The maximum low-dose RBE of 17.4 and 40 keV monochromatic X rays for the induction of dicentric chromosomes in human peripheral lymphocytes

Schmid et al. recently reported on the maximum low-dose RBE for mammography X rays (29 kV) for the induction of dicentrics in human lymphocytes. To obtain additional information on the RBE for this radiation quality, experiments with monochromatized synchrotron radiation were performed. Monochromatic 17.4 keV X rays were chosen for comparison with the diagnostic mammography X-ray spectrum to evaluate the spectral influence, while monochromatic 40 keV X rays represent a higher-energy reference radiation, within the experiment. The induction of dicentric chromosomes in human lymphocytes from one blood donor irradiated in vitro with 17.4 keV and 40 keV monochromatic X rays resulted in alpha coefficients of (3.44 +/- 0.87) x 10(-2) Gy(-1) and (2.37 +/- 0.93) x 10(-2) Gy(-1), respectively. These biological effects are only about half of the alpha coefficients reported earlier for exposure of blood from the same donor with the broad energy spectra of 29 kV X rays (mean energy of 17.4 keV) and 60 kV X rays (mean energy of 48 keV). A similar behavior is evident in terms of RBEM. Relative to weakly filtered 220 kV X rays, the RBEM for 17.4 and 40 keV monochromatic X rays is 0.86 +/- 0.23 and 0.59 +/- 0.24, respectively, which is in contrast to the RBEM of 1.64 +/- 0.27 for 29 kV X rays and 1.10 +/- 0.19 for 60 kV X rays. It is evident that the monochromatic radiations are less effective in inducing dicentric chromosomes than broad-spectrum X rays with the corresponding mean energy value. Therefore, it can be assumed that, for these X-ray qualities with broad energy spectra, a large fraction of the effects should be attributed predominantly to photons with energies well below the mean energy.     

Electron spectra and the RBE of X rays

For an assessment of the possible difference in effectiveness between mammography X rays and conventional X rays, the energy and LET spectra of the released electrons are examined. At photon energies below 20 keV and above 100 keV, the energy of the electrons increases with increasing photon energy, which implies that higher-energy photons produce less densely ionizing radiation and are therefore somewhat less effective per unit dose. However, in the intermediate energy range from 20 keV to 100 keV-the range that is relevant to medical diagnostics-the change from the photoelectric effect to the Compton effect causes a transient decrease of electron energies. The ionization density is therefore similar for 200 kVp X rays and 30 kVp mammography X rays, and the distributions of dose in LET suggest an RBE of 30 kVp mammography X rays compared to 200 kVp X rays of up to 1.3. This is in line with an earlier assessment by Brenner and Amols in terms of microdosimetric data, but it is strongly at variance with a recent claim that X rays for mammography are about four times more effective at small doses than conventional X rays and that they cause a correspondingly greater risk for breast cancer. Since LET need not be the only relevant factor, general response functions are examined here that specify-at low dose-the effect per electron of initial energy E and account, for example, for a particular role of the electron range. It is shown that, with any response per electron track that is a nondecreasing function of its starting energy, the low-dose RBE of the mammography X rays relative to the 200 kVp X rays must be substantially less than 2. The Auger electron that accompanies most photoelectrons, but only a minority of the Compton electrons, may increase the effectiveness of the mammography X rays somewhat, but it cannot explain the reported high values of the RBE.     

Microdosimetric analysis of various mammography spectra: lineal energy distributions and ionization cluster analysis

In view of recent recommendations on the frequency and the starting age of mammography screening in healthy women, it is desirable to quantify the enhanced relative biological effectiveness (RBE) of mammography X rays compared to hard X rays. While there is little doubt that the former are more potent in inducing biological damage than the latter, the magnitude of the effect is still hotly debated in the literature. We used Monte Carlo simulations and track structure analysis in micrometer and nanometer volumes to investigate differences in distributions of lineal energy and ionization clusters for a range of mammography X-ray qualities. Dose-averaged lineal energies, (yD), in breast tissue for various mammography qualities were found to result in quality factors about 40% higher than unity. Among the various mammography qualities studied, the popular molybdenum/molybdenum target/filter combination was found to have the highest (yD) in 1-microm spheres (about 5.0 keV/microm near the entrance surface of breast tissue). In 10-nm radius spheres, the mean ionization cluster order was found to be about 35% higher in mammography X rays compared to 300 keV electrons (roughly representing 60Co or 192Ir photon radiation). In even smaller spheres (2 nm radius), no significant differences were observed for the mean ionization cluster order between mammography X rays and 300 keV electrons. We conclude that the potential of mammography X rays to induce biological damage is probably not much higher than a factor of two compared to hard X rays.     

Renal damage in the mouse: the effect of d(4)-Be neutrons

A further study on the response of the mouse kidney to d(4)-Be neutrons (EN = 2.3 MeV) is described. The results confirm and augment the work published previously by Stewart et al. [Br. J. Radiol. 57, 1009-1021 (1984)]; the present paper includes the data from a "top-up" design of experiment which extends the measurements of neutron RBE (relative to 240 kVp X rays) down to X-ray doses of 0.75 Gy per fraction. The mean RBE for these neutrons increases from 5.8 to 7.3 as X-ray dose per fraction decreases from 3.0 to 1.5 Gy in the kidney. This agrees with the predictions from the linear quadratic (LQ) model, based on the renal response to X-ray doses above 4 Gy per fraction. The mean RBE estimate from a single dose group at 0.75 Gy per fraction of X rays is, however, 3.9. This is below the LQ prediction and may indicate increasing X-ray sensitivity at low doses. Data from this study and from those published previously have been used to determine more accurately the shape of the underlying response to d(4)-Be neutrons; an alpha/beta ratio of 20.5 +/- 3.7 Gy was found. The best value of alpha/beta for X rays determined from these experiments was 3.04 +/- 0.35 Gy, in agreement with previous values.     

Relative biological effectiveness of 50-MV X rays on jejunal crypt survival in vivo.

Earlier in vitro studies of relative biological effectiveness (RBE) of 50-MV X rays show an RBE of approximately 1.1 compared to 4 MV. No difference in RBE has been found for 20-MV X rays or 50-MeV electrons. The higher RBE for 50 MV can be explained to some extent by the small high linear energy transfer contribution from photonuclear reactions at high X-ray energies. To investigate the validity of the results in vitro, a study of the RBE of 50-MV X rays has been performed in vivo using the jejunal crypt microcolony assay in mice. The reference radiation used in this case was 20-MV X rays. The results confirm the earlier in vitro studies. The RBE for 50-MV X rays was estimated to be 1.06, calculated as the ratio between the slopes of the response curves.     

Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79)

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.     

Photon W value for krypton in the M-shell transition region

Absolute W values for krypton have been measured for incident X rays with energies in the range of 85 to 1000 eV, using monochromatic synchrotron radiation and a multiple-electrode ion chamber technique that yields the absolute intensity of the X-ray beam and the photoabsorption cross section. To improve the purity of the incident X rays, the electron storage ring was operated at an energy lower than the normal mode, and thin filters were used. The W values are derived from the measured photon intensity and photoabsorption cross section, using the mean charges of the residual ions obtained in previous work. A considerable oscillation of the W values with the photon energy was found in the region near the krypton 3d electron ionization edge. The results are discussed and compared with data in the literature for low-energy electrons and with the calculations from a model that includes multiple photoionization effects related to inner-shell ionization.     

Radiobiology of ultrasoft X rays. IV. Flat and round-shaped hamster cells (CHO-10B, HS-23)

The results reported earlier in this series indicated that the relative biological effectiveness (RBE) of ultrasoft X rays decreases with decreasing cell thickness, approaching unity for the thinnest cells used, plateau-phase human skin fibroblasts (HSF). The possible dependence of RBE on the configuration of the cell nucleus is investigated further in this paper using two CHO cell lines that attach well and have similar intrinsic radiosensitivities to 60Co gamma rays. One of the lines forms monolayers similar to V79 cells, while the other remains more spherical during growth. We find an increasing RBE with decreasing X-ray energy for both of these cell lines, consistent with our results using V79 cells. Also consistent with our results obtained with 10T1/2 and HSF cells, we find an increasing RBE with increasing cell thickness. The possible dependence of RBE on radiosensitivity and the use of the concept of mean dose for ultrasoft X rays is discussed.     

Enhanced neoplastic transformation by mammography X rays relative to 200 kVp X rays: indication for a strong dependence on photon energy of the RBE(M) for various end points.

The fundamental assumption implicit in the use of the atomic bomb survivor data to derive risk estimates is that the gamma rays of Hiroshima and Nagasaki are considered to have biological efficiencies equal to those of other low-LET radiations up to 10 keV/microm, including mammography X rays. Microdosimetric and radiobiological data contradict this assumption. It is therefore of scientific and public interest to evaluate the efficiency of mammography X rays (25-30 kVp) to induce cancer. In this study, the efficiency of mammography X rays relative to 200 kVp X rays to induce neoplastic cell transformation was evaluated using cells of a human hybrid cell line (CGL1). For both radiations, a linear-quadratic dose-effect relationship was observed for neoplastic transformation of CGL1 cells; there was a strong linear component for the 29 kVp X rays. The RBE(M) of mammography X rays relative to 200 kVp X rays was determined to be about 4 for doses < or = 0.5 Gy. A comparison of the electron fluences for both X rays provides strong evidence that electrons with energies of < or = 15 keV can induce neoplastic transformation of CGL1 cells. Both the data available in the literature and the results of the present study strongly suggest an increase of RBE(M) for carcinogenesis in animals, neoplastic cell transformation, and clastogenic effects with decreasing photon energy or increasing LET to an RBE(M) approximately 8 for mammography X rays relative to 60Co gamma rays.     

The induction of chromosome damage in CHO cells by beryllium and radiation given alone and in combination

Studies were conducted to determine the effects of BeSO4 or X rays, alone and in combination, on cell cycle kinetics, cell killing, and the production of chromosome aberrations in Chinese hamster ovary (CHO) cells. The concentration of BeSO4 required to kill 50% of CHO cells exposed to BeSO4 for 20 h was determined to be 1.1 mM with 95% confidence intervals of 0.72 to 1.8 mM. During the last 2 h of the 20-h beryllium treatment (0.2 and 1.0 mM), cells were exposed to 0.0, 1.0, or 2.0 Gy of X rays. Exposure to either BeSO4 or X rays produced a change in cell cycle kinetics which resulted in an accumulation of cells in the G2/M stage of the cell cycle. However, combined exposure to both agents resulted in a block similar to that observed following exposure to X rays only. The background level of chromosome damage was 0.05 +/- 0.015 aberrations/cell in the CHO cells. Seven hours after the end of exposure to 0.2 and 1.0 mM beryllium, 0.03 +/- 0.003 and 0.09 +/- 0.02 aberrations/cell, respectively, were observed. The data for chromosome aberrations following X-ray exposure were fitted to a linear model with a coefficient of 0.14 +/- 0.01 aberrations/cell/Gy. When beryllium was combined with the X-ray exposure the interactive response was predicted by a multiplicative model and was significantly higher (P less than 0.05) than predicted by an additive model. The influence of time after radiation exposure on the interaction between beryllium and X rays was also determined. No interaction between beryllium and X-ray exposure in the induction of chromosome-type aberrations (P greater than 0.05) was detected. The frequency of chromatid-type exchanges and total aberrations was significantly higher (P less than 0.05) in the radiation plus beryllium-exposed cells when compared to cells exposed to X rays only, at both 9 and 12 h after X-ray exposure. These data suggest that the multiplicative interaction may be limited to cells in the S and G2 stages of the cell cycle.     

The effect of hyperthermia on radiation-induced carcinogenesis

Ten groups of mice were exposed to either a single (30 Gy) or multiple (six fractions of 6 Gy) X-ray doses to the leg. Eight of these groups had the irradiated leg made hyperthermic for 45 min immediately following the X irradiation to temperatures of 37 to 43 degrees C. Eight control groups had their legs made hyperthermic with a single exposure or six exposures to heat as the only treatment. In mice exposed to radiation only, the postexposure subcutaneous temperature was 36.0 +/- 1.1 degrees C. Hyperthermia alone was not carcinogenic. At none of the hyperthermic temperatures was the incidence of tumors in the treated leg different from that induced by X rays alone. The incidence of tumors developing in anatomic sites other than the treated leg was decreased in mice where the leg was exposed to hyperthermia compared to mice where the leg was irradiated. A systemic effect of local hyperthermia is suggested to account for this observation. In mice given single X-ray doses and hyperthermia, temperatures of 37, 39, or 41 degrees C did not influence radiation damage as measured by the acute skin reactions. A hyperthermic temperature of 43 degrees C potentiated the acute radiation reaction (thermal enhancement factor 1.1). In the group subjected to hyperthermic temperatures of 37 or 39 degrees C and X rays given in six fractions, the skin reaction was no different from that of the group receiving X rays alone. Hyperthermic temperatures of 41 and 43 degrees C resulted in a thermal enhancement of 1.16 and 1.36 for the acute skin reactions. From Day 50 to Day 600 after treatment, the skin reactions showed regular fluctuations with a 150-day periodicity. Following a fractionated schedule of combined hyperthermia and X rays, late damage to the leg was less than that following X irradiation alone. Mice subjected to X rays and hyperthermic temperatures of 41 and 43 degrees C had a lower median survival time than the mice treated with hyperthermia alone. This effect was not associated with tumor incidence.     

Occurrence of mammary tumors in rats after exposure to tritium beta rays and 200-kVp X rays

The RBE for tritium was estimated in reference to 200-kVp X rays, using acceleration of breast tumor appearance in the female Sprague-Dawley rat as the end-point. Chronic X-ray doses of 0.3-2.0 Gy were delivered over 10 days. Intraperitoneal injections of tritiated water ranging in concentrations from 45 to 370 MBq/100 g body wt were administered, followed by four additional injections at 2-day intervals and half of the initial concentrations. Seventy-five percent of the total tritium dose was delivered to the mammary gland within the first 10 days and 95% within the first 20 days after the start of the tritium exposure. RBE estimations were based on various criteria including the tumor incidence per Gy at 450 days postirradiation and the time required to induce tumors in 50% of the animals at risk. The results suggest that tritium beta rays are about 1.1-1.3 times more effective than chronic 200-kVp X rays for acceleration of the appearance of rat mammary tumors. However, the uncertainties involved in these calculations are such that the effects of tritium beta rays could not be reliably distinguished from those of chronic 200-kVp X rays. Measured differences in RBE values were slightly larger for the comparison between acute and chronic X rays than for the comparison between chronic tritium beta rays and chronic X rays.     

Hard X-ray emission from imploding wire arrays

Results are presented from experimental studies of the generation of hard X-ray (HXR) emission with photon energies above 20 keV during the implosion of wire arrays in the Angara-5-1 facility. An analysis of X-ray images of the Z-pinch shows that the dimensions and spatial structures of the emitting regions are different for hard and soft X rays. It is found that the HXR emission peak is delayed with respect to the soft X-ray (SXR) one. The dependence of the HXR power on the material, initial diameter, and mass (implosion time) of the wire array is determined. It is shown that the HXR intensity in the spectral range >50 keV is several orders higher than the emission intensity in the high-energy tail of the SXR spectrum (assuming that this spectrum is thermal). A comparison of the time evolution and spatial localization of the HXR and SXR sources during the implosion of wire arrays indicates the presence of a new superthermal phenomenon that differs qualitatively from the processes determining the peak power of the SXR pulse. Possible mechanisms that can be responsible for the generation of HXR pulses are considered.     

Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays

Culture of human cells with human interferon alpha and beta (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IF(r) and F-IF(r) cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.     

Dependence of the yield of DNA double-strand breaks in Chinese hamster V79-4 cells on the photon energy of ultrasoft X rays

Induction of DNA DSBs by low-LET radiations reflects clustered damage produced predominantly by low-energy, secondary electron "track ends". Cell inactivation and induction of DSBs and their rejoining, assayed using pulsed-field gel electrophoresis, were determined in Chinese hamster V79-4 cells irradiated as a monolayer with characteristic carbon K-shell (CK) (0.28 keV), aluminum K-shell (AlK) (1.49 keV), and titanium K-shell (TiK) (4.55 keV) ultrasoft X rays under aerobic and anaerobic conditions. Relative to (60)Co gamma rays, the relative biological effectiveness (RBE) for cell inactivation at 10% survival and for induction of DSBs increases as the photon energy of the ultrasoft X rays decreases. The RBE values for cell inactivation and for induction of DSBs by CK ultrasoft X rays are 2.8 +/- 0.3 and 2.7 +/- 0.3, respectively, and by TiK ultrasoft X rays are 1.5 +/- 0.1 and 1.4 +/- 0.1, respectively. Oxygen enhancement ratios (OERs) of approximately 2 for cell inactivation and induction of DSBs by ultrasoft X rays are independent of the photon energy. The time scale for rejoining of DNA DSBs is similar for both ultrasoft X rays and 60Co gamma rays. From the size distribution of small DNA fragments down to 0.48 kbp, we concluded that DSBs are induced randomly by CK and AlK ultrasoft X rays. Therefore, ultrasoft X rays are more efficient per unit dose than gamma radiation at inducing DNA DSBs, the yield of which increases with decreasing photon energy.     

The induction of reciprocal translocations in rhesus monkey stem-cell spermatogonia: effects of low doses and low dose rates

The induction of reciprocal translocation in rhesus monkey spermatogonial stem cells was studied following exposure to low doses of acute X rays (0.25 Gy, 300 mGy/min) or to low-dose-rate X rays (1 Gy, 2 mGy/min) and gamma rays (1 Gy, 0.2 mGy/min). The results obtained at 0.25 Gy of X rays fitted exactly the linear extrapolation down from the 0.5 and 1.0 Gy points obtained earlier. Extension of X-ray exposure reduced the yield of translocations similar to that in the mouse by about 50%. The reduction to 40% of translocation rate after chronic gamma exposure was clearly less than the value of about 80% reported for the mouse over the same range of dose rates. Differential cell killing with ensuing differential elimination of aberration-carrying cells is the most likely explanation for the differences between mouse and monkey.     

Delayed cell cycle progression in human lymphoblastoid cells after exposure to high-LET radiation correlates with extremely localized DNA damage

To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/microm). A highly localized distribution of gammaH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.e. clustered DNA damage, after iron-ion exposure. Despite the well-established notion that clustered DNA damage is refractory to repair compared to isolated DNA lesions, there were no significant differences in the levels of clonogenic survival and apoptosis between cells treated with iron ions or X rays. Strikingly, however, cells accumulated in G(2)/M phase to a much lesser extent after iron-ion exposure than after X-ray exposure. This differential accumulation could be attributed to a much slower evacuation of the S-phase compartment in the case of cells irradiated with iron ions. Taken together, our results indicate that, relative to the situation for low-LET X rays, exposure to high-LET iron ions results in a substantially greater inhibition of S-phase progression as a result of a higher frequency of DNA replication-blocking clustered DNA damage.     

A variable-energy electron microbeam: a unique modality for targeted low-LET radiation

We have designed and constructed a low-cost, variable-energy low-LET electron microbeam that uses energetic electrons to mimic radiation damage produced by gamma and X rays. The microbeam can access lower regions of the LET spectrum, similar to conventional X-ray or 60Co gamma-ray sources. The device has two operating modes, as a conventional microbeam targeting single cells or subpopulations of cells or as a pseudo broad-beam source allowing for direct comparison with conventional sources. By varying the incident electron energy, the target cells can be selectively exposed to different parts of the energetic electron tracks, including the track ends.     

A focused ultrasoft x-ray microbeam for targeting cells individually with submicrometer accuracy.

The application of microbeams is providing new insights into the actions of radiation at the cell and tissue levels. So far, this has been achieved exclusively through the use of collimated charged particles. One alternative is to use ultrasoft X rays, focused by X-ray diffractive optics. We have developed a unique facility that uses 0.2-0.8-mm-diameter zone plates to focus ultrasoft X rays to a beam of less than 1 microm diameter. The zone plate images characteristic K-shell X rays of carbon or aluminum, generated by focusing a beam of 5-10 keV electrons onto the appropriate target. By reflecting the X rays off a grazing-incidence mirror, the contaminating bremsstrahlung radiation is reduced to 2%. The focused X rays are then aimed at selected subcellular targets using rapid automated cell-finding and alignment procedures; up to 3000 cells per hour can be irradiated individually using this arrangement.     

Radiobiological effectiveness (RBE) of megavoltage X-ray and electron beams in radiotherapy

Recent experiments indicate that significant differences exist in the microdosimetric properties (i.e., lineal energy distributions) of megavoltage X-ray and electron beams used in radiation therapy. In particular, dose averaged values of lineal energy for 18 MeV electrons are 10-30% lower than for 10 MeV bremsstrahlung X rays, which in turn are 30-60% lower than for 250 kVp X rays. Differences of this magnitude may manifest themselves in observable radiobiological effectiveness (RBE) differences between these radiations. Cell survival data have been obtained for line DLD-1 human tumor cells on all three of the above radiation sources. Results clearly demonstrate an RBE difference between orthovoltage and megavoltage radiation (P = 0.001). A small difference is also measured in RBE between megavoltage photons and megavoltage electrons, but the difference is not statistically significant (P = 0.25). All biological, dosimetric, and microdosimetric data were obtained under nearly identical geometric conditions. These data raise interesting questions vis à vis the applicability of microdosimetric theories in the interpretation of biological effects.