- Email: [email protected]
Dose-response relationship of γ-H2AX foci induction in human lymphocytes after X-rays exposure
Radiation Measurements 46 (2011) 997e999
Contents lists available at ScienceDirect
Radiation Measurements journal homepage: www.elsevier.com/locate/radmeas
Dose-response relationship of g-H2AX foci induction in human lymphocytes after X-rays exposure Tania Mandina a, Sandrine H. Roch-Lefèvre b, Pascale Voisin b, Jorge E. González a, Ana I. Lamadrid a, Ivonne Romero a, Omar García a, *, Philippe Voisin b, Laurence Roy b a b
Centro de Protección e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana, Cuba Institut de Radioprotection et de Sûreté Nucléaire (IRSN), DRPH, SRBE, LDB, BP17, 92262 Fontenay-aux-Roses, France
a b s t r a c t Keywords: g-H2AX foci X-ray Dose-response Repair kinetic Blood lymphocytes
Biological dosimeters are recommended for dose estimation in case of human overexposure to ionising radiation. Rapid measurement of g-H2AX foci as a marker of DNA double-strand breaks (DSB) induction has been recently tested with this purpose. Here we reported a dose-response relationship after X-ray irradiation at different times post-exposure. Blood samples were obtained from several healthy donors and exposed to doses between 0 and 2 Gy. After irradiation, blood samples were incubated at 37 C during 0.5 h, 5 h, and 8 h. Scoring of cells and g-H2AX foci was performed by software. The dose-response curves for different incubation times were as follows: Y(0.5h) ¼ 11.66D þ 0.15 (R2 ¼ 0.99), Y(5h) ¼ 2.44D þ 0.15 (R2 ¼ 0.99), Y(8h) ¼ 1.57D þ 0.22 (R2 ¼ 0.99). At 0.5 h post-exposure, the dose-response relationship for X-irradiated lymphocytes was similar to the one obtained after gamma-irradiation using the same protocol. On the other hand, the results were not similar after 8 h due to different kinetics after gamma- and X-irradiation. Our results confirm the possibilities of using g-H2AX foci method for dose estimation in a period from 0.5 h up to 8 h post X-irradiation and support the hypothesis of differences in g-H2AX foci kinetics after gamma- and X-irradiation in vitro. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Biological dosimetry for assessment of individuals accidentally overexposed to ionising radiation is based on the use of several biological methods (IAEA, 2001). Recently, it has been recommended to add the g-H2AX assay as another endpoint for population triage (Roy et al., 2007; Andrievsky and Wilkins, 2009; Riecke and Meineke, 2010; Roch-Lefèvre et al., 2010). The methodology allows the detection of the phosphorylated form of H2AX histone variant, known as g-H2AX foci as a response to DNA double-strand break (DSB) formation few minutes after exposure of cells to ionising radiation (Rogakou et al., 1999). g-H2AX foci response after in vitro, in vivo or ex vivo exposure of human lymphocytes to a broad dose range of ionising radiation has also been studied showing a linear doseresponse relationship (Löbrich et al., 2005; Rothkamm et al., 2007; Sak et al., 2007; Andrievsky and Wilkins, 2009; Redon et al., 2009; Roch-Lefèvre et al., 2010). The signal can be monitored in lymphocytes isolated from human blood samples until several hours after irradiation and incubation of samples at 37 C. g-H2AX foci can be
scored by visual (Löbrich et al., 2005; Rothkamm et al., 2007; Redon et al., 2009; Sak et al., 2007) or semi-automatic analysis (RochLefèvre et al., 2010). The foci disappearance after radiation exposure is a limiting factor of the g-H2AX assay that can be partially overcame by establishing dose-response relationship at different times post-exposure (Redon et al., 2009; Roch-Lefèvre et al., 2010). Furthermore, some differences in g-H2AX foci kinetics after in vitro gamma- and X-irradiation have been reported (Beels et al., 2010). Here we report the dose-response relationship in X-irradiated lymphocytes after post-exposure times of 0.5 h, 5 h and 8 h. We also compare the kinetics of foci induction and disappearance after X- and gamma- irradiation at 0.5 h and 8 h post-exposure. Our results confirm the possibilities of using g-H2AX foci method for dose estimation after different times post X-irradiation. Kinetics of foci disappearance supports the hypothesis of differences in g-H2AX foci kinetics after gamma- and X-irradiation in vitro. 2. Materials and methods 2.1. Donors, sample collection and irradiation
* Corresponding author. Fax: þ537 203 0165. E-mail address: [email protected] (O. García). 1350-4487/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.radmeas.2011.05.014
Irradiation of samples with X-rays was performed with the blood of five healthy voluntary donors. Three females D1, D2, D3
998
T. Mandina et al. / Radiation Measurements 46 (2011) 997e999
if p < 0.05) on slope, intercept and individual doses were used to compare dose-response curves obtained in the present study with those previously obtained by our team with gamma-rays (RochLefèvre et al., 2010). The comparison between the two curves was based on the use of the same protocol, the same software for semiautomatic foci counting and the similar results obtained in foci background from different donors (Roch-Lefèvre et al., (2010). Furthermore, similar foci frequencies were obtained for different donors after several doses of gamma-irradiation at 0.5 h postexposure incubation time (data not shown).
(24, 38, 58 years old respectively) and 2 males D4 and D5 (24 and 34 years old respectively) participated in our study. Informed consent of each donor accomplished Cuban Public Health Law No. 41. Blood samples were collected by vein puncture in tubes containing lithium heparin. 1 ml was used for dose irradiation. The blood was exposed to radiation using a Pantak 160 X-rays generator 100 kVp, 10 mA, with 3.4 mm Al and 4.8 mm polymethylmethacrylate (PMMA), 0.14 Gy/min. Doses of 0, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 Gy were used followed by incubation of blood (see below) for 0.5 h; while for 5 h and 8 h post-irradiation incubation the doses used were 0, 0.5, 1 and 2 Gy. Dose-response curve at 0.5 h was performed with blood samples from donors D1, D2, D3, D4 and for 5 h and 8 h with samples from donors D1, D2 and D5. Detailed information about g-irradiation conditions and donors is provided by Roch-Lefèvre et al. (2010). Briefly, the blood was split into several aliquots of 1 ml in dry tubes and irradiated at room temperature with a cobalt-60 source (ICO4000, IRSN, FRANCE) at a dose rate of 0.5 Gy/min. For each data point in the dose-response curves, at least 1000 cells from at least three different individuals were scored.
3. Results and discussion The range of basal values of g-H2AX foci for the 5 monitored subjects were similar (between 0.03 and 0.16, Mean SD: 0.1 0.07) to values previously reported (Löbrich et al., 2005; Scherthan et al., 2008; Roch-Lefèvre et al., 2010). In treated samples we observed a linear increase of the g-H2AX foci number with the X-ray dose for all post-irradiation incubation times used (See Fig. 1A, B). The equations obtained were as follows, Y(0.5h) ¼ 11.66D þ 0.15 (R2 ¼ 0.99), Y(5h) ¼ 2.44D þ 0.15 (R2 ¼ 0.99), Y(8h) ¼ 1.57D þ 0.22 (R2 ¼ 0.99). We found a mean yield of g-H2AX foci after X-irradiation and 0.5 h of post incubation of 11.66 foci/cell/Gy, very close to the previous reported 10.7 foci/cell/Gy for gamma-rays (Roch-Lefèvre et al., 2010). Other reported mean values of foci/cell/Gy for human lymphocytes are 14.7 (Rothkamm et al., 2007) and 9 (Scherthan et al., 2008) for X-irradiation and 12.6 for gamma-rays (Redon et al., 2009). Therefore, despite the possible differences in protocols applied in different laboratories a good agreement can be observed, a fact that is important for the applicability of the g-H2AX assay in biological dosimetry. Fig. 2 shows the dose-response curves correlating the number of g-H2AX foci/cell in relation with dose in blood samples incubated during 0.5 h and 8 h at 37 C after gamma- and X-rays. Dose-response relationship for X-ray at 0.5 h post-irradiation incubation time was not significantly different (p > 0.05, Student’s t test) from the relationship obtained by our team after gamma-irradiation, using the same protocol and the same post-irradiation incubation time (RochLefèvre et al., 2010). On the other hand, after 8 h post-irradiation the difference between gamma- and X-curve was significant (p < 0.05, Student’s t test). This can be explained by differences in the kinetic of disappearance of g-H2AX foci. Using X-rays, after 8 h the mean foci frequency was similar to gamma-rays at 0.5 Gy but remained higher for 1 Gy and 2 Gy than the values obtained for gamma-irradiation. The foci yield is linearly dependent of dose, consequently the kinetics of foci repair may be different at low and high doses as the number of foci to be repaired are different.
2.2. Isolation of lymphocytes, g-H2AX immunostaining and analysis The protocol described by Roch-Lefèvre et al., for immunostaining and foci analysis was used here (Roch-Lefèvre et al., 2010). Briefly, after the irradiation, the blood was diluted 1:2 with pre-warmed Roswell Park Institute medium (RPMI, Invitrogen, USA) and incubated at 37 C for, 0.5 h, 5 h and 8 h in order to obtain kinetics. Then lymphocytes were isolated using 1 ml of FicollePaque (GE Healthcare, UK), washed with phosphate buffer saline (PBS), and fixed with 2% paraformaldehyde (PFA, SigmaeAldrich, USA). Cells were centrifuged on slides and immunostained using a primary monoclonal g-H2AX antibody (Upstate-Millipore, USA) and a FITC conjugated goat anti-mouse secondary antibody (SigmaeAldrich, USA). Image analysis was performed using HistolabÔ and CartographÔ software. Parameters for focus detection and analysis were maintained constant during all the study. At least 100 cells by dose point were analysed. 2.3. Statistical analysis
14 13 12 11
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gamma-H2AX foci per cell
gamma-H2AX foci per cell
Mean values and standard errors were determined at each experimental point for dose-response curves elaboration. A regression analysis was used to analyse the adjustment of points to a linear trend. To evaluate kinetics of foci disappearance after gamma- and X-irradiation a Student’s t tests (significant differences
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Fig. 1. Dose-dependent induction of gamma-H2AX foci per cell in human lymphocytes isolated from irradiated blood incubated at 37 C during different times after X-irradiation. Left: 0.5 h (6), right: 5 h (B) and 8 h (6). Errors bars represent standard errors of the mean.
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Fig. 2. Dose-response curves showing the number of gamma-H2AX foci per cell in relation with dose in human lymphocytes isolated from blood samples incubated during 0.5 h (left) and 8 h (right) at 37 C after gamma (:) and X-rays exposure (6). Dose-response curve for gamma-rays irradiation was reproduced from Roch-Lefèvre et al. (2010).
Differences in repair kinetics after in vitro irradiation of whole blood with doses of 200 mGy of X- and gamma-irradiation have been reported recently (Beels et al., 2010). A persistence of g-H2AX foci after X-irradiation has been attributed to a decrease repair capacity of irradiated cells, when MRC-5 human fibroblasts were the target cells (Rothkamm and Löbrich, 2003). However, it seems that this is not the situation in the present study neither in the report from Beels et al. (2010). So presumably the most plausible explanation for this difference should be attributable to the differences in energy and track length of secondary particles from X-rays in comparison with gamma-rays resulting in higher local electron density for X-rays as already suggested (Beels et al., 2010). Nevertheless, this situation may have no implication during in vivo irradiation at low doses, since it has been shown normal individuals repair DSB to background levels after computed tomography examinations (Löbrich et al., 2005). 4. Conclusions In the present work we report for the first time ex vivo doseresponse curves for human lymphocytes in the interval of 0.5e8 h after 100 kV X-rays exposure of blood samples in a dose range from 0 to 2 Gy. We also first independently confirmed the previous finding about differences in repair of foci kinetics after irradiation of whole blood with X- and gamma-irradiation. Our results confirm the possibilities of using g-H2AX foci yield in a period from 0.5 h up to 8 h post X-irradiation for dose estimation purposes and support the hypothesis of differences in g-H2AX foci kinetics disappearance after gamma- and X-irradiation ex vivo. Acknowledgements This work was supported by the Institut de Radioprotection et de Sûreté Nucléaire (IRSN, France). Thanks to José A. Tamayo for technical assistance during X-irradiation.
References Andrievski, A., Wilkins, R.C., 2009. The response of g-H2AX in human lymphocytes and lymphocytes subsets measured in whole blood cultures. Int. J. Radiat. Biol. 85, 369e376. Beels, L., Werbrouck, J., Hubert, Th., 2010. Dose response and repair kinetics of c-H2AX foci induced by in vitro irradiation of whole blood and T-lymphocytes with X- and g-radiation. Int. J. Radiat. Biol. 86, 760e768. IAEA, 2001. Cytogenetic Analysis for Radiation Dose Assessment: A Manual Vienna, 127. Löbrich, M., Rief, N., Kühne, M., Heckmann, M., Fleckenstein, J., Rübe, Ch., Uder, M., 2005. In vivo formation and repair of DNA double-strand breaks after computed tomography examinations. Proc. Natl. Acad. Sci. USA 102, 8984e8989. Redon, Ch.E., Dickey, J.S., Bonner, W.M., Sedelnikova, O.A., 2009. g-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Adv. Space Res. 43, 1171e1178. Riecke, A., Ruf, Ch.G., Meineke, V., 2010. Assessment of radiation damage-the need for a multiparametric and integrative approach with the help of both clinical and biological dosimetry. Health Phys. 98, 160e167. Roch-Lefèvre, S.H., Mandina, T., Voisin, Pa., Gruel, G., González, J.E., Alves Valente, M., Bonnesoeur, P., García, O., Voisin, Ph., Roy, L., 2010. Quantification of g-H2AX foci in human lymphocytes: a method for biological dosimetry after ionizing radiation exposure. Rad. Res. 174, 185e194. Rogakou, E.P., Boon, C.H., Redon, C.H., Bonner, W.M., 1999. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905e915. Rothkamm, K., Löbrich, M., 2003. Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc. Natl. Acad. Sci. USA 100, 5057e5062. Rothkamm, K., Balroop, Sh., Shekhdar, J., Fernie, P., Goh, V., 2007. Leukocyte DNA damage alter multi-detector row CT: a quantitative biomarker of low-level radiation exposure. Radiology 242, 244e251. Roy, L., Roch-Lefèvre, S., Vaurijoux, A., Voisin, Pa., Martin, C., Grégoire, E., Voisin, Ph., 2007. Optimization of cytogenetic procedures for population triage in case of radiological emergency. Radiat. Meas. 42, 1143e1146. Sak, A., Grehl, S., Erichsen, P., Engelhard, M., Granna, A., Levegrün, S., Pöttgen, Ch., Groneberg, M., Stuschke, M., 2007. g-H2AX foci formation in peripheral blood lymphocytes of tumor patients after local radiotherapy to different sites of the body: dependence on the dose-distribution, irradiated site and time from start of treatment. Int. J. Radiat. Biol. 83, 639e652. Scherthan, H., Hieber, L., Braselmann, H., Meineke, V., Zitzelsberger, H., 2008. Accumulation of DSBs in g-H2AX domains fuel chromosomal aberrations. Biochem. Biophys. Res. Commun. 371, 694e697.
Contents lists available at ScienceDirect
Radiation Measurements journal homepage: www.elsevier.com/locate/radmeas
Dose-response relationship of g-H2AX foci induction in human lymphocytes after X-rays exposure Tania Mandina a, Sandrine H. Roch-Lefèvre b, Pascale Voisin b, Jorge E. González a, Ana I. Lamadrid a, Ivonne Romero a, Omar García a, *, Philippe Voisin b, Laurence Roy b a b
Centro de Protección e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana, Cuba Institut de Radioprotection et de Sûreté Nucléaire (IRSN), DRPH, SRBE, LDB, BP17, 92262 Fontenay-aux-Roses, France
a b s t r a c t Keywords: g-H2AX foci X-ray Dose-response Repair kinetic Blood lymphocytes
Biological dosimeters are recommended for dose estimation in case of human overexposure to ionising radiation. Rapid measurement of g-H2AX foci as a marker of DNA double-strand breaks (DSB) induction has been recently tested with this purpose. Here we reported a dose-response relationship after X-ray irradiation at different times post-exposure. Blood samples were obtained from several healthy donors and exposed to doses between 0 and 2 Gy. After irradiation, blood samples were incubated at 37 C during 0.5 h, 5 h, and 8 h. Scoring of cells and g-H2AX foci was performed by software. The dose-response curves for different incubation times were as follows: Y(0.5h) ¼ 11.66D þ 0.15 (R2 ¼ 0.99), Y(5h) ¼ 2.44D þ 0.15 (R2 ¼ 0.99), Y(8h) ¼ 1.57D þ 0.22 (R2 ¼ 0.99). At 0.5 h post-exposure, the dose-response relationship for X-irradiated lymphocytes was similar to the one obtained after gamma-irradiation using the same protocol. On the other hand, the results were not similar after 8 h due to different kinetics after gamma- and X-irradiation. Our results confirm the possibilities of using g-H2AX foci method for dose estimation in a period from 0.5 h up to 8 h post X-irradiation and support the hypothesis of differences in g-H2AX foci kinetics after gamma- and X-irradiation in vitro. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Biological dosimetry for assessment of individuals accidentally overexposed to ionising radiation is based on the use of several biological methods (IAEA, 2001). Recently, it has been recommended to add the g-H2AX assay as another endpoint for population triage (Roy et al., 2007; Andrievsky and Wilkins, 2009; Riecke and Meineke, 2010; Roch-Lefèvre et al., 2010). The methodology allows the detection of the phosphorylated form of H2AX histone variant, known as g-H2AX foci as a response to DNA double-strand break (DSB) formation few minutes after exposure of cells to ionising radiation (Rogakou et al., 1999). g-H2AX foci response after in vitro, in vivo or ex vivo exposure of human lymphocytes to a broad dose range of ionising radiation has also been studied showing a linear doseresponse relationship (Löbrich et al., 2005; Rothkamm et al., 2007; Sak et al., 2007; Andrievsky and Wilkins, 2009; Redon et al., 2009; Roch-Lefèvre et al., 2010). The signal can be monitored in lymphocytes isolated from human blood samples until several hours after irradiation and incubation of samples at 37 C. g-H2AX foci can be
scored by visual (Löbrich et al., 2005; Rothkamm et al., 2007; Redon et al., 2009; Sak et al., 2007) or semi-automatic analysis (RochLefèvre et al., 2010). The foci disappearance after radiation exposure is a limiting factor of the g-H2AX assay that can be partially overcame by establishing dose-response relationship at different times post-exposure (Redon et al., 2009; Roch-Lefèvre et al., 2010). Furthermore, some differences in g-H2AX foci kinetics after in vitro gamma- and X-irradiation have been reported (Beels et al., 2010). Here we report the dose-response relationship in X-irradiated lymphocytes after post-exposure times of 0.5 h, 5 h and 8 h. We also compare the kinetics of foci induction and disappearance after X- and gamma- irradiation at 0.5 h and 8 h post-exposure. Our results confirm the possibilities of using g-H2AX foci method for dose estimation after different times post X-irradiation. Kinetics of foci disappearance supports the hypothesis of differences in g-H2AX foci kinetics after gamma- and X-irradiation in vitro. 2. Materials and methods 2.1. Donors, sample collection and irradiation
* Corresponding author. Fax: þ537 203 0165. E-mail address: [email protected] (O. García). 1350-4487/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.radmeas.2011.05.014
Irradiation of samples with X-rays was performed with the blood of five healthy voluntary donors. Three females D1, D2, D3
998
T. Mandina et al. / Radiation Measurements 46 (2011) 997e999
if p < 0.05) on slope, intercept and individual doses were used to compare dose-response curves obtained in the present study with those previously obtained by our team with gamma-rays (RochLefèvre et al., 2010). The comparison between the two curves was based on the use of the same protocol, the same software for semiautomatic foci counting and the similar results obtained in foci background from different donors (Roch-Lefèvre et al., (2010). Furthermore, similar foci frequencies were obtained for different donors after several doses of gamma-irradiation at 0.5 h postexposure incubation time (data not shown).
(24, 38, 58 years old respectively) and 2 males D4 and D5 (24 and 34 years old respectively) participated in our study. Informed consent of each donor accomplished Cuban Public Health Law No. 41. Blood samples were collected by vein puncture in tubes containing lithium heparin. 1 ml was used for dose irradiation. The blood was exposed to radiation using a Pantak 160 X-rays generator 100 kVp, 10 mA, with 3.4 mm Al and 4.8 mm polymethylmethacrylate (PMMA), 0.14 Gy/min. Doses of 0, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 Gy were used followed by incubation of blood (see below) for 0.5 h; while for 5 h and 8 h post-irradiation incubation the doses used were 0, 0.5, 1 and 2 Gy. Dose-response curve at 0.5 h was performed with blood samples from donors D1, D2, D3, D4 and for 5 h and 8 h with samples from donors D1, D2 and D5. Detailed information about g-irradiation conditions and donors is provided by Roch-Lefèvre et al. (2010). Briefly, the blood was split into several aliquots of 1 ml in dry tubes and irradiated at room temperature with a cobalt-60 source (ICO4000, IRSN, FRANCE) at a dose rate of 0.5 Gy/min. For each data point in the dose-response curves, at least 1000 cells from at least three different individuals were scored.
3. Results and discussion The range of basal values of g-H2AX foci for the 5 monitored subjects were similar (between 0.03 and 0.16, Mean SD: 0.1 0.07) to values previously reported (Löbrich et al., 2005; Scherthan et al., 2008; Roch-Lefèvre et al., 2010). In treated samples we observed a linear increase of the g-H2AX foci number with the X-ray dose for all post-irradiation incubation times used (See Fig. 1A, B). The equations obtained were as follows, Y(0.5h) ¼ 11.66D þ 0.15 (R2 ¼ 0.99), Y(5h) ¼ 2.44D þ 0.15 (R2 ¼ 0.99), Y(8h) ¼ 1.57D þ 0.22 (R2 ¼ 0.99). We found a mean yield of g-H2AX foci after X-irradiation and 0.5 h of post incubation of 11.66 foci/cell/Gy, very close to the previous reported 10.7 foci/cell/Gy for gamma-rays (Roch-Lefèvre et al., 2010). Other reported mean values of foci/cell/Gy for human lymphocytes are 14.7 (Rothkamm et al., 2007) and 9 (Scherthan et al., 2008) for X-irradiation and 12.6 for gamma-rays (Redon et al., 2009). Therefore, despite the possible differences in protocols applied in different laboratories a good agreement can be observed, a fact that is important for the applicability of the g-H2AX assay in biological dosimetry. Fig. 2 shows the dose-response curves correlating the number of g-H2AX foci/cell in relation with dose in blood samples incubated during 0.5 h and 8 h at 37 C after gamma- and X-rays. Dose-response relationship for X-ray at 0.5 h post-irradiation incubation time was not significantly different (p > 0.05, Student’s t test) from the relationship obtained by our team after gamma-irradiation, using the same protocol and the same post-irradiation incubation time (RochLefèvre et al., 2010). On the other hand, after 8 h post-irradiation the difference between gamma- and X-curve was significant (p < 0.05, Student’s t test). This can be explained by differences in the kinetic of disappearance of g-H2AX foci. Using X-rays, after 8 h the mean foci frequency was similar to gamma-rays at 0.5 Gy but remained higher for 1 Gy and 2 Gy than the values obtained for gamma-irradiation. The foci yield is linearly dependent of dose, consequently the kinetics of foci repair may be different at low and high doses as the number of foci to be repaired are different.
2.2. Isolation of lymphocytes, g-H2AX immunostaining and analysis The protocol described by Roch-Lefèvre et al., for immunostaining and foci analysis was used here (Roch-Lefèvre et al., 2010). Briefly, after the irradiation, the blood was diluted 1:2 with pre-warmed Roswell Park Institute medium (RPMI, Invitrogen, USA) and incubated at 37 C for, 0.5 h, 5 h and 8 h in order to obtain kinetics. Then lymphocytes were isolated using 1 ml of FicollePaque (GE Healthcare, UK), washed with phosphate buffer saline (PBS), and fixed with 2% paraformaldehyde (PFA, SigmaeAldrich, USA). Cells were centrifuged on slides and immunostained using a primary monoclonal g-H2AX antibody (Upstate-Millipore, USA) and a FITC conjugated goat anti-mouse secondary antibody (SigmaeAldrich, USA). Image analysis was performed using HistolabÔ and CartographÔ software. Parameters for focus detection and analysis were maintained constant during all the study. At least 100 cells by dose point were analysed. 2.3. Statistical analysis
14 13 12 11
6
gamma-H2AX foci per cell
gamma-H2AX foci per cell
Mean values and standard errors were determined at each experimental point for dose-response curves elaboration. A regression analysis was used to analyse the adjustment of points to a linear trend. To evaluate kinetics of foci disappearance after gamma- and X-irradiation a Student’s t tests (significant differences
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Fig. 1. Dose-dependent induction of gamma-H2AX foci per cell in human lymphocytes isolated from irradiated blood incubated at 37 C during different times after X-irradiation. Left: 0.5 h (6), right: 5 h (B) and 8 h (6). Errors bars represent standard errors of the mean.
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Fig. 2. Dose-response curves showing the number of gamma-H2AX foci per cell in relation with dose in human lymphocytes isolated from blood samples incubated during 0.5 h (left) and 8 h (right) at 37 C after gamma (:) and X-rays exposure (6). Dose-response curve for gamma-rays irradiation was reproduced from Roch-Lefèvre et al. (2010).
Differences in repair kinetics after in vitro irradiation of whole blood with doses of 200 mGy of X- and gamma-irradiation have been reported recently (Beels et al., 2010). A persistence of g-H2AX foci after X-irradiation has been attributed to a decrease repair capacity of irradiated cells, when MRC-5 human fibroblasts were the target cells (Rothkamm and Löbrich, 2003). However, it seems that this is not the situation in the present study neither in the report from Beels et al. (2010). So presumably the most plausible explanation for this difference should be attributable to the differences in energy and track length of secondary particles from X-rays in comparison with gamma-rays resulting in higher local electron density for X-rays as already suggested (Beels et al., 2010). Nevertheless, this situation may have no implication during in vivo irradiation at low doses, since it has been shown normal individuals repair DSB to background levels after computed tomography examinations (Löbrich et al., 2005). 4. Conclusions In the present work we report for the first time ex vivo doseresponse curves for human lymphocytes in the interval of 0.5e8 h after 100 kV X-rays exposure of blood samples in a dose range from 0 to 2 Gy. We also first independently confirmed the previous finding about differences in repair of foci kinetics after irradiation of whole blood with X- and gamma-irradiation. Our results confirm the possibilities of using g-H2AX foci yield in a period from 0.5 h up to 8 h post X-irradiation for dose estimation purposes and support the hypothesis of differences in g-H2AX foci kinetics disappearance after gamma- and X-irradiation ex vivo. Acknowledgements This work was supported by the Institut de Radioprotection et de Sûreté Nucléaire (IRSN, France). Thanks to José A. Tamayo for technical assistance during X-irradiation.
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