Radiation response of chemically derived mitochondrial DNA-deficient AG01522 human primary fibroblasts.

Mitochondria are the main cellular source of Reactive Oxygen Species (ROS). Alterations of mitochondrial metabolism and consequent loss of mitochondrial membrane potential may lead to redox imbalance and in turn to DNA damage, chromosomal instability and apoptosis. On the other hand, impaired mitochondrial functions may either exacerbate the detrimental effects of geno- and cytotoxic agents or may bring beneficial cellular responses. To study the role of mitochondria within this framework, AG01522 human primary fibroblasts were incubated with the mitochondrial polymerase γ inhibitor 2',3'-dideoxycytidine (ddC), leading to mitochondrial DNA (mtDNA) depletion and to mitochondrial dysfunctions. The successful treatment toward mtDNA depletion was confirmed by Complex-IV subunit I (COX-I) immunofluorescence and western blot assays. mtDNA-depleted cells and their counterparts were ultrastructurally characterized by transmission electron microscopy. mtDNA-depleted cells showed dramatic mitochondrial alterations such as fragmentation and cristae disruption along with a reduction of the mitochondrial membrane potential and elevated levels of ROS. Despite increased ROS levels, we did not find any difference in telomere length between ddC-treated and untreated cells. The spontaneous rate of DNA double-strand breaks (DSBs) and chromosome aberrations was significantly enhanced in mtDNA-depleted cells whereas the induction of DSBs by low-Linear Energy Transfer (LET) (X-rays; 7.7keV/µm protons) and high-LET radiations (28.5keV/µm protons) did not differ when compared with normal cells. However, in irradiated cells impaired mitochondrial functions seemed to bring beneficial cellular responses to the detrimental effect of radiations. In fact, after X-irradiation mtDNA-depleted cells show less remaining unrejoined DSBs than normal cells and furthermore a lower induction of cytogenetic damage. Overall, these data show that active mitochondrial functions are required for the proper maintenance of cellular genome stability in primary fibroblasts.

Decreased periprosthetic bone loss in patients treated with clodronate: a 1-year randomized controlled study.

The efficacy of clodronate to reduce bone loss around uncemented stems after total hip arthroplasty(THA) was evaluated. Ninety-one patients operated with uncemented THA were randomized to receive either intramuscular clodronate at a dose of 100 mg weekly for 12 months or no treatment. Periprosthetic and contralateral bone mineral density (BMD) scans were performed and biochemical markers of bone turnover measured at baseline and at 3, 6, and 12 months. At month 12, with the exception of Gruen zones 4 and 5, patients treated with clodronate showed less bone loss at all zones, reaching statistical significance (P\0.05) in Gruen zones 2 and 6 (difference of 6.6 and 5.9%, respectively). Analysis of data according to gender revealed sex-related differences in bone loss and efficacy of treatment. After 12 months, the difference in bone loss between treated and untreated women in five out of seven Gruen zones ranged from 6.2 to 13.3% (SS at zones 2 and 6), whereas comparison between treated and untreated men showed no BMD differences in all zones(P[0.05). Median percent changes in serum levels of markers of bone metabolism by gender were consistent with BMD changes. A 1-year treatment with intramuscular clodronate determined a significant reduction of bone loss after THA. This was mainly attributed to its greater efficacy in the female population, which is at higher risk for bone loss. This observation suggests the need for the characterization of high-risk subjects as potential candidates for prevention strategies.

TOWARDS THE USE OF NANODOSIMETRY TO PREDICT CELL SURVIVAL.

Experimental nanodosimetry aims to develop a new concept of radiation quality, based on the correlation between initial features of particle tracks and late biological outcome. A direct proportionality has been observed between the cumulative probability of measuring at least k ionisations within a nanometric volume and inactivation cross sections at specific survival levels. Based on this proportionality, physical quantities which are measurable at the nanometre level can be used to estimate the alpha and beta parameters of the linear-quadratic dose-response model, provided that two proportionality factors are determined in a reference radiation field. This work describes the procedure and first results applied to published data for V79 cell survival after irradiation with protons and carbon ions.

Telomere length in mammalian cells exposed to low- and high-LET radiations.

Telomeres are specialised nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. The loss of the ends of the chromosomes due to these un-rejoined double strand breaks (DSBs) may not be lethal to the cell, but may instead result in the loss of functional telomeres, chromosome fusions and initiation of breakage/fusion/bridge cycle-induced chromosome instability. The telomeres also participate in the process of DNA repair, as evidenced by 'de novo' synthesis of telomere repeats at DSBs and by the capacity of telomeres to binding the essential components of the DNA repair machinery. Based on the observation that high-LET radiations efficiently induce chromosome aberrations, it was tested whether protons were able to affect telomere structure. Human primary fibroblasts (HFFF2) and mouse embryonic fibroblasts (MEFs) were irradiated with 4 Gy of 3 MeV protons at the radiobiology facility of the INFN-LNL. Experiments with X rays were also carried out. Cells were fixed after either 24 h or 15 d from treatment. A difference in average telomere length, measured by quantitative fluorescence in situ hybridisation (Q-FISH), between X rays and protons treatment was observed. X rays are able to modify telomere length in HFFF2 harvested at a later time. On the other hand, 3 MeV low-energy protons induced, both in HFFF2 and in MEFs, a significant increase in telomere length at short as well as at long harvesting time periods from treatment. These results seem to indicate that lesions characterised by different complexity, as those expected after low-energy protons and those induced by damage similar to that induced by sparsely ionising radiation, are able to modulate telomere elongation at different time periods.

DNA fragments induction in human fibroblasts by radiations of different qualities.

Experimental data on DNA double strand break (DSB) induction in human fibroblasts (AG1522), following irradiation with several radiation qualities, namely gamma rays, 0.84 MeV protons, 58.9 MeV u(-1) carbon ions, iron ions of 115 MeV u(-1), 414 MeV u(-1), 1 GeV u(-1), and 5 GeV u(-1), are presented. DSB yields were measured by calibrated Pulsed Field Gel Electrophoresis in the DNA fragment size range 0.023-5.7 Mbp. The DSB yields show little LET dependence, in spite of the large variation of the latter among the beams, and are slightly higher than that obtained using gamma rays. The highest yield was found for the 5 GeV u(-1) iron beam, that gave a value 30% higher than the 1 GeV u(-1) iron beam. A phenomenological method is used to parametrise deviation from randomness in fragment size spectra.

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors.

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.

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 gamma-rays and light ions. MATERIALS AND METHODS: Human fibroblasts were exposed in vitro to different doses (in the range from 40 - 200 Gy) of (60)Co gamma-rays and 0.84 MeV protons (Linear Energy Transfer, LET, in tissue 28.5 keV/microm). 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 (60)Co gamma-rays). RESULTS: For both gamma-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) x 10(-9) Gy(-1) bp(-1) for gamma-rays and (7.1 +/- 0.6) x 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 gamma-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.

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.

Calibration of GafChromic EBT3 for absorbed dose measurements in 5 MeV proton beam and (60)Co γ-rays.

PURPOSE: To study EBT3 GafChromic film in low-energy protons, and for comparison purposes, in a reference (60)Co beam in order to use it as a calibrated dosimetry system in the proton irradiation facility under construction within the framework of the Oncological Therapy with Protons (TOP)-Intensity Modulated Proton Linear Accelerator for RadioTherapy (IMPLART) Project at ENEA-Frascati, Italy. METHODS: EBT3 film samples were irradiated at the Istituto Nazionale di Fisica Nucleare-Laboratori Nazionali di Legnaro, Italy, with a 5 MeV proton beam generated by a 7 MV Van de Graaff CN accelerator. The nominal dose rates used were 2.1 Gy/min and 40 Gy/min. The delivered dose was determined by measuring the particle fluence and the energy spectrum in air with silicon surface barrier detector monitors. A preliminary study of the EBT3 film beam quality dependence in low-energy protons was conducted by passively degrading the beam energy. EBT3 films were also irradiated at ENEA-National Institute of Ionizing Radiation Metrology with gamma radiation produced by a (60)Co source characterized by an absorbed dose to water rate of 0.26 Gy/min as measured by a calibrated Farmer type ionization chamber. EBT3 film calibration curves were determined by means of a set of 40 film pieces irradiated to various doses ranging from 0.5 Gy to 30 Gy absorbed dose to water. An EPSON Expression 11000XL color scanner in transmission mode was used for film analysis. Scanner response stability, intrafilm uniformity, and interfilm reproducibility were verified. Optical absorption spectra measurements were performed on unirradiated and irradiated EBT3 films to choose the most sensitive color channel to the dose range used. RESULTS: EBT3 GafChromic films show an under response up to about 33% for low-energy protons with respect to (60)Co gamma radiation, which is consistent with the linear energy transfer dependence already observed with higher energy protons, and a negligible dose-rate dependence in the 2-40 Gy/min range. Short- and long-term scanner stabilities were 0.5% and 1.5%, respectively; film uniformity and reproducibility were better than 0.5%. CONCLUSIONS: The main purpose of this study was to implement EBT3 dosimetry in the proton low-energy radiobiology line of the TOP-IMPLART accelerator, having a maximum energy of 7 MeV. Low-energy proton and (60)Co calibrated sources were used to investigate the behavior of film response vs to be written in italicum dose. The calibration in 5 MeV protons is currently used for dose assessment in the radiobiological experiments at the TOP-IMPLART accelerator carried out at that energy value.

mFISH analysis of irradiated human fibroblasts: a comparison among radiations with different quality in the low-dose range.

The present investigation aimed to characterise the shape of dose-response curve and determining the frequency distribution of various aberration types as a function of dose and radiation quality in AG01522 primary human fibroblasts in the 0.1- to 1-Gy dose range. For this purpose, the cells were irradiated with 7.7 and 28.5 keV µm(-1) low-energy protons, 62 keV µm(-1 4)He(2+) ions (LNL Radiobiology facility) or X rays and samples collected for 24-colour mFISH analysis. X rays and 7.7 keV µm(-1) protons displayed a quadratic dose-response curve solely for total and simple exchanges, whereas for high-linear energy transfer radiations, a linear dose-response curve was observed for all the aberration categories, with the exception of complex exchanges.

Chromosome nondisjunction and loss induced by protons and X rays in primary human fibroblasts: role of centromeres in aneuploidy.

To study the origin of micronuclei induced in human primary fibroblasts by low-energy protons (7.7 and 28.5 keV/microm) and X rays, we have developed a combined antikinetochore-antibody (CREST) and FISH staining with pancentromeric probes. This technique allowed us to analyze the integrity of the kinetochore and centromeric DNA structures and to assess their role in induced aneuploidy. The effect of LET on radiation-induced chromosome nondisjunction was studied in binucleated cells with centromeric-specific DNA probes for chromosomes 7 and 11. Our results indicate that, though more than 90% of radiation-induced micronuclei were CREST(-)/FISH(-), 28.5 keV/microm protons and X rays were also able to induce statistically significant increases in the number of micronuclei that were CREST(-)/FISH(+) and CREST(+)/FISH(+), respectively. One interpretation of these results could be that the protons induced chromosome loss by kinetochore detachment or by breakage in the centromeric DNA region, whereas X rays induced aneuploidy through a non-DNA damage mechanism. Nondisjunction appears to be a far more important mechanism leading to radiation-induced aneuploidy. Irrespective of the higher frequency of micronuclei induced by 28.5 keV/microm protons, the frequency of chromosome loss was markedly higher for X rays than for 28.5 keV/microm protons, strengthening the hypothesis that non-DNA targets, such as components of the mitotic spindle apparatus, may be involved in aberrations in chromosome segregation after X irradiation.

Mutant frequency at the Hprt locus and in minisatellite sequences in Chinese hamster V79 cells irradiated with low-energy protons (31 keV/microm) and ultraviolet light (254 nm).

Ionizing radiations induce mutations which can be detected both in coding sequences (Hprt locus) by measuring the frequency of 6-thioguanine-resistant cells and in minisatellite sequences by DNA fingerprint analysis. We analyzed the effects of irradiation with low-energy protons (31 keV/pm) and, for comparison, with ultraviolet light (254 nm), for which DNA damage and repair mechanisms are better understood, on cultures of Chinese hamster V79 cells with the two methods mentioned above. The results indicate that the frequency of 6-thioguanine-resistant cells was increased significantly, although very differently, by both treatments. The analyses carried out by DNA fingerprinting with a multilocus DNA probe show that the level of induction in minisatellite sequences was higher compared to those measured at the Hprt locus after proton irradiation, but lower after treatment with ultraviolet light.

Mitotic catastrophe induced by exposure of V79 Chinese hamster cells to low-energy protons.

PURPOSE: To determine the yield of mitotic catastrophe induced after low energy proton irradiation and to compare this yield with that induced by X-rays. MATERIALS: Asynchronous Chinese hamster V79 cells were irradiated with 0.5, 1, 2, 5 and 10 Gy proton beams and X-rays. Proton LET of 7.7, 11.0 and 30.5 keV microm(-1), corresponding to energies of 5.01, 3.20 and 0.76 MeV respectively, evaluated at the cell mid-plane, were used for experiments. The occurrence and yield of mitotic catastrophe was measured as the percentage of cells exhibiting fragmented nuclei. RESULTS: Proton irradiation led to an enhanced induction of mitotic catastrophe in V79 cells. The onset of nuclear fragmentation, a hallmark of mitotic catastrophe, occurred much earlier after cell exposure to proton particles than to X-ray irradiation. CONCLUSIONS: Mitotic catastrophe is persistent in the subsequent cell generations after proton and X irradiation of V79 cells; but protons are more effective than X-rays for the induction of this phenomenon. These results are discussed in terms of their importance in space exposures and possible acquisition of genomic instability by the progeny of irradiated cells.

Low-dose hypersensitivity of V79 cells under exposure to gamma-rays and 4He ions of different energies: survival and chromosome aberrations.

PURPOSE: To examine the low-dose sensitivity of V79 cells under exposure to gamma-rays and 4He ions of different energies. MATERIALS AND METHODS: Cell survival and cytogenetic analysis using the Giemsa technique were studied following irradiation to doses of 0-3 Gy at the INFN-LNL facilities. RESULTS: Low-dose hyper-radiosensitivity (HRS) of V79 was demonstrated after irradiation with gamma-rays and alpha-particles of various linear energy transfers (LET) (58.9, 79.3 and 101.7 keV microm(-1)). Cytogenetic analysis showed an LET dependence of aberrations at a dose of 1Gy; the frequency of chromatid fragments appeared to vary with the number of alpha-particles traversing the cell nucleus. The results of both studies fit together to give a better understanding of so-called 'induced radioresistance' phenomenon. CONCLUSIONS: The mechanism of induced cellular radioresistance appears to be initiated after a certain amount of energy is deposited in the cell nucleus. This amount depends on both radiation quality and the number of particles traversing the cell.

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.

Micronuclei, CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality.

PURPOSE: To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. MATERIALS AND METHODS: Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/microm and 27.6 keV/microm protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. RESULTS: MN yield and the frequency of BN cells carrying multiple MN (> or =2) were significantly higher after exposure to 27.6 keV/microm protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/microm protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6 keV/microm protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3 Gy). CONCLUSIONS: Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.

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 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.

DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. I. Experimental results.

PURPOSE: To compare the results on DNA fragmentation induced in Chinese hamster V79 cells by various doses of gamma-rays and low-energy protons and helium-4 ions. MATERIALS AND METHODS: V79 cells were irradiated as monolayers with monoenergetic protons and helium-4 ions; gamma-rays were used as the reference radiation. DNA double-strand breaks were evaluated by calibrated pulsed-field gel electrophoresis using conditions covering the range 5.7 Mbp-23.1 kbp. RESULTS: The fragment-counting method gave double-strand breaks yields and the relative biological effectiveness higher than those obtained by the fraction of activity released method. The frequency distribution of fragments showed that protons and helium ions induced more fragments below the Mbp region than did gamma-rays at the same dose. The distributions for both the irradiated and non-irradiated samples clearly appeared to be non-random. CONCLUSION: Differences were observed in the yield and spatial correlation, at a molecular size scale characteristic of loop dimensions, of the double-strand breaks induced by gamma-rays and by light ions. These effects may have a role in the observed different cell response to these radiations.

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.