Reactive oxygen species, but not mitochondrial membrane potential, is associated with radiation-induced apoptosis of AHH-1 human lymphoblastoid cells

The aim of the study was to investigate the sensitivity of AHH-1 human lymphoblastoid cells to radiation and its relevance to intracellular events, specifically alteration in cellular energy-producing systems. AHH-1 human lymphoblastoid cells were irradiated with 6 Gy of γ radiation, and then were collected at the indicated time points. Parallel studies were conducted to assess the effects of radiation on the cell proliferation and apoptotic index. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production were monitored. A marked decrease of cell viability was observed as early as 12 h postirradiation and fraction of apoptotic cells was highest at 24 h. Intracellular ROS generation measured with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) appeared to be highest as early as 30 min postirradiation and resumed to normal level at 6 h. Unexpectedly, the fluorescence intensity of Rhodamine 123 for measuring MMP did not change during the first 3 h after radiation and exhibited an aberrant increase at 6 h. The results suggest that AHH-1 cells are sensitive to radiation-induced apoptosis and ROS generation is an early phase in the apoptosis process. Moreover, the results might cast doubts on those studies using Rhodamine 123 which hypothesized that the fall in MMP is one of the early events of apoptosis.