Evaluation of DNA damage in white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test

The present study was undertaken to contribute to the characterization of the degree of variability in baseline damage in white blood cells from control population, and to investigate how this variability is associated with external and internal factors. Altogether 170 healthy volunteers, randomly selected from the general population of the Republic of Croatia, participated in the study. Two sensitive tests: the alkaline comet assay and the chromosome aberration test were applied to study the background levels of DNA damage in their white blood cells. The results point to inter-individual differences, indicating different genome sensitivity. As revealed by both assays, the background levels of DNA damage were mostly influenced by smoking habit as well as medical exposure (especially to diagnostic X-rays). Sex and age of subjects did not significantly influence the values of DNA damage recorded in the white blood cells. Although higher levels of DNA damage were recorded in blood samples collected during winter and autumn, they were mostly influenced by medicinal exposure and smoking habit. Statistical evaluation of the data confirmed that a positive correlation exists between DNA migration and the number of long-tailed nuclei found with the comet assay and the total number of chromosome aberrations. The data obtained can serve as control values in forthcoming biomonitoring studies.     

Development of a rodent lung macrophage chromosome aberration assay

Lung macrophages are the first line of defense against inhaled xenobiotics. They are able to accumulate airborne particulates as well as having metabolic capability. They may thus be sensitive indicator cells for detecting inhalation exposure to environmental mutagens. Their usefulness as a short-term in vivo genotoxic assay has not, however, been adequately explored. We have systematically investigated the feasibility of developing a lung macrophage chromosome-aberration assay. It was found that with different types of spindle-binding chemicals (vinblastine and vincristine), and with improved harvesting procedures, an adequate number of metaphase cells can be collected from mice and Chinese hamsters. The chromosome aberration frequencies in macrophages from control mice and Chinese hamsters were found to be 1.2 +/- 2.3 and 0.75 +/- 2.2 per 100 cells respectively. These frequencies are within normal ranges for other somatic cells. After inhalation exposure to an occupational-exposure level of benzene (0, 0.1 and 1 ppm), significant dose-dependent induction of aberrations (1.2 +/- 2.3, 5.7 +/- 6.3 and 6.8 +/- 6.2 chromatid deletions per 100 cells resp.) were observed in the macrophages. Thus, these cells can be used as one of a battery of in vivo assays for inhalation exposure studies.     

Optimization of calyculin A-induced premature chromosome condensation assay for chromosome aberration studies

Calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method to assess structural and numerical chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length chromosomes would decrease the detection sensitivity of numerical and structural chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60) Co-γ rays; ～0.6 Gy/min; 0-30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 ± 2.7 and 9.9 ± 1.6-fold increases in the frequency of G(2) /M-PCC cells with extended length chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G(2) /M-PCC scoring index per PCC in 15- and 30-min treated groups was increased by 1.9 ± 0.2 (P = 0.001) and 1.8 ± 0.2 (P = 0.001) compared with the 60-min treated group over 0-20 Gy, respectively. The G(2) /M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable chromosome morphology necessary to assess structural chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.     

Comparative study of chromosome aberration formation in lymphocytes culture under pulsed and continuous neutron irradiation

Frequencies of chromosome aberration induced by prolong (continuous) neutron radiation (dose-rate 0.17 Gy/min) and pulsed neutron radiation with ultra-high dose-rate (1-4) x 10(5) Gy/s have been studied in human blood lymphocytes at G0-stage. It was demonstrated that cytogenetic efficiency of pulsed neutrons (after the substraction of approximately 50% gamma-component from the total dose) was 2 times higher than that of continuous neutron radiation.     

CORAL: Building up QSAR models for the chromosome aberration test

A high level of chromosomal aberrations in peripheral blood lymphocytes may be an early marker of cancer risk, but data on risk of specific cancers and types of chromosomal aberrations are limited. Consequently, the development of predictive models for chromosomal aberrations test is important task. Majority of models for chromosomal aberrations test are so-called knowledge-based rules system. The CORAL software (http://www.insilico.eu/coral, abbreviation of “CORrelation And Logic”) is an alternative for knowledge-based rules system. In contrast to knowledge-based rules system, the CORAL software gives possibility to estimate the influence upon the predictive potential of a model of different molecular alerts as well as different splits into the training set and validation set. This possibility is not available for the approaches based on the knowledge-based rules system. Quantitative Structure–Activity Relationships (QSAR) for chromosome aberration test are established for five random splits into the training, calibration, and validation sets. The QSAR approach is based on representation of the molecular structure by simplified molecular input-line entry system (SMILES) without data on physicochemical and/or biochemical parameters. In spite of this limitation, the statistical quality of these models is quite good.     

Comet assay with nuclear extract incubation

Alkaline comet assay is a simple sensitive method for detecting DNA strand breaks. However, at the time of cell lysis, only a fraction of the entire DNA damage appears as DNA strand breaks, while some DNA strand breaks may have been rejoined and some DNA lesions may still remain unexcised. We showed that nuclear extract (NE) prepared from human cells could excise the DNA adducts induced by UVC, X-ray, and methyl methanesulfonate (MMS). Thus, the comet assay with NE incubation allows a closer estimation of total DNA damage. Among the human urothelial carcinoma cell lines we tested, the NE of NTUB1 cells showed higher activity in excising the DNA adducts induced by UVC, but with a lower activity in excising the DNA adducts induced by MMS than the NE of BFTC905 cells. Moreover, under the same dose of X-ray irradiation, a larger difference in total DNA damage between two cell lines was revealed in comet assay incubated with NE than without NE. Therefore, the comet assay with NE incubation may be useful in the research of cancer risk, drug resistance, and DNA repair proteins.     

An intestinal cell-mediated chromosome aberration test for the detection of genotoxic agents

The use of rat-liver S9 in genotoxicity tests may not reflect true metabolism by whole cells, particularly cells of target organs. We have tested mucosal cells of the mouse small intestine for the capacity to mediate activation/inactivation of chemical carcinogens. Mucosal cells were isolated by pronase digestion. Three million cells were co-cultured with Chinese hamster ovary fibroblasts during a 3-h exposure to chemical clastogens. In the presence of the mucosal cells, aflatoxin B1 (100 microM) was activated to produce chromosome aberrations in 30% of Chinese hamster ovary cell metaphases. 4-Nitroquinoline 1-oxide was deactivated by intestinal cells, while benzo[a]pyrene and dimethylbenz[a]anthracene were not activated by the cells. The clastogenicity of the phenolic compounds caffeic acid (0.28 mg/ml) and clorogenic acid (0.25 mg/ml) was eliminated by the mouse intestinal preparation. The pyrrolizidine alkaloid monocrotaline was activated by intestinal cells. The results suggest the presence of specific activation and deactivation enzymes in the intestinal mucosa. The intestine cell-mediated chromosome aberration test could provide a means to measure tissue-specific activation and deactivation capabilities.     

Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy

The liver micronucleus test is an important method to detect pro-mutagens such as active metabolites not reaching bone marrow due to their short lifespan. We have already reported that dosing of the test compound after partial hepatectomy (PH) is essential to detect genotoxicity of numerical chromosome aberration inducers in mice [Mutat. Res. 632 (2007) 89-98]. In naive animals, the proportion of binucleated cells in rats is less than half of that in mice, which suggests a species difference in the response to chromosome aberration inducers. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the rat liver micronucleus test. Two structural chromosome aberretion inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used in the present study. PH was performed a day before or after the dosing of the test compound in 8-week old male F344 rats and hepatocytes were isolated 4 days after the PH. As a result, diethylnitrosamine and 1,2-dimethylhydrazine, structural chromosome aberration inducers, exhibited significant increase in the incidence of micronucleated hepatocyte (MNH) when given either before and after PH. Colchicine and carbendazim, numerical chromosome aberration inducers, did not result in any toxicologically significant increase in MNH frequency when given before PH, while they exhibited MNH induction when given after PH. It is confirmed that dosing after PH is essential in order to detect genotoxicity of numerical chromosome aberration inducers in rats as well as in mice. Regarding the species difference, a different temporal response to colchicine was identified. Colchicine increased the incidence of MNH 4 days after PH in rats, although such induction in mice was observed 8-10 days after PH.     

Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test

With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(?) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these investigations demonstrate the suitability of the multi-endpoint design.     

The statistical analysis of the in vitro chromosome aberration assay using Chinese hamster ovary cells

The purpose of the in vitro chromosome aberration assay (ABS) is to determine whether the test compound is a clastogen, i.e. induces structural changes in chromosomes. Details of this assay can be found in Galloway et al. [S.M. Galloway, M. Aardema, M. Ishidate Jr, J.L. Ivett, D.J. Kirkland, M. Takeshi, P. Mosesso, T. Sofuni, Mutation Res. 312 (1994) 241-261]. The standard design consists of a negative control and at least three positive dose groups. At each dose, a sample, say 200, of metaphase cells is examined microscopically and cells exhibiting at least one type of chromosome aberration are identified. Using Chinese hamster ovary cells, Margolin et al. [B.H. Margolin, M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom, E. Zeiger, Environ. Mutagen. 8 (1986) 183-204] and Richardson et al. [C. Richardson, D.A. Williams, J.A. Allen, G. Amphlett, D.O. Chanter, B. Phillips, Analysis of data from in vitro cytogenetic assays, in: D.J. Kirkland (Ed.), Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, 1989, pp. 141-154] demonstrated that a binomial sampling model could be used to describe the proportion of cells with chromosome aberrations.Statisticians and toxicologists have also suggested evaluation criteria for the dose response pattern of ABS. Margolin et al. [B.H. Margolin, M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom, E. Zeiger, Environ. Mutagen. 8 (1986) 183-204] suggested one use the Cochran-Armitage trend test. Sofuni et al. [T. Sofuni, A. Matsuoka, M. Sawada, M. Ishidate Jr, E. Zeiger, M.D. Shelby, Mutation Res. 241 (1990) 175-213] considered the dose response to be (strong) positive if it had two significant doses out of three dose groups and decided it was weakly positive if it had only one significant dose and there was a significant trend. The criterion of Galloway et al. for a positive response was a clear dose-related increase in cells with structural aberrations in one experiment or a reproducible single positive dose [S.M. Galloway, M. Aardema, M. Ishidate Jr, J.L. Ivett, D.J. Kirkland, M. Takeshi, P. Mosesso, T. Sofuni, Mutation Res. 312 (1994) 241-261].We formulate the above three procedures in terms of a Cochran-Armitage trend test and a Dunnett type test. We then compare the performance of these three procedures in terms of a Monte Carlo simulation study. We then develop a software program from the chosen procedure for its ease of use by statisticians and toxicologists.     

DNA-damage induction by eight metal compounds in TK6 human lymphoblastoid cells: results obtained with the alkaline Comet assay

Metal compounds are long-lived and can react with different macromolecules, producing a wide range of biological effects, including DNA damage. Since their reactivity is associated with their chemical structure, it is important to obtain information on more than one compound from the same metal. In this study, the DNA-damaging potential of two mercury compounds (mercury chloride and methyl mercury chloride), two nickel compounds (nickel chloride and potassium hexafluoronickelate), two palladium compounds (ammonium tetrachloropalladate and ammonium hexachloropalladate), and two tellurium compounds (sodium tellurite and sodium tellurate) was evaluated in human lymphoblastoid TK6 cells by use of the alkaline version of the Comet assay. As the use of computerized image-analysis systems to collect comet data has increased, the metric used for quantifying DNA damage was the Olive tail moment. Treatments lasted for 3h and the range of concentrations tested was different for each metal compound, depending on its toxicity. Both mercury agents produced DNA damage in TK6 cells, with mercury chloride producing considerably more DNA damage than methyl mercury chloride. Of the two nickel compounds, only nickel chloride (a Ni(II) compound) induced DNA breaks. Similarly, of the two palladium compounds, only the Pd(II) compound (ammonium tetrachloropalladate) was positive in the assay. Sodium tellurite was clearly positive, producing concentration-related increases in DNA damage, while sodium tellurate gave a negative response. In conclusion, the ability of inducing DNA damage by the selected metal compounds in human TK6 cells, when measured with the Comet assay, was dependent on the chemical form and, in general, compounds containing the metal in the lower valence state displayed the greater DNA-damaging ability.     

Environmental and occupational biomonitoring using the Comet assay

Biomonitoring of human populations exposed to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation in the development of cancer. In recent years, the Comet assay, also known as a “single cell gel” (SCG) electrophoresis assay, has become an important tool for assessing DNA damage in exposed populations. This is the method of choice for population-based studies of environmental and occupational exposure to air pollutants, metals, pesticides, radiation, and other xenobiotics as we show in this review. To appreciate the role of the Comet assay in the field of biomonitoring, we review data from 122 studies that employed the assay. These studies evaluated environmental versus occupational exposures and the levels of DNA damage in cells of individuals exposed in each case. Our review of the literature reveals the importance of the need to establish standard methodological conditions that affect unwinding and electrophoresis times and tail values (tail length, tail DNA, tail moment), with the goal of being able to compare data collected in different laboratories throughout the world. The Comet assay is susceptible to subtle artifacts of manipulation depending on the type and timing of sampling performed. Therefore, in the reporting of DNA damage detected by the Comet assay, the context of how the DNA damage was created also needs to be reported and considered in the interpretation of Comet assay results. The success of the Comet assay is reflected by its use over the past 20 years in the field of biomonitoring, and by the increasing number of studies that continue to report its use. As the shortcomings of the assay are identified and considered in the interpretation of DNA damage detection, the Comet assay will continue to provide improved reliability as a biomarker in human biomonitoring studies.     

DNA-damage induction by eight metal compounds in TK6 human lymphoblastoid cells: Results obtained with the alkaline Comet assay

Metal compounds are long-lived and can react with different macromolecules, producing a wide range of biological effects, including DNA damage. Since their reactivity is associated with their chemical structure, it is important to obtain information on more than one compound from the same metal. In this study, the DNA-damaging potential of two mercury compounds (mercury chloride and methyl mercury chloride), two nickel compounds (nickel chloride and potassium hexafluoronickelate), two palladium compounds (ammonium tetrachloropalladate and ammonium hexachloropalladate), and two tellurium compounds (sodium tellurite and sodium tellurate) was evaluated in human lymphoblastoid TK6 cells by use of the alkaline version of the Comet assay. As the use of computerized image-analysis systems to collect comet data has increased, the metric used for quantifying DNA damage was the Olive tail moment. Treatments lasted for 3 h and the range of concentrations tested was different for each metal compound, depending on its toxicity. Both mercury agents produced DNA damage in TK6 cells, with mercury chloride producing considerably more DNA damage than methyl mercury chloride. Of the two nickel compounds, only nickel chloride (a Ni(II) compound) induced DNA breaks. Similarly, of the two palladium compounds, only the Pd(II) compound (ammonium tetrachloropalladate) was positive in the assay. Sodium tellurite was clearly positive, producing concentration-related increases in DNA damage, while sodium tellurate gave a negative response. In conclusion, the ability of inducing DNA damage by the selected metal compounds in human TK6 cells, when measured with the Comet assay, was dependent on the chemical form and, in general, compounds containing the metal in the lower valence state displayed the greater DNA-damaging ability.     

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.     

Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76±1.21 (arbitrary units) for males as compared to 3.37±1.47 for females (P<0.05)], tail DNA (%) [10.2±2.96 for males as compared to 9.40±2.83 for females (P<0.05)] and tail length (μm) [59.65±9.23 for males and 49.57±14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.     

Gender-related differences in basal DNA damage in lymphocytes of a healthy Indian population using the alkaline Comet assay

The Comet assay, a sensitive, rapid and non-invasive technique, measures DNA damage in individual cells and has found wide acceptance in epidemiological and biomonitoring studies to determine the DNA damage resulting from lifestyle, occupational and environmental exposure. The present study was undertaken to measure the basal level of DNA damage in a normal, healthy Indian male and female population. Out of the 230 volunteers included in this study, 124 were male and 106 were female. All the individuals belonged to a comparable socio-economic background and aged between 20 and 30 years. They were also matched for their smoking and dietary habits. The period of sample collection was also matched. The results revealed a statistically significant higher level of DNA damage in males when compared to females as evident by an increase in the Olive tail moment [3.76+/-1.21 (arbitrary units) for males as compared to 3.37+/-1.47 for females (P<0.05)], tail DNA (%) [10.2+/-2.96 for males as compared to 9.40+/-2.83 for females (P<0.05)] and tail length (microm) [59.65+/-9.23 for males and 49.57+/-14.68 for females (P<0.001)]. To our knowledge, this report has, for the first time demonstrated significant differences in the basal level of DNA damage between males and females in a normal healthy Indian population.