International Comparison Exercise for Biological Dosimetry after Exposures with Neutrons Performed at Two Irradiation Facilities as Part of the BALANCE Project.

In the case of a radiological or nuclear event, biological dosimetry can be an important tool to support clinical decision-making. During a nuclear event, individuals might be exposed to a mixed field of neutrons and photons. The composition of the field and the neutron energy spectrum influence the degree of damage to the chromosomes. During the transatlantic BALANCE project, an exposure similar to a Hiroshima-like device at a distance of 1.5 km from the epicenter was simulated, and biological dosimetry based on dicentric chromosomes was performed to evaluate the participants ability to discover unknown doses and to test the influence of differences in neutron spectra. In a first step, calibration curves were established by irradiating blood samples with 5 doses in the range of 0-4 Gy at two different facilities in Germany (Physikalisch-Technische Bundesanstalt [PTB]) and the USA (the Columbia IND Neutron Facility [CINF]). The samples were sent to eight participating laboratories from the RENEB network and dicentric chromosomes were scored by each participant. Next, blood samples were irradiated with 4 blind doses in each of the two facilities and sent to the participants to provide dose estimates based on the established calibration curves. Manual and semiautomatic scoring of dicentric chromosomes were evaluated for their applicability to neutron exposures. Moreover, the biological effectiveness of the neutrons from the two irradiation facilities was compared. The calibration curves from samples irradiated at CINF showed a 1.4 times higher biological effectiveness compared to samples irradiated at PTB. For manual scoring of dicentric chromosomes, the doses of the test samples were mostly successfully resolved based on the calibration curves established during the project. For semiautomatic scoring, the dose estimation for the test samples was less successful. Doses >2 Gy in the calibration curves revealed nonlinear associations between dose and dispersion index of the dicentric counts, especially for manual scoring. The differences in the biological effectiveness between the irradiation facilities suggested that the neutron energy spectrum can have a strong impact on the dicentric counts.

Correction: Belchior et al. Repair Kinetics of DSB-Foci Induced by Proton and α-Particle Microbeams of Different Energies. Life 2022, 12, 2040.

In the original publication [...].

Repair Kinetics of DSB-Foci Induced by Proton and α-Particle Microbeams of Different Energies.

In this work, the induction and repair of radiation-induced 53BP1 foci were studied in human umbilical vein endothelial cells irradiated at the PTB microbeam with protons and α-particles of different energies. The data were analyzed in terms of the mean number of 53BP1 foci induced by the different ion beams. The number of 53BP1 foci found at different times post-irradiation suggests that the disappearance of foci follows first order kinetics. The mean number of initially produced foci shows the expected increase with LET. The most interesting finding of this work is that the absolute number of persistent foci increases with LET but not their fraction. Furthermore, protons seem to produce more persistent foci as compared to α-particles of even higher LET. This may be seen as experimental evidence that protons may be more effective in producing severe DNA lesions, as was already shown in other work, and that LET may not be the best suited parameter to characterize radiation quality.

The role of the construction and sensitive volume of compact ionization chambers on the magnetic field-dependent dose response.

PURPOSE: The magnetic-field correction factors k B , Q of compact air-filled ionization chambers have been investigated experimentally and using Monte Carlo simulations up to 1.5 T. The role of the nonsensitive region within the air cavity and influence of the chamber construction on its dose response have been elucidated. MATERIALS AND METHODS: The PTW Semiflex 3D 31021, PinPoint 3D 31022, and Sun Nuclear Cooperation SNC125c chambers were studied. The k B , Q factors were measured at the experimental facility of the German National Metrology Institute (PTB) up to 1.4 T using a 6 MV photon beam. The chambers were positioned with the chamber axis perpendicular to the beam axis (radial); and parallel to the beam axis (axial). In both cases, the magnetic field was directed perpendicular to both the beam axis and chamber axis. Additionally, the sensitive volumes of these chambers have been experimentally determined using a focused proton microbeam and finite element method. Beside the simulations of k B , Q factors, detailed Monte Carlo technique has been applied to analyse the secondary electron fluence within the air cavity, that is, the number of secondary electrons and the average path length as a function of the magnetic field strength. RESULTS: A nonsensitive volume within the air cavity adjacent to the chamber stem for the PTW chambers has been identified from the microbeam measurements and FEM calculations. The dose response of the three investigated ionization chambers does not deviate by more than 4% from the field-free case within the range of magnetic fields studied in this work for both the radial and axial orientations. The simulated k B , Q for the fully guarded PTW chambers deviate by up to 6% if their sensitive volumes are not correctly considered during the simulations. After the implementation of the sensitive volume derived from the microbeam measurements, an agreement of better than 1% between the experimental and Monte Carlo k B , Q factors for all three chambers can be achieved. Detailed analysis reveals that the stem of the PTW chambers could give rise to a shielding effect reducing the number of secondary electrons entering the air cavity in the presence of magnetic field. However, the magnetic field dependence of their path length within the air cavity is shown to be weaker than for the SNC125c chamber, where the length of the air cavity is larger than its diameter. For this chamber it is shown that the number of electrons and their path lengths in the cavity depend stronger on the magnetic field. DISCUSSION AND CONCLUSION: For clinical measurements up to 1.5 T, the required k B , Q corrections of the three chambers could be kept within 3% in both the investigated chamber orientations. The results reiterate the importance of considering the sensitive volume of fully guarded chambers, even for the investigated compact chambers, in the Monte Carlo simulations of chamber response in magnetic field. The resulting magnetic field-dependent dose response has been demonstrated to depend on the chamber construction, such as the ratio between length and the diameter of the air cavity as well as the design of the chamber stem.

Three-dimensional characterization of the active volumes of PTW microDiamond, microSilicon, and Diode E dosimetry detectors using a proton microbeam.

PURPOSE: The purpose of this work is the three-dimensional characterization of the active volumes of commercial solid-state dosimetry detectors. Detailed knowledge of the dimensions of the detector's active volume as well as the detector housing is of particular interest for small-field photon dosimetry. As shown in previous publications from different groups, the design of the detector housing influences the detector signal for small photon fields. Therefore, detailed knowledge of the active volume dimension and the surrounding materials form the basis for accurate Monte Carlo simulations of the detector. METHODS: A 10 MeV proton beam focused by the microbeam system of the Physikalisch-Technische Bundesanstalt was used to measure two-dimensional response maps of a synthetic diamond detector (microDiamond, type 60019, PTW Freiburg) and two silicon detectors (microSilicon, type 60023, PTW Freiburg and Diode E, type 60017, PTW Freiburg). In addition, the thickness of the active volume of the new microSilicon was measured using the method developed in a previous study. RESULTS: The analysis of the response maps leads to active area of 1.18 mm2 for the Diode E, 1.75 mm2 for the microSilicon, and 3.91 mm2 for the microDiamond detector. The thickness of the active volume of the microSilicon detector was determined to be (17.8 ± 2) µm. CONCLUSIONS: This study provides detailed geometrical data of the dosimetric active volume of three different solid-state detector types.

From Energy Deposition of Ionizing Radiation to Cell Damage Signaling: Benchmarking Simulations by Measured Yields of Initial DNA Damage after Ion Microbeam Irradiation.

Advances in accelerator technology, which have enabled conforming radiotherapy with charged hadronic species, have brought benefits as well as potential new risks to patients. To better understand the effects of ionizing radiation on tumor and surrounding tissue, it is important to investigate and quantify the relationship between energy deposition at the nanometric scale and the initial biological events. Monte Carlo track structure simulation codes provide a powerful tool for investigating this relationship; however, their success and reliability are dependent on their improvement and development accordingly to the dedicated biological data to which they are challenged. For this aim, a microbeam facility that allows for fluence control, down to one ion per cell nucleus, was used to evaluate relative frequencies of DNA damage after interaction between the incoming ion and DNA according to radiation quality. Primary human cells were exposed to alpha particles of three different energies with respective linear energy transfers (LETs) of approximately 36, 85 or 170 keV·µm-1 at the cells' center position, or to protons (19 keV·µm-1). Statistical evaluation of nuclear foci formation (53BP1/γ-H2AX), observed using immunofluorescence and related to a particle traversal, was undertaken in a large population of cell nuclei. The biological results were adjusted to consider the factors that drive the experimental uncertainties, then challenged with results using Geant4-DNA code modeling of the ionizing particle interactions on a virtual phantom of the cell nucleus with the same mean geometry and DNA density as the cells used in our experiments. Both results showed an increase of relative frequencies of foci (or simulated DNA damage) in cell nuclei as a function of increasing LET of the traversing particles, reaching a quasi-plateau when the LET exceeded 80-90 keV·µm-1. For the LET of an alpha particle ranging from 80-90 to 170 keV·µm-1, 10-30% of the particle hits did not lead to DNA damage inducing 53BP1 or γ-H2AX foci formation.

INVESTIGATION INTO THE PROBABILITY FOR MISCOUNTING IN FOCI-BASED ASSAYS.

When early radiation damage to biological systems is studied based on the formation of foci at the location of DNA double-strand breaks, the foci observed in irradiated cells either may be induced by ionizing radiation (IR) interactions or they may be due to other causes that lead to observation of foci also in unirradiated cells. Generally, to take account of the latter, additional samples are taken where the exposure to IR is skipped in the protocol. The data analysis relies on statistical independence of the frequency distributions of background and radiation-induced foci. In microscopy, however, the observed spatial patterns of foci are 2D projections of the spatial distributions of foci in the observed cell nuclei. This may lead to missing foci when scoring their number, particularly if projections of foci overlap or coincide. This paper investigates to what extent the statistical independence of the frequency distribution of the number of foci coming from IR interaction or other causes is compromised by foci overlapping.

PROSPECTS FOR METROLOGY RELATED TO BIOLOGICAL RADIATION EFFECTS OF ION BEAMS.

In recent years, several approaches have been proposed to provide an understanding of the enhanced relative biological effectiveness of ion beams based on multi-scale models of their radiation effects. Among these, the BioQuaRT project was the only one which focused on developing metrology for a multi-scale characterization of particle track structure. The progress made within the BioQuaRT project has motivated the formation of a department 'Radiation Effects' at PTB dedicated to metrological research on ionizing radiation effects. This paper gives an overview of the department's present research directions and shortly discusses ideas for the future development of metrology related to biological effects of ion beams that are based on a stakeholder consultation.

Lack of DNA Damage Response at Low Radiation Doses in Adult Stem Cells Contributes to Organ Dysfunction.

PURPOSE: Radiotherapy for head and neck cancer may result in serious side effects, such as hyposalivation, impairing the patient's quality of life. Modern radiotherapy techniques attempt to reduce the dose to salivary glands, which, however, results in low-dose irradiation of the tissue stem cells. Here we assess the low-dose sensitivity of tissue stem cells and the consequences for tissue function. EXPERIMENTAL DESIGN: Postirradiation rat salivary gland secretory function was determined after pilocarpine induction. Murine and patient-derived salivary gland and thyroid gland organoids were irradiated and clonogenic survival was assessed. The DNA damage response (DDR) was analyzed in organoids and modulated using different radiation modalities, chemical inhibition, and genetic modification. RESULTS: Relative low-dose irradiation to the high-density stem cell region of rat salivary gland disproportionally impaired function. Hyper-radiosensitivity at doses <1 Gy, followed by relative radioresistance at doses ≥1 Gy, was observed in salivary gland and thyroid gland organoid cultures. DDR modulation resulted in diminished, or even abrogated, relative radioresistance. Furthermore, inhibition of the DDR protein ATM impaired DNA repair after 1 Gy, but not 0.25 Gy. Irradiation of patient-derived salivary gland organoid cells showed similar responses, whereas a single 1 Gy dose to salivary gland-derived stem cells resulted in greater survival than clinically relevant fractionated doses of 4 × 0.25 Gy. CONCLUSIONS: We show that murine and human glandular tissue stem cells exhibit a dose threshold in DDR activation, resulting in low-dose hyper-radiosensitivity, with clinical implications in radiotherapy treatment planning. Furthermore, our results from patient-derived organoids highlight the potential of organoids to study normal tissue responses to radiation.

Comparative gene expression analysis after exposure to 123I-iododeoxyuridine, γ- and α-radiation-potential biomarkers for the discrimination of radiation qualities.

Gene expression analysis was carried out in Jurkat cells in order to identify candidate genes showing significant gene expression alterations allowing robust discrimination of the Auger emitter 123I, incorporated into the DNA as 123I-iododeoxyuridine (123IUdR), from α- and γ-radiation. The γ-H2AX foci assay was used to determine equi-effect doses or activity, and gene expression analysis was carried out at similar levels of foci induction. Comparative gene expression analysis was performed employing whole human genome DNA microarrays. Candidate genes had to show significant expression changes and no altered gene regulation or opposite regulation after exposure to the radiation quality to be compared. The gene expression of all candidate genes was validated by quantitative real-time PCR. The functional categorization of significantly deregulated genes revealed that chromatin organization and apoptosis were generally affected. After exposure to 123IUdR, α-particles and γ-rays, at equi-effect doses/activity, 155, 316 and 982 genes were exclusively regulated, respectively. Applying the stringent requirements for candidate genes, four (PPP1R14C, TNFAIP8L1, DNAJC1 and PRTFDC1), one (KLF10) and one (TNFAIP8L1) gene(s) were identified, respectively allowing reliable discrimination between γ- and 123IUdR exposure, γ- and α-radiation, and α- and 123IUdR exposure, respectively. The Auger emitter 123I induced specific gene expression patterns in Jurkat cells when compared with γ- and α-irradiation, suggesting a unique cellular response after 123IUdR exposure. Gene expression analysis might be an effective tool for identifying biomarkers for discriminating different radiation qualities and, furthermore, might help to explain the varying biological effectiveness at the mechanistic level.

Determination of the active volumes of solid-state photon-beam dosimetry detectors using the PTB proton microbeam.

PURPOSE: This study aims at the experimental determination of the diameters and thicknesses of the active volumes of solid-state photon-beam detectors for clinical dosimetry. The 10 MeV proton microbeam of the PTB (Physikalisch-Technische Bundesanstalt, Braunschweig) was used to examine two synthetic diamond detectors, type microDiamond (PTW Freiburg, Germany), and the silicon detectors Diode E (PTW Freiburg, Germany) and Razor Diode (Iba Dosimetry, Germany). The knowledge of the dimensions of their active volumes is essential for their Monte Carlo simulation and their applications in small-field photon-beam dosimetry. METHODS: The diameter of the active detector volume was determined from the detector current profile recorded by radially scanning the proton microbeam across the detector. The thickness of the active detector volume was determined from the detector's electrical current, the number of protons incident per time interval and their mean stopping power in the active volume. The mean energy of the protons entering this volume was assessed by comparing the measured and the simulated influence of the thickness of a stack of aluminum preabsorber foils on the detector signal. RESULTS: For all detector types investigated, the diameters measured for the active volume closely agreed with the manufacturers' data. For the silicon Diode E detector, the thickness determined for the active volume agreed with the manufacturer's data, while for the microDiamond detectors and the Razor Diode, the thicknesses measured slightly exceeded those stated by the manufacturers. DISCUSSION: The PTB microbeam facility was used to analyze the diameters and thicknesses of the active volumes of photon dosimetry detectors for the first time. A new method of determining the thickness values with an uncertainty of ±10% was applied. The results appear useful for further consolidating detailed geometrical knowledge of the solid-state detectors investigated, which are used in clinical small-field photon-beam dosimetry.

Analysis of Radiation-Induced Chromosomal Aberrations on a Cell-by-Cell Basis after Alpha-Particle Microbeam Irradiation: Experimental Data and Simulations.

There is a continued need for further clarification of various aspects of radiation-induced chromosomal aberration, including its correlation with radiation track structure. As part of the EMRP joint research project, Biologically Weighted Quantities in Radiotherapy (BioQuaRT), we performed experimental and theoretical analyses on chromosomal aberrations in Chinese hamster ovary cells (CHO-K1) exposed to α particles with final energies of 5.5 and 17.8 MeV (absorbed doses: ∼2.3 Gy and ∼1.9 Gy, respectively), which were generated by the microbeam at the Physikalisch-Technische Bundesanstalt (PTB) in Braunschweig, Germany. In line with the differences in linear energy transfer (approximately 85 keV/µm for 5.5 MeV and 36 keV/µm for 17.8 MeV α particles), the 5.5 MeV α particles were more effective than the 17.8 MeV α particles, both in terms of the percentage of aberrant cells (57% vs. 33%) and aberration frequency. The yield of total aberrations increased by a factor of ∼2, although the increase in dicentrics plus centric rings was less pronounced than in acentric fragments. The experimental data were compared with Monte Carlo simulations based on the BIophysical ANalysis of Cell death and chromosomal Aberrations model (BIANCA). This comparison allowed interpretation of the results in terms of critical DNA damage [cluster lesions (CLs)]. More specifically, the higher aberration yields observed for the 5.5 MeV α particles were explained by taking into account that, although the nucleus was traversed by fewer particles (nominally, 11 vs. 25), each particle was much more effective (by a factor of ∼3) at inducing CLs. This led to an increased yield of CLs per cell (by a factor of ∼1.4), consistent with the increased yield of total aberrations observed in the experiments.

Comparative study of the effects of different radiation qualities on normal human breast cells.

BACKGROUND: As there is a growing number of long-term cancer survivors, the incidence of carcinogenesis as a late effect of radiotherapy is getting more and more into the focus. The risk for the development of secondary malignant neoplasms might be significantly increased due to exposure of healthy tissue outside of the target field to secondary neutrons, in particular in proton therapy. Thus far, the radiobiological effects of these neutrons and a comparison with photons on normal breast cells have not been sufficiently characterised. METHODS: MCF10A cells were irradiated with doses of up to 2 Gy with neutrons of different energy spectra and X-rays for comparison. The biological effects of neutrons with a broad energy distribution ( = 5.8 MeV), monoenergetic neutrons (1.2 MeV, 0.56 MeV) and of the mixed field of gamma's and secondary neutrons ( = 70.5 MeV) produced by 190 MeV protons impinging on a water phantom, were analysed. The clonogenic survival and the DNA repair capacity were determined and values of relative biological effectiveness were compared. Furthermore, the influence of radiation on the sphere formation was observed to examine the radiation response of the potential fraction of stem like cells within the MCF10A cell population. RESULTS: X-rays and neutrons caused dose-dependent decreases of survival fractions after irradiations with up to 2 Gy. Monoenergetic neutrons with an energy of 0.56 MeV had a higher effectiveness on the survival fraction with respect to neutrons with higher energies and to the mixed gamma - secondary neutron field induced by proton interactions in water. Similar effects were observed for the DNA repair capacity after exposure to ionising radiation (IR). Both experimental endpoints provided comparable values of the relative biological effectiveness. Significant changes in the sphere formation were notable following the various radiation qualities. CONCLUSION: The present study compared the radiation response of MCF10A cells after IR with neutrons and photons. For the first time it was shown that monoenergetic neutrons with energies around 1 MeV have stronger radiobiological effects on normal human breast cells with respect to X rays, to neutrons with a broad energy distribution ( = 5.8 MeV), and to the mixed gamma - secondary neutron field given by interactions of 190 MeV protons in water. The results of the present study are highly relevant for further investigations of radiation-induced carcinogenesis and are very important in perspective for a better risk assessment after secondary neutron exposure in the field of conventional and proton radiotherapy.

53BP1 and MDC1 foci formation in HT-1080 cells for low- and high-LET microbeam irradiations.

An improved assessment of the biological effects and related risks of low doses of ionizing radiation is currently an important issue in radiation biology. Irradiations using microbeams are particularly well suited for precise and localized dose depositions, whereas recombinant cell lines with fluorescent proteins allow the live observation of radiation-induced foci. Living cells of the fibrosarcoma cell line HT-1080 stably expressing 53BP1 or full-length reconstituted MDC1 fused to Green Fluorescent Protein (GFP) were irradiated with protons and α-particles of linear energy transfers (LETs) of 15 and 75 keV/µm, respectively. Using a microbeam, the irradiations were carried out in line patterns, which facilitated the discrimination between undefined background and radiation-induced foci. As expected, foci formation and respective kinetics from α-particle irradiations with a high LET of 75 keV/µm could be detected in a reliable manner by both fusion proteins, as reported previously. Colocalization of γ-H2AX foci confirmed the DSB nature of the detected foci. As a novel result, the application of protons with low LET of 15 keV/µm generated 53BP1- and MDC1-mediated foci of almost equal size and slightly different kinetics. This new data expands the capability of 53BP1 and wild-type MDC1 on visible foci formation in living cells after irradiation with low-LET particles. Furthermore, the kinetics in HT-1080 cells for α-particle irradiation show a delay of about 20 s for 53BP1 foci detection compared to wild-type MDC1, confirming the hierarchical assembly of both proteins. Preliminary data for proton irradiations are shown and also these indicate a delay for 53BP1 versus MDC1.

The role of nonhomologous end joining and homologous recombination in the clonogenic bystander effects of mammalian cells after exposure to counted 10 MeV protons and 4.5 MeV alpha-particles of the PTB microbeam.

We have studied the dependence of clonogenic bystander effects on defects in the pathways of DNA double-strand break (DSB) repair and on linear energy transfer (LET). The single-ion microbeam of the Physikalisch-Technische Bundesanstalt (PTB) was used to irradiate parental Chinese hamster ovary cells or derivatives deficient in nonhomologous end joining (NHEJ) or homologous recombination (HR) in the G1-phase of the cell cycle. Cell nuclei were targeted with 10 MeV protons (LET = 4.7 keV/microm) or 4.5 MeV alpha-particles (LET = 100 keV/microm). During exposure, the cells were confluent, allowing signal transfer through both gap junctions and diffusion. When all cell nuclei were targeted with 10 MeV protons, approximately exponential survival curves were obtained for all three cell lines. When only 10% of all cell nuclei were targeted, a significant bystander effect was observed for parental and HR-deficient cells, but not for NHEJ-deficient cells. For all three cell lines, the survival data after exposure of all cell nuclei to 4.5 MeV alpha-particles could be fitted by exponential curves. When only 10% of all cell nuclei were targeted, significant bystander effects were obtained for parental and HR-deficient cells, whereas for NHEJ-deficient cells a small, but significant, bystander effect was observed only at higher doses. The data suggest that bystander cell killing is a consequence of un- or misrejoined DSB which occur in bystander cells during the S-phase as a result of the processing of oxidative bistranded DNA lesions. The relative contributions of NHEJ and HR to the repairing of DSB in the late S/G2-phase may affect clonogenic bystander effects.

Chromosome aberration analysis and the influence of mitotic delay after simulated partial-body exposure with high doses of sparsely and densely ionising radiation.

The influence of high doses of sparsely and densely ionising radiation on the yield of aberrant human peripheral lymphocytes in simulated partial-body exposures was studied by investigating radiation-induced chromosome aberration frequencies, namely dicentric and centric ring chromosomes. Peripheral blood samples from two volunteers were irradiated with high doses of 200 kV X-rays or neutrons with a mean energy of or =2.1 MeV and partial-body exposure was simulated by mixing irradiated and non-irradiated blood from the same two donors in proportions of 25, 50, and 75%. Lymphocytes were cultured and first-division metaphase cells were collected after culture times of 48, 56, and 72 h. A significant underrepresentation of dicentric and centric ring chromosomes was observed at the three highest doses of X-rays between the different culture times for nearly all proportions. After neutron irradiation, some significant differences were observed at all doses and all culture times, without however, revealing any systematic pattern. The distribution of dicentric and ring chromosomes showed overdispersion for both radiation types. After simulated partial-body exposures with 200 kV X-rays and or =2.1 MeV neutrons, strong mitotic delays could be observed, which depended on both the irradiated volume and the applied dose: the smaller the irradiated volume and the higher the dose, the higher was the selective advantage of non-irradiated cells. For the purpose of biological dosimetry after partial body exposure, an extension of the lymphocyte culture time is suggested at least for doses > or =3.0 Gy of 200 kV X-rays and > or =0.5 Gy of or =2.1 MeV neutrons in order to prevent a systematic underestimation of cytogenetic damage.

Radiation response of primary human skin fibroblasts and their bystander cells after exposure to counted particles at low and high LET.

PURPOSE: To investigate the dependence of bystander effects on linear energy transfer (LET) in the low dose region. MATERIALS AND METHODS: The single-ion microbeam of the Physikalisch-Technische Bundesanstalt (PTB) was used to irradiate confluent primary human skin fibroblasts. Cells plated on a special irradiation dish were targeted with 10 MeV protons (LET 4.7 keV/microm) and 4.5 MeV a-particles (LET 100 keV/microm). During exposure, the cells were confluent allowing signal transfers through both gap junctions and diffusion. RESULTS: For 10 MeV protons the clonogenic capability was significantly higher after exposure to 70 protons (0.31 Gy) compared with unirradiated cells. For higher doses the survival curve was exponential. Exposure of only 10% of all nuclei resulted in a similar radiation response in the low dose region. For higher doses up to 2.2 Gy no cell killing was observed. For 4.5 MeV alpha-particles an exponential survival curve was obtained. Irradiation of only 10% of all cell nuclei resulted in an survival curve as had been expected in the absence of any bystander effect. CONCLUSION: The type and extent of bystander effects turned out to be dependent on the particles' LET and are likely to depend also on the cell line used and the techniques applied.