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Measurements aimed at investigating the quenching of sensitivity of Gafchromic EBT3 films irradiated with charged particles at radiotherapy energy levels have been carried out. Films have been irradiated with beams of protons and carbon ions of interest for Hadron Therapy. The broadening of pencil beams in water phantom as a function of initial energy has been studied and the quenching of EBT3 film sensitivity has been quantified. A procedure for correcting the dose images obtained by means of EBT3 films has been improved and its application to an irradiation test has show the validity of the proposed methodology.

The various dose components generated by epithermal neutron beams in tissue-equivalent materials have different spatial distributions that also depend on the size and volume of the irradiated volume. NCT dosimetry requires the separate determination of dose components having different LET, because of their different RBE. Monte Carlo simulations have been developed concerning the absorbed doses and thermal neutron fluences, to investigate the extent of the differences that may occur by changing the shape and size of the irradiated volumes. Moreover, the doses and fluences that occur if specific isotopes (10B, 14N, 157Gd) are added to water have been evaluated.

The quenching of sensitivity of gafchromic films EBT3 with increasing radiation LET is studied and a proposed method for correcting the measured dose images is described. The method evaluates a correction coefficient in each position of the acquired image by multiplying the measured dose value for a calculated ratio of the absorbed dose to the measured one. Such calculated doses are obtained utilising knowledge about absorbed and measured doses for single Bragg peaks from protons of a generic energy E. This topic is under study and the software in MATLAB developed for achieving the correction of the measured images is continuously implemented. The first results to test the validity of the proposed method also for complex dose distributions are promising.

The Fricke gels (FG) composition has been modified over the years in order to improve their dosimetric characteristic for spatial dose evaluation in radiotherapy. Some problems, in particular those related to the diffusion of ferric ions in the gel matrix, have limited the clinical use of FG and still represent significant challenges for the scientific community working in the field of gel dosimetry. In this work, FG based on poly-vinyl alcohol (PVA) as the gelling agent, glutaraldehyde (GTA) as a cross-linker and FG based on gelatine loaded with silicate nano-clay (laponite) were developed with the aim to overcome the diffusion drawbacks affecting the traditional FG. Neither the sensitivity to the radiation dose nor the diffusion coefficient were significantly altered by the addition of laponite into the Fricke xylenol orange gel formulation employed. By contrast, lower diffusion rates were obtained with PVA-GTA gels, suggesting that this matrix could have a promising use in the field of 3D dosimetry.The Fricke gels (FG) composition has been modified over the years in order to improve their dosimetric characteristic for spatial dose evaluation in radiotherapy. Some problems, in particular those related to the diffusion of ferric ions in the gel matrix, have limited the clinical use of FG and still represent significant challenges for the scientific community working in the field of gel dosimetry. In this work, FG based on poly-vinyl alcohol (PVA) as the gelling agent, glutaraldehyde (GTA) as a cross-linker and FG based on gelatine loaded with silicate nano-clay (laponite) were developed with the aim to overcome the diffusion drawbacks affecting the traditional FG. Neither the sensitivity to the radiation dose nor the diffusion coefficient were significantly altered by the addition of laponite into the Fricke xylenol orange gel formulation employed. By contrast, lower diffusion rates were obtained with PVA-GTA gels, suggesting that this matrix could have a promising use in the field of 3D dosimetry.

The quenching of sensitivity of non-gaseous dosimeters exposed to charged particles was studied, in particular with reference to gel dosimeters and gafchromic EBT3 films. The studies were focused to dosimetry for Hadron Therapy and boron neutron capture therapy (BNCT). A correction coefficient can be more easily determined for BNCT dosimetry, while it is complex to correct the dosimeter response in the case of charged particle beams for Hadron Therapy. A proposed method for correcting the measured images is in continuous development.

Studies of the beam broadening and in-depth dose profiles in water for a pencil beams of protons and carbon ions, with energies in the range of interest for Hadron Therapy, have been carried out. Transversal images of absorbed dose have been attained and fitted with Gaussian approximation. The area of the section at half height of the fitting surface was evaluated to quantify the beam broadening. The radius R of the section was taken to describe the beam broadening and a mathematical function giving R versus depth in water, for whichever PB energy, was attained, both for protons and for carbon ions.

Purpose Gafchromic films could be valid detectors for in-phantom dose controls in hadrontherapy but the quenching of their sensitivity with increasing the linear energy transfer (LET) of radiation brings a limit to their utilisation. This effect is manifest in the case of exposures to protons but it is really noticeable for carbon ions, whose LET is high also at the beam-entrance position. A method for correcting dose images acquired with films irradiated with proton beams has been initially proposed and tested with high approximations [1] and then developed with more exhaustive procedure [2] . In this work, studies aimed at applying the method, in his final structure, to carbon ions are carried out. Methods Gafchromic EBT3 films that were suitably placed in a solid-water phantom and exposed to carbon ion beams at the synchrotron of CNAO (Pavia, Italy). Optical analyses were performed with conventional scanner or laboratory-made instrument for light-transmittance detection. The method for amending dose images evaluates, in each image point, a correction coefficient obtained evaluating, for each pencil beam (PB) giving dose contribution in the considered position, both the absorbed dose and the measured one and, finally, their ratio. Dedicated software in MATLAB has been developed for attaining the corrected images. Results Correction algorithms require 3D knowledge of both absorbed and measured doses for each PB. Measurements and calculations have been performed with PBs of various energies and fitting parameters were deduced. Conclusions The proposed method for amending the experimental images has proved to be promising and, upon completion of the software, will be suitable for the use in clinical practice.

Dosimetry in tissue exposed to the epithermal neutron beams utilized for BNCT is complex, due to the multiplicity of the possible neutron reactions and consequently of the secondary radiation that contains photons, charged particles and recoil nuclei. Owing to the different radiobiological effectiveness of the various components of the absorbed dose, it is necessary to attain the evaluation of each of them. In addition, the spatial distributions of these dose components changes considerably with size and shape of the irradiated volume. Therefore, BNCT dosimetry requires suitably developed calculations and experimental methods. In this work, Monte Carlo simulations in phantoms of different sizes and shapes have been developed. Experimental methods for separating the dose components, mainly based on gel dosimeters and thermoluminescence detectors, have been applied. Moreover, the change in the absorbed dose resulting from the addition of 157Gd was investigated. Both measurements and calculations have been done with the BNCT epithermal beam of the LVR-15 reactor.

Production of photonuclear particles in a tissue-equivalent medium has been calculated for linacs at 6, 10 and 15 MV from Varian TrueBeam. Based on the knowledge of bremsstrahlung fluence spectra and linac photon beam parameters, numerical integration was performed on the cross sections for photoparticle production of the constituent elements of tissue (2H,12C,13C,16O,17O,18O,14N,15N). At 15 MV, at the depth of photon maximum dose, the total absorbed dose due to neutrons, protons, alphas and residual nuclei from photon reactions in tissue (5.5E-05 Gy per Gy of photons) is comparable to that due to neutrons from accelerator head. Results reasonably agree with data reported in the literature using Monte Carlo models simulating linac head components. This work suggests a simple method to estimate the dose contributed by the photon-induced nuclear particles for high-energy photon beams produced by linacs in use, as it might be relevant for late stochastic effects.

Purpose The objective of this experiment was to compare the oncogenic potential of carbon ion beams and conventional photon beams for use in radiotherapy. Methods and Materials The HeLa X human skin fibroblast cell line CGL1 was irradiated with carbon ions of three different energies (270, 100, and 11.4 MeV/u). Inactivation and transformation data were compared with those for 15 MeV photons. Results Inactivation and transformation frequencies for the 270 MeV/u carbon ions were similar to those for 15-MeV photons. The maximal relative biologic effectiveness (RBE α ) values for 100MeV/u and 11.4 MeV/u carbon ions, respectively, were as follows: inactivation, 1.6 ± 0.2 and 6.7 ± 0.7; and transformation per surviving cell, 2.5 ± 0.6 and 12 ± 3. The curve for dose-transformation per cell at risk exhibited a maximum that was shifted toward lower doses at lower energies. Conclusions Transformation induction per cell at risk for carbon ions in the entrance channel was comparable to that for photons, whereas for the lower energies, 100 MeV/u and 11 MeV/u, which are representative of the energies delivered to the tumor margins and volume, respectively, the probability of transformation in a single cell was greater than it was for photons. In addition, at isoeffective doses with respect to cell killing, the 11.4-MeV/u beam was more oncogenic than were photons.

To determine the effectiveness of two UV spectra with different UVB components for cell kill and micronucleus induction in irradiated human HeLaxskin fibroblast (CGL1) hybrid cells and their progeny. To determine the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells at various post-irradiation times and their relationship with induced delayed biological effects.A commercial solar ultraviolet simulator was used. Two different filters were employed: the first transmitted radiation with lambda284nm and the second radiation with lambda293nm. The resulting spectra have different UVB components (lambda between 284 and 320nm, 19 W/m(2), and between 293 and 320nm, 13 W/m(2)) and the same UVA component (lambda between 320 and 400nm, 135 W/m(2)). CGL1 cells were irradiated with various doses. Clonogenic survival and micronucleus formation were scored in the irradiated cells and their progeny. ROS were detected by incubation of cultures at various post-irradiation times with dichlorodihydrofluorescein diacetate followed by flow cytometric measurement of the final product, dichlorofluorescein.The biological effectiveness of the lambda284nm spectrum was higher by a factor of 3 compared to the lambda293nm spectrum for cell kill, and by a factor of 5 for micronucleus induction. No delayed cell death or micronucleus formation was found in the progeny of cells exposed to lambda293nm, while a large and dose-dependent effect was found in the progeny of cells exposed to lambda284nm for both of these endpoints. ROS levels above those in unirradiated controls were found only in the progeny of cells exposed to the lambda284nm spectrum.The spectrum with lambda284nm was more effective than that with lambda293nm for induction of cell kill and micronucleus formation in the directly irradiated cells as well as induction of delayed effects in the progeny in the form of delayed reproductive death and micronucleus formation. The presence of ROS in the progeny of the irradiated cells may be the cause of the delayed effects.

To determine the relative biological effectiveness (RBE) for initial and delayed inactivation of cells by a modulated proton beam suitable for the treatment of tumours of the eye, within the spread-out Bragg peak and in its distal declining edge.Human tumour SCC25 cells were irradiated with the 65 MeV proton beam at the Cyclotron Medicyc in Nice. Perspex plates of different thickness were used to simulate five positions along the beam line: 2mm corresponding to the entrance beam; 15.6 and 25 mm in the spread-out Bragg peak; 27.2 and 27.8mm for the distal edge. At each position clonogenic survival of the irradiated cells and of their progeny were determined at various dose values. 60Co gamma-rays were used as reference radiation.RBE values evaluated at the survival level given by 2 Gy of gamma-rays increased with increasing depth from close to 1.0 at the proximal to about 1.2 at the distal part of the peak. Within the declining edge it reached the value of about 1.4 at 27.2 and about 2 at 27.8 mm. For the progeny of irradiated cells, the RBE value ranged from 1.0 to 1.1 within the spread-out Bragg peak and then increased up to a value of 2.0 at the last position. The dose-effect curves for the progeny always had a larger shoulder than for the irradiated progenitors, their alpha parameters being lower by a factor of about 4 and their beta parameters always being higher. The alpha/beta ratio was about 50 Gy for the progenitors and about 6 Gy for their progeny. The incidence of delayed effects increased with dose and with the depth within the beam.RBE values for the inactivation of cells irradiated in the spread-out Bragg peak are compatible with the value currently assumed in clinical applications. In the distal declining edge of the beam, the RBE values increased significantly to an extent that may be of concern when the region of the treatment volume is close to sensitive tissues. The yield of delayed reproductive cell death was significant at each position along the beam line.

Measurements of C3H10T1/2 and V79 cell thickness were performed on living cells by confocal laser fluorescence microscopy. Thickness distributions are reported for cells growing as a monolayer (on mylar and glass) and suspended in their medium. Mean values for cells grown on mylar (corrected for refractive index effects) are 2.9 +/- 0.6 and 6.1 +/- 1.0 microm for C3H10T1/2 and V79 cells respectively. Mean values of the diameters of cells suspended in their medium are 13.0 +/- 1.6 and 9.3 +/- 1.4 microm for C3H10T1/2 and V79 respectively. Knowledge of cell thickness, as irradiated, is of central relevance for studying the relative biological effectiveness of low energy, poorly penetrating radiations. It can be concluded, from the measured cell thickness distributions, that with C3H10T1/2 cells grown on mylar, the LET variation through the whole cell is within 20% for protons and alpha-particles with energies down to 0.6 and 2.5 MeV respectively. From a comparison with thickness values reported in the literature for living or fixed embedded cells growing on plastic substrate, mean values between 2.4 and 3.4 microm and between 6 and 7.5 microm could be assumed for C3H10T1/2 cells and for the most widely used V79 cell lines respectively.

Survival and oncogenic transformation frequencies were determined through the cell cycle in hybrid cells (HeLa x human skin fibroblasts), exposed to 0.30 and 0.15 Gy 4.3 MeV (LET= 101 keV/microm) alpha-particles. The cells were synchronized by mitotic collection and irradiated at times ranging from 2 to 10 h after collection, corresponding to G1 and early S. At 0.30 Gy the highest value in the transformation frequency (1.6 +/- 0.3) x 10(-4) transformants/survivor, occurred 4 h after mitotic collection, corresponding to mid-G1 and was about twice as high as that for the asynchronous population (0.7 +/- 0.1) x 10(-4) transformants/survivor. A similar pattern was seen at 0.15 Gy albeit less marked. The results are similar to previous findings with C3H10T1/2 exposed to 0.30 Gy where (1.8 +/- 0.4) x 10(-4) and (0.8 +/- 0.4) x 10(-4) transformants/survivor were found in mid-G1 and in the asynchronous population respectively. The results of both these studies with 101 keV/microm alpha particles indicate that mid-G1 cells may be more sensitive than asynchronous cells by up to a factor of two. However, it is unlikely that such a factor is sufficient to represent the cell cycle 'hot spot' for transformation postulated to explain the inverse dose-rate effect.

There is substantial experimental evidence that protracted exposure to high-LET radiation can have a greated effect than single exposure in inducing cell transformation, the so-called «inverse dose-rate effect». The magnitude of this enhancement is due to the complex interplay between dose, dose rate and radiation quality. We have developed a model that explains the complex trend of the experimental results. This model is based on the assumption that there is a brief period of high sensitivity to transformation in the cell cycle as proposed in the literature and takes into account the saturation observed at high doses in the dose-effect curves. Specific equations are reported for acute, protracted and fractionated irradiation. Findings with C3H10T1/2 cells were analised in the light of this model. Assuming best fitted parameters of the model obtained from acute-irradiation data, tranformation frequencies due to protracted or fractionated exposure were predicted and compared with experimental findings on fission and monoenergetic neutrons and on charged particles of LET between 20 and 150 keV/μm. The model’s predictions were found to be closely consistent with the available experimental data.

Human cell line/Carbon-ion beams/Cell inactivation/Relative Biological Effectiveness (RBE)/Linear Energy Transfer (LET). This work aimed at measuring cell-killing effectiveness of monoenergetic and Spread-Out Bragg Peak ( SOBP) carbon-ion beams in normal and tumour cells with different radiation sensitivity. Clonogenic survival was assayed in normal and tumour human cell lines exhibiting different radiosensitivity to X- or γ-rays following exposure to monoenergetic carbon-ion beams (incident LET 13–303 keV/μm) and at various positions along the ionization curve of a therapeutic carbon-ion beam, corresponding to three doseaveraged LET (LETd) values (40, 50 and 75 keV/μm). Chinese hamster V79 cells were also used. Carbon-ion effectiveness for cell inactivation generally increased with LET for monoenergetic beams, with the largest gain in cell-killing obtained in the cells most radioresistant to X- or γ-rays. Such an increased effectiveness in cells less responsive to low LET radiation was found also for SOBP irradiation, but the latter was less effective compared with monoenergetic ion beams of the same LET. Our data show the superior effectiveness for cell-killing exhibited by carbon-ion beams compared to lower LET radiation, particularly in tum our cells radioresistant to X- or γ-rays, hence the advantage of using such beams in radiotherapy. The observed lower effectiveness of SOBP irradiation compared to monoenergetic carbon beam irradiation argues against the radiobiological equivalence between dose-averaged LET in a point in the SOBP and the corresponding monoenergetic beams.

Summary Data reported in the literature on neoplastic transformation induced in cultured cells by light charged particles are compared and analyzed as a function of LET and dose protraction. Light charged particles RBE for transformation is maximum between 75 and 120 keV/μm . The majority of the data suggest that RBE values for survival and transformation are of similar magnitude. Dose protraction effects on transformation induction depend on dose, dose rate and on radiation quality.

The aim of this research was to determine the biological effectiveness for early and delayed effects of high energy, high linear energy transfer (LET) charged particles. Survival and delayed reproductive death were measured in AG1522 human fibroblast cells exposed to Fe-ion beams of energies between 0.2 and 1 GeV/n, 0.97 GeV/n Ti-ion and 0.49 GeV/n Si-ion beams. The cells were irradiated at the HIMAC accelerator in Chiba, Japan (0.2 and 0.5 GeV/n Fe and 0.49 GeV/n Si) and at the NASA Space Radiation Laboratory in Brookhaven, USA (1 GeV/n Fe and 0.97 GeV/n Ti ions). The dose-effect curves were measured in the dose range between 0.25 and 2 Gy. For comparison cells were exposed to 60Co gamma rays. Analysis of the dose-effect curves show that all the heavy ion beams induce inactivation and delayed reproductive death more effectively than 60Co gamma rays. The only exception is the 0.2 GeV/n Fe-ion beam at low doses. The progeny of the irradiated cells show delayed damage in the form of reproductive death with all the heavy ion beams with the 1 GeV/n Fe-ion beam being the most effective. The relative biological effectiveness at low doses of the iron beams is highest for LET values between 140 and 200 keV/micrometers with values of 1.6 and 3 for early and delayed reproductive death, respectively. Analysis of the fluence-effect curves shows that the cross-sections for early and delayed inactivation increase with increasing LET up to 442 keV/micrometers.

(1). To determine the biological effectiveness of two solar ultraviolet (UVB) spectra with different lower wavelength thresholds for oncogenic transformation and micronucleus induction in CGL1 cells; (2). to investigate whether the action spectra for short- and long-term effects are similar; and (3). to investigate possible links between transformation and other delayed effects.Two spectra were derived from a solar UV simulator by using two filters: the first transmitted radiation with lambda284 nm, the second with lambda293 nm. The resulting spectra have the same UVA, but different UVB components (lambda between 284 and 320 nm, 19 W m(-2), and lambda between 293 and 320 nm, 13 W m(-2)). CGL1 cells were irradiated with 466 J m(-2) with lambda284 nm and 1582 J m(-2) with lambda293 nm. These doses were approximately equilethal. The endpoints examined were oncogenic transformation, and centromere-positive and -negative micronucleus frequencies in the directly irradiated cells and in transtheir progeny.At equilethal doses, the oncogenic transformation frequency in the directly irradiated cells was greater by a factor of at least 7 for lambda284 nm irradiation compared with lambda293 nm. The micronucleus induction frequency was also significantly higher with the lambda284 spectrum. Consistent with our previous findings, no delayed micronucleus formation was found in the progeny of cells exposed to lambda293 nm, while a threefold elevation above controls was seen in the progeny of cells exposed to lambda284 nm irradiation. This was also the case for formation of micronuclei with a centromere.It was found that: (1). for equilethal doses the lambda284 nm spectrum was more biologically effective than the lambda293 nm spectrum for induction of oncogenic transformation and micronucleus formation; and (2). the higher effectiveness of the lambda284 nm spectrum found at equilethal doses for delayed effects in the progeny of irradiated cells resembles that found for transformation. The results suggest that the UVB action spectrum for cell killing is different from that of some delayed effects, and from that of transformation.

Published data on inactivation of V79 cells irradiated with monoenergetic proton and ion beams (He, C, O, Ne) have been analysed. Values for RBE alpha, RBE10% and the inactivation cross section sigma have been evaluated in the LET range between 5 and 400 keV.micron-1. RBE against LET curves and inactivation cross sections against LET and against Z*2/beta 2 curves have been studied in a comparative approach with respect to the different ion types. RBE-LET curves depend strongly on the type of ion for LET30 keV.micron-1. At LET30 keV.micron-1 and low doses protons show the greatest effectiveness; at LET30 keV.micron-1 and high doses He ions provide the most effective radiation. Apart from protons, separation among the various ion curves is less marked in the sigma against Z*2/beta 2 plot than in the sigma against LET plot. sigma against Z*2/beta 2 curves for ions with 2or = Zor = 10 and 200Z*2/beta 21500 show a common trend independent of Z and are well represented by a linear relationship.

To analyse the cell inactivation frequencies induced by low energy protons in human cells with different sensitivity to photon radiation.Four human cell lines with various sensitivities to photon irradiation were used: the SCC25 and SQ20B derived from human epithelium tumours of the tongue and larynx, respectively, and the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The cells were irradiated with y-rays and proton beams with linear energy transfer (LET) from 7 to 33 keV/microm. Clonogenic survival was assessed.Survival curves are reported for each cell line following irradiation with gamma-rays and with various proton LETs. The surviving fraction after 2 Gy of gamma-rays was 0.72 for SQ20B cells, and 0.28-0.35 for the other cell lines. The maximum LET proton effectiveness was generally greater than that of gamma-rays. In particular there was a marked increase in beam effectiveness with increasing LET for the most resistant cells (SQ20B) whose 2 Gy-survival varied from 0.72 with gamma-radiation down to 0.37 with 30 keV/microm protons. The relative biological effectiveness (RBE(2 Gy gamma)) with the 30 keV/microm beam, evaluated as the ratio of 2 Gy to the proton dose producing the same inactivation level as that given by 2 Gy of gamma-rays, was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively.RBE for inactivation with high-LET protons increased with the cellular radioresistance to gamma-rays. The cell line with the greatest resistance to gamma-rays was the most responsive to the highest LET proton beam. A similar trend has also been found in studies reported in the literature with He, C, N ions with LET in the range 20-125 keV/microm on human tumour cell lines.

For the assessment of radiation risk at low doses, it is presumed that the shape of the low-dose-response curve in humans for cancer induction is linear. Epidemiological data alone are unlikely to ever have the statistical power needed to confirm this assumption. Another approach is to use oncogenic transformation in vitro as a surrogate for carcinogenesis in vivo. In mid-1990, six European laboratories initiated such an approach using C3H 10T1/2 mouse cells. Rigid standardisation procedures were established followed by collaborative measurements of transformation down to absorbed doses of 0.25 Gy of x-radiation resulting in a total of 759 transformed foci. The results clearly support a linear dose-response relationship for cell transformation in vitro with no evidence for a threshold dose or for an enhanced, supralinear response at doses approximately 200-300 mGy. For radiological protection this represents a large dose, and the limitations of this approach are apparent. Only by understanding the fundamental mechanisms involved in radiation carcinogenesis will further knowledge concerning the effects of low doses become available. These results will, however, help validate new biologically based models of radiation cancer risk thus providing increased confidence in the estimation of cancer risk at low doses.

Purpose To determine the RBE-LET relationship for C3H10T1/2 cell inactivation by protons in the LET range 11-33 keV/microm and to compare inactivation frequencies induced in C3H10T1/2 cells by protons and deuterons at two matching LET values in the range 11-20 keV/microm. Materials and methods C3H10T1/2 cells were irradiated with protons and deuterons at the radiobiological facility set up at the 7MV Van de Graaff accelerator at the LNL, Legnaro, Padova. Gamma rays from 60Co were used as reference radiation. Results Proton RBE values (alpha/alphagamma) for inactivation of C3H10T1/2 cells are constant around a value of 2 between 11 and 20 keV/microm and then rise sharply to reach a value of 4.2+/-1.0 at 33 keV/microm. Deuteron RBE values are 1.7+/-0.4 and 2.2+/-0.6 at LET values of 13 and 18 keV/microm respectively. Conclusions Proton RBE values with C3H10T1/2 cells are significantly larger than unity at LET values as low as 11 keV/microm. No difference in effectiveness for inactivation of C3H10T1/2 has been found between protons and deuterons at two LET values in the range 10-20 keV/microm.

Cell-cycle stage radiosensitivity for the induction of chromosome aberrations has been investigated in C3H 10T1/2 cells. Exponentially growing cells were irradiated with 3 Gy X-rays (80 kVp) or 0.6 Gy alpha-particles (LET = 101 keV/micron). The two doses produce the same survival level (37%) in the asynchronous population. Cells were harvested at four different times following irradiation and cell-cycle phase at the time of irradiation was assessed by using the differential replication staining technique. The frequency of chromosome aberrations produced in a given stage of the cell cycle was not constant as a function of the sampling time, but this could not be simply related to the existence of subphases exhibiting different radiosensitivity, because of cell-cycle perturbation introduced by radiation. X-radiation induced more exchanges than deletions, whereas a predominance of isochromatid deletions was observed after alpha-irradiation. This can be interpreted on the basis of the different patterns of energy deposition of densely- and sparsely-ionizing radiation. Both X- and alpha-rays produced a significant increase in the frequency of Robertsonian translocations when cells were exposed in G1 or S phase, but not in G2 phase.

Cell survival was studied by analyzing the inactivation probability density function and its fundamental parameters. Mean D, variance sigma 2 and mode Dmode were evaluated and a set of equations relating these parameters to the usual parameters of the multitarget, multihit and linear-quadratic models Do and n, alpha and beta, kappa and lambda are reported. The multihit equation used was an extension of the usual equation, to allow parameter kappa to assume values that are not necessarily integers. In the multitarget curve, the mode of inactivation probability density function, proved to be the quasi-threshold dose Dq = Do ln(n). Relative variance, degree of asymmetry and degree of peakedness can be calculated from the shape parameters n in the multitarget model, kappa in the multihit model, and alpha/square root of beta in the linear-quadratic model. From an analysis of eight published cell survival sets of data, on C3H10T1/2 cells exposed to low LET radiations, it was found that D, sigma, and SF2 are the parameters which exhibit the least variation from experiment to experiment and the least variation in selecting the range of data available for estimation.

Oncogenic transformation of synchronized C3H 10T1/2 cells was determined after exposure to 4.3 MeV alpha particles (LET = 101 keV/microns). Two synchronization techniques were tested using basic and modified protocols: one based on the release of cells from contact inhibition and the second on the mitotic shake-off method. Progression of cells through the cycle was followed as a function of time by flow cytometric analysis, DNA labeling for passage through S phase, the growth curve for the cell number and mitotic index measurements. The conclusion is that, although the release of cells from confluence provides higher yields of synchronized cells, mitotic shake-off proved to be the best way of collecting a synchronized population of minimally perturbed cells. Cells synchronized by mitotic shake-off were irradiated with 0.30 Gy in the interval between 2 and 10 h corresponding to G1 and early S phases. For comparison asynchronous populations were irradiated in parallel. Oncogenic transformation frequency, corrected for background, in mid-G1 phase was (18 +/- 4) x 10(-5) (average values of frequencies at 4 and 6 h) compared with the value of (8 +/- 4) x 10(-5) for the asynchronous population. While these data are suggestive of a trend toward a slightly increased sensitivity in mid-G1 phase, it is not statistically significant. The surviving fraction is constant in G1 phase.

Oncogenic transformation of C3H 10T1/2 cells was determined after exposure to graded doses of 4.3-MeV α particles LET = 101 keV/μm. The source of α particles was ${}^{244}{\rm Cm}$ and the irradiation was done in an irradiation chamber built for the purpose. Graded doses in the range of 0.2 to 300 cGy were studied with special emphasis on the low-dose region, with as many as seven points in the interval up to 10 cGy. The dose-effect relationship was a complex function. Transformation frequency increased with dose up to 2 cGy; it seemed to flatten at doses between 2 and 20 cGy but increased again at higher doses. A total of 21 cGy was delivered in a single dose or in 3 or 10 equal fractions at an interval of 1.5 h. An inverse dose-protraction effect of 1.4 was found with both fractionation schemes. Measurements of the mitotic index of the population immediately before the various fractions revealed a strong effect on the rate of cell division even after very low doses of radiation. Mit...

Survival and oncogenic transformation were studied in C3H10T1/2 cells exposed to 31 MeV protons. Total doses of 0.5, 1 and 7 Gy were delivered as single and two equal fractions with various time intervals up to 10 h between doses. With split doses as compared with single doses to a total dose of 7 Gy, survival increased by a factor of 2.5 +/- 0.2, whereas the frequency of transformation per surviving cell declined by a factor of 3.1 +/- 0.5. Maximal split-dose recovery occurred within the first 5 h for both endpoints. Further, the transformation frequency decreased by factors of 3.1 +/- 0.6 and 1.5 +/- 0.3 respectively for total doses of 0.5 and 1.0 Gy split into two equal fractions. The data for 1 and 7 Gy are compatible with data in the literature for other low LET radiations.

Transformation frequencies induced in cell systems by low- and high-LET radiations are compared and analysed as a function of LET and dose protraction. At acute dose rates, the dose-response curve with low-LET radiations shows a complex shape: the transformation frequency increases with the dose by a power of less than 1 in the interval (0.25÷1.5) Gy; a power of about 2 from 1.5 Gy to 4.5 Gy, where it reaches its maximum value and then remains constant. High-LET radiation transformation frequencies increase with dose by a power of 2 from 0.1 to 2 Gy after which they remain constant. RBE values increase with LET up to 140 ke V/μm and then decrease. The transformation frequency is reduced following dose fractionation or dose rate reduction with low-LET radiation, whereas it is enhanced with high-LET radiation, such as 0.85 MeV neutrons. Values of RBE for transformation up to 50 can be evaluated for neutrons. Various models that have been proposed to explain these results are analysed and compared.

The effects of cell density on transformation frequencies were studied in C3H10T1/2 cells exposed to 0.5 and 7 Gy of 200 kVp X-rays. Initial cell density strongly influenced transformation frequency; this decreased by a factor of between 4 and 10 when the initial seeding density was changed from 50 to 2500 cells/10 cm diameter Petri dish. The data were fitted with two equations: (a) an allometric function represented on a log-log scale by a straight line and (b) a sigmoidal function with plateaux between 50 and 250 cells/dish and above 600. The two curves are compared and their probabilities discussed. Our data indicate that the region between 50 and 250 cells/dish would be the most suitable region for dose-effect measurements. A study of the growth curves at 0.5 and 8.5 Gy shows that cell growth rates are not influenced by initial cell density.

Chromosome aberrations were induced in cultured human cells by proton beams of 31, 12, and 8 MeV. The frequencies of isochromatid breaks and dicentrics have been analysed as a function of proton energy and dose. Both effects are largely dependent on proton energy; isochromatid breaks increased linearly with the dose, whereas dicentrics show a definite parabolic behaviour. The experimental data were fitted to the analytic form Y = KDn and Y = alpha D + beta D2 and the best fitted values of the parameters are reported and discussed. The values of RBE for the isochromatid breaks are in the ratio 1.7:1.3:1 for 8, 12, and 31 MeV respectively. In the case of the dicentrics the RBE values are dose-dependent function of the type CD-n. The three distributions of dicentrics among the cells do not fit a Poisson distribution.

SummaryCultured human cells of the EUE line were exposed to different doses of 12 MeV protons, plated and allowed to grow for 8 days; colonies were then scored for the presences of multinucleate cells and micronuclei. The frequency of both effects is an increasing function of the dose; the evaluated exponents of the dose-response equation (e = bDn) are n = 1·0±0·1 for multinucleate cells and n = 1·6±0·1 for micronuclei. By comparison with the results obtained with gamma irradiations, r.b.e. values were obtained for both effects. The correlation between the logarithm of the surviving fraction and the yield of the studied effects has been proved to be statistically significant.

The frequency distributions of energy deposition in microscopic volumes for proton beams of various energies and energy spreads were determined by means of a "rossi type" proportional counter. Tissue equivalent spherical volumes of 0.6, 0.72, 0.80, 1.00, 1.25, 1.50, 1.75, 2.00 micrometer diameter were simulated. Frequency distributions of energy deposition per unit pathlength are reported and their behavior as a function of the simulated pathlength, beam energy and energy spread is discussed. The results indicate that energy deposition distributions in microscopic volumes for protons in the range 8-31 MeV are skewsymmetric distributions with a tail on the high energy side, and that degraded beams behave differently from monoenergetic ones. Dose mean lineal energy values have been quoted for beams used in radiobiological experiments.

Growth curves and size of the colonies of C3H10T1/2 cells exposed to low-LET radiation (31 MeV protons) were determined after 0, 1, 3, 5, and 7 Gy. The data show that: cell density at confluence was 3.3 x 10(4) cells/cm2; the initial division delay was very small; in the first 15 h the increase in the cell number was essentially the same at all doses; at 100 h the colony size distribution was very large, ranging from 0 to 7 generations, even within the control population. The temporal dependence of the growth properties of surviving and non-surviving cells was represented by the equation N = N0(Fe(a(t - dD] + (1 - F)ea/bD(1 - e - bD(1 - e - bD(t - dD]) where F is the surviving fraction, t the time of sampling, a the growth rate, d the division delay per unit dose, b the rate per unit of dose at which the non-surviving cells lose their ability to divide. The resulting values were: a = 0.029 +/- 0.002 h-1; b = 0.0041 +/- 0.0009 h-1 Gy-1 and d = 1 +/- 0.8 h Gy-1. It was found that growth curves are affected by non-surviving progeny up to 150, 200 and 250 h after irradiation at 3, 5 and 7 Gy, whereas at longer times the population consists essentially of progeny of surviving cells.

Cells synchronized by mitotic selection were irradiated at various times during the first generation cycle with 1.75 and 3.5 Gy of 31-MeV protons and then analyzed both for lethality and for chromosome aberrations. The results showed a maximum of radiosensitivity in late G/sub 1/-early S followed by an increase in resistance to a maximum in S in both endpoints of damage. For both types of effect, early to mid G/sub 1/ is definitely less sensitive than mid to late G/sub 1/. The ratios between the maximum and minimum sensitivity were approximately 1.4 and 2 for 1.75 and 3.5 Gy, respectively. These values agree with data reported to the literature on different mammalian cell lines exposed to X rays at comparable survival levels. For both dose values, the logarithm of the surviving reaction at various times of the generation cycle is linearly related to the corresponding chromosomal aberration frequency, with 37% survival corresponding to about one aberration per cell.

Experimental data are presented on the frequencies of transformations in C3H10T1/2 cells exposed to 31 MeV protons (LET = 1.83 +/- 0.02 keV/mum in tissue) in a dose interval between 0.25 and 7.0 Gy. The transformation frequency per surviving cell curve showed a marked change in slope over the dose range used. At higher doses, above about 2 Gy, it steepened very sharply in comparison with the lower dose range. If fitted to a power of the dose, the power in the higher range was about five times that in the lower range.

The relative biological effectiveness (RBE) was determined for proton beams of 31, 12, and 8 MeV produced at the Milano University Cyclotron. Survival curves for human cells grown in monolayer at different proton energies and for γ rays from60 Co were determined. The minimum clone size to be chosen for definition of true survivors was examined. RBE values of 1.0 ± 0.1, 1.4 ± 0.2, and 1.5 ± 0.2, respectively, were found and compared with the results of other experiments in this energy range.