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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 sensitivity of FG cells to G418. FG cells were exposed to increasing concentrations of G418 for two weeks and the survival percentage of treated cells to untreated cells is calculated. Data are expressed as mean ± SD.
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biosensors-02-00318-f002: The sensitivity of FG cells to G418. FG cells were exposed to increasing concentrations of G418 for two weeks and the survival percentage of treated cells to untreated cells is calculated. Data are expressed as mean ± SD.

Mentions: As shown in Figure 2, G418 imposed obvious cytotoxic effects on FG cells in a dose-dependent manner. The lowest concentration of G418 that can kill all the exposed FG cells after a two-week exposure period was 600 μg/mL. Thus the G418 dose of 600 μg/mL was chosen in the following selection for the stably transformed FG cells, and 300 μg/mL G418 was used in the maintenance of stably transformed FG 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 sensitivity of FG cells to G418. FG cells were exposed to increasing concentrations of G418 for two weeks and the survival percentage of treated cells to untreated cells is calculated. 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-f002: The sensitivity of FG cells to G418. FG cells were exposed to increasing concentrations of G418 for two weeks and the survival percentage of treated cells to untreated cells is calculated. Data are expressed as mean ± SD.
Mentions: As shown in Figure 2, G418 imposed obvious cytotoxic effects on FG cells in a dose-dependent manner. The lowest concentration of G418 that can kill all the exposed FG cells after a two-week exposure period was 600 μg/mL. Thus the G418 dose of 600 μg/mL was chosen in the following selection for the stably transformed FG cells, and 300 μg/mL G418 was used in the maintenance of stably transformed FG 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