Project description:Biological tissues are exposed to X-rays under controlled conditions in both medical applications (diagnostic imaging and radiotherapy) and research studies (e.g. microcomputed X-ray tomography: microCT). Radiotherapy regimes may deliver doses of up to 50Gy to both the target tumour and to healthy tissues resulting in undesirable clinical side effects which can severely compromise quality of life. The substantially higher doses used in microCT imaging (in the kGy range) may, in the case of native (non-fixed tissues), impact on structure and hence function. Whilst the interaction between X-rays and cells is relatively well-characterised, X-ray-induced structural damage to the extracellular matrix (ECM) is poorly understood. In this study, we test the hypothesis that ECM proteins and ECM-rich tissues (purified collagen I and tendons) are structurally and functionally compromised by exposure to X-rays doses ranging from 50Gy (breast radiotherapy) to 495kGY (synchrotron imaging). Using protein gel electrophoresis we show that breast radiotherapy equivalent doses affect the constituent α chains of solubilised purified collagen I whilst assembly into fibrils, either in vitro or in vivo, prevents X-ray-induced fragmentation but not structural damage (as characterised by LC-MS/MS and peptide location fingerprinting: PLF). In the case of synchrotron imaging, the resultant X-ray exposure induces substantial fragmentation of both constituent collagen I α chains and the triple-helical monomer. Mass spectrometry and PLF analysis of synchrotron irradiated tendon identified structure-associated changes in collagens I, VI, XII, the large proteoglycan aggrecan, small leucine-rich proteoglycans decorin and fibromodulin and a key elastic fibre component fibulin-1. Synchrotron imaging also compromises the mechanical behaviour of tendons. We conclude, therefore, that exposure to both radiotherapy and synchrotron imaging X-rays can profoundly affect the structure of key tissue ECM components with implications for tissue function, downstream cell-matrix interactions, and the interpretation of in situ mechanical data.

Project description:The growing number of particle treatment facilities worldwide and patients treated with particles instead of X-rays marks the upcoming rearrangement of modern radiotherapy. Especially for tumors being difficult to access and for tumors that are resistant to conventional X-ray treatment particle radiotherapy is a beneficial technology. At the Heidelberg Ion Beam Therapy Center (HIT) patients are treated with this technology since 2009 as it offers clear benefits. Contrary to X-rays, which show an exponential dose decrease (after reaching electron equilibrium) with increasing tissue depth, charged particles deposit most of their energy to a small region within the tissue with a sharp dose fall-off after the so-called Bragg peak. This precise dose localization enables further dose escalation within the tumor while sparing healthy tissue. Besides physical advantages, particle radiotherapy offers additional biological advantages. In radiobiology, the term relative-biological effectiveness (RBE) is defined as the ratio of X-rays dose to an alternative irradiation modality dose which produce the same biological effect (e.g. survival or number of DSBs). While protons have a relative biological effectiveness (RBE) comparable to X-rays, carbon ions are more effective in inducing DNA damage(1, 2) and are therefore especially useful for radioresistant tumors. This is due to the fact, that carbon ions induce clustered and direct DNA damage, which is considered to be less dependent on cell cycle stage, oxygen level, genetic background and hinders DNA repair mechanisms(3–6). Nevertheless, their exact mode of action and cellular mechanisms are largely unknown. We show the first comprehensive proteomic and phosphoproteomic study elucidating the cellular response to treatment with protons, carbon ions and X-rays. We found that 2h after treatment with these radiations negligible regulation occurred at protein expression level. But 181 phosphorylation sites were deregulated by ionizing radiation contributing mainly to DNA damage response functionalities. Interestingly we found 55 phosphorylation sites being differentially regulated between the radiations. Here, we observed protons and carbon ions producing equal cellular response whereas X-rays show altered regulation for certain phosphorylation sites. A subset of 28 phosphorylation sites being involved in the DNA damage response or differentially regulated between the ionizing radiations was selected for result confirmation.

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	20230222_SherrattM_TuiengRJ_pRC4.raw	Raw
	20230222_SherrattM_TuiengRJ_pRC5.raw	Raw

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Impact of therapeutic and synchrotron x-rays on collagen I

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