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Proteomic analysis of the chronic anthracycline cardiotoxicity in rabbits
Abstracts / Toxicology Letters 180S (2008) S32–S246
O15 Proteomic analysis of the chronic anthracycline cardiotoxicity in rabbits Martin Sterba 1,∗ , Juraj Lenco 2 , Olga Popelova 1 , Alena Fucikova 2 , Tomas Simunek 3 , Yvona Mazurova 1 , Michaela Adamcova 1 , Vladimir Gersl 1 1 Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic, 2 Faculty of Military Heath Sciences, University of Defence, Hradec Kralove, Czech Republic, 3 Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Kralove, Czech Republic
The molecular basis of anthracycline (ANT) cardiotoxicity remains largely elusive, though a number of different hypotheses have been proposed. The aim of the present study was to obtain deeper molecular insight into the chronic ANT cardiotoxicity using proteomic analysis. The cardiac toxicity was induced in rabbits in a standard schedule (daunorubicin, 3 mg/kg, i.v., once weekly for 10 weeks, n = 6), while control animals received saline (n = 6). The left ventricular myocardium was harvested a week after the last administration or immediately after premature death of animals (2 of 6 in the daunorubicin group). Myocardial proteins were extracted and analysed using two-dimensional differential gel electrophoresis (2D-DIGE). The subsequent PDQuest analysis of the gels revealed the presence of approximately 875 protein spots per gel (95% matched). Further quantitative and statistical analysis found out 80 protein spots with significant change (>1.5 fold change, p < 0.01), of which 64 showed increase and 16 decrease in spot abundance. MALDI-TOF/TOF protein identification revealed different abundance of number of important mitochondrial proteins—namely those involved in the energy metabolism (e.g., complex I, IV and F1-ATP synthase subunits, S-Mt-CK). In addition, interesting changes in other mitochondrial proteins have been found (e.g., decrease in abundance of Mn-SOD or VDAC-1). Furthermore, marked up-regulation of desmin (an important intermediate filament) suggests profound remodelling of intracellular architecture of cardiac cells. Hence, this investigation brings new indices and suggests several directions for future studies on the mechanism(s) of chronic ANT cardiomyopathy as well as possibilities of its prevention. Acknowledgements: MSM0021620820.
Supported
by
Research
Project
doi:10.1016/j.toxlet.2008.06.388 O16 Creation of compound specific pathways for bioinformatics analysis of toxicogenomics datasets from EU 6th framework Carcinogenomics
S125
such as GenMAPP (www.genmapp.org), which contain predefined expert MAPPs. Comparison of in vitro toxicogenomics findings for these compounds within the context of in vivo compound-specific knowledge enables to address whether the affected processes in target organ specific in vitro models involve primarily early effects (e.g. metabolism, DNA damage processing) or downstream effects related to the potential onset of carcinogenesis (e.g. cell proliferation or apoptosis). A start has been made to create in silico pathway MAPPs for selected model compounds (thioacetamide, benzo[a]pyrene, 2-nitrofluorene). Literature based pathways are first drafted in GenMAPP format. Finally, the obtained pathway information, initially for benzo[a]pyrene, will be integrated with an existing mathematical model for in silico prediction of cancerspecific signalling pathways (Hanahan and Weinberg (2000) Cell, 100, 57-70) using the PyBioS systems biology tool (Wierling et al. (2007) Brief Funct Genomic Proteomic, 6, 240-51). In an iterative step, the outcome of these predictions for carcinogens and noncarcinogens with respect to cancer initiation and development will then be validated by experimental in vitro toxicogenomics data derived from Carcinogenomics. doi:10.1016/j.toxlet.2008.06.389 O17 Effects of methyl mercury on gene expression patterns in cerebellum and cerebrum of perinatally exposed Wistar rats Marijana Radonjic ∗ , André Wolterbeek, Didima de Groot, Rob Stierum TNO Quality of Life, Zeist, Netherlands An extended one generation reproduction study [1] was performed to study the effects of perinatal exposure to methyl mercury (MeHg) (0, 0.1, 0.4, 0.7, 1.0, 1.5, 2.0 mg MeHg/kg BW; p.o.; gestation day 6 to lactation day 10). Off-spring was studied until post natal day (PND) 70, with attention to the developing immune, hormonal and neural systems. For selected dose groups, in vivo imaging, in vitro longterm potentiation [2], toxicokinetics and toxicogenomics were performed. For toxicogenomics, rats were sacrificed on PND21 and PND70. Parts of cerebrum and cerebellum were collected. RNA was isolated using Trizol/NucleoSpin RNA II, labelled and hybridized to Affymetrix GeneChip® Rat Genome 230 2.0 arrays. For selected dose levels at each PND, microarrays were performed on RNA obtained from 3 to 5 individual animals, yielding a 68 microarray experiment. Quality control and GCRMA (slow) normalization was performed. Preliminary data analysis (principal component analysis) indicated clear differences between the two different brain regions. Gene expression patterns were distinct between PND21 and PND70, suggesting that neural biochemical changes concur with development. Analysis of the effects of methyl mercury on gene expression in cerebellum and cerebrum, in relation to developmental effects in time will be presented.
Yvonne Staal 1,∗ , Christoph Wierling 2 , Ralf Herwig 2 , Rob Stierum 1 1
TNO Quality of Life, Zeist, Netherlands, Molecular Genetics, Berlin, Germany
2
Max Planck Institute for
The EU 6th framework Carcinogenomics (www.carcinogenomics. eu) aims to improve toxicogenomics-based in vitro models for prediction of chemical carcinogenesis. To this extend, systems toxicology-based methods are developed to assess possible carcinogenic properties of compounds in in vitro models for liver, lung and kidney. Transcriptome and metabolome data will be generated within the consortium using selected model compounds (Vinken et al. (2008) Mutation Research Reviews, in press). Bioinformatics will involve, next to statistical approaches, pathway analysis tools
References Cooper, et al., 2006. Crit. Rev. Toxicol. 36 (1), 69–98. De Groot et al., Maternal exposure to MeHg during pregnancy causes life long impairment of learning and memory in the offspring, demonstrated by in vitro Long Term Potentiation in the hippocampus at juvenile and adult age, Poster abstract, Eurotox, submitted for publication.
doi:10.1016/j.toxlet.2008.06.390
O15 Proteomic analysis of the chronic anthracycline cardiotoxicity in rabbits Martin Sterba 1,∗ , Juraj Lenco 2 , Olga Popelova 1 , Alena Fucikova 2 , Tomas Simunek 3 , Yvona Mazurova 1 , Michaela Adamcova 1 , Vladimir Gersl 1 1 Faculty of Medicine in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic, 2 Faculty of Military Heath Sciences, University of Defence, Hradec Kralove, Czech Republic, 3 Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Hradec Kralove, Czech Republic
The molecular basis of anthracycline (ANT) cardiotoxicity remains largely elusive, though a number of different hypotheses have been proposed. The aim of the present study was to obtain deeper molecular insight into the chronic ANT cardiotoxicity using proteomic analysis. The cardiac toxicity was induced in rabbits in a standard schedule (daunorubicin, 3 mg/kg, i.v., once weekly for 10 weeks, n = 6), while control animals received saline (n = 6). The left ventricular myocardium was harvested a week after the last administration or immediately after premature death of animals (2 of 6 in the daunorubicin group). Myocardial proteins were extracted and analysed using two-dimensional differential gel electrophoresis (2D-DIGE). The subsequent PDQuest analysis of the gels revealed the presence of approximately 875 protein spots per gel (95% matched). Further quantitative and statistical analysis found out 80 protein spots with significant change (>1.5 fold change, p < 0.01), of which 64 showed increase and 16 decrease in spot abundance. MALDI-TOF/TOF protein identification revealed different abundance of number of important mitochondrial proteins—namely those involved in the energy metabolism (e.g., complex I, IV and F1-ATP synthase subunits, S-Mt-CK). In addition, interesting changes in other mitochondrial proteins have been found (e.g., decrease in abundance of Mn-SOD or VDAC-1). Furthermore, marked up-regulation of desmin (an important intermediate filament) suggests profound remodelling of intracellular architecture of cardiac cells. Hence, this investigation brings new indices and suggests several directions for future studies on the mechanism(s) of chronic ANT cardiomyopathy as well as possibilities of its prevention. Acknowledgements: MSM0021620820.
Supported
by
Research
Project
doi:10.1016/j.toxlet.2008.06.388 O16 Creation of compound specific pathways for bioinformatics analysis of toxicogenomics datasets from EU 6th framework Carcinogenomics
S125
such as GenMAPP (www.genmapp.org), which contain predefined expert MAPPs. Comparison of in vitro toxicogenomics findings for these compounds within the context of in vivo compound-specific knowledge enables to address whether the affected processes in target organ specific in vitro models involve primarily early effects (e.g. metabolism, DNA damage processing) or downstream effects related to the potential onset of carcinogenesis (e.g. cell proliferation or apoptosis). A start has been made to create in silico pathway MAPPs for selected model compounds (thioacetamide, benzo[a]pyrene, 2-nitrofluorene). Literature based pathways are first drafted in GenMAPP format. Finally, the obtained pathway information, initially for benzo[a]pyrene, will be integrated with an existing mathematical model for in silico prediction of cancerspecific signalling pathways (Hanahan and Weinberg (2000) Cell, 100, 57-70) using the PyBioS systems biology tool (Wierling et al. (2007) Brief Funct Genomic Proteomic, 6, 240-51). In an iterative step, the outcome of these predictions for carcinogens and noncarcinogens with respect to cancer initiation and development will then be validated by experimental in vitro toxicogenomics data derived from Carcinogenomics. doi:10.1016/j.toxlet.2008.06.389 O17 Effects of methyl mercury on gene expression patterns in cerebellum and cerebrum of perinatally exposed Wistar rats Marijana Radonjic ∗ , André Wolterbeek, Didima de Groot, Rob Stierum TNO Quality of Life, Zeist, Netherlands An extended one generation reproduction study [1] was performed to study the effects of perinatal exposure to methyl mercury (MeHg) (0, 0.1, 0.4, 0.7, 1.0, 1.5, 2.0 mg MeHg/kg BW; p.o.; gestation day 6 to lactation day 10). Off-spring was studied until post natal day (PND) 70, with attention to the developing immune, hormonal and neural systems. For selected dose groups, in vivo imaging, in vitro longterm potentiation [2], toxicokinetics and toxicogenomics were performed. For toxicogenomics, rats were sacrificed on PND21 and PND70. Parts of cerebrum and cerebellum were collected. RNA was isolated using Trizol/NucleoSpin RNA II, labelled and hybridized to Affymetrix GeneChip® Rat Genome 230 2.0 arrays. For selected dose levels at each PND, microarrays were performed on RNA obtained from 3 to 5 individual animals, yielding a 68 microarray experiment. Quality control and GCRMA (slow) normalization was performed. Preliminary data analysis (principal component analysis) indicated clear differences between the two different brain regions. Gene expression patterns were distinct between PND21 and PND70, suggesting that neural biochemical changes concur with development. Analysis of the effects of methyl mercury on gene expression in cerebellum and cerebrum, in relation to developmental effects in time will be presented.
Yvonne Staal 1,∗ , Christoph Wierling 2 , Ralf Herwig 2 , Rob Stierum 1 1
TNO Quality of Life, Zeist, Netherlands, Molecular Genetics, Berlin, Germany
2
Max Planck Institute for
The EU 6th framework Carcinogenomics (www.carcinogenomics. eu) aims to improve toxicogenomics-based in vitro models for prediction of chemical carcinogenesis. To this extend, systems toxicology-based methods are developed to assess possible carcinogenic properties of compounds in in vitro models for liver, lung and kidney. Transcriptome and metabolome data will be generated within the consortium using selected model compounds (Vinken et al. (2008) Mutation Research Reviews, in press). Bioinformatics will involve, next to statistical approaches, pathway analysis tools
References Cooper, et al., 2006. Crit. Rev. Toxicol. 36 (1), 69–98. De Groot et al., Maternal exposure to MeHg during pregnancy causes life long impairment of learning and memory in the offspring, demonstrated by in vitro Long Term Potentiation in the hippocampus at juvenile and adult age, Poster abstract, Eurotox, submitted for publication.
doi:10.1016/j.toxlet.2008.06.390