PROPOSAL FOR A EUROPEAN METROLOGY NETWORK ON BIOLOGICAL IONISING RADIATION EFFECTS.

Progress in the field of ionising radiation (IR) metrology achieved in the BioQuaRT project raised the question to what extent radiobiological investigations would benefit from metrological support of the applied methodologies. A panel of experts from the medical field, fundamental research and radiation protection attended a workshop at Physikalisch-Technische Bundesanstalt to consult on metrology needs related to biological radiation effects. The panel identified a number of metrological needs including the further development of experimental and computational techniques for micro- and nanodosimetry, together with the determination of related fundamental material properties and the establishment of rigorous uncertainty budgets. In addition to this, a call to develop a metrology support for assisting quality assurance of radiobiology experiments was expressed. Conclusions from the workshop were presented at several international conferences for further discussion with the scientific community and stakeholder groups that led to an initiative within the metrology community to establish a European Metrology Network on biological effects of IR.

State of The Art of Instrumentation in Experimental Nanodosimetry.

Nanodosimetry is a branch of dosimetry for investigation and modeling of the interaction pattern of ionizing radiation in nanometre site-sizes (at unit density), which dates back to the 1970's (Pszona S. A track ion counter. Proceedings of Fifth Symposium on Microdosimetry EUR 5452 d-e-f, Published by the Commission of the European Communities, Luxemburg, pp. 1107-1122 (1976)). To date, the different experimental approaches have lead to developing of three fully functional nanodosimeters: the Jet Counter operated at NCBJ, the Ion Counter operated at PTB and Startrack Counter operated at INFN-LNL. Descriptions of each nanodosimeter as well as of the techniques used to investigate the track structure of ionizing particles are presented.

NANODOSIMETRY: TOWARDS A NEW CONCEPT OF RADIATION QUALITY.

The biological action of ionizing charged particles is initiated at the DNA level, and the effectiveness with which the initial physical effect changes into measurable biological damage is likely ruled by the stochastics of ionizations produced by the incident ions in subcellular nanometric volumes. Based on this hypothesis, experimental nanodosimetry aims at establishing a new concept of radiation quality that builds on measurable characteristics of the particle track structure at the nanometer scale. Three different nanodosimetric detection systems have been developed to date that allow measurements of the number of ionizations produced by the passage of a primary particle in a nanometer-size gas volume (in unit density scale). Within the Italian project MITRA (MIcrodosimetry and TRAck structure), funded by the Italian Istituto Nazionale di Fisica Nucleare (INFN) and the EMRP Joint Research Project 'BioQuaRT' (Biologically Weighted Quantities in Radiotherapy), experiments have been carried out, in which the frequency distribution of ionizations produced by proton and carbon ion beams of given energy was measured with the three nanodosimetric detectors. Descriptors of the track structure can be derived from these distributions. In particular, the first moment M1, representing the mean number of ionizations produced in the target volume, and the cumulative probability Fk of measuring a number ν ≥ k of ionizations. The correlation between measured nanodosimetric quantities and experimental radiobiological data available in the literature is here presented and discussed.

Measurement of track structure parameters of low and medium energy helium and carbon ions in nanometric volumes.

Ionization cluster size distributions produced in the sensitive volume of an ion-counting wall-less nanodosimeter by monoenergetic carbon ions with energies between 45 MeV and 150 MeV were measured at the TANDEM-ALPI ion accelerator facility complex of the LNL-INFN in Legnaro. Those produced by monoenergetic helium ions with energies between 2 MeV and 20 MeV were measured at the accelerator facilities of PTB and with a 241Am alpha particle source. C3H8 was used as the target gas. The ionization cluster size distributions were measured in narrow beam geometry with the primary beam passing the target volume at specified distances from its centre, and in broad beam geometry with a fan-like primary beam. By applying a suitable drift time window, the effective size of the target volume was adjusted to match the size of a DNA segment. The measured data were compared with the results of simulations obtained with the PTB Monte Carlo code PTra. Before the comparison, the simulated cluster size distributions were corrected with respect to the background of additional ionizations produced in the transport system of the ionized target gas molecules. Measured and simulated characteristics of the particle track structure are in good agreement for both types of primary particles and for both types of the irradiation geometry. As the range in tissue of the ions investigated is within the typical extension of a spread-out Bragg peak, these data are useful for benchmarking not only 'general purpose' track structure simulation codes, but also treatment planning codes used in hadron therapy. Additionally, these data sets may serve as a data base for codes modelling the induction of radiation damages at the DNA-level as they almost completely characterize the ionization component of the nanometric track structure.

Experimental investigation of ionisation track structure of carbon ions at HIL Warsaw.

In view of the upcoming radiation therapy with carbon ions, the ionisation structure of the carbon ion track at the nanometre scale is of particular interest. Two different nanodosimeters capable of measuring track structure of ionising particles in a gas target equivalent to a nanometric site in condensed matter were involved in the presented experimental investigation, namely the NCBJ Jet Counter and the PTB Ion Counter. At the accelerator facility of the HIL in Warsaw, simulated nanometric volumes were irradiated with carbon ions of 45 and 76 MeV of kinetic energy, corresponding to a range in the tissue of ∼85 µm and ∼190 µm, respectively. The filling gas of both nanodosimeters' ionisation volume was molecular nitrogen N2, and the ionisation cluster size distributions, i.e. the statistical distribution of the number of ionizations produced by one single primary carbon ion in the filling gas, were measured for the two primary particle energies.

Future development of biologically relevant dosimetry.

Proton and ion beams are radiotherapy modalities of increasing importance and interest. Because of the different biological dose response of these radiations as compared with high-energy photon beams, the current approach of treatment prescription is based on the product of the absorbed dose to water and a biological weighting factor, but this is found to be insufficient for providing a generic method to quantify the biological outcome of radiation. It is therefore suggested to define new dosimetric quantities that allow a transparent separation of the physical processes from the biological ones. Given the complexity of the initiation and occurrence of biological processes on various time and length scales, and given that neither microdosimetry nor nanodosimetry on their own can fully describe the biological effects as a function of the distribution of energy deposition or ionization, a multiscale approach is needed to lay the foundation for the aforementioned new physical quantities relating track structure to relative biological effectiveness in proton and ion beam therapy. This article reviews the state-of-the-art microdosimetry, nanodosimetry, track structure simulations, quantification of reactive species, reference radiobiological data, cross-section data and multiscale models of biological response in the context of realizing the new quantities. It also introduces the European metrology project, Biologically Weighted Quantities in Radiotherapy, which aims to investigate the feasibility of establishing a multiscale model as the basis of the new quantities. A tentative generic expression of how the weighting of physical quantities at different length scales could be carried out is presented.

Proton-impact ionisation cross sections for nanodosimetric track structure simulations.

Monte Carlo simulations of the particle track structure require accurate ion- and electron-impact cross-section data of the medium. These data are scarce and often inconsistent when measured by different groups. In this work, literature data on ionisation cross sections (CSs) of nitrogen and propane for protons with energies 0.1-10 MeV are reviewed and implemented in the code PTra. Methane data were used to obtain proton-impact CSs of propane due to their absence in the literature. PTra is benchmarked by comparing simulated particle-track parameters to experimental results, measured with an ion-counting nanodosemeter.

Comparison of measured and Monte Carlo simulated track structure parameters in nanometric volumes.

The track structure of ionising particles in biological matter can only be assessed by simulations, since neither the type of interaction and its products nor the interaction positions in biological matter can be detected with nanometer resolution. Hence, there is a need to benchmark the deployed computer codes using suitable experimental data. For this purpose, the frequency distributions of ionisation clusters produced in the sensitive volume of the PTB ion counting nanodosemeter by monoenergetic protons and alpha particles (with energies between 0.1 and 20 MeV) were measured. C3H8 and N2 were alternately used as the working gas. The measured data were compared with the results of simulations obtained with the PTB Monte Carlo code PTra. Measured and simulated characteristics of the particle track structure are in good agreement for protons over the entire energy range investigated. For alpha particles with energies above the Bragg peak a good agreement can also be seen, whereas for energies below the Bragg peak differences of as much as 25 % occur.

Proton-induced frequency distributions of ionization cluster size in propane.

The frequency distribution of clustered ionizations produced by a proton beam was measured in a nanodosimetric volume of the size of a DNA segment by means of an ion-counting nanodosimeter in the energy range from 0.4 to 3.5 MeV. In order to meet the needs of the ion-counting nanodosimeter, the accelerator's primary beam was reduced in intensity by means of Rutherford scattering. The comparison between experimental results and Monte Carlo simulations show a good agreement in the energy dependence of the mean cluster size, while the experimental cluster size distributions show a higher amount of large ionization clusters compared with those obtained with the simulations.

Dependence of nanodosimetric spectra on the sensitive volume length and ion drift in an ion-counting nanodosemeter.

Nanodosimetric spectra, measured in a well-defined ionisation sensitive volume of an ion-counting gaseous nanodosemeter, may have a valuable predictive value of radiation damage to DNA. In such devices, the distributions of radiation-induced ions are measured after their drift in gas. The sensitive-volume size, corresponding to a DNA segment length, can be tuned by selecting an appropriate time window for ion counting; the method's accuracy depends on the velocity distribution of the drifting ions. The results of ion-drift measurements in an ion-counting nanodosemeter were used for the precise calculation of its sensitive volume length. Monte Carlo simulations of nanodosimetric spectra, performed with the obtained data, are in good agreement with experimental data. The method's limitations, arising from the spread of drift velocities, are discussed.

Post-transcriptional effect of ultraviolet light on gene expression in human cells. Stabilization of cytokine-induced and poly(I).poly(C)-induced messenger RNA.

It is well established that ultraviolet light modulates gene expression in mammalian cells, particularly at transcriptional and post-translational levels. The present study was undertaken to investigate whether the fate of mRNA is also altered in ultraviolet-light-irradiated human cells. In order to facilitate distinction between transcriptional and post-transcriptional effects, this analysis has focused on six genes whose transcription is conditional on the supply of exogenous inducers, interferon-alpha, interleukin-1 alpha or the double-stranded RNA, poly(I).poly(C). Human cells induced to express these genes were found to retain a significantly higher concentration of corresponding transcripts when irradiated with ultraviolet light at the end of the inducing treatment. This stimulation was due to dose-dependent ultraviolet-light stabilization of preformed mRNA, as shown by run-on and pulse/chase experiments. This work uncovers a new facet of the cellular response to genotoxic stresses, i.e. extension of the life-span of transcription products. Whether this stabilizing effect contributes to cell recovery by promoting gene expression remains to be determined.

Impaired recovery and mutagenic SOS-like responses in ataxia telangiectasia cells.

Radiosensitive fibroblasts from patients with ataxia telangiectasia (AT) were studied for their proficiency in two putative eukaryotic SOS-like responses, namely the enhanced reactivation (ER) and enhanced mutagenesis of damaged viruses infecting pre-irradiated versus mock-treated cells. A previous report indicated that, unlike normal human cells, a line of AT fibroblasts (AT5BIVA) could not be induced to express ER of damaged parvovirus H-1, a single-stranded DNA virus, by UV- or X-irradiation. In the present study, AT5BIVA fibroblasts were also distinguished from normal cells by the inability of the former to achieve enhanced mutagenesis of damaged H-1 virus upon cell UV-irradiation. In contrast, dose-response and time-course experiments revealed normal levels of ER of Herpes simplex virus 1, a double-stranded DNA virus, in X- or UV-irradiated AT5BIVA cells. Taken together, these data point to a possible deficiency of AT cells in a conditioned mutagenic process that contributes to a greater extent to the recovery of damaged single-stranded than double-stranded DNA. Such a defect may concern the replication of damaged DNA or the generation of signals promoting the latter process and may be related to the lack of radiation-induced delay that is typical of AT cell DNA synthesis.

Deficient expression of enhanced reactivation of parvovirus H-1 in ataxia telangiectasia cells irradiated with X-rays or u.v. light.

Cells of patients with ataxia telangiectasia (AT), an inherited disease characterized by a high propensity to cancer, are hypersensitive to ionizing radiation. We investigated whether the hyper-radiosensitivity of AT cells correlated with a defect in their constitutive and/or conditional ability to rescue a damaged exogenous virus. For that purpose, parvovirus H-1, a single-stranded DNA virus whose intranuclear replication mostly relies on host cell functions, was used as a probe. The survival of u.v.- or gamma-irradiated H-1 was measured in X-, u.v.- or mock-irradiated human cells of normal (NB-E) or AT (AT5BIVA) origin. gamma-Irradiated H-1 survived to similar extents in untreated normal and AT cell lines. Both X- and u.v.-irradiation induced normal cells to achieve an enhanced reactivation (ER) of gamma- or u.v.-damaged H-1. In contrast, neither dose-effect curves nor time course revealed significant levels of ER expression after X- or u.v.-irradiation in AT5BIVA cells. Our results suggest that the impairment of ER of damaged parvoviruses may constitute a marker of the AT cell phenotype and be related to the radiosensitivity of AT cells.

[Cells of patients with ataxia telangiectasia show a normal capacity of radio-induced reactivation of damaged HSV-1 virus]. / Les cellules de patients atteints d'ataxia telangiectasia manifestent une capacité normale de réactivation radioinduite du virus HSV-I endommagé.

Herpes Simplex Virus (HSV-1) was used to probe the expression of enhanced reactivation (ER) in cells from patients with ataxia telangiectasia (AT). The survival of UV-irradiated HSV-1 was increased as a result of UV- or X-preirradiation of both AT and normal cells. This result contrasts with our previous observation showing that contrary to normal cells AT cells are deficient for ER of a single-stranded DNA parvovirus. A difference between the molecular processes underlying ER of single- and double-stranded DNA viruses might explain these results.

[Reactivation of gamma-ray irradiated parvovirus H-1 in cells of patients with ataxia telangiectasia and Huntington's chorea]. / Réactivation du Parvovirus H-1 irradié aux Rayons gamma dans les cellules de patients atteints d'ataxia telangiectasia et de la chorée de Huntington.

Parvovirus H-1 was used to probe the cellular radiosensitivity of two human degeneration syndromes AT and HC. No difference in the survival of gamma irradiated H-1 was detected between skin fibroblasts from such patients and from a normal individual. However, AT and normal cells were distinguished by the fact that the reactivation of irradiated H-1 could be increased by UV or X-irradiation of the latter but not of the former cells.