Radiations of high linear energy transfer (LET) have long been known to have greater biological effectiveness per unit dose than those of low LET, for a wide variety of biological effects. However, values of relative biological effectiveness depend considerably on the biological system and in some instances the values are clearly below unity. The differences between high- and low-LET radiations may be due to many factors, almost all of which are related to radiation track structure in one way or another, and some can in principle lead to qualitative as well as quantitative differences between the radiations. Explanations for LET-dependent differences in effectiveness are discussed over a variety of levels from the multicellular and cellular scale down to the DNA scale, with illustrations from radiobiological data. Information from well-defined slow light ions provide particularly useful analytic data, but practical issues extend also to neutrons and fast heavy ions, which may compound high- and low-LET features. It is suggested that effectiveness of the radiation is determined predominantly by the complex clustered damage that it produces in DNA, but that for high-LET radiations long-term effects are in some instances limited by single-track-survival probabilities of the traversed cells.

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Dosimetry studies have been made for neutrons from a neutron generator at Hiroshima University (HIRRAC) which is designed for radiobiological research. Neutrons in an energy range from 0.07 to 2.7 MeV are available for biological irradiations. The produced neutron energies were measured and evaluated by a ³He-gas proportional counter. Energy spread was made certain to be small enough for radiobiological studies. Dose evaluations were performed by two different methods, namely use of tissue equivalent paired ionization chambers and activation of method with indium foils. Moreover, energy deposition spectra in small targets of tissue equivalent materials, so-called lineal energy spectrum, were also measured and are discussed. Specifications for biological irradiation are presented in terms of monoenergetic beam conditions, dose rates and deposited energy spectra.

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We have examined the neutron energy dependency of cell killing and mutation induction at the hprt locus in Chinese hamster V79 cells. Monoenergetic neutrons at 0.32, 0.57, and 1.2 MeV were generated at the Hiroshima University Radiobiological Research Accelerator (HIRRAC) Facility, and were used to irradiate cells. The variation in RBE with neutron energy for the end points of cell survival and hprt mutation induction was observed. When compared to ¹³⁷Cs γ-rays, all neutron energies were more effective at both cell killing and induction of mutation. Over the range of the neutron energies examined, we found that cytotoxicity increased as the energy decreased from 1.2 to 0.32 MeV. In comparison to γ-rays, RBEs for cell lethality at 10% survival were 5.7, 6.7, and 7.6 for 1.2, 0.57, and 0.32 MeV, respectively. Mutation induction, on the other hand, was highest at 0.57 MeV with a gradual decrease at 1.2 and 0.32 MeV. RBEs for mutation induction were 9.7, 19.4, and 13.9 for 1.2, 0.57, and 0.32 MeV neutrons. We isolated independent V79 cell mutants at the hprt locus from untreated and neutron-exposed cells and determined the genetic changes underlying the mutation by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. Preliminary results are suggestive of a specific relationship between deletion patern and neutron energy.

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Irradiations of onion seedlings with fission neutrons from bare, Pb-moderated, and Fe-moderated ²⁵²Cf sources induced micronuclei in the root-tip cells at similar rates. The rate per cGy averaged for the three sources, <b_n>, was 19 times higher than rate induced by ⁶⁰Co γ-rays. When neutron doses, D_n, were estimated from frequencies of micronuclei induced in onion seedlings after exposure to neutron- γ mixed radiation from a 1 W nuclear reactor, using the reciprocal of <b_n> as conversion factor, resulting D_n values agreed within 10% with doses measured with paired ionizing chambers. This excellent agreement was achieved by the high sensitivity of the onion system to fast neutrons relative to γ-rays and the high contribution of fast neutrons to the total dose of mixed radiation in the reactor's field.

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This study was undertaken to investigate the biological effect of monoenergetic neutrons on human lymphocyte DNA and chromosomes. Monoenergetic neutrons of 2.3, 1.0, 0.79, 0.57, 0.37 and 0.186 MeV were generated, and ²⁵²Cf neutrons and ⁶⁰Co γ-rays were also used for comparison. Biological effect was evaluated two ways. The RBE values with the comet assay were estimated as 6.3 and 5.4 at 0.37 MeV and 0.57 MeV relative to that of ⁶⁰Co γ-rays, and chromosome aberration rates were also observed in these different levels of monoenergetic neutrons. The yield of chromosome aberrations per unit dose was high at lower neutron energies with a gradual decline with 0.186 MeV neutron energy. The RBE was increased to 10.7 at 0.57 MeV from 3.9 at ²⁵²Cf neutrons and reached 16.4 as the highest RBE at 0.37 MeV, but the value decreased to 11.2 at 0.186 MeV. The response patterns of initial DNA damage and chromosome exchange were quite similar to that of LET. These results show that the intensity of DNA damage and chromosomal exchange is LET dependent. RBE of low energy neutrons is higher than that of fission neutrons. Low energy neutrons containing Hiroshima atomic bomb radiation may have created a significantly higher incidence of biological effect in atomic bomb survivors.

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A previous study of the mutagenic action of ²⁵²Cf radiation in mouse L5178Y cells showed that the mutation frequency was higher when the dose was chronic rather than acute, which was in sharp contrast to the effects reported for γ-rays (Nakamura and Sawada, 1988). A subsequent study using synchronized cells revealed that the cells at the G2/M stage were uniquely sensitive to mutation induction by ²⁵²Cf radiation but not to γ-rays (Tauchi et al., 1993). A log phase cell population was first subjected to conditioning gamma or ²⁵²Cf radiation doses at different dose-rates. The cell cycle distribution of these cells was then observed, and they were then exposed to ²⁵²Cf radiation, and the mutation rate was determined. The G2/M fraction increased by 3- to 4-fold when the conditioning doses (2 Gy of gamma or 1 Gy of ²⁵²Cf radiation) were delivered chronically over 10 h , but only slightly when the same doses were delivered over a 1 h period or less. Subsequent ²⁵²Cf irradiation gave higher mutation frequencies in the cells pre-irradiated with γ-rays over a protracted period of time than in those exposed with the higher dose-rate γ-rays. These results suggest that the radiation-induced G2 block could be at least partly (but not totally) responsible for this reverse dose-rate effect (Tauchi et al. 1996). Possible factors which cause the hyper-sensitivity of G2/M cells to mutation induction by neutrons will be discussed.

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Both cell lethality and neoplastic transformation were assessed for C3H10Tl/2 cells exposed to neutrons with energies from 0.040 to 13.7 MeV. Monoenergetic neutrons with energies from 0.23 to 13.7 MeV and two neutron energy spectra with average energies of 0.040 and 0.070 MeV were produced with a Van de Graaff accelerator at the Radiological Research Accelerator Facility (RARAF) in the Center for Radiological Research of Columbia University. For determination of relative biological effectiveness (RBE), cells were exposed to 250 kVp X rays. With exposures to 250 kVp X rays, both cell survival and radiation-induced oncogenic transformation were curvilinear. Irradiation of cells with neutrons at all energies resulted in linear responses as a function of dose for both biological endpoints. Results indicate a complex relationship between RBE_m and neutron energy. For both survival and transformation, RBE_m was greatest for cells exposed to 0.35 MeV neutrons. RBE_m was significantly less at energies above or below 0.35 MeV. These results are consistent with microdosimetric expectation. These results are also compatible with current assessments of neutron radiation weighting factors for radiation protection purposes. Based on calculations of dose-averaged LET, 0.35 MeV neutrons have the greatest LET and therefore would be expected to be more biologically effective than neutrons of greater or lesser energies.

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Ultrasoft X-rays have been extensively used to explore radiobiological mechanisms surrounding cell killing. These studies for the most part have been linked to a small number of X-ray energies. Recently, this field of study has been broadened by the availability of synchrotron-produced ultrasoft X-rays which can be produced at any desired energy. We have taken advantage of the University of Wisconsin Synchrotron to reexamine two fundamental radiobiological questions: Dose RBE vary with different ultrasoft X-ray energies? Dose the fraction of the nuclear volume exposed to equal total X-ray energy modify cell cytotoxicity? The first study focuses on the survival of Chinese hamster V79 and mouse C3H10T1/2 cells irradiated with synchrotron-produced 273 eV and 860 eV ultrasoft X-rays. These two energies, which are available by multilayer monochromatization of the synchrotron output spectrum, exhibit equal attenuation within living cells. Such an isoattenuating energy pair allows the direct examination of how biological effectiveness varies with the energy of the ultrasoft X-rays. In comparing survival results, we find similar biological effectiveness of these two energies for both the C3H10T1/2 and the V79 cells. These results are no consistent with previous findings of increasing RBE with decreasing ultrasoft X-ray energies. In addition, after correcting for mean nuclear dose based on measurements of cell thickness obtained with confocal microscopy, we find no significant differences in survival between the two ultrasoft X-ray energies and 250 kVp X-rays. These results suggest that RBE does not increase with decreasing energy of ultrasoft X-ray between 860 eV and 273 eV. In a second study we introduced an method which allows partial-volume irradiation of live cells using synchrotron-produced ultrasoft X-rays and micro-fabricated irradiation masks. The masks were made by X-ray lithography at the University of Wisconsin Synchrotron Radiation Center, and they consist of 1.85-μm-wide stripes of gold 1.35 μm apart plated onto thin silicon nitrate membranes. When placed adjacent to mylar on which live cells are plated, these masks allow cells to be irradiated in a striped pattern with dimensions much smaller than the cell nuclei. Using 1340 eV synchrotron-produced X-rays, we compare the survival of cells subjected to uniform irradiation and cells subjected to partial-volume irradiation. Our results show that, at equal mean dose to the nucleus (i.e. equal total energies deposited), survival is not statistically different for the two treatments over a wide range of doses. Thus, imparting equal energies to smaller intranuclear volumes does not appear to modulate cell killing.

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A brief back ground of high-LET radiotherapy developments and its current status will be presented. In addition, based on my experience in developing a program on cancer prevention and early detection in rural India, the gulf between sophisticated developments in cancer diagnosis and treatment and the basic needs in the developing world will be presented with some possible solutions.

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The paper considers the relationship between the quality of radiation and biological lesions produced by ionizing radiation. The paper provides a brief review of the modelling of induction of strand breakage, chromosome aberration, revertant mutation in bacteria and Drosophila melanogaster. Experimental data are presented for the relative biological effectiveness of helium ions and α-particles for mutation induction and genome lethality in Escherichia coli. The paper examines the relationship between the mutational events and LET. The RBE-LET values for T4 phage, E. coli WP2 and mwh (multiple wing hair) show dependency on LET while the wⁱ (white-ivory) allele mutants show no dependency.

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The relative biological effectiveness (RBE) of ²⁵²Cf neutrons and synchrotron-generated high-energy charged particles for mutation induction was evaluated as a function of linear energy transfer (LET), using the loss of heterozygosity for wing-hair mutations and the reversion of the mutant white-ivory eye-color in Drosophila melanogaster. Loss of heterozygosity for wing-hair mutations results predominantly from mitotic crossing over induced in wing anlage cells of larvae, while the reverse mutation of eye-color is due to an intragenic structural change (2.96 kb-DNA excision) in the white locus on the X-chromosome. The measurements were performed in a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-color clone can be detected simultaneously in the same individual. Larvae were irradiated at the age of 3 days post oviposition with ²⁵²Cf neutrons, carbon beam or neon beam. For the neutron irradiation, the RBE values for wing-hair mutations were larger than that for eye-color mutation by about 7 fold. The RBE of carbon ions for producing the wing-hair mutations increased with increase in LET. The estimated RBE values were found to be in the range 2 to 6.5 for the wing-hair. For neon beam irradiation, the RBE values for wing-hair mutations peak near 150 keV/μm and decrease with further increase in LET. On the other hand, the RBE values for the induction of the eye-color mutation are nearly unity in ²⁵²Cf neutrons and both ions throughout the LET range irradiated. We discuss the relationships between the initial DNA damage and LET in considering the mechanism of somatic mutation induction.

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An interest in the possible cancer causing ability of neutrons began soon after their discovery. Early use of neutrons from radioactive sources and from cyclotrons led to a need to define risk for such exposures. This need was soon followed by a more tangible need to define risk to the general population of high LET radiation from nuclear fall out and use of the Atomic bomb and possible use of the H-bomb. Neutrons were soon found to be very effective cell killing agents compared to conventional ionizing radiation. However High LET radiation sources and neutrons in particular, come in many different energies and from many types of sources. I will survey the differences between different energy neutrons and conventional types of radiation, particularly with respect to the dose rate of exposures and the influence of repair or lack thereof and more recently the effect of cell cycle distribution on the carcinogenic outcome. I will illustrate these ideas with examples of carcinogenicity studies and mutation studies from my own laboratory and in some cases from the work of others. Lastly I will introduce some possible avenues for molecular studies of neutron effects that might answer such vexing questions as the real risk at very low doses, is repair error free or error prone, do neutrons cause genetic instability for many cell generations after exposure, and others? There remain many questions about the biology of neutron action that require answers if we are to protect the ever increasing number of people exposed to them because of their growing use in medicine, in the military and in commercial industry.

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Following the hormonal treatment of rats with high prolactin levels and glucocorticoid deficiency (Prl+/Glc-) for 48 days (Day+48), total recoverable mammary DNA was increased by more than sevenfold, tritiated thymidine uptake by nearly fourfold, and total mammary clonogens by about fivefold. Irradiation with 4, 40, and 80 cGy X-rays on Day +48 increased total mammary carcinomas per rat-day-at-risk linearly with dose, and 40 and 80 cGy significantly decreased first carcinoma latency. A dose of 40 cGy X-rays before hormone treatment (Day-1) yielded tumor latencies and frequencies insignificantly different from unirradiated controls but significantly different from those when the dose was given on Day +48. Total carcinomas per rat-day-at-risk were fitted better by a function of dose to the power 0.4 than by a linear function after exposure to 1, 10. and 20 cGy fission neutrons, and 10 and 20 cGy significantly shortened the time to appearance of the first cancer. In contrast to results with X-rays, 10 cGy neutrons on Day -l yielded tumor frequencies and latencies insignificantly different from 10 cGy neutrons on Day +48. The carcinogenic action of X-rays, but not of neutrons, was thus influenced by total clonogen numbers and/or proliferation rates.

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Residual radioactivity data of ¹⁵²Eu, ⁶⁰Co and ³⁶Cl have been accumulated and it has been revealed in the thermal neutron region that a systematic discrepancy exists between the measured data and activation calculation based on the DS86 neutrons in Hiroshima. Recently ⁶³Ni produced in copper samples by the fast neutron reaction ⁶³Cu(n,p) ⁶³Ni has been of interest for evaluation of fast neutrons. Reevaluation of atomic-bomb neutrons and prospects based on residual activity measurements have been discussed.

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There has been a large discrepancy between the Dosimetry system 1986 (DS86)¹⁾ and measured data, some of which data in Hiroshima at about 1.5 km ground distance from the hypocenter are about 10 times larger than the calculation. Therefore its causes have long been discussed^2-10), since it will change the estimated radiation risks obtained based on the Hiroshima and Nagasaki data. In this study the contradiction was explained by a bare-fission-neutron leakage model through a crack formed at the time of neutron emission. According to the present calculation, the crack has a 3 cm parallel spacing, which is symmetric with respect to the polar axis from the hypocenter to the epicenter of the atomic bomb. We made also an asymmetric opening closing 3/4 of this symmetric geometry, because there are some data which shows asymmetry^5,12,13). In addition, the height of the neutron emission point was elevated 90 m. By using the asymmetric calculation, especially for long distant data located more than 1 km, it was verified that all of the activity data induced by thermal and fast neutrons, were simultaneously explained within the data scattering. The neutron kerma at a typical 1.5 km ground distance increases 3 and 8 times more than DS86 based on the symmetric and asymmetric model, respectively.

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Seventy eight atomic bomb survivors were examined for biological dosimetry using chromosome abnormality. They had been exposed within 500 meters from the hypocenter in heavily shielded conditions and were found from NHK-RIRBM joint study carried out from 1966 to 1971. Estimation of the exposure doses for these survivors was made under the following steps; 1) calculation by DS86 system (physically estimated doses) in survivors who had been exposed within 1,500 meters and had precise records of exposure conditions. RBE for the neutron was defined as 10. 2) setting of exposure dose-chromosome aberration curve, and 3) observation of chromosome aberrations in the proximally exposed survivors, for whom biological doses were estimated. Estimation of the exposure doses were possible from the aberration rate of chromosome in the peripheral lymphocytes, even 25-40 years after the exposure. Of the 78 survivors, 96% were estimated to have exposed more than one Sv. Detection of transforming gene(s) of N and K RAS genes in DNAs from non-leukemic survivors was carried out as one of the biological investigations for these heavily exposed survivors. All four survivors examined showed N or K RAS gene mutation. Three of the four healthy survivors had cancer or leukemia 7-10 years after the examination. Further continuous follow-up study of these heavily exposed people will give us more information on the late effects of A-bomb radiation, which may arise in the future.

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Since the first proposal by Brenner and Sachs (Radiation Res. 140, 134-142, 1994), the F-value, the ratio of inter- to intra-chromosomal interchanges, as a biomarker for the quality of radiation has been a matter of repeated discussion. Controversies seem to stem from the selection of data which are heterogeneous in terms of chromosome scoring criteria and dose range. In the context of the critical evaluation of the validity of the F-value, the cytogenetic data obtained in our laboratory from the in vitro irradiation of human peripheral blood lymphocytes have been re-assessed for the F-value. The results were consistent with the original contention that the densely ionizing radiations showed lower F-value. The differential F-value was more pronounced in the low-dose range and disappeared with the increase of the dose, or more precisely with the number of charged particles passing through the cell nucleus. The range of charged particles also plays a role, which makes the F-value of neutrons insensitive to their energy due to a wide variation of the kinetic energy of recoil protons.

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