Abstract
p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.
Highlights
The ocean is a final sink of environmental pollutants including genotoxicants, which are often transported into the sea by rainfall and rivers
A Prerequisite for flounder gill (FG) Cells to Be Engineered into a Genotoxicity Biosensor System Is to Hold a
The integrity and validity of the p53-signaling pathway is indispensible for the maintenance of genomic stability [55,56]
Summary
The ocean is a final sink of environmental pollutants including genotoxicants, which are often transported into the sea by rainfall and rivers. The others monitor the activation of DNA damage-induced over-expression of reporter genes including GFP (green fluorescent protein), eGFP (enhanced green fluorescent protein) and luciferase, which are driven by the promoter of DNA damage repair genes like RAD54 [20,21,22] These yeast-based reporter gene assays show a high specificity and sensitivity in the genotoxicity detection of various compounds and are adaptable to high throughput screening [23,24,25,26]. Mammalian cell-based genotoxicity tests are more useful for risk assessment in humans They are often designed to detect DNA damage, gene mutation or cellular DNA damage response. The obtained results showed that this fish cell biosensor system could be used for rapid and high throughput screening of genotoxic agents
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