CAAT Research Activities in Developmental NeurotoxicityIntroductionThe apparently increasing incidence of neurodevelopmental disorders in children—e.g., neurodevelopmental delays, learning disabilities, autism, and attention deficit and hyperactivity disorder (ADHD)—is a matter of serious concern. Although the diagnosis and reporting of such disorders has improved over the last few years, scientific evidence is accumulating that exposures to environmental and industrial chemicals during early life contribute to this increase. The developing central nervous system (CNS) is much more vulnerable to chemical-induced injury than the adult CNS. Several factors contribute to this high susceptibility, including the still immature blood brain barrier, increased absorption versus low body weight, and diminished ability to detoxify exogenous chemicals. CNS development is a complex process involving a large number of events in a strictly controlled time frame, e.g., differentiation of progenitor cells, cell proliferation and migration, synaptogenesis, myelination, cell death, synthesis of neurotransmitters, and formation of receptors. Once neurodevelopment is disturbed the potential for repair is minimal, and permanent health effects often result. The critical lack of knowledge when it comes to the developmental neurotoxicity (DNT) effects of environmental and industrial chemicals further complicates the issue. Current regulatory DNT testing guidelines are not suitable for large-scale DNT assessment of the approximately 80,000 chemicals now produced, primarily due to the high costs: about $1.4 million per chemical. Moreover, there are scientific concerns regarding the relevance of the applied in vivo studies for human health effects. Testing is performed at high doses that are not relevant for human exposure scenarios, and the interpretation of the behavioral and histology data can be difficult and the prediction of human health effects limited. Consequently, current DNT guidelines often do not provide sufficient information to facilitate regulatory decision-making. In response, experts in the field, guided by the Center for Alternatives to Animal Testing (CAAT), have organized a series of International workshops to discuss the current status of DNT assessment and identify promising alternative approaches. Based on the recommendations made during these meetings, CAAT initiated its DNT research activities. Research teamThe research team, headed by Thomas Hartung, currently includes five research associates (Mounir Bouhifd, Helena Hogberg, Andre Kleensang, Lena Smirnova, and Liang Zhang), one technician (Shelly Odwin-DaCosta), and one graduate student (Tyna Dao). The laboratory facilities are shared with the research groups of Jim Yager and Winnie Tang. Joint lab meetings are organized to discuss potential difficulties, progress, and research strategy. The CAAT laboratory currently is studying pathways of toxicity in a rat primary cell culture model using genomics and metabolomics approaches. Research goalsThe main goal of CAAT’s research is to implement the NAS vision on toxicity testing in the 21st century for the assessment of DNT. More specific aims include the identification of toxicity pathways using genomics, metabolomics, and bioinformatics technologies. We intend to identify accurate concentrations at which DNT chemicals trigger such toxicity pathways and to evaluate whether these concentrations are relevant for the assessment of DNT using pharmacokinetic modeling and existing in vivo or human data. Three in vitro models will be used to perform these studies: rat primary three-dimensional aggregating brain cell cultures, which were previously shown to closely reproduce the morphology and biochemical cell signaling of the CNS; human embryonic stem cells, which cover the early processes of neurodevelopment, and LUHMES human neuronal precursor cells, which are non-transformed human fetal cells that can be differentiated into neurons. The human models were extensively characterized previously by our collaborator, the research group of Marcel Leist at Konstanz University, Germany. Processes of neurodevelopment in the rat and human in vitro models will be studied by the quantification of gene expression relevant for CNS development. First results of RT-PCR analysis in aggregating brain cell cultures demonstrated the expression patterns of nestin expressed in neuroprecurser cells, neurofilament-200 expressed in neurons, S100? expressed in astrocytes and myelin basic protein expressed in oligodendrocytes during 35 day in vitro. Neurodevelopment also will be studied by mass spectrometry-based metabolomics, which identifies and quantifies low molecular weight metabolites. Agilent technologies awarded the “Thought Leader Award” to Thomas Hartung, which included a liquid chromatography/mass spectrometry based metabolomics system. The intracellular and extracellular metabolome will be analyzed over time to identify metabolites relevant for neurodevelopment. These studies will be performed in close collaboration with the biotech company Stemina, which has experience in such metabolomics studies. So far, twelve compounds have been tested for DNT effects in primary aggregating brain cell cultures. Compounds include pesticides (Chlorpyrifos, Carbaryl, Lindane, Maneb), metals (Cadmium chloride, Lead chloride), drugs (Valproic acid, Lamotrigine), chemicals (Bisphenol A, Trichlorethylene, PFOA), and food additives (Aspartame). All compounds except Lamotrigine and Aspartame induced DNT effects, as demonstrated by the alterations of gene expression of a single or several CNS-specific genes at relevant µMolar concentrations. Currently, the DNT effects of the compounds are studied by a mass spectrometry metabolomics system. First results showed that lead chloride and trichloroethylene induce metabolic perturbations in the intra-cellular metabolome in a concentration response manner. Metabolites will be identified using a commercial database using their retention time and accurate mass. Once these are identified, bioinformatics will be used to identify relationships between metabolites leading to potential pathways of toxicity. Using sensitive dose-response experiments, concentrations will be established that trigger these pathways. The gene expression data also will be used to verify these concentrations, since it is known to be a sensitive endpoint for developmental neurotoxicity. Expected outcomeIf the research project is successful, a number of toxicity pathways will be identified that are relevant for DNT. Based on these pathways, a study will be set up to evaluate the DNT potential of chemicals for which there is currently no DNT information. The data will give insight into the usefulness of an in vitro pathway-based testing approach to more efficiently identify DNT chemicals that might contribute to the increasing incidence of neurodevelopmental disorders in children. | |
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