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      Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach

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      Archives of Toxicology
      Springer-Verlag
      Methylmercury, Valproic acid, Transcription factor, Reproductive toxicity, Alternative testing strategies

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          Abstract

          Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)’ European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large ‘common response’ to VPA and MeHg could be distinguished from ‘compound-specific’ responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

          Electronic supplementary material

          The online version of this article (doi:10.1007/s00204-012-0967-3) contains supplementary material, which is available to authorized users.

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          Most cited references56

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          Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei.

          The underlying brain injury that leads to autism has been difficult to identify. The diagnostic criteria of the disease are not readily associated with any brain region or system, nor are they mimicked by vascular accidents, tumors, or degenerative neurological diseases occurring in adults. Fortuitously, a recent report of autism induced by thalidomide exposure provides evidence that the disease originates by an injury at the time of closure of the neural tube. The human data suggest that the initiating lesion includes the motor cranial nerve nuclei. To test this hypothesis, we first examined motor nuclei in the brainstem of a human autistic case. The autopsy brain exhibited near-complete absence of the facial nucleus and superior olive along with shortening of the brainstem between the trapezoid body and the inferior olive. A similar deficit has been reported in Hoxa-1 gene knockout mice in which pattern formation of the hindbrain is disrupted during neurulation. Alternatively, exposure to antimitotic agents just after neural tube closure could produce the observed pattern of deficits. Thus, the lesions observed in the autopsy case appear to match those predicted by the thalidomide cases in both time of origin and central nervous system (CNS) location. To produce similar brain lesions experimentally, we exposed rat embryos to valproic acid, a second teratogen newly linked to autism. Dams received 350 mg/kg of valproic acid (VPA) on day 11.5 (the day of neural tube closure), day 12, or day 12.5 gestation. Each treatment significantly reduced the number of motor neurons counted in matched sections of the earliest-forming motor nuclei (V, XII), and progressively later exposures affected the VIth and IIIrd cranial nerve nuclei. All treatments spared the facial nucleus, which forms still later. Counts from the mesencephalic nucleus of trigeminal, the dorsal motor nucleus of the vagus, and the locus ceruleus were not affected by exposure to VPA, even though these nuclei form during the period when exposure occurred. Despite its effects on the motor nuclei, valproic acid exposure did not alter the further development of the brain in any obvious way. Treated animals were robust and had no external malformations. The autopsy data and experimental data from rats confirm that CNS injuries occurring during or just after neural tube closure can lead to a selective loss of neurons derived from the basal plate of the rhombencephalon. The results add two new lines of evidence that place the initiating injury for autism around the time of neural tube closure.
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            Integration of dosimetry, exposure, and high-throughput screening data in chemical toxicity assessment.

            High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, clearance, and exposure. Hepatic metabolic clearance and plasma protein binding were experimentally measured for 239 ToxCast Phase I chemicals. The experimental data were used in a population-based in vitro-to-in vivo extrapolation model to estimate the daily human oral dose, called the oral equivalent dose, necessary to produce steady-state in vivo blood concentrations equivalent to in vitro AC(50) (concentration at 50% of maximum activity) or lowest effective concentration values across more than 500 in vitro assays. The estimated steady-state oral equivalent doses associated with the in vitro assays were compared with chronic aggregate human oral exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. A total of 18 (9.9%) chemicals for which human oral exposure estimates were available had oral equivalent doses at levels equal to or less than the highest estimated U.S. population exposures. Ranking the chemicals by nominal assay concentrations would have resulted in different chemicals being prioritized. The in vitro assay endpoints with oral equivalent doses lower than the human exposure estimates included cell growth kinetics, cytokine and cytochrome P450 expression, and cytochrome P450 inhibition. The incorporation of dosimetry and exposure provide necessary context for interpretation of in vitro toxicity screening data and are important considerations in determining chemical testing priorities.
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              A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin κ C as a compatible prognostic marker in human solid tumors.

              Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell-derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non-small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining. ©2012 AACR.
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                Author and article information

                Contributors
                +49-221-4787373 , +49-221-4786965 , a.sachinidis@uni-koeln.de
                Journal
                Arch Toxicol
                Arch. Toxicol
                Archives of Toxicology
                Springer-Verlag (Berlin/Heidelberg )
                0340-5761
                1432-0738
                21 November 2012
                21 November 2012
                January 2013
                : 87
                : 1
                : 123-143
                Affiliations
                [ ]Department of Biology, University of Konstanz (UKN), 78457 Constance, Germany
                [ ]OÜ Quretec (Qure), Limited Liability Company, 51003 Tartu, Estonia
                [ ]Institute of Computer Science, University of Tartu, 50409 Tartu, Estonia
                [ ]Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne (UKK), Robert-Koch-Str. 39, 50931 Cologne, Germany
                [ ]Leibniz Research Centre for Working Environment and Human Factors (IfADo), Technical University of Dortmund, 44139 Dortmund, Germany
                [ ]Commission of the European Communities (JRC) Joint Research Centre, 1049 Brussels, Belgium
                [ ]Department of Pathology and Immunology, Geneva Medical Faculty, University of Geneva (UNIGE), 1211 Geneva 4, Switzerland
                [ ]Brunel University (Brunel), Uxbridge, UB8 3PH UK
                [ ]Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek (TNO), 2628 VK Delft, The Netherlands
                [ ]Gottfried Wilhelm Leibniz University (LUH), Institute for Biostatistics, 30167 Hannover, Germany
                [ ]Department of Statistics, TU Dortmund University , 44221 Dortmund, Germany
                Article
                967
                10.1007/s00204-012-0967-3
                3535399
                23179753
                adc99b62-b4b6-4d44-b6f5-9ecce52ccb78
                © The Author(s) 2012
                History
                : 18 October 2012
                : 24 October 2012
                Categories
                Toxicogenomics
                Custom metadata
                © Springer-Verlag Berlin Heidelberg 2013

                Toxicology
                methylmercury,valproic acid,transcription factor,reproductive toxicity,alternative testing strategies

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