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The role of functional human aryl hydrocarbon receptor in estrogenicity of polycyclic aromatic hydrocarbons
Abstracts / Toxicology Letters 280S (2017) S82–S91
drives alterations in cellular proces e.g. differentiation, proliferation, apoptosis and it also modifies the AhR pathway itself. However, MAPK involvemet in b[a]p metabolic activation and toxicity in lung tissues is not well understood. Here we used BEAS-2B cell line (SV40-immortalized normal bronchial epithelium cells) to study the participation of ERK1/2 kinases in the b[a]p-related genotoxic effects. Our results indicate that b[a]p is not cytotoxic to BEAS-2B cells at relatively low concentrations. It enhances CYP1A1 gen trancription and protein induction. Additionally, b[a]p promotes ERK1/2 phosphorylation. Accordingly, inhibition of ERK1/2 decreases CYP1A1 protein induction and production of b[a]p adducts. Together, these data suggest a crosstalk between AhR and MAPK pathways, which are essential in the modulation of CYP1A1 enzyme and b[a]p-related adduct production in BEAS-2B cells. http://dx.doi.org/10.1016/j.toxlet.2017.07.233 P-01-01-11 Global changes in expression and functional profile of lung adenocarcinoma cells exposed to various toxic AhR ligands Jirina Prochazkova 1 , Martina Hyzdalova 1 , Simona Strapacova 1 , Lucie Svrzkova 1 , Eva Hruba 1 , Josef Masek 1 , Helena Libalova 2 , Jiri Klema 3 , Jan Topinka 2 , Jan Vondracek 4 , Miroslav Machala 1 1
Department of Chemistry and Toxicology, Veterinary Research Institute, v.v.i., Brno, Czech Republic 2 Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, v.v.i., Prague, Czech Republic 3 Department of Computer Science, Czech Technical University in Prague, Prague, Czech Republic 4 Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, v.v.i., Brno, Czech Republic The lung epithelium represents one of a first-wave target tissues, which has to deal with both acute and chronic exposure to various environmental toxicants and carcinogens presented in i.e. cigarette smoke. Here, we focused on elucidation of molecular and cellular mechanisms underlying the role of aryl hydrocarbon receptor (AhR) signaling in pro-carcinogenic effects of its toxic ligands. We employed lung adenocarcinoma cells (A549) and exposed them to benzo[a]pyrene (BaP; a genotoxic, easily metabolized AhR ligand), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; persistent AhR agonist) and CH223191 (synthetic AhR antagonist) for various time intervals. We observed that the ‘early’ AhR-dependent global gene signature (up to 72 h) was generally common for both TCDD and BaP, while the ‘late’ (2 weeks) global gene signatures were only partially overlapping, and they were enriched in biological processes (e.g. cellular proliferation, lipid metabolism) and in TNFa, NFkB or p21 signaling pathways. Besides, A549 cells exposed to BaP underwent epithelial-to-mesenchymal transition (EMT) and gained mesenchymal phenotype together with enhanced migratory potential, while their TCDD-treated counterparts did not. In contrast, chronic TCDD exposure led to cell growth progression and increased cellular numbers. These findings suggest that AhR signaling could be engaged during all stages of lung carcinogenesis induced by toxic compounds, including tumor progression, and that AhR may crosstalk with distinct sets of signaling pathways within lung epithelial cells in a ligand type-dependent manner. Supported by the Czech Science Foundation (project no. 17-27669S). http://dx.doi.org/10.1016/j.toxlet.2017.07.234
S85
P-01-01-12 PCB 153 increases degradation of connexin 43 via induction of autophagy in liver progenitor cells ˇ Pavlína Simeˇ cková 1 , Kateˇrina Pˇenˇcíková 1 , Josef Maˇsek 2 , Miroslav Machala 1 1
Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic 2 Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic Non-dioxin-like PCB 153 (2,2 ,4,4 ,5,5 - hexachlorobiphenyl) is the most abundant PCB congener found in the environment. Previously, we have reported that PCB 153 inhibits gap junctional intercellular communication (GJIC) and enhances both internalisation and degradation of connexin 43 (Cx43) protein forming gap junctions in rat liver WB-F344 epithelial cells. The aim of the present study was to provide an insight into the role of autophagy in the observed suppression of Cx43 protein elicited by PCB 153. Western blotting analysis revealed that PCB 153 increased level of LC3B protein, a commonly used marker of autophagy. We also detected increased lysosomal content after PCB 153, by measuring the uptake of fluorescent acidotropic probe LysoTracker by flow-cytometry, which might be a result of enhanced autophagy. Furthermore, we found co-localization of Cx43 with LC3B in the cytoplasm after PCB 153 treatment, suggesting that internalised Cx43 could be targeted to autophagosomes. These data seem to support the hypothesis that PCB 153, a highly lipophilic environmental contaminant, induces autophagy in rat liver progenitor cells, which provide the degradation pathway of Cx43 after PCB 153 treatment. This toxic mode of action may contribute to negative impact of PCB 153 on cell-to-cell communication in liver cells. This work was supported by the Ministry of Education, Youth and Sports OPVVV PO1 project “FIT” (Pharmacology, Immunotherapy, nanoToxicology) CZ.02.1.01/0.0/0.0/15 003/0000495. http://dx.doi.org/10.1016/j.toxlet.2017.07.235 P-01-01-13 The role of functional human aryl hydrocarbon receptor in estrogenicity of polycyclic aromatic hydrocarbons Martina Hyzdalova 1,2 , Jakub Pivnicka 1 , Ondrej Zapletal 1 , Gerardo Vazquez-Gomez 1 , Jason Matthews 3 , Jiri Neca 2 , Miroslav Machala 2 , Jan Vondracek 1 1 Department of Cytokinetics, Institute of Biophysics of the CAS, Brno, Czech Republic 2 Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic 3 University of Oslo, Oslo, Norway
It remains unclear, whether various aspects of estrogenicity of polycyclic aromatic hydrocarbons (PAHs) are linked to PAHs themselves, or to their metabolites. The aryl hydrocarbon receptor (AhR) controls expression of cytochrome P450 family 1 (CYP1) enzymes mediating PAH metabolism. Here, we used AhR knockout variant (AhRKO ) and wild-type (AhRwt ) estrogen-sensitive MCF-7 human breast cancer cells to investigate the role of AhR-mediated metabolism in estrogenic effects of benzo[a]pyrene (BaP) and/or benz[a]anthracene (BaA). AhRKO have significantly reduced basal CYP1A1 and CYP1B1 expression, and BaP failed to induce either of those two enzymes in AhRKO cells. This led to a near complete
S86
Abstracts / Toxicology Letters 280S (2017) S82–S91
inhibition of BaP metabolism — at 24 h exposure, e.g. levels of five hydroxyBaP (its known estrogenic metabolites), were negligible in AhRKO cells. When investigating cell cycle progression in MCF-7 cells that were synchronized using charcoal-stripped serum, we found that 17b estradiol, but neither BaP nor BaA increased percentage of cells in S-phase in AhRKO cells. The same trend was observed, when estimating cell proliferation using WST1 assay. Thus, AhRKO cells are sensitive to endogenous estrogen, but not to parental PAHs. Moreover, while PAHs were found, in a time-dependent manner, to moderately increase activity of an ER-dependent luciferase reporter gene in AhRwt cells, they failed to stimulate luciferase activity in AhRKO cells. Together, our data suggest that a metabolism of PAHs may contribute significantly to their impact on estrogenic signaling. Supported by the Czech Science Foundation (project no. 1617085S). http://dx.doi.org/10.1016/j.toxlet.2017.07.236 P-01-01-14 Time-dependent alterations of sphingolipid metabolism elicited by PCB 153 (2,2 ,4,4 ,5,5 -hexachlorobiphenyl) in liver progenitor cells Katerina Pencikova 1 , Josef Slavík 1 , Lucie Svrzkova 1 , Jan Vondracek 2 , Miroslav Machala 1 1
Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic 2 Cytokinetics, Institute of Biophysics of the CAS, Brno, Czech Republic Significant changes of sphingolipid metabolism were observed in rat liver progenitor-like WB-F344 cells after exposure to PCB 153, a highly abundant PCB congener found both in the environment and in living organisms. After short-term exposure (1–3 h), the intracellular concentrations of ceramide and hexosylceramide decreased significantly, while the levels of dihydroceramide and dihydrosphingomyelin were strongly increased. Using ceramide 12/0, an artificial substrate for dihydroceramide desaturase (DES), we found that DES activity was suppressed after PCB 153 treatment. Fenretinide, a small molecular inhibitor of DES, mimicked the effects of PCB 153. These results suggest that DES activity can be rapidly suppressed by PCB 153, which is a novel mode of action of this important environmental contaminant. In contrast to the short-term exposure, longer incubation of cells with PCB 153 (24 h) increased concentrations of ceramide, hexosylceramide and dihydroceramide, thus suggesting a more complex, dynamic deregulation of sphingolipid metabolism by PCB 153. This is the first study showing significantly altered metabolism of sphingolipids, lipid signaling molecules involved in regulation of many cellular functions. The present results suggest that there might exist links between modulation of sphingolipid metabolism and further cellular effects of non-dioxin-like PCB congeners, including disruption of cell-cell communication and modulations of intracellular signaling pathways. This work is supported by the Czech Science Foundation (project no. 17-27669S). http://dx.doi.org/10.1016/j.toxlet.2017.07.237
P-01-01-15 The effects of puwainaphycins F on Caco-2 cell line as a model of the intestinal barrier Ondˇrej Vaˇsíˇcek 1 , Pavel Hrouzek 2 , Jan Hájek 2 , Lukáˇs Kubala 1 , 1,3 ˇ ˇ Pavel Babica 3 , Lenka Svihálková Sindlerová 1
Departement of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, v. v. i., Brno, Czech Republic 2 Institute of Microbiology, Centre Algatech, The Czech Academy of Sciences, Tˇrebon, ˇ Czech Republic 3 Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno, Czech Republic Cyanobacteria are a prolific source of structurally diverse secondary metabolites with a wide spectrum of bioactivities. Puwainaphycins are cyclic lipopeptides of cyanobacterial origin and are composed of nine amino acid units and a -amino fatty acid. Current knowledge indicates that puwainaphycins F/G are able to induce necrosis, increased Ca2+ influx and relocate the actin filaments. In this study, we focused on effect of 4 nature modifications of puwainaphycin F–PUW1118, PUW1146, PUW1188 and PUW1190 on Caco-2 cell line as a model of the intestinal barrier. Cytotoxicity was detected by lactate dehydrogenase release and total protein level. Concentration of interleukin 8 (IL-8) was measured by ELISA. Dextran-FITC trans-well assay was used to detect changes in permeability of differentiated Caco-2 monolayer. Expression of tight-junction proteins (claudin, occludin, ZO-1) was measured by western blotting. Our findings show that PUW1146 was the most cytotoxic metabolite, PUW1118 and PUW1188 were slightly cytotoxic and PUW1190 was very low cytotoxic. The nontoxic concentrations of all PUWs (except PUW1190) were able to increase IL-8 production after 24 h of exposure in dose-dependent manner. The permeability of differentiated Caco-2 monolayer was increased after the addition of PUWs. Expression of all studied tight junction proteins was slightly changed after the exposure to PUWs. In conclusion, cyanobacterial secondary metabolite puwainaphycin F has cytotoxic and pro-inflammatory effects dependent on its structural modifications. It seems that the effect on intestinal barrier permeability is not primarily caused by the remodeling of tight junctions but more likely by decreased membrane elasticity and fluidity. This study was supported by Czech Science Foundation (1624949S). http://dx.doi.org/10.1016/j.toxlet.2017.07.238
drives alterations in cellular proces e.g. differentiation, proliferation, apoptosis and it also modifies the AhR pathway itself. However, MAPK involvemet in b[a]p metabolic activation and toxicity in lung tissues is not well understood. Here we used BEAS-2B cell line (SV40-immortalized normal bronchial epithelium cells) to study the participation of ERK1/2 kinases in the b[a]p-related genotoxic effects. Our results indicate that b[a]p is not cytotoxic to BEAS-2B cells at relatively low concentrations. It enhances CYP1A1 gen trancription and protein induction. Additionally, b[a]p promotes ERK1/2 phosphorylation. Accordingly, inhibition of ERK1/2 decreases CYP1A1 protein induction and production of b[a]p adducts. Together, these data suggest a crosstalk between AhR and MAPK pathways, which are essential in the modulation of CYP1A1 enzyme and b[a]p-related adduct production in BEAS-2B cells. http://dx.doi.org/10.1016/j.toxlet.2017.07.233 P-01-01-11 Global changes in expression and functional profile of lung adenocarcinoma cells exposed to various toxic AhR ligands Jirina Prochazkova 1 , Martina Hyzdalova 1 , Simona Strapacova 1 , Lucie Svrzkova 1 , Eva Hruba 1 , Josef Masek 1 , Helena Libalova 2 , Jiri Klema 3 , Jan Topinka 2 , Jan Vondracek 4 , Miroslav Machala 1 1
Department of Chemistry and Toxicology, Veterinary Research Institute, v.v.i., Brno, Czech Republic 2 Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, v.v.i., Prague, Czech Republic 3 Department of Computer Science, Czech Technical University in Prague, Prague, Czech Republic 4 Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, v.v.i., Brno, Czech Republic The lung epithelium represents one of a first-wave target tissues, which has to deal with both acute and chronic exposure to various environmental toxicants and carcinogens presented in i.e. cigarette smoke. Here, we focused on elucidation of molecular and cellular mechanisms underlying the role of aryl hydrocarbon receptor (AhR) signaling in pro-carcinogenic effects of its toxic ligands. We employed lung adenocarcinoma cells (A549) and exposed them to benzo[a]pyrene (BaP; a genotoxic, easily metabolized AhR ligand), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; persistent AhR agonist) and CH223191 (synthetic AhR antagonist) for various time intervals. We observed that the ‘early’ AhR-dependent global gene signature (up to 72 h) was generally common for both TCDD and BaP, while the ‘late’ (2 weeks) global gene signatures were only partially overlapping, and they were enriched in biological processes (e.g. cellular proliferation, lipid metabolism) and in TNFa, NFkB or p21 signaling pathways. Besides, A549 cells exposed to BaP underwent epithelial-to-mesenchymal transition (EMT) and gained mesenchymal phenotype together with enhanced migratory potential, while their TCDD-treated counterparts did not. In contrast, chronic TCDD exposure led to cell growth progression and increased cellular numbers. These findings suggest that AhR signaling could be engaged during all stages of lung carcinogenesis induced by toxic compounds, including tumor progression, and that AhR may crosstalk with distinct sets of signaling pathways within lung epithelial cells in a ligand type-dependent manner. Supported by the Czech Science Foundation (project no. 17-27669S). http://dx.doi.org/10.1016/j.toxlet.2017.07.234
S85
P-01-01-12 PCB 153 increases degradation of connexin 43 via induction of autophagy in liver progenitor cells ˇ Pavlína Simeˇ cková 1 , Kateˇrina Pˇenˇcíková 1 , Josef Maˇsek 2 , Miroslav Machala 1 1
Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic 2 Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic Non-dioxin-like PCB 153 (2,2 ,4,4 ,5,5 - hexachlorobiphenyl) is the most abundant PCB congener found in the environment. Previously, we have reported that PCB 153 inhibits gap junctional intercellular communication (GJIC) and enhances both internalisation and degradation of connexin 43 (Cx43) protein forming gap junctions in rat liver WB-F344 epithelial cells. The aim of the present study was to provide an insight into the role of autophagy in the observed suppression of Cx43 protein elicited by PCB 153. Western blotting analysis revealed that PCB 153 increased level of LC3B protein, a commonly used marker of autophagy. We also detected increased lysosomal content after PCB 153, by measuring the uptake of fluorescent acidotropic probe LysoTracker by flow-cytometry, which might be a result of enhanced autophagy. Furthermore, we found co-localization of Cx43 with LC3B in the cytoplasm after PCB 153 treatment, suggesting that internalised Cx43 could be targeted to autophagosomes. These data seem to support the hypothesis that PCB 153, a highly lipophilic environmental contaminant, induces autophagy in rat liver progenitor cells, which provide the degradation pathway of Cx43 after PCB 153 treatment. This toxic mode of action may contribute to negative impact of PCB 153 on cell-to-cell communication in liver cells. This work was supported by the Ministry of Education, Youth and Sports OPVVV PO1 project “FIT” (Pharmacology, Immunotherapy, nanoToxicology) CZ.02.1.01/0.0/0.0/15 003/0000495. http://dx.doi.org/10.1016/j.toxlet.2017.07.235 P-01-01-13 The role of functional human aryl hydrocarbon receptor in estrogenicity of polycyclic aromatic hydrocarbons Martina Hyzdalova 1,2 , Jakub Pivnicka 1 , Ondrej Zapletal 1 , Gerardo Vazquez-Gomez 1 , Jason Matthews 3 , Jiri Neca 2 , Miroslav Machala 2 , Jan Vondracek 1 1 Department of Cytokinetics, Institute of Biophysics of the CAS, Brno, Czech Republic 2 Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic 3 University of Oslo, Oslo, Norway
It remains unclear, whether various aspects of estrogenicity of polycyclic aromatic hydrocarbons (PAHs) are linked to PAHs themselves, or to their metabolites. The aryl hydrocarbon receptor (AhR) controls expression of cytochrome P450 family 1 (CYP1) enzymes mediating PAH metabolism. Here, we used AhR knockout variant (AhRKO ) and wild-type (AhRwt ) estrogen-sensitive MCF-7 human breast cancer cells to investigate the role of AhR-mediated metabolism in estrogenic effects of benzo[a]pyrene (BaP) and/or benz[a]anthracene (BaA). AhRKO have significantly reduced basal CYP1A1 and CYP1B1 expression, and BaP failed to induce either of those two enzymes in AhRKO cells. This led to a near complete
S86
Abstracts / Toxicology Letters 280S (2017) S82–S91
inhibition of BaP metabolism — at 24 h exposure, e.g. levels of five hydroxyBaP (its known estrogenic metabolites), were negligible in AhRKO cells. When investigating cell cycle progression in MCF-7 cells that were synchronized using charcoal-stripped serum, we found that 17b estradiol, but neither BaP nor BaA increased percentage of cells in S-phase in AhRKO cells. The same trend was observed, when estimating cell proliferation using WST1 assay. Thus, AhRKO cells are sensitive to endogenous estrogen, but not to parental PAHs. Moreover, while PAHs were found, in a time-dependent manner, to moderately increase activity of an ER-dependent luciferase reporter gene in AhRwt cells, they failed to stimulate luciferase activity in AhRKO cells. Together, our data suggest that a metabolism of PAHs may contribute significantly to their impact on estrogenic signaling. Supported by the Czech Science Foundation (project no. 1617085S). http://dx.doi.org/10.1016/j.toxlet.2017.07.236 P-01-01-14 Time-dependent alterations of sphingolipid metabolism elicited by PCB 153 (2,2 ,4,4 ,5,5 -hexachlorobiphenyl) in liver progenitor cells Katerina Pencikova 1 , Josef Slavík 1 , Lucie Svrzkova 1 , Jan Vondracek 2 , Miroslav Machala 1 1
Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic 2 Cytokinetics, Institute of Biophysics of the CAS, Brno, Czech Republic Significant changes of sphingolipid metabolism were observed in rat liver progenitor-like WB-F344 cells after exposure to PCB 153, a highly abundant PCB congener found both in the environment and in living organisms. After short-term exposure (1–3 h), the intracellular concentrations of ceramide and hexosylceramide decreased significantly, while the levels of dihydroceramide and dihydrosphingomyelin were strongly increased. Using ceramide 12/0, an artificial substrate for dihydroceramide desaturase (DES), we found that DES activity was suppressed after PCB 153 treatment. Fenretinide, a small molecular inhibitor of DES, mimicked the effects of PCB 153. These results suggest that DES activity can be rapidly suppressed by PCB 153, which is a novel mode of action of this important environmental contaminant. In contrast to the short-term exposure, longer incubation of cells with PCB 153 (24 h) increased concentrations of ceramide, hexosylceramide and dihydroceramide, thus suggesting a more complex, dynamic deregulation of sphingolipid metabolism by PCB 153. This is the first study showing significantly altered metabolism of sphingolipids, lipid signaling molecules involved in regulation of many cellular functions. The present results suggest that there might exist links between modulation of sphingolipid metabolism and further cellular effects of non-dioxin-like PCB congeners, including disruption of cell-cell communication and modulations of intracellular signaling pathways. This work is supported by the Czech Science Foundation (project no. 17-27669S). http://dx.doi.org/10.1016/j.toxlet.2017.07.237
P-01-01-15 The effects of puwainaphycins F on Caco-2 cell line as a model of the intestinal barrier Ondˇrej Vaˇsíˇcek 1 , Pavel Hrouzek 2 , Jan Hájek 2 , Lukáˇs Kubala 1 , 1,3 ˇ ˇ Pavel Babica 3 , Lenka Svihálková Sindlerová 1
Departement of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, v. v. i., Brno, Czech Republic 2 Institute of Microbiology, Centre Algatech, The Czech Academy of Sciences, Tˇrebon, ˇ Czech Republic 3 Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno, Czech Republic Cyanobacteria are a prolific source of structurally diverse secondary metabolites with a wide spectrum of bioactivities. Puwainaphycins are cyclic lipopeptides of cyanobacterial origin and are composed of nine amino acid units and a -amino fatty acid. Current knowledge indicates that puwainaphycins F/G are able to induce necrosis, increased Ca2+ influx and relocate the actin filaments. In this study, we focused on effect of 4 nature modifications of puwainaphycin F–PUW1118, PUW1146, PUW1188 and PUW1190 on Caco-2 cell line as a model of the intestinal barrier. Cytotoxicity was detected by lactate dehydrogenase release and total protein level. Concentration of interleukin 8 (IL-8) was measured by ELISA. Dextran-FITC trans-well assay was used to detect changes in permeability of differentiated Caco-2 monolayer. Expression of tight-junction proteins (claudin, occludin, ZO-1) was measured by western blotting. Our findings show that PUW1146 was the most cytotoxic metabolite, PUW1118 and PUW1188 were slightly cytotoxic and PUW1190 was very low cytotoxic. The nontoxic concentrations of all PUWs (except PUW1190) were able to increase IL-8 production after 24 h of exposure in dose-dependent manner. The permeability of differentiated Caco-2 monolayer was increased after the addition of PUWs. Expression of all studied tight junction proteins was slightly changed after the exposure to PUWs. In conclusion, cyanobacterial secondary metabolite puwainaphycin F has cytotoxic and pro-inflammatory effects dependent on its structural modifications. It seems that the effect on intestinal barrier permeability is not primarily caused by the remodeling of tight junctions but more likely by decreased membrane elasticity and fluidity. This study was supported by Czech Science Foundation (1624949S). http://dx.doi.org/10.1016/j.toxlet.2017.07.238