Zebrafish and nematodes as whole organism models to measure developmental neurotoxicity.

Despite the growing epidemiological evidence of an association between toxin exposure and developmental neurotoxicity (DNT), systematic testing of DNT is not mandatory in international regulations for admission of pharmaceuticals or industrial chemicals. However, to date around 200 compounds, ranging from pesticides, pharmaceuticals and industrial chemicals, have been tested for DNT in the current OECD test guidelines (TG-443 or TG-426). There are calls for the development of new approach methodologies (NAMs) for DNT, which has resulted in a DNT testing battery using in vitro human cell-based assays. These assays provide a means to elucidate the molecular mechanisms of toxicity in humans which is lacking in animal-based toxicity tests. However, cell-based assays do not represent all steps of the complex process leading to DNT. Validated models with a multi-organ network of pathways that interact at the molecular, cellular and tissue level at very specific timepoints in a life cycle are currently missing. Consequently, whole model organisms are being developed to screen for, and causally link, new molecular targets of DNT compounds and how they affect whole brain development and neurobehavioral endpoints. Given the practical and ethical restraints associated with vertebrate testing, lower animal models that qualify as 3 R (reduce, refine and replace) models, including the nematode (Caenorhabditis elegans) and the zebrafish (Danio rerio) will prove particularly valuable for unravelling toxicity pathways leading to DNT. Although not as complex as the human brain, these 3 R-models develop a complete functioning brain with numerous neurodevelopmental processes overlapping with human brain development. Importantly, the main signalling pathways relating to (neuro)development, metabolism and growth are highly conserved in these models. We propose the use of whole model organisms specifically zebrafish and C. elegans for DNT relevant endpoints.

A prioritization strategy for functional alternatives to bisphenol A in food contact materials.

The use of bisphenol A (BPA), a substance of very high concern, is proposed to be banned in food contact materials (FCMs) in the European Union. To prevent regrettable substitution of BPA by alternatives with similar or unknown hazardous properties, it is of importance to gain the relevant toxicological information on potential BPA alternative substances and monitor them adequately. We created an inventory of over 300 substances mentioned as potential BPA alternatives in regulatory reports and scientific literature. This study presents a prioritization strategy to identify substances that may be used as an alternative to BPA in FCMs. We prioritized 20 potential BPA alternatives of which 10 are less familiar. We subsequently reviewed the available information on the 10 prioritized less familiar substances regarding hazard profiles and migration potential obtained from scientific literature and in silico screening tools to identify a possible risk of the substances. Major data gaps regarding the hazard profiles of the prioritized substances exist, although the scarce available data give some indications on the possible hazard for some of the substances (like bisphenol TMC, 4,4-dihydroxybenzophenone, and tetrachlorobisphenol A). In addition, very little is known about the actual use and exposure to these substances. More toxicological research and monitoring of these substances in FCMs are, therefore, required to avoid regrettable substitution of BPA in FCM.

Adopting Mechanistic Molecular Biology Approaches in Exposome Research for Causal Understanding.

Through investigating the combined impact of the environmental exposures experienced by an individual throughout their lifetime, exposome research provides opportunities to understand and mitigate negative health outcomes. While current exposome research is driven by epidemiological studies that identify associations between exposures and effects, new frameworks integrating more substantial population-level metadata, including electronic health and administrative records, will shed further light on characterizing environmental exposure risks. Molecular biology offers methods and concepts to study the biological and health impacts of exposomes in experimental and computational systems. Of particular importance is the growing use of omics readouts in epidemiological and clinical studies. This paper calls for the adoption of mechanistic molecular biology approaches in exposome research as an essential step in understanding the genotype and exposure interactions underlying human phenotypes. A series of recommendations are presented to make the necessary and appropriate steps to move from exposure association to causation, with a huge potential to inform precision medicine and population health. This includes establishing hypothesis-driven laboratory testing within the exposome field, supported by appropriate methods to read across from model systems research to human.

Prolonged Differentiation of Neuron-Astrocyte Co-Cultures Results in Emergence of Dopaminergic Neurons.

Dopamine is present in a subgroup of neurons that are vital for normal brain functioning. Disruption of the dopaminergic system, e.g., by chemical compounds, contributes to the development of Parkinson's disease and potentially some neurodevelopmental disorders. Current test guidelines for chemical safety assessment do not include specific endpoints for dopamine disruption. Therefore, there is a need for the human-relevant assessment of (developmental) neurotoxicity related to dopamine disruption. The aim of this study was to determine the biological domain related to dopaminergic neurons of a human stem cell-based in vitro test, the human neural progenitor test (hNPT). Neural progenitor cells were differentiated in a neuron-astrocyte co-culture for 70 days, and dopamine-related gene and protein expression was investigated. Expression of genes specific for dopaminergic differentiation and functioning, such as LMX1B, NURR1, TH, SLC6A3, and KCNJ6, were increasing by day 14. From day 42, a network of neurons expressing the catecholamine marker TH and the dopaminergic markers VMAT2 and DAT was present. These results confirm stable gene and protein expression of dopaminergic markers in hNPT. Further characterization and chemical testing are needed to investigate if the model might be relevant in a testing strategy to test the neurotoxicity of the dopaminergic system.

Occupational exposure to hexavalent chromium. Part I. Hazard assessment of non-cancer health effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review focuses on non-cancer health effects. It was concluded that occupational exposure to Cr(VI) can cause perforation of the nasal septum by chromium ulcers, chronic lung diseases, including asthma, rhinitis, pulmonary fibrosis and COPD, skin ulcers and allergic contact dermatitis in humans. It is currently insufficiently clear if Cr(VI) can cause irreversible diseases due to disturbances of the immune system (other than allergic contact eczema, allergic asthma and rhinitis and chronic lung diseases) or adverse effects on fertility or prenatal development in humans.

Occupational exposure to hexavalent chromium. Part II. Hazard assessment of carcinogenic effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities on NATO equipment. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review provides the hazard assessment for occupational exposure to Cr(VI) and carcinogenic effects by integrating and weighting evidence provided by international agencies complemented with newly published studies. It was concluded that occupational exposure to Cr(VI) can cause lung cancer, nose and nasal sinus cancer in humans. Cr(VI) is suspected to cause stomach cancer and laryngeal cancer in humans. It is currently insufficiently clear if Cr(VI) can cause cancer of the small intestine, oral cavity, pancreas, prostate or bladder in humans.

An update on the hazard of and exposure to diethyl hexyl phthalate (DEHP) alternatives used in medical devices.

The use of the plasticizer diethyl hexyl phthalate (DEHP) in PVC medical devices is being questioned due to its potential reprotoxic effects in patients exposed as a result from migration from the device. This article reviews new information on migration and toxicity data of eleven alternative plasticizers that have previously been evaluated by the Danish EPA and the EU SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks). The new toxicity data did not justify the reconsideration of the critical NOAELs as established by SCENIHR and Danish EPA. The dataset on oral toxicity studies is rather complete for most substances; however, in particular for reproductive toxicity and endocrine disruption, data gaps still exist for many alternatives. Toxicity data on intravenous exposure are lacking and these are essential to conclude on hazard characteristics of alternatives that are poorly absorbed via the oral exposure route. Migration data are emerging for a few alternatives but still sparse for the majority of the alternatives. Taking all data on migration and toxicity in consideration, 1,2-cyclohexanedicarboxylic acid, diisononylester (DINCH), and tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate display a more favorable profile compared to DEHP. For these promising alternatives, a risk assessment for use in medical devices should be conducted. As a next step, we recommend the (further) generation of relevant migration data and, where needed, relevant toxicity data for the alternative substances, in order to be able to conduct a benefit-risk analysis of DEHP and the alternatives as obligatory in the new European Union Medical Device Regulation.

Substitution of bisphenol A: a review of the carcinogenicity, reproductive toxicity, and endocrine disruption potential of alternative substances.

The use of bisphenol A (BPA) is restricted due to its reproductive toxicity and endocrine disrupting (ED) properties. The public concern and regulatory restrictions on BPA stimulated the development of alternative substances to replace BPA. The aim of this study is to review the available data on carcinogenic, mutagenic, reproductive toxicity, and ED properties of BPA alternatives used in consumer products. The focus is on the potential hazard for (young) children and/or pregnant women. An inventory of known potential alternative substances (n = 99) was made, of which 20 were prioritized based on reported use by the general population. For all the selected alternatives, data on ED potential, carcinogenicity and reproductive toxicity was very limited or even absent (i.e. Tefacid Stearic 95, Bisphenol C, AP, and P). For the alternative substances bisphenol S (BPS), bisphenol AF (BPAF), p-tert-butylphenol and to a lesser extent bisphenol F (BPF), fluorine-9-bisphenol (BHPF), bisphenol E, M, and Z (BPE, BPM, BPZ), Irganox 1076, and butylated hydroxytoluene (BHT), the data indicates a reproductive toxicity hazard with a possible ED mode of action. 3,3',5,5'-Tetrabromobisphenol A (TBBPA) tested positive for carcinogenicity. Data gaps are present for most of these substances. In this study, data on reproductive toxicity and/or ED potential were only negative, although not complete, for benzoic acid and Irganox 1010, tetra methyl bis phenol F (TMBPF) and bisphenol-A bis(diphenyl phosphate) (BDP). A full evaluation of all data, including in vitro data, is recommended to guide targeted testing prioritization.

Design and validation of an ontology-driven animal-free testing strategy for developmental neurotoxicity testing.

Developmental neurotoxicity entails one of the most complex areas in toxicology. Animal studies provide only limited information as to human relevance. A multitude of alternative models have been developed over the years, providing insights into mechanisms of action. We give an overview of fundamental processes in neural tube formation, brain development and neural specification, aiming at illustrating complexity rather than comprehensiveness. We also give a flavor of the wealth of alternative methods in this area. Given the impressive progress in mechanistic knowledge of human biology and toxicology, the time is right for a conceptual approach for designing testing strategies that cover the integral mechanistic landscape of developmental neurotoxicity. The ontology approach provides a framework for defining this landscape, upon which an integral in silico model for predicting toxicity can be built. It subsequently directs the selection of in vitro assays for rate-limiting events in the biological network, to feed parameter tuning in the model, leading to prediction of the toxicological outcome. Validation of such models requires primary attention to coverage of the biological domain, rather than classical predictive value of individual tests. Proofs of concept for such an approach are already available. The challenge is in mining modern biology, toxicology and chemical information to feed intelligent designs, which will define testing strategies for neurodevelopmental toxicity testing.

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. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

Mapping of a FEB3 homologous febrile seizure locus on mouse chromosome 2 containing candidate genes Scn1a and Scn3a.

Febrile seizures (FS) are the most common seizure type in children. Recurrent FS are a risk factor for developing temporal lobe epilepsy later in life and are known to have a strong genetic component. Experimental FS (eFS) can be elicited in mice by warm-air induced hyperthermia. We used this model to screen the chromosome substitution strain (CSS) panel derived from C57BL/6J and A/J for FS susceptibility and identified C57BL/6J-Chr2A /NaJ (CSS2), as the strain with the strongest FS susceptibility phenotype. The aim of this study was to map FS susceptibility loci and select candidate genes on mouse chromosome 2. We generated an F2 population by intercrossing the hybrids (F1 ) that were derived from CSS2 and C57BL/6J mice. All CSS2-F2 individuals were genotyped and phenotyped for eFS susceptibility, and QTL analysis was performed. Candidate gene selection was based on bioinformatics analyses and differential brain expression between CSS2 and C57BL/6J strains determined by microarray analysis. Genetic mapping of the eFS susceptibility trait identified two significant loci: FS-QTL2a (LOD-score 3.6) and FS-QTL2b (LOD-score 6.2). FS-QTL2a contained 44 genes expressed in the brain at post natal day 14. Four of these (Arl6ip6, Cytip, Fmnl2 Ifih1) contained a non-synonymous SNP comparing CSS2 and C57BL/6J, six genes (March7, Nr4a2, Gpd2, Grb14, Scn1a, Scn3a) were differentially expressed between these strains. A region within FS-QTL2a is homologous to the human FEB3 locus. The fact that we identify mouse FS-QTL2a with high FEB3 homology is strong support for the validity of the eFS mouse model to study genetics of human FS.

Developmental immunotoxicity of chemicals in rodents and its possible regulatory impact.

Around 25% of the children in developed countries are affected with immune-based diseases. Juvenile onset diseases such as allergic, inflammatory and autoimmune diseases have shown increasing prevalences in the last decades. The role of chemical exposures in these phenomena is unclear. It is thought that the developmental immune system is more susceptible to toxicants than the mature situation. Developmental immunotoxicity (DIT) testing is nowadays not or minimally included in regulatory toxicology requirements. We reviewed whether developmental immune parameters in rodents would provide relatively sensitive endpoints of toxicity, whose inclusion in regulatory toxicity testing might improve hazard identification and risk assessment of chemicals. For each of the nine reviewed toxicants, the developing immune system was found to be at least as sensitive or more sensitive than the general (developmental) toxicity parameters. Functional immune (antigen-challenged) parameters appear more affected than structural (non-challenged) immune parameters. Especially, antibody responses to immune challenges with keyhole limpet hemocyanine or sheep red blood cells and delayed-type hypersensitivity responses appear to provide sensitive parameters of developmental immune toxicity. Comparison with current tolerable daily intakes (TDI) and their underlying overall no observed adverse effect levels showed that for some of the compounds reviewed, the TDI may need reconsideration based on developmental immune parameters. From these data, it can be concluded that the developing immune system is very sensitive to the disruption of toxicants independent of study design. Consideration of including functional DIT parameters in current hazard identification guidelines and wider application of relevant study protocols is warranted.

Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats.

Mesiotemporal sclerosis (MTS), the most frequent form of drug-resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)-induced changes in expression of key components in the glutamate-glutamine cycle, known to be affected in MTS patients. SE was induced by Li(+) /pilocarpine injection in 21-day-old rats. At 2-19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15-18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin-positive GABAergic interneurons. Hilar neuron loss (or end-folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)-immunoreactivity from 2 (-15%) to 19 weeks (-33.5%) after SE. Immunoreactivity for excitatory amino-acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21-day-old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate-glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis.

Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1A.

Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36 × 10(-9), odds ratio (A) = 1.42, 95% confidence interval: 1.26-1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures.

How neurobehavior and brain development in alternative whole-organism models can contribute to prediction of developmental neurotoxicity.

Developmental neurotoxicity (DNT) is not routinely evaluated in chemical risk assessment because current test paradigms for DNT require the use of mammalian models which are ethically controversial, expensive, and resource demanding. Consequently, efforts have focused on revolutionizing DNT testing through affordable novel alternative methods for risk assessment. The goal is to develop a DNT in vitro test battery amenable to high-throughput screening (HTS). Currently, the DNT in vitro test battery consists primarily of human cell-based assays because of their immediate relevance to human health. However, such cell-based assays alone are unable to capture the complexity of a developing nervous system. Whole organismal systems that qualify as 3 R (Replace, Reduce and Refine) models are urgently needed to complement cell-based DNT testing. These models can provide the necessary organismal context and be used to explore the impact of chemicals on brain function by linking molecular and/or cellular changes to behavioural readouts. The nematode Caenorhabditis elegans, the planarian Dugesia japonica, and embryos of the zebrafish Danio rerio are all suited to low-cost HTS and each has unique strengths for DNT testing. Here, we review the strengths and the complementarity of these organisms in a novel, integrative context and highlight how they can augment current cell-based assays for more comprehensive and robust DNT screening of chemicals. Considering the limitations of all in vitro test systems, we discuss how a smart combinatory use of these systems will contribute to a better human relevant risk assessment of chemicals that considers the complexity of the developing brain.

Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response.

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disorder characterized by recurrent seizures. The pathogenic mechanisms underlying mTLE may involve defects in the post-transcriptional regulation of gene expression. MicroRNAs (miRNAs) are non-coding RNAs that control the expression of genes at the post-transcriptional level. Here, we performed a genome-wide miRNA profiling study to examine whether miRNA-mediated mechanisms are affected in human mTLE. miRNA profiles of the hippocampus of autopsy control patients and two mTLE patient groups were compared. This revealed segregated miRNA signatures for the three different patient groups and 165 miRNAs with up- or down-regulated expression in mTLE. miRNA in situ hybridization detected cell type-specific changes in miRNA expression and an abnormal nuclear localization of select miRNAs in neurons and glial cells of mTLE patients. Of several cellular processes implicated in mTLE, the immune response was most prominently targeted by deregulated miRNAs. Enhanced expression of inflammatory mediators was paralleled by a reduction in miRNAs that were found to target the 3'-untranslated regions of these genes in reporter assays. miR-221 and miR-222 were shown to regulate endogenous ICAM1 expression and were selectively co-expressed with ICAM1 in astrocytes in mTLE patients. Our findings suggest that miRNA changes in mTLE affect the expression of immunomodulatory proteins thereby further facilitating the immune response. This mechanism may have broad implications given the central role of astrocytes and the immune system in human neurological disease. Overall, this work extends the current concepts of human mTLE pathogenesis to the level of miRNA-mediated gene regulation.

Identification of candidate genes for developmental colour agnosia in a single unique family.

Colour agnosia is a disorder that impairs colour knowledge (naming, recognition) despite intact colour perception. Previously, we have identified the first and only-known family with hereditary developmental colour agnosia. The aim of the current study was to explore genomic regions and candidate genes that potentially cause this trait in this family. For three family members with developmental colour agnosia and three unaffected family members CGH-array analysis and exome sequencing was performed, and linkage analysis was carried out using DominantMapper, resulting in the identification of 19 cosegregating chromosomal regions. Whole exome sequencing resulted in 11 rare coding variants present in all affected family members with developmental colour agnosia and absent in unaffected members. These variants affected genes that have been implicated in neural processes and functions (CACNA2D4, DDX25, GRINA, MYO15A) or that have an indirect link to brain function, development or disease (MAML2, STAU1, TMED3, RABEPK), and a remaining group lacking brain expression or involved in non-neural traits (DEPDC7, OR1J1, OR8D4). Although this is an explorative study, the small set of candidate genes that could serve as a starting point for unravelling mechanisms of higher level cognitive functions and cortical specialization, and disorders therein such as developmental colour agnosia.

Mapping an X-linked locus that influences heat-induced febrile seizures in mice.

PURPOSE: Febrile seizures (FS) are the most common seizure type in children between the age of 6 months and 5 years. Although FS are largely benign, recurrent FS are a major risk factor for developing temporal lobe epilepsy (TLE) later in life. The mechanisms underlying FS are largely unknown; however, family and twin studies indicate that FS susceptibility is under complex genetic control. We have recently developed a phenotypic screen to study the genetics of FS susceptibility in mice. Using this screen in a phenotype-driven genetic strategy we analyzed the C57BL/6J-Chr #(A)/NaJ chromosome substitution strain (CSS) panel. In each CSS line one chromosome of the A/J strain is substituted in a genetically homogeneous C57BL/6J background. The analysis of the CSS panel revealed that A/J chromosomes 1, 2, 6, 10, 13, and X carry at least one quantitative trait locus (QTL) for heat-induced FS susceptibility. The fact that many X-linked genes are highly expressed in the brain and have been implicated in human developmental disorders often presenting with seizures (like fragile X mental retardation) prompted us to map the chromosome X QTL. METHODS: C57BL/6J mice were mated with C57BL/6J-Chr X(A) /NaJ (CSSX) to generate F(2)-generations-CXBL6 and BL6CX-originating from CSSX or C57BL/6J mothers, respectively. Heat-induced FS were elicited on postnatal day 14 by exposure to a controlled warm airstream of 50°C. The latency to heat-induced FS is our phenotype. This phenotype has previously been validated by video-electroencephalography (EEG) monitoring. After phenotyping and genotyping the F(2)-population, QTL analysis was performed using R/QTL software. KEY FINDINGS: QTL analysis revealed a significant peak with an LOD-score of 3.25. The 1-LOD confidence interval (149,886,866-158,836,462 bp) comprises 52 protein coding genes, of which 34 are known to be brain expressed. Two of these brain-expressed genes have previously been linked to X-linked epilepsies, namely Cdkl5 and Pdha1. SIGNIFICANCE: Our results show that the mouse genetics of X-linked FS susceptibility is complex, and that our heat-induced FS-driven genetic approach is a powerful tool for use in unraveling the complexities of this trait in mice. Fine-mapping and functional studies will be required to further identify the X-linked FS susceptibility genes.

Neuronal differentiation pathways and compound-induced developmental neurotoxicity in the human neural progenitor cell test (hNPT) revealed by RNA-seq.

There is an increased awareness that the use of animals for compound-induced developmental neurotoxicity (DNT) testing has limitations. Animal-free innovations, especially the ones based on human stem cell-based models are pivotal in studying DNT since they can mimic processes relevant to human brain development. Here we present the human neural progenitor test (hNPT), a 10-day protocol in which neural progenitor cells differentiate into a neuron-astrocyte co-culture. The study aimed to characterise differentiation over time and to find neurodevelopmental processes sensitive to compound exposure using transcriptomics. 3992 genes regulated in unexposed control cultures (p ≤ 0.001, log2FC ≥ 1) showed Gene Ontology (GO-) term enrichment for neuronal and glial differentiation, neurite extension, synaptogenesis, and synaptic transmission. Exposure to known or suspected DNT compounds (acrylamide, chlorpyrifos, fluoxetine, methyl mercury, or valproic acid) at concentrations resulting in 95% cell viability each regulated unique combinations of GO-terms relating to neural progenitor proliferation, neuronal and glial differentiation, axon development, synaptogenesis, synaptic transmission, and apoptosis. Investigation of the GO-terms 'neuron apoptotic process' and 'axon development' revealed common genes that were responsive across compounds, and might be used as biomarkers for DNT. The GO-term 'synaptic signalling', on the contrary, whilst also responsive to all compounds tested, showed little overlap in gene expression regulation patterns between the conditions. This GO-term may articulate compound-specific effects that may be relevant for revealing differences in mechanism of toxicity. Given its focus on neural progenitor cell to mature multilineage neuronal cell maturation and its detailed molecular readout based on gene expression analysis, hNPT might have added value as a tool for neurodevelopmental toxicity testing in vitro. Further assessment of DNT-specific biomarkers that represent these processes needs further studies.

Exploring the biological domain of the neural embryonic stem cell test (ESTn): Morphogenetic regulators, Hox genes and cell types, and their usefulness as biomarkers for embryotoxicity screening.

Animal-free assessment of compound-induced developmental neurotoxicity will most likely be based on batteries of multiple in vitro tests. The optimal battery is built by combining tests with complementary biological domains that together ideally cover all relevant toxicity pathways. Thus, biological domain definition, i.e. which biological processes and cell types are represented, is an important assay characteristic for determining the place of assays in testing strategies. The murine neural embryonic stem cell test (ESTn) is employed to predict the developmental neurotoxicity of compounds. The aim of this study was to explore the biological domain of ESTn according to three groups of biomarker genes of early (neuro)development: morphogenetic regulators, Hox genes and cell type markers for the ectodermal and neural lineages. These biomarker groups were selected based on their crucial regulatory role in (neuro)development. Analysis of these genes in a series of previously generated whole transcriptome datasets of ESTn showed that at day 7 in culture cell differentiation resembled hindbrain/branchial/thoracic development between E6.5-E12.5 in vivo, with subsequent development into a mixed cell culture containing different neural subtypes, astrocytes and oligodendrocytes by day 13. In addition, the selected biomarkers showed common and distinct responses to compound exposure. Monitoring the biological domain of ESTn through gene expression patterns of morphogenetic regulators, Hox genes and cell type markers proved instrumental in providing mechanistic understanding of compound effects on neural differentiation in ESTn, and can aid in positioning of the test in a battery of complementary in vitro tests in integrated approaches to testing and assessment.