There exists critical concern that exposures to drugs and chemicals during early life contribute to the increasing incidence of neurodevelopmental disorders, such as lowered IQ, learning disabilities, autism and attention deficit and hyperactivity disorder (ADHD). The developing brain is more susceptible to substance-induced injury compared to the adult brain due to the complex developmental processes, the absence of a functional blood/brain-barrier and a diminished ability to detoxify chemicals. Demanding animal tests have been devised, but because of low frequencies of hazardous substances and manifestations, as well as complex underlying mechanisms, limitations of current approaches are enormous. The area is therefore of key interest for new approaches, as outlined in the NRC vision document for a toxicology in the 21st century and the critical path initiative. The applicants have steering such a paradigm change. As a pilot reproductive toxicity concern, we propose to address the developmental neurotoxicity area, since this is a stand-alone health issue not adequately covered by current testing strategies. It represents a key element in the current transition from the two-generation study to an extended one-generation study and profits from an explosion of knowledge from basic science. In a 6-year process with critical involvement of the applicants, including three international conferences and a workshop co-organized, promising models and prototypic test substances have been identified. Extending our recent metabolomics and genomics approaches, a systems toxicology approach shall be applied now, in order to identify and validate critical pathways of toxicity (PoT). PoT-specific reporter gene models shall then be established allowing higher test throughput. We will make use of our rat primary three- dimensional organotypic in vitro model, i.e. aggregating brain cell cultures, which was shown to closely reproduce the in vivo situation of the CNS and neurotoxic mechanisms. Moreover, neurodevelopment processes have been well characterized, making the model relevant for DNT studies. Cell cultures will be exposed to DNT reference compounds during development and the expression levels of cell-specific genes will be quantified by real-time PCR. Furthermore, low molecular weight metabolites relevant for neurodevelopment will be quantified by mass spectrometry based metabolomics. Results shall give insight into the specific cell types targeted and the neurodevelopmental alterations induced. The creation of reporter gene assays in murine embryonic stem cells shall make PoT-specific testing with higher throughput possible. This will advance our understanding of DNT and test capabilities.
A 3D-organotypic rat brain cell model will be combined with mass-spectrum-based metabolomics to identify pathways of developmental neurotoxicity. After pathway validation, pathway-specific reporter gene assays are constructed to allow higher throughput testing. Such assays promise to overcome some limitations of current reproductive and neurodevelopmental toxicity testing.
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