O.V. Belyakov

Studies of bystander effects in human fibroblasts using a charged particle microbeam.

PURPOSE To determine the role of cell-to-cell mediated effects in the response of cells to different radiations by using a charged particle microbeam capable of targeting individual cells with counted particles. MATERIALS AND METHODS Primary human fibroblasts were irradiated conventionally with X-rays or alpha-particles and scored for the induction of micronucleated or apoptotic cell formation at various times after irradiation. Cells were also individually irradiated with helium-3 particles from a microbeam and the distribution of damaged cells between hit and non-hit cells scored. RESULTS Conventionally exposed fibroblasts showed a dose-dependent production of micronucleated and apoptotic cells 3 days after irradiation for both X-rays and alpha-particles, with a high RBE value for alpha-particles at low doses. Targeting individual alpha-particles, using a microbeam, to four cells within a population produced more micronucleated and apoptotic cells than expected on the basis of a direct effect only. CONCLUSIONS This study provides direct evidence for the production of transmissible, cell-to-cell effects between hit and non-hit cells individually exposed to charged particles.

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts. We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600–800 cells were targeted with between 1 and 15 helium ions (effectively, α-particles). The charged particles were delivered through the centre of the nucleus with an accuracy of ± 2 μm and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80–100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell. Similar results of a 2–3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2–3-fold increase in the damage level in an unexposed quadrant of the dish. This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved.

Non-targeted effects of ionising radiation-Implications for low dose risk

Non-DNA targeted effects of ionising radiation, which include genomic instability, and a variety of bystander effects including abscopal effects and bystander mediated adaptive response, have raised concerns about the magnitude of low-dose radiation risk. Genomic instability, bystander effects and adaptive responses are powered by fundamental, but not clearly understood systems that maintain tissue homeostasis. Despite excellent research in this field by various groups, there are still gaps in our understanding of the likely mechanisms associated with non-DNA targeted effects, particularly with respect to systemic (human health) consequences at low and intermediate doses of ionising radiation. Other outstanding questions include links between the different non-targeted responses and the variations in response observed between individuals and cell lines, possibly a function of genetic background. Furthermore, it is still not known what the initial target and early interactions in cells are that give rise to non-targeted responses in neighbouring or descendant cells. This paper provides a commentary on the current state of the field as a result of the non-targeted effects of ionising radiation (NOTE) Integrated Project funded by the European Union. Here we critically examine the evidence for non-targeted effects, discuss apparently contradictory results and consider implications for low-dose radiation health effects.

A proliferation-dependent bystander effect in primary porcine and human urothelial explants in response to targeted irradiation.

The aim of this study was to test whether radiation-induced bystander effects are involved in the response of multicellular systems to targeted irradiation. A primary explant technique was used that reconstructed the in vivo microarchitecture of normal urothelium with proliferating and differentiated cells present. Sections of human and porcine ureter were cultured as explants and irradiated on day 7 when the urothelial outgrowth formed a halo around the tissue fragment. The Gray Cancer Institute charge particle microbeam facility allowed the irradiation of individual cells within the explant outgrowth with a predetermined exact number of 3He2+ ions (which have very similar biological effectiveness to α-particles). A total of 10 individual cell nuclei were irradiated with 10 3He2+ ions either on the periphery, where proliferating cells are located, or at the centre of the explant outgrowth, which consisted of terminally differentiated cells. Samples were fixed 3 days after irradiation, stained and scored. The fraction of apoptotic and micronucleated cells was measured and a significant bystander-induced damage was observed. Approximately 2000–6000 cells could be damaged by the irradiation of a few cells initially, suggesting a cascade mechanism of cell damage induction. However, the fraction of micronucleated and apoptotic cells did not exceed 1–2% of the total number of the cells within the explant outgrowth. It is concluded that the bystander-induced damage depends on the proliferation status of the cells and can be observed in an in vitro explant model.

Classical radiation biology, the bystander effect and paradigms: a reply.

Although, in retrospect, it can be seen that the bystander effect and the related effect of genomic instability were observed well before they were recognized as such, they have not been able to be accommodated within the existing understanding of how radiation causes late effects, which provides the basis for radiological protection standards. It is argued here that before these effects can be fully researched and there can be full confidence in radiological protection, a paradigm shift that provides a framework in which these effects can be considered alongside the well established effects of radiation is needed. In particular this framework will encompass the epigenetic as well as genetic aspects of radiation biology. Examples of how this might be achieved are given.

A 238Pu irradiator for exposure of cultured cells with alpha-radiation: Construction, calibration and dosimetry

An alpha-particle irradiator that can facilitate investigations of alpha-radiation effects on human cells in radiation protection, carcinogenesis and radioimmunotherapy was constructed. The irradiator was based on a 1.3 GBq (238)Pu source, housed in a stainless steel tube flushed with helium. Radiation provided by (238)Pu consists mainly of alpha-particles with energy of 5.5 MeV. The alpha-particle fluence and energy spectra were measured with a silicon semiconductor detector. Monte Carlo simulations were used to estimate the mean number of alpha-particles and the mean absorbed alpha-particle dose to cells for various irradiation times and distances between cells and source. There was a linear dependence between exposure time and alpha-particle fluence for exposure times above 1s. The alpha-particle activity concentration varied with a factor 2.7 over the source area, while the variation in energy peak position was <4%. At the cell nucleus position and with a distance of 45 mm between the source and the mylar dish surface, the alpha-fluence was 4.6 x 10(4)counts/(mm(2)s), the average incident alpha-particle energy was 2.5 MeV and the average linear energy transfer was 167 keV/microm. The average dose rate to the cells, with 5 microm diameter nucleus, was 1.2 Gy/s. The (238)Pu alpha-particle irradiator is feasible for irradiation of cells and it can be used for studies of both direct effects and bystander effects of alpha-radiation.