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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 cytotoxicity of bleomycin, mitomycin C and ethanol to flounder gill (FG) cells as determined by thiazolyl blue tetrazolium bromide (MTT) assay. Data are expressed as mean ± SD.
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biosensors-02-00318-f001: The cytotoxicity of bleomycin, mitomycin C and ethanol to flounder gill (FG) cells as determined by thiazolyl blue tetrazolium bromide (MTT) assay. Data are expressed as mean ± SD.

Mentions: After two days’ recovery, the transiently transformed FG cells were then exposed to G418 selection for two weeks. At the end, the selective medium was replaced with regular medium and all the survival FG cells were then challenged with DNA damaging reagent of bleomycin (30 μg/mL) for 4 h. The above-mentioned dose and exposure time of bleomycin were selected for validation purposes, as they were high enough to initiate the p53-dependent DNA damaging response in mammalian cells [47,48,49], but imposed acceptable cytotoxicity on FG cells (Figure 1). After that, the challenged FG cells were collected and lysed. The activities of both firefly luciferase and Renilla luciferase of the obtained lysates were sequentially measured using the Dual-Luciferase® Reporter Assay System (Promega, USA, Cat.#E1910) with a luminometer (GloMax® 20/20, Promega, USA). The protocol for the dual reporter assay is presented in detail in Section 2.8. Intact FG cells (not transformed) were seeded and performed simultaneously as control.


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 cytotoxicity of bleomycin, mitomycin C and ethanol to flounder gill (FG) cells as determined by thiazolyl blue tetrazolium bromide (MTT) assay. 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-f001: The cytotoxicity of bleomycin, mitomycin C and ethanol to flounder gill (FG) cells as determined by thiazolyl blue tetrazolium bromide (MTT) assay. Data are expressed as mean ± SD.
Mentions: After two days’ recovery, the transiently transformed FG cells were then exposed to G418 selection for two weeks. At the end, the selective medium was replaced with regular medium and all the survival FG cells were then challenged with DNA damaging reagent of bleomycin (30 μg/mL) for 4 h. The above-mentioned dose and exposure time of bleomycin were selected for validation purposes, as they were high enough to initiate the p53-dependent DNA damaging response in mammalian cells [47,48,49], but imposed acceptable cytotoxicity on FG cells (Figure 1). After that, the challenged FG cells were collected and lysed. The activities of both firefly luciferase and Renilla luciferase of the obtained lysates were sequentially measured using the Dual-Luciferase® Reporter Assay System (Promega, USA, Cat.#E1910) with a luminometer (GloMax® 20/20, Promega, USA). The protocol for the dual reporter assay is presented in detail in Section 2.8. Intact FG cells (not transformed) were seeded and performed simultaneously as control.

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