No indications of an enhanced UV-light-induced unscheduled DNA synthesis in splenocytes of mice following a low-dose irradiation in vivo or in vitro

One of the open questions regarding the adaptive response to ionizing radiation is whether it can be induced in G₀ lymphocytes. In the majority of experiments in which an adaptive response in G₀ lymphocytes was observed, the adapting dose was applied in vivo. In order to investigate whether there is some in vivo component of adaptive response, mouse splenocytes of the C57BL/6 strain were irradiated with 0.1 Gy x-rays either in vivo or in vitro, and their UV-light-induced unscheduled DNA synthesis (UDS) levels were determined autoradiographically. An augmented UV-light-induced UDS following an adapting dose applied in vivo has previously been described by several authors in splenocytes of C57BL/6 mice, indicating that the adapting dose enhanced the DNA repair capacity of lymphocytes. In the present investigation, however, no evidence of an adaptive response could be seen regardless of whether the adapting dose was given in vivo or in vitro. Those results present a further indication for the fact that the adaptive response to ionizing radiation is not always inducible, even in lymphocytes of an inbred mouse strain in which its existence has been reported before.     

Influence of in vitro low-level gamma-radiation on the UV-induced DNA repair capacity of human lymphocytes – analysed by unscheduled DNA synthesis (UDS) and comet assay

Unscheduled DNA synthesis (UDS) induced by ultraviolet radiation (UV) was studied in human lymphocytes after exposing blood samples in vitro to doses ranging between 1 and 10 mGy gamma-radiation, by way of measuring tritiated thymidine (³H-TdR) uptake in the DNA of these lymphocytes. The results indicate that samples pre-exposed to gamma-ray doses ranging between 2.5 and 4 mGy show higher UDS levels compared with those pre-exposed to doses of less than 2.5 or more than 4 mGy. These results were verified by studying the rate of removal of UV-induced photoproducts using the comet assay. The reason for the increase in DNA repair capacity in this dose range is discussed in comparison with earlier reports on this phenomenon. The DNA repair capacity with respect to inter-individual variability and age is also analysed. The study implies that the comet assay is a simple and sensitive visual method to track nucleotide excision repair and hence can be used to estimate UV-induced DNA repair in the place of the more reliable yet cumbersome and time-consuming, grain-counting autoradiographic technique. Received: 28 April 1998 / Accepted in revised form: 1 September 1998     

Study of reparative DNA synthesis in lymphocytes of persons occupationally exposed to radiation

The UDS efficiency in lymphocytes of professionals chronically exposed to gamma/neutron radiation, as well as for a control cohort was estimated. A credible reduction of UV-induced UDS (KUV) index as compared to the control was demonstrated. This shows an invalid repair state of blood cells in professionals. As for the control cohort, the decreasing tendency of UDS index with age was found. The correlative analysis of UDS index dependence upon an absorbed dose (based on physical dosimetry data) revealed the trend towards repair index reduction along with a higher total absorbed doze, that is followed by UDS index coming onto a plateau. It was also demonstrated that after a sharp and/or accidental irradiation UDS index reduces as compared to permanent portioned irradiation. It was not observed the influence of smoking upon UDS efficiency in blood lymphocytes neither for control, nor for experimental group. The analysis of vitamin therapy of the both cohorts showed that such therapy raises UDS index of the control (i.e., non-professional) group. Besides, it was found out an additional positive effect of vitamin therapy, i.e. doze leveling in long-term perspective after irradiation. The most expressed KUV/doze correlation was characteristic for professionals, who do not take vitamins and do not smoke. This proves that in blood cells of professionals it is preserved a dependence of repair system invalidity upon the absorbed doze without similar external factors.     

In vivo and in vitro ethylene oxide exposure of human lymphocytes assessed by chemical stimulation of unscheduled DNA synthesis

Factory workers exposed to ethylene oxide (EO), 0.5–1.0 ppm in factory air, together with matched controls from the same factory, were examined for evidence of toxic exposure by measurement of unscheduled DNA synthesis (UDS) induced by N-acetoxy-2-acetylaminofluorene (NA-AAF) and of chromosome aberrations in peripheral lymphocytes.The total chromatid gaps plus breaks were significantly elevated and NA-AAF-induced UDS was significantly reduced in the EO-exposed group as compared with the unexposed control group. The NA-AAF-induced UDS values negatively correlated to the duration (yr) of EO exposure (r = ?0.45, p < 0.02) and the number of chromosome breaks (r = ?0.61, p < 0.05), indicating an inhibition in vivo of DNA-repair capacity by EO. These data were verified in vitro by biochemical and autoradiographic studies of EO-induced UDS in human blood cells. Above 2 mM EO, UDS was inhibited in lymphocytes whether they were cultured for 24 or 122 h after alkylation with EO. Even at the subtoxic EO dose of 0.1 mM, lymphocytes were sensitized to additional exposures of NA-AAF, so that cytotoxicity was increased to 40% compared with 5% for the controls even though UDS was unaffected.It is concluded that EO was toxic to lymphocytes, even when they were sensitized at non-toxic EO doses to the cytotoxic action of other mutagens (e.g. NA-AAF), and the cells that did survive above 2 mM EO were inhibited in their DNA-repair capacity as judged by reduced UDS.     

Estimation of relative biological effectiveness of tritium according to chromosome aberration frequency in human blood lymphocytes

The goal of this work was to determine the relative biological effectiveness (RBE) of tritium β-radiation according to the chromosome aberration frequency in the peripheral blood lymphocytes after in vitro and in vivo radiation exposures. The experimental RBE assessment of tritium β-radiation relative to ⁶⁰Co γ-radiation according to unstable chromosome aberration frequency in the peripheral blood lymphocytes under particular conditions is described. It has been demonstrated that tritium β-radiation is, in general, more effective in the dose range of up to 1 Gy, which is most pronounced at low doses. The RBE value of tritium β-radiation at minimum doses reached 2.2 and decreased at higher doses (1 Gy) to 1.25. The data on comparative analysis of the frequency of stable chromosome aberrations in the blood lymphocytes of professional nuclear workers (Sarov, Russia) after long-term chronic exposure to tritium β-radiation, as compared with γ-irradiation, are reported for the first time. The higher biological effectiveness of tritium β-radiation was demonstrated and was estimated as 2.5.     

Evaluation of DNA damage in a population of bats (Chiroptera) residing in an abandoned monazite mine

Ionising radiation has the ability to induce DNA damage. While the effects of high doses of radiation of short duration have been well documented, the biological effects of long-term exposure to low doses are poorly understood. This study evaluated the clastogenic effects of low dose ionising radiation on a population of bats (Chiroptera) residing in an abandoned monazite mine. Bats were sampled from two chambers in the mine, where external radiation levels measured around 20 microSv/h (low dose) and 100 microSv/h (higher dose), respectively. A control group of bats was sampled from a cave with no detectable radiation above normal background levels. The micronucleus assay was used to evaluate residual radiation damage in binucleated lymphocytes and showed that the micronucleus frequency per 500 binucleated lymphocytes was increased in the lower radiation-exposed group (17.7) and the higher radiation-exposed group (27.1) compared to the control group (5.3). This study also showed that bats exposed to radiation presented with an increased number of micronuclei per one thousand reticulocytes (2.88 and 10.75 in the lower and high radiation-exposed groups respectively) when compared to the control group (1.7). The single-cell gel electrophoresis (comet) assay was used as a means of evaluating clastogenecity of exposure to radiation at the level of individual cells. Bats exposed to radiation demonstrated increased DNA damage as shown by the length of the comet tails and showed an increase in cumulative damage. The results of the micronucleus and the comet assays indicated not only a statistically significant difference between test and control groups (P<0.001), but also a dose-dependent increase in DNA damage (P<0.001). These assays may thus be useful in evaluating the potential clastogenecity of exposure to continuous low doses of ionising radiation.     

The participation of oxygen in the process of the interphase death of T-lymphocytes following irradiation in vivo

The ability of lymphocytes to inhibit proliferation of non-syngeneic stem cells decreases differently after exposure in vivo and in vitro. The causes of the observed differences and the mechanism of radiation impairment of this function under different irradiation conditions have been investigated. Cells exposed in vivo die in the interphase irreversibly. The newly formed lymphocytes start the repair process as late as one month after irradiation. The injury to in vivo exposed cells is severer due to the presence of oxygen in tissues. A definite time interval is needed for the damaging effect of oxygen radicals to be implemented: the effect is maximum as early as 4 h following irradiation. With in vivo exposure under hypoxic conditions the functional activity of lymphocytes is the same as that of lymphocytes irradiated in vitro with the same dose. In vitro irradiation of lymphocytes at a high oxygen content causes a decrease in the functional activity of cells.     

Ultraviolet radiation rapidly induces tyrosine phosphorylation and calcium signaling in lymphocytes.

UV radiation is known to induce lymphocyte nonresponsiveness both in vitro and in vivo. We have found that UV radiation rapidly induced tyrosine phosphorylation and calcium signaling in normal human peripheral blood lymphocytes. In the leukemic T cell line Jurkat and the Burkitt's lymphoma cell line Ramos, UV rapidly induced tyrosine phosphorylation in a wavelength-dependent manner, giving strong signals after UVB and UVC, but not UVA, irradiation. Similarly, in Jurkat cells UV-induced calcium signals were dependent on the dose of UVB or UVC irradiation over a range of 150-1200 J/m2, but only a small signal was observed for UVA at a dose of 1200 J/m2. The UV-induced calcium signals were blocked by the tyrosine kinase inhibitor herbimycin A, indicating that they were dependent on tyrosine phosphorylation. Phospholipase C (PLC) gamma 1 was tyrosine phosphorylated in response to UV irradiation but to a lesser extent than observed after CD3 cross-linking. However, PLC gamma 1-associated proteins demonstrated to bind to the PLC gamma 1 SH2 domain were tyrosine phosphorylated strongly after UV irradiation. A similar dose response was observed for the inhibition by herbimycin A of UV-induced calcium signals and UV-induced tyrosine phosphorylation of PLC gamma 1 and associated proteins. We propose that in contrast to CD3/Ti stimulation, UV aberrantly triggers lymphocyte signal transduction pathways by a mechanism that bypasses normal receptor control.     

Indications of an adaptive response in C57BL mice pre-exposed in vivo to low doses of ionizing radiation

C57BL/6 mice were whole-body irradiated with 5 cGy/day (‘adapting dose’) on 4 consecutive days and their spleens removed on day 1, 3, 7, 12, 19 or 26 after the last irradiation. In vitro UV-light-induced unscheduled DNA synthesis (UDS) and mitomycin C (MMC)-induced sister-chromatid exchanges (SCEs) were scored in lymphocytes (UV-light and MMC being the ‘challenging agents’), yielding higher UDS values and lower frequencies of induced SCEs than cells of non-adapted animals. On day 12 this effect could only be seen in half, on days 19 and 26 in none of the performed experiments. The results support those published by Tuschl et al. (1980, 1983) and Liu et al. (1987), showing that it is possible to induce the adaptive response in vivo.     

Reduced DNA repair in progeria cells and effects of gamma-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33-50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by gamma-ray irradiation to the cells before UV irradiation. The dose of gamma-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, gamma-ray irradiation further reduced UDS with a lower gamma-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a 'built-in' defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by gamma-ray irradiation.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.     

Analysis of chromosome damage in circulating lymphocytes of radiological workers affected by thyroid nodules

The aim of our study was to assess whether or not thyroid nodularity in combination with occupational exposure to low levels of ionising radiation would be correlated with chromosome damage in peripheral lymphocytes. Conventional chromosome-aberration analysis was performed on a group of 92 hospital workers with or without thyroid nodules. On the basis of measurements of their exposure levels, the workers were classified into a low (mean total level=0.03 mSv), medium (mean total level=1.04 mSv) or high (mean total level=8.60 mSv) exposure category. Our results indicate that among workers with thyroid nodules, the high-exposed workers showed significantly higher levels of both total (2.35+/-0.34 per 100 cells) and chromosome-type aberrations (1.46+/-0.20 per 100 cells) than medium-exposed (0.98+/-0.42 and 0.68+/-0.25 per 100 cells, respectively) or low-exposed workers (1.11+/-0.29 and 0.58+/-0.17 per 100 cells, respectively). Workers without thyroid nodules had comparable frequencies of chromosome aberrations among the three exposure categories. To our knowledge, this is the first study revealing a slight, but significant increase of chromosome damage in peripheral lymphocytes from hospital workers who developed thyroid nodules under conditions of occupational exposure to radiation well below the threshold limit for the workplace. The existence of a possible association between chromosome aberrations and development of thyroid nodularity will be discussed.     

Diethyldithiocarbamate inhibits scheduled and unscheduled DNA synthesis of rat thymocytes in vitro and in vivo--dose-effect relationships and mechanisms of action

In vitro as well as in animal models, diethyldithiocarbamate (DDC) modifies the tumoricidal activity of some antineoplastic agents. To gain further information about the mechanism of action of DDC, we measured (i) in vitro and (ii) in vivo changes in DNA synthesis of rat thymocytes. (i) In vitro, the scheduled (SDS) and unscheduled (UDS) incorporation of [3H]thymidine ([3H]dT) into DNA of rat thymic cells were biphasically inhibited in a dose range of 1-1000 micrograms DDC/ml. The UV-induced UDS was totally suppressed by 10 and 100 micrograms DDC/ml. (ii) In vivo, 1-4 h following intraperitoneal administration of 250-1000 mg DDC per kg body wt., SDS and UDS were inhibited up to about 80% in a dose-dependent manner. Nucleoid sedimentation, uptake of [3H]dT into the cells, and the pattern of phosphorylation of the intracellular [3H]dT following DDC treatment did not reveal any differences to the controls. A possible effect of DDC treatment on the ribonucleotide reductase and the DNA polymerase alpha is suggested.     

Induction of chromosome aberrations and micronuclei in human peripheral blood lymphocytes at low dose of radiation

The chromosome damage induced by the doses of y-irradiation 6)Co in peripheral blood lymphocytes was studied using different cytogenetic assays. Isolated lymphocytes were exposed to 0.01-1.0 Gy, stimulated by PHA, and analysed for chromosome aberrations at 48 h postirradiation by metaphase method, at 49 h--by the anaphase method, at 58 h by micronucleus assay with cytochalasin B and, additionally, micronuclei were counted at 48 h on the slides prepared for the metaphase analysis without cytochalasin B. Despite of the quantitative differences in the amount of chromosome damage revealed by different methods all of them demonstrated complex nonlinear dose dependence of the frequency of aberrant cells and aberrations. At the dose range from 0.01 Gy to 0.05-0.07 Gy the cells had the highest radiosensitivity mainly due to chromatid-type aberration induction. With dose increasing the frequency of the aberrant cells and aberrations decreased significantly (in some cases to the control level). At the doses up to 0.5-0.7 Gy the dose-effect curves have become linear with the decreased slope compare to initial one (by factor of 5 to 10 for different criteria) reflecting the higher radioresistance of cells. These data confirm the idea that the direct linear extrapolation of high dose effect to low dose range--the procedure routinelly used to estimate genetic risk of low dose irradiation--cannot be effective and may lead to underestimation of chromosome damage produced by low radiation doses. Preferences and disadvantages of used cytogenetic assays and possible mechanisms of low ionising radiation doses action were discussed.     

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843)

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1–40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.     

The effect of recombinant human interleukin-1 beta on the repopulation of bone-marrow colony-forming cells in mice subjected to the action of radiation and burns]

The radioprotective and restorative (therapeutic) effects of human recombinant interleukin-1 beta (IL-1 beta) on the population of bone marrow CFU-S of mice, subjected to either sublethal doses of ionising irradiation itself or the same irradiation in combination with thermal burn, are investigated. Both the effects of the agent are registered under both in vitro and in vivo irradiation in semi-, syn- and allogeneic animals. If the irradiation was combined with thermal burn, the "therapeutic" effect of the agent was demonstrated at irradiation dose equal to 3.06 Gy rather than to 6.12 Gy. If the bone marrow cells were irradiated in vitro in dose 3.06 Gy with the following heat shock at 42 degrees C for 10-20 min, the "therapeutic" effect of IL-1 beta was seen only if it was added to cells before rather than after irradiation. The radioprotective effect of IL-1 beta is maintained under in vitro, as well as in vivo conditions in the allogeneic system of transplantation of the CBA donor bone marrow to the C57BL mice.     

Ratio of γ-H2AX level in lymphocytes to that in granulocytes detected using flow cytometry as a potential biodosimeter for radiation exposure

This study aims to assess utilisation of the ratio of γ-H2AX in lymphocytes to that in granulocytes (RL/G of γ-H2AX) in blood as a rapid method for population triage and dose estimation during large-scale radiation emergencies. Blood samples from healthy volunteers exposed to 0–10 Gy of ⁶⁰Co irradiation were collected. The samples were cultured for 0–24 h and then analysed using flow cytometry to measure the levels of γ-H2AX in lymphocytes and granulocytes. The basal RL/G levels of γ-H2AX in healthy human blood, the response of RL/G of γ-H2AX to ionising radiation and its relationship with doses, time intervals after exposure and individual differences were also analysed. The level of γ-H2AX in lymphocytes increased in a dose-dependent manner after irradiation, whereas the level in granulocytes was not affected. A linear dose–effect relationship with low inter-experimental and inter-individual variations was observed. The RL/G of γ-H2AX may be used as a biomarker for population triage and dose estimation during large-scale radiation emergencies if blood samples can be collected within 24 h.     

Protection from radiation-induced apoptosis by the radioprotector amifostine (WR-2721) is radiation dose dependent

The radioprotective agent amifostine is a free radical scavenger that can protect cells from the damaging effects of ionising radiation when administered prior to radiation exposure. However, amifostine has also been shown to protect cells from chromosomal mutations when administered after radiation exposure. As apoptosis is a common mechanism by which cells with mutations are removed from the cell population, we investigated whether amifostine stimulates apoptosis when administered after radiation exposure. We chose to study a relatively low dose which is the maximum radiation dose for radiation emergency workers (0.25 Gy) and a high dose relevant to radiotherapy exposures (6 Gy). Mice were administered 400 mg/kg amifostine 30 min before, or 3 h after, whole-body irradiation with 0.25 or 6 Gy X-rays and apoptosis was analysed 3 or 7 h later in spleen and bone marrow. We observed a significant increase in radiation-induced apoptosis in the spleen of mice when amifostine was administered before or after 0.25 Gy X-rays. In contrast, when a high dose of radiation was used (6 Gy), amifostine caused a reduction in radiation-induced apoptosis 3 h post-irradiation in spleen and bone marrow similar to previously published studies. This is the first study to investigate the effect of amifostine on radiation-induced apoptosis at a relatively low radiation dose and the first to demonstrate that while amifostine can reduce apoptosis from high doses of radiation, it does not mediate the same effect in response to low-dose exposures. These results suggest that there may be a dose threshold at which amifostine protects from radiation-induced apoptosis and highlight the importance of examining a range of radiation doses and timepoints.     

Long-term effects of ionising radiation on the brain: cause for concern?

There is no clear evidence proving or disproving that ionising radiation is causally linked with neurodegenerative diseases such as Parkinson’s and Alzheimer’s. However, it is known that high doses of ionising radiation to the head (20–50 Gy) lead to severe learning and memory impairment which is characteristical for Alzheimer’s. The cumulative doses of ionising radiation to the Western population are accruing, mostly due to the explosive growth of medical imaging procedures. Children are in particular prone to ionising radiation as the molecular processes within the brain are not completely finished. Furthermore, they have a long lifespan under risk. We wish to open a debate if such low doses of radiation exposure may lead to delayed long-term cognitive and other defects, albeit at a lower frequency than those observed during application of high doses. Further, we want to sensitise the society towards the risks of ionising radiation. To achieve these aims, we will recapitulate the known symptoms of Parkinson’s and Alzheimer’s on the molecular level and incorporate data of mainly low- and moderate-ionising radiation (<5 Gy). Thus, we want to highlight in general the potential similarities of both the neurodegenerative and radiation-induced pathways. We will propose a mechanistic model for radiation-induced neurodegeneration pointing out mitochondria as a key element. This includes effects of oxidative stress and neuroinflammation—all fundamental players of neurodegenerative diseases.     

Gamma-H2AX-based dose estimation for whole and partial body radiation exposure

Most human exposures to ionising radiation are partial body exposures. However, to date only limited tools are available for rapid and accurate estimation of the dose distribution and the extent of the body spared from the exposure. These parameters are of great importance for emergency triage and clinical management of exposed individuals. Here, measurements of γ-H2AX immunofluorescence by microscopy and flow cytometry were compared as rapid biodosimetric tools for whole and partial body exposures. Ex vivo uniformly X-irradiated blood lymphocytes from one donor were used to generate a universal biexponential calibration function for γ-H2AX foci/intensity yields per unit dose for time points up to 96 hours post exposure. Foci--but not intensity--levels remained significantly above background for 96 hours for doses of 0.5 Gy or more. Foci-based dose estimates for ex vivo X-irradiated blood samples from 13 volunteers were in excellent agreement with the actual dose delivered to the targeted samples. Flow cytometric dose estimates for X-irradiated blood samples from 8 volunteers were in excellent agreement with the actual dose delivered at 1 hour post exposure but less so at 24 hours post exposure. In partial body exposures, simulated by mixing ex vivo irradiated and unirradiated lymphocytes, foci/intensity distributions were significantly over-dispersed compared to uniformly irradiated lymphocytes. For both methods and in all cases the estimated fraction of irradiated lymphocytes and dose to that fraction, calculated using the zero contaminated Poisson test and γ-H2AX calibration function, were in good agreement with the actual mixing ratios and doses delivered to the samples. In conclusion, γ-H2AX analysis of irradiated lymphocytes enables rapid and accurate assessment of whole body doses while dispersion analysis of foci or intensity distributions helps determine partial body doses and the irradiated fraction size in cases of partial body exposures.