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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 time-course responses of the stable p21FGLuc cells to model genotoxic and non-genotoxic agents. The stable p21FGLuc cells were exposed to 30 μg /mL bleomycin, 10 μg /mL mitomycin C and 30% (v/v) ethanol, respectively. The same stable p21FGLuc cells, not exposed to any toxicants but with the same volume of PBS, were used as control. ** Shows the highly significant difference (p < 0.01). Data are expressed as mean ± SD.
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biosensors-02-00318-f004: The time-course responses of the stable p21FGLuc cells to model genotoxic and non-genotoxic agents. The stable p21FGLuc cells were exposed to 30 μg /mL bleomycin, 10 μg /mL mitomycin C and 30% (v/v) ethanol, respectively. The same stable p21FGLuc cells, not exposed to any toxicants but with the same volume of PBS, were used as control. ** Shows the highly significant difference (p < 0.01). Data are expressed as mean ± SD.

Mentions: FG cells have been successfully co-transfected with three expression plasmids of pGL3-p21-luc, pRL-CMV and pcDNA3.1. As shown in Figure 4, Figure 5 and Figure 6, the obtained stable p21FGLuc cells have a constant basal level of firefly luciferase and Renilla luciferase expression even after one year of selection and maintenance with G418. Challenge of genotoxicants of bleomycin and mitomycin C can significantly up-regulate the expression of firefly luciferase in the stable p21FGLuc cells.


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 time-course responses of the stable p21FGLuc cells to model genotoxic and non-genotoxic agents. The stable p21FGLuc cells were exposed to 30 μg /mL bleomycin, 10 μg /mL mitomycin C and 30% (v/v) ethanol, respectively. The same stable p21FGLuc cells, not exposed to any toxicants but with the same volume of PBS, were used as control. ** Shows the highly significant difference (p < 0.01). 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-f004: The time-course responses of the stable p21FGLuc cells to model genotoxic and non-genotoxic agents. The stable p21FGLuc cells were exposed to 30 μg /mL bleomycin, 10 μg /mL mitomycin C and 30% (v/v) ethanol, respectively. The same stable p21FGLuc cells, not exposed to any toxicants but with the same volume of PBS, were used as control. ** Shows the highly significant difference (p < 0.01). Data are expressed as mean ± SD.
Mentions: FG cells have been successfully co-transfected with three expression plasmids of pGL3-p21-luc, pRL-CMV and pcDNA3.1. As shown in Figure 4, Figure 5 and Figure 6, the obtained stable p21FGLuc cells have a constant basal level of firefly luciferase and Renilla luciferase expression even after one year of selection and maintenance with G418. Challenge of genotoxicants of bleomycin and mitomycin C can significantly up-regulate the expression of firefly luciferase in the stable p21FGLuc cells.

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