Alterations in Phosphate Metabolism during Cellular Recovery of Radiation Damage in Yeast

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Abstract
We have examined alterations in phosphate pools during cellular recovery from radiation damage in intact, wild-type diploid yeast cells using 31P nuclear magnetic resonance (NMR) spectroscopy. Concurrent cell survival analysis was determined following exposure to 60Co .gamma.-radiation. Cells held in citrate-buffered saline (CBS) showed increased survival with increasing time after irradiation (liquid holding recovery, LHR) with no further recovery beyond 48 h. Addition of 100 mmol dm-3 glucose to the recovery medium resulted in greater recovery. In the presence of 5 mmol dm-3 2-deoxyglucose (2-DG), LHR was completely inhibited. NMR analyses were done on cells perfused in agarose threads and maintained under conditions similar to those in the survival studies. ATP was observable by NMR only when glucose was present in the recovery medium. In control cells, ATP concentrations increased and plateaued with increasing recovery time. With increasing radiation dose the increase in ATP was of lesser magnitude, and after 2000 Gy no increase was observed. These observations suggest that either the production of ATP in irradiated cells is suppressed or there is enhanced ATP utilization for repair of radiation damage. In CBS with 100 mmol dm-3 glucose, a dose-dependent decrease in polyphosphate (polyP) was detectable with no concurrent increase in inorganic phosphate (Pi). In the absence of an external energy source, such as glucose, there was a slight increase in Pi. This suggests that polyP may be used as an alternative energy supply. When 2-DG was present in the recovery medium, polyP decreased, but there was a simultaneous increase in Pi with increasing radiation dose and recovery time. This suggests that the polyP are hydrolyzed as a source of phosphates for repair of radiation damage.