title: Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression.
abstract: BACKGROUND
  Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents.
  
  
  METHODS
  Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay.
  
  
  RESULTS
  The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability.
  
  
  CONCLUSIONS
  The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.
authors.affiliation: Institute of Oncology Ljubljana, Department of Radiotherapy and Department of Experimental Oncology, Ljubljana, Slovenia.
authors.forename: Valerija
authors.initials: V
authors.lastname: Zager
authors.forename: Maja
authors.initials: M
authors.lastname: Cemazar
authors.forename: Irena
authors.initials: I
authors.lastname: Hreljac
authors.forename: Tamara T
authors.initials: TT
authors.lastname: Lah
authors.forename: Gregor
authors.initials: G
authors.lastname: Sersa
authors.forename: Metka
authors.initials: M
authors.lastname: Filipic