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

本提案的目的是发现为什么电离辐射的非靶向效应（NTE），包括旁观者效应（BE）和基因组不稳定性（GI）在细胞或生物体暴露于中子后没有被观察到。在过去的30年里，申请人和其他人使用不同的中子源进行了几次实验，证实了这一点，但没有进行系统的研究来理解其中的原因。BE -即对邻近细胞的影响和GI -即对远端后代的影响，不是直接照射的目标，是电离辐射和紫外线的低剂量效应。它们在急性低LET辐射暴露的2mGy-0.5Gy剂量范围内占主导地位，并且被认为是由暴露细胞中的氧化应激产生（ROS）介导的，ROS在未受照射的细胞中开启激活应激反应的信号机制。有许多端点可以确定旁观者信号的发生，如细胞死亡的测量、染色体损伤、钙通量、DNA修复机制的上调和ROS的过度产生。目前的提案将调查围绕5个问题建立的三个假设，试图确定为什么中子不会产生旁观者效应。Q1。是中子永远不会产生NTE，还是我们只是错过了有效窗口？Q2。什么能阻止中子暴露的细胞产生NTE？信号产生是否受到影响？如果是，在机制的哪一点？第三季。大多数中子束都有伽马成分——中子的存在会抵消或抵消任何伽马产生的信号吗？第四季度。中子放射生物学显示了辐射转化（体外致癌潜力测定）的“逆剂量率效应”，其中，较低剂量率比高剂量率更有效地产生损害。缺乏NTE是造成这种情况的原因吗？Q5。中子和α粒子的细胞存活曲线都没有肩（低剂量保留），但据报道α粒子产生NTE，质子效应是模棱两可的。这是真的吗？这对理解中子NTE放射生物学有什么意义？这些问题被提炼成3个可检验的假设，简要地列在这里：a .单个细胞受到中子撞击的剂量如此之高，以至于不能产生信号。这将使用剂量学和生物学方法进行测试，以确定对细胞的剂量，以及该剂量是否阻断钙离子通量、活性氧和血清素结合等信号产生机制。测试将雇用3名博士生，并将回答问题1、2、3和4。B.除了触发NTE的低LET和低剂量阈值外，还有一个高剂量阈值，超过这个阈值信号的产生就会受到抑制。这将采用旨在比较不同辐射质量的系统方法进行测试。这将额外雇用一名博士生，并将解决问题3和5。x射线和伽马射线与细胞膜的相互作用导致膜的直接激发和电离，并激活离子门控膜泵，从而触发信号级联。某些类型的中子可能不会引起这种激发，这取决于它们是否引起间接电离。这将通过在暴露于伽马射线或热中子或快中子之前用膜稳定剂处理细胞来进行测试。这也将解决问题2和3。这些方法应该回答为什么中子不产生NTE的问题，并且应该提供一般低剂量放射生物学，特别是中子放射生物学中重要的机制信息。由于中子是CANDU反应堆的副产品，研究结果也可能有利于那些关注核电工业辐射防护问题的人。

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.