Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the in vitro micronucleus test: Determination of No-Observed-Effect Levels

The existence of thresholds for indirect DNA-damaging agents has been widely accepted in the last few years. In contrast, DNA-reactive agents have been assumed to have a non-threshold mode of action, as they directly induce DNA lesions that have the potential to be converted into mutations. However, this does not take into account protective factors acting to reduce or repair genotoxic damage. Among the compounds acting through possible threshold-mechanisms, some of them induce DNA damage by oxidative stress. In this context, the aim of our study was to investigate the dose–response relationship of well-known DNA-oxidizing agents acting through different mechanisms of oxidative stress, viz. potassium bromate, bleomycin and hydrogen peroxide (by the action of glucose oxidase) by assessing the induction of chromosomal damage using the in vitro micronucleus test (MNT) on the human lymphoblastoid cell line TK6. In order to provide a first characterization of their genotoxic mechanism, two treatment schedules were applied. Cells received both short-term treatment followed by a recovery time (1 + 23 h, 2 + 22 h, 3 + 21 h or 6 + 18 h) and long-term treatment (24 h continually). Our results show interesting non-linear dose–effect relationships starting with a range of non-mutagenic very low doses allowing the determination of a No-Observed-Effect Level (NOEL) and going step-wise up to higher doses. After a short exposure, three different plateaus were observed suggesting complex activations and interactions of different cellular mechanisms whose nature and efficiency were dose-dependent. In contrast, after a long treatment, the dose–response curves were different depending on the test compound investigated. Therefore, the in vitro MNT seems to be an appropriate predictive test to establish the NOEL(s) of DNA-oxidizing agents. In order to confirm and to determine the origin of the different cellular step-wise responses observed, additional mechanistic studies would be required, especially by means of other genotoxicity endpoints and gene-expression profiling.     

Genotoxicity evaluation of polluted ground water in human peripheral blood lymphocytes using the comet assay

This paper presents the genotoxicity experiments with the ground water collected from an area under the influence of textile dyeing and bleaching industries in Tirupur, Tamilnadu, India. The alkaline single cell gel electrophoresis (SCGE) assay was performed in vitro with human peripheral blood lymphocytes. The cells were exposed to two doses of non-volatile organic agents extracted from ground water samples. Ground water samples were collected from 12 locations distributed in and around Tirupur and extracts were taken at different pHs (without pH adjustment and acidic pH 2.0). The persistence of the DNA damage after exposure to the organic extracts was also studied. All the samples were found to contain substances capable of inducing DNA damage in human lymphocytes. Extracts from acidified waters (pH=2.0) were found to induce more DNA damage than extracts from without pH adjustment (natural pH). The DNA damage was not fully repaired after incubation for 2h at 37 degrees C. The chemical characterization of the sub-fractions revealed the existence of aromatic amines in the extracts, which may be responsible for the DNA damaging activity of the water samples. The results of this investigation demonstrate the application of the comet assay in environmental monitoring studies.     

Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated. Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested. The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage. Results were available from both the alkaline unwinding assay and the mouse lymphoma assay for 61 compounds; they showed a concordance between the 2 assays of 77%. Of the 47 compounds that were positive or equivocal in the alkaline unwinding assay, only carbon tetrachloride and prednisolone were negative in the mouse lymphoma assay, while 12 of the 19 compounds that were negative in the alkaline unwinding assay were positive in the mouse lymphoma assay. These included 3 compounds that interfere with nucleic acid metabolism, and 3 crosslinking agents, which would be expected to produce mutations to a greater extent than strand breaks. The other 6 compounds were anthranilic acid, benzoquinone, p-chloroaniline, diethylmaleate, glucose and procarbazine HCl. Of these only the last is a known carcinogen. It is concluded from the present study that there was good overall agreement between the results of the DNA alkaline unwinding and mouse lymphoma TK locus assays, but that the sensitivity of the alkaline unwinding assay is lower for some classes of compounds. Bearing this in mind, the alkaline unwinding assay is considered suitable as a rapid screen for genotoxic activity in eukaryotic cells.     

Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins

The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.     

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.     

DNA-damaging ability of isoprene and isoprene mono-epoxide (EPOX I) in human cells evaluated with the comet assay

Isoprene is produced in combustion processes and is widely used as an industrial chemical. It is a natural product emitted by plants and endogenously produced by humans and other mammals. Therefore, exposure to isoprene from both endogenous and exogenous sources is unavoidable and occurs during the entire human life. Based on evaluations of the International Agency for Research on Cancer (IARC), isoprene has been classified in Group 2B (possibly carcinogenic to humans). In the present work, we have demonstrated, by use of the single-cell gel electrophoresis assay (SCGE or comet assay), that isoprene is able to induce DNA damage in peripheral blood mononuclear cells (PBMCs) in the presence of metabolic activation. In addition, treatment of cells with the main isoprene mono-epoxide (EPOX I) induced time- and dose- dependent DNA damage in both PBMCs and human leukaemia cells (HL60). The metabolic activation system, represented by rat liver post-mitochondrial fractions (S9), was obtained from rats that had been treated - or not - with inducing agents such as phenobarbital and ethanol. The inclusion of S9 fractions (4mg protein/mL) from non-induced or phenobarbital-induced rats resulted in a statistically significant enhancement of isoprene genotoxicity. A different pattern was obtained by the addition of ethanol-induced S9, which appeared highly genotoxic by itself even in the absence of isoprene. Reducing the concentration of ethanol-induced S9 to 0.25mg protein/mL resulted in a considerable enhancement of isoprene genotoxicity. In the absence of clear epidemiological evidence of the carcinogenicity of isoprene in humans, the results of this study seem to be particularly important since they add new findings to support the classification of this chemical as possibly carcinogenic to humans.     

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.     

Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds

Despite recent improvements in genotoxicity protocols, we have observed an increase in the occurrence of positive results, particularly in chromosomal aberration tests in vitro, yet very few of these are accompanied by positive responses in vivo. Thus, the positive results may not be biologically relevant either for rodents or humans in vivo, but how should we determine "biological relevance"? Chemicals that produce thresholded dose-responses may well not pose a genotoxic risk at low (relevant to human) exposures, but thresholds should not just be "seen"; there must be an explanation and understanding of the underlying mechanism. In addition to extremes of pH, ionic strength and osmolality, as have been identified previously, such mechanisms include indirect genotoxicity resulting from interaction with non-DNA targets, chemicals/metabolites which are inherently genotoxic but which, at low concentrations, are effectively conjugated and unable to form adducts, and production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. If such thresholded mechanisms can be identified at exposures which are well in excess of expected human exposure, then there may be a strong argument that the positive results are not biologically relevant.     

Application of Euglena gracilis cells to comet assay: evaluation of DNA damage and repair

Alkaline single-cell gel electrophoresis (comet assay) enables sensitive detection of DNA damage in eukaryotic cells induced by genotoxic agents. We performed a comet assay of unicellular green alga Euglena gracilis that was exposed to genotoxic chemicals, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), benzo[a]pyrene (BAP), mitomycin C (MMC) and actinomycin D (AMD). Tail length and tail moment in migrated DNA were measured as indications of DNA damage. MNNG and BAP were found to cause concentration-dependent increases in DNA damage. The responses were more sensitive than those of human lymphocytes under the same treatment conditions. MMC and AMD showed no positive response, as reported elsewhere. The comet assays performed at specified times after treatment revealed that the DNA damaged by MNNG and gamma-ray irradiation was repaired during the initial 1h. The results clearly show that the comet assay is useful for evaluating chemically-induced DNA damage and repair in E. gracilis. Given the ease of culturing and handling E. gracilis as well as its sensitivity, the comet assay of this alga would undoubtedly prove to be a useful tool for testing the genotoxicity of chemicals and monitoring of environmental pollution.     

Genotoxicity testing of PLGA-PEO nanoparticles in TK6 cells by the comet assay and the cytokinesis-block micronucleus assay

The in vitro genotoxicity of PLGA-PEO (poly-lactic-co-glycolic acid-polyethylene oxide copolymer) nanoparticles was assessed in TK6 cells using the comet assay as well as cytokinesis-block micronucleus (CBMN) assay. The cells were exposed to 0.12-75μg/cm2 of PLGA-PEO nanoparticles during 2 and 24h for analysis in the comet assay, and to 3-75μg/cm2 of these nanoparticles during 4, 24, 48 and 72h, respectively, for analysis in the CBMN assay. Two different protocols for treatment with cytochalasin B were used. We found that PLGA-PEO was neither cytotoxic (measured by relative cell growth activity and cytokinesis-block proliferation index (CBPI)), nor did it induce DNA strand-breaks (detected by the comet assay) or oxidative DNA lesions (measured by the comet assay modified with lesion-specific enzyme formamidopyrimidine-DNA-glycosylase). There were no statistically significant differences in the frequencies of micronucleated binucleated cells (MNBNCs) between untreated and treated cells in either of the conditions used. This suggests that PLGA-PEO did not have potential genotoxicity. However, using two experimental protocols of the micronucleus assay, PLGA-PEO nanoparticles showed a weak but significant increase in the level of MN in mononucleated cells, in cells treated for 48h with PLGA-PEO nanoparticles when cytochalasin B was added for the last 24h (1st protocol), and in cells treated for 24h with PLGA-PEO nanoparticles followed by washing of NPs and addition of cytochalasin B for another 24h (2nd protocol). It remains unclear whether the increase of MNMNC after treatment with PLGA-PEO nanoparticles is the effect of a possible, weak aneugenic potential or early effect of these particles, or due to another reason. These results suggest that aneugenicity in addition to clastogenicity may be considered as an important biomarker when assessing the genotoxic potential of polymeric nanoparticles.     

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.     

Potassium bromate treatment predominantly causes large deletions, but not GC>TA transversion in human cells

Potassium bromate (KBrO(3)) is strongly carcinogenic in rodents and mutagenic in bacteria and mammalian cells in vitro. The proposed genotoxic mechanism for KBrO(3) is oxidative DNA damage. KBrO(3) can generate high yields of 8-hydroxydeoxyguanosine (8OHdG) DNA adducts, which cause GC>TA transversions in cell-free systems. In this study, we investigated the in vitro genotoxicity of KBrO(3) in human lymphoblastoid TK6 cells using the comet (COM) assay, the micronucleus (MN) test, and the thymidine kinase (TK) gene mutation assay. After a 4h treatment, the alkaline and neutral COM assay demonstrated that KBrO(3) directly yielded DNA damages including DNA double strand breaks (DSBs). KBrO(3) also induced MN and TK mutations concentration-dependently. At the highest concentration (5mM), KBrO(3) induced MN and TK mutation frequencies that were over 30 times the background level. Molecular analysis revealed that 90% of the induced mutations were large deletions that involved loss of heterozygosity (LOH) at the TK locus. Ionizing-irradiation exhibited similar mutational spectrum in our system. These results indicate that the major genotoxicity of KBrO(3) may be due to DSBs that lead to large deletions rather than to 8OHdG adducts that lead to GC>TA transversions, as is commonly believed. To better understand the genotoxic mechanism of KBrO(3), we analyzed gene expression profiles of TK6 cells using Affymetrix Genechip. Some genes involved in stress, apoptosis, and DNA repair were up-regulated by the treatment of KBrO(3). However, we could not observe the similarity of gene expression profile in the treatment of KBrO(3) to ionizing-irradiation as well as oxidative damage inducers.     

Enhanced prediction of potential rodent carcinogenicity by utilizing comet assay and apoptotic assay in combination

The comet assay has been recently validated as a sensitive and specific test system for the quantification of DNA damage. The objectives of this study are to investigate the utility of comet assay for detecting mutagens with 11 substances that demonstrated positive results in at least one test among four standard short-term genotoxicity tests, and to evaluate its ability to predict rodent carcinogenicity. Out of 11 test substances, positive comet results were obtained for colchicine, hydroxyurea and actinomycin D. No effect on DNA migration, determined as the tail moment, was found with theophylline or 2,4-dinitrophenol. Bisphenol A, vinblastine, paclitaxel and p-anisidine appeared cytotoxic clastogens because these induced tail moment at concentrations showing 60% or less cell survival. In addition, among three test substances showing the bimodal distribution of DNA damage, which is a characteristic of apoptosis, true apoptosis result was obtained for camptothecin and dexamethasone with the Annexin V affinity assay. With this limited data-set, an investigation into the predictive value of these short-term genotoxicity tests for determining the carcinogenicity showed that comet assay has relatively high sensitivity and superior specificity to other four short-term genotoxicity assay. Therefore, our data suggest that comet assay, especially in combination with apoptotic assay, would be a good predictive test to minimize false-positives in evaluation of the potential rodent carcinogenicity.     

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.     

Use of the alkaline comet assay for the detection of transplacental genotoxins in newborn mice

Several lines of evidence show that in utero exposure to different toxicants has greater consequences than their exposure during adult life. This may be due to involvement of critical developmental stages, physiological immaturity and the long later-life span over which disease may initiate, develop and progress. The in vivo alkaline comet (single-cell gel electrophoresis) assay has been favoured by the scientific community for the evaluation of genotoxins. The objective of this study was to demonstrate the suitability of alkaline comet assay in detecting transplacental genotoxins using newborn mice. Here, we report the successful use of the comet assay in detecting multi-organ genotoxicity of known transplacental genotoxins in newborn mice. Three well known transplacental genotoxic agents, cyclophosphamide (CP), mitomycin-C (MMC) and zidovudine (AZT) were tested in pregnant Swiss mice. These compounds were administered in the late gestational period (16-20th days of pregnancy) and the comet assay was performed with lymphocytes, bone marrow, liver and kidney cells of newborn mice. Significant DNA damage was observed in all the tissues with tested transplacental genotoxins. The results of the comet assay were confirmed by the micronucleus (MN) assay of the peripheral blood of newborn mice. The results of this study provide sufficient evidence that the comet assay can be applied successfully for the detection of transplacental genotoxins in newborn mice.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

Comet assay: rapid processing of multiple samples

The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 microg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 microg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

No evidence for genotoxic effects from 24 h exposure of human leukocytes to 1.9 GHz radiofrequency fields

The current study extends our previous investigations of 2-h radiofrequency (RF)-field exposures on genotoxicity in human blood cell cultures by examining the effect of 24-h continuous-wave (CW) and pulsed-wave (PW) 1.9 GHz RF-field exposures on both primary DNA damage and micronucleus induction in human leukocyte cultures. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures was maintained at 37.0 +/- 1.0 degrees C for the duration of the 24-h exposure period. No significant differences in primary DNA damage were observed between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures when processed immediately after the exposure period by the alkaline comet assay. Similarly, no significant differences were observed in the incidence of micronuclei, incidence of micronucleated binucleated cells, frequency of binucleated cells, or proliferation index between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures. In conclusion, the current study found no evidence of 1.9 GHz RF-field-induced genotoxicity in human blood cell cultures after a 24-h exposure period.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.