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Targeted whole transcriptome gene expression profiling for mechanistic toxicology
Abstracts / Toxicology Letters 280S (2017) S294–S296
P-09-05-03 Transcriptomic profile alterations in organotypic cultures of bronchial tissues indirectly exposed to cigarette smoke and novel tobacco product vapor Kazushi Matsumura, Shinkichi Ishikawa, Shigeaki Ito Scientific Product Assessment Center, Japan Tobacco Inc., 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa, Japan Novel tobacco products, such as heat-not-burn type of products, normally generate lower levels of potentially harmful constituents than cigarettes, and they are expected to show different tissuespecific biological impacts from conventional cigarette. So we compared the effects of the vapor from our novel tobacco vapor product (NTV) and smoke from combustible tobacco on transcriptomic profiles by indirectly exposing organotypic cultures of human bronchial epithelium to smoke and vapor extracts for 4 and 24 h. A dose-dependent increase in the number of differentially expressed genes was observed in both exposures. Furthermore, the differentially expressed gene profiles varied between 4 h and 24 h exposures, suggesting distinct early responses and later responses. We also carried out pathway analyses, and found that the 3R4F exposure perturbed cellular functions and pathways in the early phase, but the pathways related to stress and inflammatory responses is emphasized after 24 h exposure. In contrast, the NTV exposure induced a downregulation in the expression of genes related to several cellular functions and pathways in the early phase, but not in the later phase. It is suggested that different mechanisms of action underlie the effects of smoke from the 3R4F cigarette and vapor from the NTV, and that the effects of the cigarette smoke are partially persistent, while those of the vapor of NTV are transient and recoverable. We consider that our approach is useful for elucidating tissue-specific biological impacts of cigarette smoke, as well as for a comparative study of novel tobacco products. http://dx.doi.org/10.1016/j.toxlet.2017.07.826 P-09-05-04 Targeted whole transcriptome gene expression profiling for mechanistic toxicology Harper VanSteenhouse, Peter Shepard, Joanne Yeakley, Bruce Seligmann BioSpyder Technologies, Carlsbad, United States Gene expression profiling is a powerful and sensitive method to characterize biological responses to chemical or drug treatments, and for read-across evaluations. However, traditional RNA-seq is cost and effort prohibitive for large-scale studies. Whole transcriptome TempO-Seq is a simple and cost effective method covering the whole human transcriptome targeting 18,886 genes. It is highly correlated with RNA-seq for measuring differential expression, and exhibits single base specificity and single cell sensitivity. To assess its utility for detecting compound-induced changes in expression, we treated cells with Trichostatin A and cell lysates were assayed directly without RNA purification or reverse transcription. In addition to accurately identifying overlapping gene sets due to TSA treatment of MCF7 cells in the CMAP database, other strongly overlapping gene sets were TSA treatment of other cell-types. Thus, a consistent TSA signature was evident despite large differences in baseline expression of very different cell lines, implying TSA effects may be more specific than previously reported. TempO-Seq sensitivity was demonstrated by identification of additional genes that
S295
did not appear in any of the 26 MCF7 TSA studies in the CMAP database, despite high levels of expression and fold differences. Among the most significant overlaps of these genes with GSEA were associated with UV irradiation, a previously unreported effect of TSA alone. These results suggest that TempO-Seq detects changes in important mechanisms and pathways that other methods fail to detect, highly useful for monitoring gene expression responses to compounds. http://dx.doi.org/10.1016/j.toxlet.2017.07.827 P-09-05-05 Effect of subtoxic and toxic concentrations of galactosamine in the metabolome of primary mouse hepatocytes Ana Margarida Araújo 1 , Ana Rita Lima 1 , Maria de Lourdes Bastos 1 , Félix Carvalho 1 , Márcia Carvalho 1,2 , Paula Guedes de Pinho 1 1 Department of Biological Sciences, Faculty of Pharmacy, University of Porto, UCIBIO, REQUIMTE, Porto, Portugal 2 UFP Energy, Environment and Health Research Unit (FP-ENAS), University Fernando Pessoa, Porto, Portugal
Galactosamine (GalN) is a classic hepatotoxin that has been widely used in laboratory studies. However, the mechanisms by which GalN induces liver damage still require further clarification. The aim of the present study was to identify metabolic changes caused by GalN in primary mouse hepatocytes (PMH), in order to investigate the toxicity-related metabolic pathways. For this purpose, metabolomics represents a valuable strategy to monitor, in a non-targeted manner, the changes that are at the base of the hepatotoxicity mechanism. Cell viability assessed by the MTT assay showed a concentration-dependent toxic effect of GalN on PMH. The exometabolome (the extracellular metabolome) and endometabolome (metabolites within the cell) analysis of PMH exposed for 24 h to three low concentrations of GalN (0.66, 1.37 and 2.61 mM corresponding to LC01, LC10 and LC30 levels, respectively) was performed using gas chromatography/mass spectrometry (GC/MS). Results obtained showed that metabolic patterns of GalN exposed cells are separated from control in a concentration-dependent manner. Among the discriminatory metabolites, sugars, organic acids, amino acids, fatty acids, among others (endometabolome), as well various volatile organic compounds (VOCs), namely aldehydes and ketones (exometabolome), suffered significant alterations, suggesting that GalN induces marked metabolic alterations at low concentrations, even in the absence of evident liver toxicity. A.M.A. thanks FCT for her PhD grant (SFRH/BD/107708/2015). This work was financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI/01/0145/FEDER/007728). http://dx.doi.org/10.1016/j.toxlet.2017.07.828
P-09-05-03 Transcriptomic profile alterations in organotypic cultures of bronchial tissues indirectly exposed to cigarette smoke and novel tobacco product vapor Kazushi Matsumura, Shinkichi Ishikawa, Shigeaki Ito Scientific Product Assessment Center, Japan Tobacco Inc., 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa, Japan Novel tobacco products, such as heat-not-burn type of products, normally generate lower levels of potentially harmful constituents than cigarettes, and they are expected to show different tissuespecific biological impacts from conventional cigarette. So we compared the effects of the vapor from our novel tobacco vapor product (NTV) and smoke from combustible tobacco on transcriptomic profiles by indirectly exposing organotypic cultures of human bronchial epithelium to smoke and vapor extracts for 4 and 24 h. A dose-dependent increase in the number of differentially expressed genes was observed in both exposures. Furthermore, the differentially expressed gene profiles varied between 4 h and 24 h exposures, suggesting distinct early responses and later responses. We also carried out pathway analyses, and found that the 3R4F exposure perturbed cellular functions and pathways in the early phase, but the pathways related to stress and inflammatory responses is emphasized after 24 h exposure. In contrast, the NTV exposure induced a downregulation in the expression of genes related to several cellular functions and pathways in the early phase, but not in the later phase. It is suggested that different mechanisms of action underlie the effects of smoke from the 3R4F cigarette and vapor from the NTV, and that the effects of the cigarette smoke are partially persistent, while those of the vapor of NTV are transient and recoverable. We consider that our approach is useful for elucidating tissue-specific biological impacts of cigarette smoke, as well as for a comparative study of novel tobacco products. http://dx.doi.org/10.1016/j.toxlet.2017.07.826 P-09-05-04 Targeted whole transcriptome gene expression profiling for mechanistic toxicology Harper VanSteenhouse, Peter Shepard, Joanne Yeakley, Bruce Seligmann BioSpyder Technologies, Carlsbad, United States Gene expression profiling is a powerful and sensitive method to characterize biological responses to chemical or drug treatments, and for read-across evaluations. However, traditional RNA-seq is cost and effort prohibitive for large-scale studies. Whole transcriptome TempO-Seq is a simple and cost effective method covering the whole human transcriptome targeting 18,886 genes. It is highly correlated with RNA-seq for measuring differential expression, and exhibits single base specificity and single cell sensitivity. To assess its utility for detecting compound-induced changes in expression, we treated cells with Trichostatin A and cell lysates were assayed directly without RNA purification or reverse transcription. In addition to accurately identifying overlapping gene sets due to TSA treatment of MCF7 cells in the CMAP database, other strongly overlapping gene sets were TSA treatment of other cell-types. Thus, a consistent TSA signature was evident despite large differences in baseline expression of very different cell lines, implying TSA effects may be more specific than previously reported. TempO-Seq sensitivity was demonstrated by identification of additional genes that
S295
did not appear in any of the 26 MCF7 TSA studies in the CMAP database, despite high levels of expression and fold differences. Among the most significant overlaps of these genes with GSEA were associated with UV irradiation, a previously unreported effect of TSA alone. These results suggest that TempO-Seq detects changes in important mechanisms and pathways that other methods fail to detect, highly useful for monitoring gene expression responses to compounds. http://dx.doi.org/10.1016/j.toxlet.2017.07.827 P-09-05-05 Effect of subtoxic and toxic concentrations of galactosamine in the metabolome of primary mouse hepatocytes Ana Margarida Araújo 1 , Ana Rita Lima 1 , Maria de Lourdes Bastos 1 , Félix Carvalho 1 , Márcia Carvalho 1,2 , Paula Guedes de Pinho 1 1 Department of Biological Sciences, Faculty of Pharmacy, University of Porto, UCIBIO, REQUIMTE, Porto, Portugal 2 UFP Energy, Environment and Health Research Unit (FP-ENAS), University Fernando Pessoa, Porto, Portugal
Galactosamine (GalN) is a classic hepatotoxin that has been widely used in laboratory studies. However, the mechanisms by which GalN induces liver damage still require further clarification. The aim of the present study was to identify metabolic changes caused by GalN in primary mouse hepatocytes (PMH), in order to investigate the toxicity-related metabolic pathways. For this purpose, metabolomics represents a valuable strategy to monitor, in a non-targeted manner, the changes that are at the base of the hepatotoxicity mechanism. Cell viability assessed by the MTT assay showed a concentration-dependent toxic effect of GalN on PMH. The exometabolome (the extracellular metabolome) and endometabolome (metabolites within the cell) analysis of PMH exposed for 24 h to three low concentrations of GalN (0.66, 1.37 and 2.61 mM corresponding to LC01, LC10 and LC30 levels, respectively) was performed using gas chromatography/mass spectrometry (GC/MS). Results obtained showed that metabolic patterns of GalN exposed cells are separated from control in a concentration-dependent manner. Among the discriminatory metabolites, sugars, organic acids, amino acids, fatty acids, among others (endometabolome), as well various volatile organic compounds (VOCs), namely aldehydes and ketones (exometabolome), suffered significant alterations, suggesting that GalN induces marked metabolic alterations at low concentrations, even in the absence of evident liver toxicity. A.M.A. thanks FCT for her PhD grant (SFRH/BD/107708/2015). This work was financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI/01/0145/FEDER/007728). http://dx.doi.org/10.1016/j.toxlet.2017.07.828