Investigation of direct and bystander effects following neutron irradiation of skin cells

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The aim of this proposal is to discover why non-targeted effects (NTE) of ionising radiation including bystander effects (BE) and genomic instability (GI) are not seen following exposure of cells or organisms to neutrons. Several experiments over the last 30 years by the applicant and others using different neutron sources have confirmed this but no systematic study has been done to understand why. BE - i.e. effects in neighbouring cells and GI i.e. effects in the distant progeny, not targeted by direct irradiation are very well characterised low dose effects of ionising radiation and UV. They predominate in the dose range 2mGy-0.5Gy of acute low LET radiation exposure and are thought to be mediated by oxidative stress generation (ROS) in exposed cells which turns on signalling mechanisms activating stress responses in un-hit cells. There are many endpoints for determining that bystander signalling has occurred such as measurement of cell death, chromosome damage, calcium flux, up-regulation of DNA repair mechanisms and over production of ROS. The current proposal will investigate three hypotheses built around 5 questions, to try to determine why neutrons do not produce bystander effects. Q1. Is it true that neutrons NEVER produce NTE or have we just missed the effective window? Q2. What is stopping neutron exposed cells from producing NTE? Is signal production affected? If so at what point in the mechanism? Q3. Most neutron beams have a gamma component - does the presence of neutrons cancel or otherwise neutralise any gamma-produced signal? Q4. Neutron radiobiology shows an “inverse dose rate effect” for radiation transformation (in vitro assay for carcinogenic potential) where, lower dose rates are more effective at producing damage than high dose rates. Is the lack of NTE responsible for this? Q5. Neither neutron nor alpha particle cell survival curves have a shoulder (low dose sparing) but alpha particles are reported to produce NTE, protons effects are equivocal. Is this true and what are the implications for understanding neutron NTE radiobiology? These questions have been distilled into 3 testable hypotheses briefly listed here: A. The dose to the individual cell receiving a neutron hit is so high that it cannot generate a signal. This will be tested using dosimetric and biological approaches to determine the dose to the cell and whether that dose blocks mechanisms involved in signal production such as calcium ion flux, ROS and serotonin binding. Testing this will employ 3 PhD students and will address questions 1,2,3 and 4. B. In addition to a low LET, low dose threshold for triggering NTE there is also a high dose threshold above which signal production is inhibited. This will be tested using systematic approaches aimed at comparing different radiation qualities. This will employ one additional PhD student and will address questions 3 and 5. C. The interaction of x- and gamma rays with cell membranes causes direct excitation and ionisation of the membrane and activates ion-gated membrane pumps to trigger the signalling cascade. Some types of neutrons may not cause this excitation depending on whether they cause indirect ionisation or not. This will be tested by treating cells before exposure to gamma rays or thermal or fast neutrons, with membrane stabilising agents. This will also address question 2 and 3. These approaches should answer the question of why neutrons do not produce NTE and should provide mechanistic information of importance in low dose radiobiology in general and neutron radiobiology in particular. Because neutrons are a by-product in CANDU reactors, the results may also benefit those concerned with radiation protection issues in the nuclear power industry.