Developing A Transgenic Fish Cell For Rapid Investigation Of The Genotoxicity Of Environmental Pollutants

The p53signal pathway can activate the expression of p53protein and induceup-regulation of the level of p53protein when responses to celluar DNA damage, thenthe p53protein will regulate the transcription of downstream multiple target genesinvolving in the induction of cell cycle arrest, DNA repair and apoptosis, so the cellswill be in the status of growth arrest and giving themselves enough time to repairDNA damage. If the DNA damage is too serious to repair, the p53protein will induceapoptosis, so as to ensure the integrity of the genome. The cyclin-dependent kinase(Cdks) inhibitor p21(WAF1/Cip1) is the major downstream target gene of activatedp53and is responsible for causing cell arrest following DNA damage. Here wereported the development of a fish cell biosensor system for genotoxicity detection bythe stable integration of two reporter plasmids of pGL3-p21-luc (human p21promoterlinked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renillaluciferase) into the marine flatfish cell line of FG (flounder gill), named as p21FGLuccells. The trans-activated expression of firefly luciferase in the p21FGLuc cells ispositively correlated with the cellular DNA damage response to genotoxicants, whilethe expression of Renilla luciferase is constitutive and serves as an internal control,normalizing the experimental variability. so we planed to treat this transgenic fish cellgenotoxicity biosensor system based on dual-luciferase reporter system withenvironmental pollutants and drugs, in order to check whether the chemicals weregenetic toxicity, if they were, then the relative fluerescent signals(fireflyluciferases/renilla luciferases) of p21FGLuc cells would be stronger.Firstly, we optimized the transforming conditions of FG cells, results showedthat the optimal transforming conditions were: LipofectamineTMLTX Reagents was the best option because of its lower cytotoxicity and higher efficiency; the maximumDNA was500ng/well in24-well mutiplates; in the co-trancfection experiment, theexperimental vector and co-reporter vector combination at the ratio of5:1could givethe strongest fluerescent signal.Secondly, in order to test the entirety of the p53signalling pathway in the FGcells, we transformed plasmids of pGL3-p53-luc (human p53promoter linked tofirefly luciferase) and pGL3-p21-luc (human p21promoter linked to firefly luciferase)into the marine flatfish cell line of FG (flounder gill)，respectively. Then the transienttransformed FG cells were challenged with genotoxic agent of bleomycin. Theefficient trans-activation of pGL3-p21-luc plasmid showed that FG cells still heldwild-type p53proteins and p53proteins of FG cells were able to recognize andtrans-activate the promoter of human p21gene and activate the expression of fireflyluciferase, making the system an appropriate model for development of the testsystem based on the p53-mediated DNA damage response.Thirdly, we tested the sensitivity of FG cells to G418, and determined theconcentration of600μg/ml as the screening concentration for transformed FG cells,and the maintain concentration was300μg/ml. Based on this result, we co-transfectedthe three plasmids of pGL3-p21-luc, pRL-CMV and pcDNA3.1at the ratio of5:1:1,after more than2week’s selection of G418, finally we obtained the stablytransformed FG cell, called as p21FGLuc.At last, p21FGLuc cells were exposed to genotoxic agents of different geneticmechanism and non-genotoxic agent in order to check the specificity, sensitivity andreliability of this biosensor system. The results showed that, they specificallyresponded to the challenge of genotoxic agents of bleomycin and mitomycin C in adose-dependent manner, but did not respond to the stress of non-genotoxic agent ofethanol; the response speed of this biosensor system to genotoxic agents was fast andthe induced expression level of reporter genes was relatively stable among thedifferent exposure periods tested; under similar cytotoxicity, bleomycin exhibitedhigher genotoxicity to the p21FGLuc cells than mitomycin C, indicating its ability inthe classification of genotoxic grade. In short, it is suggested that this fish cell biosensor system showed very good specificity.Di(2-ethylhexyl) phthalate (DEHP), a kind of plasticizer, was one of the mostwidespread environmental pollutants in the world. DEHP always gives negativeresults not only in the bacterial, yeast and mammalian cells-based bioassay systemsbut also in micronucleus assay and chromosome aberration experiment, but it isreported that DEHP can cause liver cancer in exposed rodents. In this paper, DEHPshowed positive result in the genotoxicity test of p21FGLuc cells, we first providedevidence about genotoxicity of DEHP, but the specific mechanism was unclear. At theenvironmental dose (0.005μg/ml), DEHP obviously induced DNA damage responseof p21FGLuc cells, at50μg/ml, the DNA damage response went to a peak. It wasthus clear that, p21FGLuc biosensor system was more sensitive than othergenotoxicity bioassay systems. Cyclophosphamide itself was not the role of mutagenbut after metabolic activation in liver it would produced alkylating metabolites whichcould cause DNA damage.When using metabolically incompetent cells, theindirectly-acting genotoxic agents had to be tested in the presence of exogenousmetabolic activation,usually S9liver extracts.The results showed thatcyclophosphamide was negative in the test only gave a statistically significantinduction of fluorescence in the presence of S9mix.The acyclic polyol D-mannitolwas a kind if non-genotoxic agent and had no effect on fluorescence induction in thetest.These results indicated this fish cell biosensor system showed very goodsensitivity.But the obtained fish cell (p21FGLuc) biosensor system for the detection ofgenotoxicity has a deficiency. That is, The p21FGLuc cells will respond to thetreatment of non-genotoxic agent of sodium butyrate and give a false positive resultsince sodium butyrate can trans-activated the expression of p21gene viap53-independent pathway. This make it necessary to establish another biosensorsystem based on the transactivation of p53gene in a p53-/-cells.In short, we still believed that this fish cell biosensor system may become aspecific and sensitive tool for genotoxicity detection of new chemicals and drugs. Inaddition, the obtained p21FGLuc cells can be easily maintained at room temperature and a sub-confluent monolayer can survive over1month at15℃without mediumreplacement. This will facilitate the marketing of this fish cell biosensor system.