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The effect of selected furanocoumarins on the spontaneous motoric activity of rat isolated intestine strips
Abstracts / Toxicology Letters 221S (2013) S59–S256
intended to evaluate and discriminate personal care products within the mild irritancy range. The methodology relies on the use of a standard viability test, on the measurement of inflammatory markers expression and on the standardized exposure techniques according to physical state (powder, liquid, sticky, etc.). Furthermore, the need for modified contact periods compared with the raw material time application is based on the mimicking of human behavior. The first step of the internal validation is the testing of a group of pure substances with a known in vivo score and classification according to the United Nations Globally Harmonized System by following internal standard operating procedures. The second step is the selection and the evaluation of personal care products for leave-on application in order to correlate results with human patch test data. Results and highlights of the mains steps of the internal validation and the results obtained with finished cosmetics formulations are presented. http://dx.doi.org/10.1016/j.toxlet.2013.05.341
P12-75 Systems-toxicology approach to evaluate the biological impact of a subset of harmful/potentially harmful constituents of tobacco smoke Ignacio Gonzalez-Suarez 1,∗ , Carole Mathis 1 , Emmanuel Guedj 1 , Paul Walker 2 , Samantha Ellis 2 , Heather Woodhouse 2 , Remi Dulize 1 , Sandra Wagner 1 , Florian Martin 1 , Nikolai Ivanov 1 , Julia Hoeng 1 , Manuel C. Peitsch 1
S161
P12-76 The effect of environmentally relevant concentration of selected PPCPs on fish cell lines J. Kolarova ∗ , V. Zlabek, R. Grabic, O. Golovko, K. Grabicova, V. Burkina, T. Randak University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic Nowadays, there are several thousands of pharmaceuticals and personal care products (PPCPs) for human and veterinary use. Although most of PPCPs are present in water at trace concentrations, there is a concern that they can have possible adverse effects on sensitive non-target organisms constantly exposed in the aquatic environment. Six tested PPCPs were selected based on relevant ecotoxicological criteria. Results of surface water analysis and fish steady state plasma concentration model were considered for selection of relevant PPCPs and tested concentrations. The effects of diltiazem, clotrimazol, sucralose, atenolol, 2-phenylbenzimidazole-5-sulfonic acid (PBSA) and verapamil were tested in vivo and in vitro on fish and fish cell lines respectively. The toxicity was evaluated in vitro by NR test using RTG, EPC and FHM stable fish cell lines. LOEC of all tested PPCPs for selected cell lines was higher than environmentally relevant concentration in surface water or in fish plasma. However, adverse effects in fish were found even for environmentally relevant concentration tested in vivo. Further research is needed to reveal toxic effects of emerging polar contaminants in aquatic organisms. http://dx.doi.org/10.1016/j.toxlet.2013.05.343
1
Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland, 2 Cyprotex, 15 Beech Lane, SK10 2DR Macclesfield, UK Exposure to cigarette smoke (CS) increases the risk of developing lung diseases including chronic obstructive pulmonary disease (COPD) and cancer. CS is a complex aerosol with over 5000 chemicals, thus it is difficult to determine individual contributions to overall toxicity, as well as the molecular mechanisms by which smoke constituents exert their effects. We selected 14 well-known smoke constituents and established a High Content Screening method in normal human bronchial epithelial cells (NHBE). The impact of the constituents was investigated using 13 multi-parametric indicators of cellular toxicity and complemented with microarray-based transcriptome analysis followed by a computational approach leveraging mechanistic network models to identify and quantify perturbed molecular pathways. Smoke constituents were evaluated in a wide range of concentrations and at different time points. Most constituents induced cellular death only at high doses. Most important toxicity mechanisms were: DNA damage/growth arrest, oxidative stress, mitochondrial stress and apoptosis/necrosis. In summary, combination of standard toxicological endpoints with a systems-based impact assessment allows for a more robust scientific basis for toxicological assessment of smoke constituents, while gaining insight on the molecular mechanisms activated upon exposure This allowed us to establish an in vitro Systems Toxicology platform which will now be applied to a broader selection of smoke constituents and their mixtures and can serve as the basis for testing the impact of other airborne toxicants on NHBEs. http://dx.doi.org/10.1016/j.toxlet.2013.05.342
P12-77 The effect of selected furanocoumarins on the spontaneous motoric activity of rat isolated intestine strips Marta Mendel 1,∗ , Magdalena Chlopecka 1 , Natalia Dziekan 1 , Wojciech Karlik 1 , Krystyna Skalicka-Wozniak 2 Warsaw University of Life Sciences, Poland, 2 Medical University of Lublin, Poland
1
Furanocoumarins are secondary metabolites produced by plants like Garden Angelica, Bishops’ flower or lovage, which are common in moderate climate and are potential reasons of poisonings in human and animals. There is only little knowledge about their effect on gut motility. On the other hand, the traditional indication of using those plants includes gastrointestinal disorders suggesting a possible impact of furanocoumarins on gastrointestinal smooth muscle. Thus, the study was aimed at evaluating the effect of imperatorin and bergapten on gut smooth muscle. All experiments were conducted on rat isolated duodenum and jejunum strips under isotonic conditions. The obtained results revealed that imperatorin caused miorelaxant response of jejunum and duodenum strips when applied in a concentration range of 1–100 M or 10–100 M, respectively. The force of the relaxation was dose-dependent and exceeded the size of reaction to isoproterenol if furanocoumarin was administered in the highest doses (75 and 100 M). In case of bergapten, a relaxant response of duodenum and jejunum strips was observed in the concentration range of 0.0001 up to 10 or 50 M, respectively. Interestingly, the strength of the miorelaxation was similar in case of lower doses (0.0001–1 M) and became weaker or was displaced
S162
Abstracts / Toxicology Letters 221S (2013) S59–S256
by a contraction if bergapten was applied in higher concentrations (10 and 50 M). Concluding, both imperatorin and bergapten affect the motoric activity of rat isolated intestine strips. However, character and strength of evoked reactions depend on the particular furanocoumarin and its concentration. http://dx.doi.org/10.1016/j.toxlet.2013.05.344
P12-78 The gastrointestinal uptake of TiO2 nanoparticles and associated toxicity Constantinos Gitrowski ∗ , Aliaa Al-Jubory, Richard Handy University of Plymouth, UK The gastrointestinal uptake of different crystal structures of TiO2 nanoparticles (NPs) was investigated using Caco-2 intestinal cells. Caco-2 monolayers exhibited time-dependent, saturable uptake of TiO2 for exposures of 1 mg l−1 over 24 h, which was influenced by crystal type. Initial uptake rates of TiO2 (Bulk and NPs) ranged between 3.7 and 5.3 nmol mg−1 protein h−1 . The bulk (amorphous, particle size > 100 nm) of TiO2 exhibited the greatest accumulation at 24 h (14.1 nmol mg−1 protein), whilst nano-rutile TiO2 exhibited the least (6.93 nmol mg−1 protein). All TiO2 exposures caused elevations of Ti in the cells relative to the control, with a crystal structure effect in the order bulk > P25 > anatase > rutile. Electron microscopy of the cells showed that both NPs and bulk TiO2 accumulated below the apical membrane inside vesicles within the cells. X-ray microanalysis confirmed the presence of TiO2 . Incubating cells with 120 IU nystatin or 100 mol l−1 vanadate in the presence of TiO2 caused significant increases in Ti metal accumulation for exposure to all crystal types relative to controls (ANOVA P < 0.05). Incubating cells with 90 mol l−1 genistein or 27 mol l−1 chloropromazine caused a large decrease in cell Ti concentrations compared to controls (ANOVA P < 0.05). Cell viability measures were generally good (low LDH leak, normal cell morphology). However, there were increases in calcium concentration in cells exposed to all TiO2 types (ANOVA P < 0.05). Overall the data suggest that TiO2 accumulation by Caco-2 cells is crystal structure-dependent, and that the mechanism(s) involves endocytosis of intact particles. http://dx.doi.org/10.1016/j.toxlet.2013.05.345
P12-79 The importance of understanding drivers of irritation in vivo for selection of chemicals used in the development and evaluation of in vitro eye irritation assays: Cosmetics Europe analysis J. Barroso 1,∗ , N. Alépée 2 , A. De Smedt 3 , B. De Wever 4 , J. Hibatallah 5 , P. McNamee 6 , K. Mewes 4 , M. Millet 7 , U. Pfannenbecker 8 , M. Tailhardat 9 , M. Templier 7 Cosmetics Europe, Brussels, Belgium, 2 L’Oréal, Aulnay, France, Janssen Research & Development, Beerse, Belgium, 4 Henkel AG & Co. KGaA, Düsseldorf, Germany, 5 Chanel Parfums Beauté, Neuilly sur Seine, France, 6 The Procter & Gamble Company, Egham, United Kingdom, 7 Pierre Fabre, Castres, France, 8 Beiersdorf AG, Hamburg, Germany, 9 LVMH Recherche, St. Jean de Braye, France
1
3
Cosmetics Europe’s Task Force Eye Irritation (TFEI) is actively involved in the development of alternative methods to assess eye irritation potential of cosmetic ingredients using in vitro methods.
Selecting chemicals for use in the development and evaluation of in vitro eye irritation assays based on a understanding of what drives irritation in classification of ocular effects of chemicals in the in vivo Draize test is therefore a critical element that enables identification and evaluation of predictive capacity and applicability domain at an early stage of in vitro methods development. An in depth analysis of the data available from external databases containing in vivo eye irritation data for chemicals tested in the Draize eye irritation test has been undertaken. This analysis is based on having good quality in vivo data that has allowed a clear understanding of the different ocular tissues effects that drive classification. These include corneal opacity, iritis, conjunctival redness, conjunctival chemosis, days to clear and/or persistence of effects. In addition, all chemicals were screened for their commercial availability, assurance that they cover the whole range of irritation potential and represent relevant classes and physical states. The work presented here is focused on a subset of chemicals and the approach used to define the drivers of irritation is described. http://dx.doi.org/10.1016/j.toxlet.2013.05.346
P12-80 The survival of murine dendritic cells is controlled by the transcriptional factor Nrf2 in response to contact sensitizers Zeina El Ali ∗ , Marc Pallardy, Saadia Kerdine-Römer Université Paris Sud – INSERM UMR-996, Faculté de Pharmacie, 5 rue J.B.Clément, F-92296 Châtenay-Malabry, France Dendritic cells (DC) are professional antigen presenting cells playing a major role in the induction of primary immune response. Contact sensitizers, such as dinitrochlorobenzene (DNCB) or cinnamaldehyde (CinA) are known to induce reactive oxygen species (ROS). These compounds generate a chemical or oxidative stress that can be perceived as a danger signal by DC. The Nrf2/Keap1 pathway is central for detoxification. In the absence of a chemical stress, Keap1 associates with Nrf2 and targets it to degradation. In the presence of an electrophilic compound, Keap1’s conformation is modified leading to Nrf2 translocation to the nucleus and transcription of its target genes. We have demonstrated that contact sensitizers like DNCB or CinA induce Nrf2 accumulation in human DC. In order to elucidate the role of Nrf2 in cell survival, nrf2 WT or KO bone marrow-derived dendritic cells (BM-DC) were treated with DNCB or CinA for 24 h. AnV and 7-AAD staining were used to measure apoptotic and necrotic cells respectively. Results show (1) an increase of late apoptotic cells and total apoptotic cells, and (2) a decrease in living cells in absence of Nrf2. An increase of caspase 3 activity, a rapid loss of mitochondrial transmembrane potential ( m ) and a higher ROS production are observed in nrf2−/− DC compared to nrf2+/+ DC in response to DNCB or CinA. Furthermore, our results show that Nrf2 positively regulates bcl2 gene expression in response to DNCB or CinA. These results suggest that Nrf2 plays a role in cell survival and protects DC from contact sensitizers. http://dx.doi.org/10.1016/j.toxlet.2013.05.347
intended to evaluate and discriminate personal care products within the mild irritancy range. The methodology relies on the use of a standard viability test, on the measurement of inflammatory markers expression and on the standardized exposure techniques according to physical state (powder, liquid, sticky, etc.). Furthermore, the need for modified contact periods compared with the raw material time application is based on the mimicking of human behavior. The first step of the internal validation is the testing of a group of pure substances with a known in vivo score and classification according to the United Nations Globally Harmonized System by following internal standard operating procedures. The second step is the selection and the evaluation of personal care products for leave-on application in order to correlate results with human patch test data. Results and highlights of the mains steps of the internal validation and the results obtained with finished cosmetics formulations are presented. http://dx.doi.org/10.1016/j.toxlet.2013.05.341
P12-75 Systems-toxicology approach to evaluate the biological impact of a subset of harmful/potentially harmful constituents of tobacco smoke Ignacio Gonzalez-Suarez 1,∗ , Carole Mathis 1 , Emmanuel Guedj 1 , Paul Walker 2 , Samantha Ellis 2 , Heather Woodhouse 2 , Remi Dulize 1 , Sandra Wagner 1 , Florian Martin 1 , Nikolai Ivanov 1 , Julia Hoeng 1 , Manuel C. Peitsch 1
S161
P12-76 The effect of environmentally relevant concentration of selected PPCPs on fish cell lines J. Kolarova ∗ , V. Zlabek, R. Grabic, O. Golovko, K. Grabicova, V. Burkina, T. Randak University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic Nowadays, there are several thousands of pharmaceuticals and personal care products (PPCPs) for human and veterinary use. Although most of PPCPs are present in water at trace concentrations, there is a concern that they can have possible adverse effects on sensitive non-target organisms constantly exposed in the aquatic environment. Six tested PPCPs were selected based on relevant ecotoxicological criteria. Results of surface water analysis and fish steady state plasma concentration model were considered for selection of relevant PPCPs and tested concentrations. The effects of diltiazem, clotrimazol, sucralose, atenolol, 2-phenylbenzimidazole-5-sulfonic acid (PBSA) and verapamil were tested in vivo and in vitro on fish and fish cell lines respectively. The toxicity was evaluated in vitro by NR test using RTG, EPC and FHM stable fish cell lines. LOEC of all tested PPCPs for selected cell lines was higher than environmentally relevant concentration in surface water or in fish plasma. However, adverse effects in fish were found even for environmentally relevant concentration tested in vivo. Further research is needed to reveal toxic effects of emerging polar contaminants in aquatic organisms. http://dx.doi.org/10.1016/j.toxlet.2013.05.343
1
Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland, 2 Cyprotex, 15 Beech Lane, SK10 2DR Macclesfield, UK Exposure to cigarette smoke (CS) increases the risk of developing lung diseases including chronic obstructive pulmonary disease (COPD) and cancer. CS is a complex aerosol with over 5000 chemicals, thus it is difficult to determine individual contributions to overall toxicity, as well as the molecular mechanisms by which smoke constituents exert their effects. We selected 14 well-known smoke constituents and established a High Content Screening method in normal human bronchial epithelial cells (NHBE). The impact of the constituents was investigated using 13 multi-parametric indicators of cellular toxicity and complemented with microarray-based transcriptome analysis followed by a computational approach leveraging mechanistic network models to identify and quantify perturbed molecular pathways. Smoke constituents were evaluated in a wide range of concentrations and at different time points. Most constituents induced cellular death only at high doses. Most important toxicity mechanisms were: DNA damage/growth arrest, oxidative stress, mitochondrial stress and apoptosis/necrosis. In summary, combination of standard toxicological endpoints with a systems-based impact assessment allows for a more robust scientific basis for toxicological assessment of smoke constituents, while gaining insight on the molecular mechanisms activated upon exposure This allowed us to establish an in vitro Systems Toxicology platform which will now be applied to a broader selection of smoke constituents and their mixtures and can serve as the basis for testing the impact of other airborne toxicants on NHBEs. http://dx.doi.org/10.1016/j.toxlet.2013.05.342
P12-77 The effect of selected furanocoumarins on the spontaneous motoric activity of rat isolated intestine strips Marta Mendel 1,∗ , Magdalena Chlopecka 1 , Natalia Dziekan 1 , Wojciech Karlik 1 , Krystyna Skalicka-Wozniak 2 Warsaw University of Life Sciences, Poland, 2 Medical University of Lublin, Poland
1
Furanocoumarins are secondary metabolites produced by plants like Garden Angelica, Bishops’ flower or lovage, which are common in moderate climate and are potential reasons of poisonings in human and animals. There is only little knowledge about their effect on gut motility. On the other hand, the traditional indication of using those plants includes gastrointestinal disorders suggesting a possible impact of furanocoumarins on gastrointestinal smooth muscle. Thus, the study was aimed at evaluating the effect of imperatorin and bergapten on gut smooth muscle. All experiments were conducted on rat isolated duodenum and jejunum strips under isotonic conditions. The obtained results revealed that imperatorin caused miorelaxant response of jejunum and duodenum strips when applied in a concentration range of 1–100 M or 10–100 M, respectively. The force of the relaxation was dose-dependent and exceeded the size of reaction to isoproterenol if furanocoumarin was administered in the highest doses (75 and 100 M). In case of bergapten, a relaxant response of duodenum and jejunum strips was observed in the concentration range of 0.0001 up to 10 or 50 M, respectively. Interestingly, the strength of the miorelaxation was similar in case of lower doses (0.0001–1 M) and became weaker or was displaced
S162
Abstracts / Toxicology Letters 221S (2013) S59–S256
by a contraction if bergapten was applied in higher concentrations (10 and 50 M). Concluding, both imperatorin and bergapten affect the motoric activity of rat isolated intestine strips. However, character and strength of evoked reactions depend on the particular furanocoumarin and its concentration. http://dx.doi.org/10.1016/j.toxlet.2013.05.344
P12-78 The gastrointestinal uptake of TiO2 nanoparticles and associated toxicity Constantinos Gitrowski ∗ , Aliaa Al-Jubory, Richard Handy University of Plymouth, UK The gastrointestinal uptake of different crystal structures of TiO2 nanoparticles (NPs) was investigated using Caco-2 intestinal cells. Caco-2 monolayers exhibited time-dependent, saturable uptake of TiO2 for exposures of 1 mg l−1 over 24 h, which was influenced by crystal type. Initial uptake rates of TiO2 (Bulk and NPs) ranged between 3.7 and 5.3 nmol mg−1 protein h−1 . The bulk (amorphous, particle size > 100 nm) of TiO2 exhibited the greatest accumulation at 24 h (14.1 nmol mg−1 protein), whilst nano-rutile TiO2 exhibited the least (6.93 nmol mg−1 protein). All TiO2 exposures caused elevations of Ti in the cells relative to the control, with a crystal structure effect in the order bulk > P25 > anatase > rutile. Electron microscopy of the cells showed that both NPs and bulk TiO2 accumulated below the apical membrane inside vesicles within the cells. X-ray microanalysis confirmed the presence of TiO2 . Incubating cells with 120 IU nystatin or 100 mol l−1 vanadate in the presence of TiO2 caused significant increases in Ti metal accumulation for exposure to all crystal types relative to controls (ANOVA P < 0.05). Incubating cells with 90 mol l−1 genistein or 27 mol l−1 chloropromazine caused a large decrease in cell Ti concentrations compared to controls (ANOVA P < 0.05). Cell viability measures were generally good (low LDH leak, normal cell morphology). However, there were increases in calcium concentration in cells exposed to all TiO2 types (ANOVA P < 0.05). Overall the data suggest that TiO2 accumulation by Caco-2 cells is crystal structure-dependent, and that the mechanism(s) involves endocytosis of intact particles. http://dx.doi.org/10.1016/j.toxlet.2013.05.345
P12-79 The importance of understanding drivers of irritation in vivo for selection of chemicals used in the development and evaluation of in vitro eye irritation assays: Cosmetics Europe analysis J. Barroso 1,∗ , N. Alépée 2 , A. De Smedt 3 , B. De Wever 4 , J. Hibatallah 5 , P. McNamee 6 , K. Mewes 4 , M. Millet 7 , U. Pfannenbecker 8 , M. Tailhardat 9 , M. Templier 7 Cosmetics Europe, Brussels, Belgium, 2 L’Oréal, Aulnay, France, Janssen Research & Development, Beerse, Belgium, 4 Henkel AG & Co. KGaA, Düsseldorf, Germany, 5 Chanel Parfums Beauté, Neuilly sur Seine, France, 6 The Procter & Gamble Company, Egham, United Kingdom, 7 Pierre Fabre, Castres, France, 8 Beiersdorf AG, Hamburg, Germany, 9 LVMH Recherche, St. Jean de Braye, France
1
3
Cosmetics Europe’s Task Force Eye Irritation (TFEI) is actively involved in the development of alternative methods to assess eye irritation potential of cosmetic ingredients using in vitro methods.
Selecting chemicals for use in the development and evaluation of in vitro eye irritation assays based on a understanding of what drives irritation in classification of ocular effects of chemicals in the in vivo Draize test is therefore a critical element that enables identification and evaluation of predictive capacity and applicability domain at an early stage of in vitro methods development. An in depth analysis of the data available from external databases containing in vivo eye irritation data for chemicals tested in the Draize eye irritation test has been undertaken. This analysis is based on having good quality in vivo data that has allowed a clear understanding of the different ocular tissues effects that drive classification. These include corneal opacity, iritis, conjunctival redness, conjunctival chemosis, days to clear and/or persistence of effects. In addition, all chemicals were screened for their commercial availability, assurance that they cover the whole range of irritation potential and represent relevant classes and physical states. The work presented here is focused on a subset of chemicals and the approach used to define the drivers of irritation is described. http://dx.doi.org/10.1016/j.toxlet.2013.05.346
P12-80 The survival of murine dendritic cells is controlled by the transcriptional factor Nrf2 in response to contact sensitizers Zeina El Ali ∗ , Marc Pallardy, Saadia Kerdine-Römer Université Paris Sud – INSERM UMR-996, Faculté de Pharmacie, 5 rue J.B.Clément, F-92296 Châtenay-Malabry, France Dendritic cells (DC) are professional antigen presenting cells playing a major role in the induction of primary immune response. Contact sensitizers, such as dinitrochlorobenzene (DNCB) or cinnamaldehyde (CinA) are known to induce reactive oxygen species (ROS). These compounds generate a chemical or oxidative stress that can be perceived as a danger signal by DC. The Nrf2/Keap1 pathway is central for detoxification. In the absence of a chemical stress, Keap1 associates with Nrf2 and targets it to degradation. In the presence of an electrophilic compound, Keap1’s conformation is modified leading to Nrf2 translocation to the nucleus and transcription of its target genes. We have demonstrated that contact sensitizers like DNCB or CinA induce Nrf2 accumulation in human DC. In order to elucidate the role of Nrf2 in cell survival, nrf2 WT or KO bone marrow-derived dendritic cells (BM-DC) were treated with DNCB or CinA for 24 h. AnV and 7-AAD staining were used to measure apoptotic and necrotic cells respectively. Results show (1) an increase of late apoptotic cells and total apoptotic cells, and (2) a decrease in living cells in absence of Nrf2. An increase of caspase 3 activity, a rapid loss of mitochondrial transmembrane potential ( m ) and a higher ROS production are observed in nrf2−/− DC compared to nrf2+/+ DC in response to DNCB or CinA. Furthermore, our results show that Nrf2 positively regulates bcl2 gene expression in response to DNCB or CinA. These results suggest that Nrf2 plays a role in cell survival and protects DC from contact sensitizers. http://dx.doi.org/10.1016/j.toxlet.2013.05.347