Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures.

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (60Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters ß/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

Cytogenetic Damage Induced by Radioiodine Therapy: A Follow-Up Case Study.

The risk of toxicity attributable to radioiodine therapy (RIT) remains a subject of ongoing research, with a whole-body dose of 2 Gy proposed as a safe limit. This article evaluates the RIT-induced cytogenetic damage in two rare differentiated thyroid cancer (DTC) cases, including the first follow-up study of a pediatric DTC patient. Chromosome damage in the patient's peripheral blood lymphocytes (PBL) was examined using conventional metaphase assay, painting of chromosomes 2, 4, and 12 (FISH), and multiplex fluorescence in situ hybridization (mFISH). Patient 1 (female, 1.6 y.o.) received four RIT courses over 1.1 years. Patient 2 (female, 49 y.o.) received 12 courses over 6.4 years, the last two of which were examined. Blood samples were collected before and 3-4 days after the treatment. Chromosome aberrations (CA) analyzed by conventional and FISH methods were converted to a whole-body dose accounting for the dose rate effect. The mFISH method showed an increase in total aberrant cell frequency following each RIT course, while cells carrying unstable aberrations predominated in the yield. The proportion of cells containing stable CA associated with long-term cytogenetic risk remained mostly unchanged during follow-up for both patients. A one-time administration of RIT was safe, as the threshold of 2 Gy for the whole-body dose was not exceeded. The risk of side effects projected from RIT-attributable cytogenetic damage was low, suggesting a good long-term prognosis. In rare cases, such as the ones reviewed in this study, individual planning based on cytogenetic biodosimetry is strongly recommended.

Modeling of chromosome aberration response functions induced by particle beams with different LET.

This study is based on our already published experimental data (Kowalska et al. in Radiat Environ Biophys 58:99-108, 2019) and is devoted to modeling of chromosome aberrations in human lymphocytes induced by 22.1 MeV/u 11B ions, 199 MeV/u 12C ions, 150 MeV and spread-out Bragg peak (SOBP) proton beams as well as by 60Co γ rays. The curvature of the dose-effect curves determined by the linear-quadratic model was considered in the frame of a simple analytical approach taking into account increase in the irradiation dose due to overlapping interaction regions of ion tracks. The model enabled to estimate effective interaction radius which could be compared with the physical expectations. The results were also compared to the Amorphous Track Structure Model of Katz which allows to get some additional information about the ion track structure. The analysis showed that the curvature of the experimental dose-effect curves mainly results from highly efficient repair processes of the DNA damage.

Production and distribution of chromosome aberrations in human lymphocytes by particle beams with different LET.

We investigated induction of chromosome aberrations (CA) in human lymphocytes when exposed to 150 MeV and spread out Bragg peak (SOBP) proton beams, and 199 MeV/u carbon beam which are currently widely used for cancer treatment and simultaneously are important components of cosmic radiation. For a comparison, the boron ions of much lower energy 22 MeV/u and a 60Co γ rays were used. Dose-effect curves as well as the distributions of CA were studied using Poisson and Neyman type A statistics. Systematics of experimentally determined parameters, their dependence on applied doses and irradiation quality are presented.

Modeling cell response to low doses of photon irradiation--Part 1: on the origin of fluctuations.

Intra- and inter-individual variability is a well-known aspect of biological responses of cells observed at low doses of radiation, whichever the phenomenon considered (adaptive response, bystander effects, genomic instability, etc.). There is growing evidence that low-dose phenomena are related to cell mechanisms other than DNA damage and misrepair, meaning that other cellular structures may play a crucial role. Therefore, in this study, a series of calculations at low doses was carried out to study the distribution of specific energies from different irradiation doses (3, 10 and 30 cGy) in targets of different sizes (0.1, 1 and 10 µm) corresponding to the dimensions of different cell structures. The results obtained show a strong dependence of the probability distributions of specific energies on the target size: targets with dimensions comparable to those of the cell show a Gaussian-like distribution, whereas very small targets are very likely to not be hit. A statistical analysis showed that the level of fluctuations in the fraction of aberrant cells is only related to the fraction of aberrant cells and the number of irradiated cells, regardless of, for instance, the heterogeneity in cell response.

Modeling cell response to low doses of photon irradiation: Part 2--application to radiation-induced chromosomal aberrations in human carcinoma cells.

The biological phenomena observed at low doses of ionizing radiation (adaptive response, bystander effects, genomic instability, etc.) are still not well understood. While at high irradiation doses, cellular death may be directly linked to DNA damage, at low doses, other cellular structures may be involved in what are known as non-(DNA)-targeted effects. Mitochondria, in particular, may play a crucial role through their participation in a signaling network involving oxygen/nitrogen radical species. According to the size of the implicated organelles, the fluctuations in the energy deposited into these target structures may impact considerably the response of cells to low doses of ionizing irradiation. Based on a recent simulation of these fluctuations, a theoretical framework was established to have further insight into cell responses to low doses of photon irradiation, namely the triggering of radioresistance mechanisms by energy deposition into specific targets. Three versions of a model are considered depending on the target size and on the number of targets that need to be activated by energy deposition to trigger radioresistance mechanisms. These model versions are applied to the fraction of radiation-induced chromosomal aberrations measured at low doses in human carcinoma cells (CAL51). For this cell line, it was found in the present study that the mechanisms of radioresistance could not be triggered by the activation of a single small target (nanometric size, 100 nm), but could instead be triggered by the activation of a large target (micrometric, 10 µm) or by the activation of a great number of small targets. The mitochondria network, viewed either as a large target or as a set of small units, might be concerned by these low-dose effects.

Chromosome aberration measurements in mitotic and G2-PCC lymphocytes at the standard sampling time of 48 h underestimate the effectiveness of high-LET particles.

The relationship between heavy-ion-induced cell cycle delay and the time-course of aberrations in first-cycle metaphases or prematurely condensed G(2)-cells (G(2)-PCC) was investigated. Lymphocytes of the same donor were irradiated with X-rays or various charged particles (carbon, iron, xenon, and chromium) covering an LET range of 2-3,160 keV/µm. Chromosome aberrations were measured in samples collected at 48, 60, 72, and 84 h postirradiation. Linear-quadratic functions were fitted to the data, and the fit parameters α and ß were determined. At any sampling time, α values derived from G(2)-cells were higher than those from metaphases. The α value derived from metaphase analysis at 48 h increased with LET, reached a maximum around 155 keV/µm, and decreased with a further rise in LET. At the later time-points, higher α values were estimated for particles with LET > 30 keV/µm. Estimates of α values from G(2)-cells showed a similar LET dependence, yet the time-dependent increase was less pronounced. Altogether, our data demonstrate that heavily damaged lymphocytes suffer a prolonged G(2)-arrest that is clearly LET dependent. For this very reason, the standard analysis of aberrations in metaphase cells 48 h postirradiation will considerably underestimate the effectiveness of high-LET radiation. Scoring of aberrations in G(2)-PCC at 48 h as suggested by several authors will result in higher aberration yields. However, when particles with a very high-LET value (LET > 150 keV/µm) are applied, still a fraction of multiple damaged cells escape detection by G(2)-analysis 48 h postirradiation.

Chromosome Aberrations in Lymphocytes of Patients Undergoing Radon Spa Therapy: An Explorative mFISH Study.

In the present exploratory study, we aim to elucidate the action of radon in vivo and to assess the possible health risks. Chromosome aberrations were analyzed in lymphocytes of two patients (P1, P2) undergoing radon spa therapy in Bad Steben (Germany). Both patients, suffering from painful chronic degenerative disorders of the spine and joints, received nine baths (1.2 kBq/L at 34 °C) over a 3-week period. Chromosome aberrations were analyzed before and 6, 12 and 30 weeks after the start of therapy using the high-resolution multiplex fluorescence in situ hybridization (mFISH) technique. For comparison, the lymphocytes from two healthy donors (HD1, HD2) were examined. P1 had a higher baseline aberration frequency than P2 and both healthy donors (5.3 ± 1.3 vs. 2.0 ± 0.8, 1.4 ± 0.3 and 1.1 ± 0.1 aberrations/100 analyzed metaphases, respectively). Complex aberrations, biomarkers of densely ionizing radiation, were found in P1, P2 and HD1. Neither the aberration frequency nor the fraction of complex aberrations increased after radon spa treatment, i.e., based on biological dosimetry, no increased health risk was found. It is worth noting that a detailed breakpoint analysis revealed potentially clonal aberrations in both patients. Altogether, our data show pronounced inter-individual differences with respect to the number and types of aberrations, complicating the risk analysis of low doses such as those received during radon therapy.

Complex exchanges are responsible for the increased effectiveness of C-ions compared to X-rays at the first post-irradiation mitosis.

The purpose of the present study was to investigate as to what extent differences in the linear energy transfer (LET) are reflected at the chromosomal level. For this study human lymphocytes were exposed to 9.5 MeV/u C-ions (1 or 2 Gy, LET=175 keV/microm) or X-rays (1-6 Gy), harvested at 48, 72 or 96 h post-irradiation and aberrations were scored in first cycle metaphases using 24 color fluorescence in situ hybridization (mFISH). Additionally, in selected samples aberrations were measured in prematurely condensed G2-phase cells. Analysis of the time-course of aberrations in first cycle metaphases showed a stable yield of simple and complex exchanges after X-ray irradiation. In contrast, after C-ion exposure the yields profoundly increased with harvesting time complicating the estimation of the frequency of aberrations produced by high LET particles within the entire cell population. This is especially true for the yield of complex exchanges. Complex aberrations dominate the aberration spectrum produced by C-ions. Their fraction was about 50% for the two measured doses. In contrast, isodoses of X-rays induced smaller proportions of complex aberrations (i.e. 5% and 15%, respectively). For both radiation qualities the fraction of complexes did not change with harvesting time. As expected from the different dose deposition of high and low LET radiation, complex exchanges produced by high LET C-ions involved more breaks and more chromosomes than those induced by isodoses of X-rays. Noteworthy, C-ions but not X-rays induced a small number of complex chromatid-isochromatid exchanges that are not expected for cells exposed in the G0-phase. The results obtained so far for cells arrested in G2-phase confirm these patterns. Altogether our data show that the increased effectiveness of C-ions for the induction of aberrations in first cycle cells is determined by complex exchanges, whereas for simple exchanges the relative biological effectiveness is about one.

Radiation-induced premature senescence is associated with specific cytogenetic changes.

In the present study, we set out to investigate cytogenetic changes in the progeny of two normal human fibroblast cell strains after exposure to sparsely or densely ionizing irradiation (X-rays or 9.8 MeV u(-1) carbon ions). The cells were regularly subcultured up to senescence. The transition to senescence was determined by measurement of population doubling numbers and senescence associated (SA) beta-galactosidase activity. Chromosomal changes (structural aberrations, tetraploidy) were investigated by solid staining. In temporal proximity to senescence, we observed for all populations of the two fibroblasts cell strains an increase in the fraction of cells with structural and numerical aberrations. The observed changes in the yield of structural chromosomal aberrations were similar for the progeny of controls and irradiated cells, except that a previous irradiation with a high, fractionated X-ray dose resulted in a stronger increase. Noteworthy, delayed tetraploidy in the descendants of irradiated cells exceeded the level in control cells. In addition, tetraploidy and the time of onset of senescence were significantly correlated for all populations, regardless of a preceding radiation exposure. However, the time of the onset of senescence depends on previous exposure to radiation. We conclude that the occurrence of tetraploidy is associated with senescence independently of exposure to radiation.

Biomedical Research Programs at Present and Future High-Energy Particle Accelerators.

Biomedical applications at high-energy particle accelerators have always been an important section of the applied nuclear physics research. Several new facilities are now under constructions or undergoing major upgrades. While the main goal of these facilities is often basic research in nuclear physics, they acknowledge the importance of including biomedical research programs and of interacting with other medical accelerator facilities providing patient treatments. To harmonize the programs, avoid duplications, and foster collaboration and synergism, the International Biophysics Collaboration is providing a platform to several accelerator centers with interest in biomedical research. In this paper, we summarize the programs of various facilities in the running, upgrade, or construction phase.

Modeling radiation-induced cell cycle delays.

Ionizing radiation is known to delay the cell cycle progression. In particular after particle exposure significant delays have been observed and it has been shown that the extent of delay affects the expression of damage, such as chromosome aberrations. Thus, to predict how cells respond to ionizing radiation and to derive reliable estimates of radiation risks, information about radiation-induced cell cycle perturbations is required. In the present study we describe and apply a method for retrieval of information about the time-course of all cell cycle phases from experimental data on the mitotic index only. We study the progression of mammalian cells through the cell cycle after exposure. The analysis reveals a prolonged block of damaged cells in the G2 phase. Furthermore, by performing an error analysis on simulated data valuable information for the design of experimental studies has been obtained. The analysis showed that the number of cells analyzed in an experimental sample should be at least 100 to obtain a relative error <20%.

Relationship between radioadaptive response and individual radiosensitivity to low doses of gamma radiation: an extended study of chromosome damage in blood lymphocytes of three donors.

PURPOSE: Our study aimed at evaluating: 1) whether well-established variability in radioadaptive response (AR) in various donor blood lymphocytes might be attributed to inter-individual differences in radiosensitivity to different low dose levels; 2) whether AR is reproducibly present over time in the lymphocytes of AR-positive individuals. Experimental procedure: Whole blood samples of three donors were exposed to low doses (2-30 cGy) of γ-radiation alone (G0 phase) or followed by a 1 Gy challenge dose (late S/early G2 phase), and chromosome aberration were scored to assess the dose-response relationship and adaptive response, correspondingly. Three experiments were performed on blood samples of the same donors at six month intervals. RESULTS: Significant differences in dose response relationship for blood lymphocytes were found among individuals. In most cases, the donors exhibited initial low-dose hypersensitivity (HRS) followed by an increase in radioresistance (IRR). AR could be successfully induced by some particular priming doses in the lymphocytes of each donor; however, the doses resulting in a protective response were quite different for all three donors. These protective doses could equally belong to either HRS or IRR region on the individual dose-response curves. In most cases, no clear AR outcome dependence on the priming dose was found at all. Moreover, pre-exposure to the same low dose could result in opposite effects in the lymphocytes of the same donor in different experiments. CONCLUSIONS: AR variability in human lymphocytes is not attributed to variation in radiosensitivity among individuals and is more drastic than was believed. It seems doubtful that AR is a universal phenomenon which has a consistent impact on the effects of radiation exposure on humans.

Persistence of radiation-induced aberrations in patients after radiotherapy with C-ions and IMRT.

BACKGROUND AND PURPOSE: Chromosomal aberrations in peripheral blood lymphocytes are a biomarker for radiation exposure and are associated with an increased risk for malignancies. To determine the long-term cytogenetic effect of radiotherapy, we analyzed the persistence of different aberration types up to 2.5 years after the treatment. MATERIALS AND METHODS: Cytogenetic damage was analyzed in lymphocytes from 14 patients that had undergone C-ion boost + IMRT treatment for prostate cancer. Samples were taken immediately, 1 year and 2.5 years after therapy. Aberrations were scored using the multiplex fluorescence in situ hybridization technique and grouped according to their transmissibility to daughter cells. RESULTS: Dicentric chromosomes (non-transmissible) and translocations (transmissible) were induced with equal frequencies. In the follow-up period, the translocation yield remained unchanged while the yield of dicentrics decreased to ≈40% of the initial value (p = 0.011 and p = 0.001 for 1 and 2.5 years after compared to end of therapy). In 2 patients clonal aberrations were observed; however they were also found in samples taken before therapy and thus were not radiotherapy induced. CONCLUSION: The shift in the aberrations spectrum towards a higher fraction of translocations indicates the exposure of hematopoietic stem and progenitor cells underlining the importance of a careful sparing of bone marrow during radiotherapy to minimize the risk for secondary cancers.

Interrelation amongst differentiation, senescence and genetic instability in long-term cultures of fibroblasts exposed to different radiation qualities.

BACKGROUND AND PURPOSE: The goal of the present study was to investigate aging and genetic instability in the progeny of human fibroblasts exposed to X-rays and carbon ions. MATERIALS AND METHODS: Following irradiation, cells were regularly subcultured until senescence. At selected time-points BrdU-labelling index, expression of cell cycle related proteins, cell differentiation pattern and chromosome aberrations were assessed. RESULTS: After exposure, an immediate cell cycle arrest occurred followed by a period of a few weeks where premature differentiation and senescence were observed. In all cultures cycling cells expressing low levels of cell cycle inhibiting proteins were present and finally dominated the populations. About 5months after exposure, the cellular and molecular changes attributed to differentiation and senescence reappeared and persisted. Concurrently, genetic instability was observed, but the aberration yields and types differed between repeated experiments. The descendants of cells exposed to carbon ions did not senesce earlier and displayed a similar rate of genetic instability as the X-ray progeny. For high doses an impaired cell cycle regulation and extended life span was observed, but finally cell proliferation ceased in all populations. CONCLUSIONS: The descendants of irradiated fibroblasts undergo stepwise senescence and differentiation. Genetic instability is frequent and an extension of the life span may occur.

Ionizing Radiation Alters Human Embryonic Stem Cell Properties and Differentiation Capacity by Diminishing the Expression of Activin Receptors.

Exposure of the embryo to ionizing radiation (IR) is detrimental as it can cause genotoxic stress leading to immediate and latent consequences such as functional defects, malformations, or cancer. Human embryonic stem (hES) cells can mimic the preimplantation embryo and help to assess the biological effects of IR during early development. In this study, we describe the alterations H9 hES cells exhibit after X-ray irradiation in respect to cell cycle progression, apoptosis, genomic stability, stem cell signaling, and their capacity to differentiate into definitive endoderm. Early postirradiation, hES cells responded with an arrest in G2/M phase, elevated apoptosis, and increased chromosomal aberrations. Significant downregulation of stem cell signaling markers of the TGF beta-, Wnt-, and Hedgehog pathways was observed. Most prominent were alterations in the expression of activin receptors. However, hES cells responded differently depending on the culture conditions chosen for maintenance. Enzymatically passaged cells were less sensitive to IR than mechanically passaged ones showing fewer apoptotic cells and fewer changes in the stem cell signaling 24 h after irradiation, but displayed higher levels of chromosomal aberrations. Even though many of the observed changes were transient, surviving hES cells, which were differentiated 4 days postirradiation, showed a lower efficiency to form definitive endoderm than their mock-irradiated counterparts. This was demonstrated by lower expression levels of SOX17 and microRNA miR-375. In conclusion, hES cells are a suitable tool for the IR risk assessment during early human development. However, careful choice of the culture methods and a vigorous monitoring of the stem cell quality are mandatory for the use of these cells. Exposure to IR influences the stem cell properties of hES cells even when immediate radiation effects are overcome. This warrants consideration in the risk assessment of radiation effects during the earliest stages of human development.

Impact of Charged Particle Exposure on Homologous DNA Double-Strand Break Repair in Human Blood-Derived Cells.

Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment.

Chromosome fragmentation after irradiation with C ions.

The premature chromosome condensation (PCC) technique has been used to compare chromatin breakage and repair in non-cycling CHO-K1 cells following high LET (C ions) and low LET (X-rays) irradiation. For both radiation qualities the average initial number of excess PCC fragments increases linearly with dose. However, the frequency of chromatin breaks follows the pattern of energy deposition and at higher LET values reveals clustering due to the large number of ionizing events being concentrated in a small volume of the cell nucleus. In consequence, the distribution of PCC chromosomes plus excess fragments among cells has followed Poisson statistics after X-ray irradiation while the overdispersion of the frequencies has been observed after C-irradiation indicating that a single particle traversal through a cell nucleus can produce multiple chromatin lesions.

Relationship between aberration yield and mitotic delay in human lymphocytes exposed to 200 MeV/u Fe-ions or X-rays.

The time-course of Fe-ion (200 MeV/u, 440 keV/microm) and X-ray induced chromosomal damage was investigated in human lymphocytes. After cells were exposed in G0 and stimulated to grow, aberrations were measured in first-cycle metaphases harvested 48, 60 and 72 h post-irradiation. Additionally, lesions were analysed in G2 and mitotic (M) cells collected at 48 h using calyculin A-induced premature chromosome condensation (PCC). Following X-irradiation, similar aberration yields were found in all of the samples scored. In contrast, after Fe-ion exposure a drastic increase in the aberration frequency with sampling time was observed, i.e. cells arriving late at the first mitosis carried more aberrations than those arriving at earlier times. The PCC data indicate that the delayed entry of heavily damaged cells into mitosis observed after Fe-ion irradiation resulted from a prolonged arrest in G2. Altogether these experiments provide further evidence that in the case of high-LET exposure cell-cycle delays of severely damaged cells have to be taken into account for any meaningful quantification of chromosomal damage and, consequently, for an accurate estimate of the RBE.

Optimal self-calibration of tomographic reconstruction parameters in whole-body small animal optoacoustic imaging.

In tomographic optoacoustic imaging, multiple parameters related to both light and ultrasound propagation characteristics of the medium need to be adequately selected in order to accurately recover maps of local optical absorbance. Speed of sound in the imaged object and surrounding medium is a key parameter conventionally assumed to be uniform. Mismatch between the actual and predicted speed of sound values may lead to image distortions but can be mitigated by manual or automatic optimization based on metrics of image sharpness. Although some simple approaches based on metrics of image sharpness may readily mitigate distortions in the presence of highly contrasting and sharp image features, they may not provide an adequate performance for smooth signal variations as commonly present in realistic whole-body optoacoustic images from small animals. Thus, three new hybrid methods are suggested in this work, which are shown to outperform well-established autofocusing algorithms in mouse experiments in vivo.