The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information.

Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.

To investigate the neurotoxicity profile of glycidol and its effect on developmental hippocampal neurogenesis, pregnant Sprague Dawley rats were given drinking water containing 0, 100, 300, or 1000 ppm glycidol from gestational day 6 until weaning on day 21 after delivery. At 1000 ppm, dams showed progressively worsening gait abnormalities, and histopathological examination showed generation of neurofilament-L(+) spheroids in the cerebellar granule layer and dorsal funiculus of the medulla oblongata, central chromatolysis in the trigeminal nerve ganglion cells, and axonal degeneration in the sciatic nerves. Decreased dihydropyrimidinase-like 3(+) immature granule cells in the subgranular zone (SGZ) and increased immature reelin(+) or calbindin-2(+) γ-aminobutyric acid-ergic interneurons and neuron-specific nuclear protein (NeuN)(+) mature neurons were found in the dentate hilus of the offspring of the 1000 ppm group on weaning. Hilar changes remained until postnatal day 77, with the increases in reelin(+) and NeuN(+) cells being present at ≥ 300 ppm, although the SGZ change disappeared. Thus, glycidol caused axon injury in the central and peripheral nervous systems of adult rats, suggesting that glycidol targets the newly generating nerve terminals of immature granule cells, resulting in the suppression of late-stage hippocampal neurogenesis. The sustained hilar changes may be a sign of continued aberrations in neurogenesis and migration. The no-observed-adverse-effect level was determined to be 300 ppm (48.8mg/kg body weight/day) for dams and 100 ppm (18.5mg/kg body weight/day) for offspring. The sustained developmental exposure effect on offspring neurogenesis was more sensitive than the adult axonal injury.

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Valproic acid (VPA) is used to establish models of experimental autism. The present study investigated the developmental exposure effect of VPA on postnatal hippocampal neurogenesis in accordance with the exposure scheme of OECD Test Guideline 426 adopted for developmental neurotoxicity. Pregnant rats were administered drinking water containing 0, 667, or 2000 ppm VPA from gestational day 6 until day 21 post-delivery. In the subgranular zone (SGZ) and granule cell layer (GCL) of offspring, the number of granule cell lineage subpopulations remained unchanged upon weaning. However, in the hilus of the dentate gyrus, the number of reelin interneurons decreased at ≥667 ppm, and the number of PVALB or GAD67 interneurons decreased at 2000 ppm. Conversely, Reln and Gad1 transcript levels increased at 2000 ppm, but Pvalb and Grin2d decreased, in the dentate gyrus. At the adult stage, PCNA proliferating SGZ cells, NeuN postmitotic SGZ/GCL neurons, and ARC or COX2 GCL neurons increased at ≥667 ppm. In the dentate hilus, decreases in GAD67 interneuron subpopulations and Grin2d transcript levels sustained at 2000 ppm. These results suggested that VPA primarily targets interneurons by developmental exposure, and this is followed by late effects on granule cell lineages, likely by influencing SGZ cell proliferation and synaptic plasticity. A reduced population of reelin or PVALB interneurons did not affect distribution of granule cell lineage subpopulations upon weaning. The late effect on neurogenesis, which resulted in increased GCL neurons, might be the result of a sustained decrease in GAD67 interneurons expressing NR2D encoded by Grin2d.

-For molecular analysis in anatomically-specific brain regions for rodent studies, it is necessary to establish a fast and accurate procedure for tissue sampling to achieve high integrity and expression fidelity of extracted molecules. The present study was performed to examine suitability of whole brain fixation with methacarn and subsequent tissue sampling using punch-biopsy devices for gene expression analysis in rats. After fixation, each specific region, i.e., hippocampal dentate gyrus, corpus callosum, cingulate cortex or cerebellar vermis was collected, and the integrity and variability of expression data of extracted total RNAs and polypeptides were examined. Methacarn fixation, acetone fixation, and unfixed tissues were compared. Methacarn fixation resulted in high integrity of total RNAs sufficient for conducting global expression analysis and superior in terms of uniformity in the integrity among brain regions to that of acetone fixation. Extracted polypeptide after methacarn fixation revealed similar integrity to that without fixation or with acetone fixation. Methacarn fixation resulted in lower mRNA expression variability between samples than acetone fixation in microarray analysis. The fidelity of polypeptide expression was mostly equivalent between methacarn and acetone fixation in 2-dimensional differential in-gel electrophoresis, although the expression levels of a small number of polypeptides from acetonefixed tissues were affected. These results suggest that whole brain fixation with methacarn retains advantages for global analyses of mRNAs and polypeptides in rodent studies. Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.38, No.3, 431-443, 2013 Vol. 38 No. 3 431 throughput and use of fixed brain samples may be preferable for this purpose.Methacarn is a protein-precipitating and non-crosslinking organic solvent fixative (Mitchell et al., 1985;Puchtler et al., 1970), and fixation with this solution enables preservation of tissue morphology for histopathological assessment in paraffin-embedded tissues (Delfour et al., 2006;Srinivasan et al., 2002). Methacarn generally gives superior immunohistochemical results over aldehyde-based cross-linking fixatives (Banks, 1979;Orstavik et al., 1981;Rognum et al., 1980) because antigenicity is usually maintained (Mitchell et al., 1985). We previously found that methacarn fixation yields high quality DNA, RNA and protein even in paraffin-embedded sections for analyses of genomic DNA, expression microarrays, real-time RT-PCR and western blotting (Shibutani et al., 2000;Shibutani and Uneyama, 2002;Takagi et al., 2004;.For global assessment of genes and proteins in anatomically-specific brain regions in animal studies using rodents, both high integrity of extracted molecules and minimal inter-animal variability in the expression of extracted macromolecules are essential. Therefore, establishment of a procedure for quick and accurate tissue sampling and processing which can overcome these hurdles is necessary. For th...