DNA damage induced by micro- and nanoparticles--interaction with FPG influences the detection of DNA oxidation in the comet assay

Reliable methods for evaluation of toxicity from particles, such as manufactured nanoparticles, are needed. One promising tool is the comet assay, often used to measure DNA breaks (strand breaks and alkali-labile sites) as well as oxidatively damaged DNA, the latter by addition of specific DNA repair enzymes such as formamidopyrimidine DNA glycosylase (FPG). The aim of this study was to investigate the use of the comet assay for analysis of DNA oxidation by a range of micro- and nanoparticles in the lung cell lines A549 and BEAS-2B and to test the hypothesis that nanoparticles present in the cells during the assay performance may interact with FPG. This was done by investigating the ability of micro- and nanoparticles (stainless steel, subway particles, MnO(2), Ag, CeO(2), Co(3)O(4), Fe(3)O(4), NiO and SiO(2)) to induce DNA breaks, oxidatively damaged DNA (FPG sites, dominantly 8-oxoguanine), intracellular production of reactive oxygen species (ROS) and non-cellular oxidation of the DNA base guanine, as well as by studying interactions of the particles and their released ions with FPG. Several particles caused DNA breaks, but low levels of FPG sites. The ability of FPG to detect DNA oxidation induced by a photosensitiser was however shown. An oxidative capacity of the particles was indicated by increased levels of intracellular ROS, and especially Ag and subway particles caused non-cellular oxidation of guanine. Incubation of FPG with the particles led to less FPG activity, particularly with nanoparticles of Ag but also with CeO(2), Co(3)O(4) and SiO(2). Further investigations of these particles revealed that for Ag, the decreased activity was mainly due to released Ag ions, whereas for CeO(2) and Co(3)O(4), FPG interactions were due to the particles. We conclude that measurement of oxidatively damaged DNA in cells exposed to nanoparticles may be underestimated in the comet assay due to interactions with FPG.     

DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization

The comet assay is a sensitive method for measuring DNA strand breaks in eukaryotic cells. After embedding in agarose, cells are lysed and electrophoresed at high pH. DNA loops containing breaks (in which supercoiling is relaxed) escape from the nucleoid comet head to form a tail. Oligonucleotide probes were designed for 5' and 3' regions of the genes for dihydrofolate reductase (DHFR) and O6-methylguanine DNA methyltransferase (MGMT), both from the Chinese hamster, and the human tumour suppressor p53 gene. Alternate ends were labelled with either biotin or fluorescein. These probes were hybridized to the DNA of comets from Chinese hamster ovary (CHO) cells or human lymphocytes treated with H2O2 or photosensitizer plus light to induce oxidative damage. Amplification with Texas red- and fluorescein-tagged antibodies led, in the case of p53 in human cells, to red and green signals located in the comet tail (as well as in the head), indicating the presence of breaks in the vicinity of the gene. However, only one end of the MGMT gene appeared in the tail and almost no signals from the DHFR gene, either red or green, were in the tail of comets from CHO cells. Restriction on movement from the head to tail may result from the presence of a 'matrix-associated region' in the gene. The kinetics of repair of oxidative damage were followed; strand breaks in the p53 gene were repaired more rapidly than total DNA. Thus, fluorescent in situ hybridization in combination with the comet assay provides a powerful method for studying repair of specific genes in relation to chromatin structure.     

Adaptation to alkylation damage in DNA measured by the comet assay

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.     

Predictors of esophageal cancer risk: assessment of susceptibility to DNA damage using comet assay

Individuals' susceptibility to DNA damage could be identified by mutagen-challenged assays. We tested the hypothesis that susceptibility to DNA damage, measured by comet assay, may be associated with increased esophageal cancer (EC) risk. We recruited 102 subjects with previously untreated EC and 112 healthy controls. Baseline (untreated), benzo[a]pyrene diol epoxide (BPDE)-induced, and gamma-radiation-induced DNA damage were quantified by the Olive tail moment parameter. The mean tail moment was significantly higher in cases than in controls at baseline (case vs. control: 2.6 vs. 1.9, P < 0.01), after BPDE induction (case vs. control: 3.8 vs. 2.7, P < 0.01), and after gamma-radiation-induction (case vs. control: 5.0 vs. 3.8, P < 0.01). When data were dichotomized with the median values in the controls, a significantly increased risk for EC was observed for high baseline tail moment [odds ratio (OR) = 5.7, 95% confidence interval (CI) = 2.9-11.4], high BPDE-induced tail moment (OR = 5.8, 95% CI = 2.9-11.8), and high gamma-radiation-induced tail moment (OR = 4.6, 95% CI = 2.4-8.8). Further, the association between DNA damage and EC was stronger in never smokers than in ever smokers. Compared with subjects not sensitive to both mutagens, individuals sensitive to only one mutagen showed a 3.4-fold risk for EC and those sensitive to both mutagens showed an 8.7-fold risk for EC. Thus, we conclude that susceptibility to DNA damage as assessed by comet assay might help identify individuals with high EC risk.     

Analysis of UV-induced damage and repair in young and senescent human dermal fibroblasts using the comet assay

A major cause of ageing is thought to be the accumulation of damage to macromolecules. Accumulation to DNA damage in cells therefore presupposes that aged cells are unable to repair this damage. We have used the in vitro model of cellular ageing to test the idea that senescent cells are deficient in some aspect of DNA repair. Using the alkaline single cell gel electrophoresis assay (comet assay), we have determined the responses of young and senescent human dermal fibroblasts to DNA damage caused by exposure to UVC light. At low doses of UVC, senescent cells generate smaller comets than young cells whilst at medium doses the situation is reversed. At high doses, young and senescent cells respond similarly to one another. Time course experiments revealing repair of DNA damage show that senescent cells generate larger comets than young cells at early stages of repair suggesting that either senescent cells bear more damage per genome than do young cells or that senescent cells are more efficient at excising bulky adducts from DNA. Cells maintained in low levels of serum irrespective of age are less able to repair DNA damage compared with cells maintained in high levels of serum, and furthermore young and senescent cells maintained in high levels of serum are equally able to repair DNA damage. Our data, therefore, reveal both age-dependent and age-independent responses to UV-induced DNA damage. Use of the comet assay highlights the heterogeneity of cellular responses to genotoxic stress.     

Assessment of chemotherapy-induced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline comet assay.

The alkaline comet assay was employed to assess the pre- and post-treatment levels of in vivo DNA damage in peripheral blood leukocytes of cancer patients. During the study all patients were given antineoplastic drugs, mainly as polychemotherapy. To quantify the DNA damage, two different comet parameters were evaluated: the tail length and the tail moment. Our results indicate marked interindividual variations between baseline DNA damage in peripheral blood leukocytes recorded among cancer patients prior to the chemotherapy. After intravenous administration of various antineoplastic drugs, a significantly increased level of DNA damage in all cancer patients compared to their pre-treatment values was recorded The highest level of DNA damage was seen following administration of 5-fluorouracil, adriamycin, and cisplatin (FAP protocol). The results indicate that administration of antineoplastic drugs in standard protocols is accompanied by significant DNA damage in peripheral blood leukocytes. In order to diminish the potential risks of developing second neoplasms, a continuous biomonitoring of cancer patients after the ending of chemotherapy becomes important. Despite their limitations, present results confirm the usefulness of the alkaline comet assay as a sensitive biomarker of exposure that enables rapid and simple detection of primary DNA damage in peripheral blood leukocytes of cancer patients. Together with standard cytogenetic endpoints, the comet assay provides a powerful technique for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.     

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.     

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.     

Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay

The single cell gel electrophoresis (SCGE) or comet assay, whichmeasures DNA strand breaks in individual cells, was used toanalyse DNA damage and repair induced by the SN₁-type alkylatingcarcinogens N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosoureain CHO cells. The comet assay was comparable in sensitivityto the alkaline elution assay. The alkyl-adducts detected asDNA single-strand breaks (ssb) by this technique were completelyrepaired within 24 h after treatment. These data indicate thatlong-lived lesions, such as alkylphosphotriesters, are not convertedinto ssb under the standard SCGE alkaline conditions (pH 13.5).The lesions revealed by the comet assay are mainly apurinic/apyrimidinic(AP) sites and breaks formed as intermediates in the base excisionrepair process of N-alkylpurines. When SCGE was performed atpH 12.5 instead of pH 13.5 a lower level of ssb was detectedand these breaks were completely resealed within 2 h after treatmentThese data suggest that different subsets of lesions are detectedunder different pH conditions. The SCGE combined with inclusionwithin the cells of endo-nuclease III revealed that a high portionof AP sites induced by alkylation damage were not convertedinto ssb by alkali. The level of endonuclease III-sensitivesites decreased as a function of the repair time and by 24 hafter treatment no sites were left on the DNA. The use of thismodified SCGE assay allows the estimation of the total amountof unrepaired AP sites present on DNA. Alkylation-induced ssbas detected by the comet assay should be regarded as an indicatorof repair rate and balance more than a measure of actual DNAdamage. ³To whom correspondence should be addressed     

Detection of DNA-crosslinking agents with the alkaline comet assay

The single cell gel electrophoresis, or comet assay, under alkaline conditions is a sensitive, simple and rapid method for the detection of DNA damage at the individual cell level. Its applicability as an indicator for the DNA crosslinking potency of a test substance was investigated in human white blood cells by combined treatment with the DNA damaging agent methyl methanesulphonate (MMS) for 2 hr at 37°C. The known crosslinking agents cisplatinum, mitomycin C and formaldehyde, and the formaldehyde releasers diazolidinyl urea and dimethylol urea, were shown to reduce MMS-induced DNA migration in the comet assay in a concentration-dependent manner. Two other protocols, adding MMS to the cells before or after treatment with a crosslinking agent, were carried out and achieved similar results. The results of this study indicate that the comet assay is a useful tool for the detection of crosslinking agents. Advantages and limitations of this method compared to the alkaline elution technique are discussed. © 1996 Wiley-Liss, Inc.     

Evaluation of manual and image analysis quantification of DNA damage in the alkaline comet assay

The alkaline comet assay or single cell microgel electrophoresisassay is a sensitive method of detecting DNA strand breaks andalkali labile sites in individual cells. The results of thisassay can be analysed by different methods. In this study wecompared analyses of the same slides by a manual method andby image analysis, post-treatment of clone 707 Friend erythroleukaemiacells with H₂O₂. The parameters which were found to be particularlyuseful were comet area and comet length (measured manually)and percentage tail DNA, tail moment, tail length and tail length/headradius (L/H), measured using image analysis. The manual methodfor comet analysis presented in this paper would appear to providegood and reliable comet data. However, the image analysis cometsystem described offers an alternative analysis method whichavoids the need for photomicrographs and tedious manual analysis.The image analysis parameters: % tail DNA, tail moment, taillength and L/H give good consistent results and for large-scaleanalysis it will, therefore, conceivably be the method of choice. ¹To whom correspondence should be addressed     

Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.     

In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test

The genus Miconia comprises approximately 1000 species belonging to the Melastomataceae family. Several crude plant extracts from Miconia and their isolated compounds have shown biological activities, such as analgesic and anti-neoplastic action; however, no studies concerning their effects on DNA are available. The present study aimed to evaluate, in vivo, the genotoxic and mutagenic effects of four species of plants from Miconia genus using the comet assay and micronucleus test. Their possible protective effects were also evaluated in experiments associating the plant extracts with cyclophosphamide (CPA). The methanolic extracts of Miconia albicans, Miconia cabucu, Miconia rubiginosa, Miconia stenostachya and the chloroformic extract of M. albicans were investigated. For genotoxic and mutagenic evaluations, three concentrations were tested, 200, 400 and 540 mg/kg body weight (bw), based on the solubility limit of the extract in distilled water. For the protective effects, only the highest dose was evaluated against 40 mg/kg bw of CPA. Blood was removed from mice tails pre- (T0) and post-treatment (T1-30 h) for the micronucleus test and 24 h post-treatment for the comet assay. The Student's t-test was used to compare data obtained at T0 and T1, the analysis of variance-Tukey test was used to compare between groups in the micronucleus test and the Kruskal-Wallis and Dunn's test were used to compare different groups in the comet assay. All the extracts induced alterations in DNA migration (comet assay); however, no mutagenic effect was observed in the micronucleus assay. All extracts showed a protective effect against CPA in both assays. Our study showed that the use of crude extracts could be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.     

Repair of X-ray induced DNA damage measured by the comet assay in roots of Vicia faba

The comet assay was used to measure DNA damage and repair in nuclei released from 1 cm root ends of Vicia faba after X-ray irradiation. Irradiation induced a linear increase of DNA content in comet tail with doses under various denaturation and electrophoretic conditions. The pH of the electrophoresis solution played the most important role in the detection of DNA damage. After irradiation with 30 Gy of X-rays, most of the DNA damage was removed during the first 20 min, even in the presence of DNA repair inhibitors. This first, rapid phase of DNA repair was not affected by incubation on ice, but was partially blocked by 3-aminobenzamide. When DNA was exposed to alkali (0.3 M NaOH) and electrophoresed at neutral pH, all DNA damage was removed in 2 hr, even in the presence of 3-aminobenzamide. Complete repair was inhibited by incubation on ice (30% of DNA remaining in tail) and partially by aphidicolin (13% DNA remaining in tail). Under alkaline (0.3 M NaOH) pretreatment and electrophoresis, more than 20% of detected DNA damage remained unrepaired after 2 hr of postirradiation incubation with and without 3-aminobenzamide at room temperature. Aphidicolin and incubation on ice inhibited the removal of DNA damage to 33% and 39% DNA, respectively. Moreover, aphidicolin treatment attenuated the first phase of damage removal. Environ. Mol. Mutagen. 32:281–285, 1998 © 1998 Wiley-Liss, Inc.     

Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay

Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose—effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D₀ value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D₀ values = 0.12-0.60 Gy). In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkalilabile sites was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stromal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as “comets” by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. © 1996 Wiley-Liss, Inc.     

Aflatoxin exposure and DNA damage in the comet assay in individuals from the Gambia, West Africa.

The single cell gel electrophoresis assay (Comet assay) was used to measure DNA damage in peripheral lymphocytes from a group of individuals from The Gambia in order to determine whether such damage could be associated with increased exposure to aflatoxin in this population. Responses obtained were correlated to responses previously obtained [1] in a cross-sectional study in the same individuals of various cytogenetic alterations [chromosomal aberrations, micronuclei (crest positive and negative staining), and sister chromatid exchanges], and aflatoxin-albumin adducts. Analysis of variance methods were used to assess the effects of smoking, GSTM1 genotype, sex, age, and smoking status. A comparison was also made between The Gambian individuals and a group of healthy, non-smoking volunteers in the United Kingdom where aflatoxin exposure would be expected to be low. From the earlier study [1], it was determined that the levels of the sister chromatid exchanges and micronuclei were higher in The Gambian group than in a European group where aflatoxin exposure was lower, but that there were no correlations between the adduct levels and the cytogenetic abnormalities at the individual level. In the present study, DNA damage as measured in the Comet assay was not significantly higher than in the healthy United Kingdom volunteers. In addition, there were no associations between cytogenetic damage, GSTM1 genotype, age, sex, lifestyle factors (smoking and aflatoxin exposure), and Comet response at the individual level. Comet response was higher in females than males in The Gambia if one outlier was excluded from analysis and not taking into account other sources of variability. It would appear that DNA damage as measured in the Comet assay in peripheral blood lymphocytes is not a sensitive genotoxic marker of aflatoxin exposure in this population.     

Assessment of aniline derivatives-induced DNA damage in the liver cells of B6C3F1 mice using the alkaline single cell gel electrophoresis ("comet") assay.

The alkaline single cell gel electrophoresis (SCGE) or "comet" assay under alkaline conditions was used to measure DNA damage in the liver cells of B6C3F1 male mice exposed to 2,4-dimethylaniline and 2, 4,6-trimethylaniline. Cells embedded in agarose were lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. The effect of 2,4-dimethylaniline and 2,4,6-trimethylaniline was studied after a single intraperitoneal injections at doses equal to 100, 200 mg/kg and 150, 300 mg/kg b.w., respectively. It was found that 2, 4-dimethylaniline and 2,4,6-trimethylaniline were able to damage DNA in the liver cells of the mice. As has been published elsewhere, the DNA damaging effect of the studied compounds were also observed in bone marrow cells of the mice. In conclusion, taking into account the results mentioned above and the results obtained by other researchers who reported mutagenic activity of 2,4-dimethylaniline and 2,4,6-trimethylaniline in Salmonella typhimurium assay and in a DNA repair test using Chinese hamster hepatocytes, it can be stated that both aromatic amines are genotoxic. Teratogenesis Carcinog. Mutagen. 19:323-327, 1999.     

Alkaline comet assay study with breast cancer patients: evaluation of baseline and chemotherapy-induced DNA damage in non-target cells

Abstract The sensitivity of the alkaline comet assay for the evaluation of baseline and treatment-induced DNA damage in white blood cells of breast cancer patients receiving adjuvant chemotherapy according to three conventional anthracycline- and cyclophosphamide-containing protocols was investigated. Additionally, baseline DNA damage in cancer patients was compared with the levels of DNA damage recorded in healthy women. Altogether 30 patients with diagnosed breast cancer and 30 female blood donors with no known familial history of breast cancer participated in the study. Alkaline comet assay was performed according to standard protocol and DNA migration in peripheral blood leukocytes was measured by a computer-based image analysis system. For each subject the frequency of “damaged” cells, i.e., long-tailed nuclei (LTN) with tail length exceeding 95th percentile for the considered parameter among controls, is also reported. Breast cancer patients had significantly increased background levels of DNA damage in their peripheral blood leukocytes as compared to healthy women. Prior to the chemotherapy, a majority of patients showed a statistically significant increase in the number of LTN compared to healthy blood donors. Marked interindividual variations in baseline DNA damage among patients were recorded, some of them related to the disease stage and status. The present study confirmed the alkaline comet assay as a sensitive technique able to detect significantly elevated DNA migration in blood cells of patients already one hour after completion of the first cycle of chemotherapy. Administration of antineoplastic drugs in three chemotherapy protocols studied induced a similar increase of primary DNA damage in nontarget cells. The evaluation of the LTN frequencies indicates the best response to the protocol containing cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Our results point to the significance of simultaneous evaluation of DNA migration and frequency of LTN in the same subject and approved the use of alkaline comet assay as a suitable method for the routine detection of critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.     

A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay

Baseline DNA damage in spermatozoa from fertile and infertilemen was compared using a modified alkali single cell gel electrophoresis(comet) assay. Semen from normozoospermic fertile, normozoospermicinfertile and asthenozoospermic infertile (World Health Organizationcriteria, 1992) samples were studied. No significant differencewas observed in levels of baseline damage between the threegroups. A median value for baseline damage of     

Biomonitoring with the comet assay of Greek greenhouse workers exposed to pesticides

The pesticides in use in Greek greenhouses include a number of agents known to be mutagens and carcinogens. In the present study, we evaluated whether occupational exposure of agricultural workers to a complex mixture of pesticides resulted in a significant increase in DNA damage in human peripheral blood lymphocytes (PBLs). A total of 116 healthy individuals were divided into groups based on exposure to pesticides, smoking status, and gender. Alkaline comet assays performed on PBLs from these individuals indicated no statistically significant differences in basal DNA damage between the study groups. In addition, exposure of PBLs to a dose of hydrogen peroxide led to a similar degree of DNA damage and subsequent repair for all the study populations. The results of the study indicate that the agricultural workers who participated in this study had no detectable increase in DNA damage or alteration in the cellular response to DNA damage.