Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression

Abstract

BackgroundHuman 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.MethodsMetabolically 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.ResultsThe 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.ConclusionsThe 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.

Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

“…Recently, Zager et al . reported the development of a p21 -mediated eGFP reporter gene bioassay system for genotoxicity detection in human hepatoma HepG2 cells [ 39 ]. Challenging this, stably transformed HepG2 cells (p21HepG2GFP) by genotoxic agents with known mechanisms of action showed that this human cell biosensor system can be used for simple and fast detection of genotoxic agents.…”

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system

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“…Though prior work has examined the gross effects (e.g., viability, proliferation) of analytes on cells313637, these gross assays do not reveal more subtle effects that mask or alter particular phenotypes of interest, such as the activation of signaling pathways. Recently, methods based on the reporting of analytes by fluorescence reaction in engineered cells offer a simple and nondestructive option33343538. Living cells used as biosensors are typically produced with a constructed plasmid, in which genes that code for the bio-reporter are placed under control of a promoter that recognizes the analyte of interest.…”

Acute radiation syndrome and Fukushima: A watershed moment?

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