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

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.

Cell Based-Green Fluorescent Biosensor Using Cytotoxic Pathway for Bacterial Lipopolysaccharide Recognition

“…Because LPS can enter the circulatory system, causing a systemic inflammatory response that is detrimental to the host, using whole living cells for LPS recognition would be a best choice and enable direct functional information to be obtained regarding the effects of it on a living system. Methods based on reporting of analytes by fluorescence reactions in engineered cells have the potential to be reagent-free, simple, and nondestructive. − Living cells used as biosensors are typically propagated with a plasmid containing the genes that code for the bioreporter and are placed under control of a promoter that recognizes the analytes of interest, and inducers activate the promoter genes, providing a genetic signal transducer that triggers and regulates the bioreporter expression . What is more, mammalian cell-based biosensors for LPS have a significant advantage in reflecting cellular physiological action rather than quantitative detection, because external stimuli or changes in cellular microenvironment can disturb the “normal” physiological activities of mammalian cells and can provide insight into the mechanism of action of LPS.…”

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

“…Rider et al reported the first cell sensor that used B lymphocytes to recognize specific bacteria with the help of membrane-bound IgM antibodies32. Mammalian cell-based biosensors have a distinct advantage in reflecting cellular physiological action rather than just quantitative detection and therefore can provide insight into mechanism of action of analytes333435. More importantly, tests based on human cells rather than other cell types, are more likely to detect contaminants that can cause adverse effects in humans.…”

“…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.…”

Micro and Nanotechnologies Enhanced Biomolecular Sensing

“…Such development aims beyond improving upon current techniques in terms of speed and sensitivity, but strives to enable scientists to conduct studies and clinicians to conduct tests that were unfeasible or impractical with previous technologies. This special issue focuses on the contributions related to the development or use of micro and nanomaterials, devices, and techniques for biomolecular sensing research and applications [ 1 , 2 , 3 , 4 , 5 ].…”

“…The use of biosensors can also be applied to the detection and monitoring of environmental pollutants including genotoxicants. Geng et al [ 5 ] reported a fish cell biosensor system capable of genotoxicity detection in a high throughput manner. The sensor was developed by integrating two report plasmids into marine flatfish flounder gill cells and was validated by a p21-mediated luciferase reporter gene bioassay system for genotoxicity.…”