M. A. Tabocchini

RBE-LET relationships for cell inactivation and mutation induced by low energy protons in V79 cells: further results at the LNL facility.

PURPOSE RBE-LET relationships for cell inactivation and hprt mutation in V79 cells have been studied with mono-energetic low-energy proton beams at the radiobiological facility of the INFN-Laboratori Nazionali di Legnaro (LNL), Padova, Italy. MATERIALS AND METHODS V79 cells were irradiated in mono-layer on mylar coated stainless steel petri dishes, in air. Inactivation data were obtained at 7.7, 34.6 and 37.8 keV/microm and hprt mutation was studied at 7 7 and 37.8 keV/microm. Additional data were also collected for both the end points with the proton LET already considered in our previous publications, namely 11.0, 20.0 and 30.5 keV/microm. RESULTS A maximum in the RBE-LET relationship for cell inactivation was found at around 31 keV/microm, while the RBE for mutation induction increased continuously with LET. CONCLUSIONS The proton RBE-LET relationship for cell inactivation is shifted to lower LET values compared with that for heavier ions. For mutation induction, protons of LET equal to 7.7keV/microm gave an RBE value comparable with that obtained by helium ions of about 20 keV/microm. Mutagenicity and lethality caused by protons at low doses in the LET range 7.7-31 keV/microm were proportional, while the data at 37.8 keV/microm suggest that this may not hold at higher LET values.

RBE-LET Relationship for the Survival of V79 Cells Irradiated with Low Energy Protons

The survival of V79 Chinese hamster cells irradiated with proton beams with energies of 0.73, 0.84, 1.16, 1.70 and 3.36 MeV, corresponding to LET values, evaluated at the cell midplane, of 34.5, 30.4, 23.9, 17.8 and 10.6 keV/micron respectively, have been studied in the dose range 0.5-6.0 Gy. As a reference, the survival curve obtained with 200 kV X-rays was used. The initial shoulder, typical of survival curves obtained with sparsely ionizing radiation, decreases as the LET increases and completely disappears at 23.9 keV/micron. This value corresponds to the maximum of the RBE, expressed as the initial slope ratio. In the energy range we have considered, the RBEs for protons are higher than those reported for other ions of comparable LET and the RBE-LET relationship results shifted to lower LET values. Our data seem to indicate that the RBE-LET curve depends on the type of radiation and this could imply that LET is not a good reference for the dose-effectiveness relationship.

Inactivation and Mutation Induction in V79 Cells by Low Energy Protons: Re-evaluation of the Results at the LNL Facility

During the upgrading of the radiobiological facility at the Laboratori Nazionali di Legnaro (LNL) we found that uncorrected values of the proton energy were used in the past. This circumstance prompted us to perform the re-evaluation of the physical parameters for all the proton beams used in our previous radiobiological investigations (Belli et al. 1987) and, subsequently, the re-evaluation of all our previous dose-response curves for inactivation and mutation induction (Belli et al. 1989, 1991). This re-evaluation leads to significant changes in the dose-response curves and in the RBE-LET relationships only at the two lowest energies (highest LET) used. These two points are not reliable for the identification of a peak in RBE-LET relationship for cell inactivation. In spite of that, the extent of the changes is not such as to modify the general conclusion previously drawn, pointing out that there is a LET range where protons are more effective than alpha-particles.

Direct Comparison of Biological Effectiveness of Protons and Alpha-particles of the Same LET. III. Initial Yield of DNA Double-strand Breaks in V79 Cells

The results reported form part of a series of experiments to substantiate and extend the findings by Belli et al. (1989) that protons are more biologically effective at cell killing than alpha-particles of the same LET. The irradiations were carried out using the Variable Energy Cyclotron (VEC) at the Harwell Laboratories. V79-4 Chinese hamster cells were exposed to alpha-particles and protons with LETs of 20 and 23 keV microns-1 in the dose range 40-150 Gy. X-rays were also used for comparison. Two methods were used for measurement of initial DNA double-strand breaks: sedimentation and DNA precipitation assays. The dose-response relationships were found to be well fitted by straight lines in all cases. With the sedimentation assay a slightly lower yield of dsb was found from protons than from alpha-particles of the same LET. The yield from X-rays was not significantly different from either. The precipitation assay showed similar yields of DNA damage from both particle types but significantly higher yields from X-rays. This may reflect a difference in the type of lesions scored by the two methods. Since the initial amount of dsb does not account for the observed differences in cellular response to radiations of different qualities, it is likely that these are related to the nature of the dsb (affecting reparability) or to the occurrence of other types of molecular damage.

Proton irradiation facility for radiobiological studies at a 7 MV Van de Graaff accelerator

Abstract This paper describes the proton irradiation facility built up at the 7 MV Van de Graaff accelerator of the INFN-Laboratori Nazionali di Legnaro for radiobiological studies. The system mainly consists of two vacuum chambers, a fast tantalum shutter for intercepting the beam and a beam line containing the beam collimator arrangement and ending with a thin window for the beam extraction in air. Each chamber houses a Ta beam collimator, an Au scatterer foil for the broadening and uniformization of the proton beam over the cell samples and a solicon surface barrier detector for beam monitoring purposes. The test of the facility performance is discussed. With the double scattering system a counting rate in air as low as hundreds of protons per second and a fluence heterogeneity less than 5% within a circular field of 15 mm diameter were obtained. The first results of survival studies of Chinese hamster lung cells (V79-753B) irradiated with a 1.1 MeV proton beam are presented. For comparison, results obtained irradiating the cells with 200 kV X-rays are reported, too.

DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study.

Purpose : To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). Materials and methods : V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). Results : The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. Conclusions : The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.PURPOSE To study the induction and the time-course of rejoining of DNA double strand breaks (DSB) in V79 cells irradiated with light ions with different linear energy transfer (LET). MATERIALS AND METHODS V79 cells were irradiated in monolayer with monoenergetic proton, deuteron, helium-3 or helium-4 ion beams, each at two different energy values. Gamma rays were used as reference radiation. DSB have been measured by constant field gel electrophoresis (CFGE). RESULTS The initial yield depended little on the particle type and LET. The amount of DSB left unrejoined for up to 2 h incubation time could be roughly described by a decreasing exponential function with a final plateau, although more complex functions cannot be excluded. Radiation quality had little effect on the rejoining rate but affected the plateau. The amount of residual DSB after 2 h was higher for densely than for sparsely ionizing radiation, and for the same particle was dependent on LET. The corresponding RBE ranged from 1.8 to 6.0. CONCLUSIONS The results support the hypothesis that complex, less reparable DSB are induced in higher proportion by light ions with respect to gamma-rays and that, for the same ion, increasing LET leads to an increase in this proportion.

Relationships Between Cell Killing, Mutation Induction and DNA Damage in X-irradiated V79 Cells: The Influence of Oxygen and DMSO

The relationships between cell killing, mutation induction and DNA double (dsb) and single (ssb) strand breaks have been studied in V79 cells irradiated with X-rays under oxic and anoxic conditions in the presence and in the absence of dimethylsulphoxide (DMSO). Curvilinear relationships were found between all pairs of endpoints, except for dsb versus ssb. Statistical analysis of experimental data has shown that in the absence of DMSO there is evidence of good correlations between cell killing, mutation induction and dsb in oxic and anoxic conditions. However, when DMSO was present, no significant correlation was found. In the presence of oxygen DMSO always exerts a protective effect while in anoxia it is generally much less protective and induces a strong sensitization with respect to mutation induction. Possibly DMSO acts not only as a radical scavenger but also as an agent inducing chromatin relaxation and/or under anoxia, forming highly mutagenic short-term radicals. The present data suggest that lethal and mutational events are at least partially independent and not proportional to the initial number of DNA breaks. This may imply that either other kinds of lesions are involved in cell lethality and mutability, or dose-dependent repair mechanisms of dsb have to be considered.

Direct Comparison of Biological Effectiveness of Protons and Alpha-particles of the Same LET. II. Mutation Induction at the HPRT Locus in V79 Cells

Mutation induction at the hprt locus has been studied in V79-4 Chinese hamster cells irradiated with mono-energetic protons and alpha-particles with LET of 20.3 and 23 keV microns-1. The mutation frequency increased linearly with the dose for all the four radiation qualities investigated, so that effectiveness for mutation induction could be expressed by the slope of the relevant curve. This effectiveness did not significantly change with the small change in LET of each kind of particle, while sizeable differences were found between particles. Protons were more effective in mutation induction than alpha-particles with the same LET by a factor of about 2. This finding is similar to, although slightly larger than, the factors 1.5-1.8 found for inactivation of the same cells in the same series of experiments.

Induction and Repair of DNA Double-Strand Breaks in Human Cells: Dephosphorylation of Histone H2AX and its Inhibition by Calyculin A

Abstract Antonelli, F., Belli, M., Cuttone, G., Dini, V., Esposito, G., Simone, G., Sorrentino, E. and Tabocchini, M. A. Induction and Repair of DNA Double-Strand Breaks in Human Cells: Dephosphorylation of Histone H2AX and its Inhibition by Calyculin A. Radiat. Res. 164, 514–517 (2005). Phosphorylation of histone H2AX at serine 139 (γ-H2AX) represents one of the earliest steps in DNA DSB signaling and repair, but the mechanisms of coupling this histone modification to DSB processing remain to be established. In this work, H2AX phosphorylation-dephosphorylation kinetics induced by low doses of γ rays in MRC-5 human fibroblasts was studied. The number of γ-H2AX foci increased rapidly, with the maximum reached 20 min after irradiation. Using calyculin A, a protein phosphatase inhibitor, no significant dephosphorylation was found in this time. At longer times, no further induction of γ-H2AX foci occurred. This indicates that the number of γ-H2AX foci scored at 20 min can be used as representative of the initial number of DSBs. Pulsed-field gel electrophoresis (PFGE) was also used to determine whether calyculin A-mediated inhibition of γ-H2AX dephosphorylation and DSB rejoining are independent phenomena. We found that the maintenance of the phosphate group at Ser 139 in γ-H2AX does not represent an obstacle for DSB rejoining. Preliminary experiments performed with 62 MeV/nucleon carbon ions have shown a longer persistence of γ-H2AX foci with respect to γ rays, consistent with the induction of damage that is more severe and difficult to repair.

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.