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Inhibition of apoptotic DNA-fragmentation by 2,3,7,8-tetrachlorodibenzo-p-dioxin
Abstracts / Toxicology Letters 189S (2009) S57–S273
regulated through -catenin transcriptional activity, we employed in vitro model of rat liver progenitor cells, in order to explore the impact of canonical Wnt signaling on AhR-dependent targets. In rat liver progenitor WB-F344 cell line, AhR has been shown to regulate not only the expression of xenobiotic-metabolizing enzymes, but also a number of other cellular processes, including contact inhibition and proteins involved in cell-to-cell communication. We found that recombinant Wnt3a protein, a model trigger of canonical Wnt signaling, potently up-regulated AhR mRNA and protein levels. Moreover, Wnt3a synergized with 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) to up-regulate both cytochrome P450 1A1 and 1B1 expression, as well as additional enzymes regulated by both signaling pathways, such as cyclooxygenase-2. Nevertheless, AhR ligands were also found to induce a time-dependent degradation of both total -catenin pool and the active -catenin form, dephosphorylated at residues Ser37 and Thr41, in rat liver progenitor cell line. Additional analysis of global gene expression, using Affymetrix Rat Exon 1.0 arrays, revealed that AhR ligand suppressed expression levels of some model -catenin transcriptional targets, such as Ccnd1 and Axin2. Given the importance of -catenin-dependent processes in both liver biology and hepatocarcinogenesis, it is important to further analyze the role of AhR ligands, efficient liver carcinogens, in the above described processes. Our ongoing studies attempt to characterize the nature of the observed dual crosstalk between AhR and -catenin in liver progenitor cells. Acknowledgement: This study is funded by the Czech Science Foundation, grant no. 524/09/1337. 夽 Selected for Oral Presentation. doi:10.1016/j.toxlet.2009.06.316
N03 Inhibition
of
apoptotic
DNA-fragmentation 夽 tetrachlorodibenzo-p-dioxin
by
2,3,7,8-
Martin Chopra 1,∗ , Gregor Meiss 2 , Arunasalam Dharmarajan 3 , Dieter Schrenk 1 1 TU Kaiserslautern, Food Chemistry and Toxicology, Kaiserslautern, Germany, 2 Justus-Liebig-Universität, Biochemistry, Gießen, Germany, 3 University of Western Australia, Anatomy and Human Biology, Crawley, Australia
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous contaminant which is/has been formed in the production of chloro-organics or during incineration processes. It can be found in soil, water and the food chain where it accumulates due to its chemical stability and lipophilicity. Despite not being directly genotoxic, TCDD was classified as a class 1 carcinogen by the IARC and it is one of the most potent tumor promotors in rodent liver. It has been hypothesized that TCDD promotes tumor growth by inhibiting apoptosis of initiated cells. Therefore, we tested which targets along apoptotic pathways are affected by TCDD treatment. Primary rat hepatocytes and human hepatoma cells Huh-7 were irradiated with UVC light to induce apoptosis. After subsequent treatment with TCDD, up-stream apoptotic events like changes in the expression of bcl-2 proteins, mitochondrial membrane depolarization, caspase-activity and caspase-substrate cleavage were not inhibited whereas the extend of apoptotic DNA-fragmentation was decreased in both cells types. Using arylhydrocarbon receptor (AhR) antagonists, this effect could be linked to AhR activation.
S97
We are currently looking at various chromatin modifications that might affect the activity of apoptotic nucleases and translate the apoptosis-inhibiting effect of TCDD. It appears plausible that TCDD inhibits apoptotic DNA-cleavage in order to retain genomic integrity, allowing the cell to deal with xenobiotics despite an initiation of apoptosis. These cells could recover from an apoptotic insult, harbouring genetic aberrations. This could explain the tumor promoting potential of TCDD and might be an underlying mechanism for other tumor-promotors which induce xenobiotic metabolism. 夽 Selected for Oral Presentation. doi:10.1016/j.toxlet.2009.06.317
N04 Transitioning to mode of action based testing: The critical role of frameworks for human relevance analysis of modes of action Bette Meek University of Ottawa, McLaughlin Centre for Population Health Risk Assessment, Ottawa, Canada Mode of action is defined as a series of key biological events leading to an observed toxicological effect (for example, metabolism to a toxic entity, cell death, regenerative repair and tumours). While a hypothesized mode of action is supported by experimental observations and related mechanistic data, it contrasts with mechanism of action, which generally involves a detailed understanding of the molecular basis for an effect. An international framework to consider the weight of evidence for hypothesized modes of action in animals and their relevance to humans developed based on input from scientists worldwide, has been widely adopted and used by government agencies and international organizations. The framework, which was developed and refined through its application in case studies for principally non-DNA reactive carcinogens, has more recently been extended to DNA reactive carcinogens, non-cancer endpoints and different life stages. In addition to increasing transparency, use of the framework promotes consistency in decision-making concerning adequacy of weight of evidence, facilitates peer input and review and identifies critical research needs. Iterative use of the framework also facilitates communication between assessors and researchers on key data gaps and encourages early assimilation of mechanistic data in hazard characterization and dose–response analysis. Systematic consideration of key events in modes of action in application of the framework also constitutes a pragmatic first step in transitioning to more progressive testing strategies such as those envisaged under REACH or proposed in the US National Research Council Report on Toxicity Testing in the 21st Century. Principal elements of the framework will be presented and illustrated by way of example. doi:10.1016/j.toxlet.2009.06.318
regulated through -catenin transcriptional activity, we employed in vitro model of rat liver progenitor cells, in order to explore the impact of canonical Wnt signaling on AhR-dependent targets. In rat liver progenitor WB-F344 cell line, AhR has been shown to regulate not only the expression of xenobiotic-metabolizing enzymes, but also a number of other cellular processes, including contact inhibition and proteins involved in cell-to-cell communication. We found that recombinant Wnt3a protein, a model trigger of canonical Wnt signaling, potently up-regulated AhR mRNA and protein levels. Moreover, Wnt3a synergized with 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) to up-regulate both cytochrome P450 1A1 and 1B1 expression, as well as additional enzymes regulated by both signaling pathways, such as cyclooxygenase-2. Nevertheless, AhR ligands were also found to induce a time-dependent degradation of both total -catenin pool and the active -catenin form, dephosphorylated at residues Ser37 and Thr41, in rat liver progenitor cell line. Additional analysis of global gene expression, using Affymetrix Rat Exon 1.0 arrays, revealed that AhR ligand suppressed expression levels of some model -catenin transcriptional targets, such as Ccnd1 and Axin2. Given the importance of -catenin-dependent processes in both liver biology and hepatocarcinogenesis, it is important to further analyze the role of AhR ligands, efficient liver carcinogens, in the above described processes. Our ongoing studies attempt to characterize the nature of the observed dual crosstalk between AhR and -catenin in liver progenitor cells. Acknowledgement: This study is funded by the Czech Science Foundation, grant no. 524/09/1337. 夽 Selected for Oral Presentation. doi:10.1016/j.toxlet.2009.06.316
N03 Inhibition
of
apoptotic
DNA-fragmentation 夽 tetrachlorodibenzo-p-dioxin
by
2,3,7,8-
Martin Chopra 1,∗ , Gregor Meiss 2 , Arunasalam Dharmarajan 3 , Dieter Schrenk 1 1 TU Kaiserslautern, Food Chemistry and Toxicology, Kaiserslautern, Germany, 2 Justus-Liebig-Universität, Biochemistry, Gießen, Germany, 3 University of Western Australia, Anatomy and Human Biology, Crawley, Australia
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous contaminant which is/has been formed in the production of chloro-organics or during incineration processes. It can be found in soil, water and the food chain where it accumulates due to its chemical stability and lipophilicity. Despite not being directly genotoxic, TCDD was classified as a class 1 carcinogen by the IARC and it is one of the most potent tumor promotors in rodent liver. It has been hypothesized that TCDD promotes tumor growth by inhibiting apoptosis of initiated cells. Therefore, we tested which targets along apoptotic pathways are affected by TCDD treatment. Primary rat hepatocytes and human hepatoma cells Huh-7 were irradiated with UVC light to induce apoptosis. After subsequent treatment with TCDD, up-stream apoptotic events like changes in the expression of bcl-2 proteins, mitochondrial membrane depolarization, caspase-activity and caspase-substrate cleavage were not inhibited whereas the extend of apoptotic DNA-fragmentation was decreased in both cells types. Using arylhydrocarbon receptor (AhR) antagonists, this effect could be linked to AhR activation.
S97
We are currently looking at various chromatin modifications that might affect the activity of apoptotic nucleases and translate the apoptosis-inhibiting effect of TCDD. It appears plausible that TCDD inhibits apoptotic DNA-cleavage in order to retain genomic integrity, allowing the cell to deal with xenobiotics despite an initiation of apoptosis. These cells could recover from an apoptotic insult, harbouring genetic aberrations. This could explain the tumor promoting potential of TCDD and might be an underlying mechanism for other tumor-promotors which induce xenobiotic metabolism. 夽 Selected for Oral Presentation. doi:10.1016/j.toxlet.2009.06.317
N04 Transitioning to mode of action based testing: The critical role of frameworks for human relevance analysis of modes of action Bette Meek University of Ottawa, McLaughlin Centre for Population Health Risk Assessment, Ottawa, Canada Mode of action is defined as a series of key biological events leading to an observed toxicological effect (for example, metabolism to a toxic entity, cell death, regenerative repair and tumours). While a hypothesized mode of action is supported by experimental observations and related mechanistic data, it contrasts with mechanism of action, which generally involves a detailed understanding of the molecular basis for an effect. An international framework to consider the weight of evidence for hypothesized modes of action in animals and their relevance to humans developed based on input from scientists worldwide, has been widely adopted and used by government agencies and international organizations. The framework, which was developed and refined through its application in case studies for principally non-DNA reactive carcinogens, has more recently been extended to DNA reactive carcinogens, non-cancer endpoints and different life stages. In addition to increasing transparency, use of the framework promotes consistency in decision-making concerning adequacy of weight of evidence, facilitates peer input and review and identifies critical research needs. Iterative use of the framework also facilitates communication between assessors and researchers on key data gaps and encourages early assimilation of mechanistic data in hazard characterization and dose–response analysis. Systematic consideration of key events in modes of action in application of the framework also constitutes a pragmatic first step in transitioning to more progressive testing strategies such as those envisaged under REACH or proposed in the US National Research Council Report on Toxicity Testing in the 21st Century. Principal elements of the framework will be presented and illustrated by way of example. doi:10.1016/j.toxlet.2009.06.318