Effect of changing the radiation dose range on the in vitro cytogenetic dose response to gamma-rays.

PURPOSE: To examine the distortion of the linear quadratic (LQ) model of in vitro cytogenetic dose response over an extended range of γ-ray doses by analyzing the available literature data, and to establish the dose ranges, in which the LQ dose response curve (DRC) can be most accurately fitted for biological dosimetry. MATERIALS AND METHODS: Data on yields of dicentrics (Dic) or dicentrics plus centric rings (Dic + CR) induced in vitro in human lymphocytes by acute γ-rays were extracted from 108 open sources. The overall dose response dataset in the dose range up to 50 Gy was fitted to a fractional-rational (FR) model, which included a 'basic' LQ function in the numerator, and a reduction factor dependent on the square of the dose in the denominator. Cytogenetic dose response data obtained at Grigoriev Institute for Medical Radiology, Kharkiv, Ukraine (GIMRO) in the range 0.1 - 20.3 Gy acute γ-rays were fitted to the LQ model with the progressive changing minimum or maximum radiation dose. RESULTS: The overall dose response, as expected, followed the LQ function in the dose range ≤5 Gy, but in the extended dose range appeared to be S-shaped, with intensive saturation and a plateau at doses ≥22 Gy. Coefficients of the 'basic' LQ equation in FR model were very close to many published DRCs; calculated asymptote was 17. Fitting of the GIMRO dataset to the LQ model with the shift of the dose range showed the increase in linear coefficient with the increment of either minimum or maximum radiation dose, while the decline of the quadratic coefficient was regulated mostly by the increase of the highest dose. The best goodness of fit, assessed by lower χ2 values, occurred for dose ranges 0.1 - 1.0 Gy; 0.5 - 5.9 Gy; 1.0 - 7.8 Gy; 2.0 - 9.6 Gy, 3.9 - 16.4 Gy and 5.9 - 20.3 Gy. The 'see-saw' effect in changes of LQ coefficients was confirmed by re-fitting datasets published by other laboratories. CONCLUSIONS: The classical LQ model with fixed coefficients appears to have limited applicability for cytogenetic dosimetry at radiation doses >5 Gy due to the saturation of the dose response. Different response of the LQ coefficients to the changes of the dose range must be considered during the DRC construction. Proper selection of minimum and maximum dose in calibration experiments makes it possible to improve the goodness of fit of the LQ DRC.

Transcriptional Dynamics of DNA Damage Responsive Genes in Circulating Leukocytes during Radiotherapy.

External beam radiation therapy leads to cellular activation of the DNA damage response (DDR). DNA double-strand breaks (DSBs) activate the ATM/CHEK2/p53 pathway, inducing the transcription of stress genes. The dynamic nature of this transcriptional response has not been directly observed in vivo in humans. In this study we monitored the messenger RNA transcript abundances of nine DNA damage-responsive genes (CDKN1A, GADD45, CCNG1, FDXR, DDB2, MDM2, PHPT1, SESN1, and PUMA), eight of them regulated by p53 in circulating blood leukocytes at different time points (2, 6-8, 16-18, and 24 h) in cancer patients (lung, neck, brain, and pelvis) undergoing radiotherapy. We discovered that, although the calculated mean physical dose to the blood was very low (0.038-0.169 Gy), an upregulation of Ferredoxin reductase (FDXR) gene transcription was detectable 2 h after exposure and was dose dependent from the lowest irradiated percentage of the body (3.5% whole brain) to the highest, (up to 19.4%, pelvic zone) reaching a peak at 6-8 h. The radiation response of the other genes was not strong enough after such low doses to provide meaningful information. Following multiple fractions, the expression level increased further and was still significantly up-regulated by the end of the treatment. Moreover, we compared FDXR transcriptional responses to ionizing radiation (IR) in vivo with healthy donors' blood cells exposed ex vivo and found a good correlation in the kinetics of expression from the 8-hours time-point onward, suggesting that a molecular transcriptional regulation mechanism yet to be identified is involved. To conclude, we provided the first in vivo human report of IR-induced gene transcription temporal response of a panel of p53-dependant genes. FDXR was demonstrated to be the most responsive gene, able to reliably inform on the low doses following partial body irradiation of the patients, and providing an expression pattern corresponding to the % of body exposed. An extended study would provide individual biological dosimetry information and may reveal inter-individual variability to predict radiotherapy-associated adverse health outcomes.

Clinical Applications of Biological Dosimetry in Patients Exposed to Low Dose Radiation Due to Radiological, Imaging or Nuclear Medicine Procedures.

Radiation dosimetric biomarkers have found applications beyond radiation protection area and now are actively introduced into clinical practice. Cytogenetic assays appeared to be a valuable tool for individualized quantifying radiation effects in patients, with high capability for assessing genotoxicity of various medical exposure modalities and providing meaningful radiation dose estimates for prognoses of radiation-related cancer risk. This review summarized current data on the use of biological dosimetry methods in patients undergoing various medical irradiations to low doses. The highlighted topics include basic aspects of biological dosimetry and its limitations in the range of low radiation doses, and main patterns of in vivo induction of radiation biomarkers in clinical exposure scenarios, occurring in X-ray diagnostics, computed tomography, interventional radiology, low dose radiotherapy, and nuclear medicine (internally administered 131I and other radiopharmaceuticals). Additionally, several specific issues, examined by biodosimetry techniques, are analysed, such as contrast media effect, radiation response in pediatric patients, impact of magnetic resonance imaging, evaluation of radioprotectors, detection of patients' abnormal intrinsic radiosensitivity and dose estimation in persons involved in medical radiation incidents. A prognosis of possible directions for further improvements in this area includes the automation of cytogenetic analysis, introduction of molecular biodosimeters and development of multiparametric biodosimetry platforms. A potential approach to the advanced biodosimetry of internal exposure and/or low dose external irradiation is suggested; this can be a multiparametric platform based on the combination of the γ-H2AX foci, dicentric, and translocation assays, each applied in the optimum postexposure time range, with the amalgamation of the dose estimates. The study revealed the necessity of further research, which might clarify medical radiation safety concerns for patients via using stringent biodosimetry methodology.

Prognostic assessment of the zone of occurrence of radiation combined injuries within a nuclear blast area.

BACKGROUND: A detonation of nuclear weapon (NW) is considered as one of the most devastating radiological scenarios in the list of modern global threats. An essential proportion of victims in a mass casualty radiation event may require an immediate medical care due to radiation combined injuries (RCI). Surprisingly, there is a lack of clear guidance for quantitative prognosis of the spatial distribution of expected RCI casesin a given nuclear explosion scenario. PURPOSE: This work is aimed at the presentation of a new, improved model, allowing more confident evaluation of the contributions from different NW destructive forces to RCI formation, thus leading to more accurate approximation of the zone around the epicenter for a guided search for RCI cases. MATERIALS AND METHODS: The model is made compatible with a classic approach and provides the estimates of radial distance from the epicenter, at which NW explosion can produce RCI. Mathematical formalism comprises a set of equations for the reciprocal assessment of a distance-effect for radiation dose (separately for neutrons and gamma-rays), thermal wave and blast shock wave depending on the NW type, detonation yield and altitude, environmental conditions (i.e. season) and shielding factors. The model's capabilities were demonstrated using an example of the RCI grade causing a profound operational performance decrement of military personnel in two marginal scenarios: Troops deployed in an open area or a tank crew. RESULTS: A remarkable difference in the expected radial zones of possible RCI occurrence was found between the actions of a 'historical' atomic bomb, thermonuclear weapons, and low-yield neutron munitions, also with a noticeable impact of the season factor (summer/winter). For a tank crew the clinically manageable RCI are possible only in very high yield explosion scenarios, while the damage caused by radiation alone possess much higher risk. CONCLUSIONS: Suggested formalism may provide guidance for a preliminary planning of countermeasures, targeting of radiation reconnaissance, and clarification of triage results in a broad range of radiological scenarios based on NW detonation. Further improvement of the model is possible by considering neutrons' and gamma-rays' relative biological efficacy, possible shielding factors, and a synergetic effect of NW's destructive forces.

Prediction of the Acute or Late Radiation Toxicity Effects in Radiotherapy Patients Using Ex Vivo Induced Biodosimetric Markers: A Review.

A search for effective methods for the assessment of patients' individual response to radiation is one of the important tasks of clinical radiobiology. This review summarizes available data on the use of ex vivo cytogenetic markers, typically used for biodosimetry, for the prediction of individual clinical radiosensitivity (normal tissue toxicity, NTT) in cells of cancer patients undergoing therapeutic irradiation. In approximately 50% of the relevant reports, selected for the analysis in peer-reviewed international journals, the average ex vivo induced yield of these biodosimetric markers was higher in patients with severe reactions than in patients with a lower grade of NTT. Also, a significant correlation was sometimes found between the biodosimetric marker yield and the severity of acute or late NTT reactions at an individual level, but this observation was not unequivocally proven. A similar controversy of published results was found regarding the attempts to apply G2- and γH2AX foci assays for NTT prediction. A correlation between ex vivo cytogenetic biomarker yields and NTT occurred most frequently when chromosome aberrations (not micronuclei) were measured in lymphocytes (not fibroblasts) irradiated to relatively high doses (4-6 Gy, not 2 Gy) in patients with various grades of late (not early) radiotherapy (RT) morbidity. The limitations of existing approaches are discussed, and recommendations on the improvement of the ex vivo cytogenetic testing for NTT prediction are provided. However, the efficiency of these methods still needs to be validated in properly organized clinical trials involving large and verified patient cohorts.

Radiation Exposure Biomarkers in the Practice of Medical Radiology: Cooperative Research and the Role of the International Atomic Energy Agency (IAEA) Biodosimetry/Radiobiology Laboratory.

The strategy toward personalized medicine in radiation oncology, nuclear medicine, and diagnostic and interventional radiology demands a specific set of assays for individualized estimation of radiation load for safety concerns and prognosis of normal tissue reactions caused by ionizing radiation. Apparently, it seems reasonable to use validated radiation dosimetric biomarkers for these purposes. However, a number of gaps in knowledge and methodological limitations still have to be resolved until dosimetric biomarkers will start to play a valuable role in clinical practice beyond radiation protection and radiation medicine. An extensive international multicenter research is necessary to improve the methodology of clinical applications of biodosimetry. That became a rationale for launching the IAEA Coordinated Research Project E35010 MEDBIODOSE: "Applications of Biological Dosimetry Methods in Radiation Oncology, Nuclear Medicine, and Diagnostic and Interventional Radiology." At the 2 Coordination Meeting on MEDBIODOSE (18-22 February 2019, Recife, Brazil), participants reported progress in the usage of biological dosimetry for genotoxicity assessment and/or individualization of radiotherapy treatment plans. Another avenue of research was the prognosis of normal tissue toxicity and cancer risk prediction using biomarkers' yield measured in vivo or after ex vivo irradiation of patients' cells. Other important areas are mechanisms of cytogenetic radiation response, validation of new radiation biomarkers, development of innovative techniques, automated and high-throughput assays for biodosimetry, and the overall improvement of biodosimetry service. An important aspect of clinical application of biodosimetry is standardization of techniques and unification of approaches to data interpretation. The new IAEA Biodosimetry/Radiobiology Laboratory, which is being established, will provide support for this activity. The declared lab's mission includes, among other tasks, a harmonization of the biodosimetry applications with relevant international standards, guidelines on good laboratory practice, and the IAEA EPR-Biodosimetry manual.

The assessment of radiation hazardous areas considering the spectral analysis of the neutron component of a tactical neutron bomb detonation.

This study provides an improved background for the operational assessment of the radiation hazardous areas with induced radioactivity, based on the analysis of spectral characteristics of the neutron component of the penetrating radiation. The interrelationships between the nuclear munitions explosion parameters, environmental conditions and dosimetric characteristics of the secondary gamma-ray emission from the activated soil are analyzed. The interrelationships of these parameters are used in the methodology of the assessment of the dosimetric characteristics of the radiation hazardous areas.

CLINICAL APPLICATIONS OF BIOMARKERS OF RADIATION EXPOSURE: LIMITATIONS AND POSSIBLE SOLUTIONS THROUGH COORDINATED RESEARCH.

Dosimetric biomarkers have been effectively and intensively used for a long time in the area of radiation protection. In contrast to that, no robust standards or widely accepted protocols for application of these end-points in radiotherapy, diagnostic and interventional radiology and nuclear medicine exist to date. The International Atomic Energy Agency (IAEA) organized the review of the available data on the possibilities of the use of dosimetric biomarkers in medical irradiation scenarios. The resultant Technical Report also contains a summary of identified problems, gaps in knowledge, limitations in methodology and recommendations for their overcoming. This work provided a conceptual background for the initiation of a new IAEA Coordinated Research Project E35010, MEDBIODOSE (2017-21), which is aimed specifically at the development and improvement of applications of biodosimetric markers in clinical practice.

FDXR is a biomarker of radiation exposure in vivo.

Previous investigations in gene expression changes in blood after radiation exposure have highlighted its potential to provide biomarkers of exposure. Here, FDXR transcriptional changes in blood were investigated in humans undergoing a range of external radiation exposure procedures covering several orders of magnitude (cardiac fluoroscopy, diagnostic computed tomography (CT)) and treatments (total body and local radiotherapy). Moreover, a method was developed to assess the dose to the blood using physical exposure parameters. FDXR expression was significantly up-regulated 24 hr after radiotherapy in most patients and continuously during the fractionated treatment. Significance was reached even after diagnostic CT 2 hours post-exposure. We further showed that no significant differences in expression were found between ex vivo and in vivo samples from the same patients. Moreover, potential confounding factors such as gender, infection status and anti-oxidants only affect moderately FDXR transcription. Finally, we provided a first in vivo dose-response showing dose-dependency even for very low doses or partial body exposure showing good correlation between physically and biologically assessed doses. In conclusion, we report the remarkable responsiveness of FDXR to ionising radiation at the transcriptional level which, when measured in the right time window, provides accurate in vivo dose estimates.

Optimizing the Microscopy Time Schedule for Chromosomal Dosimetry of High-dose and Partial-body Irradiations.

The methodology of cytogenetic triage can be improved by optimizing a schedule of microscopy for different exposure scenarios. Chromosome aberrations were quantified by microscopy in human blood lymphocytes irradiated in vitro to ~2, 4, and 12 Gy acute 60Co γ-rays mixed with the unirradiated blood simulating 10%, 50%, 90%, and 100% exposure and in along with a sample from a homogeneous exposure to ~20 Gy. Biodosimetry workload was statistically modeled assuming that 0.5, 1, 5, or 25 h was available for scoring one case or for analysis of up to 1000 cells or 100 dicentrics plus centric rings by one operator. A strong negative correlation was established between the rates of aberration acquisition and cell recording. Calculations showed that the workload of 1 case per operator per·day (5 h of scoring by microscopy) allows dose estimates with high accuracy for either 90%-100% irradiations of 2 Gy or 50%-90% irradiations of 4-12 Gy; lethal homogeneous (100%) exposures of 12 and 20 Gy can be evaluated with just 1 h of microscopy. Triage analysis of 0.5 h scoring per case results in the minimum tolerable accuracy only for partial- and total-body exposure of 4-20 Gy. Time-related efficacy of conventional biodosimetry depends primarily on the aberration yield in the sample, which is dependent on the radiation dose and its distribution in the patient's body. An optimized schedule of microscopy scoring should be developed for different exposure scenarios in each laboratory to increase their preparedness to radiological emergencies.

A new Bayesian model applied to cytogenetic partial body irradiation estimation.

A new zero-inflated Poisson model is introduced for the estimation of partial body irradiation dose and fraction of body irradiated. The Bayes factors are introduced as tools to help determine whether a data set of chromosomal aberrations obtained from a blood sample reflects partial or whole body irradiation. Two examples of simulated cytogenetic radiation exposure data are presented to demonstrate the usefulness of this methodology in cytogenetic biological dosimetry.

Review of Bayesian statistical analysis methods for cytogenetic radiation biodosimetry, with a practical example.

Classical methods of assessing the uncertainty associated with radiation doses estimated using cytogenetic techniques are now extremely well defined. However, several authors have suggested that a Bayesian approach to uncertainty estimation may be more suitable for cytogenetic data, which are inherently stochastic in nature. The Bayesian analysis framework focuses on identification of probability distributions (for yield of aberrations or estimated dose), which also means that uncertainty is an intrinsic part of the analysis, rather than an 'afterthought'. In this paper Bayesian, as well as some more advanced classical, data analysis methods for radiation cytogenetics are reviewed that have been proposed in the literature. A practical overview of Bayesian cytogenetic dose estimation is also presented, with worked examples from the literature.

CytoBayesJ: software tools for Bayesian analysis of cytogenetic radiation dosimetry data.

A number of authors have suggested that a Bayesian approach may be most appropriate for analysis of cytogenetic radiation dosimetry data. In the Bayesian framework, probability of an event is described in terms of previous expectations and uncertainty. Previously existing, or prior, information is used in combination with experimental results to infer probabilities or the likelihood that a hypothesis is true. It has been shown that the Bayesian approach increases both the accuracy and quality assurance of radiation dose estimates. New software entitled CytoBayesJ has been developed with the aim of bringing Bayesian analysis to cytogenetic biodosimetry laboratory practice. CytoBayesJ takes a number of Bayesian or 'Bayesian like' methods that have been proposed in the literature and presents them to the user in the form of simple user-friendly tools, including testing for the most appropriate model for distribution of chromosome aberrations and calculations of posterior probability distributions. The individual tools are described in detail and relevant examples of the use of the methods and the corresponding CytoBayesJ software tools are given. In this way, the suitability of the Bayesian approach to biological radiation dosimetry is highlighted and its wider application encouraged by providing a user-friendly software interface and manual in English and Russian.

A comparison of six statistical distributions for analysis of chromosome aberration data for radiation biodosimetry.

The Poisson distribution is the most widely recognised and commonly used distribution for cytogenetic radiation biodosimetry. However, it is recognised that, due to the complexity of radiation exposure cases, other distributions may be more properly applied. Here, the Poisson, gamma, negative binomial, beta, Neyman type-A and Hermite distributions are compared in terms of their applicability to 'real-life' radiation exposure situations. The identification of the most appropriate statistical model in each particular exposure situation more correctly characterises data. The results show that for acute, homogeneous (whole-body) exposures, the Poisson distribution can still give a good fit to the data. For localised partial-body exposures, the Neyman type-A model was found to be the most robust. Overall, no single distribution was found to be universally appropriate. A distribution-specific method of analysis of cytogenetic data is therefore recommended. Such an approach may lead potentially to more accurate biological dose estimates.

Cytogenetic dose-response in vitro for biological dosimetry after exposure to high doses of gamma-rays.

The dose response for dicentrics plus centric rings and total unstable chromosome-type aberrations was studied in the first mitoses of cultured human peripheral blood lymphocytes irradiated in vitro to doses of ∼2, 4, 6, 8, 10, 16 and 20 Gy of acute (60)Со gamma-rays. A dose-dependent increase of aberration yield was accompanied by a tendency to the underdispersion of dicentrics and centric rings among cells distributions compared with Poisson statistics at doses ≥6 Gy. The formal fitting of the data to a linear-quadratic model resulted in an equation with the linear and quadratic coefficients ranged 0.098-0.129×cell(-1)×Gy(-1) and 0.039-0.034×cell(-1)×Gy(-2), respectively, depending on the fitting method. The actual radiation-induced aberration yield was markedly lower than expected from a calibration curve, generated earlier within a lower dose range. Interlaboratory variations in reported dicentric yields induced by medium-to-high radiation doses in vitro are discussed.

Bystander apoptosis in human cells mediated by irradiated blood plasma.

Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G(0)-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40Gy acute γ-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24h at 37°C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2±1.8% in plasma-free cultures, 21.6±1.1% in cultures treated with plasma from unirradiated blood, 20.2±1.4% in cultures with plasma from blood given 2-4Gy and 16.7±3.2% in cultures with plasma from blood given 6-10Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

Limitations associated with analysis of cytogenetic data for biological dosimetry.

The scientific literature concerning cytogenetic biodosimetry has been reviewed to identify the range of scenarios of radiation exposure where biodosimetry has been carried out. Limitations in the existing standardized statistical methodology have been identified and categorized, and the reasons for these limitations have been explored. Statistical problems generally occur due to either low numbers of aberrations leading to large uncertainties or deviations in aberration-per-cell distributions leading to over- or under-dispersion with respect to the Poisson model. A number of difficulties also stem from limitations of the classical statistical methodology, which requires that chromosome aberration yields be considered as something "fixed" and thus provides a deterministic estimate of radiation dose and associated confidence limits (because an assignment of a probability to an event is based solely on the observed frequency of occurrence of the event). Therefore, it is suggested that solutions to the listed problems should be based in the Bayesian framework. This will allow the investigator to take a probabilistic approach to analysis of cytogenetic data, which can be considered highly appropriate for biological dose estimation.

Delayed chromosomal instability in lymphocytes of cancer patients after radiotherapy.

PURPOSE: To assess possible delayed chromosomal instability (DCI) expressed as elevated chromatid breakage in cells containing previously formed chromosome type aberrations in cultured blood lymphocytes of cancer patients after radiotherapy (RT). MATERIALS AND METHODS: Twenty patients treated for uterine cancer with external Co(60) RT, without chemotherapy, were selected. Blood was taken before, 1-2 days after RT and one year later. Lymphocytes were cultured for 50 and 100 h. Metaphases were stained with fluorescence-plus-Giemsa and analysed for chromosome and chromatid aberrations in 1st (M1) and 3rd plus later (M3+) mitoses. RESULTS: RT caused a significant increase of radiation-specific chromosome aberrations in patients' lymphocytes together with DCI, which was observed as an excessive yield of cells containing both chromosome and chromatid aberrations (defined as C(acs&act)). This DCI passed successfully through mitoses in vitro, and at the end of RT a mean yield of 'extra' C(acs&act) was 3 x 10(-3) x cell(-1) amongst either M1 or M3+ cells. At the end of RT and one year later DCI in M1 lymphocytes appeared at random amongst patients, but some inter-individual variation was found for DCI presence in M3+ cells at both post-irradiation samplings. As time passed, the mean yield of lymphocytes exhibiting DCI decreased in vivo and one year after RT reached the pre-treatment level of 1 x 10(-3) x cell(-1). CONCLUSIONS: DCI was demonstrated in descendants of human lymphocytes after therapeutic irradiation. The effect diminished one year later, suggesting that the progeny of patients' irradiated stem cells did not produce new daughter lymphocytes exhibiting DCI during the studied post-irradiation period.