Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

French, C. Tenley; Ross, Carley D.; Keysar, Stephen B.; Joshi, Dhanashree D.; Lim, Chang-Uk; Fox, Michael H.

doi:10.1016/j.mrfmmm.2006.07.009

Cited by 10 publications

(19 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When originally developing the FCMA, we defined the mutant region as 97% of unstained control cells which we later changed to 1% of the mean of the positive peak [2]. Here we propose to set the standard mutant region for the FCMA at 10% of the mean of the CD59 positive peak.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were treated with 8 mM EMS (CAS number 62-50-0) diluted in F12 medium for 3 h in 37 °C incubators. This dose results in 20% survival of cells [2]. After the treatment, cells were washed twice with warm PBS and F12 medium was replaced.…”

Section: Methodsmentioning

confidence: 99%

“…One million cells were stained for CD59 using phycoerythrin (PE)-conjugated mouse anti-human CD59 monoclonal antibodies (Caltag Laboratories, Burlingame, CA) as previously described [1,2]. …”

Section: Methodsmentioning

confidence: 99%

“…We previously defined the mutant region of the FCMA as cells with fluorescence less than 1% of the mean fluorescence of the positive peak [2]. This was a conservative measurement and we subsequently showed that many cells mutated in CD59 by the clastogen gamma radiation [10] were not being scored in that region [11], suggesting that a larger region should be used.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

EMS mutant spectra generated by multi-parameter flow cytometry

Keysar

Fox

2009

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

Self Cite

View full text Add to dashboard Cite

The CHO AL cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59− regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59− clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

EMS mutant spectra generated by multi-parameter flow cytometry

Keysar

Fox

2009

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mutagenic procedures can be optimized in terms of type of mutagen, dose and temperature. EMS is an alkylating agent that induces point mutations by A-T transition to G-C 18 . This mutagenic agent has been used to increase glucose oxidase activity, 19 phytase, 20 pectinase, 21 catalase, 19 lipase, 22 laccase 23 and citric acid production in Aspergillus niger , 24 , 25 as well as to improve recombinant strains of Saccharomyces cerevisiae 26 and Trichoderma viride , 27 among others.…”

Section: Introductionmentioning

confidence: 99%

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

et al. 2013

View full text Add to dashboard Cite

To improve the general secretion ability of the biotechnologically relevant fungus Ashbya gossypii, random mutagenesis with ethyl methane sulfonate (EMS) was performed. The selection and screening strategy followed revealed mutants with improved secretion of heterologous Trichoderma reesei endoglucanase I (EGI), native α-amylase and/or native β-glucosidase. One mutant, S436, presented 1.4- to 2-fold increases in all extracellular enzymatic activities measured, when compared with the parent strain, pointing to a global improvement in protein secretion. Three other mutants exhibited 2- to 3-fold improvements in only one (S397, B390) or two (S466) of the measured activities. A targeted genetic approach was also followed. Two homologs of the Saccharomyces cerevisiae GAS1, AgGAS1A (AGL351W) and AgGAS1B (AGL352W), were deleted from the A. gossypii genome. For both copies deletion, a new antibiotic marker cassette conferring resistance to phleomycin, BLE3, was constructed. GAS1 encodes an β-1,3-glucanosyltransglycosylase involved in cell wall assembly. Higher permeability of the cell wall was expected to increase the protein secretion capacity. However, total protein secreted to culture supernatants and secreted EGI activity did not increase in the Aggas1AΔ mutants. Deletion of the AgGAS1B copy affected cellular morphology and resulted in severe retardation of growth, similarly to what has been reported for GAS1-defficient yeast. Thus, secretion could not be tested in these mutants.

show abstract

Kinetics of CHO A_L mutant expression after treatment with γ radiation, EMS, and asbestos

Keysar

Fox

2009

Cytometry Pt A

Self Cite

View full text Add to dashboard Cite

The flow cytometry mutation assay (FCMA) uses hybrid CHO A L cells to measure mutations of the cd59 gene located on human chromosome 11 by the absence of fluorochrome-conjugated antibody binding to the CD59 surface antigen. Mutant expression peaks between 6 and 12 days, then decreases to a stable plateau, instead of a constant mutant fraction obtained by clonogenic assays. To evaluate this variable mutant expression time, cells were treated with radiation, EMS or asbestos and cell proliferation and survival were measured at times leading up to peak mutant expression. Potential doubling time (T pot ) values increased by at least 75% for each agent by 3 h after treatment but returned to control levels after only 3 days. Survival returned to 90% of control within a week, close to the peak expression day for all three agents. The survival of CD59 2 cells sorted on the peak day of expression was roughly half that of CD59 1 cells. Cloned EMS-treated CD59 2 cells had a doubling time of 16.7 vs. 14.1 h for CD59 1 cells. Triple mutants (CD59 2 /CD44 2 /CD90 2 ) were preferentially lost from the population over time, while the proportion of CD59 2 /CD90 2 mutants increased. In conclusion, the peak day of mutant expression occurs only when cells recover from the toxic effects of the mutagen. A fraction of cells originally quantified as mutants are lost over time due to lethal deletions and slower growth. ' 2009 International Society for Advancement of Cytometry Key terms mutation; CD59; CHO; flow cytometry; EMS; radiation; asbestos PEOPLE may be exposed to a wide range of potentially genotoxic substances in the environment. Furthermore, regulatory agencies require many commercial compounds to be tested for mutagenicity prior to production (1). As it was discovered that many mutagens are also carcinogens, there have been efforts to develop more sensitive methods to detect mutations caused by various agents, including mammalian cell assays such as the mouse lymphoma assay and Chinese hamster ovaryhuman hybrid (CHO A L ) assay (2-4). We and others have developed a flow cytometry mutation assay (FCMA) using the hybrid CHO A L cell line that measures mutant yield in less than 2 weeks (5-7). Mutations in the cd59 gene located on human chromosome 11 are measured by the absence of fluorochrome-conjugated antibody binding to the CD59 antigen on the cell surface, instead of relying on clonogenic survival.We have previously characterized the sensitivity of the FCMA to detect mutations induced by a wide range of genotoxic agents, including low responders, while discriminating between true mutagens and false positives (8). Because of the significant differences in mutant detection between clonogenic assays and the FCMA, it is important to determine if mutant fractions generated by the two methods are similar. Significantly larger mutant fractions for the same agents have been measured using the FCMA when compared with clonogenic assays such as the mouse lymphoma assay (MLA) or the HPRT, which is expected since only a small reg...

show abstract

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

Cited by 10 publications

References 54 publications

EMS mutant spectra generated by multi-parameter flow cytometry

EMS mutant spectra generated by multi-parameter flow cytometry

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

Kinetics of CHO A_L mutant expression after treatment with γ radiation, EMS, and asbestos

Contact Info

Product

Resources

About

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

Cited by 10 publications

References 54 publications

EMS mutant spectra generated by multi-parameter flow cytometry

EMS mutant spectra generated by multi-parameter flow cytometry

Random and direct mutagenesis to enhance protein secretion inAshbya gossypii

Kinetics of CHO AL mutant expression after treatment with γ radiation, EMS, and asbestos

Contact Info

Product

Resources

About

Kinetics of CHO A_L mutant expression after treatment with γ radiation, EMS, and asbestos