Omar García

Quantification of γ-H2AX Foci in Human Lymphocytes: A Method for Biological Dosimetry after Ionizing Radiation Exposure

Abstract Recent studies have suggested that visualization of γ-H2AX nuclear foci can be used to estimate exposure to very low doses of ionizing radiation. Although this approach is widely used for various purposes, its suitability for individual human biodosimetry has not yet been assessed. We therefore conducted such an assessment with the help of available software for observing and automatically scoring γ-H2AX foci. The presence of γ-H2AX foci was evaluated in human peripheral blood lymphocytes exposed ex vivo to γ rays in a dose range of 0.02 to 2 Gy. We analyzed the response of γ-H2AX to ionizing radiation in relation to dose, time after exposure, and individual variability. We constructed dose–effect calibration curves at 0.5, 8 and 16 h after exposure and evaluated the threshold of detection of the technique. The results show the promise of automatic γ-H2AX scoring for a reliable assessment of radiation doses in a dose range of 0.6 Gy to 2 Gy up to 16 h after exposure. This γ-H2AX-based assay may be useful for biodosimetry, especially for triage to distinguish promptly among individuals the ones who have received negligible doses from those with significantly exposures who are in need of immediate medical attention. However, additional in vivo experiments are needed for validation.

Intercomparison in cytogenetic dosimetry among five laboratories from Latin America

As part of a regional International Atomic Energy Agency (IAEA) collaborative project within Latin America, five countries participated in an intercomparison in cytogenetic dosimetry. Coded slides for chromosomal aberrations and micronucleus analyses were prepared by the coordinator laboratory which organized the exercise and sent to the other participating laboratories. For estimates of dose, each laboratory scored the frequency of dicentrics in metaphases and the frequency of micronuclei in binucleated cells. The lymphocytes were irradiated with 60Co gamma-rays (0, 0.75, 1.5 and 3.0 Gy). Eleven of the 15 estimates of dose based on dicentrics and nine of the 12 based on micronuclei fell within +/- 30% of the true dose. When considering the uncertainties of the dose estimates, the true dose fell within the 95% confidence limits of the estimates on eight of the 15 occasions for dicentrics and four of the 12 for micronuclei.

Rapid assessment of high-dose radiation exposures through scoring of cell-fusion-induced premature chromosome condensation and ring chromosomes.

Analysis of premature chromosome condensation (PCC) mediated by fusion of G0-lymphocytes with mitotic CHO cells in combination with rapid visualization and quantification of rings (PCC-Rf) is proposed as an alternative technique for dose assessment of radiation-exposed individuals. Isolated lymphocytes or whole blood from six individuals were γ-irradiated with 5, 10, 15 and 20Gy at a dose rate of 0.5Gy/min. Following either 8- or 24-h post-exposure incubation of irradiated samples at 37°C, chromosome spreads were prepared by standard PCC cytogenetic procedures. The protocol for PCC fusion proved to be effective at doses as high as 20Gy, enabling the analysis of ring chromosomes and excess PCC fragments. The ring frequencies remained constant during the 8-24-h repair time; the pooled dose relationship between ring frequency (Y) and dose (D) was linear: Y=(0.088±0.005)×D. During the repair time, excess fragments decreased from 0.91 to 0.59 chromatid pieces per Gy, revealing the importance of information about the exact time of exposure for dose assessment on the basis of fragments. Compared with other cytogenetic assays to estimate radiation dose, the PCC-Rf method has the following benefits: a 48-h culture time is not required, allowing a much faster assessment of dose in comparison with conventional scoring of dicentrics and rings in assays for chemically-induced premature chromosome condensation (PCC-Rch), and it allows the analysis of heavily irradiated lymphocytes that are delayed or never reach mitosis, thus avoiding the problem of saturation at high doses. In conclusion, the use of the PCC fusion assay in conjunction with scoring of rings in G0-lymphocytes offers a suitable alternative for fast dose estimation following accidental exposure to high radiation doses.

Radiosensitization induced by the anti-epidermal growth factor receptor monoclonal antibodies cetuximab and nimotuzumab in A431 cells

Epidermal growth factor receptors (EGFR) are overexpressed in a wide range of malignancies including head and neck, colon, and breast cancers. It has been identified that carcinomas with high expression levels of EGFR are more resistant to radiotherapy. Therefore, inhibiting nuclear translocation of EGFR to increase the radiosensitivity of malignant cells expressing EGFR offers the potential for increasing the therapeutic index of radiotherapy. The purpose of the present study was to quantify and to compare the radiosensitizing properties of the well-known anti-EGFR antibodies, cetuximab and nimotuzumab in human epidermoid A431 overexpressing EGFR cells. Cells were treated with two concentrations of the antibodies and then irradiated with a single dose of 4 Gy. The results indicated that the two antibodies induced radiosensitization increasing the percentage of dead/dying cells and the yield of γ-H2AX foci 24 h after irradiation. Whereas cetuximab exhibited a significant increase in radiosensitization at the highest concentration, the effects of nimotuzumab were more modest. A correlation between γ-H2AX foci signals and dead/dying cells was observed. The disparity in modulation of radiation-induced DNA damage by the two antibodies could be associated with the level of their respective intrinsic cytotoxic properties. Overall, the findings highlight the potential therapeutic benefit of combination therapy with anti-EGFR antibodies and radiotherapy for relevant carcinomas.

Visual estimation of the percentage of DNA in the tail in the comet assay: Evaluation of different approaches in an intercomparison exercise

One of the difficulties in the comparison of results between laboratories working with the comet assay is the great diversity of parameters used to express DNA damage and the lack of conversion factors between the majority of them. Here we report a scorer-independent conversion curve to transform the values of DNA damage reported in arbitrary units (AU) into estimated percentage of DNA in the tail (E%T), and the results obtained in an intercomparison exercise where the effectiveness of this curve and two others proposed in the literature (E%T=AU/4 and E%T=(AU/5)+10) were tested. To obtain the conversion curve, human lymphocytes were first treated with radiation or H(2)O(2). Percentage of DNA in tail (%T) was then measured in 2100 comets (300 comets per treatment) using Casp image analysis software. Subsequently, using these values of %T, categories of 0, 1, 2, 3, and 4 were assigned to comets with %T [0-1), [1-25), [25-45), [45-70), and >70, and DNA damage was calculated in AU, as usual. DNA damage was induced in the interval 24-315AU (1.54-65.23%T). The best-fit conversion curve obtained by regression analysis was E%T=(AU-25.87)/4.46. In the intercomparison exercise, ten scorers from nine laboratories analyzed the same comet images (recorded on a compact disc) visually. The values reported in comet categories were transformed into AU and subsequently into E%T, using the three approaches mentioned above. The best agreement between E%T and %T measured by the software (S%T) was obtained with the conversion curve reported here, where the slope of E%T versus S%T from the ten scorers was not different from 1. Using this conversion curve, the overall mean difference between E%T and S%T was 1.4±2.62 and 57 (81%) of E%T values differ from S%T by less than 5 units. These findings show the strength of the scorer-independent conversion curve as a tool to compare results reported in AU or %T by different laboratories.

Induction of γ-H2AX foci in human exfoliated buccal cells after in vitro exposure to ionising radiation

Purpose: To test the γ-H2AX (Histone 2AX phosphorylation of serine 139) foci assay for the detection of ionising radiation-induced DNA damage in buccal exfoliated cells. Materials and methods: Buccal mucosa cells from five individuals (three females, two males, aged 26–47 years) were exposed to 0, 0.5, 1, 2 and 4 Gy of gamma-rays. DNA damage and DNA damage removal were measured using the γ-H2AX foci assay. Lymphocytes from one donor and the nuclear antigen H2B were used as a positive control to test the staining protocol. Results: In the absence of radiation exposure, no significant differences for both H2B and γ-H2AX signals were detected when comparing buccal cells and lymphocytes. The γ-H2AX foci rate per cell in non-irradiated buccal cells was 0.08 ± 0.02. The number of γ-H2AX foci increased linearly with ionising radiation dose in the interval from 0–4 Gy, and reached a foci rate per cell of 0.82 ± 0.22 at 4 Gy. Incubation experiments after in vitro gamma irradiation revealed that the number of γ-H2AX foci did not show a significant decrease 5 h post exposure under the experimental conditions used. Conclusion: Data suggest that it is possible to apply the γ-H2AX foci assay for the detection of ionising radiation-induced DNA damage in buccal exfoliated cells. The low removal of ionising radiation induced γ-H2AX foci in buccal cells is a potential advantage for a biological dosimetry application.

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA)

Abstract Purpose: Two quality controlled inter-laboratory exercises were organized within the EU project ‘Realizing the European Network of Biodosimetry (RENEB)’ to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. Materials and methods: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. Results: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. Conclusions: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners.

Automatic analysis of silver-stained comets by CellProfiler software.

The comet assay is one of the most widely used methods to evaluate DNA damage and repair in eukaryotic cells. The comets can be measured by software, in a semi-automatic or automatic process. In this paper, we apply the CellProfiler open-source software for automatic analysis of comets from digitized images, reporting the percentage of tail DNA. A side-by-side comparison of CellProfiler with CASP software demonstrated good agreement between the two packages. Our work demonstrates that automatic measurement of silver-stained comets with open-source software is possible, providing significant time savings.

Prematurely Condensed Chromosome Rings after Neutron Irradiation of Human Lymphocytes

Calibration curves for fission spectrum neutrons and other high LET radiations are scarce in cytogenetic dosimetry and particularly for Prematurely Condensed Chromosome Rings (PCC-ring). Here we analyzed the behavior of the PCC-ring frequency and PCC index after neutron irradiation in a broad dose interval from 1 to 26 Gy. PCC-rings were induced in lymphocytes with Calyculin A. 6455 PCC cells in G1, G2/M and M/A stages were analyzed. The best fitting between the frequency of PCC ring (Y) and the Dose (D) was obtained with the equation Y = (0.059 ± 0.003) D. The saturation of the PCC-ring was observed after around 4 Gy, but it was still possible to analyze cells exposed up to 26 Gy. The distribution of rings by cell follows Poisson or Neyman type distribution for all doses. This PCC-ring dose effect curve can be used in case of accidental overexposure to neutron radiation, allowing a dose assessment in a reliable way. Additionally, the PCC index seems to be well correlated with radiation dose and decrease in a dose dependent manner from 13% in non exposed sample down to 0.2%. This observation allows the possibility to perform a quick classification of victims exposed to high doses of both gamma and neutron radiations. The PCC assay can then be used for both neutron dose estimation up to 4 Gy and for the rapid classification of victims exposed to higher doses. This assay could be included in the multiparametric approach developed to optimize the medical treatment of radiation victims.

Biodosimetry estimation using the ratio of the longest:shortest length in the premature chromosome condensation (PCC) method applying autocapture and automatic image analysis

The combination of automatic image acquisition and automatic image analysis of premature chromosome condensation (PCC) spreads was tested as a rapid biodosimeter protocol. Human peripheral lymphocytes were irradiated with 60Co gamma rays in a single dose of between 1 and 20 Gy, stimulated with phytohaemaglutinin and incubated for 48 h, division blocked with Colcemid, and PCC-induced by Calyculin A. Images of chromosome spreads were captured and analysed automatically by combining the Metafer 4 and CellProfiler platforms. Automatic measurement of chromosome lengths allows the calculation of the length ratio (LR) of the longest and the shortest piece that can be used for dose estimation since this ratio is correlated with ionizing radiation dose. The LR of the longest and the shortest chromosome pieces showed the best goodness-of-fit to a linear model in the dose interval tested. The application of the automatic analysis increases the potential use of the PCC method for triage in the event of massive radiation causalities.