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At least 217 records · Page 12Linked to original sources
PubMed2001

Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay.

We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug candidates. A dataset of more than 250 tests carried out with 75 drug candidates of various chemical classes was analyzed to elucidate the influence of cytotoxicity and compound precipitation on DNA migration in the comet assay. Using a V79 Chinese hamster cell line, 38 of the compounds were negative and 37 were positive in the comet assay. The reproducibility of test results between repeat experiments was 85%. Data on 72 tests with a negative call in which the compounds were tested up to highly cytotoxic concentrations demonstrated that cytotoxicity, as determined by Trypan blue dye exclusion and occurrence of cells with completely fragmented chromatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the absence of excessive cytotoxicity. Compound precipitation was observed in 84 tests. In 88.1% of these cases, the test result at the precipitating concentration did not differ from that found at the highest soluble concentration. Half of the remaining 11.9% of contrary results (most of them weak effects) were not reproducible in the respective repeat experiment, indicating no or only a negligible influence of precipitation on test results. The data indicate that using V79 cells, the comet assay specifically detects genotoxic effects and is not confounded by cytotoxicity or compound precipitation under the conditions used.

Animals↗
PubMed1999

A comparative evaluation of aflatoxin B1 genotoxicity in fish models using the Comet assay.

Aflatoxin B1 (AFB1) is classified as a Group I hepatocarcinogen in humans by the International Agency for Research on Cancer (IARC). The alkaline Comet assay is a simple and rapid method by which DNA damage can be demonstrated as a function of tail moment. The present work is the first to evaluate the genotoxicity of AFB1 in fish using the Comet assay. Two different species of fish were selected as models due to previously established sensitivity to AFB1: rainbow trout (sensitive) and channel catfish (resistant). Fish were i.p. injected with 0.5 mg AFB1/1 ml DMSO/1 kg body weight. The Comet assay was performed after 4 and 24 h on whole blood, liver, and kidney cells of both species. Trout blood and kidney tissue tested displayed significant (p < 0.05) and extensive DNA damage (shown by increased tail moment) after 4 h which then decreased by 24 h. In liver cells, damage progressively increased over time. Conversely, similarly treated catfish showed no elevation in DNA damage over controls at the same doses. These results suggest that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB1 and for evaluating organ specific effects of these agents in different species.

Aflatoxin B1↗
PubMed2006

Radiosensitivity in breast cancer assessed by the Comet and micronucleus assays.

Spontaneous and radiation-induced genetic instability of peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=50) was examined using the single-cell gel electrophoresis (Comet) assay and a modified G2 micronucleus (MN) test. Cells from apparently healthy donors (n=16) and from cancer patients (n=9) with an adverse early skin reaction to radiotherapy (RT) served as references. Nonirradiated cells from the three tested groups exhibited similar baseline levels of DNA fragmentation assessed by the Comet assay. Likewise, the Comet analysis of in vitro irradiated (5 Gy) cells did not reveal any significant differences among the three groups with respect to the initial and residual DNA fragmentation, as well as the DNA repair kinetics. The G2 MN test showed that cells from cancer patients with an adverse skin reaction to RT displayed increased frequencies of both spontaneous and radiation-induced MN compared to healthy control or the group of unselected BC patients. Two patients from the latter group developed an increased early skin reaction to RT, which was associated with an increased initial DNA fragmentation in vitro only in one of them. Cells from the other BC patient exhibited a striking slope in the dose-response curve detected by the G2 MN test. We also found that previous RT strongly increased both spontaneous and in vitro radiation-induced MN levels, and to a lesser extent, the radiation-induced DNA damage assessed by the Comet assay. These data suggest that clinical radiation may provoke genetic instability and/or induce persistent DNA damage in normal cells of cancer patients, thus leading to increased levels of MN induction and DNA fragmentation after irradiation in vitro. Therefore, care has to be taken when blood samples collected postradiotherapeutically are used to assess the radiosensitivity of cancer patients.

Adolescent↗
PubMed2004

Intra-laboratory comet assay sample scoring exercise for determination of formamidopyrimidine DNA glycosylase sites in human mononuclear blood cell DNA.

Oxidative DNA damage detected by the comet assay as formamidopyrimidine DNA glycosylase (FPG) sensitive sites, almost as a rule is reported as comet assay score rather than numerical sites in the genome, probably because the latter requires X-ray calibration. We compared the ability of five experienced and five inexperienced comet assay investigators to detect a dose-response relationship in irradiated A549 lung epithelial cell culture samples (0, 10 Gy and three samples of 5 Gy), based on an arbitrary five class scoring system. The samples were scored on three different occasions, thus allowing determination of the variation in sample scoring. All investigators qualitatively distinguished between samples in a dose-dependent manner, albeit with large variation in the slope and intercept of dose-response curves. There was a tendency that investigators with experience in scoring A549 cells had more consistent results than experienced investigators who had only scored lymphocytes or inexperienced investigators. The inexperienced investigators improved their scoring ability during the three sessions. Subsequently we showed that the variation in baseline level of FPG modifications in mononuclear blood cells of five healthy humans was lower when investigators used their individual X-ray calibration curve as compared to a common calibration curve. In conclusion, this study showed that comet assay investigators score differently when using a five class scoring system, which indicates that more consistent estimations of FPG sites in the genome are obtained by use of investigators' individual X-ray calibrations.

Calibration↗
PubMedDate not supplied

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model.

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics+/-antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3+/-1.7% TI versus 10.2+/-4.1% TI, n=19), but not of non-smokers (8.6+/-2.8% TI versus 8.3+/-2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4+/-2.9% TI versus 18.9+/-13.1% TI, n=15) but not in smokers (15.5+/-10.7% TI versus 20.4+/-14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread+/-antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.

Adult↗
PubMed2005

Biomonitoring of the genotoxic potential of draining water from dredged sediments, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (Mutatox and Ames tests).

Management of contaminated dredged sediments is a matter of great human concern. The present investigation evaluates the genotoxic potential of aqueous extracts of five sediments from French channels (draining water from dredged sediments), using larvae of the frog Xenopus laevis. Two genotoxic endpoints were analyzed in larvae: clastogenic and/or aneugenic effects (micronucleus induction after 12 d of exposure) and DNA-strand breaking potency (comet assay after 1 and 12 d of exposure) in the circulating blood. Additionally, in vitro bacterial assays (Microtox and Ames tests) were carried out and the results were compared with those obtained with larvae. Physicochemical analyses were also taken into account. Analytical analyses highlighted in the five draining waters a heavy load of contaminants such as metals and hydrocarbons. The results obtained with the micronucleus test established the genotoxicity of three draining waters. The comet assay showed that all 5 draining waters were genotoxic after 1 d of exposure. Although 3 of them were still genotoxic after 12 d of exposure, DNA damage globally decreased between d 1 and 12. The comet assay can be considered as a genotoxicity-screening tool. Data indicate that both tests should be used in conjunction in Xenopus. Bacterial tests (Ames) revealed genotoxicity for only one draining water. The results confirm the relevance of the amphibian model and the need to resort to bioassays in vivo such as the Xenopus micronucleus and comet assays for evaluation of the ecotoxicological impact, an essential complement to the physicochemical data.

Animals↗
PubMed2002

Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters.

BACKGROUND: Standard semen parameters have proven poor at predicting the outcomes of IVF treatment cycles. As recent studies suggest that the male genome may play an important role in early embryogenesis, this study attempts to correlate the level of sperm DNA damage in fresh semen and prepared sperm with the outcomes of conventional IVF treatment cycles. METHODS: Forty patients embarking on IVF treatment were recruited into this prospective observational study. Both fresh semen and PureSperm-prepared sperm were processed using a modified comet assay 3-6 months prior to the patients' IVF treatment cycles. Comet head DNA (mean and integrated head density) and tail DNA parameters (length and moment) were measured separately. RESULTS: Significant correlations between total sperm concentration and between comet length, moment, mean head density with embryo quality were detected in fresh semen and prepared sperm. Surprisingly, no significant correlations between head and tail parameters were detected. CONCLUSIONS: Comet head and tail DNA parameters appear to be potentially useful as predictors of embryo quality and IVF outcomes, especially in couples with unexplained subfertility. The lack of correlation between head and tail parameters may be due to a different mechanism of DNA damage within these two compartments.

Adult↗
PubMed2003

Genetic instability in bladder cancer assessed by the comet assay.

BACKGROUND: Latent genetic instability has been associated with an increased risk for several cancers. We used the comet assay (single-cell gel electrophoresis) to assess whether genetic instability, as reflected by susceptibility to DNA damage, was associated with the risk of bladder cancer in a case-control study. METHODS: We used the comet assay to measure baseline and benzo[a]pyrene diol epoxide (BPDE)- and gamma-radiation-induced DNA damage in individual peripheral blood lymphocytes from 114 incident case patients with bladder cancer and 145 matched healthy control subjects. All subjects provided personal information, including smoking history. DNA damage was visualized with the comet assay and quantified by the Olive tail moment parameter, a relative measure. Multivariable analysis was used to assess relative risks for bladder cancer associated with DNA damage. All statistical tests were two-sided. RESULTS: Baseline levels of DNA damage were statistically significantly higher in case patients (tail moment = 1.40) than in control subjects (tail moment = 1.21) (difference = 0.19, 95% confidence interval [CI] = 0.04 to 0.32; P =.015), as were gamma-radiation-induced (tail moment = 4.76 versus 4.22; difference = 0.54, 95% CI = 0.11 to 0.96; P =.013) and BPDE-induced (tail moment = 4.06 versus 3.45; difference = 0.61, 95% CI = 0.23 to 0.99; P =.002) DNA damage. When data were dichotomized at the median value for DNA damage in control subjects and adjusted for age, sex, ethnicity, and smoking status, an increased estimated relative risk of bladder cancer was statistically significantly associated with DNA damage at baseline (odds ratio [OR] = 1.84, 95% CI = 1.07 to 3.15) and after gamma-radiation (OR = 1.81, 95% CI = 1.04 to 3.14) but not after BPDE treatment (OR = 1.69, 95% CI = 0.98 to 2.93). CONCLUSION: Latent genetic instability as measured by the comet assay is associated with an increased estimated relative risk of bladder cancer.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Application of the alkaline comet assay in human biomonitoring for genotoxicity: a study on Croatian medical personnel handling antineoplastic drugs.

The alkaline comet assay was used to evaluate the genotoxicity towards peripheral blood lymphocytes of medical personnel regularly handling various antineoplastic drugs with different safety precautions. The study population consisted of 50 exposed subjects working in the oncology, pulmonology, gynaecology and haematology units of nine Croatian hospitals and 20 unexposed control subjects. Peripheral blood lymphocytes from the subjects were embedded in agarose on a microscope slide and lysed; the DNA was unwound and subjected to electrophoresis at pH 13. Staining with a fluorescent dye was used to identify cells with DNA damage, as judged by increased migration of genetic material from the cell nucleus. DNA damage was quantified by measuring the displacement between the genetic material of the nucleus and the resulting tail using an image analysis system. Three parameters were used as indicators of DNA damage: i.e. tail length, percentage of DNA in the tail and tail moment. Statistically significant differences in all three parameters were observed between the exposed and control groups. Within the exposed group, there were marked differences between individuals in the comet tail parameters. In the majority of exposed subjects an effect on DNA damage of age or duration of occupational exposure could be excluded. In the exposed group, the highest level of DNA damage was recorded in subjects who used only latex gloves in their work with antineoplastic drugs. The observed DNA damage was lower in exposed subjects who used more than one type of protective equipment and who worked in a well-ventilated safety cabinet. No statistically significant differences were found between the mean values of comet tail parameters for smoking and non-smoking subpopulations from the exposed group. In view of the results obtained, the alkaline comet assay, as a simple, rapid and sensitive method, appears to be a promising additional test for biomonitoring purposes in human populations.

Adult↗
PubMed2003

Evaluation of genetic damage in workers employed in pesticide production utilizing the Comet assay.

The use of pesticides has been increasing in recent years, resulting in the need for increased production of pesticides. However, some pesticides may represent a hazard to human health, especially by causing cancer. Genotoxicity tests form an important part of cancer research and risk assessment of potential carcinogens. Therefore, in the current study the potential DNA damage associated with exposure to pesticides of Indian pesticide production workers was assessed using the single cell gel electrophoresis assay or Comet assay. Blood leukocytes of a group of 54 pesticide workers and an equal number of control subjects were examined for genotoxicity in this study. The two groups had similar mean ages and smoking prevalences. The mean comet tail length was used to measure DNA damage. The exposed workers had significantly greater mean comet tail lengths than those of controls (mean +/- SD 19.17 +/- 2.467 versus 8.938 +/- 2.889, P < 0.001). Smokers had significantly larger mean tail lengths than non-smokers (19.75 +/- 2.52 versus 18.26 +/- 2.13, P = 0.024). Analysis of covariance showed that occupational exposure (P < 0.05) and smoking (P < 0.05) had significant effects on mean tail length, whereas age and gender had no effect on DNA damage. The present study suggests that occupational exposure to pesticides and smoking can cause DNA damage. This investigation confirms the sensitivity of the Comet assay.

Adult↗
PubMed2003

The alkaline Comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation.

The alkaline Comet assay was selected as a biomarker of exposure to evaluate the ongoing exposure to ionizing radiation of 50 medical workers occupationally exposed to ionizing radiation and 50 corresponding unexposed control subjects. The primary DNA damage was evaluated by measuring the extent of DNA migration in peripheral blood leukocytes. The inter-individual differences in DNA damage between exposed subjects were compared with their dosimeter readings and occupation. It was found that medical workers who were occupationally exposed to ionizing radiation for different periods of time showed highly significant increases in levels of DNA damage compared with controls. However, influences of the different occupational settings and doses absorbed on the levels of DNA damage, assessed by use of the Comet assay, might be excluded in the majority of subjects. Differences in comet parameters measured due to smoking and gender were not statistically significant in either exposed or control subjects. The results obtained have confirmed the usefulness of the alkaline Comet assay as an additional complement to standard biodosimetric methods. By detection of momentary DNA damage and/or repair activity, it reflects the concurrent exposure and the actual levels of DNA damage present in peripheral blood leukocytes of the radiological workers at the moment of blood sampling.

Biomarkers↗
PubMed2004

Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations.

The alkaline Comet assay was used to investigate the in vivo genotoxicity of 17 compounds. Altogether 21 studies were conducted with these compounds. The investigations were triggered for various reasons. The main reason for performing the studies was to evaluate the in vivo relevance of in vitro genotoxicity findings with 10 compounds. Eight of these compounds showed no effects in the in vivo Comet assay while two compounds induced altered DNA migration patterns in specific organs. The remaining seven compounds were tested to follow up on neoplastic/preneoplastic or chronic toxicity changes as detected in specific target organs identified in rodent studies, to investigate the possibility of site-of-contact genotoxicity and to test the liver as a target organ for a suspected reactive metabolite. For the studies, various organs of rodents were analyzed, depending on the suspected properties of the compounds, including liver, jejunum, leukocytes, stomach mucosa, duodenum, lung and kidney. All tissues were amenable to investigation by gel electrophoresis after simple disaggregation of organs by means of mincing or, in the case of epithelial cells from the gastrointestinal tract, scraping off cells from the epithelium. In conclusion, the Comet assay was found to be a reliable and robust test to investigate in vivo genotoxicity in a variety of rodent organs. Therefore, it is concluded that in vivo Comet assay data are useful for elucidating positive in vitro genotoxicity findings and to evaluate genotoxicity in target organs of toxicity.

Animals↗
PubMed2004

Genotoxic evaluation of welders occupationally exposed to chromium and nickel using the Comet and micronucleus assays.

Chromium (Cr) and nickel (Ni) are widely used industrial chemicals. Welders in India are inclined to possible occupational Cr and Ni exposure. The carcinogenic potential of metals is a major issue in defining human health risk from exposure. Hence, in the present investigation, 102 welders and an equal number of control subjects were monitored for DNA damage in blood leucocytes utilizing the Comet assay. The two groups had similar mean ages and smoking prevalences. A few subjects were randomly selected for estimation of Cr and Ni content in whole blood by inductively coupled plasma mass spectrometry. The Comet assay was carried out to quantify basal DNA damage. The mean comet tail length was used to measure DNA damage. Welders had higher Cr and Ni content when compared with controls (Cr, 151.65 versus 17.86 micro g/l; Ni 132.39 versus 16.91 micro g/l; P < 0.001). The results indicated that the welders had a larger mean comet tail length than that of the controls (mean +/- SD, 23.05 +/- 3.86 versus 8.94 +/- 3.16; P < 0.001). In addition, the micronucleus test on buccal epithelial cells was carried out in a few randomly selected subjects. Welders showed a significant increase in micronucleated cells compared with controls (1.30 versus 0.32; P < 0.001). Analysis of variance revealed that occupational exposure (P < 0.05) had a significant effect on DNA mean tail length, whereas smoking and age had no significant effect on DNA damage. The current study suggested that chronic occupational exposure to Cr and Ni during welding could lead to increased levels of DNA damage.

Adolescent↗
PubMed2004

Parallel evaluation of doxorubicin-induced genetic damage in human lymphocytes and sperm using the comet assay and spectral karyotyping.

In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm significantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 micro M DX. At 1.6 micro M the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a significant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 micro M DX, continuing at the same level to a final concentration of 1.6 micro M. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 micro M DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the findings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the first reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different end-points, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a 'parallelogram' approach in human cells.

Adult↗
PubMed2006

hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII.

The European Standards Committee on Oxidative DNA Damage (ESCODD) recommended the use of the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG) in the comet assay to detect oxidative DNA damage. In the present study, FPG was compared with endonuclease III (ENDOIII) and human 8-hydroxyguanine DNA-glycosylase (hOGG1) for the ability to modify the sensitivity of the comet assay. Mouse lymphoma L5178Y cells were treated with dimethyl sulphoxide (DMSO) as a standard solvent or reference agents known to induce oxidative damage (gamma irradiation and potassium bromate) or alkylation (methyl methanesulfonate, MMS; ethylnitrosurea, ENU). Using DMSO even up to toxic concentrations, no increase in breaks was seen with FPG, ENDOIII or hOGG1. With gamma irradiation (1-10 Gy), dose-related increases in breaks were seen with all three enzymes. FPG and hOGG1 gave similar increases in breaks after potassium bromate treatment between 0.25 and 2.5 mmol/l, but ENDOIII showed an increase only at the highest concentration, 2.5 mmol/l. Following MMS treatment (5-23 micromol/l), FPG induced a dramatic increase in breaks compared with control levels and ENDOIII also showed a significant but smaller increase; in marked contrast, hOGG1 gave no increase. With ENU (0.5-2.0 mmol/l), increases in breaks were seen with FPG and ENDOIII at 1 and 2 mmol/l but, again, no increase was observed with hOGG1. These data indicate that all three endonucleases recognize oxidative DNA damage and, in addition, FPG and ENDOIII also recognize alkylation damage. Therefore, caution should be taken when using FPG and ENDOIII in the comet assay with an agent that has an unknown mode of action since any additional strand breaks induced by either enzyme cannot necessarily be ascribed to oxidative damage. The use of hOGG1 in the modified comet assay offers a useful alternative to FPG and is apparently more specific for 8-oxoguanine and methyl-fapy-guanine.

Animals↗
PubMed2006

Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes.

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

Adenomatous Polyposis Coli Protein↗
PubMed1999

Detection of hypoxic cells in murine tumors using the comet assay: comparison with a conventional radiobiological assay.

The comet (single-cell electrophoresis) assay has been developed as a method for measuring DNA damage in single cells after irradiation. We have developed our own methods and image analysis system for the comet assay to identify hypoxic fractions. In vitro, we tested our system using a cultured tumor cell line (SCCVII). In vivo, we compared the hypoxic fractions detected by this assay with those determined by the in vivo-in vitro clonogenic assay using two rodent tumors (SCCVII/ C3H, EMT6/KU/balb/c), which exhibit different types of hypoxia: acute and chronic. In vitro, our method could differentiate hypoxic cells from oxic cells, using the parameter of tail moment. In vivo, there were good correlations between the hypoxic fractions determined by the comet assay and by the clonogenic assay, in SCCVII/C3H (r=0.85) and in EMT6/KU/balb/c (r=0.75) tumors. By comparison of the two methods in chronically hypoxic and acutely hypoxic tumors, we further confirmed that the comet assay is clinically useful for estimating hypoxic fractions of solid tumors.

Animals↗
PubMed2000

Capability for identification of gamma-irradiated bovine liver by new high sensitivity comet assay.

DNA in food will sustain damage by gamma radiation. The detection capability of the high sensitivity comet assay was studied using fluorescence-microscopy. Beef liver was irradiated at a range of 1 Gy to 8 kGy. Single cells were obtained from the irradiated liver, then analyzed by agaros-gel electrophoresis. The pH of the buffer for electrophoresis was pH 13, which is generally utilized for sensitive detection of DNA damage. The pattern formed by DNA was visualized by staining with ethidium bromide. The resulting comets were evaluated with a scale we developed, and Influence Scores were calculated based on the Tice method. It is possible to detect irradiation damage to beef liver at 10 Gy. Together with Influence Score, histogram of comet type is used for detection of irradiation. We elucidated those histograms were useful for distinguishing damage caused by irradiation from that of others. DNA damage can be caused not only by irradiation, but also by the other treatments. Therefore, the respective influences of freezing, preservation, irradiating temperature, atmosphere of irradiation, cooking, and homogenizing devices were also examined. This new comet assay will be a useful method of detecting DNA damage to identify irradiated foods.

Animals↗