Impact of therapeutic X-ray exposure on collagen I and associated proteins

Biological tissues are exposed to X-rays in medical applications (such as diagnosis and radiotherapy) and in research studies (for example microcomputed X-ray tomography: microCT). Radiotherapy may deliver doses up to 50Gy to both tumour and healthy tissues, resulting in undesirable clinical side effects which can compromise quality of life. Whilst cellular responses to X-rays are relatively well-characterised, X-ray-induced structural damage to the extracellular matrix (ECM) is poorly understood. This study tests the hypotheses that ECM proteins and ECM-rich tissues (purified collagen I and rat tail tendons respectively) are structurally compromised by exposure to X-ray doses used in breast radiotherapy. Protein gel electrophoresis demonstrated that breast radiotherapy equivalent doses can induce fragmentation of the constituent α chains in solubilised purified collagen I. However, assembly into fibrils, either in vitro or in vivo, prevented X-ray-induced fragmentation but not structural changes (as characterised by LC-MS/MS and peptide location fingerprinting: PLF). In subsequent experiments exposure to higher (synchrotron) X-ray doses induced substantial fragmentation of solubilised and fibrillar (chicken tendon) collagen I. LC-MS/MS and PLF analysis of synchrotron-irradiated tendon identified structure-associated changes in collagens I, VI, XII, proteoglycans including aggrecan, decorin, and fibromodulin, and the elastic fibre component fibulin-1. Thus, exposure to radiotherapy X-rays can affect the structure of key tissue ECM components, although additional studies will be required to understand dose dependent effects. STATEMENT OF SIGNIFICANCE: Biological systems are routinely exposed to X-rays during medical treatments (radiotherapy) and in imaging studies (microCT). Whilst the impact of ionising radiation on cells is well characterised, the interactions between X-rays and the extracellular matrix are not. Here, we show that relatively low dose breast radiotherapy X-rays are sufficient to affect the structure of collagen I in both its solubilised and fibrillar forms. Although the impact of intermediate X-ray doses on extracellular proteins was not determined, the high dose exposures which are achievable using a synchrotron source had an even greater effect on the structure of collagen I molecules and, in tendon, on the structures of many accessory extracellular matrix proteins, The unwanted side effects of radiotherapy may therefore be due to not only cellular damage but also damage to the surrounding matrix.