The implausibility of systemic genotoxic effects measured by the comet assay in rats exposed to formaldehyde

A recent publication reported genotoxic effects in the alkaline comet assay in lymphocytes and liver cells of rats exposed to formaldehyde (FA) by inhalation (Im,H.; et al. J. Proteome Res. 2006, 5, 1354-1366). Rats were exposed to 5 and 10 ppm FA for 2 weeks in inhalation chambers. A similar dose-related increase in DNA migration was measured in both cell types. These results are inconsistent with published data concerning FA toxicity: (i) Systemic genotoxic effects are reported, whereas previous animal experiments indicated only local effects. (ii) Similar effects were observed in liver cells and lymphocytes despite administration by inhalation. (iii) Increased DNA migration was measured, whereas FA-induced DNA-protein cross-links (DPX) should reduce DNA migration. These three aspects are critically discussed, and the plausibility of the results published by Im and colleagues is questioned.     

DNA damage evaluated by the comet assay in lymphocytes of children with 137Cs internal contamination caused by the Chernobyl accident

The comet assay is one of the most versatile and popular tools for evaluating DNA damage. Its sensitivity to low dose radiation has been tested in vitro, but there are limited data showing its application and sensitivity in chronic exposure situations. The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 56 Ukrainian children. The study was performed during 2003 on children with demonstrable 137Cs internal contamination caused by food consumption. The children were selected for the study immediately after a 137Cs whole body counter measurement of internal contamination. The minimal detectable amount of 137Cs was 75 Bq. The control group included 29 children without detectable internal contamination, while in the exposed group 27 children with measured activity between 80 and 4037 Bq and committed effective dose between 54 and 3155 microSv were included. Blood samples were taken by a finger prick. The alkaline version of the comet assay was used, in combination with silver stained comets and arbitrary units (AU), for comet measurement. Factors such as disease, medical treatment, surface contamination of children's living location, etc., were considered in the study. Non-significant differences (p > 0.05) in DNA damage in control (9.0 +/- 5.7 AU) versus exposed (8.5 +/- 4.8 AU) groups were found. These results suggest that low doses of 137Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. Further studies considering effects of high exposure should be performed on chronically exposed people using this assay.     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.     

Lack of direct DNA damage in human blood leukocytes and lymphocytes after in vitro exposure to high power microwave pulses

Currently, the potential genotoxicity of high power microwave pulses (HPMP) is not clear. Using the alkaline single cell gel electrophoresis assay, also known as the alkaline comet assay, we studied the effects of HPMP (8.8 GHz, 180 ns pulse width, peak power 65 kW, pulse repetition frequency 50 Hz) on DNA of human whole-blood leukocytes and isolated lymphocytes. The cell suspensions were exposed to HPMP for 40 min in a rectangular waveguide. The average SAR calculated from the temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The steady-state temperature rise in the 50 microl samples exposed to HPMP was 3.5 +/- 0.1 degrees C. In independent experiments, we did not find any statistically significant DNA damage manifested immediately after in vitro HPMP exposure of human blood leukocytes or lymphocytes or after HPMP exposure of leukocytes subsequently incubated at 37 degrees C for 30 min. Our results indicate that HPMP under the given exposure conditions did not induce DNA strand breaks, alkali-labile sites, and incomplete excision repair sites, which could be detected by the alkaline comet assay.     

Detection of oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the alkaline comet assay

Oxaliplatin is frequently used in the therapy of cancer. In DNA, oxaliplatin induces, like cisplatin, the formation of crosslinks, which are commonly accepted as being responsible for the cytotoxicity of platinum agents. The detection of oxaliplatin-induced DNA crosslink formation and repair could be a good measure of assessing how a patient is responding to the agent. In this study, we used a validated modification of the alkaline comet assay for detecting the presence of these crosslinks in vitro and in cancer patients. The H460 tumour cell line was treated in vitro with a range of oxaliplatin and cisplatin doses, and the subsequent crosslink formation and repair compared between the two agents. In addition, lymphocytes from cancer patients undergoing oxaliplatin-based chemotherapy were assayed for the formation and repair of oxaliplatin-induced crosslinks. A dose-response was observed in the in vitro samples, with cisplatin producing more crosslinks than oxaliplatin at equimolar concentrations and lesions induced by both agents showing different repair efficiencies. Furthermore, evidence of crosslink formation and repair was observed in the peripheral blood lymphocytes of all cancer patients studied, along with the detection of interindividual variability in crosslink formation and repair efficiencies. To the best of our knowledge, this is the first time that oxaliplatin DNA crosslinks have been detected either in vitro or in patient samples using the alkaline comet assay. Due to its sensitivity, rapidity, small cell sample and low cost, the alkaline comet assay is a good method for the detection of oxaliplatin-induced crosslinks and their subsequent repair and, in future clinical studies, could prove to be a valuable tool in assessing/predicting a patient's response to chemotherapy.     

Assessment of DNA sensitivity in peripheral blood leukocytes after occupational exposure to microwave radiation: the alkaline comet assay and chromatid breakage assay

DNA sensitivity in peripheral blood leukocytes of radar-facility workers daily exposed to microwave radiation and an unexposed control subjects was investigated. The study was carried out on clinically healthy male workers employed on radar equipment and antenna system service within a microwave field of 10 μW/cm²–20 mW/cm² with frequency range of 1,250–1,350 MHz. The control group consisted of subjects of similar age. The evaluation of DNA damage and sensitivity was performed using alkaline comet assay and chromatid breakage assay (bleomycin-sensitivity assay). The levels of DNA damage in exposed subjects determined by alkaline comet assay were increased compared to control group and showed inter-individual variations. After short exposure of cultured lymphocytes to bleomycin cells of subjects occupationally exposed to microwave (MW) radiation responded with high numbers of chromatid breaks. Almost three times higher number of bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes were determined in comparison with control group. The difference in break per cell (b/c) values recorded between smokers and non-smokers was statistically significant in the exposed group. Regression analyses showed significant positive correlation between the results obtained with two different methods. Considering the correlation coefficients, the number of metaphase with breaks was a better predictor of the comet assay parameters compared to b/c ratio. The best correlation was found between tail moment and number of chromatid with breaks. Our results indicate that MW radiation represents a potential DNA-damaging hazard using the alkaline comet assay and chromatid breakage assay as sensitive biomarkers of individual cancer susceptibility.     

DNA damage evaluated by the comet assay in lymphocytes of children with Cs internal contamination caused by the Chernobyl accident

The comet assay is one of the most versatile and popular tools for evaluating DNA damage. Its sensitivity to low dose radiation has been tested in vitro, but there are limited data showing its application and sensitivity in chronic exposure situations. The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 56 Ukrainian children. The study was performed during 2003 on children with demonstrable ¹³⁷Cs internal contamination caused by food consumption. The children were selected for the study immediately after a ¹³⁷Cs whole body counter measurement of internal contamination. The minimal detectable amount of ¹³⁷Cs was 75 Bq. The control group included 29 children without detectable internal contamination, while in the exposed group 27 children with measured activity between 80 and 4037 Bq and committed effective dose between 54 and 3155 μSv were included. Blood samples were taken by a finger prick. The alkaline version of the comet assay was used, in combination with silver stained comets and arbitrary units (AU), for comet measurement. Factors such as disease, medical treatment, surface contamination of children's living location, etc., were considered in the study. Non-significant differences (p > 0.05) in DNA damage in control (9.0 ± 5.7 AU) versus exposed (8.5 ± 4.8 AU) groups were found. These results suggest that low doses of ¹³⁷Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. Further studies considering effects of high exposure should be performed on chronically exposed people using this assay.     

Primary DNA damage in human blood lymphocytes exposed in vitro to 2450 MHz radiofrequency radiation

Human peripheral blood samples collected from three healthy human volunteers were exposed in vitro to pulsed-wave 2450 MHz radiofrequency (RF) radiation for 2 h. The RF radiation was generated with a net forward power of 21 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The average power density at the position of the cells in the flask was 5 mW/cm(2). The mean specific absorption rate, calculated by finite difference time domain analysis, was 2.135 (+/-0.005 SE) W/kg. Aliquots of whole blood that were sham-exposed or exposed in vitro to 50 cGy of ionizing radiation from a (137)Cs gamma-ray source were used as controls. The lymphocytes were examined to determine the extent of primary DNA damage (single-strand breaks and alkali-labile lesions) using the alkaline comet assay with three different slide-processing schedules. The assay was performed on the cells immediately after the exposures and at 4 h after incubation of the exposed blood at 37 +/- 1 degrees C to allow time for rejoining of any strand breaks present immediately after exposure, i.e. to assess the capacity of the lymphocytes to repair this type of DNA damage. At either time, the data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to the comet tail length, fluorescence intensity of the migrated DNA in the tail, and tail moment. The conclusions were similar for each of the three different comet assay slide-processing schedules examined. In contrast, the response of lymphocytes exposed to ionizing radiation was significantly different from RF-radiation- and sham-exposed cells. Thus, under the experimental conditions tested, there is no evidence for induction of DNA single-strand breaks and alkali-labile lesions in human blood lymphocytes exposed in vitro to pulsed-wave 2450 MHz radiofrequency radiation, either immediately or at 4 h after exposure.     

DNA damage in mouse lymphocytes exposed to curcumin and copper

Dietary polyphenolics, such as curcumin, have shown antioxidant and anti-inflammatory effects. Some antioxidants cause DNA strand breaks in excess of transition metal ions, such as copper. The aim of this study was to evaluate thein vitro effect of curcumin in the presence of increasing concentrations of copper to induce DNA damage in murine leukocytes by the comet assay. Balb-C mouse lymphocytes were exposed to 50 μM curcumin and various concentrations of copper (10 μM, 100 μM and 200 μM). Cellular DNA damage was detected by means of the alkaline comet assay. Our results show that 50 μM curcumin in the presence of 100–200 μM copper induced DNA damage in murine lymphocytes. Curcumin did not inhibit the oxidative DNA damage caused by 50 μM H₂O₂ in mouse lymphocytes. Moreover, 50 μM curcumin alone was capable of inducing DNA strand breaks under the tested conditions. The increased DNA damage by 50 μM curcumin was observed in the presence of various concentrations of copper, as detected by the alkaline comet assay.     

Chromosomal instability at the 7q11.23 region impacts on DNA-damage response in lymphocytes from Williams-Beuren syndrome patients

Williams-Beuren syndrome (WBS) is the chromosomal disorder arising from a hemizygous microdeletion at 7q11.23. The present study was focused on a comparative investigation of genomic integrity in WBS patients by use of cytogenetic methods and the alkaline comet assay. Lymphocytes of whole peripheral blood were cultured and metaphases were examined for frequency and spectrum of chromosome aberrations. A WBS-related microdeletion was detected by means of the FISH (fluorescence in situ hybridization) technique. The blood samples from patients who were carriers of this microdeletion, were tested in the comet assay. For this purpose, freshly collected lymphocytes were exposed to hydrogen peroxide (100μM, 1min, 4°C). The frequencies of endogenous and exogenous DNA damage, and the kinetics and efficiency of DNA repair were measured during three subsequent hours of incubation. Comparison of the two data sets in this group of patients demonstrated a slightly elevated average frequency of chromosome aberrations, significantly increased levels of endogenous and H(2)O(2)-induced DNA damage, and somewhat impaired DNA repair. The relationship between an abnormal DNA-damage response and the 7q11.23 hemizygous microdeletion was confirmed experimentally when comparing the comet assay data in FISH-positive and FISH-negative lymphocytes from WBS-suspected patients. Briefly, our results indicate the impact of chromosomal instability within this region on susceptibility towards DNA damage, which may contribute to pathogenesis of this disease. It was shown also that the comet assay, as well as an experimental design proposed here, seem to be useful tools for estimating genome integrity in WBS patients.     

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.     

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.     

Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.     

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.     

Evaluation of DNA damage in workers occupationally exposed to pesticides using single-cell gel electrophoresis (SCGE) assay. Pesticide genotoxicity revealed by comet assay

The comet assay, also called the single-cell gel electrophoresis (SCGE) assay, is a rapid and sensitive method for the detection of DNA damage (strand breaks and alkali-labile sites) in individual cells. The assay is based on the embedding of cells in agarose, their lysis in alkaline buffer and finally subjection to an electric current. In the present study, alkaline SCGE was used to evaluate the extent of primary DNA damage and DNA repair in peripheral blood lymphocytes of workers employed in pesticide production. After the period of high pesticide exposure, lymphocytes of the occupationally exposed workers manifested increased tail length and tail moment compared to the control group. After the workers spent 6 months out of the pesticide exposure zone, both endpoints were still above that of the control but significantly decreased as compared to the results of the first analysis.     

Extended-term cultures of human T-lymphocytes and the comet assay: a useful combination when testing for genotoxicity in vitro?

Extended-term cultures of human lymphocytes provide a source of uniform human cells that can be used for several experiments performed over a long time, avoiding the variability arising from taking blood samples for individual experiments. The use of extended-term cultures of human T-lymphocytes in the alkaline single-cell gel electrophoresis assay (comet assay) was evaluated as a test for the potential genotoxicity of chemicals. The DNA-damaging effects of five DNA-reactive mutagens and clastogens (benzo[a]pyrene, cyclophosphamide, formaldehyde, 4-nitroquinoline-N-oxide (4NQO) and N-nitrosopiperidine) was determined and compared with the effects of one non-DNA-reactive mutagen (5-hydroxyurea), and one non-mutagenic agent (ethanol). The alkylating and/or DNA-adduct forming agents N-nitrosopiperidine, cyclophosphamide, 4-nitroquinoline-N-oxide and benzo[a]pyrene increased the DNA migration in a dose-dependent manner. In contrast, the DNA/protein-crosslinking agent formaldehyde decreased the migration of DNA during the electrophoresis. The lowest observed effect levels (LOELs) under the experimental conditions used in the present study, were: 0.0001 mM (4-nitroquinoline-N-oxide without S9), 0.05 mM (benzo[a]pyrene with S9), 0.1mM (formaldehyde without S9), 0.25 mM (cyclophosphamide with S9), and 0.5mM (N-nitrosopiperidine with S9), respectively. The antimetabolite 5-hydroxyurea was also found to increase the tail moment, but only in cells that had been exposed to rather high concentrations (> or =10mM) of the compound. Ethanol did not affect the tail moment, not even in cells that had been exposed to an apparently cytotoxic concentration (500 mM). The results of the present study are in qualitative agreement with those obtained using other cells in the alkaline comet assay and it is therefore concluded that extended-term cultures of human T-lymphocytes and the alkaline version of the single-cell gel electrophoresis assay is a useful combination when testing for the potential genotoxicity of chemicals.     

Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay

The alkaline single cell gel electrophoresis (comet) assay was applied to study genotoxic properties of two inhalation anesthetics-halothane and isoflurane-in human peripheral blood lymphocytes (PBL). The cells were exposed in vitro to either halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) at concentrations 0.1-10 mM in DMSO. The anesthetics-induced DNA strand breaks as well as alkali-labile sites were measured as total comet length (i.e., increase of a DNA migration). Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner. In experiments conducted at two different electrophoretic conditions (0. 56 and 0.78 V/cm), halothane was able to increase DNA migration to a higher extent than isoflurane. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA degradation due to cell death. For this reason a contribution of toxicity in the observed effects was examined. We tested whether the exposed PBL were able to repair halothane- and isoflurane-induced DNA damage. The treated cells were incubated in a drug-free medium at 37 degrees C for 120 min to allow processing of the induced DNA damage. PBL exposed to isoflurane at 1 mM were able to complete repair within 60 min whereas for halothane a similar result was obtained at a concentration lower by one order of magnitude: the cells exposed to halothane at 1 mM removed the damage within 120 min only partly. We conclude that the increase of DNA migration induced in PBL by isoflurane at 1 mM and by halothane at 0.1 mM was not a result of cell death-associated DNA degradation but was caused by genotoxic action of the drugs. The DNA damage detected after the exposure to halothane at 1 mM was in part a result of DNA fragmentation due to cell death.     

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.     

Comet assay to sense neutron 'fingerprint'

The suitability of comet assay to identify DNA damage induced by neutrons of varying energy was tested. For this purpose, monoenergetic neutrons from Hiroshima University Radiobiological Research Accelerator (HIRRAC) were used to induce DNA damage in irradiated human peripheral blood lymphocytes. The level of damage was computed as tail moment for different doses (0.125-1 Gy) and compared with the effects resulting from irradiation with (60)Co gamma. The neutron-irradiated cells exhibited longer comet tails consisting of tiny pieces of broken DNA in contrast to the streaking tails generated by (60)Co gamma. The peak biological effectiveness occurred at 0.37 and 0.57 MeV; a further increase or decrease in neutron energy led to a reduced RBE value. The RBE values, as measured by the comet assay, were 6.3, 5.4, 4.7, 4.3, 2.6, and 1.7 for 0.37, 0.57, 0.79, 0.186, 1, and 2.3 MeV neutrons. The lower RBE value obtained by the comet assay when compared to that for other biological end points is discussed. This study reports the usefulness of the alkaline comet assay for identifying DNA damage induced by neutrons of the same radiation weighting factor. The comet assay is a potential tool for use in neutron therapy, as well as a method for the rapid screening of samples from individuals accidentally exposed to radiation.     

Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis.

The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet assay. The maximal comet length corresponded to 2 Mb DNA loops. At high concentrations of EtBr, 2 mg/ml, significant reduction in AVTD below control level was seen that suggested hypercondensation of chromatin. The hypercondensation was directly observed with the neutral comet assay. EtBr did not induce DNA strand breaks as measured by the alkaline comet assay. The hypercondensed nuclei could be decondensed by irradiation with gamma-rays or exposure to light. The data provide evidence that EtBr at high concentrations resulted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops and AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condensation of chromatin was verified. Further, the data show that the comet assay at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation.