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The aryl hydrocarbon receptor-mediated disruption of differentiation status of liver progenitor cells
S240
Abstracts / Toxicology Letters 205S (2011) S180–S300
ting Results: In addition to B(a)P, various PAHs, including pyrene and benzo(e)pyrene, known to not or only very poorly interact with AhR, similarly up-regulated [Ca2+ ]i in human endothelial HMEC-1 cells. Moreover, a-naphthoflavone, a flavonoïd antagonist of AhR, was also able to induce [Ca2+ ]i . Knocking-down AhR expression in HMEC-1 cells through transfection of siRNAs, was finally demonstrated to not prevent B(a)P-mediated induction of [Ca2+ ]i , whereas it efficiently counteracted B(a)P-mediated induction of the referent AhR target gene cytochrome P-450 1B1. Taken together, these data demonstrate that environmental PAHs trigger [Ca2+ ]i induction in an AhR-independent manner. doi:10.1016/j.toxlet.2011.05.818
P2195 Beta-Amyloid induces toxic degenerative pathways on human retinal cells with P2X7 cell death receptor activation: Role in Age-Related Macular Degeneration (AMD) P. Rat 1,∗ , A. Waks 1 , M. Dutot 1 , B. De Moucheron 1 , O. Laprévote 2 , J. Warnet 2 1
Laboratoire de Toxicologie Analytique et Cellulaire (c-tac), Faculté de Pharmacie, Université Paris Descartes, Paris, France, 2 Laboratoire de Chimie et Toxicologie Analytique et Cellulaire, EA 4463, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France Age-Related Macular Degeneration (AMD) is a degenerative retinal disease, defined by a chronic, progressive and incapacitating disorder, characterized by alteration of the central retina. BetaAmyloid peptide is known in Alzheimer’s disease to be present in senile plaques; it had been also found more recently in retinal extracellular deposits observed in AMD patients. Beta-Amyloid seems to be important in neurodegenerative disorders (brain, retina) but mechanisms need to be explored in retina. Our first aim was to investigate and compare cellular effects of Beta-Amyloid in fibrillar and non-fibrillar form. Our second aim was to explore the toxic mechanism of degenerative pathway and modulate those effects using docosahexaenoic acid (DHA). Beta-Amyloid was incubated on human retinal cells at 25 M for 48 h. Beta-Amyloid fibrillation was confirmed using Thioflavin-T test. Cell viability was assessed using LDH assay; oxidative stress using MTT and H2-DCFDA assays; P2X7 cell death receptor activation using YOPRO-1 probe; lysosomal activity using acid phosphatase assay and autophagy using monodansylcadaverine assay. We observed a significant loss of cell viability after incubation with fibrillar Beta-Amyloid but not with non-fibrillar. Beta-Amyloid fibrillation seems to be a major step in AMD development. Fibrillar Beta-Amyloid induces an oxidative stress increase, mitochondrial injury and P2X7 cell death and degeneration receptor activation. Moreover, autophagy seems to be involved in Beta-Amyloid effects. DHA protected retinal cells from Beta-Amyloid cytotoxicity and apoptosis. Moreover P2X7 cell death receptor appeared to be an interesting therapeutic target in AMD and degenerative retina disease. doi:10.1016/j.toxlet.2011.05.819
P2196 The aryl hydrocarbon receptor-mediated disruption of differentiation status of liver progenitor cells J. Vondracek 1,∗ , J. Prochazkova 1 , M. Kabatkova 1 , L. Umannova 1 , V. Bryja 1 , A. Kozubik 1 , M. Machala 2 1 Department of Cytokinetics, Institute of Biophysics AS CR, Brno, Czech Republic, 2 Department of Toxicology, Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic
WB-F344 liver epithelial cells constitute a defined in vitro model of liver progenitors, sensitive to the aryl hydrocarbon receptor (AhR)-mediated deregulation of cell cycle control and disruption of intercellular communication. Recently, we found that AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), disrupt the canonical Wnt/-catenin signaling pathway, which plays an important role in liver biology, in WB-F344 cell model. Therefore, we examined the impact of TCDD on the differentiation of liver progenitor cells, as well as the potential role of AhR in this process. Downregulation of cytokeratins 14 and 19 in rat adult liver stem cells can be associated with cell progression towards a more differentiated phenotype. Activation of Wnt/-catenin signaling increased Krt19 mRNA and partly also Krt14 mRNA. In contrast, AhR activation by TCDD markedly reduced both Krt14 and Krt19 mRNAs, and simultaneously increased expression of Krt8, a major hepatocyte type 2 cytokeratin. TCDD also reduced expression of other adult hepatic stem cell markers, such as Kitl/SCF or Ncam1. AhR knock-down deregulated Krt19 expression, thus confirming the functional role of AhR in the observed effects. The present data indicate that activated AhR may deregulate differentiation of liver progenitors, which may affect liver regeneration. These effects could be partly related to a reduced -catenin signaling. This study was funded by the Czech Science Foundation, Grant No. 524/09/1337. doi:10.1016/j.toxlet.2011.05.820 Regulatory toxicology P2197 Determination of the toxicological relevance of pesticide metabolites in groundwater and in food residues A. Cavelier ∗ , E. Bracq, A. Fastier Regulated Products Department (dpr) – Toxicology Evaluation Unit For Pesticides and Fertilisers, French Agency for Food, Environmental and Occupational Health Safety (Anses), Maisons-Alfort Cedex, France The toxicological relevance of certain metabolites of plant protection products found in groundwater and/or in food commodities must be determined for the purpose of regulatory decision-making at national or European Union level. Currently, the toxicological relevance of a metabolite can have different definitions depending on the compartment in which it is found. A European Food Safety Authority guidance document on the assessment of the relevance of metabolites in groundwater is available and is used to identify and assess relevant metabolites; no similar document is currently available for metabolites found in food residues. Data on the metabolites have to be taken into account and compared to the toxicological profile of the active substance. For groundwater metabolites, a tiered approach is available and toxicological tests may be required, depending on the toxicological profile of the active substance and the level of contamination in groundwater. For food residues, a
Abstracts / Toxicology Letters 205S (2011) S180–S300
ting Results: In addition to B(a)P, various PAHs, including pyrene and benzo(e)pyrene, known to not or only very poorly interact with AhR, similarly up-regulated [Ca2+ ]i in human endothelial HMEC-1 cells. Moreover, a-naphthoflavone, a flavonoïd antagonist of AhR, was also able to induce [Ca2+ ]i . Knocking-down AhR expression in HMEC-1 cells through transfection of siRNAs, was finally demonstrated to not prevent B(a)P-mediated induction of [Ca2+ ]i , whereas it efficiently counteracted B(a)P-mediated induction of the referent AhR target gene cytochrome P-450 1B1. Taken together, these data demonstrate that environmental PAHs trigger [Ca2+ ]i induction in an AhR-independent manner. doi:10.1016/j.toxlet.2011.05.818
P2195 Beta-Amyloid induces toxic degenerative pathways on human retinal cells with P2X7 cell death receptor activation: Role in Age-Related Macular Degeneration (AMD) P. Rat 1,∗ , A. Waks 1 , M. Dutot 1 , B. De Moucheron 1 , O. Laprévote 2 , J. Warnet 2 1
Laboratoire de Toxicologie Analytique et Cellulaire (c-tac), Faculté de Pharmacie, Université Paris Descartes, Paris, France, 2 Laboratoire de Chimie et Toxicologie Analytique et Cellulaire, EA 4463, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France Age-Related Macular Degeneration (AMD) is a degenerative retinal disease, defined by a chronic, progressive and incapacitating disorder, characterized by alteration of the central retina. BetaAmyloid peptide is known in Alzheimer’s disease to be present in senile plaques; it had been also found more recently in retinal extracellular deposits observed in AMD patients. Beta-Amyloid seems to be important in neurodegenerative disorders (brain, retina) but mechanisms need to be explored in retina. Our first aim was to investigate and compare cellular effects of Beta-Amyloid in fibrillar and non-fibrillar form. Our second aim was to explore the toxic mechanism of degenerative pathway and modulate those effects using docosahexaenoic acid (DHA). Beta-Amyloid was incubated on human retinal cells at 25 M for 48 h. Beta-Amyloid fibrillation was confirmed using Thioflavin-T test. Cell viability was assessed using LDH assay; oxidative stress using MTT and H2-DCFDA assays; P2X7 cell death receptor activation using YOPRO-1 probe; lysosomal activity using acid phosphatase assay and autophagy using monodansylcadaverine assay. We observed a significant loss of cell viability after incubation with fibrillar Beta-Amyloid but not with non-fibrillar. Beta-Amyloid fibrillation seems to be a major step in AMD development. Fibrillar Beta-Amyloid induces an oxidative stress increase, mitochondrial injury and P2X7 cell death and degeneration receptor activation. Moreover, autophagy seems to be involved in Beta-Amyloid effects. DHA protected retinal cells from Beta-Amyloid cytotoxicity and apoptosis. Moreover P2X7 cell death receptor appeared to be an interesting therapeutic target in AMD and degenerative retina disease. doi:10.1016/j.toxlet.2011.05.819
P2196 The aryl hydrocarbon receptor-mediated disruption of differentiation status of liver progenitor cells J. Vondracek 1,∗ , J. Prochazkova 1 , M. Kabatkova 1 , L. Umannova 1 , V. Bryja 1 , A. Kozubik 1 , M. Machala 2 1 Department of Cytokinetics, Institute of Biophysics AS CR, Brno, Czech Republic, 2 Department of Toxicology, Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic
WB-F344 liver epithelial cells constitute a defined in vitro model of liver progenitors, sensitive to the aryl hydrocarbon receptor (AhR)-mediated deregulation of cell cycle control and disruption of intercellular communication. Recently, we found that AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), disrupt the canonical Wnt/-catenin signaling pathway, which plays an important role in liver biology, in WB-F344 cell model. Therefore, we examined the impact of TCDD on the differentiation of liver progenitor cells, as well as the potential role of AhR in this process. Downregulation of cytokeratins 14 and 19 in rat adult liver stem cells can be associated with cell progression towards a more differentiated phenotype. Activation of Wnt/-catenin signaling increased Krt19 mRNA and partly also Krt14 mRNA. In contrast, AhR activation by TCDD markedly reduced both Krt14 and Krt19 mRNAs, and simultaneously increased expression of Krt8, a major hepatocyte type 2 cytokeratin. TCDD also reduced expression of other adult hepatic stem cell markers, such as Kitl/SCF or Ncam1. AhR knock-down deregulated Krt19 expression, thus confirming the functional role of AhR in the observed effects. The present data indicate that activated AhR may deregulate differentiation of liver progenitors, which may affect liver regeneration. These effects could be partly related to a reduced -catenin signaling. This study was funded by the Czech Science Foundation, Grant No. 524/09/1337. doi:10.1016/j.toxlet.2011.05.820 Regulatory toxicology P2197 Determination of the toxicological relevance of pesticide metabolites in groundwater and in food residues A. Cavelier ∗ , E. Bracq, A. Fastier Regulated Products Department (dpr) – Toxicology Evaluation Unit For Pesticides and Fertilisers, French Agency for Food, Environmental and Occupational Health Safety (Anses), Maisons-Alfort Cedex, France The toxicological relevance of certain metabolites of plant protection products found in groundwater and/or in food commodities must be determined for the purpose of regulatory decision-making at national or European Union level. Currently, the toxicological relevance of a metabolite can have different definitions depending on the compartment in which it is found. A European Food Safety Authority guidance document on the assessment of the relevance of metabolites in groundwater is available and is used to identify and assess relevant metabolites; no similar document is currently available for metabolites found in food residues. Data on the metabolites have to be taken into account and compared to the toxicological profile of the active substance. For groundwater metabolites, a tiered approach is available and toxicological tests may be required, depending on the toxicological profile of the active substance and the level of contamination in groundwater. For food residues, a