p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures
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The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.
Keywordsp53 tumor suppression protein p53 pathway DNA damage Genotoxicity Cytotoxicity Reporter gene cell line bioassay
Thanks to the RWTH colleague Simone Hotz for the support during the establishment of the cell line and assay in our laboratory. Thanks to BioDetection Systems BV (BDS, Amsterdam, The Netherlands) for supplying the cell line and respective culture and method protocols. Thanks to Promega GmbH, Germany, and to Tecan Group Ltd., Switzerland, for their contribution to this study as a partner of the Students Lab “Fascinating Environment” at Aachen Biology and Biotechnology (ABBt). This study was supported by the EDA-EMERGE ITN project within the EU Seventh Framework Program (FP7-PEOPLE-2011-ITN) under the grant agreement number 290100.
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