Phytochemicals protect human pancreatic β-cells against oxidative damage
1
Abstracts / Toxicology Letters 229S (2014) S40–S252 S135 transformation, on PON-1 activity and to unveil its potential bioprotective role. Funding: EXPL/DTP-FTO/0204/2012(CGD);SFRH/BD/92191/ 2013(ATM). http://dx.doi.org/10.1016/j.toxlet.2014.06.476 P-2.130 Phytochemicals protect human pancreatic -cells against oxidative damage Amma Adomako-Bonsu ∗ , Margaret Pratten, Jeffrey Fry, Sue Chan University of Nottingham, Nottingham, UK Oxidative stress is produced under conditions of chronic hyper- glycaemia and is likely to be involved in progression of pancreatic -cell dysfunction in diabetes. Thus, there is considerable inter- est in the potential use of natural antioxidants in the management of diabetes. From previous reports, caffeic acid, curcumin and quercetin possess strong antioxidant properties in vitro (Adomako- Bonsu et al., 2013). Using the novel 1.1B4 human pancreatic -cells, cytoprotection by caffeic acid, curcumin and quercetin was inves- tigated. Sensitivity of 1.1B4 -cells to tert-butyl hydroperoxide (tBHP) induced-cytotoxicity was determined using Neutral Red viability assay. -Cell protection by quercetin (Q), caffeic acid (CA) and curcumin (Cur) was then assessed in 5hr co-exposure and 20hr pre-exposure experiments with 0.5 mM tBHP (>95% cell death). tBHP caused a concentration-dependent decrease in cell viabil- ity (mean EC 50 0.0628 mM), indicating sensitivity of 1.1B4 human -cells to redox imbalance. Quercetin (mean EC 50 48.6 ± 8.9 M) and curcumin (maximum protection at 67.9 M) exhibited potent radical scavenging activities in the 5 h co-exposure cytoprotection assay, while CA was less effective (mean EC 50 831.8 ± 16.4 M). Curcumin (maximum protection at 70.9 M) was equi-potent with quercetin (mean EC 50 241.5 ± 26.0 M) as indirectly acting antiox- idants, with no -cell protection by CA. At high concentrations, Q, CA and Cur were toxic. Quercetin and curcumin showed strong -cell protective activ- ity, whereas caffeic acid exhibited only modest direct antioxidant activity. Future studies will examine the activities of these natural antioxidants in -cells exposed to high glucose (glucotoxicity). Reference Adomako-Bonsu, A., et al., 2013. Protection against oxidative stress by rosmarinic acid and its major metabolites in hepatic cells. Planta Med. 79, PJ3. http://dx.doi.org/10.1016/j.toxlet.2014.06.477 P-2.131 Embryonic stem cells carrying a transgenic BMP-reporter construct: A useful tool for the identification and analysis of teratogenic compounds in vitro Josephine Kugler 1 , Julian Tharmann 1 , Susana M. Chuva de Sousa Lopez 2 , Mummery Christine 2 , Kemler Rolf 3 , Andreas Luch 1 , Michael Oelgeschläger 1 , Andrea Hasse 1,∗ 1 Federal Institute for Risk Assessment, Berlin, Germany, 2 Leiden University Medical Center; Department of Anatomy & Embryology, Leiden, Belgium, 3 Max Planck Institute of Immunobiology and Epigenetic, Department of Molecular Embryology, Freiburg, Germany Embryonic stem cells (ESC) are used as a tool for the identi- fication of teratogenic activities in the analysis of chemicals or pharmaceuticals. In particular, the embryonic stem cell test (EST) has been scientifically validated some time ago and is now used for screening purposes. The differentiation of embryonic stem cells in various tissues is regulated by a set of essential signal transduc- tion pathways, including the TGF, Wnt, and Shh pathway as well as tyrosine kinase receptors mediating FGF or EGF signaling. For some of these essential signal transduction pathways, transgenic reporter mice have been developed that allow the in vivo analysis of pathway activity during embryonic development. We have isolated and characterized embryonic stem cells from transgenic mice carrying an EGFP transgene under the control of a BMP responsive element that has been shown to nicely reca- pitulate in vivo BMP activity (Monteiro et al., 2008). During the differentiation of ESC into cardiomyocytes, we analyzed the dif- ferentiation process using quantitative RT-PCR, FACS, quantitative EGFP measurements, and the functional analysis of contracting car- diomyocytes. Our results show that the activity of the reporter gene can be used for the detection of teratogenic activities of val- proic acid and 6-aminonicotinamide in vitro. In addition these cells provide a useful tool to characterize the molecular mecha- nism underlying the activity of chemicals or pharmaceuticals on the differentiation process during early embryonic development. Reference Monteiro, R.M., de Sousa Lopes, S.M., Bialecka, M., de Boer, S., Zwijsen, A., Mummery, C.L., 2008. Real time monitoring of BMP Smads transcriptional activity during mouse development. Genesis 46, 335–346. http://dx.doi.org/10.1016/j.toxlet.2014.06.478 P-2.132 In-house validation and technology transfer of the GARD assay for prediction of sensitizing compounds Andy Forreryd ∗ , Henrik Johansson, Frida Rydnert, Ann-Sofie Albrekt, Carl Borrebaeck, Malin Lindstedt Department of Immunotechnology, Lund, Sweden Background: Allergic contact dermatitis is caused by an adverse immune response towards chemical haptens. The disease affects a significant proportion of the population, leading to a substantial economic burden for society. New legislations on the registration and use of chemicals within cosmetic industry require develop- ment of high-throughput, in vitro assays for the prediction of sensitization, to replace current animal-based experiments. Methods: We have developed a cell-based assay for prediction of sensitizing chemicals, called Genomic Allergen Rapid Detection, GARD. Analyzing the transcriptome of the MUTZ-3 cell line after 24 h stimulation, using well characterized skin sensitizing chem- icals and controls, we identified a genomic biomarker signature with potent discriminatory ability. To further adapt the assay to high-throughput screening mode, we evaluated the performance of three non-array based platforms using a restricted set of probes from the biomarker signature. Results: Prediction accuracy of the assay was assessed in three separate in-house, validation studies, and is thus far 89%. Results from the evaluation of platforms mimicked previous data from genome wide transcriptome analysis in terms of reproducibility while alternative platforms proved to be superior in terms of cost efficiency, sample throughput and simplified protocols. Conclusions: GARD was demonstrated to have potent ability to predict sensitization. Changing the technical platform for gene
Transcript of Phytochemicals protect human pancreatic β-cells against oxidative damage
Abstracts / Toxicology Letters 229S (2014) S40–S252 S135
transformation, on PON-1 activity and to unveil its potentialbioprotective role.
Funding: EXPL/DTP-FTO/0204/2012(CGD);SFRH/BD/92191/2013(ATM).
http://dx.doi.org/10.1016/j.toxlet.2014.06.476
P-2.130Phytochemicals protect human pancreatic�-cells against oxidative damage
Amma Adomako-Bonsu ∗, Margaret Pratten, Jeffrey Fry, Sue Chan
University of Nottingham, Nottingham, UK
Oxidative stress is produced under conditions of chronic hyper-glycaemia and is likely to be involved in progression of pancreatic�-cell dysfunction in diabetes. Thus, there is considerable inter-est in the potential use of natural antioxidants in the managementof diabetes. From previous reports, caffeic acid, curcumin andquercetin possess strong antioxidant properties in vitro (Adomako-Bonsu et al., 2013). Using the novel 1.1B4 human pancreatic �-cells,cytoprotection by caffeic acid, curcumin and quercetin was inves-tigated.
Sensitivity of 1.1B4 �-cells to tert-butyl hydroperoxide (tBHP)induced-cytotoxicity was determined using Neutral Red viabilityassay. �-Cell protection by quercetin (Q), caffeic acid (CA) andcurcumin (Cur) was then assessed in 5hr co-exposure and 20hrpre-exposure experiments with 0.5 mM tBHP (>95% cell death).
tBHP caused a concentration-dependent decrease in cell viabil-ity (mean EC50 0.0628 mM), indicating sensitivity of 1.1B4 human�-cells to redox imbalance. Quercetin (mean EC50 48.6 ± 8.9 �M)and curcumin (maximum protection at 67.9 �M) exhibited potentradical scavenging activities in the 5 h co-exposure cytoprotectionassay, while CA was less effective (mean EC50 831.8 ± 16.4 �M).Curcumin (maximum protection at 70.9 �M) was equi-potent withquercetin (mean EC50 241.5 ± 26.0 �M) as indirectly acting antiox-idants, with no �-cell protection by CA. At high concentrations, Q,CA and Cur were toxic.
Quercetin and curcumin showed strong �-cell protective activ-ity, whereas caffeic acid exhibited only modest direct antioxidantactivity. Future studies will examine the activities of these naturalantioxidants in �-cells exposed to high glucose (glucotoxicity).
Reference
Adomako-Bonsu, A., et al., 2013. Protection against oxidative stress by rosmarinicacid and its major metabolites in hepatic cells. Planta Med. 79, PJ3.
http://dx.doi.org/10.1016/j.toxlet.2014.06.477
P-2.131Embryonic stem cells carrying a transgenicBMP-reporter construct: A useful tool for theidentification and analysis of teratogeniccompounds in vitro
Josephine Kugler 1, Julian Tharmann 1, Susana M. Chuva de SousaLopez 2, Mummery Christine 2, Kemler Rolf 3, Andreas Luch 1,Michael Oelgeschläger 1, Andrea Hasse 1,∗
1 Federal Institute for Risk Assessment, Berlin, Germany, 2 LeidenUniversity Medical Center; Department of Anatomy & Embryology,Leiden, Belgium, 3 Max Planck Institute of Immunobiology andEpigenetic, Department of Molecular Embryology, Freiburg, Germany
Embryonic stem cells (ESC) are used as a tool for the identi-fication of teratogenic activities in the analysis of chemicals orpharmaceuticals. In particular, the embryonic stem cell test (EST)has been scientifically validated some time ago and is now usedfor screening purposes. The differentiation of embryonic stem cellsin various tissues is regulated by a set of essential signal transduc-tion pathways, including the TGF�, Wnt, and Shh pathway as wellas tyrosine kinase receptors mediating FGF or EGF signaling. Forsome of these essential signal transduction pathways, transgenicreporter mice have been developed that allow the in vivo analysisof pathway activity during embryonic development.
We have isolated and characterized embryonic stem cells fromtransgenic mice carrying an EGFP transgene under the control ofa BMP responsive element that has been shown to nicely reca-pitulate in vivo BMP activity (Monteiro et al., 2008). During thedifferentiation of ESC into cardiomyocytes, we analyzed the dif-ferentiation process using quantitative RT-PCR, FACS, quantitativeEGFP measurements, and the functional analysis of contracting car-diomyocytes. Our results show that the activity of the reportergene can be used for the detection of teratogenic activities of val-proic acid and 6-aminonicotinamide in vitro. In addition thesecells provide a useful tool to characterize the molecular mecha-nism underlying the activity of chemicals or pharmaceuticals onthe differentiation process during early embryonic development.
Reference
Monteiro, R.M., de Sousa Lopes, S.M., Bialecka, M., de Boer, S., Zwijsen, A., Mummery,C.L., 2008. Real time monitoring of BMP Smads transcriptional activity duringmouse development. Genesis 46, 335–346.
http://dx.doi.org/10.1016/j.toxlet.2014.06.478
P-2.132In-house validation and technology transfer ofthe GARD assay for prediction of sensitizingcompounds
Andy Forreryd ∗, Henrik Johansson, Frida Rydnert, Ann-SofieAlbrekt, Carl Borrebaeck, Malin Lindstedt
Department of Immunotechnology, Lund, Sweden
Background: Allergic contact dermatitis is caused by an adverseimmune response towards chemical haptens. The disease affectsa significant proportion of the population, leading to a substantialeconomic burden for society. New legislations on the registrationand use of chemicals within cosmetic industry require develop-ment of high-throughput, in vitro assays for the prediction ofsensitization, to replace current animal-based experiments.
Methods: We have developed a cell-based assay for predictionof sensitizing chemicals, called Genomic Allergen Rapid Detection,GARD. Analyzing the transcriptome of the MUTZ-3 cell line after24 h stimulation, using well characterized skin sensitizing chem-icals and controls, we identified a genomic biomarker signaturewith potent discriminatory ability. To further adapt the assay tohigh-throughput screening mode, we evaluated the performanceof three non-array based platforms using a restricted set of probesfrom the biomarker signature.
Results: Prediction accuracy of the assay was assessed in threeseparate in-house, validation studies, and is thus far 89%. Resultsfrom the evaluation of platforms mimicked previous data fromgenome wide transcriptome analysis in terms of reproducibilitywhile alternative platforms proved to be superior in terms of costefficiency, sample throughput and simplified protocols.
Conclusions: GARD was demonstrated to have potent abilityto predict sensitization. Changing the technical platform for gene
