The effects of the modification of energy metabolism on cellular response to ionizing radiation

Doctoral Thesis


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University of Cape Town

It is generally accepted that energy is required for repair of radiation-induced damage in living cells. Some of this energy is probably provided by adenosine triphosphate (ATP), which is derived from energy substrates via energy metabolism. This dissertation follows two general avenues. The first explores the effect of radiation on ATP levels after irradiation of cells. The second investigates the effect of inhibitors of certain pathways associated with energy metabolism on radiation response. It was proposed that ATP levels might be raised after irradiation in some systems and that this rise in ATP might be due to compensatory mechanisms related to repair. Experiments were conducted using B16 melanoma cells in vitro and using normal murine liver and CaNT tumours in vivo. ATP concentration was measured in extracts of these cells after irradiation using the luciferase-luciferin method. No major changes from unirradiated controls were found. Several types of substrates exist from which cells can derive energy, including glucose and glutamine which are initially metabolised via glycolysis and glutaminolysis, respectively, before their products are further metabolised in respiration. Since energy is necessary for repair of radiation damage, it has been proposed that the inhibition of energy metabolism might alter the radiation response of cells. An inhibitor of glycolysis, 2-deoxyglucose (2DG), and an inhibitor of glutaminolysis, aminooxyacetic acid (AOA), were administered to CHO cells in vitro to determine the effects of these substances on cellular radiosensitivity and repair. Repair was assessed by means of a split radiation dose experiment. The design of such an experiment required that cells be exposed to inhibitory test media for different times between two fractions of radiation. Any changes in clonogenic survival with time between tween fractions could, therefore be as a result of repair effects or as a result of changes in radiosensitivity. A method of estimating and subtracting the effects of radiosensitivity to make conclusions concerning repair is presented and discussed. Most combinations of 2DG, AOA, glucose omission and glutamine omission in culture media resulted in reductions in repair rate but the extent of repair was found to vary from one medium variation to the next. In addition, the effects of various culture media on glycolysis/PPP (glycolysis/pentose phosphate pathway) and glutaminolysis were investigated by determining the production of CO2 and lactate from radiolabelled-glucose and -glutamine substrates. It was apparent that the presence of either of the inhibitors, 2DG or AOA, could inhibit the activity of glutaminolysis and reduce oxygen consumption. 2DG was shown to inhibit glycolysis/PPP but AOA was shown to stimulate glycolysis/PPP, suggesting a regulatory link between glutaminolysis and glycolysis/PPP. The presence of either inhibitor resulted in a reduction in the rate of radiation damage repair. The medium which had the most significant effect in respect of repair inhibition and increased radiosensitivity was medium lacking both glucose and glutamine and containing both 2DG and AOA. This medium was shown to inhibit oxygen consumption and to result in a depression of both cellular glycolysis/PPP and glutaminolysis. The effect of 2DG on the rate of growth and radiation induced growth delay of three murine tumours in vivo was assessed. 2DG alone inhibited the growth of B16 tumours. However, 2DG alone produced little if any change in the rates of growth of Fib/T tumours and rhabdomyosarcomas but the combination of 2DG and AOA produced an inhibition of growth in the Fib/T tumour. 2DG appeared to enhance the effects of radiation in the Fib/T and B16 tumours but not in the rhabdomyosarcoma, although, in the Fib/T, the combination of AOA, 2DG and radiation was less effective in inhibiting tumour growth than was radiation alone. The effects of radiation and 2DG did not appear to be additive in the Fib/T tumour and the B16 tumours which may imply an influence of 2DG on repair or radiosensitivity. This work suggests that the effects of radiation can be altered by manipulation of metabolic pathways associated with the supply of energy. However, a complex interaction of pathways is probably also involved and it is the detail of this interaction which may partially determine the severity of radiation response.