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Omics technologies for the prediction of immunmodulatory effects
S230
Abstracts / Toxicology Letters 238S (2015) S56–S383
tions initiated by exposure to environmental stressors; and (b) identify which of these perturbations overcome the homeostasis barrier, resulting in observed alterations of the cell/tissue environment and eventually to pathologic phenotypes. Towards this aim, integrated exposure biology provides the methodological elements for the surveillance of changes at different levels of biological organization through the use of the full array of -omics and post-omics technologies including epigenomics. Starting from untargeted transcriptomics and metabolomics we proceed with joint analysis of biological processes induced by exposure to xenobiotics at the molecular level and of metabolic processes induced in parallel. Dynamic flux balance analysis is a key element in the joint interpretation of gene expression data and metabolite profiles. This allows us to identify putative pathways of toxicity, which need to be verified by targeted multi-omics and functional assays. Identification of the functional links among the data derived from different high throughput testing platforms and their proper interpretation are supported by advanced bioinformatics such as support vector machines and clustering algorithms and systems biology models. Examples of the dawn of the exposure biology era are given and the future perspectives discussed in the context of supporting efficiently exposome studies.
state” broadens the view of chronic toxicity with a way to directly compare with the chemical dosing studies using gene modified mice. http://dx.doi.org/10.1016/j.toxlet.2015.08.681
P10-012 Omics technologies for the prediction of immunmodulatory effects P. Zwicker 1,∗ , N. Schultze 1 , S. Niehs 2 , K. Methling 2 , M. Wurster 2 , J. Bernhardt 3 , G. Wachlin 3 , M. Lalk 2 , R. Preissner 4 , U. Lindequist 1 , B. Haertel 1 1
University of Greifswald, Institute of Pharmacy, Greifswald, Germany 2 University of Greifswald, Institute of Biochemistry, Greifswald, Germany 3 University of Greifswald, Institute of Microbiology, Greifswald, Germany 4 Charité University Medicine, Institute of Physiology and ECRC, Berlin, Germany
http://dx.doi.org/10.1016/j.toxlet.2015.08.680
P10-011 Percellome toxicogenomics for mechanistic analysis towards chronic toxicity by a newly designed repeated dose study J. Kanno ∗ , S. Kitajima, K.-I. Aisaki National Institute of Health Sciences, Division of Cellular & Molecular Toxicology, Tokyo, Japan The Percellome Project aims at reinforcing and eventually replacing the “safety factor (uncertainty factor)” currently used for the extrapolation of experimental animal data to humans. Our project attempts to comprehensively identify the transcriptomic networks induced by xenobiotics. For this attempt, we adopted phenotype-independent approach, because not all changes in mRNA expression can be anchored to overt phenotypes. Consequently, there emerged a need to pile up certain amount of transcriptomic data. This situation is analogous to when the electron microscope had invented; to gain consensus on its new images, database for textbooks and atlases was needed. For this need, “Percellome” method was developed (BMC Genomics 7:64, 2006) to generate absolute copy numbers of each mRNA per one cell (in average). By this method, data from the Affymetrix MOE430 2.0 GeneChip are absolutized and visualized in 3-D graphs (time × dose × copy number per cell). Up to now, datasets of mouse liver (4 time points × 4 dose levels, triplicate, 48 GeneChip data per chemical) on more than 100 chemicals are compiled. Here, we report the effect of pretreatment of mice with repeated dose of carbon tetrachloride (CCl4 ) on single dose of various chemicals at 2, 4, 8 and 24 h. This study design is a derivative of a single dose study using gene knockout mouse. Instead of using knockout mouse, mouse pretreated with repeated dose of a chemical is considered to be in a “chemically-induced transgenic state”. Various chemicals include CCl4 itself, clofibrate and phenobarbital. Repeated dose of CCl4 induced suppression of baseline expression levels of genes related to ER stress and attenuated the effect of chemicals given single dose. The mechanisms suggested to be related to this phenomenon included mTOR signaling. Analysis strategy by the new concept of repeated dose study, i.e. “chemically-induced transgenic
Ethical concerns and low predictability of animal data to humans gave rise for the development of different in vitro assays for the evaluation of immunotoxicity. In this study, a proteomic approach, combined with metabolomics and functional assays was developed to evaluate immunmodulatory effects of natural compounds on a human T cell line (Jurkat) in vitro. For the proteomic analysis, a 2D gel electrophoresis based assay together with an antibody array was used. Additionally, intra- and extracellular metabolites were measured with GC–MS and 1 H NMR. Functional tests as apoptosis, cell cycle and intracellular reactive oxygen species (iROS) assays were an additional tool for the interpretation of the obtained data. The measurement of cytokine levels will also be part of the approach. The cytotoxic and immunosuppressive substances Vincristine (VCR), Cannabidiol (CBD), Deoxynivalenol (DON) and the immunostimulating compound Tulipalin A (TUPA) were investigated. General cytotoxic effects of the substances were measured using the MTT assay. Concentrations that lead to a decrease in cell viability of 10% (IC10 ) were used for further investigations. This decrease is achieved by concentrations of 0.45 nM (VCR), 8.65 M (CBD), 0.47 M (DON) and 26.8 M (TUPA). Jurkat cells treated with the substances in low doses (IC10 ) revealed alterations in the abundance of different proteins. The reaction to the treatment with CBD has similarities to the reaction to TUPA. Affected biological processes are for example cell cycle, stress response and apoptosis. In contrast, VCR showed only few shifts in protein formation and low similarity to the affected processes after treatment with CBD and TUPA. The metabolomic analysis indicates a similar tendency in the quantity of altered extra- and intracellular metabolites in comparison to proteomic data. Functional assays support alterations in cell cycle progression and apoptosis. Changes in cytokine levels have to be validated. In summary, a comprehensive approach to investigate immunmodulatory effects is established. However, more substances have to be screened for their effects on different human cell lines. Final aim is the creation of a database for the prediction of immunotoxic effects. http://dx.doi.org/10.1016/j.toxlet.2015.08.682
Abstracts / Toxicology Letters 238S (2015) S56–S383
tions initiated by exposure to environmental stressors; and (b) identify which of these perturbations overcome the homeostasis barrier, resulting in observed alterations of the cell/tissue environment and eventually to pathologic phenotypes. Towards this aim, integrated exposure biology provides the methodological elements for the surveillance of changes at different levels of biological organization through the use of the full array of -omics and post-omics technologies including epigenomics. Starting from untargeted transcriptomics and metabolomics we proceed with joint analysis of biological processes induced by exposure to xenobiotics at the molecular level and of metabolic processes induced in parallel. Dynamic flux balance analysis is a key element in the joint interpretation of gene expression data and metabolite profiles. This allows us to identify putative pathways of toxicity, which need to be verified by targeted multi-omics and functional assays. Identification of the functional links among the data derived from different high throughput testing platforms and their proper interpretation are supported by advanced bioinformatics such as support vector machines and clustering algorithms and systems biology models. Examples of the dawn of the exposure biology era are given and the future perspectives discussed in the context of supporting efficiently exposome studies.
state” broadens the view of chronic toxicity with a way to directly compare with the chemical dosing studies using gene modified mice. http://dx.doi.org/10.1016/j.toxlet.2015.08.681
P10-012 Omics technologies for the prediction of immunmodulatory effects P. Zwicker 1,∗ , N. Schultze 1 , S. Niehs 2 , K. Methling 2 , M. Wurster 2 , J. Bernhardt 3 , G. Wachlin 3 , M. Lalk 2 , R. Preissner 4 , U. Lindequist 1 , B. Haertel 1 1
University of Greifswald, Institute of Pharmacy, Greifswald, Germany 2 University of Greifswald, Institute of Biochemistry, Greifswald, Germany 3 University of Greifswald, Institute of Microbiology, Greifswald, Germany 4 Charité University Medicine, Institute of Physiology and ECRC, Berlin, Germany
http://dx.doi.org/10.1016/j.toxlet.2015.08.680
P10-011 Percellome toxicogenomics for mechanistic analysis towards chronic toxicity by a newly designed repeated dose study J. Kanno ∗ , S. Kitajima, K.-I. Aisaki National Institute of Health Sciences, Division of Cellular & Molecular Toxicology, Tokyo, Japan The Percellome Project aims at reinforcing and eventually replacing the “safety factor (uncertainty factor)” currently used for the extrapolation of experimental animal data to humans. Our project attempts to comprehensively identify the transcriptomic networks induced by xenobiotics. For this attempt, we adopted phenotype-independent approach, because not all changes in mRNA expression can be anchored to overt phenotypes. Consequently, there emerged a need to pile up certain amount of transcriptomic data. This situation is analogous to when the electron microscope had invented; to gain consensus on its new images, database for textbooks and atlases was needed. For this need, “Percellome” method was developed (BMC Genomics 7:64, 2006) to generate absolute copy numbers of each mRNA per one cell (in average). By this method, data from the Affymetrix MOE430 2.0 GeneChip are absolutized and visualized in 3-D graphs (time × dose × copy number per cell). Up to now, datasets of mouse liver (4 time points × 4 dose levels, triplicate, 48 GeneChip data per chemical) on more than 100 chemicals are compiled. Here, we report the effect of pretreatment of mice with repeated dose of carbon tetrachloride (CCl4 ) on single dose of various chemicals at 2, 4, 8 and 24 h. This study design is a derivative of a single dose study using gene knockout mouse. Instead of using knockout mouse, mouse pretreated with repeated dose of a chemical is considered to be in a “chemically-induced transgenic state”. Various chemicals include CCl4 itself, clofibrate and phenobarbital. Repeated dose of CCl4 induced suppression of baseline expression levels of genes related to ER stress and attenuated the effect of chemicals given single dose. The mechanisms suggested to be related to this phenomenon included mTOR signaling. Analysis strategy by the new concept of repeated dose study, i.e. “chemically-induced transgenic
Ethical concerns and low predictability of animal data to humans gave rise for the development of different in vitro assays for the evaluation of immunotoxicity. In this study, a proteomic approach, combined with metabolomics and functional assays was developed to evaluate immunmodulatory effects of natural compounds on a human T cell line (Jurkat) in vitro. For the proteomic analysis, a 2D gel electrophoresis based assay together with an antibody array was used. Additionally, intra- and extracellular metabolites were measured with GC–MS and 1 H NMR. Functional tests as apoptosis, cell cycle and intracellular reactive oxygen species (iROS) assays were an additional tool for the interpretation of the obtained data. The measurement of cytokine levels will also be part of the approach. The cytotoxic and immunosuppressive substances Vincristine (VCR), Cannabidiol (CBD), Deoxynivalenol (DON) and the immunostimulating compound Tulipalin A (TUPA) were investigated. General cytotoxic effects of the substances were measured using the MTT assay. Concentrations that lead to a decrease in cell viability of 10% (IC10 ) were used for further investigations. This decrease is achieved by concentrations of 0.45 nM (VCR), 8.65 M (CBD), 0.47 M (DON) and 26.8 M (TUPA). Jurkat cells treated with the substances in low doses (IC10 ) revealed alterations in the abundance of different proteins. The reaction to the treatment with CBD has similarities to the reaction to TUPA. Affected biological processes are for example cell cycle, stress response and apoptosis. In contrast, VCR showed only few shifts in protein formation and low similarity to the affected processes after treatment with CBD and TUPA. The metabolomic analysis indicates a similar tendency in the quantity of altered extra- and intracellular metabolites in comparison to proteomic data. Functional assays support alterations in cell cycle progression and apoptosis. Changes in cytokine levels have to be validated. In summary, a comprehensive approach to investigate immunmodulatory effects is established. However, more substances have to be screened for their effects on different human cell lines. Final aim is the creation of a database for the prediction of immunotoxic effects. http://dx.doi.org/10.1016/j.toxlet.2015.08.682