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

Geng D, Zhang Z, Guo H - Biosensors (Basel) (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biology, Ocean University of China, Qingdao 266003, China. doarey@126.com.

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.

No MeSH data available.


Related in: MedlinePlus

The responses of the p21FGLuc cells to varied concentrations of cyclophosphamide, di(2-ethylhexyl) phthalate (DEHP), D-mannitol and sodium butyrate for 24 h. S9+ and S9− refer to results with and without metabolic activation by the S9 mixture, respectively. Data are expressed as mean ± SD.
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biosensors-02-00318-f007: The responses of the p21FGLuc cells to varied concentrations of cyclophosphamide, di(2-ethylhexyl) phthalate (DEHP), D-mannitol and sodium butyrate for 24 h. S9+ and S9− refer to results with and without metabolic activation by the S9 mixture, respectively. Data are expressed as mean ± SD.

Mentions: As shown in Figure 7, after metabolically activated by rat liver S9 mixture, cyclophosphamide could significantly induce the up-regulation of the expression of firefly luciferase in the exposed p21FGLuc cells in a dose-dependent manner, but not for the cyclophosphamide without S9 mixture pretreatment. Similar positive responses were observed in the p21FGLuc cells exposed to increasing concentrations of DEHP. Even at an environmental dose level of 0.005 μg/mL DEHP, obvious up-regulation of the expression of firefly luciferase was observed in the exposed p21FGLuc cells. At the concentration of 50 μg/mL DEHP, the induced expression level of firefly luciferase in the exposed cells reached a peak level. In contrast, D-mannitol did not induce any up-regulation of firefly luciferase expression in the exposed cells like ethanol.


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

Geng D, Zhang Z, Guo H - Biosensors (Basel) (2012)

The responses of the p21FGLuc cells to varied concentrations of cyclophosphamide, di(2-ethylhexyl) phthalate (DEHP), D-mannitol and sodium butyrate for 24 h. S9+ and S9− refer to results with and without metabolic activation by the S9 mixture, respectively. Data are expressed as mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263550&req=5

biosensors-02-00318-f007: The responses of the p21FGLuc cells to varied concentrations of cyclophosphamide, di(2-ethylhexyl) phthalate (DEHP), D-mannitol and sodium butyrate for 24 h. S9+ and S9− refer to results with and without metabolic activation by the S9 mixture, respectively. Data are expressed as mean ± SD.
Mentions: As shown in Figure 7, after metabolically activated by rat liver S9 mixture, cyclophosphamide could significantly induce the up-regulation of the expression of firefly luciferase in the exposed p21FGLuc cells in a dose-dependent manner, but not for the cyclophosphamide without S9 mixture pretreatment. Similar positive responses were observed in the p21FGLuc cells exposed to increasing concentrations of DEHP. Even at an environmental dose level of 0.005 μg/mL DEHP, obvious up-regulation of the expression of firefly luciferase was observed in the exposed p21FGLuc cells. At the concentration of 50 μg/mL DEHP, the induced expression level of firefly luciferase in the exposed cells reached a peak level. In contrast, D-mannitol did not induce any up-regulation of firefly luciferase expression in the exposed cells like ethanol.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biology, Ocean University of China, Qingdao 266003, China. doarey@126.com.

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.

No MeSH data available.


Related in: MedlinePlus