The new technology of laser-driven ion acceleration (LDA) has shown the potential for driving highly brilliant particle beams. Laser-driven ion acceleration differs from conventional proton sources by its ultra-high dose rate, whose radiobiological impact should be investigated thoroughly before adopting current clinical dose concepts. The growth of human FaDu tumors transplanted onto the hind leg of nude mice was measured sonographically. Tumors were irradiated with 20 Gy of 23 MeV protons at pulsed mode with single pulses of 1 ns duration or continuous mode (∼100 ms) in comparison to controls and to a dose-response curve for 6 MV photons. Tumor growth delay and the relative biological effectiveness (RBE) were calculated for all irradiation modes. The mean target dose reconstructed from Gafchromic films was 17.4 ± 0.8 Gy for the pulsed and 19.7 ± 1.1 Gy for the continuous irradiation mode. The mean tumor growth delay was 34 ± 6 days for pulsed, 35 ± 6 days for continuous protons, and 31 ± 7 days for photons 20 ± 1.2 Gy, resulting in RBEs of 1.22 ± 0.19 for pulsed and 1.10 ± 0.18 for continuous protons, respectively. In summary, protons were found to be significantly more effective in reducing the tumor volume than photons ( P < 0.05). Together with the results of previous in vitro experiments, the in vivo data reveal no evidence for a substantially different radiobiology that is associated with the ultra-high dose rate of protons that might be generated from advanced laser technology in the future.

There is rapidly increasing information on the issue of three-dimensional nuclear architecture, according to which chromosomes are organized in localized territories and chromosome arms in exclusive domains within a given territory. The aim of the present study was to investigate the impact of different cell exposure conditions on cytogenetic damage induced by high-LET radiation. To this end the yield ratio of dicentrics to centric rings ( F value) induced by 241 Am α particles was analyzed in monolayer cultures of human lymphocytes that were either settled or attached to foils, simulating a rounded or spread out cellular geometry, respectively. Monolayers were exposed in special irradiation chambers to 0.1 and 1.0 Gy and subsequently analyzed for chromosome aberrations. Independent of these different dose levels, significantly different F values of 10.07 ± 1.73 and 4.27 ± 0.44 have been determined in attached and settled lymphocytes, respectively. Since the diameter of nuclei vertically traversed by α particles in attached cells is about one-half that in settled cells, these F values support the postulate that proximity effects regarding the chromatin geometry in flattened or spherical human lymphocytes influence the formation of high-LET radiation-induced dicentrics and centric rings. A comparison with our earlier data sets obtained for both in vitro and in vivo exposure of human lymphocytes to α particles or 137 Cs γ rays supports the notion that the F value depends on the radiation quality when investigations are confined to spherical human lymphocytes. Thus the F value should not be ruled out as a practical chromosomal “fingerprint” for past exposure to high-LET radiation.

Laser accelerated radiotherapy is a potential cancer treatment with proton and carbon-ion beams that is currently under development. Ultra-fast high-energy laser pulses will accelerate ion beams that deliver their dose to a patient in a “pulsed mode” that is expected to differ from conventional irradiation by increasing the dose delivery rate to a tissue voxel by approximately 8 orders of magnitude. In two independently performed experiments at the ion microprobe SNAKE of the 14 MV Munich tandem accelerator, A L cells were exposed either to protons with 1-ns pulse durations or to protons applied over 150 ms in continuous irradiation mode. A slightly but consistently lower aberration yield was observed for the pulsed compared to the continuous mode of proton irradiation. This difference was not statistically significant when each aberration type was analyzed separately ( P values between 0.61 and 0.85 in experiment I and P values between 0.32 and 0.64 in experiment II). However, excluding the total aberrations, which were not analyzed as independent radiation-induced effects, the mean ratio of the yields of dicentrics, centric rings and excess acentrics scored together showed (with 95% CI) a significant difference of 0.90 (0.81; 0.98) between the pulsed and the continuous irradiation modes. A similar tendency was also determined for the corresponding RBE values relative to 70 kV X rays. Since the different findings for the comparisons of individual chromosome aberration types and combined comparisons could be explained by different sample sizes with the consequence that the individual comparisons had less statistical power to identify a difference, it can be concluded that 20 MeV protons may be slightly less effective in the pulsed mode.

The aim of this study was to investigate and quantify the production of spindle disturbances in A L cells, a human-hamster hybrid cell line, by 0.106 THz radiation (continuous wave). Monolayer cultures in petri dishes were exposed for 0.5 h to 0.106 THz radiation with power densities ranging from 0.043 mW/cm 2 to 4.3 mW/cm 2 or were kept under sham conditions (negative control) for the same period. As a positive control, 100 µg/ml of the insecticide trichlorfon, which is an aneuploidy-inducing agent, was used for an exposure period of 6 h. During exposure, the sample containers were kept at defined environmental conditions in a modified incubator as required by the cells. Based on a total of 6,365 analyzed mitotic cells, the results of two replicate experiments suggest that 0.106 THz radiation is a spindle-acting agent as predominately indicated by the appearance of spindle disturbances at the anaphase and telophase (especially lagging and non-disjunction of single chromosomes) of cell divisions. The findings in the present study do not necessarily imply disease or injury but may be important for evaluating possible underlying mechanisms.

To obtain greater insight into the future potential of tumor radiotherapy using proton beams generated from high-intensity lasers, it is important to characterize the ionization quality of the new beams by measuring the relative biological effectiveness (RBE) under conditions where the full dose at one irradiation site will be deposited by a few proton pulses less than 1 ns in duration. HeLa cells attached to a Mylar foil were irradiated with 70 kV X rays to obtain a reference dose–response curve or with 3 Gy of 20 MeV protons at the Munich tandem accelerator (Garching), either using a continuous mode where a cell sample was irradiated within a 100-ms time span or using a pulsed mode where radiation was given in a single proton pulse of about 1 ns. After irradiation cytochalasin B was added; 24 h later cells were fixed and stained with acridine orange and micronuclei were counted. The X-ray dose–response curve for the production of micronuclei in HeLa cells followed a linear-quadratic model. The corresponding RBE values for 20 MeV protons in pulsed and continuous irradiation modes were 1.07 ± 0.08 and 1.06 ± 0.10 in the first proton experiment and 1.09 ± 0.08 and 1.05 ± 0.11 in the second, respectively. There was no evidence for a difference in the RBE for pulsed and continuous irradiation of HeLa cells with 20 MeV protons.

Gomolka, M., Rössler, U., Hornhardt, S., Walsh, L., Panzer, W. and Schmid, E. Measurement of the Initial Levels of DNA Damage in Human Lymphocytes Induced by 29 kV X Rays (Mammography X Rays) Relative to 220 kV X Rays and γ Rays. 163, 510–519 (2005). Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137 Cs γ rays and 60 Co γ rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F d = A /( t + A ), where F d is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the γ rays of 137 Cs and 60 Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150–250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.

Schmid, E., Krumrey, M., Ulm, G., Roos, H. and Regulla, D. The Maximum Low-Dose RBE of 17.4 and 40 keV Monochromatic X Rays for the Induction of Dicentric Chromosomes in Human Peripheral Lymphocytes. Radiat. Res. 160, 499–504 (2003). Schmid et al. ( Radiat. Res. 158, 771–777, 2002) 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 α coefficients of (3.44 ± 0.87) × 10 –2 Gy –1 and (2.37 ± 0.93) × 10 –2 Gy –1 , respectively. These biological effects are only about half of the α 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 RBE M . Relative to weakly filtered 220 kV X rays, the RBE M 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 RBE M 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.

Schmid, E., Regulla, D., Kramer, H-M. and Harder, D. The Effect of 29 kV X Rays on the Dose Response of Chromosome Aberrations in Human Lymphocytes. Radiat. Res. 158, 771–777 (2002). The induction of chromosome aberrations in human lymphocytes irradiated in vitro with X rays generated at a tube voltage of 29 kV was examined to assess the maximum low-dose RBE (RBE M ) relative to higher-energy X rays or 60 Co γ rays. Since blood was taken from the same male donor whose blood had been used for previous irradiation experiments using widely varying photon energies, the greatest possible accuracy was available for such an estimation of the RBE M , avoiding the interindividual variations in sensitivity or differences in methodology usually associated with interlaboratory comparisons. The magnitude of the linear coefficient α of the linear-quadratic dose–effect relationship obtained for the production of dicentric chromosomes by 29 kV X rays (α = 0.0655 ± 0.0097 Gy −1 ) confirms earlier observations of a strong increase in α with decreasing photon energy. Relating this value to previously published values of α for the dose–effect curves for dicentrics obtained in our own laboratory, RBE M values of 1.6 ± 0.3 in comparison with weakly filtered 220 kV X rays, 3.0 ± 0.7 compared to heavily filtered 220 kV X rays, and 6.1 ± 2.5 compared to 60 Co γ rays have been obtained. These data emphasize that the choice of the reference radiation is of fundamental importance for the RBE M obtained. A special survey of the RBE M values obtained by different investigators in the narrow quality range from about 30 to 350 kV X rays indicates that the present RBE is in fairly good agreement with previously published findings for the induction of chromosome aberrations or micronuclei in human lymphocytes but differs from recently published findings for neoplastic transformation in a human hybrid cell line.

Regulla, D., Schmid, E., Friedland, W., Panzer, W., Heinzmann, U. and Harder, D. Enhanced Values of the RBE and H Ratio for Cytogenetic Effects Induced by Secondary Electrons from an X-Irradiated Gold Surface. Radiat. Res. 158, 505–515 (2002). The low-energy secondary electrons emerging from the entrance surface of an X-irradiated gold foil increase the dose to cells in contact with or at micrometer distances from this surface ( Radiat. Res. 150, 92–100, 1998). We examined the effect of the spectrum of these low-energy electrons on the RBE for cytogenetic effects and showed that this RBE was increased. A monolayer of surface-attached human T lymphocytes was exposed to 60 kV X rays in the absence or presence of a gold foil positioned immediately behind the cell layer or separated from it by a Mylar foil 0.9 or 2 μm thick. The enhancement of dose in the cell nuclei caused by the photoelectrons and Auger electrons emerging from the entrance surface of the gold foil was measured by TSEE dosimetry. Dose enhancement factors of 55.7, 46.6 and 37.5 were obtained with 0, 0.9 and 2 μm of Mylar inserted between the gold surface and the cell layer. This large enhancement results from the photoelectric effect in the gold foil, as shown by the accompanying Monte Carlo calculations of the secondary electron spectra at the gold surface. Auger electrons from the gold foil generally were not able to penetrate into the cell nuclei except for that fraction of the cells that had a very thin (< 0.7 μm) layer of cytoplasm and membranes between gold surface and cell nucleus. The dose–yield curves for dicentric chromosomes plus centric rings and for acentric fragments obtained after exposures without or with the gold foil were linear-quadratic. The coefficient α, the slope of the linear yield component, was increased in the presence of the gold foil and showed RBE values ranging from 1.7 to 2.2 compared to exposures in absence of the gold foil. The ratio of the yield of interstitial deletions and dicentrics (H ratio) was significantly increased from about 0.17 in the absence of the gold foil to about 0.22 in the presence of the gold foil. The increases in the RBE and the H ratio are interpreted in microdosimetric terms: The preferred occurrence of electron track ends in the vicinity of the gold surface causes an increase in the dose-mean restricted linear energy transfer in cell nuclei exposed to the photoelectrons and Auger electrons.

Schmid, E., Regulla, D., Guldbakke, S., Schlegel, D. and Roos, M. Relative Biological Effectiveness of 144 keV Neutrons in Producing Dicentric Chromosomes in Human Lymphocytes Compared with 60 Co Gamma Rays under Head-to-Head Conditions. Radiat. Res. 157, 453–460 (2002). The RBE for neutrons was assessed in a head-to-head experiment in which cultures of lymphocytes from the same male donor were irradiated simultaneously with 144 keV neutrons and with 60 Co γ rays as the reference radiation and evaluated using matched time, culture conditions, and the end point of chromosomal aberrations to avoid potential confounding factors that would influence the outcome of the experiment. In addition, the irradiation time was held constant at 2 h for the high-dose groups for both radiation types, which resulted in rather low dose rates. For the induction of dicentric chromosomes, the exposure to the 144 keV neutrons was found to be almost equally as effective (yield coefficient α dic = 0.786 ± 0.066 dicentrics per cell per gray) as that found previously for irradiation with monoenergetic neutrons at 565 keV (α dic = 0.813 ± 0.052 dicentrics per cell per gray) under comparable exposure and culture conditions ( Radiat. Res. 154, 307–312, 2000). However, the values of the maximum low-dose RBE (RBE m ) relative to 60 Co γ rays that were determined in the present and previous studies show an insignificant but conspicuous difference: 57.0 ± 18.8 and 76.0 ± 29.5, respectively. This difference is mainly due to the difference in the α dic value of the 60 Co γ rays, the reference radiation, which was 0.0138 ± 0.0044 Gy −1 in the present study and 0.0107 ± 0.0041 Gy −1 in the previous study. In the present experiment, irradiations with 144 keV neutrons and 60 Co γ rays were both performed at 21°C, while in the earlier experiment irradiations with 565 keV neutrons were performed at 21°C and the corresponding reference irradiation with γ rays was performed at 37°C. However, the temperature difference between 21°C and 37°C has a minor influence on the yield of chromosomal alterations and hence RBE values. The large cubic PMMA phantom that was used for the γ irradiations in the present study results in a larger dose contribution from Compton-scattered photons compared to the mini-phantom used in the earlier experiments. The contribution of these scattered photons may explain the large value of α dic for γ irradiation in the present study. These results indicate that the yield coefficient α dic for 144 keV neutrons is similar to the one for 565 keV neutrons, and that modification of the α dic value of the low-LET reference radiation, due to changes in the experimental conditions, can influence the RBE m . Consequently, α dic values cannot be shared between cytogenetic laboratories for the purpose of assessment of RBM m without verification of the comparability of the experimental conditions.

Regulla, D., Panzer, W., Schmid, E., Stephan, G. and Harder, D. Detection of Elevated RBE in Human Lymphocytes Exposed to Secondary Electrons Released from X-Irradiated Metal Surfaces. Radiat. Res. 155, 744–747 (2001). Monolayers of human lymphocytes, attached to a 2-μm Mylar film, were irradiated with 60 kV X rays in the presence and absence of a 150-μm gold film backing the Mylar film. With the gold film present, the absorbed dose imparted to the cells was increased by a factor of 45.4 due to the release of photoelectrons from the gold film. The frequencies of dicentric chromosomes and centric rings as well as of excess acentric fragments were increased in agreement with this dose enhancement, and in addition an RBE of about 1.7 compared to the frequencies observed in the absence of the gold film was found. These radiation effects, which contribute to risk considerations in radiology, are interpreted in terms of the increased dose-mean restricted LET of the photoelectrons backscattered from the metal and slowed down in the Mylar film before they enter the cell layer.

Schmid, E., Regulla, D., Guldbakke, S., Schlegel, D. and Bauchinger, M. The Effectiveness of Monoenergetic Neutrons at 565 keV in Producing Dicentric Chromosomes in Human Lymphocytes at Low Doses. The induction of dicentric chromosomes in human lymphocytes from one individual irradiated in vitro with monoenergetic neutrons at 565 keV was examined to provide additional data for an improved evaluation of neutrons with respect to radiation risk in radioprotection. The resulting linear dose–response relationship obtained (0.813 ± 0.052 dicentrics per cell per gray) over the dose range of 0.0213–0.167 Gy is consistent with published results obtained for irradiation with neutrons from different sources and with different spectra at energies lower than 1000 keV. Comparing this value to previously published “average” dose–response curves obtained by different laboratories for 60 Co γ rays and orthovoltage X rays resulted in maximum RBEs (RBE m ) of about 37 ± 8 and 16 ± 4, respectively. However, when our neutron data were matched to low-LET dose responses that were constructed several years earlier for lymphocytes from the same individual, higher values of RBE m resulted: 76.0 ± 29.5 for 60 Co γ rays and 54.2 ± 18.4 for 137 Cs γ rays; differentially filtered 220 kV X rays produced values of RBE m between 20.3 ± 2.0 or 37.0 ± 7.1. The results highlight the dependence of RBE m on the choice of low-LET reference radiation and raise the possibility that differential individual response to low-LET radiations may need to be examined more fully in this context.

The debate on the validity of the ratios of radiation-induced yields of chromosome aberrations, in particular the F value (dicentrics/ring chromosomes), as a chromosomal fingerprint of radiation quality is still in progress. From a recent analysis of their experimental data, Sasaki et al. (Radiat. Res. 150, 253-258, 1998) noted that despite a considerable variability in the data, the limiting F value at the lowest doses, or the F 0 value, obviously decreased with increasing LET, indicating that the LET could be a factor that determines the F value. We have reassessed here our own 13 cytogenetic data sets that cover a range of dose-averaged LET of 0.5 to 150 keV/μm in terms of this <tex-math>$F_{0}\text{-value}$</tex-math> approach, but we could not confirm such a dependence on LET at very low doses. The validity of the F value as a biomarker therefore remains questionable. For a final evaluation, scoring of a far greater number of cells at low doses would be necessary to reduce the large error ranges of F values.

A selection of published data on the ratio, F, of interchromosomal to intrachromosomal stable (reciprocal translocations and pericentric inversions) and unstable (dicentric chromosomes and centric rings) exchange aberrations in human lymphocytes has recently been presented as evidence for F values of about 15 for X and γ rays and about 6 for neutrons and α particles (D. J. Brenner and R. K. Sachs, Radiat. Res. 140, 134-142, 1994). On this basis it was proposed that low F values could serve as a chromosomal "fingerprint" of densely ionizing radiation. In the present commentary it is shown that some of the quoted data sets provide little support for this concept. It is further demonstrated that our own data, including a "head-to-head" experiment with γ rays and α particles, reveal no LET dependence, even in the comparison of F values from low-LET radiation with those from low doses of high-LET radiation. In this context it is pointed out that a change in F values cannot be expected at doses of high-LET radiation where the linear component of the dose-effect relationship for exchange aberrations prevails. Additional data for the effects of high- and low-LET radiation which have not been considered in the discussion so far confirm that support of the concept of F-ratio "fingerprinting" by experimental data is insufficient.

Human peripheral lymphocytes were irradiated with different doses of 3-MeV electrons. The dosimetry was performed according to Fricke. The frequency of different aberration types was determined and the dose-response relation was calculated. The data were fitted by least-squares regression analysis to different models. The dicentric, dicentric + centric ring, and different acentric data gave the best fit to the linear quadratic model. The RBE of 3-MeV electrons compared with 220-kV x-rays increases with increasing dose.