Development and use of an adoptive transfer method for detecting radiation-induced bystander effects in vivo

Author: Benjamin John Blyth

Blyth, Benjamin John, 2009 Development and use of an adoptive transfer method for detecting radiation-induced bystander effects in vivo, Flinders University, School of Medicine

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Ionising radiation can cause damage to DNA that can result in gene mutations contributing to carcinogenesis. Radiation-protection policy currently estimates cancer risks from exposures to radiation in terms of excess risk per unit dose. At very low radiation dose-rates, where not all cells are absorbing radiation energy, this formula carries the inherent assumption that risk is limited to those cells receiving direct energy depositions. Numerous studies have now called this assumption into question. Such low dose-rates are in the relevant range that the public receives from natural background and man-made sources, and, if this fundamental assumption proves unfounded, current estimations of radiation-induced cancer risk at low doses will be incorrect. Accurate predictions of stochastic cancer risks from low-dose radiation exposures are crucial to evaluating the safety of radiation-based technologies for industry, power generation and the increasing use of radiation for medical diagnostic and screening purposes. This thesis explores phenomena known as radiation-induced bystander effects. The term bystander effects, as used here, describes biological responses to ionising radiation (hitherto observed in vitro) in cells not directly traversed by an ionising track, due to intercellular signals received from neighbouring cells that did receive energy depositions. This study aimed to determine whether radiation effects are communicated between irradiated and unirradiated cells in vivo, and if so, whether this effect alters current estimations of cancer risk following low-dose radiation exposures. In order to answer these questions, an in vivo experimental system for studying bystander effects in mice was developed. The method was based on the adoptive transfer of irradiated splenocytes into unirradiated hosts with simultaneous identification of irradiated donor cells, and biological endpoints in unirradiated bystander cells in situ using fluorescence microscopy and image analysis. Splenocytes from donor mice were radiolabelled with 3H-thymidine or received an acute X-ray dose. The irradiated donor cells, labelled with a fluorescent probe, were then adoptively transferred into unirradiated recipient mice via the tail vein, whilst control mice received sham-irradiated donor cells. A proportion of the cells lodged in the recipient mouse spleens where they remained for a period before the tissues were cryopreserved. The locations of donor cells were identified in frozen spleen sections by the fluorescent probe, and the levels of apoptosis and proliferation were simultaneously evaluated in situ in the surrounding unirradiated bystander cells using fluorescence-based assays. Transgenic pKZ1 recipient mice were also used to quantify chromosomal inversions in bystander cells. Since three-dimensional spatial relationships were preserved, responses could be measured in the local area surrounding irradiated cells as well as further afield. Following the development of the irradiated-cell adoptive transfer protocol and validation of the sensitivity and reproducibility of the biological assays in situ, a series of experiments was performed. In the initial experiments, 500,000 radiolabelled cells (0.33 mBq.cell-1) were injected into recipient mice and the spleen tissues were isolated 22 h later. No changes in apoptosis or proliferation were detected in local bystander spleen cells or throughout the spleen, compared to mice receiving sham-radiolabelled donor cells. In subsequent experiments, the effects of a number of experimental conditions were explored including the injection of tenfold more donor cells, analysis of spleen tissues after three days lodging in vivo, radiolabelling of donor cells with 100-fold higher 3H dose-rate and irradiation of donor cells ex vivo with 0.1 or 1 Gy X-rays. In each case, no changes in apoptosis or proliferation were observed. The in vivo method described here was designed to simulate the conditions of a bystander scenario from low dose-rate exposures relevant to public radiation protection. Contrary to the many reports of bystander effects in vitro, experiments using this sensitive method for examining the in vivo responses of unirradiated cells to neighbouring low-dose irradiated cells, have so far shown no changes in bystander cells in the spleen. This adoptive transfer method is the first in vivo method for examining the effects of known irradiated cells exposed to low radiation doses at low dose-rates, on neighbouring cells in situ that are truly unirradiated. Both the irradiated and bystander cells are normal, non-transformed primary spleen cells functioning in their natural environment. This in vivo experimental system allows the examination of tens of thousands of bystander cells and has shown a remarkable sensitivity, with statistical power to rule out changes in apoptosis <10% from the control. The relevance of in vitro bystander findings is unclear. Many reported bystander effects are more analogous to the systemic communication of abscopal effects from highly irradiated tissues. Disagreement between experimental systems and difficulty in reproducing key results between laboratories further complicate the translation of bystander data in vitro to human risk-estimation. The radiation protection community has expressed its need for in vivo validation of the bystander phenomenon before it can be included into the appraisal of carcinogenic risk. This adoptive transfer method is now available to study a range of bystander endpoints and potential signalling mechanisms in vivo, and provides a way to translate the wealth of data previously collected in vitro into findings directly relevant to human risk-estimation.

Keywords: Bystander Effects,Radiation,Low Dose,Non targeted effects,Spleen,Apoptosis

Subject: Haematology thesis, Pathology thesis

Thesis type: Doctor of Philosophy
Completed: 2009
School: School of Medicine
Supervisor: Associate Professor Pamela J Sykes