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Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation.

ABSTRACT: Bladder tumours show a variable response to radiotherapy with only about 50% showing good local control; currently there is no test to predict outcome prior to treatment. We have used five bladder tumour cell lines (T24, UM-UC-3, TCC-SUP, RT112, HT1376) to investigate the potential of the alkaline comet assay (ACA) to predict radiosensitivity. Radiation-induced DNA damage and repair were compared to clonogenic survival. When the five cell lines were irradiated and initial DNA damage was plotted against cell survival, at all doses (0-6 Gy), a significant correlation was found (r2=0.9514). Following 4 Gy X-irradiation, all cell lines, except T24, showed a correlation between SF2 vs half-time for repair and SF2 vs residual damage at 5, 10, 20 and 30 min. The T24 cell line showed radioresistance at low doses (0-2 Gy) and radiosensitivity at higher doses (4-6 Gy) using both cell survival and ACA end points, explaining the lack of correlation observed for this cell line. These data indicate that initial DNA damage and residual damage can be used to predict for radiosensitivity. Our data suggest that predictive tests of radiosensitivity, appropriate to the clinical situation, may require the use of test doses in the clinical range.

SUBMITTER: McKeown SR

PROVIDER: S-EPMC2395295 | biostudies-other | 2003 Dec

REPOSITORIES: biostudies-other

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Evaluating nanobiomaterial-induced DNA strand breaks using the alkaline comet assay.

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| S-EPMC9360128 | biostudies-literature

Assessing Genotoxicity of Ten Different Engineered Nanomaterials by the Novel Semi-Automated FADU Assay and the Alkaline Comet Assay.

Project description:Increased engineered nanomaterial (ENM) production and incorporation in consumer and biomedical products has raised concerns about the potential adverse effects. The DNA damaging capacity is of particular importance since damaged genetic material can lead to carcinogenesis. Consequently, reliable and robust in vitro studies assessing ENM genotoxicity are of great value. We utilized two complementary assays based on different measurement principles: (1) comet assay and (2) FADU (fluorimetric detection of alkaline DNA unwinding) assay. Assessing cell viability ruled out false-positive results due to DNA fragmentation during cell death. Potential structure-activity relationships of 10 ENMs were investigated: three silica nanoparticles (SiO2-NP) with varying degrees of porosity, titanium dioxide (TiO2-NP), polystyrene (PS-NP), zinc oxide (ZnO-NP), gold (Au-NP), graphene oxide (GO) and two multi-walled carbon nanotubes (MWNT). SiO2-NPs, TiO2-NP and GO were neither cytotoxic nor genotoxic to Jurkat E6-I cells. Quantitative interference corrections derived from GO results can make the FADU assay a promising screening tool for a variety of ENMs. MWNT merely induced cytotoxicity, while dose- and time-dependent cytotoxicity of PS-NP was accompanied by DNA fragmentation. Hence, PS-NP served to benchmark threshold levels of cytotoxicity at which DNA fragmentation was expected. Considering all controls revealed the true genotoxicity for Au-NP and ZnO-NP at early time points.

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The use of comet assay in plant toxicology: recent advances.

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Use of the comet-FISH assay to compare DNA damage and repair in p53 and hTERT genes following ionizing radiation.

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Agricultural exposures and DNA damage in PBMC of female farmers measured using the alkaline comet assay.

Project description:ObjectiveSeveral studies investigated the link between agricultural occupational exposures and DNA damage, in an attempt to bring elements of biological plausibility to the increased cancer risk associated with them. However, only a few of these studies focused on females.MethodsThe comet assay was performed on PBMC (Peripheral Blood Mononuclear Cells) samples from 245 females working in open field farming and cattle raising, located in the Normandy area of France. Individual questionnaires on tasks performed were administered at the time of sampling to directly assess exposures. Environmental exposures were issued from a questionnaire assessing the farm productions. Linear regression analyses were done using the DNA damage scores.ResultsRegarding direct exposures, several tasks associated with exposure to potentially harmful chemicals were not associated with DNA damage, but a longer duration of use of herbicide on meadows (p = 0.05) or of cleaning and upkeep of agricultural equipment (p = 0.06) revealed higher DNA damage levels, although the number of exposed women was low. Several indirect and/or environmental exposures were associated with DNA damage in multivariate analyses: a larger surface of meadows (p = 0.006) or the presence of poultry (p = 0.03) was associated with less DNA damage, while the presence of swine (p = 0.01) was associated with higher DNA damage. Smokers and former smokers had less DNA damage than non-smokers (p = 0.0008 and p = 0.03).ConclusionsWe report modified levels of DNA damage for those environmentally exposed to meadows, poultry and pig farming, underlining the need for a better knowledge of the potential health risks experienced by females in this setting.

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New applications of the Comet assay: Comet-FISH and transcription-coupled DNA repair.

Project description:Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30kb. The single cell gel electrophoresis (Comet) assay allows detection of single- or double-strand breaks at a 10-100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet "heads", and that Comet "tails" consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR.

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Use of fractional exhaled nitric oxide as a potential predictor of bronchodilator response.

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Optimized protocol for single-cell isolation and alkaline comet assay to detect DNA damage in cells of Drosophila wing imaginal discs.

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| S-EPMC11804111 | biostudies-literature

B-Comet Assay (Comet Assay on Buccal Cells) for the Evaluation of Primary DNA Damage in Human Biomonitoring Studies.

Project description:Many subjects perceive venous blood collection as too invasive, and thus moving to better-accepted procedures for leukocytes collection might be crucial in human biomonitoring studies (e.g., biomonitoring of occupational or residential exposure to genotoxins) management. In this context, primary DNA damage was assessed in buccal lymphocytes (BLs), fresh whole venous, and capillary blood leukocytes, and compared with that in peripheral blood lymphocytes (PBLs)-the most frequently used cells-in 15 young subjects. Mouthwashes were collected after the volunteers rinsed their mouths with normal saline, and BLs were isolated by density gradient centrifugation. Blood samples were collected by venipuncture or by lancet. Anthropometric and lifestyle information was obtained by the administration of a structured questionnaire. As shown in the Bland-Altman plots, the level of agreement between BLs and PBLs lied within the accepted range, we thus enrolled a wider population (n = 54) to assess baseline DNA damage in BLs. In these cells, mean values of tail length (µm), tail intensity (%), and tail moment were 25.7 ± 0.9, 6.7 ± 0.4 and 1.0 ± 0.1, respectively. No significant association was observed between sex and smoking habit with any of the DNA damage parameters. Conversely, underweight subjects displayed significantly higher genomic instability compared with normal weight group (p < 0.05). In conclusion, we successfully managed to set up and update a non-invasive and well-accepted procedure for the isolation of BLs from saliva that could be useful in upcoming biomonitoring studies.

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