Low Dose Studies with Focused X-Rays in cell and Tissue Models: Mechanisms of Bystander and Genomic Instability Responses

The management of the risks of exposure of people to ionizing radiation is important in relation to its uses in industry and medicine, also to natural and man-made radiation in the environment. The vase majority of exposures are at a very low level of radiation dose. The risks are of inducing cancer in the exposed individuals and a smaller risk of inducing genetic damage that can be indicate that they are low. As a result, the risks are impossible to detect in population studies with any accuracy above the normal levels of cancer and genetic defects unless the dose levels are high. In practice, this means that our knowledge depends very largely on the information gained from the follow-up of the survivors of the atomic bombs dropped on Japanese cities. The risks calculated from these high-dose short-duration exposures then have to be projected down to the low-dose long-term exposures that apply generally. Recent research using cells in culture has revealed that the relationship between high- and low-dose biological damage may be much more complex than had previously been thought. The aims of this and other projects in the DOE's Low-Dose Program are to gain an understanding of the biological actions of low-dose radiation, ultimately to provide information that will lead to more accurate quantification of low-dose risk. Our project is based on the concept that the processes by which radiation induces cancer start where the individual tracks of radiation impact on cells and tissues. At the dose levels of most low-dose exposures, these events are rare and any individual cells only ''sees'' radiation tracks at intervals averaging from weeks to years apart. This contrasts with the atomic bomb exposures where, on average, each cell was hit by hundreds of tracks instantaneously. We have therefore developed microbeam techniques that enable us to target cells in culture with any numbers of tracks, from one upwards. This approach enables us to study the biological ha sis of the relationship between high- and low-dose exposures. The targeting approach also allows us to study very clearly a newly recognized effect of radiation, the ''bystander effect'', which appears to dominate some low-dose responses and therefore may have a significant role in low-dose risk mechanisms. Our project also addresses the concept that the background of naturally occurring oxidative damage that takes place continually in cells due to byproducts of metabolism may play a role in low-dose radiation risk. This project therefore also examines how cells are damaged by treatments that modify the levels of oxidative damage, either alone or in combination with low-dose irradiation. In this project, we have used human and rodent cell lines and each set of experiments has been carried out on a single cell type. However, low-dose research has to extend into tissues because signaling between cells of different types is likely to influence the responses. Our studies have therefore also included microbeam experiments using a model tissue system that consists of an explant of a small piece of pig ureter grown in culture. The structure of this tissue is similar to that of epithelium and therefore it relates to the tissues in which carcinoma arises. Our studies have been able to measure bystander-induced changes in the cells growing out from the tissue fragment after it has been targeted with a few radiation tracks to mimic a low-dose exposure

A Variable-Energy Soft X-Ray Microprobe to Investigate Mechanisms of the Radiation-Induced Bystander Effect.

The Gray Cancer Institute has pioneered the use of X ray focussing techniques to develop systems for micro irradiating individual cells and sub cellular targets in vitro. Cellular micro irradiation is now recognised as a highly versatile technique for understanding how ionising radiation interacts with living cells and tissues. The strength of the technique lies in its ability to deliver precise doses of radiation to selected individual cells (or sub cellular targets). The application of this technique in the field of radiation biology continues to be of great interest for investigating a number of phenomena currently of concern to the radiobiological community. One important phenomenon is the so called ‘bystander effect’ where it is observed that unirradiated cells can also respond to signals transmitted by irradiated neighbours. Clearly, the ability of a microbeam to irradiate just a single cell or selected cells within a population is well suited to studying this effect. Our prototype ‘tabletop’ X-ray microprobe was optimised for focusing 278 eV C-K X rays and has been used successfully for a number of years. However, we have sought to develop a new variable energy soft X-ray microprobe capable of delivering focused CK (0.28 keV), Al-K (1.48 keV) and notably, Ti-K (4.5 keV) X rays. Ti-K X rays are capable of penetrating several cell layers and are therefore much better suited to studies involving tissues and multi cellular layers. In our new design, X-rays are generated by the focussed electron bombardment of a material whose characteristic-K radiation is required. The source is mounted on a 1.5 x 1.0 metre optical table. Electrons are generated by a custom built gun, designed to operate up to 15 kV. The electrons are focused using a permanent neodymium iron boron magnet assembly. Focusing is achieved by adjusting the accelerating voltage and by fine tuning the target position via a vacuum position feedthrough. To analyze the electron beam properties, a custom built microscope is used to image the focussed beam on the target, through a vacuum window. The X-rays are focussed by a zone plate optical assembly mounted to the end of a hollow vertical tube that can be precisely positioned above the X ray source. The cell finding and positioning stage comprises an epi-fluorescence microscope and a feedback controlled 3 axis cell positioning stage, also mounted on the optical table. Independent vertical micro positioning of the microscope objective turret allows the focus of the microscope and the X ray focus to coincide in space (i.e. at the point where the cell should be positioned for exposure). The whole microscope stage assembly can be precisely raised or lowered, to cater for large differences in the focal length of the X ray zone plates. The facility is controlled by PC and the software provides full status and control of the source and makes use of a dual-screen for control and display during the automated cell finding and irradiation procedures

Development and applications of a gamma-ray tomographic scanner.

Computerised Tomography (CT) is now an established technique in medicine, but has yet to see any widespread applications in industry, partly because of the high cost of medical machines. This thesis examines the requirements and limitations of a gamma-ray tomographic scanner for industrial and research applications. The basic theory of CT reconstruction is outlined, together with a description of how this has been practically realised by means of a stepping motor controlled table that accurately positions the sample within a fixed radiation beam. Several features of the scanner are described in detail; namely the development of the isotope sources and their associated collimators, and the microcomputer control. The performance is analysed and many possible applications are discussed, accompanied by examples derived from the scanner. It has been shown that worthwhile results can be obtained with only a modest amount of equipment. Some suggestions for improvements are made, many of which may be implemented in a future programme of work

Development and applications of a gamma-ray tomographic scanner.

Computerised Tomography (CT) is now an established technique in medicine, but has yet to see any widespread applications in industry, partly because of the high cost of medical machines. This thesis examines the requirements and limitations of a gamma-ray tomographic scanner for industrial and research applications. The basic theory of CT reconstruction is outlined, together with a description of how this has been practically realised by means of a stepping motor controlled table that accurately positions the sample within a fixed radiation beam. Several features of the scanner are described in detail; namely the development of the isotope sources and their associated collimators, and the microcomputer control. The performance is analysed and many possible applications are discussed, accompanied by examples derived from the scanner. It has been shown that worthwhile results can be obtained with only a modest amount of equipment. Some suggestions for improvements are made, many of which may be implemented in a future programme of work

The use of microbeams to investigate radiation damage in living cells.

The micro-irradiation technique continues to be highly relevant to a number of radiobiological studies in vitro. In particular, studies of the bystander effect show that direct damage to cells is not the only trigger for radiation-induced effects, but that unirradiated cells can also respond to signals from irradiated neighbours. Furthermore, the bystander response can be initiated even when no energy is deposited in the genomic DNA of the irradiated cell (i.e. by targeting just the cytoplasm)

Vojnovic B: The use of microbeams to investigate radiation damage in living cells. ApplRadiat Isot 2010

a b s t r a c t The micro-irradiation technique continues to be highly relevant to a number of radiobiological studies in vitro. In particular, studies of the bystander effect show that direct damage to cells is not the only trigger for radiation-induced effects, but that unirradiated cells can also respond to signals from irradiated neighbours. Furthermore, the bystander response can be initiated even when no energy is deposited in the genomic DNA of the irradiated cell (i.e. by targeting just the cytoplasm)