Abstracts / Toxicology Letters 229S (2014) S40–S252
transformation, on PON-1 activity and to unveil its potential bioprotective role. Funding: 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 hyperglycaemia and is likely to be involved in progression of pancreatic ␤-cell dysfunction in diabetes. Thus, there is considerable interest 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 (AdomakoBonsu et al., 2013). Using the novel 1.1B4 human pancreatic ␤-cells, cytoprotection by caffeic acid, curcumin and quercetin was investigated. 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 viability (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 potent radical scavenging activities in the 5 h co-exposure cytoprotection assay, while CA was less effective (mean EC50 831.8 ± 16.4 M). Curcumin (maximum protection at 70.9 M) was equi-potent with quercetin (mean EC50 241.5 ± 26.0 M) as indirectly acting antioxidants, with no ␤-cell protection by CA. At high concentrations, Q, CA and Cur were toxic. Quercetin and curcumin showed strong ␤-cell protective activity, 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 identiﬁcation 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 transduction 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 recapitulate in vivo BMP activity (Monteiro et al., 2008). During the differentiation of ESC into cardiomyocytes, we analyzed the differentiation process using quantitative RT-PCR, FACS, quantitative EGFP measurements, and the functional analysis of contracting cardiomyocytes. Our results show that the activity of the reporter gene can be used for the detection of teratogenic activities of valproic acid and 6-aminonicotinamide in vitro. In addition these cells provide a useful tool to characterize the molecular mechanism 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 development 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 chemicals 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