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The cell-based biosensor system HepG2CDKN1A-DsRed for rapid and simple detection of genotoxic agents
Blagus, Tanja ...
The regulatory requirements for genotoxicity testing rely on a battery of genotoxicity tests, which generally consist of bacterial and mammalian cell assays for detection of gene mutations and ...chromosomal aberrations. However, for rapid screening, these methods are not appropriate. We have developed a new cell-based biosensor system that provides rapid and simple detection of genotoxic substances. This is based on stable transfection of human hepatoma HepG2 cells with a plasmid that encodes the red fluorescent protein DsRed2 under the control of the CDKN1A promoter (HepG2CDKN1A-DsRed cells). As the major downstream target gene of activated TP53, the tumour-suppressor gene CDKN1A is responsible for cell-cycle arrest following DNA damage, and it has been shown to be specifically up-regulated by genotoxic carcinogens. The assay is optimised for a 96-well microplate format and spectrofluorimetric quantification of induced DsRed expression. The assay was evaluated by testing direct-acting and indirect-acting genotoxic compounds with different mechanisms of action, along with non-genotoxic compounds. Of 25 compounds that are known to be genotoxic in vitro and in vivo, 21 (84%) were detected as positive at noncytotoxic doses, whereas of 12 compounds not considered genotoxic, 11 (92%) were negative. These data indicate the high sensitivity and specificity of our biosensor system. Based on its simplicity and sensitivity, this biosensor developed with HepG2CDKN1ADsRed cells has the potential to become a valuable tool for genotoxicity screening for chemical safety evaluation, as well as for environmental and occupational monitoring of exposure to genotoxic agents and their complex mixtures.
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