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We previously used the γ-H2AX assay as a biodosimeter for total-body-irradiation (TBI) exposure (γ-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant γ-H2AX responses days after total-body exposure to 1–8.5 Gy (60Co γ-rays at 55 cGy min−1). Here, we introduce a partial-body exposure analysis method, Qγ-H2AX, which is based on the number of γ-H2AX foci per damaged cells as evident by having one or more γ-H2AX foci per cell. Results from the rhesus monkey – TBI study were used to establish Qγ-H2AX dose-response calibration curves to assess acute partial-body exposures. γ-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay’s utility for dose assessment in various body regions. The quantitation of γ-H2AX may provide a robust biodosimeter for analyzing partial body exposures to ionizing radiation in humans.
Measurement of in vivo DNA damage responses to various treatments is providing useful information to clinicians for improving human health. Hundreds of H2AX molecules are rapidly phosphorylated on serine 139 (γ-H2AX) in response to the formation of each DNA double-strand break (DSB) (Rogakou et al., 1999; Rogakou et al., 1998). The most sensitive assays of γ-H2AX utilize immunocyto- and immunohisto-chemistry techniques to stain for γ-H2AX foci, intranuclear structures composed of the hundreds of γ-H2AX molecules, which form at each DSB site in cells and tissues (Bonner et al., 2008). Exposure of humans to ionizing radiation during various radiological diagnostic or therapeutic treatments (Kuefner et al., 2009; Lobrich et al., 2005; Porcedda et al., 2006; Sak et al., 2007) and space travel (Ohnishi et al., 2009) lead to DSB formation that can be measured by counting the γ-H2AX foci in many tissues including lymphocytes, buccal cells, and skin biopsies; see reviews by (Redon et al., 2010b; Rothkamm and Horn, 2009).
Thus, counting γ-H2AX foci in cells of persons exposed to irradiation may be useful in determining the extent of exposure and in providing optimum medical care. In addition to radiation exposure from medical procedures and/or diagnostics, the risk of accidental irradiation to the general public and emergency responders has also increased in recent years. We recently utilized a non-human primate (NHP) model to further validate the γ-H2AX assay following TBI with both non-lethal and lethal radiation doses (Redon et al., 2010a). Measuring γ-H2AX foci in NHP lymphocytes and plucked hairs enabled us to assess the subject’s exposure several days after irradiation at doses in the medical triage range, making γ-H2AX biodosimetry a robust tool for measuring TBI in macaques for several days after irradiation.
However, a serious consideration is that many radiation accidents are likely to involve inhomogeneous exposures or partial-body irradiation (PBI) (Prasanna et al.), the extent of which may be unknown both in terms of the fraction of the body irradiated and dose delivered to the irradiated fraction. In cases of acute exposure, the determination that a fraction of the blood volume had escaped irradiation would indicate that a portion of the body, and hence the bone marrow, may have also escaped lethal irradiation. Such knowledge may be critical for guiding decisions on optimal medical treatment for the victim, for example, whether a bone-marrow transplant is necessary for survival.
A method (Qdr) to evaluate PBI in individuals was originally demonstrated by Sasaki and Miyata (Sasaki and Miyata, 1968) utilizing the frequency of chromosomal aberrations (dicentric and ring chromosomes) in lymphocyte metaphase spreads taken from exposed subjects. It was later adapted to utilize the frequency of chromosome fragments present in lymphocytes subjected to premature chromosome condensation (Blakely et al., 1995; Prasanna, et al.). Here, we extend this method to utilize the levels of γ-H2AX foci in the lymphocyte population of the exposed subject to assess dose and fraction of body exposed to PBI.
We also briefly introduce the use of plucked hairs as a mean to assess PBI. It may take a few minutes for blood to make a complete bodily circuit, hence, the fraction of the blood volume exposed and the dose received by that fraction may differ considerably from that received by other portions of the body, depending in large part on the dose rate and length of exposure. Exposure of sufficient duration to any part of a body could lead to irradiation of the complete blood volume. However, in these cases, measurements of γ-H2AX foci levels in plucked hair bulbs could help determine the extent of partial-body exposure.
The data used to develop the method for PBI calculations were obtained from a previous study by Redon et al. (2010) using γ-H2AX foci levels in macaque lymphocytes for TBI biodosimetry (Redon, et al., 2010a). This method, based on the Sasaki and Miyata method (Sasaki and Miyata, 1968), may provide useful estimates of the fraction of the body exposed to radiation. Considering a γ-H2AX focus to denote a damaged chromosome, the term Qγ-H2AX, the mean number of γ-H2AX foci per cell in the cells containing foci, ignoring the foci-free cells, can be calculated. Similarly, we use the term Fγ-H2AX to refer to the fraction of lymphocyte population containing damaged cells. Fγ-H2AX and Qγ-H2AX can be calculated with the following formula:
∑fγ-H2AX is the total number of γ-H2AX foci in all cells observed; NγH2AX is the number of damaged cells (with at least one γ-H2AX focus); Nt, the total number of cells observed and N0 is the number of cells without foci.
We determined both Fγ-H2AX and Qγ-H2AX values vs doses from the TBI data, values that allowed us to establish standard curves (Fig. 2A and 2B). Following PBI, both experimental Qγ-H2AX and experimental Fγ-H2AX can be determined after γ-H2AX examination in lymphocytes. Both experimental Qγ-H2AX and experimental Fγ-H2AX and the standard curves obtained from TBI will allow the estimate of the irradiation dose and of the fraction of the body that was exposed.
As an example, shown graphically in Fig. 2A and 2B and mathematically here, we consider a hypothetical case in which a blood sample collected 2 days after a possible PBI was found to yield the experimental values Qγ-H2AX =2 and Fγ-H2AX = 0.40. Using the standard curve for 2 days after TBI in Fig. 2A, the value of Qγ-H2AX yields an estimated dose of ~4.4 Gy to the exposed portion of the body. In Fig. 2B, a dose of 4.4-Gy TBI would yield an Fγ-H2AX value after 2 days of 0.63, considerably greater than the value in the hypothetical example of 0.4, a difference that would indicate PBI. PBI can be calculated by the ratios after subtracting the background values as follows:
Plucked hairs preparation for immunohistochemistry and γ-H2AX detection were performed as previously described (Redon, et al., 2010a).
The method utilizing γ-H2AX foci distribution frequencies is modeled for TBI and simulated PBI in Fig. 1A. In an idealized acute PBI scenario, the lymphocyte population would have two components, one from the irradiated fraction exhibiting cells with foci (Fig 1A, blue line), and the other from the unirradiated fraction exhibiting cells with few if any foci (Fig 1A, red line). The fraction of each of these two components represented in the lymphocyte population would depend on the fraction of the blood volume irradiated. Figure 1B presents two modeled situations, one with 80% PBI (80% of cells from TBI samples and 20% of cells from unirradiated samples) and 20% PBI (20% of cells from TBI samples and 80% of cells from unirradiated samples). By first focusing on the damaged cell component of the TBI data, the number of foci per damaged cell, named Qγ-H2AX after the Sasaki and Miyata method, can be calculated. This term gives information on the dose received by the exposed body portion, largely independent of the fraction of the body exposed (Fig 2A). Second, the fraction of total cells with foci, Fγ-H2AX, when used in conjunction with Fγ-H2AX calibration curves determined for TBI, gives information on the fraction of the body exposed (Fig 2B). Calibration curves for the application of the Qγ-H2AX and Fγ-H2AX analysis method for the case of TBI are illustrated in Figure 2A and 2B.
Results from an illustrative example, as described in the Methods paragraph above, is shown graphically utilizing the TBI calibration graphs for Qγ-H2AX and Fγ-H2AX (Figs. 2A and 2B). In this example, blood drawn from a putative acute PBI victim 2 days after exposure yielded values for Qγ-H2AX of 2 foci per cell, and a Fγ-H2AX of 0.4, which is significantly less than that a TBI value of 0.63. The resulting estimate from these input values is that 59% of the body received ~4.4 Gy, and more importantly, 41% of the body was not exposed to radiation (see Materials and Methods paragraph for details of the calculation). To check for internal consistency, computer PBI simulations of γ-H2AX foci distributions for 5, 12.5, 33, 55, 80 and 95% of total-body exposure to doses of 1, 3.5 6.5 and 8.5 Gy at 0.3, 1, 2 and 4 days post exposure were prepared as modeled in Fig. 3A. Fγ-H2AX values were calculated for the simulated γ-H2AX foci distributions and plotted vs the input PBI values (Fig. 3). When the result of the hypothetical example (59% of total body exposed to ~4.4 Gy) is plotted in the “2 d post-irradiation” panel (Fig. 3, 3rd panel from top), an Fγ-H2AX value of 0.4–0.45 is obtained, in good agreement with the input value of 0.40. The 41% of the body that received little or no irradiation exposure may contain sufficient undamaged bone marrow to replenish the blood cell population without the need for a transplant.
We previously showed that plucked hairs may be a useful surrogate tissue for assessing exposures to ionizing radiation (Redon, et al., 2010a). Other studies show the use of hair follicles can be applied to monitor radiation exposure (Kyoizumi et al., 1998; Sieber et al., 1992) as well as to use γ-H2AX as a marker for DNA damage in patients undergoing chemotherapy (Fong et al., 2009). γ-H2AX residual foci in plucked hairs are dose and time dependent (Fig. 4). The incidence of γ-H2AX foci after 1 Gy-TBI is low as signals disappear after 2 days TBI. In contrast γ-H2AX signals were still detected after 2 days for 3.5 and 6.5 Gy-, and 8.5 Gy-TBI, after 9 days for 8.5 Gy-TBI.
Radiation accidents are more commonly characterized by acute partial-body or inhomogeneous rather than homogeneous TBI radiation exposures. Dose inhomogeneity can be a major confounder in dose assessment using biodosimetry (Kolanko et al., 2000; Prasanna, et al.). Two chromosome aberrations assays, metaphase-spread dicentric and ring and premature chromosome condensation or PCC, along with analysis methods, Qdr (Sasaki and Miyata, 1968) and Qpcc (Blakely, et al., 1995) respectively, for partial-body exposure assessment have been proposed for this purpose. Sak and colleagues evaluated the yield of γ-H2AX foci formation to allow estimation of the applied integral body dose after local radiotherapy to different sites of the body (Sak, et al., 2007). In 2007, a study by Rothkamm et al. described the application of a related cytogenetic data analysis method, the contaminated Poisson method by Dolphin, to detect partial body exposure using γ-H2AX-based biodosimetry in patients undergoing CT scans (Rothkamm et al., 2007). The authors observed an overdispersion of the γ-H2AX focus distribution from the Poison distributions after CT examinations that revealed a partial body exposure to radiation. Here, we introduce a partial-body analysis methodology (i.e., Qγ-H2AX, Fγ-H2AX), similar to that done for dicentrics (Qdr) and PCC (QPCC) assays, utilizing the gamma-H2AX foci assay. Utilizing γ-H2AX foci levels for these calculations has the advantage over micronuclei and damaged chromosomes of not requiring cell culturing and stimulation, since the foci are formed in the quiescent lymphocytes. Representing an alternative and rapid approach both for the assessment of dose intensity and inhomogeneity following acute ionizing radiation exposures, this approach could be useful for several practical biodosimetry applications.
However, we are aware that this method is most useful when the partial irradiation time is much less than the rate of blood circulation so that the blood pool renewal in the irradiated part of the body is limited. In addition, further analysis will be necessary to evaluate the influence of potential confounders on our method (i.e., inter-individual variations in background foci levels, gradient dose distributions rather than just 'clean' partial body irradiation, number of cells scored, response of special populations, etc). Because of the fast signal lost, we believe that Qγ-H2AX could be used in the first hours but less than a day following low irradiation doses (< 1Gy). Thus, Qγ-H2AX could be limited to high PBI doses. PBI doses higher than 3.5 Gy would result in long lasting γ-H2AX foci in irradiated lymphocytes with PBI doses ≥ 6.5 Gy resulting in a population of lymphocytes with “stable” γ-H2AX foci (non-repairable of DSBs)(Redon, et al., 2010a). With such high PBI irradiation doses, we could expect to use Qγ-H2AX for a few days after exposure (time that would also depend on the volume of irradiated body). Therefore, future studies will need to be performed to validate this model. Finally, because tissues from other parts of the body such as nails and teeth may be useful for biodosimetry (Fattibene and Callens, 2010; Romanyukha et al., 2010), their analysis together with γ-H2AX detection in other parts of the body, i.e., hairs plucked from the scalp and elsewhere, may develop into a robust tool for PBI dose assessment. Since non-clinical personnel could pluck hairs, this tissue will be well suited for large scale sample collection.
This research was supported by the NIAID Radiation/Nuclear Countermeasures Program, the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, NIH and the Armed Forces Radiobiology Research Institute under research work unit number BD-13 (RBB4AR). Views presented in this paper are those of the authors; no endorsement by the Department of Defense has been give or should be inferred. The authors gratefully acknowledge Dr. Terry C. Pellmar (Silver Spring, MD) and Dr. Andrea L. DiCarlo-Cohen (NIAID) for their efforts to enable these studies, the assistance of the AFRRI Veterinarian, Dr. Jennifer Mitchell, and her colleagues in AFRRI’s Veterinary Science Department, radiation exposure and dosimetry support from AFRRI’s dosimetrist, Dr. Vitaly Nagy, and his colleagues in AFRRI’s Radiation Science Department, professional assistance in the rhesus macaque radiation model from Drs. Natalia I. Ossetrova and Gregory L. King, and technical support by David J. Sandgren, HM2 Sergio Gallego, Katya Kranopolsky, and Yvonne Eudy. We also thank Dr. O. Sedelnikova, NCI, for critical reading of the manuscript.
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