Chromosome aberrations in human fibroblasts induced by monoenergetic neutrons. I. Relative biological effectiveness

The relative biological effectiveness (RBE) of neutrons for many biological end points varies with neutron energy. To test the hypothesis that the RBE of neutrons varies with respect to their energy for chromosome aberrations in a cell system that does not face interphase death, we studied the yield of chromosome aberrations induced by monoenergetic neutrons in normal human fibroblasts at the first mitosis postirradiation. Monoenergetic neutrons at 0.22, 0.34, 0.43, 1, 5.9 and 13.6 MeV were generated at the Accelerator Facility of the Center for Radiological Research, Columbia University, and were used to irradiate plateau-phase fibroblasts at low absorbed doses from 0.3 to 1.2 Gy at a low dose rate. The reference low-LET, low-dose-rate radiation was 137Cs-gamma rays (0.66 MeV). A linear dose response (Y = alphaD) for chromosome aberrations was obtained for all monoenergetic neutrons and for the gamma rays. The yield of chromosome aberrations per unit dose was high at low neutron energies (0.22, 0.34 and 0.43 MeV) with a gradual decline with the increase in neutron energy. Maximum RBE (RBEm) values varied for the different types of chromosome aberrations. The highest RBE (24.3) for 0.22 and 0.43 MeV neutrons was observed for intrachromosomal deletions, a category of chromosomal change common in solid tumors. Even for the 13.6 MeV neutrons the RBEm (11.1) exceeded 10. These results show that the RBE of neutrons varies with neutron energy and that RBEs are dissimilar between different types of asymmetric chromosome aberrations and suggest that the radiation weighting factors applicable to low-energy neutrons need firmer delineation. This latter may best be attained with neutrons of well-defined energies. This would enable integrations of appropriate quality factors with measured radiation fields, such as those in high-altitude Earth atmosphere. The introduction of commercial flights at high altitude could result in many more individuals being exposed to neutrons than occurs in terrestrial workers, emphasizing the necessity for better-defined estimates of risk.     

Characteristics of neutron beam generated by 500 MeV proton beam

A neutron irradiation facility was constructed at PARMS, University of Tsukuba to produce an ultrahigh energy neutron beam with a depth dose distribution superior to an x-ray beam generated by a modern linac. This neutron beam was produced from the reaction on a thick uranium target struck by a 500 MeV proton beam from the booster synchrotron of the High Energy Physics Laboratory. The percentage depth dose of this neutron beam was nearly equivalent to that of x-rays around 20 MV and the dose rate was 15 cGy per minute. The relative biological effectiveness (RBE) of this neutron beam has been estimated using the cell inactivation effect and the HMV-I cell line. The survival curve of cells after neutron irradiation has a shoulder with n and Dq of 8 and 2.3 Gy, respectively. The RBE value at the 10(-2) survival level for the present neutron beam as compared with 137Cs gamma rays was 1.24. The results suggest that the biological effects of ultrahigh energy neutrons are not large enough to be useful, although the depth dose distribution of neutrons can be superior to that of high energy linac x-rays.     

Radiobiology of alpha particles. IV. Cell inactivation by alpha particles of energies 0.4-3.5 MeV

In this report the effectiveness of low-energy alpha particles in the range 0.4 to 3.5 MeV for cell killing is investigated. Four cell lines of different nuclear dimensions (AG1522, C3H 10T1/2, CHO-10B, and HS-23) are studied. Monte Carlo simulations are carried out to interpret the experimental results. They are presented as a function of dose to the nucleus, the total track length of alpha particles in the nucleus, and other parameters. It is found that the effectiveness of alpha particles for cell killing decreases with decreasing alpha-particle energy. The maximum RBE value is found to extend to LET values as high as 180 keV/microns. Although the LET might be the same, the effectiveness of alpha particles for cell killing is higher in the ascending part of the Bragg curve compared to descending part of the Bragg curve. The terminal tracks of alpha particles are observed to be less effective for cell killing.     

Changes in RBE of 14-MeV (d + T) neutrons for V79 cells irradiated in air and in a phantom: is RBE enhanced near the surface?

PURPOSE: The relative biological effectiveness (RBE) for inactivation of V79 cells was determined as function of dose at the Heidelberg 14-MeV (d + T) neutron therapy facility after irradiation with single doses in air and at different depths in a therapy phantom. Furthermore, to assess the reproducibility of RBE determinations in different experiments we examined the relationship between the interexperimental variation in radiosensitivity towards neutrons with that towards low LET 60Co photons. METHODS: Clonogenic survival of V79 cells was determined using the colony formation assay. The cells were irradiated in suspension in small volumes (1.2 ml) free in air or at defined positions in the perspex phantom. Neutron doses were in the range, Dt = 0.5-4 Gy. 60Co photons were used as reference radiation. RESULTS: The radiosensitivity towards neutrons varied considerably less between individual experiments than that towards photons and also less than RBE. However, the mean sensitivity of different series was relatively constant. RBE increased with decreasing dose per fraction from RBE = 2.3 at 4 Gy to RBE = 3.1 at 0.5 Gy. No significant difference in RBE could be detected between irradiation at 1.6 cm and 9.4 cm depth in the phantom. However, an approximately 20% higher RBE was found for irradiation free in air compared with inside the phantom. Combining the two effects, irradiation with 0.5 Gy free in air yielded an approximately 40% higher RBE than a dose of 2 Gy inside the phantom. CONCLUSION: The measured values of RBE as function of dose per fraction within the phantom is consistent with the energy of the neutron beam. The increased RBE free in air, however, is greater than expected from microdosimetric parameters of the beam and may be due to slow recoil protons produced by interaction of multiply scattered neutrons or to an increased contribution of alpha particles from C(n, alpha) reactions near the surface. An enhanced RBE in subcutaneous layers of skin combined with an increase in RBE at low doses per fraction outside the target volume could potentially have significant consequences for normal tissue reactions in radiotherapy patients treated with fast neutrons.     

A comparison between combined neutron- and telecobalt-therapy with telecobalt-therapy alone for cancer of the bronchus

The difference in response of human tumours to high and low LET radiation has been investigated in a series of inoperable, histologically confirmed bronchial carcinomas. One hundred and forty-nine were treated with low LET radiation alone (60Co gamma rays) and 108 with a combination of gamma rays and fast neutrons of mean energy 6 MeV, one-fifth to one-third of the effective dose being from neutrons. The response was analysed by histological examination of the autopsy specimens. Tumour cell destruction was found to be significantly greater in the neutron-treated series. The two series were not strictly randomized but were closely similar to terms of tumour volume, histological grade and total treatment time. The sequence of treatments with neutrons and gamma rays (N-gamma, gamma-N, gamma-N -gamma) was found to have no influence on the results.     

The hadron therapy project

The neologism "hadrontherapy" means radiotherapy with hadrons, which are the particles constituted by quarks, such as protons, neutrons and ions. The theoretical considerations about the clinical advantages this treatment modality can yield and the results obtained at the centers where it has already been used justify the proposal to project a center of this kind also in our Country. To this purpose, two of the authors of this paper (U. Amaldi, G. Tosi) founded the TERA Group formed by physicists, engineers and radiotherapists who work in close collaboration on a feasibility study for a hadrontherapy facility. The first aim of the Hadrontherapy Project is to design a center equipped with a synchrotron which, at the beginning, will accelerate negative hydrogen ions (H-) which will first produce 70-250 MeV proton beams and, then accelerate light ions (up to 16O) to 430 MeV/amu. This accelerator will serve four or five treatment rooms where patients can be irradiated simultaneously. Two rooms will be equipped with a fixed horizontal beam for the treatment of eye, head and neck tumors; the others will be equipped with rotating gantries to administer, in any clinical situation, really adequate treatment. Such a unit, when enough experience is fained, will allow at least 1000 patients to be treated yearly. The synchrotron injector will be designed so as to allow, parallel to the radiotherapy activities, other applications of medical and biological interest such as: the production of radioisotopes for diagnostic use (especially positron emitters), the analysis of trace elements through the PIXE technique and the production of thermal and epithermal neutrons for boron neutron capture therapy.     

Frequencies and types of exchange aberrations induced by X-rays and neutrons in Chinese hamster splenocytes detected by FISH using chromosome-specific DNA libraries

PURPOSE: To estimate the frequencies of radiation- (low and high LET) induced chromosome aberrations in Chinese hamster splenocytes by two-colour fluorescence in situ hybridization using DNA painting probes specific for chromosomes 2, 3, 8, X and Y and to determine (1) the ratio of radiation-induced translocations and dicentrics; (2) the spectrum of exchange aberrations induced by X-rays and neutrons; and (3) the relative involvement of the different chromosomes in the formation of aberrations. MATERIALS AND METHODS: Isolated splenocytes from the Chinese hamster were irradiated in vitro with different doses of 200 kV X-rays (0.75, 1.5, 3.0 Gy) and 1 MeV fast neutrons (0.25, 0.5, 1.0 Gy). Conventional analysis of chromosome aberrations was carried out in Giemsa-stained preparations. Chromosome aberrations involving chromosomes 2, 3, 8, X and Y were analysed in first division metaphases using two-colour FISH. RESULTS: The results indicate that when all types of translocations are taken into account both X-rays and neutrons induce more translocations than dicentrics, the ratio between the two types of exchanges being 1.4 and 1.8 respectively. The ratio of 'apparently simple' reciprocal translocations and reciprocal complete dicentrics was close to 1 for both types of radiation. The RBE of neutrons for induction of exchanges was found to be between 5 and 8. Neutron irradiation was more efficient at inducing insertions. Among the chromosomes studied, an increased involvement was observed for chromosome 8 in dicentrics and translocations than that expected on the basis of its chromosome length. The high content of interstitial telomeric sequences in chromosome 8 may be responsible for the observed sensitivity of this chromosome. CONCLUSIONS: The results obtained in this study indicate that: (1) more translocations are found than dicentrics; (2) heterogeneity exists among Chinese hamster chromosomes for involvement in radiation-induced exchanges; (3) the spectrum and distribution of exchange aberrations are different between X-rays and neutrons; and (4) the relative frequencies of insertions could be used as a 'fingerprint' for exposure to high LET radiation.     

Conformal radiation therapy with hadron beams and the programs of the TERA Foundation

Proposed fifty years ago, tumor therapy with charged hadron beams has been under rapid development since 1993-94. Indeed hadrontherapy was born in 1938, when neutron beams have been used in cancer therapy, but it has become an accepted therapeutical modality only in the last five years. Fast neutrons are still in use, even if their limitations are now apparent. Charged hadron beams are more favorable, since the largest specific energy deposition occurs at the end of their range in matter. The most used hadrons are at present protons and carbon ions. Both allow a dose deposition which conforms to the tumor target. Radiobiology experiments and the results of the first clinical trials indicate that carbon ions have, on top of this macroscopic property, a different way of interacting with cells at the microscopic level. There are thus solid hopes to use carbon beams of about 4500 MeV to control tumors which are radioresistant both to X-rays and protons. After discussing these macroscopic and microscopic properties of hadrontherapy, the twelve dedicated hadrontherapy centres, which will be treating patients from 2001-2002, are shortly described. Five of them are in the USA and seven in Japan, while no hospital based centre for deep protontherapy is fully financed in Europe. The second part of this review is devoted to the Italian hadrontherapy programme, based on the development of the network RITA, the construction in Rome by the "Istituto Superiore di Sanità" of a novel proton accelerator based on a 3 GHz linac, the design of a linac to boost the energy of protons extracted from a 50-70 MeV cyclotron and the construction in Mirasole, near Milano, of a center for protons and ions known as "CNAO". This center will have a synchrotron, which is under design at CERN in the framework of a collaboration of TERA with AUSTRON and GSI which is called PIMMS (Proton Ion Medical Machine Study) and is headed by Dr. Phyl Bryant.     

Joining of correct and incorrect DNA ends at double-strand breaks produced by high-linear energy transfer radiation in human fibroblasts

DNA double-strand breaks (DSBs) were measured within a 3.2-Mbp NotI fragment on chromosome 21 of cells of a normal human fibroblast cell line. Correct rejoining of DSBs was followed by measuring reconstitution of the original-size NotI fragment, and this was compared to total rejoining as measured by a conventional pulsed-field gel electrophoresis technique (FAR assay). After 80 Gy of particle irradiations with LETs in the range of 7-150 keV/microm, it was found that the repair kinetics was generally slower after irradiation with high-LET particles compared to X irradiation and that a larger proportion of the breaks remained unrepaired after 24 h. On the other hand, the misrejoining frequency as measured by the difference between correct and total rejoining after 24 h did not change with LET, but was approximately the same for all radiations at this dose, equal to 25-30% of the initial breaks. This result is discussed in relation to formation of chromosomal aberrations, deletion mutations and other biological end points.     

Dose and LET distributions due to neutrons and photons emitted from stopped negative pions

Computer calculations are made of the dose and LET distributions due to neutrons and photons produced when negative pions are stopped in a phantom. When negative pions are stopped in a material they undergo nuclear capture, resulting in the disintegration of the nucleus and the emission of short range charged particles and longer range neutrons and photons. The uncharged radiation constitutes a potentially large source of dose outside the treatment volume. A simple phantom consisting of a 0-25 m cube of either tissue or bone-equivalent material is set up with a 0-05 m cube in the centre to represent the treatment volume. Neutrons and photons are started in this central volume and transported across the phantom using Monte Carlo transport codes. Several different initial energy spectra for the neutrons are used, taken from experimental and theoretical data. These different spectra are found to give significant differences in dose, though the distance to the 80% dose level is always about 0-015 m. Order of magnitude differences in some LET regions are also found. The dose deposited by neutrons in bone is about 24% less than in soft tissue, the photon dose being small compared with the neutron dose.     

The present system of quantities and units for radiation protection

The International Commission on Radiation Units and Measurements has over the last decade developed operational quantities, the ambient, directional and personal dose equivalent, suitable for the measurement of radiation fields in a variety of circumstances. Experience with the use of these quantities to represent the dose limitation quantities defined by the International Commission on Radiological Protection in 1977 has been an important part of recent radiation protection metrology. The definition by International Commission on Radiological Protection in 1991 of new limitation quantities, the equivalent dose and the effective dose has necessitated a redirection of this work. The metrology field has made good progress, however. It has found that for photons, at least above 50 keV, the effective dose can be measured by the ambient dose equivalent about as well as the former effective dose equivalent. Unfortunately, for neutrons the existing and already quite severe complications have been made somewhat worse by the new quantities although not any worse in the important region between 0.1 and 1 MeV. Neutron measurements over a broad energy range are the subject of extensive evaluation and some new suggestions as the metrology field wrestles with these problems. Values of wR constitute an important part of the International Commission on Radiological Protection recommendations. A brief history of the development of higher relative biological effectiveness values for fission neutrons and alpha particles leading to the selection of 20 for wR in each case, is provided.     

Initial recombination in a parallel-plate ionization chamber exposed to heavy ions

For exact determination of absorbed dose in heavy-ion irradiation fields which are used in radiation therapy and biological experiments, ionization chambers have been characterized with defined heavy-ion beams and correction factors. The LET (linear energy transfer) dependence of columnar recombination in a parallel-plate ionization chamber has been examined. Using 135 MeV/u carbon and neon beams, the ion collection efficiency was measured for several gases (air, carbon dioxide, argon and tissue-equivalent gas). 95 MeV/u argon beams and 90 MeV/u iron beams were also used for measurements of columnar recombination in air. As expected by Jaffe theory, the inverse of the ratio of the ionization charge to the saturated ionization charge had a linear relationship with the inverse of the electric field strength in the region below 0.002 V(-1) cm. The gradient of the line increases as the LET of the heavy ions increases. A strong LET dependence of the gradient was observed in air and carbon dioxide. The LET dependence was not observed in tissue-equivalent gas, nitrogen or argon. The exact depth-dose distribution of the heavy-ion beam was obtained by this correction of the initial recombination effect for the collected ionization charge. The columnar recombination in air was analysed using Jaffe theory; the obtained parameter b (a track radius) should be in the range between 0.001 cm and 0.005 cm, whereas the value obtained by Jaffe is 0.00179 cm. The value of the parameter b should increase as the LET of the heavy-ion beam increases in order to reproduce the experimental values of the initial recombination.     

The direct effect of heavy ions and electrons on thymidine in the solid state

PURPOSE: To study the direct effect of heavy ions and electrons on thymidine. MATERIALS AND METHODS: The thymidine samples in the solid state were exposed to a beam of O7+ heavy ions with an energy of 10.6 MeV/u (LET approximately 500keV/microm) and to electrons of 2MeV (LET approximately 0.18keV/microm). The major decomposition products of thymidine were purified by high performance liquid chromatography (HPLC) and identified by extensive spectrometric measurements (UV, mass spectroscopy, 1H and 13C NMR). RESULTS: The main degradation products of thymidine were isolated and characterized. Reaction mechanisms, involving transient radical species, are proposed to explain the heavy ion-mediated formation of the modified products. Furthermore, a semi-quantitative comparison of the modifications induced within thymidine by the two types of radiations was performed. CONCLUSION: Several new radiation-induced thymidine decomposition products have been isolated and characterized. The comparison of the effects induced by heavy ions and electrons on thymidine in the solid state clearly indicates several significant differences in the mechanisms of action. A relative increase in the extent of the modifications of the sugar moiety with respect to those of the base is observed with the heavy ions by comparison with electrons.     

Non-random distribution of DNA double-strand breaks induced by particle irradiation

Induction of DNA double-strand breaks (dsbs) in mammalian cells is dependent on the spatial distribution of energy deposition from the ionizing radiation. For high LET particle radiations the primary ionization sites occur in a correlated manner along the track of the particles, while for X-rays these sites are much more randomly distributed throughout the volume of the cell. It can therefore be expected that the distribution of dsbs linearly along the DNA molecule also varies with the type of radiation and the ionization density. Using pulsed-field gel and conventional gel techniques, we measured the size distribution of DNA molecules from irradiated human fibroblasts in the total range of 0.1 kbp-10 Mbp for X-rays and high LET particles (N ions, 97 keV/microns and Fe ions, 150 keV/microns). On a mega base pair scale we applied conventional pulsed-field gel electrophoresis techniques such as measurement of the fraction of DNA released from the well (FAR) and measurement of breakage within a specific NotI restriction fragment (hybridization assay). The induction rate for widely spaced breaks was found to decrease with LET. However, when the entire distribution of radiation-induced fragments was analysed, we detected an excess of fragments with sizes below about 200 kbp for the particles compared with X-irradiation. X-rays are thus more effective than high LET radiations in producing large DNA fragments but less effective in the production of smaller fragments. We determined the total induction rate of dsbs for the three radiations based on a quantitative analysis of all the measured radiation-induced fragments and found that the high LET particles were more efficient than X-rays at inducing dsbs, indicating an increasing total efficiency with LET. Conventional assays that are based only on the measurement of large fragments are therefore misleading when determining total dsb induction rates of high LET particles. The possible biological significance of this non-randomness for dsb induction is discussed.     

R.b.e. for d(42MeV)-Be neutrons based on chromosome-type aberrations induced in human lymphocytes and scored in cells at first division

The irradiation of human lymphocytes with five doses of 250 kV X-rays and five doses of d(42MeV)-Be neutrons was performed in order to obtain dose-response curves for both radiation qualities. By using the FPC technique, aberration scoring was confined to first division cells only and dose-response curves were obtained at three sampling times. Sampling time independence was observed for chromosome-type aberrations and common curves could be fitted, confirming homogeneity of the initial lymphocyte population. R.b.e. values between 1x1 and 8x8 were obtained for dicentric yields between 0x01 and 2x0 per cell. Some variabilities were encountered for other aberration types. Mitotic delay showed an r.b.e. of 1x75. There was no evidence for any induction of SCE by either X-rays or neutrons, up to the highest doses used.     

A proton dose calculation algorithm for conformal therapy simulations based on Molière's theory of lateral deflections

An algorithm is developed for computing proton dose distributions in the therapeutic energy range (100-250 MeV). The goal is to provide accurate pencil beam dose distributions for two-dimensional or three-dimensional simulations of possible intensity-modulated proton therapy delivery schemes. The algorithm is based on Molière's theory of lateral deflections, which accurately describes the distribution of lateral deflections suffered by incident charged particles. The theory is applied to nonuniform targets through the usual pencil beam approximation which assumes that all protons from a given pencil beam pass through the same material at each depth. Fluence-to-dose conversion is made via Monte Carlo calculated broad-field central-axis depth-dose curves, which accounts for attenuation due to nuclear collisions and range straggling. Calculation speed is enhanced by using a best-fit Gaussian approximation of the radial distribution function at depth. Representative pencil beam and spread-out Bragg-peak computations are presented at 250 MeV and 160 MeV in water. Computed lateral full-widths-at-half-maximum's in water, at the Bragg peak, agree with the expected theoretical lateral values to within 1% at 160 MeV and to within 3% at 250 MeV. This algorithm differs from convolution methods in that the effect of the depth of any inhomogeneities in density or atomic composition are accounted for in a rigorous fashion. The algorithm differs from Fermi-Eyges based methods by accounting in a rigorous way for the effect of nonsmall-angle scattering and screening due to atomic electrons. The computational burden is only slightly greater than that expected using the less-rigorous Fermi-Eyges theory.     

Monte Carlo and analytical calculation of proton pencil beams for computerized treatment plan optimization

Proton pencil beams in water, in a format suitable for treatment planning algorithms and covering the radiotherapy energy range (50-250 MeV), have been calculated using a modified version of the Monte Carlo code PTRAN. A simple analytical model has also been developed for calculating proton broad-beam dose distributions which is in excellent agreement with the Monte Carlo calculations. Radial dose distributions are also calculated analytically and narrow proton pencil-beam dose distributions derived. The physical approximations in the Monte Carlo code and in the analytical model together with their limitations are discussed. Examples showing the use of the calculated set of proton pencil beams as input to an existing photon treatment planning algorithm based on biological optimization are given for fully 3D scanned proton pencil beams; these include intensity modulated beams with range shift and scanning in the transversal plane.     

The differential induction of collateral resistance to 62.5 MeV (p-->Be+) neutrons and 4 MeV photons by exposure to cis-platinum

PURPOSE: To determine the relative sensitivity to cis-platinum, 4 MeV photons and 62.5 MeV (p-->Be+) neutrons in five human tumor cell lines, and their cis-platinum resistant variants. METHODS AND MATERIALS: The degree of cross-resistance of five human in-vitro cell lines to photons or fast neutrons was analysed for both cisplatinum-sensitive and resistant variants. RESULTS: The development of acquired cis-platinum resistance conferred collateral resistance to 62.5 MeV (p--Be+) neutrons in all five cell lines, but did not consistently decrease the photon sensitivity of these same cells. CONCLUSION: The reduction in photon and neutron sensitivity following the development of acquired cis-platinum resistance may possibly be regulated by different mechanisms. The reduction in neutron sensitivity was primarily due to a 1.3-1.7 fold reduction in the magnitude of the initial slope (alpha), which was independent of the degree of platinum resistance induced, suggesting a non-stochiometric relationship between the mechanisms responsible for acquired cis-platinum, and 62.5 MeV (p-->Be+) neutron resistance.     

The present and future of heavy charged particle therapy

Introduction of heavy charged particles (protons and heavy-ions) is a promising approach in cancer treatment, permitting selective irradiation to the tumor while minimizing irradiation to the surrounding normal tissues. Additionally, the efficiency of heavy-ions will be further augmented by an increased biological effectiveness caused by high-LET components. At the University of Tsukuba, treatment with 250 MeV proton beams is being performed and, unlike other facilities in the world, successful results have been obtained in the thoraco-abdominal and pelvic tumors. Heavy-ion therapy was initiated at the University of California, and in 1993 the first heavy-ion synchrotron complex dedicated to medical use in a hospital environment was completed at the National Institute of Radiological Sciences. The carbon-ion therapy was begun in June 1994 which is expected to provide optimal results in various type of tumors. A construction of heavy-ion facility is also under consideration in Germany (GSI), Austria (AUSTRON), Italy (TERA) and Japan (Hyogo prefecture).