The DNA damage response to radiological imaging: from ROS and γH2AX foci induction to gene expression responses in vivo.

Candidate ionising radiation exposure biomarkers must be validated in humans exposed in vivo. Blood from patients undergoing positron emission tomography-computed tomography scan (PET-CT) and skeletal scintigraphy (scintigraphy) was drawn before (0 h) and after (2 h) the procedure for correlation analyses of the response of selected biomarkers with radiation dose and other available patient information. FDXR, CDKN1A, BBC3, GADD45A, XPC, and MDM2 expression was determined by qRT-PCR, DNA damage (γH2AX) by flow cytometry, and reactive oxygen species (ROS) levels by flow cytometry using the 2', 7'-dichlorofluorescein diacetate test in peripheral blood mononuclear cells (PBMC). For ROS experiments, 0- and 2-h samples were additionally exposed to UVA to determine whether diagnostic irradiation conditioned the response to further oxidative insult. With some exceptions, radiological imaging induced weak γH2AX foci, ROS and gene expression fold changes, the latter with good coherence across genes within a patient. Diagnostic imaging did not influence oxidative stress in PBMC successively exposed to UVA. Correlation analyses with patient characteristics led to low correlation coefficient values. γH2AX fold change, which correlated positively with gene expression, presented a weak positive correlation with injected activity, indicating a radiation-induced subtle increase in DNA damage and subsequent activation of the DNA damage response pathway. The exposure discrimination potential of these biomarkers in the absence of control samples as frequently demanded in radiological emergencies, was assessed using raw data. These results suggest that the variability of the response in heterogeneous populations might complicate identifying individuals exposed to low radiation doses.

Small is beautiful: low activity alpha and gamma sources for small-scale radiation protection research experiments.

PURPOSE: Uncertainties regarding the magnitude of health effects following exposure to low doses of ionizing radiation remain a matter of concern both for professionals and for the public. There is consensus within the international radiation research community that more research is required on biological effects of radiation doses below 100 mGy applied at low dose rates. Moreover, there is a demand for increasing education and training of future radiation researchers and regulators. Research, education and training is primarily carried out at universities but university-based radiation research is often hampered by limited access to radiation sources. The aim of the present report is to describe small and cost-effective low activity gamma and alpha sources that can easily be installed and used in university laboratories. METHODS AND RESULTS: A gamma radiation source was made from an euxenite-(Y) rock (Y,Ca,Ce,U,Th)(Nb,Ta,Ti)2O6) that was found in an abandoned mine in Sweden. It allows exposing cells grown in culture dishes to radiation at a dose rate of 50 µGy/h and lower. Three alpha sources were custom-made and yield a dose rate of 1 mGy/h each. The construction, dosimetry and cellular effects of the sources are described. CONCLUSIONS: We hope that the report will stimulate research and training activities in the low dose field by facilitating access to radiation sources.

Studies of Element Concentration in the Lymphocytes, Erythrocytes, and Plasma of Healthy Human Donors Using Total Reflection X-ray Fluorescence.

In this article, the total reflection X-ray fluorescence (TXRF) analysis was used for determination of element concentrations in three isolated peripheral blood components: lymphocytes, erythrocytes, and plasma, collected from 36 healthy donors (15 men and 21 women) from eastern Poland. The studied blood components were isolated from whole peripheral blood using Histopaque-1077 density gradient centrifugation. In the lymphocytes, the following elements were measured: P, S, Cl, K, Ca, Fe, Zn, Br, Sr, and Pb. In the erythrocytes: P, S, Cl, K, Cr, Mn, Fe, Cu, Zn, Se, Br, Rb, and Pb, while in the plasma samples: P, S, Cl, K, Ti, V, Cr, Fe, Co, Ni, Cu, Zn, Br, Se, Sr, and Pb. The descriptive statistics parameters of concentration such as: mean value, standard deviation, median, first and third quartiles, 10th and 90th percentiles were calculated for all samples of the studied blood components, as well as separately for male and female groups. The measured element concentrations and calculated parameters can be used as the reference values. Element concentration distributions for male and female groups were statistically compared using the non-parametric Mann-Whitney test and statistical significance differences (α = 0.05) were found for: P (in lymphocytes), Se and Rb (in erythrocytes), and V (in plasma). The multigroup statistical comparison of element concentration distribution for different blood components was also done (Mann-Whitney and Kruskal-Wallis tests). The statistical tests show that the concentration levels are usually different, except in the following cases: Zn, Fe (lymphocytes and plasma, Fe only for female group), Cr (erythrocytes and plasma). The obtained concentration ranges were compared with literature-based data available for element concentration in lymphocytes, erythrocytes, and plasma. In this work, the application of the TXRF method allowed additionally for the simultaneous determination of the concentrations of such elements as: P, S, Cl, Br, Sr and Pb in lymphocytes, S, Cl and Br in erythrocytes, and P, S, Cl, K, Ti, Br and Pb in plasma. From an analytical point of view, the article describes in detail the measurement procedure including the isolation of blood components and samples preparation for TXRF measurements, and later the detection limit of the method is discussed.

Analysis of elements secreted by CHO-K1 cells exposed to gamma radiation under different treatments.

Purpose: The aim of the study was to determine the concentration of elements using the two methods: total reflection X-ray fluorescence (TXRF) and wavelength dispersive X-ray fluorescence (WD-XRF) in two media, DMEM + and PBS+.Materials and methods: Tests were carried out at 37 and 0 °C, irradiated by gamma radiation doses of 0, 0.25, 0.5, 5 Gy, both with and without contact with CHO-K1 cells. The survival of non-irradiated CHO-K1 cells was determined after transmission of media from irradiated CHO-K1.Results: Normalized concentrations of elements as a percentage of control data (i.e. 0 Gy dose) for Al, P, S, Cl, K, Ca, Zn, Br, were determined using the TXRF method and for Na, P, S, Cl, K, Ca determined using the WD-XRF method in DMEM + and PBS + without and with contact with cells at two temperatures, 37 and 0 °C, and three absorbed doses of 0.25, 0.5 and 5 Gy. Concentration of elements, presented on the coordinates of the two principal components (PC) for media without contact with cells, determined by the TXRF method and in contact with cells, determined by the TXRF and WD-XRF methods were presented. Treatments to which the media were subjected, presented as co-ordinates determined by the first two PC when media were without and in contact with cells (TXRF method) and for media in contact with cells (WD-XRF method) were shown.Conclusions: The results showed that a statistically significant difference occurred in elemental concentrations for media in contact with the cells at the temperatures used. From principal component analysis (PCA), it was observed that the concentrations of elements such as Al, K, Ca, Zn, Br were similar to each other, in contrast to the concentrations of P, Cl, S, both with contact and without contact with cells. A high correlation between the treatment of media within the group at doses of 0.25 Gy and for the group with 0.5 and 5 Gy doses was confirmed. Numerous correlations were observed between the concentrations of elements for media that were in contact with cells, which were not observed in media without contact with cells. The survival of non-irradiated CHO-K1 cells, was determined after transmission of media from irradiated CHO-K1 cells showing no statistically significant differences.

Monte Carlo dosimetry using Fluka code and experimental dosimetry with Gafchromic EBT2 and XR-RV3 of self-built experimental setup for radiobiological studies with low-energy X-rays.

Purpose: The aim of this research was to simulate self-built experimental setup for radiobiological research using X-ray diffraction C-tech tube and PW 3830 generator (PANalytical, Netherlands) and to calculate absorbed dose and to compare it with experimental dose measurements. The maximum X-ray energy was 60 keV.Materials and methods: Petri dish was specially adapted to hold biological cells during the irradiation process. Rotation of Petri dish ensured radiation homogeneity and effectiveness of rotation process was confirmed using EBT2 Gafchromic film. Monte Carlo simulation using Fluka 2011 2c.4 was used to model the setup and to calculate dose absorbed by live cells. The EBT2 and XR-RV3 Gafchromic films were used to estimate relative experimental absorbed dose.Results: The radiation homogeneity provided values with maximum deviation equal to ±3.5% from the average value and the absorbed dose rate was 0.9 Gy/min using simulation process and 1 Gy/min or 0.8 Gy/min using experimental methods (XR-RV3 and EBT2 Gafchromic film, respectively). All dose rate values show metrological compatibility.Conclusions: Influence of specially constructed Petri dish on absorbed dose was determined using simulations that showed that low-energy photons, emitted as characteristic line from borosilicate glass forming component of Petri dish, were source of increase in dose absorbed by cells. This experimental setup will be used to conduct radiobiological research.HighlightsA low-energy X-ray system constructed for radiobiological studies was used.Dosimetry was based on a Monte Carlo simulation using Fluka 2011 code version 2c.4.A specially designed rotating Petri dish ensured the uniformity of the radiation distribution.Gafchromic EBT2 and XR-RV3 films were used to experimental dosimetry.Monte Carlo and experimental dosimetry showed metrological compatibility.

CALIBRATION OF LOW ENERGY X-RAY EXPERIMENTAL SETUP WITH STRONGLY FILTERED BEAM USING DATA FROM A SEMICONDUCTOR AND A THERMOLUMINESCENT DETECTORS.

The calibration of low energy X-ray experimental setup with strongly filtered beam dedicated to radiobiological research was performed using the absorbed dose calculated from the data collected by two types detectors. For this purpose a semiconductor (Amptek, USA) and a thermoluminescent (Institute of Nuclear Physics, Krakow, Poland) detectors were applied. The absorbed dose in water values estimated by both detectors are in good agreement.

Biological effects of mixed-ion beams. Part 2: The relative biological effectiveness of CHO-K1 cells irradiated by mixed- and single-ion beams.

The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236-300 and 461-470 keV/µm for 12C and 16O ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (12C) and 470 keV/µm (16O).

Biological effects of mixed-ion beams. Part 1: Effect of irradiation of the CHO-K1 cells with a mixed-ion beam containing the carbon and oxygen ions.

Carbon and oxygen ions were accelerated simultaneously to estimate the effect of irradiation of living cells with the two different ions. This mixed ion beam was used to irradiate the CHO-K1 cells, and a survival test was performed. The type of the effect of the mixed ion beam on the cells was determined with the isobologram method, whereby survival curves for irradiations with individual ion beams were also used. An additive effect of irradiation with the two ions was found.

Simultaneous breast cancer and DLBCL lymphoma - role of PET/CT examination with 18F-FDG and 18F-FES.

Breast cancer is the most common cancer in women in the western world. The estrogen receptor (ER) is expressed in two thirds of newly diagnosed breast cancers, so hormonal treatment is performed only in the receptor positive patients. The most successful ER imaging radiopharmaceutical in PET techniques is 16α-[18F]-flouro-17ß-estradiol (¹8F-FES). The diffuse large B cell lymphoma (DLBCL) is one of the most common NHL, however, non-Hodgkin lymphoma constitutes only 4% of all primary cancers in women. The typical staging of disease is done using 18-fluorodeoksyglukose (¹8F-FDG) PET/CT.

Scintigraphic assessment of renal function in steel plant workers occupationally exposed to lead.

OBJECTIVES: Occupational exposure to lead may produce kidney damage, but existing data on the dose range associated with nephrotoxicity are inconclusive. We here assessed renal function under conditions of low to moderate lead exposure using renal scintigraphy. METHODS: Fifty-three male foundrymen (exposed group) and fourty male office workers (control group) from a steel plant were included in the study. Glomerular and tubular renal function were assessed by means of (99m)Tc-DTPA and (99m)Tc-EC clearance, respectively. Urinary markers of glomerular dysfunction (albumin) and tubular damage (α1-microglobulin (α1M), ß2-microglobulin (ß2M), retinol-binding protein (RBP), N-acetyl-ß-glucosaminidase (NAG) activity) were determined using latex beads tests or colorimetry. The lead concentration in blood was measured with atomic absorption spectrometry. RESULTS: The blood lead concentrations were 145.8 (121.3-175.3) and 39.3 (35.1-44.1) µg/l (geometric mean, 95(th) CI, p<0.001) in the exposed and control groups, respectively. Subjects exposed to lead presented with increased (99m)Tc-DTPA clearance (158.3 (148.4-168.8) vs. 135.9 (127.9-144.4) ml/min; p<0.01) and urinary albumin excretion (7.61 (6.28-9.22) vs. 4.78 (4.05-5.65) mg/g creatinine; p<0.001). (99m)Tc-EC clearance and excretion of α1M, ß2M, RBP and NAG were not significantly different between the groups. Significant correlations between (99m)Tc-DTPA clearance and blood lead concentrations (r=0.45; p<0.01) and between urinary albumin excretion and blood lead concentrations (r=0.71; p<0.001) were noted. CONCLUSIONS: Use of renal scintigraphy in present study revealed measurable alterations of renal function under the conditions of low-level lead exposure and suggest that increased glomerular filtration may be an early indicator of kidney damage in subjects occupationally exposed to lead.

Analysis of copper concentration in human serum by application of total reflection X-ray fluorescence method.

The chemotherapy and photon radiotherapy are the most often applied methods in treatment of the cancer diseases because of their effectiveness and high cure rates. Apart from eligible destruction of the tumour, one of the side effects of these treatment methods is possible modification of main and trace element concentration in different human tissues and fluids. In this paper, the copper (Cu) level in human serum was determined by total reflection X-ray fluorescence method in 142 chemotherapy patients and in 44 healthy persons being a control group. The Cu concentration in the chemotherapy group was found to be on the level 1.78 ± 0.909 mg/L, while in the control group, it was 1.08 ± 0.551 mg/L. Performed measurements allowed for calculation of the parameters of copper concentration distribution (mean value, standard deviation, median) for both analysed groups. The theoretical nature of the concentration distribution was tested and found as a log-normal distribution (control group) and a log-stable distribution (chemotherapy group). The copper concentration distributions for both studied group were statistically compared using Kolmogorov-Smirnov test, and the conclusion was that the distributions are statistically different. Serum Cu levels were significantly higher in the chemotherapy group than in the control group. Taking into account the results for the control group, the copper concentration reference quantile ranges in human serum were obtained. The values of the mean, median and other quantiles determined in this case can be applied in two-group comparison studies. The obtained results can be used as a diagnostic tool for chemotherapy patients.

Investigation of the bystander effect in CHO-K1 cells.

AIM: Investigation of the bystander effect in Chinese Hamster Ovary cells (CHO-K1) co-cultured with cells irradiated in the dose range of 0.1-4 Gy of high LET 12C ions and X-rays. BACKGROUND: The radiobiological effects of charged heavy particles on a cellular or molecular level are of fundamental importance in the field of biomedical applications, especially in hadron therapy and space radiation biology. MATERIALS AND METHODS: A heavy ion 12C beam from the Heavy Ion Laboratory of the University of Warsaw (HIL) was used to irradiate CHO-K1 cells. Cells were seeded in Petri dishes specially designed for irradiation purposes. Immediately after irradiation, cells were transferred into transwell culture insert dishes to enable co-culture of irradiated and non-irradiated cells. Cells from the membrane and well shared the medium but could not touch each other. To study bystander effects, a clonogenic survival assay was performed. RESULTS: The survival fraction of cells co-cultured with cells irradiated with 12C ions and X-rays was not reduced. CONCLUSIONS: The bystander effect was not observed in these studies.

The dose-response relationship for dicentric chromosomes and γ-H2AX foci in human peripheral blood lymphocytes: influence of temperature during exposure and intra- and inter-individual variability of donors.

PURPOSE: Hypothermia during in vitro irradiation of human peripheral blood lymphocytes (PBL) affects the level of chromosome aberrations. The molecular mechanisms of this phenomenon are not fully understood. The aim of our study was to examine the effect of hypothermia on the dose-response relationship for dicentric chromosomes and the level of γ-H2AX (phosphorylated histone H2AX) foci. In addition, the inter- and intra-individual variability was assessed in relation to temperature. MATERIALS AND METHODS: PBL were kept at 0.8, 20 and 37°C and then exposed to gamma-rays (from 0-3 Gy). Dicentric chromosomes were scored in first post-treatment mitoses. γ-H2AX foci were scored 15, 30, 60, 120 min and 24 h post irradiation. RESULTS: Our results revealed that the frequency of dicentric chromosomes in cells exposed at 37°C to gamma-rays was higher than after exposure at 0.8 and 20°C. No effect of temperature was observed on the number of γ-H2AX foci as well as on the intra- and inter-individual variations of the dicentric yield and the number of γ-H2AX foci. CONCLUSIONS: Temperature at exposure to ionizing radiation has a pronounced effect on the level of cytogenetic damage but not γ-H2AX foci.

Micronuclei in human peripheral blood lymphocytes exposed to mixed beams of X-rays and alpha particles.

The purpose of this study was to analyse the cytogenetic effect of exposing human peripheral blood lymphocytes (PBL) to a mixed beam of alpha particles and X-rays. Whole blood collected from one donor was exposed to different doses of alpha particles ((241)Am), X-rays and a combination of both. All exposures were carried out at 37 °C. Three independent experiments were performed. Micronuclei (MN) in binucleated PBL were scored as the endpoint. Moreover, the size of MN was measured. The results show that exposure of PBL to a mixed beam of high and low linear energy transfer radiation led to significantly higher than expected frequencies of MN. The measurement of MN size did not reveal any differences between the effect of alpha particles and mixed beam. In conclusion, a combined exposure of PBL to alpha particles and X-rays leads to a synergistic effect as measured by the frequency of MN. From the analysis of MN distributions, we conclude that the increase was due to an impaired repair of X-ray-induced DNA damage.

Characterisation of a setup for mixed beam exposures of cells to 241Am alpha particles and X-rays.

Exposure of humans to mixed fields of high- and low-linear energy transfer (LET) radiation occurs in many situations-for example, in urban areas with high levels of indoor radon as well as background gamma radiation, during airplane flights or certain forms of radiation therapy. From the perspective of health risk associated with exposure to mixed fields, it is important to understand the interactions between different radiation types. In most cellular investigations on mixed beams, two types of irradiations have been applied sequentially. Simultaneous irradiation is the desirable scenario but requires a dedicated irradiation facility. The authors have constructed a facility where cells can be simultaneously exposed to (241)Am alpha particles and 190-kV X-rays at 37°C. This study presents the technical details and the dosimetry of the setup, as well as validates the performance of the setup for clonogenic survival in AA8 Chinese hamster ovary cells. No significant synergistic effect was observed. The relative biological effectiveness of the alpha particles was 2.56 for 37 % and 1.90 for 10 % clonogenic survival.

Bone metastases diagnosis possibilities in studies with the use of 18F-NaF and 18F-FDG.

Scintigraphy of the skeletal system is performed mainly with the use of 99mTc-MDP. There are other radiopharmaceuticals showing affinity to bone lesions: 99mTc-MIBG, 201Tl, 131I (in the case of papillary or follicular thyroid cancers), and 99mTc (V) DMSA (in the case of medullary cancer). Currently, positron emitted radioisotopes are also used in clinical practice. It seems that 18F-NaF/PET is a highly sensitive and specific modality for detection of metastases and other bone lesions. Certain data concerning the role of 18F-FDG, 18F-choline, or 68Ga-DOTATATE can be found. The aim of this article is to review the role of 18F-NaF and 18F-FDG in diagnosis of bone metastases.

The yield of radiation-induced micronuclei in early and late-arising binucleated cells depends on radiation quality.

There are conflicting data regarding the effect of culturing time of human peripheral blood lymphocytes on the yield of chromosomal aberrations induced by sparsely ionising radiation in the G0 phase of the cell cycle. While some authors find that the yield of aberrations does not change with time, others find increased frequencies of aberrations with harvesting time. The reasons for the conflicting results are not known, but the majority of studies were performed with lymphocytes of a single donor collected at one time point. We performed a study to verify if individual variability could be a confounding factor. As a positive control, lymphocytes were also exposed to high LET radiation (neutrons and alpha-rays), where an effect of harvesting time on the level of damage is expected to be seen. Blood was drawn from a total of 8 donors at two time points and exposed to X-rays, 6 MeV neutrons or alpha particles generated by an Am-241 source. Whole blood cultures were set up and micronuclei (Mn) were scored in binucleated cells harvested after 72, 96 and 120 h of culture time. The results show that in lymphocytes exposed to X-rays, the frequency of Mn was generally not influenced by the culture time while for both neutrons and alpha particles consistently increased micronucleus frequencies with culture time were detected. Some individual variability was detected and the conflicting results regarding the relationship between the yield of cytogenetic damage and lymphocyte culture time can, at least partly, be due to this variability.

Biological effectiveness of (12)C and (20)Ne ions with very high LET.

PURPOSE: To determine the relationship between the relative biological effectiveness (RBE) for cell inactivation and linear energy transfer (LET) in the Bragg peak region of (12)C and (20)Ne ions. MATERIALS AND METHODS: Chinese hamster ovary (CHO-K1) cells were exposed to high LET (12)C (33.2 MeV, 20.3 MeV, 9.1 MeV at cell entrance) and (20)Ne ions (56.2 MeV, 34.7 MeV, 15 MeV at cell entrance) and to low LET x-rays. Technical details of the irradiation facility are presented which is based on the Monte Carlo simulation of the lateral spread of heavy ions as a result of the multiscattering small-angle process in physical conditions of the experimental set-up. RESULTS: RBE has been measured for LET values close to the Bragg peak maximum, i.e., 440-830 keV/microm for (12)C and for 1020-1600 keV/microm for (20)Ne ions. RBE values at several levels of survival were estimated and were found to decrease with increasing LET. The inactivation cross sections were calculated from the final slope of dose-response curves and were found to increase with increasing LET. CONCLUSIONS: The RBE decreases with increasing LET in the range between 440 and 1600 keV/microm for the two types of radiations forming a single line when plotted together, pointing towards LET as the single determinant of RBE. The inactivation cross section describing the killing efficiency of a single particle at the end of particle range comes close to the size of the cell nucleus.

Trace element concentration distributions in breast, lung and colon tissues.

The concentrations of Fe, Cu, Zn and Se in cancerous and benign tissues of breast, lung and intestine (colon) have been determined. In the cases when the element concentration has not been determined in all samples the Kaplan-Meier method has been used for the reconstruction of the original concentration distributions and estimation of the true mean concentrations and medians. Finally, the log-rank test has been applied to compare the elemental concentration distributions between cancerous and benign tissues of the same organ, between cancerous tissues and between benign tissues taken from different organs. Comparing benign and malignant neoplastic tissues, statistically significant differences have been found between Fe and Se concentration distributions of breast as well as for Cu and Zn in the case of lung tissues and in the case of colon tissues for Zn. The concentrations of all elements have been found to be statistically different in cancer tissues as well as in benign ones when comparing the different organs, i.e. groups 'breast-colon' and 'breast-lung'. Concentrations of Fe and Cu have been found to be statistically different in lung and colon cancerous tissues. For benign tissues of lung and colon a statistically significant difference has been found only for Zn.