Impact of medical imaging on the epigenome - low-dose exposure in the course of computed tomography does not induce detectable changes of DNA-methylation profiles in peripheral blood cells.

BACKGROUND: Computed tomography (CT) is a main contributor to artificial low-dose exposure. Understanding the biological effects induced by CT exposure and their dependency on the characteristics of photon spectra is essential for knowledge-driven risk assessment. In a previous gene expression study, we have identified upregulation of AEN, BAX, DDB2, EDA2R and FDXR after ex vivo exposure with single-energy CT and dual-energy CT (DECT). In this study, we focused on CT-induced changes of DNA methylation. This epigenetic modification of DNA is a central regulator of gene expression and instrumental in preserving genome integrity. Previous studies reported focal hypermethylation and global hypomethylation after exposure with doses above 100 mSv, however, the effect of low dose exposure on DNA methylation is hardly explored. MATERIALS AND METHODS: DNA was isolated from peripheral blood of three healthy individuals 6 h after ex vivo exposition to single-energy (80 kV and 150 kV) and DECT (80 kV/Sn150 kV) with a calculated effective dose of 7.0 ± 0.08 mSv. The experimental setting was identical to the one used in our previous gene expression study enabling a direct comparison of gene expression results with changes of DNA methylation identified in this study. DNA methylation was analyzed by high-throughput sequencing of bisulfite-treated DNA targeted methylation sequencing. RESULTS: Unsupervised hierarchical clustering based on DNA methylation profiles of all samples created three distinct clusters. Formation of these three clusters was solely determined by the origin of samples, indicating the absence of prominent irradiation-associated changes of DNA methylation. In line with this observation, inter-individual comparison of non-irradiated samples revealed 1163, 1224 and 4550 significant differentially methylated regions (DMRs), respectively, whereas the pairwise comparison of irradiated and non-irradiated samples failed to identify irradiation-induced DMRs in any of the three probands. This even applied to the genomic regions harboring AEN, BAX, DDB2, EDA2R and FDXR, the five genes known to be upregulated by CT exposure. CONCLUSIONS: CT exposure with various photon spectra did not result in detectable changes of DNA methylation. However, minor effects in a subpopulation of irradiated cells cannot be ruled out. Thus, future studies with extended observation intervals are needed to investigate DNA methylation changes that are induced by indirect effects at later points of time or become detectable by clonal expansion of affected cells. Moreover, our data suggest that DNA methylation analysis is less sensitive in detecting immediate effects of low-dose irradiation when compared to gene expression analysis.

MODIFICATION OF THE TUMOR/INDUCED BYSTANDER EFFECT BY IRRADIATION UNDER COCULTIVATION OF LYMPHOCYTES FROM PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA AND LYMPHOCYTES FROM HEALTHY DONORS. / MODYFIKATsIIa OPROMINENNIaM PUKhLYNNO/INDUKOVANOGO EFEKTU SVIDKA PRY SUMISNO/ROZDIL'NOMU KUL'TYVUVANNI LIMFOTsYTIV KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEIKEMIIu Z LIMFOTsYTAMY ZDOROVYKh DONORIV.

OBJECTIVE: Study the tumor-induced bystander effect of blood cells from chronic lymphocytic leukemia (CLL)patients on non-transformed bystander cells (peripheral blood lymphocytes (PBL) of conditionally healthy individ-uals) and the possibility of its modification after the impact of ionizing radiation. MATERIALS AND METHODS: We carried out cocultivation and separate cultivation of blood samples from conditionallyhealthy volunteers and patients with CLL according to our technique. Using the Comet assay, the relative level ofDNA damage was evaluated. RESULTS: A statistically significant increase (р < 0.001) in the level of DNA damage in PBL culture of conditionallyhealthy individuals after co-cultivation with malignant cells of CLL patients was observed. After irradiation, a drop in the level of cells with a high degree of DNA damage was noted, which was connected with an increase in the frequency of cells that were delayed in division at the S stage of the cell cycle. An increase in apoptotic activity in cultures of bystander cells was observed in all variants of the experiment (р < 0.001). CONCLUSION: The influence of irradiated blood cells of patients with CLL results in an enhancement of the tumor-induced bystander effect manifestation in the PBL of conditionally healthy individuals.

Whole blood gene expression within days after total-body irradiation predicts long term survival in Gottingen minipigs.

Gottingen minipigs mirror the physiological radiation response observed in humans and hence make an ideal candidate model for studying radiation biodosimetry for both limited-sized and mass casualty incidents. We examined the whole blood gene expression profiles starting one day after total-body irradiation with increasing doses of gamma-rays. The minipigs were monitored for up to 45 days or time to euthanasia necessitated by radiation effects. We successfully identified dose- and time-agnostic (over a 1-7 day period after radiation), survival-predictive gene expression signatures derived using machine-learning algorithms with high sensitivity and specificity. These survival-predictive signatures fare better than an optimally performing dose-differentiating signature or blood cellular profiles. These findings suggest that prediction of survival is a much more useful parameter for making triage, resource-utilization and treatment decisions in a resource-constrained environment compared to predictions of total dose received. It should hopefully be possible to build such classifiers for humans in the future.

The cytokinesis-block micronucleus assay for cryopreserved whole blood.

PURPOSE: The cytokinesis-block micronucleus (MN) assay is a widely used technique in basic radiobiology research, human biomonitoring studies and in vitro radiosensitivity testing. Fresh whole blood cultures are commonly used for these purposes, but immediate processing of fresh samples can be logistically challenging. Therefore, we aimed at establishing a protocol for the MN assay on cryopreserved whole blood, followed by a thorough evaluation of the reliability of this assay for use in radiosensitivity assessment in patients. MATERIALS AND METHODS: Whole blood samples of 20 healthy donors and 4 patients with a primary immunodeficiency disease (PID) were collected to compare the results obtained with the MN assay performed on fresh versus cryopreserved whole blood samples. MN yields were scored after irradiation with 220 kV X-rays (dose rate 3 Gy/min), with doses ranging from 0.5-2 Gy. RESULTS: The application of the MN assay on cryopreserved blood samples was successful in all analyzed samples. The radiation-induced MN and NDI scores in fresh and cryopreserved blood cultures were found to be similar. Acceptable inter-individual and intra-individual variabilities in MN yields were observed. Repeated analysis of cryopreserved blood cultures originating from the same blood sample, thawed at different time points, revealed that MN values remain stable for cryopreservation periods up to one year. Finally, radiosensitive patients were successfully identified using the MN assay on cryopreserved samples. CONCLUSIONS: To our knowledge, this study is the first report of the successful use of cryopreserved whole blood samples for application of the MN assay. The data presented here demonstrate that the MN assay performed on cryopreserved whole blood is reliable for radiosensitivity testing. Our results also support its wider use in epidemiological, biomonitoring and genotoxicity studies. The presented method of cryopreservation of blood samples might also benefit other assays.

Late Health Effects of Partial Body Irradiation Injury in a Minipig Model Are Associated with Changes in Systemic and Cardiac IGF-1 Signaling.

Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig is an emerging model for studying radiation effects given its high analogy to human anatomy and physiology. Here we use a minipig model to study late health effects of radiation by exposing male Göttingen minipigs to 1.9-2.0 Gy X-rays (lower limb tibias spared). Animals were monitored for 120 days following irradiation and blood counts, body weight, heart rate, clinical chemistry parameters, and circulating biomarkers were assessed longitudinally. Collagen deposition, histolopathology, IGF-1 signaling, and mRNA sequencing were evaluated in tissues. Our findings indicate a single exposure induced histopathological changes, attenuated circulating IGF-1, and disrupted cardiac IGF-1 signaling. Electrolytes, lipid profiles, liver and kidney markers, and heart rate and rhythm were also affected. In the heart, collagen deposition was significantly increased and transforming growth factor beta-1 (TGF-beta-1) was induced following irradiation; collagen deposition and fibrosis were also observed in the kidney of irradiated animals. Our findings show Göttingen minipigs are a suitable large animal model to study long-term effects of radiation exposure and radiation-induced inhibition of IGF-1 signaling may play a role in development of late organ injuries.

Acute radiation syndrome-related gene expression in irradiated peripheral blood cell populations.

PURPOSE: In a nuclear or radiological event, an early diagnostic tool is needed to distinguish the worried well from those individuals who may later develop life-threatenFing hematologic acute radiation syndrome. We examined the contribution of the peripheral blood's cell populations on radiation-induced gene expression (GE) changes. MATERIALS AND METHODS: EDTA-whole-blood from six healthy donors was X-irradiated with 0 and 4Gy and T-lymphocytes, B-lymphocytes, NK-cells and granulocytes were separated using immunomagnetic methods. GE were examined in cell populations and whole blood. RESULTS: The cell populations contributed to the total RNA amount with a ratio of 11.6 for T-lymphocytes, 1.2 for B-cells, 1.2 for NK-cells, 1.0 for granulocytes. To estimate the contribution of GE per cell population, the baseline (0Gy) and the radiation-induced fold-change in GE relative to unexposed was considered for each gene. The T-lymphocytes (74.8%/80.5%) contributed predominantly to the radiation-induced up-regulation observed for FDXR/DDB2 and the B-lymphocytes (97.1%/83.8%) for down-regulated POU2AF1/WNT3 with a similar effect on whole blood gene expression measurements reflecting a corresponding order of magnitude. CONCLUSIONS: T- and B-lymphocytes contributed predominantly to the radiation-induced up-regulation of FDXR/DDB2 and down-regulation of POU2AF1/WNT3. This study underlines the use of FDXR/DDB2 for biodosimetry purposes and POU2AF1/WNT3 for effect prediction of acute health effects.

Expression of genes and pathways associated with the B7-CD28 superfamily in response to irradiation of blood cells using 137Cs.

PURPOSE: DNA damage is one of the main consequences of exposure to ionizing irradiation (IR). Recent studies indicate that IR can modulate the expression of immune system-related genes. However, the effects of IR on the expression of genes and pathways of the B7-CD28 superfamily remain poorly defined. The aim of this study was to evaluate the modulation of genes and pathways related to the B7-CD28 superfamily in response to IR. MATERIALS AND METHODS: In this study, we used transcriptome data available from the Gene Expression Omnibus (GEO) database to investigate the modulation of the response of genes and pathways of samples of human peripheral blood irradiated with doses of 150, 300, and 600 cGy. The data were obtained at 6 and 24 h after irradiation. The relationship between genes and pathways was established through the Reactome database. The behavior of these pathways was analyzed using mathematical methods based on relative activity and diversity. Analysis of variance (ANOVA) followed by multiple comparisons tests (Bonferroni and Tamhanes) was used to identify differentially expressed genes. Data on transcriptomes were analyzed through ViaComplex V.1.0 and IBM SPSS Statistics 22. RESULTS: For the pathways investigated in this study, we observed that the effects produced by these doses significantly modified the behavior of five pathways associated with the immune system. Also, the dose of 300 cGy might trigger signaling for the activation of T cells through the negative regulation (p < .05) of the co-inhibitory PDCD1LG2 gene. Positive regulation caused by 300 cGy (p < .05) of the CD80 receptor was observed by us, which might be related to a stimulatory signal. According to our findings, this dose induced the production of cytokines and genes that are associated with the activation and differentiation of T cells. CONCLUSIONS: Our findings indicate that the irradiation modulated the organization of the biological system, suggesting that 300 cGy is more efficient in activating the immune system.

Assessment of the results and hematological side effects of 3D conformal and IMRT/ARC therapies delivered during craniospinal irradiation of childhood tumors with a follow-up period of five years.

BACKGROUND: Craniospinal irradiation (CSI) of childhood tumors with the RapidArc technique is a new method of treatment. Our objective was to compare the acute hematological toxicity pattern during 3D conformal radiotherapy with the application of the novel technique. METHODS: Data from patients treated between 2007 and 2014 were collected, and seven patients were identified in both treatment groups. After establishing a general linear model, acute blood toxicity results were obtained using SPSS software. Furthermore, the exposure dose of the organs at risk was compared. Patients were followed for a minimum of 5 years, and progression-free survival and overall survival data were assessed. RESULTS: After assessment of the laboratory parameters in the two groups, it may be concluded that no significant differences were detected in terms of the mean dose exposures of the normal tissues or the acute hematological side effects during the IMRT/ARC and 3D conformal treatments. Laboratory parameters decreased significantly compared to the baseline values during the treatment weeks. Nevertheless, no significant differences were detected between the two groups. No remarkable differences were confirmed between the two groups regarding the five-year progression-free survival or overall survival, and no signs of serious organ toxicity due to irradiation were observed during the follow-up period in either of the groups. CONCLUSION: The RapidArc technique can be used safely even in the treatment of childhood tumors, as the extent of the exposure dose in normal tissues and the amount of acute hematological side effects are not higher with this technique.

Automated Dicentric Aberration Scoring for Triage Dose Assessment: 60Co Gamma Ray Dose-response at Different Dose Rates.

The objective of this study was to establish radiation dose-response calibration curves using automated dicentric scoring to support rapid and accurate cytogenetic triage dose-assessment. Blood was drawn from healthy human volunteers and exposed to Co gamma rays at several dose rates (i.e., 1.0, 0.6, and 0.1 Gy min). After radiation, the blood was placed for 2 h in a 37 °C incubator for repair. Blood was then cultured in complete media to which a mitogen (i.e., phytoghemagglutinin, concentration 4%) was added for 48 h. Colcemid was added to the culture at a final concentration of 0.2 µg mL after 24 h for the purpose of arresting first-division metaphase mitotics. Cells were harvested at the end of 48 h. Samples were processed using an automated metaphase harvester and automated microscope metaphase finder equipped with a suite of software including a specialized automated dicentric scoring application. The data obtained were used to create dose-response tables of dicentric yields. The null hypothesis that the data is Poisson-distributed could not be rejected at the significance level of α = 0.05 using results from a Shiny R Studio application (goodness-of-fit Poisson). Calibration curves based on linear-quadratic fits for Co gamma rays at the three different dose rates were generated using these data. The calibration curves were used to detect blind test cases. In conclusion, using the automated harvester and automated microscope metaphase finder with associated automated dicentric scoring software demonstrates high-throughput with suitable accuracy for triage radiation dose assessment.

Effect of Different Antioxidants on X-ray Induced DNA Double-strand Breaks Using γ-H2AX in Human Blood Lymphocytes.

Ionizing radiation exposure produces direct or indirect biological effects on genomic DNA. The latter are ionizing radiation mediated by induction of free radicals and oxygen species (ROS). The study was conducted to evaluate the dose-effect/time-effect of antioxidant treatments in reducing the induction of double-strand breaks in human blood lymphocytes. Human peripheral blood samples of 2 mL each from healthy donors were irradiated with 10 mGy after pre-incubation with different antioxidants (ß-carotene, vitamin E, vitamin C, N-acetyl L-cysteine). In order to assess their efficiency as prophylactic therapy for irradiation, various concentrations and combinations of antioxidants, as well as different incubation times, have been evaluated. To assess double-strand breaks induced by ionizing radiation, the phosphorylated histone γ-H2AX has been used. A significant reduction (p < 0.001) in double-strand breaks studied with a γ-H2AX assay was observed with N-acetyl L-cysteine with a 1-h incubation time, followed by vitamin C, vitamin E, and ß-carotene. The use of antioxidants, especially N-acetyl L-cysteine before irradiation, significantly decreased the occurrence of double-strand breaks, demonstrating the potential radiological protection for exposure to ionizing radiation.

CT Irradiation-induced Changes of Gene Expression within Peripheral Blood Cells.

Computed tomography (CT) is a crucial element of medical imaging diagnostics. The widespread application of this technology has made CT one of the major contributors to medical radiation burden, despite the fact that doses per individual CT scan steadily decrease due to the advancement of technology. Epidemiological risk assessment of CT exposure is hampered by the fact that moderate adverse effects triggered by low doses of CT exposure are likely masked by statistical fluctuations. In light of these limitations, there is need of further insights into the biological processes induced by CT scans to complement the existing knowledge base of risk assessment. This prompted us to investigate the early transcriptomic response of ex vivo irradiated peripheral blood of three healthy individuals. Samples were irradiated employing a modern dual-source-CT-scanner with a tube voltage of 150 kV, resulting in an estimated effective dose of 9.6 mSv. RNA was isolated 1 h and 6 h after exposure, respectively, and subsequently analyzed by RNA deep sequencing. Differential gene expression analysis revealed shared upregulation of AEN, FDXR, and DDB2 6 h after exposure in all three probands. All three genes have previously been discussed as radiation responsive genes and have already been implicated in DNA damage response and cell cycle control after DNA damage. In summary, we substantiated the usefulness of AEN, FDXR, and DDB2 as RNA markers of low dose irradiation. Moreover, the upregulation of genes associated with DNA damage reminds one of the genotoxic nature of CT diagnostics even with the low doses currently applied.

Identification of Potential Biomarkers of Radiation Exposure in Blood Cells by Capillary Electrophoresis Time-of-Flight Mass Spectrometry.

Biodosimetry is a useful method for estimating personal exposure doses to ionizing radiation. Studies have identified metabolites in non-cellular biofluids that can be used as markers in biodosimetry. Levels of metabolites in blood cells may reflect health status or environmental stresses differentially. Here, we report changes in the levels of murine blood cell metabolites following exposure to X-rays in vivo. Levels of blood cell metabolites were measured by capillary electrophoresis time-of-flight mass spectrometry. The levels of 100 metabolites were altered substantially following exposure. We identified 2-aminobutyric acid, 2'-deoxycytidine, and choline as potentially useful markers of radiation exposure and established a potential prediction panel of the exposure dose using stepwise regression. Levels of blood cell metabolites may be useful biomarkers in estimating exposure doses during unexpected radiation incidents.

New Approaches for Quantitative Reconstruction of Radiation Dose in Human Blood Cells.

In the event of a nuclear attack or large-scale radiation event, there would be an urgent need for assessing the dose to which hundreds or thousands of individuals were exposed. Biodosimetry approaches are being developed to address this need, including transcriptomics. Studies have identified many genes with potential for biodosimetry, but, to date most have focused on classification of samples by exposure levels, rather than dose reconstruction. We report here a proof-of-principle study applying new methods to select radiation-responsive genes to generate quantitative, rather than categorical, radiation dose reconstructions based on a blood sample. We used a new normalization method to reduce effects of variability of signal intensity in unirradiated samples across studies; developed a quantitative dose-reconstruction method that is generally under-utilized compared to categorical methods; and combined these to determine a gene set as a reconstructor. Our dose-reconstruction biomarker was trained using two data sets and tested on two independent ones. It was able to reconstruct dose up to 4.5 Gy with root mean squared error (RMSE) of ± 0.35 Gy on a test dataset using the same platform, and up to 6.0 Gy with RMSE of ± 1.74 Gy on a test set using a different platform.

Radiosafe micro-computed tomography for longitudinal evaluation of murine disease models.

Implementation of in vivo high-resolution micro-computed tomography (µCT), a powerful tool for longitudinal analysis of murine lung disease models, is hampered by the lack of data on cumulative low-dose radiation effects on the investigated disease models. We aimed to measure radiation doses and effects of repeated µCT scans, to establish cumulative radiation levels and scan protocols without relevant toxicity. Lung metastasis, inflammation and fibrosis models and healthy mice were weekly scanned over one-month with µCT using high-resolution respiratory-gated 4D and expiration-weighted 3D protocols, comparing 5-times weekly scanned animals with controls. Radiation dose was measured by ionization chamber, optical fiberradioluminescence probe and thermoluminescent detectors in a mouse phantom. Dose effects were evaluated by in vivo µCT and bioluminescence imaging read-outs, gold standard endpoint evaluation and blood cell counts. Weekly exposure to 4D µCT, dose of 540-699 mGy/scan, did not alter lung metastatic load nor affected healthy mice. We found a disease-independent decrease in circulating blood platelets and lymphocytes after repeated 4D µCT. This effect was eliminated by optimizing a 3D protocol, reducing dose to 180-233 mGy/scan while maintaining equally high-quality images. We established µCT safety limits and protocols for weekly repeated whole-body acquisitions with proven safety for the overall health status, lung, disease process and host responses under investigation, including the radiosensitive blood cell compartment.

A Novel Application of [18F]Fluorothymidine-PET ([18F]FLT-PET) in Clinical Practice to Quantify Regional Bone Marrow Function in a Patient With Treatment-Induced Cytopenias and to Guide "Marrow-Sparing" Radiotherapy.

F-Fluorothymidine-positron emission tomography with CT fusion ([F]FLT-PET/CT) offers a unique and non-invasive method for three-dimensional localization and quantification of functional bone marrow. [F]FLT-PET/CT has potential application in radiotherapy planning when risk of marrow toxicity is a significant clinical concern. In this patient with chemo-refractory, transformed lymphoma and treatment-induced cytopenias, [F]FLT-PET/CT was a novel and useful adjunct to (1) image the distribution of functional bone marrow reserve, (2) guide "marrow-sparing" radiotherapy planning, and (3) quantify the effects of radiotherapy-induced bone marrow suppression both in-field and out-of-field.

[Predictive assays for responses of tumors and normal tissues in radiation oncology]. / Outils pour la prédiction de la réponse tumorale et des tissus sains en oncologie radiothérapie.

The impact of curative radiotherapy depends mainly on the total dose delivered homogenously in the target volume. Tumor sensitivity to radiotherapy may be particularly inconstant depending on location, histology, somatic genetic parameters and the capacity of the immune system to infiltrate the tumor. In addition, the dose delivered to the surrounding healthy tissues may reduce the therapeutic ratio of many radiation treatments. In a same population treated in one center with the same technique, it appears that individual radiosensitivity clearly exists, namely in terms of late side effects that are in principle non-reversible. This review details the different radiobiological approaches that have been developed to better predict the tumor response but also the radiation-induced late effects.

Biological Dosimetry Network in Africa: Establishment of a Dose-Response Curve Using Telomere and Centromere Staining.

PURPOSE: Biological dosimetry, based on the relationship between the absorbed dose after exposure to ionizing radiation and the frequency of scored aberrations, has been and continues to be an important tool for estimating the dose after exposure. Dicentric chromosomes are considered to be the most specific and sensitive aberration related to radiation exposure. Here, we established the dose-response curve following in vitro irradiation of circulating lymphocytes from healthy donors from three African countries after scoring unstable chromosomal aberrations. MATERIALS AND METHODS: Blood samples from 16 African donors were exposed to various doses (0 to 4 Gy) using an X-RAD320 x-ray system with a maximum photon energy of 250 kV at a dose rate of 0.1 Gy min. Blood lymphocytes were cultured for 48 h, and chromosomal aberrations were scored during the first mitosis by telomere and centromere staining. The distribution of dicentric chromosomes was determined. RESULTS: No dicentric chromosomes were found after the analysis of 2,669 first-division metaphases before in vitro exposure. We established a linear-quadratic dose-response curve based on the frequency of dicentric and ring chromosomes and calculated double-strand breaks, taking into account all scored aberrations. CONCLUSION: The generation of a specific dose-response curve for African donors will allow the practice of precise biological dosimetry in these countries. This work is the first step towards realizing an African biodosimetry network and the establishment of a biological dosimetry laboratory, which could play a major role in the application of radioprotection norms.

COMPREHENSIVE EXPRESSION ANALYSIS OF MIRNAS IN MICE EXPOSED TO LETHAL RADIATION AND/OR RADIO-MITIGATIVE DRUG.

New minimally invasive indicators that are capable of predicting the biological effects and radiation damage to various organs and systems are urgently needed for the development of optimal treatment protocols for victims of radiation accidents. In the present study, we focused on microRNA (miRNA) that have recently emerged as biomarkers for predicting and diagnosing various pathological conditions and identified the serum miRNA signatures. All of the mice treated with lethal radiation alone strongly expressed certain serum miRNAs detectable for 24 h after radiation exposure, whereas the administration of radio-mitigative drug immediately after irradiation suppressed these miRNA expressions to the same levels as in control mice. These results suggest that serum miRNAs may reflect the degree of radiation damage and can be used to predict the radiation-mitigative information in victims of accidental radiation exposure.

Bone Marrow and Peripheral Blood Cells Toxicity of a Single 2.0 Gy Cobalt60 Ionizing Radiation: An Animal Model.

BACKGROUND: Bone marrow is extremely vulnerable to damage caused by radiation therapy. Hence, bone marrow suppression is an important side effect of radiotherapy. Effective use of radiotherapy is therefore compromised by radiation-related injuries. MATERIAL AND METHODS: Six Guinea-pigs were recruited for the study of which three were subjected to total body irradiation with Co60 while the other three served as controls. Bone marrow and peripheral blood samples were collected before and at days 9, 14 and 21, post irradiation. Manual and automated counts were performed for bone marrow nucleated cells and peripheral blood cells respectively. RESULTS: Declining bone marrow cellularity was evident immediately post irradiation. Mean ± SD of marrow cell counted per mm3 were 121,924±281, 87,603±772, 121,367±375 and 122,750±1000 pre-irradiation and days 9, 14 and 21, postirradiation (p-values 0.10, 0.27 and 0.29 respectively). Significant drops in counts were noticed on day 9 post-irradiation for all red cell parameters (p-values <0.05), for Total White Blood Cell Count and Neutrophil count (p-values <0.05) and also on days 14 and 21 for Lymphocytes (p-values <0.05) and on day 21 for Eosinophil/Basophil/Monocytes (p-value <0.05). A significant drop in platelets counts was also noticed on day 9 (p-value <0.05) which significantly increased above pre-irradiation value on day 21. CONCLUSION: Total body irrradiation with Co60 significantly affects the bone marrow with maximum reductions in marrow nucleated cells and peripheral blood cells counts on day 9 post irradiation.

Assessment of epigenetic mechanisms and DNA double-strand break repair using laser micro-irradiation technique developed for hematological cells.

BACKGROUND: Certain tumors rely heavily on their DNA repair capability to survive the DNA damage induced by chemotherapeutic agents. Therefore, it is important to monitor the dynamics of DNA repair in patient samples during the course of their treatment, in order to determine whether a particular drug regimen perturbs the DNA repair networks in cancer cells and provides therapeutic benefits. Quantitative measurement of proteins and/or their posttranslational modification(s) at DNA double strand breaks (DSBs) induced by laser microirradiation provides an applicable diagnostic approach to examine DNA repair and its dynamics. However, its use is restricted to adherent cell lines and not employed in suspension tumor cells that include the many hematological malignancies. METHODS: Here, we report the development of an assay to laser micro-irradiate and quantitatively measure DNA repair transactions at DSB sites in normal mononuclear cells and a variety of suspension leukemia and lymphoma cells including primary patient samples. FINDINGS: We show that global changes in the H3K27me3-ac switch modulated by inhibitors of Class I HDACs, EZH2 methyltransferase and (or) H3K27me3 demethylases do not reflect the dynamic changes in H3K27me3 that occur at double-strand break sites during DNA repair. INTERPRETATION: Results from our mechanistic studies and proof-of-principle data with patient samples together show the effectiveness of using the modified micro-laser-based assay to examine DNA repair directly in suspension cancer cells, and has important clinical implications by serving as a valuable tool to assess drug efficacies in hematological cancer cells that grow in suspension.