In Vitro and in Vivo 31 P Nuclear Magnetic Resonance Measurements of Metabolic Changes Post Radiation 1

Radiation-induced metabolic changes previously observed in tumors using phosphorus nuclear magnetic resonance spectroscopy include changes in the relative amounts of the phospholipid precursors phosphoethanolamine and phosphocholine, increases in membrane catabolites, and increases in energy status. To elucidate the degree to which these in vivo alterations are a result of intrinsic cellular changes versus radiationinduced systemic effects, the Radiation-Induced Fibrosarcoma-1 tumor model was studied before and over the course of 7 days after a single dose of 17 Gy. In vivo studies were performed with tumors implanted in OH/IKmice; in vitro studies used cells that were perfused in agarose gel threads after being grown, radiated, and maintained in monolayer. The statistically significant increases in the downfield component of the phosphomonoester peak, which consists primarily of phosphoethanolamine, compared to the upfield component, phosphocholine, were qual itatively similar in vivo and in vitro post radiation. Statistically significant increases in the membrane catabolite glycerophosphocholine, a phosphodiester, were also observed in both tumors and cell culture after irradiation, with a greater percentage change in vitro. This suggests that changes in the phosphomonoester and phosphodiester concentrations are primarily an intrinsic effect of radiation on cellular metabolism, modu lated to a lesser degree by systemic effects. In contrast, the statistically significant increases in energy status after the 17-Gy dose showed mark edly different temporal responses in the two systems. Therefore, energy status changes observed in vivo are due largely to systemic changes, such as changes in blood flow. Flow cytometry data obtained from the cultured cells showed a sustained increase in the G2-M fraction starting at 24 h, the first time point measured after irradiation, which continued for the 7 days studied post radiation. These data indicate that the in vivo changes de tected by nuclear magnetic resonance in phospholipid precursors and catabolites occur directly at the cellular level and may reflect cell death or growth inhibition after antineoplastic therapy. anisms of observed changes allows for more rational treatment design and therapeutic modality combinations. Changes we have previously noted after tumor irradiation in the in vivo 31P NMR3 spectra of the murine MCa tumor model include increases in PME', compared to the upfield component, phosphocho line; increases in the membrane catabolites seen in the phosphodiester region of the spectra; and increases in high-energy phosphates (1, 2, 8). PME' is used instead of PE throughout this article to represent the downfield component of the PME peak, because it contains minor contributions from other compounds in addition to phosphoethanolamine in both the MCa and RIF-1 tumors.4 To determine if these changes are direct cellular effects of radiation or if they are physio logically induced, a comparison of in vitro and in vivo models was necessary. Since the MCa tumor line could not be grown in cell culture, we have studied the RIF-1 fibrosarcoma both in vivo and in vitro after 17 Gy of radiation. Tumors were implanted on the feet of C3H/He mice and were followed serially over the course of 7 days after radiation for the in vivo studies. Cells were grown and radiated in monolayer, with data for each time point post radiation obtained from a different set of cells, for the in vitro studies. Cell cycle data from the cultured cells were also used to determine the relationship between metabolic and cell cycle changes. The effects of radiation on the metabolic response of the RIF-1 tumor model have been studied previously in vivo (9-11) and in vitro (12). We undertook the current study because the increased spectral resolution obtained using susceptibility matching (13) allowed the resolution of the PME peak into two components for both in vivo and in vitro states. We present data showing changes in the PME and PDE resonances, which represent changes in phospholipid precursors and catabolites, not previously detected post radiation in vivo and in vitro for a single tumor model.