CHANGE IN THE FREQUENCY OF APOPTOSIS AFTER LOW-DOSE AND HIGH-DOSE X-IRRADIATION OF HUMAN-LYMPHOCYTES

The purpose of the present study was to correlate the type and frequency of cell death in human lymphocytes receiving variable doses of X-irradiation. Monocyte-depleted lymphocyte fractions were exposed in vitro to variable doses of X-rays of 0-20 Gy (0-2000 rads) and incubated for 4 and 16 h. An assessment of the mode of cell death (apoptosis vs. classical necrosis) was carefully evaluated using a multidisciplinary approach using light, fluorescence and electron microscopy (EM), and dye exclusion assays. Eosin Y exclusion assays indicated the absence of classical necrosis occurring in short-term cultures (4 h postirradiation). An assessment of cell counts, however, revealed a mean decrease of 4% at 0 Gy and 13% at 10-20 Gy (1000-2000 rads). The predominant mode of cell death was apoptosis, but the percent apoptotic cells (determined by EM) did not parallel this increase in cell loss with increasing radiation and actually decreased at doses above 5 Gy (500 rads). The discrepancy between percent cell loss and percent apoptosis was explained by a proposed change in overall duration of the apoptotic process. In long-term cultures (16 h postirradiation), a combination of classical necrosis, classical apoptosis, and combined apoptosis and necrosis (secondary necrosis of apoptotic cells) was apparent and was associated with a marked decrease in viability. Irradiation effects on lymphocytes showing none of the morphologic features of apoptosis or classical necrosis in short-term culture were evidenced by an increase in nuclear lobation. The results of this study indicate that the vast majority of peripheral blood lymphocytes are radioresistant. The use of irradiation in an in vitro model to study the biochemical events of the apoptotic process is also evaluated.