The DNA damage response to radiological imaging: from ROS and γH2AX foci induction to gene expression responses in vivo.

Candidate ionising radiation exposure biomarkers must be validated in humans exposed in vivo. Blood from patients undergoing positron emission tomography-computed tomography scan (PET-CT) and skeletal scintigraphy (scintigraphy) was drawn before (0 h) and after (2 h) the procedure for correlation analyses of the response of selected biomarkers with radiation dose and other available patient information. FDXR, CDKN1A, BBC3, GADD45A, XPC, and MDM2 expression was determined by qRT-PCR, DNA damage (γH2AX) by flow cytometry, and reactive oxygen species (ROS) levels by flow cytometry using the 2', 7'-dichlorofluorescein diacetate test in peripheral blood mononuclear cells (PBMC). For ROS experiments, 0- and 2-h samples were additionally exposed to UVA to determine whether diagnostic irradiation conditioned the response to further oxidative insult. With some exceptions, radiological imaging induced weak γH2AX foci, ROS and gene expression fold changes, the latter with good coherence across genes within a patient. Diagnostic imaging did not influence oxidative stress in PBMC successively exposed to UVA. Correlation analyses with patient characteristics led to low correlation coefficient values. γH2AX fold change, which correlated positively with gene expression, presented a weak positive correlation with injected activity, indicating a radiation-induced subtle increase in DNA damage and subsequent activation of the DNA damage response pathway. The exposure discrimination potential of these biomarkers in the absence of control samples as frequently demanded in radiological emergencies, was assessed using raw data. These results suggest that the variability of the response in heterogeneous populations might complicate identifying individuals exposed to low radiation doses.

Hypothermia differentially modulates the formation and decay of NBS1, γH2AX and 53BP1 foci in U2OS cells exposed to gamma radiation.

In studies on the mechanism of DNA damage response where ionizing radiation is used as the DNA damaging agent, cells are often exposed to ionizing radiation on melting ice (corresponding to 0.8 °C). The purpose of this procedure is to inhibit cellular processes i.e. DNA repair. Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage, but its mechanisms of action are poorly understood. The aim of the study was to analyze the effect of hypothermia at the level of formation and decay of NBS1, γH2AX, and 53BP1 foci, micronuclei, survival, cell cycle progression and oxidative stress in U2OS cells. The results show that hypothermia alone induced oxidative stress and foci. When applied in combination with radiation but only during the exposure time, it potentiated the formation of γH2AX and 53BP1 but not of NBS1 foci. When applied during irradiation and subsequent repair time, 53BP1 and NBS1 foci formed and decayed, but the levels were markedly lower than when repair was carried out at 37 °C. The frequency of micronuclei was elevated in cells irradiated at 0.8 °C, but only when analysed 20 h after irradiation which is likely due to a reduced G2 cell cycle block. Hypothermia reduced cell survival, both with and without radiation exposure. The temperature effect should be considered when cooling cells on melting ice to inhibit DNA repair in the induction of DNA damage.

Could veterinarians be immune to contracting SARS-CoV-2?

Introduction: The aim of the study was to assess the epidemic situation among veterinarians of the Swietokrzyskie Voivodeship, Poland, in relation to the control group. Material and methods: The research was divided into 3 stages. Stage I involved the selection of subjects. In stage II, flow cytometry for immunophenotyping was performed and the percentage of the sub-population of CD4 cells and CD8 cells was assessed. Stage III involved collection of nasopharyngeal swab samples in order to determine the canine coronavirus CR-CoV mRNA with the rT-PCR method. Results: The percentage of the CD4 and CD8 lymphocyte subpopulation in relation to the total lymphocyte population in veterinarians did not differ statistically from the percentage in the control group. The CD4/CD8 ratio in the group of veterinarians was on average 1.93, and 2.04 in the control group. There was no statistically significant difference between the groups, p = 0.591. Canine CR-CoV mRNA was not detected in any of the veterinarians or in the control group. Conclusions: None of the veterinarians had a significant increase in T lymphocytes, which could be an effective defense against SARS-CoV-2.

Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy-RC) and nine cervical cancer patients (treatment with radiotherapy alone-R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

Coralyne Radiosensitizes A549 Cells by Upregulation of CDKN1A Expression to Attenuate Radiation Induced G2/M Block of the Cell Cycle.

Coralyne is a synthetic analog of berberine related to protoberberine-isoquinoline alkaloids. Isoquinoline derivatives and analogs are renowned as potent radiosensitizers with potential medical application. In the present study, we investigated the effect of coralyne on the cell death, cytoskeletal changes and cell cycle progression of irradiated A549 cells. A clonogenic assay revealed that coralyne pretreatment decreased the viability of A549 cells in a time- and dose-dependent manner. Moreover, exposure to coralyne and ionizing radiation (IR) markedly altered the filamentous actin cytoskeletal architecture and integrin-ß binding sites of A549 cells. Treatment with 1-25 µM coralyne in combination with 2 Gy of IR significantly reduced the percentage of cells in G2/M phase compared with 2 Gy IR alone. These results indicate that coralyne is a potent radiosensitizing agent that may find an application in medicine.

Targeted Radionuclide Therapy of Prostate Cancer-From Basic Research to Clinical Perspectives.

Prostate cancer is the most commonly diagnosed malignancy in men and the second leading cause of cancer-related deaths in Western civilization. Although localized prostate cancer can be treated effectively in different ways, almost all patients progress to the incurable metastatic castration-resistant prostate cancer. Due to the significant mortality and morbidity rate associated with the progression of this disease, there is an urgent need for new and targeted treatments. In this review, we summarize the recent advances in research on identification of prostate tissue-specific antigens for targeted therapy, generation of highly specific and selective molecules targeting these antigens, availability of therapeutic radionuclides for widespread medical applications, and recent achievements in the development of new-generation small-molecule inhibitors and antibody-based strategies for targeted prostate cancer therapy with alpha-, beta-, and Auger electron-emitting radionuclides.

Selective cytotoxicity and antifungal properties of copper(II) and cobalt(II) complexes with imidazole-4-acetate anion or 1-allylimidazole.

The physicochemical properties of metal complexes determine their potential applications as antitumor agents. In this study, the antitumor properties of mononuclear cobalt(II) and copper(II) coordination compounds (stoichiometry: [Co(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Co(1-allim)6](NO3)2 (1-allim = 1-allylimidazole), [Cu(iaa)2H2O] and [Cu(1-allim)4(NO3)2]) and their ligands have been evaluated on human lung carcinoma A549 cells and normal bronchial BEAS-2B cells. Designing the chemical structure of new antitumor agents the possible interactions with macromolecules such as DNA or proteins should be take into account. PCR gene tlr4 product served as DNA model, whereas lysozyme and phage-derived endolysin (both peptidoglycan degrading enzymes) were applied as protein/enzyme model. The interactions were analysed using PCR-HRM and circular dichroism, FT-IR, spectrophotometry, respectively. Additionally, the antimicrobial properties of the complexes at a non-cytotoxic concentration were analyzed against S. aureus, E. coli, P. aeruginosa and C. albicans strains. The results obtained in this study showed the selective cytotoxicity of metal complexes, mainly [Cu(1-allim)4(NO3)2] towards tumor cells. From all tested compounds, only [Co(iaa)2(H2O)2].H2O non-covalently interacts with DNA. Cu(II) and Co(II) complexes did not affect the secondary conformation of tested proteins but modified the hydrolytic activity of enzymes (lysozyme and endolysin). Moreover, only [Co(iaa)2(H2O)2].H2O exhibited the antifungal properties. In conclusion, Co(II) and Cu(II) metal complexes bearing two imidazole-4-acetate ligands seemed to be promising antitumor and antifungal agents for future drug design and application.

Hypothermia modulates the DNA damage response to ionizing radiation in human peripheral blood lymphocytes.

PURPOSE: Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage. It was suggested to be due to an effective transformation of DNA damage to chromosomal damage at low temperature. The purpose of the study was to analyze the kinetics of aberration formation during the first hours after exposing human peripheral blood lymphocytes to ionizing radiation at 0.8 °C and 37 °C. MATERIALS AND METHODS: To this end, we applied the technique of premature chromosome condensation. In addition, DNA damage response was analyzed by measuring the levels of phosphorylated DNA damage responsive proteins ATM, DNA-PK and p53 and mRNA levels of the radiation-responsive genes BBC3, FDXR, GADD45A, XPC, MDM2 and CDKN1A. RESULTS: A consistently lower frequency of chromosomal breaks was observed in cells exposed at 0.8 °C as compared to 37 °C already after 30 minutes postexposure. This effect was accompanied by elevated levels of phosphorylated ATM and DNA-PK proteins and a reduced immediate level of phosphorylated p53 and of the responsive genes. CONCLUSIONS: Low temperature at exposure appears to promote DNA repair leading to reduced transformation of DNA damage to chromosomal aberrations.

Genotoxic potential of diesel exhaust particles from the combustion of first- and second-generation biodiesel fuels-the FuelHealth project.

Epidemiological data indicate that exposure to diesel exhaust particles (DEPs) from traffic emissions is associated with higher risk of morbidity and mortality related to cardiovascular and pulmonary diseases, accelerated progression of atherosclerotic plaques, and possible lung cancer. While the impact of DEPs from combustion of fossil diesel fuel on human health has been extensively studied, current knowledge of DEPs from combustion of biofuels provides limited and inconsistent information about its mutagenicity and genotoxicity, as well as possible adverse health risks. The objective of the present work was to compare the genotoxicity of DEPs from combustion of two first-generation fuels, 7% fatty acid methyl esters (FAME) (B7) and 20% FAME (B20), and a second-generation 20% FAME/hydrotreated vegetable oil (SHB: synthetic hydrocarbon biofuel) fuel. Our results revealed that particulate engine emissions from each type of biodiesel fuel induced genotoxic effects in BEAS-2B and A549 cells, manifested as the increased levels of single-strand breaks, the increased frequencies of micronuclei, or the deregulated expression of genes involved in DNA damage signaling pathways. We also found that none of the tested DEPs showed the induction of oxidative DNA damage and the gamma-H2AX-detectable double-strand breaks. The most pronounced differences concerning the tested particles were observed for the induction of single-strand breaks, with the greatest genotoxicity being associated with the B7-derived DEPs. The differences in other effects between DEPs from the different biodiesel blend percentage and biodiesel feedstock were also observed, but the magnitude of these variations was limited.

A comparative analysis of in vitro toxicity of diesel exhaust particles from combustion of 1st- and 2nd-generation biodiesel fuels in relation to their physicochemical properties-the FuelHealth project.

Biodiesels represent more carbon-neutral fuels and are introduced at an increasing extent to reduce emission of greenhouse gases. However, the potential impact of different types and blend concentrations of biodiesel on the toxicity of diesel engine emissions are still relatively scarce and to some extent contradictory. The objective of the present work was to compare the toxicity of diesel exhaust particles (DEP) from combustion of two 1st-generation fuels: 7% fatty acid methyl esters (FAME; B7) and 20% FAME (B20) and a 2nd-generation 20% FAME/HVO (synthetic hydrocarbon biofuel (SHB)) fuel. Our findings indicate that particulate emissions of each type of biodiesel fuel induce cytotoxic effects in BEAS-2B and A549 cells, manifested as cell death (apoptosis or necrosis), decreased protein concentrations, intracellular ROS production, as well as increased expression of antioxidant genes and genes coding for DNA damage-response proteins. The different biodiesel blend percentages and biodiesel feedstocks led to marked differences in chemical composition of the emitted DEP. The different DEPs also displayed statistically significant differences in cytotoxicity in A549 and BEAS-2B cells, but the magnitude of these variations was limited. Overall, it seems that increasing biodiesel blend concentrations from the current 7 to 20% FAME, or substituting 1st-generation FAME biodiesel with 2nd-generation HVO biodiesel (at least below 20% blends), affects the in vitro toxicity of the emitted DEP to some extent, but the biological significance of this may be moderate.

Modification biological activity of S and R forms of Proteus mirabilis and Burkholderia cepacia lipopolysaccharides by carrageenans.

The modification of biological features of S and R forms of Proteus mirabilis and Burkholderia cepacia LPS by kappa/iota and kappa/beta carrageenans was shown in Limulus activation test, ELISA, human complement activation and apoptotic assay. The role of positively charged substituent Ara4N in lipid A was evaluated as a suspected major domain for interactions with sulphate groups of carrageenans.The experiments obtained by three serological methods indicated that not only lipid A part of LPS but also polysaccharide elements such as core and O-specific chain are involved in interaction with carrageenes. Carrageenans turned out to be non-cytotoxic for A549 cells and were able to inhibit the apoptotic effect caused by lipid A of P. mirabilis and B. cepacia.

The effects of nickel(II) complexes with imidazole derivatives on pyocyanin and pyoverdine production by Pseudomonas aeruginosa strains isolated from cystic fibrosis.

Pseudomonas aeruginosa infection is problematic in patients with cystic fibrosis (CF). P. aeruginosa secretes a diversity of pigments, such as pyocyanin and pyoverdine. The aim of this study was to evaluate the effects of complexes of nickel(II) ([Ni(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Ni(1-allim)6](NO3)2 (1-allim = 1-allylimidazole) and NiCl2 on pyocyanin and pyoverdine production by 23 strains of P. aeruginosa isolated from cystic fibrosis under growth conditions specific for the CF respiratory system. The antibacterial effects and biophysical properties of the tested substances were measured by spectrofluorometric techniques, as well as by laser interferometry, confocal and atomic force microscopy. The cytotoxic properties of all compounds were measured by Annexin/IP assay against A549 cells. All tested compounds have no effect on pyocyanin production and decrease the pyoverdine secretion in about 40% of tested P. aeruginosa strains at non-cytotoxic range of concentrations. Imidazole-4-acetate anion and 1-allylimidazole have good diffusion properties in the mature P. aeruginosa PAO1 biofilm. In conclusion, the tested nickel(II) complexes do not have clinical implications in P. aeruginosa eradication in cystic fibrosis. The diffusion properties of 1-allylimidazole and imidazole-4-acetate and their lack of effect on A549 cells suggest that they might be considered for chemical synthesis with other transition metals.

Modulation of radiation-induced cytogenetic damage in human peripheral blood lymphocytes by hypothermia.

PURPOSE: Recent studies have shown that low temperature (hypothermia) at exposure can act in a radio-protective manner at the level of cytogenetic damage. The mechanisms of this phenomenon are not understood, but it was suggested to be due to hypothermia-induced perturbations of the cell cycle. The purpose of the present study was to detect whether a reduced frequency of micronuclei is observed in peripheral blood lymphocytes (PBL) irradiated at low temperature and harvested sequentially at 3 time points. Additionally, the level of apoptosis was estimated by microscopic analysis of the MN slides. MATERIALS AND METHODS: Experiments were carried out with blood drawn from three donors at the Stockholm University and from three donors at the Jan Kochanowski University. Prior to irradiation, blood samples were incubated for 20min and irradiated at the respective temperature (0°C and 37°C) with gamma rays. Whole blood cultures were set up, cytochalasin B was added after 44h of irradiation and the samples were harvested after 72, 96 and 120h of incubation time. RESULTS AND CONCLUSIONS: The frequency of micronuclei was markedly lower in PBL harvested at 72h, 96h and 120h following irradiation at 0°C as compared to 37°C. This indicates that the temperature effect observed in peripheral blood lymphocytes after irradiation is not related to a temporary perturbation of the cell cycle. Also, it is not due to selective elimination of damaged cells by apoptosis.

Defining Blood Processing Parameters for Optimal Detection of γ-H2AX Foci: A Small Blood Volume Method.

Biodosimetric methods used to measure the effects of radiation are critical for estimating the health risks to irradiated individuals or populations. The direct measurement of radiation-induced γ-H2AX foci in peripheral blood lymphocytes is one approach that provides a useful end point for triage. Despite the documented advantages of the γ-H2AX assay, there is considerable variation among laboratories regarding foci formation in the same exposure conditions and cell lines. Taking this into account, the goal of our study was to evaluate the influence of different blood processing parameters on the frequency of γ-H2AX foci and optimize a small blood volume protocol for the γ-H2AX assay, which simulates the finger prick blood collection method. We found that the type of fixative, temperature and blood processing time markedly affect the results of the γ-H2AX assay. In addition, we propose a protocol for the γ-H2AX assay that may serve as a potential guideline in the event of large-scale radiation incidents.

Effect of hypothermia on radiation-induced micronuclei and delay of cell cycle progression in TK6 cells.

PURPOSE: Low temperature (hypothermia) during irradiation leads to a reduced frequency of micronuclei in TK6 cells and it has been suggested that perturbation of cell cycle progression is responsible for this effect. The aim of the study was to test this hypothesis. MATERIALS AND METHODS: Human lymphoblastoid TK6 cells were treated by a combination of hypothermia (0.8°C) and ionizing radiation in varying order (hypothermia before, during or after irradiation) and micronuclei were scored. Growth assay and two-dimensional flow cytometry was used to analyze cell cycle kinetics following irradiated of cells at 0.8°C or 37.0°C. RESULTS: The temperature effect was observed at the level of micronuclei regardless of whether cells were cooled during or immediately before or after the radiation exposure. No indication of cell cycle perturbation by combined exposure to hypothermia and radiation could be detected. CONCLUSIONS: The protective effect of hypothermia observed at the level of cytogenetic damage was not due to a modulation of cell cycle progression. A possible alternative mechanism and experiments to test it are discussed.

The dose-response relationship for dicentric chromosomes and γ-H2AX foci in human peripheral blood lymphocytes: influence of temperature during exposure and intra- and inter-individual variability of donors.

PURPOSE: Hypothermia during in vitro irradiation of human peripheral blood lymphocytes (PBL) affects the level of chromosome aberrations. The molecular mechanisms of this phenomenon are not fully understood. The aim of our study was to examine the effect of hypothermia on the dose-response relationship for dicentric chromosomes and the level of γ-H2AX (phosphorylated histone H2AX) foci. In addition, the inter- and intra-individual variability was assessed in relation to temperature. MATERIALS AND METHODS: PBL were kept at 0.8, 20 and 37°C and then exposed to gamma-rays (from 0-3 Gy). Dicentric chromosomes were scored in first post-treatment mitoses. γ-H2AX foci were scored 15, 30, 60, 120 min and 24 h post irradiation. RESULTS: Our results revealed that the frequency of dicentric chromosomes in cells exposed at 37°C to gamma-rays was higher than after exposure at 0.8 and 20°C. No effect of temperature was observed on the number of γ-H2AX foci as well as on the intra- and inter-individual variations of the dicentric yield and the number of γ-H2AX foci. CONCLUSIONS: Temperature at exposure to ionizing radiation has a pronounced effect on the level of cytogenetic damage but not γ-H2AX foci.

Effect of surface modification of silica nanoparticles on toxicity and cellular uptake by human peripheral blood lymphocytes in vitro.

Silica nanoparticles have an interesting potential in drug delivery, gene therapy and molecular imaging due to the possibility of tailoring their surface reactivity that can be obtained by surface modification. Despite these potential benefits, there is concern that exposure of humans to certain types of silica nanomaterials may lead to significant adverse health effects. The motivation of this study was to determine the kinetics of cellular binding/uptake of the vinyl- and the aminopropyl/vinyl-modified silica nanoparticles into peripheral blood lymphocytes in vitro, to explore their genotoxic and cytotoxic properties and to compare the biological properties of modified silica nanoparticles with those of the unmodified ones. Size of nanoparticles determined by SEM varied from 10 to 50 nm. The average hydrodynamic diameter and zeta potential also varied from 176.7 nm (+18.16 mV) [aminopropyl/vinyl-modified] and 235.4 nm (-9.49 mV) [vinyl-modified] to 266.3 (-13.32 mV) [unmodified]. Surface-modified silica particles were internalized by lymphocytes with varying efficiency and expressed no cytotoxic nor genotoxic effects, as determined by various methods (cell viability, apoptosis/necrosis, oxidative DNA damage, chromosome aberrations). However, they affected the proliferation of the lymphocytes as indicated by a decrease in mitotic index value and cell cycle progression. In contrast, unmodified silica nanoparticles exhibited cytotoxic and genotoxic properties at high doses as well as interfered with cell cycle.

Effects of saponins against clinical E. coli strains and eukaryotic cell line.

Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 µg/mL and in the range of 12-50 µg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 µg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells.

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20nm and 200nm) and titanium dioxide (21nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact.

FociCounter: a freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci.

Gamma-H2AX foci are sensitive and specific indicator for the induction of DNA double-strand breaks (DSBs) and an immunocytochemical assay with antibodies recognizing gamma-H2AX has become the gold standard for the detection of this type of DNA lesion. Quantification of gamma-H2AX foci can be achieved by various methods such as Western blotting, flow cytometry, visual analysis and computational analysis with a fluorescence microscope. The best sensitivity is achieved by computer analysis. Since no freeware programme for the analysis of gamma-H2AX foci exists for a PC platform, the aim of our study was to develop a simple and user-friendly public-domain software. The algorithm applied in our programme allows determination of the number of foci in a single cell, a focus intensity per cell, as well as a cell intensity. Its graphical user interface is based on a GTK+ library and the whole application can be run under a variety of operating systems, including MS Windows and Linux. The programme called FociCounter is publicly available at http://focicounter.sourceforge.net. Application of the programme was tested by analysing gamma-H2AX foci in CHO and MO59K cells irradiated in vitro with X-rays and validated by comparing the results obtained with the outcome of automated image analysis and flow cytometry.