Massimo Pinto

Abstract: Low intensity radioactive sources emitting low energy photons are used in the clinic for low dose rate brachytherapy treatments of tumours. The dosimetry of these sources is based on reference air kerma rate measurements. The absorbed dose rate to water at the reference depth d0=1 cm, � , is then obtained by a conversion procedure with a large relative standard uncertainty of about 5%. This paper describes a primary standard developed at ENEA-INMRI to directly measure Dw,1 cm due to LDR sources. The standard is based on a large-angle and variable-volume ionization chamber, embedded in a graphite phantom and operating in �wall-less air chamber� conditions. A set of correction and conversion factors, based on experiments and Monte Carlo simulations are determined to obtain the value of Dw,1 cm from measurements of increment of ionization current with the chamber volume increase. The relative standard uncertainty on � is 2.3%, which is appreciably lower than the current uncertainty. Characteristics of the standard, its associated uncertainty budget, and some experimental results are given for 125I BEBIG I25.S16.C brachytherapy seeds. Finally, results of the experimental determination of the dose rate constant �1cm, traceable to the Dw,1 cm and the low energy air kerma ENEA-INMRI standards, are given. The relative standard uncertainty on Dw,1 cm is 2.6%, appreciably lower than the typical uncertainty (4.8%) of the values available in literature.

Abstract: The study of radiation-induced bystander effects in normal human cells maintained in three-dimensional (3D) architecture provides more in vivo-like conditions and is relevant to human risk assessment. Linear energy transfer, dose and dose rate have been considered as critical factors in propagating radiation-induced effects. This investigation uses an in vitro 3D tissue culture model in which normal AG1522 human fibroblasts are grown in a carbon scaffold to investigate induction of a G1 arrest in bystander cells that neighbor radiolabeled cells. Cell cultures were co-pulse-labeled with [3H]deoxycytidine (3HdC) to selectively irradiate a minor fraction of cells with 1�5 keV/µm β particles and bromodeoxyuridine (BrdU) to identify the radiolabeled cells using immunofluorescence. The induction of a G1 arrest was measured specifically in unlabeled cells (i.e. bystander cells) using a flow cytometry-based version of the cumulative labeling index assay. To investigate the relationship between bystander effects and adaptive responses, cells were challenged with an acute 4 Gy γ-radiation dose after they had been kept under the bystander conditions described above for several hours, and the regulation of the radiation-induced G1 arrest was measured selectively in bystander cells. When the average dose rate in 3HdC-labeled cells (<16% of population) was 0.04�0.37 Gy/h (average accumulated dose 0.14�10 Gy), no statistically significant stressful bystander effects or adaptive bystander effects were observed as measured by magnitude of the G1 arrest, micronucleus formation, or changes in mitochondrial membrane potential. Higher dose rates and/or higher LET may be required to observe stressful bystander effects in this experimental system, whereas lower dose rates and challenge doses may be required to detect adaptive bystander responses.

Abstract: In this paper we present results from an experiment aimed at investi- gating whether living cells are influenced by background ionizing radiation. Parallel human cell cultures were set-up in two separate laboratories and maintained for sev- eral months under identical conditions but for a 80� different level of background ionizing radiation. Periodically, the cell cultures were monitored for the onset of di- vergences in biochemical behavior, using two distinct cellular biology assays, namely micronuclei induction and activity of enzymes implicated in the management of ox- idative stress. To reveal any subtle modifications, responses were also amplified by subjecting cell cultures to acute stress induced by exposure to moderately high doses of ionizing radiation. Compared to reference radiation background condi- tions, cultures maintained in a reduced background radiation environment handled the consequences of acute stress with diminished efficacy.

Abstract: Adaptive response (AR) is a term that has been generally accepted to describe the ability of a low �priming� radiation dose to decrease the cell response to a subsequent higher �challenging� dose. The main proposed mechanisms to explain AR are: increased efficiency of DNA repair and induction of antioxidant enzymes. A model that considers a modulation of the efficiency of DNA repair activity and of the level of antioxidant enzymes, starting from the framework of a lethal�potentially lethal (LPL) model is proposed. The LPL model has been extended with the inclusion of the dynamic variables representing the efficiency of repair, the levels of radiation induced radicals and of antioxidant enzymes. The model used here is able to describe the protective effect of a priming dose. Moreover, in agreement with the data in the literature, the simulations show that the AR happens in given priming dose and priming dose-rate ranges only, and requires at least 4 h to develop. In order to get more insights into the role of cell�cell communication as factors affecting the AR, experimental studies were planned using sparse or confluent AG1522 cell monolayer. The results obtained after gamma irradiation suggest that cell density is a crucial factor for observing an AR.

Abstract: We have previously reported that yeast and Chinese Hamster V79 cells cultured under reduced levels of environmental ionizing radiation background show enhanced susceptibility to damage caused by acute doses of genotoxic agents. Reduction of environmental radiation dose rate was achieved by setting up an underground laboratory at Laboratori Nazionali del Gran Sasso, central Italy. We now report on the extension of our studies to a human cell line. We have maintained human lymphoblastoid TK6 cells under identical in vitro culture conditions for six continuous months, at different environmental ionizing radiation levels. Compared to "reference" environmental radiation conditions, we found that cells cultured in the underground laboratories were more sensitive to acute exposures to radiation, as measured both at the level of DNA damage and oxidative metabolism. Our results are compatible with the hypothesis that ultra-low dose rate ionizing radiation, i.e. environmental radiation, may act as a conditioning agent in the classic radiation-induced adaptive response.

Abstract: Some years ago we performed two experiments aimed at studying the influence of the background radiation on living matter by exploiting the low radiation background environment in the underground Gran Sasso Laboratory of the INFN. Their results were consistent with the hypothesis that the �normal� background radiation determines an adaptive response, although they cannot be considered conclusive. PULEX-3 (the third experiment of the series) is aimed at comparing the effects of different background radiation environments on metabolism of cultured mammalian cells, with substantial improvements with respect to the preceding ones. The experiment was designed to minimize variabilities, by maintaining two cultures of Chinese hamster V79 cells in exponential growth for up to ten months in the underground Gran Sasso Laboratory (LNGS), while two other cultures were maintained in parallel in a biological laboratory installed at the LNGS outside the tunnel. Exposure due to γ-rays was reduced by a factor of about 10 in the underground laboratory while the Rn concentration was small in both cases. After ten months the cells grown in the underground laboratory, compared to those grown in the external one, exhibited: i) a significantly lower capacity to scavenge reactive oxygen species (ROS), and ii) an increased sensitivity to the mutagenic effect of rays. Since the probability that this finding is due to casual induction of radiosensitive mutants is extremely low, it corroborates the hypothesis that cells grown in a �normal� background radiation environment exhibit an adaptive response when challenged with genotoxic agents, which is lost after many generations in a low background radiation environment.

Abstract: Targeted therapies result in heterogeneous drug delivery, often with highly variable drug uptake in the targeted cells and significant numbers of cells that are essentially untargeted. However both the variably targeted cells and neighboring bystander cells may respond to the treatment. Using ionizing radiation as an example of a targeted therapeutic agent, we describe a quantitative immunofluorescence-based approach for concomitant quantification of exposure and measurement of biological responses in both targeted and bystander cells. Cultures of human skin fibroblasts are co-pulse-labeled with 3H-deoxycytidine (3H-dC) and bromodeoxyuridine (BrdU). The labeled cells, identified by BrdU immunofluorescence, are internally irradiated by low-energy beta-particles emitted by incorporated 3H-dC. BrdU immunofluorescence intensity is proportional to radioactivity incorporated and, therefore, to radiation dose rate. Cell-cycle arrest in G2 is measured in labeled cells as function of dose rate. Stress responses in bystander cells, indicated by a G1 checkpoint, are concomitantly measured with a flow cytometric-cumulative labeling index (FCM-CLI) assay. The overall approach presented herein may be useful in the context of evaluating responses to targeted drug delivery.

Abstract: Research on the radiation-induced bystander effect has been carried out mainly in 2-D tissue culture systems. This study uses a 3-D model, wherein apparently normal human diploid fibroblasts (AG1522) are grown in a carbon scaffold, to investigate the induction of a G(1) checkpoint in bystander cells present alongside radiolabelled cells. Cultures were simultaneously pulse-labelled with (3)H-deoxycytidine ((3)HdC) to selectively irradiate a minor fraction of cells, and bromodeoxyuridine (BrdU) to identify the radiolabelled cells. After thorough washing of cultures, iododeoxyuridine (IdU) was administered to detect proliferating bystander cells. The cultures were harvested at various times thereafter, and cells were reacted with two monoclonal antibodies specific to IdU/BrdU or BrdU, respectively, stained with propidium iodide, and subjected to multi-parameter flow cytometry. Cell-cycle progression was followed in radiolabelled cells (BrdU(+)) that were chronically irradiated by low energy beta particles emitted by DNA-incorporated (3)H, and in unlabelled bystander cells (BrdU(-)) by a flow cytometry based cumulative labelling index assay. As expected, radiolabelled cells were delayed, in a dose-dependent manner, in G(2) and subsequently G(1). No delay occurred in progression of bystander cells through G(1), when the labelled cells were irradiated at dose rates up to 0.32 Gy h(-1).

Abstract: Prediction of risks and therapeutic outcome in nuclear medicine largely rely on calculation of the absorbed dose. Absorbed dose specification is complex due to the wide variety of radiations emitted, non-uniform activity distribution, biokinetics, etc. Conventional organ absorbed dose estimates assumed that radioactivity is distributed uniformly throughout the organ. However, there have been dramatic improvements in dosimetry models that reflect the substructure of organs as well as tissue elements within them. These models rely on improved nuclear medicine imaging capabilities that facilitate determination of activity within voxels that represent tissue elements of approximately 0.2-1 cm(3). However, even these improved approaches assume that all cells within the tissue element receive the same dose. The tissue element may be comprised of a variety of cells having different radiosensitivities and different incorporated radioactivity. Furthermore, the extent to which non-uniform distributions of radioactivity within a small tissue element impact the absorbed dose distribution is strongly dependent on the number, type, and energy of the radiations emitted by the radionuclide. It is also necessary to know whether the dose to a given cell arises from radioactive decays within itself (self-dose) or decays in surrounding cells (cross-dose). Cellular response to self-dose can be considerably different than its response to cross-dose from the same radiopharmaceutical. Bystander effects can also play a role in the response. Evidence shows that even under conditions of 'uniform' distribution of radioactivity, a combination of organ dosimetry, voxel dosimetry and dosimetry at the cellular and multicellular levels can be required to predict response.

Abstract: The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high-LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9+/-0.5 h and a slow component with a half-life of 16+/-9 h. For alpha particles, a fast component with a half-life of 0.7+/-0.4 h and a slow component with a half-life of 12+/-5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37+/-0.12) relative to low-LET radiation (0.22+/-0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites.

Abstract: In studies of radiation-induced DNA fragmentation and repair, analytical models may provide rapid and easy-to-use methods to test simple hypotheses regarding the breakage and rejoining mechanisms involved. The random breakage model, according to which lesions are distributed uniformly and independently of each other along the DNA, has been the model most used to describe spatial distribution of radiation-induced DNA damage. Recently several mechanistic approaches have been proposed that model clustered damage to DNA. In general, such approaches focus on the study of initial radiation-induced DNA damage and repair, without considering the effects of additional (unwanted and unavoidable) fragmentation that may take place during the experimental procedures. While most approaches, including measurement of total DNA mass below a specified value, allow for the occurrence of background experimental damage by means of simple subtractive procedures, a more detailed analysis of DNA fragmentation necessitates a more accurate treatment. We have developed a new, relatively simple model of DNA breakage and the resulting rejoining kinetics of broken fragments. Initial radiation-induced DNA damage is simulated using a clustered breakage approach, with three free parameters: the number of independently located clusters, each containing several DNA double-strand breaks (DSBs), the average number of DSBs within a cluster (multiplicity of the cluster), and the maximum allowed radius within which DSBs belonging to the same cluster are distributed. Random breakage is simulated as a special case of the DSB clustering procedure. When the model is applied to the analysis of DNA fragmentation as measured with pulsed-field gel electrophoresis (PFGE), the hypothesis that DSBs in proximity rejoin at a different rate from that of sparse isolated breaks can be tested, since the kinetics of rejoining of fragments of varying size may be followed by means of computer simulations. The problem of how to account for background damage from experimental handling is also carefully considered. We have shown that the conventional procedure of subtracting the background damage from the experimental data may lead to erroneous conclusions during the analysis of both initial fragmentation and DSB rejoining. Despite its relative simplicity, the method presented allows both the quantitative and qualitative description of radiation-induced DNA fragmentation and subsequent rejoining of double-stranded DNA fragments.

Abstract: PURPOSE: To assess the applicability of methods of quantification of double-strand breaks (DSB) based on the random breakage paradigm, measuring yield and distribution of DSB induced by varying radiation quality. MATERIAL AND METHODS: 240 kVp X-rays and (238)Pu alpha-particles were used to induce DSB in AG01522B primary human fibroblasts. DNA molecular weight distributions were resolved by means of three pulsed-field gel-electrophoresis (PFGE) protocols, which, when combined together, allowed separation and quantification of double-stranded fragments between 5.7 Mbp and 12 kbp. Several analytical methods quantified the DSB yields. RESULTS: Data showed significant differences in the fragmentation patterns according to radiation quality. For both X-rays and alpha-particles, it was observed that the shape of the fragmentation profiles deviates from the prediction of a random breakage mechanism. This is in contrast to other studies where sparsely ionizing radiations appeared to distribute breaks uniformly throughout the genome. Deviations from random breakage were more evident after high linear energy transfer (LET) radiation, which showed an excess of breaks <1 Mbp and a deficit in the production of fragments >1 Mbp, a value that could be dose-dependent. CONCLUSIONS: Current methods of DNA fragmentation analysis after induction of DSB may lead to contradictory conclusions on both DSB yields and distributions. This study showed that the application of different DSB quantification methods, derived from random breakage or supported by its concepts, resulted in different radiation biological effectivenesses (RBE) for the induction of DSB, depending on how these methods were employed. To compare experimental results from different laboratories, care should be taken to provide as many details as possible about the application of methods of quantification of DNA damage. For all the methods used, total DSB yields resulted in RBE less than those for mutation induction or reproductive cell death, suggesting that total DSB yields only gave a limited indication of the severity of the inflicted damage. Production of correlated breaks on the chromatin loop structures by single particle-track traversals may explain the deviations observed between experimental data and the predictions of the random breakage paradigm.

Abstract: When a charged-particle track intercepts the chromatin fibre in DNA of mammalian cells, clustered damage is induced depending on the DNA conformation, local environment and track structure. Intra-track correlated DNA damage may have a higher probability of being mis-repaired or left un-repaired. Fragment size-distributions of DNA double strand breaks (DSBs) induced in primary human fibroblasts by 240 kVp X rays and 238Pu alpha particles (110 keV.micron-1) were resolved using pulsed-field gel electrophoresis (PFGE). By monitoring DSB rejoining kinetics and changes in the fragment size distribution with repair time, the relevance of spatial association of DSBs in determining rejoining kinetics was investigated. Rejoining kinetics appeared bi-phasic and independent of the size of the DNA fragments for both radiation qualities, with high LET radiation-induced DSBs repairing more slowly. Results suggest that local complexity of individual DSBs, rather than spatial association with other breaks is more significant in the determination of rejoining kinetics.

Abstract: Underpinning current models of the mechanisms of the action of radiation is a central role for DNA damage and in particular double-strand breaks (DSBs). For radiations of different LET, there is a need to know the exact yields and distributions of DSBs in human cells. Most measurements of DSB yields within cells now rely on pulsed-field gel electrophoresis as the technique of choice. Previous measurements of DSB yields have suggested that the yields are remarkably similar for different types of radiation with RBE values < or = 1.0. More recent studies in mammalian cells, however, have suggested that both the yield and the spatial distribution of DSBs are influenced by radiation quality. RBE values for DSBs induced by high-LET radiations are greater than 1.0, and the distributions are nonrandom. Underlying this is the interaction of particle tracks with the higher-order chromosomal structures within cell nuclei. Further studies are needed to relate nonrandom distributions of DSBs to their rejoining kinetics. At the molecular level, we need to determine the involvement of clustering of damaged bases with strand breakage, and the relationship between higher-order clustering over sizes of kilobase pairs and above to localized clustering at the DNA level. Overall, these studies will allow us to elucidate whether the nonrandom distributions of breaks produced by high-LET particle tracks have any consequences for their repair and biological effectiveness.

Abstract: PURPOSE: To analyse the currently existing methods to infer the extent of cellular DNA damage induced by ionizing radiation when the pulsed field gel electrophoresis (PFGE) technique is used. RESULTS AND CONCLUSIONS: PFGE is currently the method of choice for the measurement of radiation-induced double-strand breaks (dsb). For accurate determination of both the yields and distributions of breaks, separation of a large range of fragment sizes is required. In the conventional analysis of PFGE experiments, the background distribution of fractionated molecules is, normally, simply subtracted from the irradiated measured distribution, for each molecular weight region available. This work shows that this approach may lead to incorrect estimation of the breakage frequencies. An alternative approach based on correcting the fitting functions for the actual nonrandom damage present in the control unirradiated samples has been developed.

Abstract: A key comparison has been made between the air-kerma standards of the ENEA-INMRI, Italy and the BIPM in the low-energy x-ray range. The results show the standards to be in agreement at the level of the standard uncertainty for the comparison of 1.9 parts in 103. No significant trend with radiation quality is observed. The results are analysed and presented in terms of degrees of equivalence, suitable for entry in the BIPM key comparison database.

Abstract: