to developmental neurotoxicity, risk assessors should be aware that chemical-induced neurotoxicity in adults may not always be a good predictor of developmental neurotoxicity. Adverse effects on the developing nervous system can occur prior to conception up to the time of sexual maturity, depend on the time of exposure relative to a critical state of nervous system development, can be seen at any time during the lifespan of the organism, may lead to delayed onset or latent effects, and may elicit compensatory mechanisms that obscure underlying neurotoxicity. Adverse effects include persistent alterations in function or structure of the nervous system or a change in the time or appearance of any endpoint. Relative to neurotoxicity in adult animals, there are several special concerns in hazard characterization of developmental studies, including maternal toxicity, the use of the litter as the statistical unit, and time of exposure relative to the ontogeny of various structural or functional endpoints. Dose-response evaluation of data from developmental studies is similar to that for adults, although a safety factor of 10 may be applied to protect children's health. The guidelines also note that exposure patterns of children differ from those of adults resulting in a greater intake of chemicals on a per body weight basis. The guidelines note several research needs, including more information on mechanisms of developmental neurotoxicity, mechanistically based dose-response models, impact of early exposure to chemicals on late-onset disease, studies on threshold, and experiments on potential interactions between chemicals in mixtures.     

Single-Cell ELISA and Flow Cytometry as Methods for Highlighting Potential Neuronal and Astrocytic Toxicant Specificity

The timeline imposed by recent worldwide chemical legislation is not amenable to conventional in vivo toxicity testing, requiring the development of rapid, economical in vitro screening strategies which have acceptable predictive capacities. When acquiring regulatory neurotoxicity data, distinction on whether a toxic agent affects neurons and/or astrocytes is essential. This study evaluated neurofilament (NF) and glial fibrillary acidic protein (GFAP) directed single-cell (S-C) ELISA and flow cytometry as methods for distinguishing cell-specific cytoskeletal responses, using the established human NT2 neuronal/astrocytic (NT2.N/A) co-culture model and a range of neurotoxic (acrylamide, atropine, caffeine, chloroquine, nicotine) and non-neurotoxic (chloramphenicol, rifampicin, verapamil) test chemicals. NF and GFAP directed flow cytometry was able to identify several of the test chemicals as being specifically neurotoxic (chloroquine, nicotine) or astrocytoxic (atropine, chloramphenicol) via quantification of cell death in the NT2.N/A model at cytotoxic concentrations using the resazurin cytotoxicity assay. Those neurotoxicants with low associated cytotoxicity are the most significant in terms of potential hazard to the human nervous system. The NF and GFAP directed S-C ELISA data predominantly demonstrated the known neurotoxicants only to affect the neuronal and/or astrocytic cytoskeleton in the NT2.N/A cell model at concentrations below those affecting cell viability. This report concluded that NF and GFAP directed S-C ELISA and flow cytometric methods may prove to be valuable additions to an in vitro screening strategy for differentiating cytotoxicity from specific neuronal and/or astrocytic toxicity. Further work using the NT2.N/A model and a broader array of toxicants is appropriate in order to confirm the applicability of these methods.     

A novel in vitro metabolomics approach for neurotoxicity testing, proof of principle for methyl mercury chloride and caffeine

There is a need for more efficient methods giving insight into the complex mechanisms of neurotoxicity. Testing strategies including in vitro methods have been proposed to comply with this requirement. With the present study we aimed to develop a novel in vitro approach which mimics in vivo complexity, detects neurotoxicity comprehensively, and provides mechanistic insight. For this purpose we combined rat primary re-aggregating brain cell cultures with a mass spectrometry (MS)-based metabolomics approach. For the proof of principle we treated developing re-aggregating brain cell cultures for 48 h with the neurotoxicant methyl mercury chloride (0.1-100 microM) and the brain stimulant caffeine (1-100 microM) and acquired cellular metabolic profiles. To detect toxicant-induced metabolic alterations the profiles were analysed using commercial software which revealed patterns in the multi-parametric dataset by principal component analyses (PCA), and recognised the most significantly altered metabolites. PCA revealed concentration-dependent cluster formations for methyl mercury chloride (0.1-1 microM), and treatment-dependent cluster formations for caffeine (1-100 microM) at sub-cytotoxic concentrations. Four relevant metabolites responsible for the concentration-dependent alterations following methyl mercury chloride treatment could be identified using MS-MS fragmentation analysis. These were gamma-aminobutyric acid, choline, glutamine, creatine and spermine. Their respective mass ion intensities demonstrated metabolic alterations in line with the literature and suggest that the metabolites could be biomarkers for mechanisms of neurotoxicity or neuroprotection. In addition, we evaluated whether the approach could identify neurotoxic potential by testing eight compounds which have target organ toxicity in the liver, kidney or brain at sub-cytotoxic concentrations. PCA revealed cluster formations largely dependent on target organ toxicity indicating possible potential for the development of a neurotoxicity prediction model. With such results it could be useful to perform a validation study to determine the reliability, relevance and applicability of this approach to neurotoxicity screening. Thus, for the first time we show the benefits and utility of in vitro metabolomics to comprehensively detect neurotoxicity and to discover new biomarkers.     

Low Glucose Utilization and Neurodegenerative Changes Caused by Sodium Fluoride Exposure in Rat’s Developmental Brain

Fluorine, a toxic and reactive element, is widely prevalent throughout the environment and can induce toxicity when absorbed into the body. This study was to explore the possible mechanisms of developmental neurotoxicity in rats treated with different levels of sodium fluoride (NaF). The rats’ intelligence, as well as changes in neuronal morphology, glucose absorption, and functional gene expression within the brain were determined using the Morris water maze test, transmission electron microscopy, small-animal magnetic resonance imaging and Positron emission tomography and computed tomography, and Western blotting techniques. We found that NaF treatment-impaired learning and memory in these rats. Furthermore, NaF caused neuronal degeneration, decreased brain glucose utilization, decreased the protein expression of glucose transporter 1 and glial fibrillary acidic protein, and increased levels of brain-derived neurotrophic factor in the rat brains. The developmental neurotoxicity of fluoride may be closely associated with low glucose utilization and neurodegenerative changes.     

Chemotherapy-induced neuropathy

Abstract Neurotoxic side effects of cancer therapy are second in frequency to hematological toxicity. Unlike hematological side effects that can be treated with hematopoietic growth factors, neuropathies cannot be treated and protective treatment strategies have not been effective. For the neurologist, the diagnosis of a toxic neuropathy is primarily based on the case history, the clinical and electrophysiological findings, and knowledge of the pattern of neuropathy associated with specific agents. In most cases, toxic neuropathies are length‐dependent, sensory, or sensorimotor neuropathies often associated with pain. The platinum compounds are unique in producing a sensory ganglionopathy. Neurotoxicity is usually dependent on cumulative dose. Severity of neuropathy increases with duration of treatment and progression stops once drug treatment is completed. The platinum compounds are an exception where sensory loss may progress for several months after cessation of treatment (“coasting”). As more effective multiple drug combinations are used, patients will be treated with several neurotoxic drugs. Synergistic neurotoxicity has not been extensively investigated. Pre‐existent neuropathy may influence the development of a toxic neuropathy. Underlying inherited or inflammatory neuropathies may predispose patients to developing very severe toxic neuropathies. Other factors such as focal radiotherapy or intrathecal administration may enhance neurotoxicity. The neurologist managing the cancer patient who develops neuropathy must answer a series of important questions as follows: (1) Are the symptoms due to peripheral neuropathy? (2) Is the neuropathy due to the underlying disease or the treatment? (3) Should treatment be modified or stopped because of the neuropathy? (4) What is the best supportive care in terms of pain management or physical therapy for each patient? Prevention of toxic neuropathies is most important. In patients with neuropathy, restorative approaches have not been well established. Symptomatic and other management are necessary to maintain and improve quality of life.     

Antiretroviral neurotoxicity

Combination antiretroviral therapy (CART) has proven to effectively suppress systemic HIV burden, however, poor penetration into the central nervous system (CNS) provides incomplete protection. Although the severity of HIV-associated neurocognitive disorders (HAND) has been reduced, neurological disease is expected to exert an increasing burden as HIV-infected patients live longer. Strategies to enhance penetration of antiretroviral compounds into the CNS could help to control HIV replication in this reservoir but also carries an increased risk of neurotoxicity. Efforts to target antiretroviral compounds to the CNS will have to balance these risks against the potential gain. Unfortunately, little information is available on the actions of antiretroviral compounds in the CNS, particularly at concentrations that provide effective virus suppression. The current studies evaluated the direct effects of 15 antiretroviral compounds on neurons to begin to provide basic neurotoxicity data that will serve as a foundation for the development of dosing and drug selection guidelines. Using sensitive indices of neural damage, we found a wide range of toxicities, with median toxic concentrations ranging from 2 to 10,000?ng/ml. Some toxic concentrations overlapped concentrations currently seen in the CSF but the level of toxicity was generally modest at clinically relevant concentrations. Highest neurotoxicities were associated with abacavir, efavarenz, etravirine, nevaripine, and atazanavir, while the lowest were with darunavir, emtracitabine, tenofovir, and maraviroc. No additive effects were seen with combinations used clinically. These data provide initial evidence useful for the development of treatment strategies that might reduce the risk of antiretroviral neurotoxicity.