RESUMO
During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.
Assuntos
Linfócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes para Micronúcleos/métodos , Testes de Mutagenicidade/métodos , Mutagênicos/farmacologia , Dano ao DNA , Previsões , Humanos , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Testes para Micronúcleos/tendências , Radiação IonizanteRESUMO
AIM: Investigation of the bystander effect in Chinese Hamster Ovary cells (CHO-K1) co-cultured with cells irradiated in the dose range of 0.1-4 Gy of high LET 12C ions and X-rays. BACKGROUND: The radiobiological effects of charged heavy particles on a cellular or molecular level are of fundamental importance in the field of biomedical applications, especially in hadron therapy and space radiation biology. MATERIALS AND METHODS: A heavy ion 12C beam from the Heavy Ion Laboratory of the University of Warsaw (HIL) was used to irradiate CHO-K1 cells. Cells were seeded in Petri dishes specially designed for irradiation purposes. Immediately after irradiation, cells were transferred into transwell culture insert dishes to enable co-culture of irradiated and non-irradiated cells. Cells from the membrane and well shared the medium but could not touch each other. To study bystander effects, a clonogenic survival assay was performed. RESULTS: The survival fraction of cells co-cultured with cells irradiated with 12C ions and X-rays was not reduced. CONCLUSIONS: The bystander effect was not observed in these studies.
RESUMO
There are conflicting data regarding the effect of culturing time of human peripheral blood lymphocytes on the yield of chromosomal aberrations induced by sparsely ionising radiation in the G0 phase of the cell cycle. While some authors find that the yield of aberrations does not change with time, others find increased frequencies of aberrations with harvesting time. The reasons for the conflicting results are not known, but the majority of studies were performed with lymphocytes of a single donor collected at one time point. We performed a study to verify if individual variability could be a confounding factor. As a positive control, lymphocytes were also exposed to high LET radiation (neutrons and alpha-rays), where an effect of harvesting time on the level of damage is expected to be seen. Blood was drawn from a total of 8 donors at two time points and exposed to X-rays, 6 MeV neutrons or alpha particles generated by an Am-241 source. Whole blood cultures were set up and micronuclei (Mn) were scored in binucleated cells harvested after 72, 96 and 120 h of culture time. The results show that in lymphocytes exposed to X-rays, the frequency of Mn was generally not influenced by the culture time while for both neutrons and alpha particles consistently increased micronucleus frequencies with culture time were detected. Some individual variability was detected and the conflicting results regarding the relationship between the yield of cytogenetic damage and lymphocyte culture time can, at least partly, be due to this variability.
Assuntos
Partículas alfa/efeitos adversos , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Nêutrons/efeitos adversos , Raios X/efeitos adversos , Células Cultivadas , Humanos , Transferência Linear de Energia , Radiação , Fase de Repouso do Ciclo Celular , Fatores de TempoRESUMO
The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236-300 and 461-470â¯keV/µm for 12C and 16O ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (12C) and 470â¯keV/µm (16O).
Assuntos
Radioterapia com Íons Pesados , Íons/uso terapêutico , Eficiência Biológica Relativa , Animais , Células CHO , Sobrevivência Celular/efeitos da radiação , Cricetulus , Radioterapia com Íons Pesados/estatística & dados numéricos , Íons Pesados , Transferência Linear de Energia , Neoplasias/radioterapia , OxigênioRESUMO
PURPOSE: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of (12)C and (20)Ne ions. MATERIALS AND METHODS: Chinese hamster ovary (CHO-K1) cells were exposed to high LET (12)C (33.2 MeV, 20.3 MeV, 9.1 MeV at cell entrance) and (20)Ne ions (56.2 MeV, 34.7 MeV, 15 MeV at cell entrance) and to low LET x-rays. Technical details of the irradiation facility are presented which is based on the Monte Carlo simulation of the lateral spread of heavy ions as a result of the multiscattering small-angle process in physical conditions of the experimental set-up. RESULTS: RBE has been measured for LET values close to the Bragg peak maximum, i.e., 440-830 keV/microm for (12)C and for 1020-1600 keV/microm for (20)Ne ions. RBE values at several levels of survival were estimated and were found to decrease with increasing LET. The inactivation cross sections were calculated from the final slope of dose-response curves and were found to increase with increasing LET. CONCLUSIONS: The RBE decreases with increasing LET in the range between 440 and 1600 keV/microm for the two types of radiations forming a single line when plotted together, pointing towards LET as the single determinant of RBE. The inactivation cross section describing the killing efficiency of a single particle at the end of particle range comes close to the size of the cell nucleus.
Assuntos
Carbono/farmacologia , Transferência Linear de Energia , Neônio/farmacologia , Animais , Células CHO , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Relação Dose-Resposta à Radiação , Isótopos , Fótons , Radiometria , Eficiência Biológica Relativa , Raios XRESUMO
BACKGROUND: Restenosis is the complete occlusion of the blood vessel leading to such complications as ischemia/angina, myocardial infarction, and death. It can be managed by endovascular brachytherapy with both gamma and beta sources. Endovascular brachytherapy is performed worldwide on several thousands of cases per year. The gamma-emitter 192Ir as well as the beta-emitters 32P and 90Sr are mainly used. The dose to the occluded endothelial wall is 20 Gy. Interestingly, no information with respect to the dose absorbed by the blood during the course of the treatment exists. The aim of the present investigation was to verify if the micronucleus test is suitable to detect the dose absorbed by lymphocytes in the course of endovascular brachytherapy with 32P. MATERIALS AND METHODS: Blood was drawn from 16 patients immediately before and 1 day after the treatment. Frequencies of micronuclei were assessed. In order to ensure that the micronuclei did not arise due to fluoroscopy or reperfusion, we analyzed lymphocytes of 16 control patients who underwent interventional cardiology with balloon angioplasty only. RESULTS AND CONCLUSIONS: Enhanced frequencies of micronuclei were observed in lymphocytes of some donors following brachytherapy. No correlation could be detected between the level of induced micronuclei and the absorbed dose. Also, no effect of fluoroscopy or reperfusion was seen. Thus, although brachytherapy of restenosis with 32P leads to weak enhancement of the micronucleus frequency in lymphocytes, the effect was not seen in all patients; the reason for this heterogeneous response remains to be elucidated.
Assuntos
Angioplastia Coronária com Balão/métodos , Braquiterapia/métodos , Reestenose Coronária/radioterapia , Linfócitos/ultraestrutura , Micronúcleo Germinativo/efeitos da radiação , Células Cultivadas , Reestenose Coronária/sangue , Seguimentos , Humanos , Linfócitos/efeitos da radiação , Micronúcleo Germinativo/ultraestrutura , Resultado do TratamentoRESUMO
Carbon and oxygen ions were accelerated simultaneously to estimate the effect of irradiation of living cells with the two different ions. This mixed ion beam was used to irradiate the CHO-K1 cells, and a survival test was performed. The type of the effect of the mixed ion beam on the cells was determined with the isobologram method, whereby survival curves for irradiations with individual ion beams were also used. An additive effect of irradiation with the two ions was found.
Assuntos
Carbono/uso terapêutico , Radioterapia com Íons Pesados , Oxigênio/uso terapêutico , Eficiência Biológica Relativa , Animais , Células CHO , Carbono/administração & dosagem , Carbono/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Relação Dose-Resposta à Radiação , Transferência Linear de Energia , Oxigênio/administração & dosagem , Oxigênio/efeitos da radiação , RadiometriaRESUMO
Sirtuins (type III histone deacetylases) are an important member of a group of enzymes that modify chromatin conformation. We investigated the role of sirtuin inhibitor, GPI 19015, in double strand break (DSB) repair in CHO-K1 wt and xrs-6 mutant cells. The latter is defective in DNA-dependent protein kinase (DNA-PK)-mediated non-homologous end-joining (D-NHEJ). DSB were estimated by the neutral comet assay and histone gammaH2AX foci formation. We observed a weaker effect of GPI 19015 treatment on the repair kinetics in CHO wt cells than in xrs6. In the latter cells the increase in DNA repair rate was most pronounced in G1 phase and practically absent in S and G2 cell cycle phases. The decrease in the number of histone gammaH2AX foci was faster in xrs6 than in CHO-K1 cells. The altered repair rate did not affect survival of X-irradiated cells. Since in G1 xrs6 cells DNA-PK-dependent non-homologous end-joining, D-NHEJ, does not operate, these results indicate that inhibition of sirtuins modulates DNA-PK-independent (backup) non-homologous end-joining, B-NHEJ, to a greater extent than the other DSB repair system, D-NHEJ.
Assuntos
Dano ao DNA , Reparo do DNA , Sirtuínas/antagonistas & inibidores , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Histona Desacetilase 2 , Inibidores de Histona Desacetilases , Cinética , Mutação , Proteínas Repressoras/antagonistas & inibidoresRESUMO
Biological dosimetry relies on the assessment of dose in peripheral blood lymphocytes (PBL) of a victim. Variability in the individual radiosensitivity of PBL has an impact on the precision of dose estimate and radiation-induced micronuclei show a strong individual variability. A factor which can influence the radiosensitivity of PBL is the hormonal status of female donors, which shows a regular pattern during the menstrual cycle. The aim of the present investigation was to verify whether the position within the menstrual cycle has an impact on the level of micronuclei in PBL. Blood was collected from 19 donors during the first and second half of the menstrual cycle and exposed to 2 Gy. Although statistically significant differences between the MN frequencies in PBL collected during the different time points were observed in the case of some donors, no reproducible trend that could find application in biological dosimetry could be detected.
Assuntos
Linfócitos/efeitos da radiação , Ciclo Menstrual/efeitos da radiação , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Adulto , Relação Dose-Resposta à Radiação , Feminino , Humanos , Testes para Micronúcleos , Pessoa de Meia-Idade , FumarRESUMO
PURPOSE: In the framework of RENEB, several biodosimetry exercises were conducted analyzing different endpoints. Among them, the analysis of translocations is considered the most useful method for retrospective biodosimetry due to the relative stability of their frequency with post irradiation time. The aim of this study was to harmonize the accuracy of translocation-based biodosimetry within the RENEB consortium. MATERIALS AND METHODS: An initial telescoring exercise analyzing FISH metaphase images was done to harmonize chromosome aberration descriptions. Then two blind intercomparison exercises (IE) were performed, by sending irradiated blood samples to each partner. Samples were cultured and stained by each partner using their standard protocol and translocation frequency was used to produce dose estimates. RESULTS: The coefficient of variation in the 1st IE (CV = 0.34) was higher than in the 2nd IE (CV = 0.16 and 0.23 in the two samples analyzed), for the genomic frequency of total translocations. Z-score analysis revealed that eight out of 10 and 17 out of 20 dose estimates were satisfactory in the 1st and 2nd IE, respectively. CONCLUSIONS: The results obtained indicate that, despite the problems identified in few partners, which can be corrected, the RENEB consortium is able to carry out retrospective biodosimetry analyzing the frequency of translocations by FISH.
Assuntos
Bioensaio/métodos , Hibridização in Situ Fluorescente/métodos , Garantia da Qualidade dos Cuidados de Saúde , Exposição à Radiação/análise , Monitoramento de Radiação/métodos , Translocação Genética/efeitos da radiação , Bioensaio/normas , Europa (Continente) , Humanos , Hibridização in Situ Fluorescente/normas , Linfócitos/efeitos da radiação , Monitoramento de Radiação/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Translocação Genética/genéticaRESUMO
PURPOSE: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. MATERIALS AND METHODS: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. RESULTS: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. CONCLUSIONS: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.
Assuntos
Bioensaio/métodos , Aberrações Cromossômicas/efeitos da radiação , Testes para Micronúcleos/métodos , Garantia da Qualidade dos Cuidados de Saúde , Exposição à Radiação/análise , Monitoramento de Radiação/métodos , Bioensaio/normas , Europa (Continente) , Humanos , Linfócitos/citologia , Linfócitos/efeitos da radiação , Monitoramento de Radiação/normas , Reprodutibilidade dos Testes , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/efeitos da radiação , Sensibilidade e EspecificidadeRESUMO
PURPOSE: In the framework of the 'Realizing the European Network of Biodosimetry' (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. MATERIALS AND METHODS: Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. RESULTS: The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (≤ 0.94 Gy). For higher dose points (≥ 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. CONCLUSION: The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories.
Assuntos
Bioensaio/métodos , Aberrações Cromossômicas/efeitos da radiação , Testes para Micronúcleos/métodos , Garantia da Qualidade dos Cuidados de Saúde , Exposição à Radiação/análise , Monitoramento de Radiação/métodos , Bioensaio/normas , Europa (Continente) , Humanos , Linfócitos/efeitos da radiação , Monitoramento de Radiação/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
PURPOSE: RENEB, 'Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,' is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. MATERIALS AND METHODS: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones. RESULTS: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. CONCLUSIONS: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios.
Assuntos
Bioensaio/métodos , Planejamento em Desastres/métodos , Laboratórios , Exposição à Radiação/análise , Monitoramento de Radiação/métodos , Gestão da Segurança/métodos , União Europeia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Integração de SistemasRESUMO
PURPOSE: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. MATERIALS AND METHODS: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. RESULTS: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). CONCLUSIONS: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested.
Assuntos
Planejamento em Desastres/organização & administração , Monitoramento de Radiação/métodos , Liberação Nociva de Radioativos , Radiobiologia/educação , Gestão da Segurança/organização & administração , Triagem/organização & administração , Europa (Continente)RESUMO
PURPOSE: To investigate if deviations from DNA-proportional distribution of radiation-induced chromosomal aberrations are individually variable. MATERIALS AND METHODS: Peripheral blood lymphocytes were collected from seven healthy donors and exposed to different doses of gamma rays. Chromosomes 2, 8 and 14 were painted in different colors and aberrations scored with the help of an image-analysis system. RESULTS: Chromosome 2 was generally less sensitive than expected on the basis of DNA-proportional distribution and the extent of inter-donor variability was minimal. A higher than expected frequency of aberrations was found in chromosome 14 of five donors, while a higher than expected frequency of aberrations was found in chromosome 8 of two donors. CONCLUSIONS: Inter-donor variability may explain some of the controversies regarding the inter-chromosomal distribution of radiation-induced aberrations.
Assuntos
Aberrações Cromossômicas/efeitos da radiação , Cromossomos Humanos Par 14/efeitos da radiação , Cromossomos Humanos Par 2/efeitos da radiação , Cromossomos Humanos Par 8/efeitos da radiação , Adulto , Técnicas de Cultura de Células , DNA/análise , Dano ao DNA , Feminino , Humanos , Linfócitos/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.
Assuntos
Neoplasias da Mama/radioterapia , Aberrações Cromossômicas , Elétrons/uso terapêutico , Linfócitos/efeitos da radiação , Micronúcleos com Defeito Cromossômico , Liberação Nociva de Radioativos , Adulto , Neoplasias da Mama/genética , Feminino , Humanos , Linfócitos/ultraestrutura , Pessoa de Meia-IdadeRESUMO
We examined radiosensitizing properties of two novel platinum complexes (ethylenediamine(L-malato)platinum(II)), Pt1 and bis(1-ethylimidazole(L-malato)platinum(II)), Pt4. Initial double strand break (DSB) level and DSB rejoining were measured, using pulse field gel electrophoresis (PFGE) in human G1 phase lymphocytes subjected to Pt complex treatment alone and in combination with 10Gy of X-rays. Effects of Pt complex pre-treatment followed by X-irradiation were examined on survival (clonogenic ability) and growth (48 h growth tests) in Chinese hamster ovary (CHO-K1), xrs6 and L5178Y (LY) cells (LY-R and LY-S sublines). Cell cycle distributions of CHO cells after drug treatment were determined with the use of flow cytometry. Pt1 slowed down rejoining of X-ray induced DSB. It exerted a more than additive lethal effect on CHO-K1 cells but not on L5178Y cells subjected to combined Pt complex treatment and X-irradiation. In xrs6 cells the effect of combined Pt1+X treatment was additive. We conclude that, as earlier proposed for other Pt complexes, the radiosensitizing effect of Pt1 is connected with converting repairable DNA damage into irrepairable one (mode (i) of action). The requirements for this mode of sensitization are functional DNA repair systems (nucleotide excision repair (NER) and non-homologous end-joining (NHEJ)). Pt4 does not slow down DSB rejoining. It shows a considerable ability to arrest cells in G2 phase. We assume that Pt4 pre-treatment arrests cells in G2 phase and thus sensitizes to X-rays these cells that have a radiosensitive G2 phase (mode (ii) of action).
Assuntos
Compostos Organoplatínicos/química , Compostos Organoplatínicos/toxicidade , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/química , Radiossensibilizantes/toxicidade , Compostos de Sulfidrila/química , Adulto , Animais , Células CHO/efeitos dos fármacos , Terapia Combinada , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/toxicidade , Adutos de DNA/química , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , DNA de Neoplasias/química , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/efeitos da radiação , Relação Dose-Resposta a Droga , Humanos , Leucemia L5178 , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Masculino , CamundongosRESUMO
Comet assay under neutral conditions allows the detection of DNA double-strand breaks (DSB), considered to be the biologically relevant radiation-induced lesion. In this report, we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA DSB in X-irradiated mammalian cells in culture. The analysis carried out according to this protocol takes less time than those most often applied. Also, the use of lysis at 50 degrees C is avoided; this is important in view of the presence of heat-labile sites in the chromatin of irradiated cells, recently reported by Rydberg [Radiation-induced heat-labile sites that convert into DNA double-strand breaks, Radiation Research 153 (2000) 805-812]. The comets have well-defined, sharp limits, suitable for image analysis. The chromatin of the hydrogen peroxide-treated or UV-C-irradiated cell remains condensed similarly to that of the control cells. We checked the neutral comets for the presence of single-stranded DNA by means of a specific antibody. The results point to a satisfactory sensitivity of the modified neutral comet assay and its specificity for DSB. The minimum detection level of the modified neutral comet assay is about 5 Gy.