Tests for measuring DNA damage following exposure to ionising radiation and genotoxicity testing of chemicals and products such as pharmaceuticals, pesticides, food additives, and cosmetics are important for the assessment of health hazard and are also regulatory requirements. We developed a new genotoxicity test – a cell-based biosensor system - based on a p21-dependent DsRED reporter gene assay with stably transformed human hepatoma HepG2 cells. The cell-based biosensor system was prepared by transfection of HepG2 cells with a plasmid encoding red fluorescent protein (DsRed) under the control of the p21 promoter (p21HepG2DsRed). The performance and sensitivity of our cell-based biosensor system for the detection of DNA damage has been evaluated with genotoxic agents with known mechanisms of action, such as radiation and chemical agents cisplatin, methyl methanesulphonate (MMS), benzo(a)pyrene (BaP) and vinblastine (VLB). The increase in fluorescence intensity at different post-exposure time points, due to p21-mediated DsRed expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. Our preliminary results show that radiation, as well as all tested agents, induces the increase in DsRED fluorescence above a certain dose (concentration) in a dose-dependent manner. The increase in fluorescence intensity was detected already at 48 h after exposure at doses that were not cytotoxic. Based on the results, we demonstrated that this novel assay can be used as a fast and simple biosensor system for the detection of genetic damage.
COBISS.SI-ID: 1366651