Apoptosis can be a confusing factor in in vitro clastogenic assays

Meintières, Sophie

doi:10.1093/mutage/16.3.243

Cited by 40 publications

(31 citation statements)

References 25 publications

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“…Consistent with our previous work [Meintieres et al, 2001], all the apoptosis inducers that are not genotoxins did not induce apoptosis in CTLL-2 bcl2 cells. Induction of micronucleated cells occurred only in the nontransfected cell line concurrently with the appearance of apoptosis.…”

Section: Ctll-2/ctll-2 Bcl2 Cell Lines Allow Distinction Between Apopsupporting

confidence: 92%

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Using CTLL‐2 and CTLL‐2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test

Meintières

Biola

Pallardy

et al. 2003

Environ and Mol Mutagen

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In vitro assays for chromosome aberrations (i.e., in vitro micronucleus and in vitro metaphase analysis tests) frequently produce false-positive or exaggerated-positive results. Our previous work suggested that apoptosis interferes with these tests, producing misleading results. These previous studies were conducted by performing the in vitro micronucleus test in CTLL-2 cells and a CTLL-2 cell derivative stably transfected with the apoptosis inhibitor gene bcl2. In the present study, these previous observations were extended by examining micronucleus induction with a larger number of compounds in both CTLL-2 and CTLL-2 bcl2 cells and measuring apoptosis with annexin V-FITC. Both cell lines were treated with different classes of compounds that were anticipated to be exclusively apoptosis inducers, or compounds known to be clastogens or aneugens, some of which were anticipated to be both genotoxic and apoptotic. We were able to confirm that compounds that are only apoptogenic induced micronuclei in CTLL-2 but not CTLL-2 bcl2 cells, indicating that the positive responses are due to apoptosis in CTLL-2 cells. Some genotoxins (clastogens and aneugens) did not produce apoptosis by the annexin V assay and gave similar responses in CTLL-2 and CTLL-2 bcl2 cells. Finally, higher responses were induced in CTLL-2 cells compared to CTLL-2 bcl2 cells that were treated with aneugens or clastogens that were also apoptosis inducers, suggesting that the greater response in CTLL-2 cells is a consequence of both genotoxicity and apoptosis. Finally, it was demonstrated that just eliminating CTLL-2 cells having three or more micronuclei from scoring was not adequate for correctly evaluating agents that only produce apoptosis. The results indicate that coupling the in vitro micronucleus test in both CTLL-2 and CTLL-2 bcl2 cells with the measurement of apoptosis is able to distinguish the genotoxic effects of a test compound from its apoptotic potential and is able to avoid interference from apoptosis in the in vitro micronucleus test. These observations may provide the basis for a useful genotoxicity assay.

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Section: Ctll-2/ctll-2 Bcl2 Cell Lines Allow Distinction Between Apopsupporting

confidence: 92%

“…The purpose of this present study was to confirm and extend the results obtained in our previous study [Meintieres et al, 2001] by using additional agents. Furthermore, we wanted to improve the MN test performed in CTLL-2 and CTLL-2 bcl2 cells in order to develop a standardized protocol.…”

Section: Introductionsupporting

confidence: 84%

Using CTLL‐2 and CTLL‐2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test

Meintières

Biola

Pallardy

et al. 2003

Environ and Mol Mutagen

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“…Clearly the number and classes of compounds analyzed needs to be expanded. Even so, these results are very encouraging, as the non-genotoxic cytotoxicants DEX and STS tend to be extreme challenges to mammalian cell genotoxicity assays [16][17]. Furthermore, we chose 24 hrs of continuous treatment as a particularly challenging experimental design to study, as previous work by lab L4 had demonstrated that assay specificity is reduced in this case [23].…”

Section: Discussionmentioning

confidence: 98%

“…Reports by Mentieres et al [16][17] and others suggest that the apoptotic program, perhaps especially nuclear fragmentation, can lead to the presence of MN-like events that may enhance the rate of false positive results. STS data presented here may represent a case in point, as elevated MN frequencies were observed by microscopy (L3).…”

Section: Apoptosis As a Confoundermentioning

confidence: 97%

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) [56][57][58][59][60][61][62][63][64][65][66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei-the genotoxicants mitomycin C, etoposide, and vinblastine, and the non-genotoxicants sucrose, staurosporine, or dexamethasone. The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow ™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 hrs over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy-and flow cytometry-based scoring methods detected concentrationdependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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“…The frequencies of micronucleated erythrocytes and leukocyte types were determined by randomly moving the field of view and counting 1000 erythrocytes on each slide using 10009 magnification under oil immersion. A micronucleus was identified as a small body that was adjacent but not connected to the principle nucleus (Meintieres et al 2001, Fig. 1).…”

Section: Micronucleus and White Blood Cell Testmentioning

confidence: 99%

Toxicity of coal-tar pavement sealants and ultraviolet radiation to Ambystoma Maculatum

2010

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Polycyclic aromatic hydrocarbons (PAHs) can affect amphibians in lethal and many sublethal ways. There are many natural and anthropogenic sources of PAHs in aquatic environments. One potentially significant source is run off from surfaces of parking lots and roads that are protected with coal tar sealants. Coal tar is 50% or more PAH by wet weight and is used in emulsions to treat these surfaces. Break down of sealants can result in contamination of nearby waters. The toxicity of PAHs can be greatly altered by simultaneous exposure to ultraviolet radiation. This study exposes larvae of the spotted salamander (Ambystoma maculatum) to determine if coal tar sealant can have negative effects on aquatic amphibians and if coal tar toxicity is influenced by ultraviolet radiation. Spotted salamanders were exposed to 0, 60, 280 and 1500 mg coal tar sealant/kg sediment for 28 days. Half of the animals were exposed to conventional fluorescent lighting only and half were exposed to fluorescent lighting plus ultraviolet radiation. No significant mortality occurred during the experiment. Exposure to sealants resulted in slower rates of growth, and diminished ability to swim in a dose-dependent fashion. Exposure to ultraviolet radiation affected the frequencies of leukocytes and increased the incidence of micronucleated erythrocytes. There was an interactive effect of sealant and radiation on swimming behavior. We conclude that coal-tar sealant and ultraviolet radiation increased sublethal effects in salamanders, and may be a risk to salamanders under field conditions.

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Apoptosis can be a confusing factor in in vitro clastogenic assays

Cited by 40 publications

References 25 publications

Using CTLL‐2 and CTLL‐2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test

Using CTLL‐2 and CTLL‐2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Toxicity of coal-tar pavement sealants and ultraviolet radiation to Ambystoma Maculatum

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