Infrared Microspectroscopic Characteristics of Radiation-Induced Apoptosis in Human Lymphocytes

Infrared microspectroscopic characterization of radiation-induced apoptosis was used as a new analytical tool to study the kinetics of apoptosis in human peripheral blood lymphocytes at the molecular level. This vibrational technique, which has already been used to investigate biomolecules in normal and tumor cells, allows the simultaneous detection of the biochemical changes in the various subcellular compartments. Normal circulating lymphocytes from five healthy human donors were given a single dose of 6 Gy ((60)Co) and deposited on ZnS windows for infrared spectral acquisition 1, 2 and 4 days after irradiation. Apoptosis was assessed simultaneously by flow cytometry analysis of lymphocytes displaying annexin V-positive staining, and by detection of the DNA laddering that is characteristic of apoptosis. The flow cytometry study showed that about 80% of sham-irradiated lymphocytes were annexin V(neg)/PI(neg) at 1, 2 and 4 days. One day after irradiation, 46% of irradiated lymphocytes were annexin V(neg)/PI(neg), 48% were annexin V(pos)/PI(neg), 5% were annexin V(pos)/PI(pos), and 1% were annexin V(neg)/PI(pos). These mean percentages were respectively 31, 59, 9 and 1 at day 2 and 23, 36, 30, and 11 at day 4. Irradiated lymphocytes presented a DNA laddering pattern characteristic of apoptosis from day 1 after irradiation. In the infrared spectra of irradiated lymphocytes, qualitative and quantitative changes were observed from days 1 and 2, respectively. In the range of 960-1245 cm(-1) mainly attributed to nucleic acids, changes corresponding to conformational changes in DNA were associated with a decrease in the amount of detectable DNA. Conformational changes were also observed in secondary protein structures, in particular an increase in the amount of beta structures. These DNA and protein changes were associated with an increase in the detectable amount of lipids at day 4 after irradiation. These results showed that DNA is probably the first cellular target of radiation-induced apoptosis, which, however, also requires conformational changes and synthesis of cell proteins. Our results are in agreement with biochemical and morphological data on radiation-induced apoptosis of normal human circulating lymphocytes, and they demonstrate that infrared microspectroscopy may be useful for assessing the process of apoptosis at the molecular level.