S. Gerardi

DNA DSB induced in human cells by charged particles and gamma rays: Experimental results and theoretical approaches

Purpose:To quantify the role played by radiation track structure and background fragments in modulating DNA fragmentation in human cells exposed to γ-rays and light ions. Materials and methods: Human fibroblasts were exposed in vitro to different doses (in the range from 40 – 200 Gy) of 60Co γ-rays and 0.84 MeV protons (Linear Energy Transfer, LET, in tissue 28.5 keV/μm). The resulting DNA fragments were scored under two electrophoretic conditions, in order to optimize separation in the size ranges 0.023 – 1.0 Mbp and 1.0 – 5.7 Mbp. In parallel, DNA fragmentation was simulated both with a phenomenological approach based on the “generalized broken-stick” model, and with a mechanistic approach based on the PARTRAC (acronym of PARticle TRACk) Monte Carlo code (1.32 MeV photons were used for the simulation of 60Co γ-rays). Results: For both γ-rays and protons, the experimental dose response in the range 0.023 – 5.7 Mbp could be approximated as a straight line, the slope of which provided a yield of (5.3 ± 0.4) • 10−9 Gy−1 bp−1 for γ-rays and (7.1 ± 0.6) • 10−9 Gy−1 bp−1 for protons, leading to a Relative Biological Effectiveness (RBE) of 1.3 ± 0.2. From both theoretical analyses it appeared that, while γ-ray data were consistent with double-strand breaks (DSB) random induction, protons at low doses showed significant deviation from randomness, implying enhanced production of small fragments in the low molecular weight part of the experimental range. The theoretical analysis of fragment production was then extended to ranges where data were not available, i.e. to fragments larger than 5.7 Mbp and smaller than 23 kbp. The main outcome was that small fragments (<23 kbp) are produced almost exclusively via non-random processes, since their number is considerably higher than that produced by a random insertion of DSB. Furthermore, for protons the number of these small fragments is a significant fraction (about 20%) of the total number of fragments; these fragments remain undetected in these experiments. Calculations for 3.3 MeV alpha particle irradiation (for which no experimental data were available) were performed to further investigate the role of fragments smaller than 23 kbp; in this case, besides the non-random character of their production, their number resulted to be at least as much as half of the total number of fragments. Conclusion: Comparison between experimental data and two different theoretical approaches provided further support to the hypothesis of an important role of track structure in modulating DNA damage. According to the theoretical approaches, non-randomness of fragment production was found for proton irradiation for the smaller fragments in the experimental size range and, in a significantly larger extent, for fragments of size less than 23 kbp, both for protons and alpha particles.

Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death.

The spatial distribution of radiation-induced DNA breaks within the cell nucleus depends on radiation quality in terms of energy deposition pattern. It is generally assumed that the higher the radiation linear energy transfer (LET), the greater the DNA damage complexity. Using a combined experimental and theoretical approach, we examined the phosphorylation-dephosphorylation kinetics of radiation-induced γ-H2AX foci, size distribution and 3D focus morphology, and the relationship between DNA damage and cellular end points (i.e., cell killing and lethal mutations) after exposure to gamma rays, protons, carbon ions and alpha particles. Our results showed that the maximum number of foci are reached 30 min postirradiation for all radiation types. However, the number of foci after 0.5 Gy of each radiation type was different with gamma rays, protons, carbon ions and alpha particles inducing 12.64 ± 0.25, 10.11 ± 0.40, 8.84 ± 0.56 and 4.80 ± 0.35 foci, respectively, which indicated a clear influence of the track structure and fluence on the numbers of foci induced after a dose of 0.5 Gy for each radiation type. The γ-H2AX foci persistence was also dependent on radiation quality, i.e., the higher the LET, the longer the foci persisted in the cell nucleus. The γ-H2AX time course was compared with cell killing and lethal mutation and the results highlighted a correlation between cellular end points and the duration of γ-H2AX foci persistence. A model was developed to evaluate the probability that multiple DSBs reside in the same gamma-ray focus and such probability was found to be negligible for doses lower than 1 Gy. Our model provides evidence that the DSBs inside complex foci, such as those induced by alpha particles, are not processed independently or with the same time constant. The combination of experimental, theoretical and simulation data supports the hypothesis of an interdependent processing of closely associated DSBs, possibly associated with a diminished correct repair capability, which affects cell killing and lethal mutation.

Transient Activation of the ALT Pathway in Human Primary Fibroblasts Exposed to High-LET Radiation

Abstract It is well established that high-LET radiations efficiently induce chromosome aberrations. However, data on the effect of protons on telomere maintenance, as involved in genomic stability, are scarce and contradictory. Here we demonstrate that high-LET protons induce telomere lengthening in human primary fibroblasts and that this elongation does not involve the telomerase enzyme, supporting the hypothesis that high-LET radiations are able to activate a telomerase-independent mechanism. In tumor cells that lack telomerase, one or more non-telomerase mechanisms for telomere maintenance are present, which are termed alternative lengthening of telomeres (ALT). Since ALT cells are characterized by recombinational events at telomeres, known as telomeric-sister chromatid exchanges (T-SCE), and colocalization of telomeres and premyelocytic leukemia protein (PML), we analyzed both T-SCE and PML. Our results show that high-LET protons induce a 2.5-fold increase of T-SCE and a colocalization of PML protein and telomeric DNA. Furthermore, our data show that the ALT pathway can be activated in human primary cells after induction of severe DNA damage. Thus, since telomeres are known to be involved in chromosome maintenance, the present work may contribute in the elucidation of the mechanism by which ionizing radiation induces genomic instability.

The role of telomere length modulation in delayed chromosome instability induced by ionizing radiation in human primary fibroblasts

Telomere integrity is important for chromosome stability. The main objective of our study was to investigate the relationship between telomere length modulation and mitotic chromosome segregation induced by ionizing radiation in human primary fibroblasts. We used X‐rays and low‐energy protons because of their ability to induce different telomeric responses. Samples irradiated with 4 Gy were fixed at different times up to 6 days from exposure and telomere length, anaphase abnormalities, and chromosome aberrations were analyzed. We observed that X‐rays induced telomere shortening in cells harvested at 96 hrs, whereas protons induced a significant increase in telomere length at short as well as at long harvesting times (24 and 96 hrs). Consistent with this, the analysis of anaphase bridges at 96 hrs showed a fourfold increase in X‐ray‐ compared with proton‐irradiated samples, suggesting a correlation between telomere length/dysfunction and chromosome missegregation. In line with these findings, the frequency of dicentrics and rings decreased with time for protons whereas it remained stable after X‐rays irradiation. Telomeric FISH staining on anaphases revealed a higher percentage of bridges with telomere signals in X‐ray‐treated samples than that observed after proton irradiation, thus suggesting that the aberrations observed after X‐ray irradiation originated from telomere attrition and consequent chromosome end‐to‐end fusion. This study shows that, beside an expected “early” chromosome instability induced shortly after irradiation, a delayed one occurs as a result of alterations in telomere metabolism and that this mechanism may play an important role in genomic stability. Environ. Mol. Mutagen. 54:172–179, 2013.

TELOMERE ALTERATIONS AND GENOMIC INSTABILITY IN LONG-TERM CULTURES OF NORMAL HUMAN FIBROBLASTS IRRADIATED WITH X RAYS AND PROTONS

Telomeres are the end of linear chromosomes, responsible for chromosome stability and cell viability. It is well known that radiations are able to induce chromosome instability but it has not yet been investigated whether telomere structure is affected by the radiation exposure and if radiations with different quality act in a different way on telomeres. The effect of radiations with different quality on telomere structure and chromosome instability was analysed in human primary fibroblasts exposed to X rays or low-energy protons (28.5 keV μm(-1)). Telomere length was evaluated at different harvesting times from 24 h up to 360 h (15 days), whereas chromosome instability was evaluated in terms of sister chromatid exchanges (SCEs) (48 h from irradiation) and chromosome painting (360 h from irradiation). Results indicated a delayed telomere lengthening 360 h after X-ray treatment, whereas protons were able to induce such a lengthening shortly from irradiation as well as at longer harvesting times. Data obtained from chromosome instability analysis indicated an increase of SCE frequency only after proton irradiation, but, on the contrary, at the longer harvesting time chromosome painting analysis displayed a higher frequency of aberrations after X-ray treatment, suggesting a role of selective process against highly damaged cells.

A Microcollimated Ion Beam Facility for Investigations of the Effects of Low-Dose Radiation

Abstract Gerardi, S., Galeazzi, G. and Cherubini, R. A Microcollimated Ion Beam Facility for Investigations of the Effects of Low-Dose Radiation. Radiat. Res. 164, 586–590 (2005). Charged-particle microbeams are unique tools to mimic low-dose exposure in vitro by delivering a defined number of particles to single mammalian cells down to only one particle per cell or group of cells. A horizontal single-ion microbeam facility has been built at the INFN-Laboratori Nazionali di Legnaro 7 MV Van de Graaff accelerator. Different light ions (1H+, 2H+, 3He2+, 4He2+) are available covering a wide range of LET from 7 to 150 keV/μm. Collimators of different geometries and materials have been tested, and beam spots 2–3 μm in diameter have been obtained using a tantalum disc. Cell visualization and recognition are performed with a phase-contrast optical microscope coupled with dedicated software. One unique characteristic of such a system is that neither cell staining nor UV light is used. Cells are automatically positioned on the beam spot through remotely controlled precision XY translation stages. A particle detector is positioned downstream of a specially designed petri dish to perform energy measurements and count particles crossing the cell. A particle counting rate of less than 1 ion/s can be reached. This feature, combined with a fast beam deflection system, ensures high reproducibility in administering a preset number of particles per cell.

Cyogenetics effects in AG01522 human primary fibroblasts exposed to low doses of radiations with different quality

Abstract Purpose: Biological effects produced by low doses of ionizing radiations, though relevant for the risk assessment, have not been fully elucidated. The aim of the present work was to evaluate cytogenetic endpoints, as telomere dysfunctions and chromosome instability in the low-dose range as a function of radiation quality. In particular, we analyzed whether the telomere length was modulated, as well as the involvement of telomeres in chromosomal alterations at anaphase, and the yield of stable simple and complex chromosome aberrations. Materials and methods: AG01522 human primary fibroblasts were irradiated with 0.1–1 Gy of X-rays, protons (28.5 keV/μm), and 4He2+ ions (62 keV/μm). Frequency of chromosome bridges carrying or not telomeric signals and telomere length were measured in irradiated samples up to 72 h. Moreover, chromosome instability was measured using multicolor fluorescence in situ hybridization (mFISH). Results: The results evidenced a linear energy transfer (LET)- and dose-dependent response in the frequency of anaphase bridges induction and in their persistence as a function of time. However, neither variation in telomere length and telomere loss, nor in the proportion of bridges bearing telomeric signals, was detected, thus indicating a minor role of telomeres in the generation of the radiation-induced chromosome bridges. Chromosome instability followed a linear-dependence with dose and LET, showing a far higher extent of complex translocations in helium-ion-irradiated cells than in proton- or X-ray-irradiated samples. Conclusions: Altogether, the results indicated the lack of telomere involvement in cytogenetic effects induced by low-dose ionizing radiation. On the contrary, chromosome aberration yield and spectrum were LET- and dose-dependent.

Single-ion microbeam as a tool for low-dose radiation effects investigations

Practical assessment of human radiation exposure risk deserves particular attention especially for low doses (and low dose rates), which concern environmental and occupational exposure. At these dose levels ionizing radiation exposures involve mainly isolated charged particle tracks, which strike individual cells at time intervals averaging from weeks to several years apart. Accelerator-based microbeam irradiation technique offers a unique tool to mimic such an exposure, allowing irradiating single cells individually with micrometer precision and with a preset number of charged particles down to one particle per cell. A horizontal single-ion microbeam facility for single-cell irradiations has been designed and set up at the INFN-LNL 7MV CN Van de Graaff accelerator. The light ion beam is collimated in air down to a section of 2-3µm in diameter by means of appropriate pinholes. Semi-automatic cell visualization and automatic cell positioning and revisiting system, based on an inverted phase contrast optical microscope and on X-Y translation stages with 0.1µm positioning precision, has been developed. An in-house-written software allows to control remotely the irradiation protocol. As a distinctive feature of the facility, cell recognition is performed without using fluorescent staining and UV light. Particle detection in air, behind the biological sample, is based on a silicon detector while in-air beam profile and precise hit position measurements are accomplished by a custom-made cooled-CCD camera and Solid State Nuclear Track detectors, respectively. A particle counting rate of less than 1 ion/sec can be reached.

Hyper-radiosensitivity and induced radioresistance and bystander effects in rodent and human cells as a function of radiation quality.

In the past two decades, a body of experimental evidences in vitro has shown the presence of a plethora of phenomena occurring after low-dose irradiation [including hypersensitivity and induced radioresistance (IRR), adaptive response, bystander effect (BE) and genomic instability], which might imply a non-linear behaviour of cancer risk curves in the low-dose region and question the validity of the linear no-threshold model for cancer risk assessment in such a dose region. In this framework, a systematic investigation have been undertaken on non-linear effects at low doses as a function of different radiation quality and cellular radiosensitivity and in terms of different biological end points. The present article reports the recent results on hyper-radiosensitivity and IRR and BE phenomena, in terms of clonogenic survival in V79 Chinese hamster cells and T98G human glioblastoma cells irradiated with protons and carbon ions with different energy, as a function of dose (and fluence).

mBAND and mFISH analysis of chromosomal aberrations and breakpoint distribution in chromosome 1 of AG01522 human fibroblasts that were exposed to radiation of different qualities.

High-resolution multicolour banding FISH (mBAND) and multiplex FISH (mFISH) were used to analyse the aberrations of chromosome 1 in irradiated-AG01522 human primary fibroblasts. The cells were exposed to 1Gy of a panel of radiation of different qualities, such as X-rays, low-energy protons (28keV/μm), helium-ions (62keV/μm) and carbon-ions (96 and 252keV/μm). mBAND and mFISH analysis in calyculin-A G2-condensed chromosome spreads allowed us to detect intra- and interchromosome aberrations involving chromosome 1, including simple and complex-type exchanges, inversions (both para- and pericentric ones), deletions and rings. The data indicate that the induction of chromosomal exchanges was influenced by both Linear energy transfer (LET) and particle types. Moreover, the complex-to-simple exchanges ratio (C-ratio) and interchromosome to intrachromosome exchanges ratio (F-ratio) were evaluated by mFISH and mBAND techniques, respectively. Our results indicate that the C-ratio is a more reliable marker of radiation quality, with values that increased linearly in an LET-dependent manner. In addition, by means of mBAND analysis, the distribution of radiation-induced breakpoints along chromosome 1 was analyzed and compared with the expected distributions of the breaks. The expected values were calculated assuming a random distribution of the breakpoints. The data indicate that, irrespective of the radiation that was used, the breakpoints were non-randomly distributed along chromosome 1. In particular, breaks in the pericentromeric region were encountered at a higher frequency than expected. A deeper analysis revealed that breaks were not located in the constitutive heterochromatin (G-bands 1p11/1q11 and 1q12), but rather in a region comprised between 1p11.2 and 1p22.1, which includes G-light and G-dark bands.