Assessment of ROS production using DCFH-DA dye in CHO cells after application of ionizing radiation

Abstract

The research on x-ray effect on cell behavior is important for improving clinical radiotherapy application. Studies show that part of ionizing radiation induced DNA damage is done indirectly by generating reactive oxygen species [1] that interact with cells’ DNA and other cellular macromolecular materials that leads to decreased cell proliferation and signaling abilities [2]. ROS also increases the risk of cell transformation into cancer cells ((carcinogenesis))) and impair the functionality and efficiency of enzymes that are needed to repair damaged DNA [4]. In this study Chinese hamster ovary (CHO) cells were irradiated using medical linear accelerator Varian Clinac DMX (X-rays, 6Mev and 3Gy/min) with doses ranging from 0.5 to 10 Gy.50μMconcentration of dichlorodihydrofluorescein diacetate (DCFH-DA) dye was used as a probe to monitor ROS generated inside irradiated cells and in a growth medium. The DCF production dynamics in cells was observed 1, 3, 6, 12 and 24h after cell irradiation. To assess DNA damage and cell viability dependence on irradiated dose COMET and clonogenic assays were performed. Results show linear delivered dose dependency with DCF fluorescence measured one hour after irradiation. Cells irradiated with 4 and 8 Gy produced higher additional DCF fluorescence 3 and 6 h post irradiation compared to lower doses. Therefore results suggest that 4 Gy dose is a threshold at which ROS production increases significantly after 3 and 6 h. DNA damage and cell viability results support this observation. Cell irradiation at 2, 4, 6, and 8 Gy increased DNA damage to 5.1±0.6, 24.8±2.4, 32.0±3.2, 45.6±3.5% respectively. Cell viability decreased to 82±5% after 2 Gy and to 48±1.8% after 4 Gy irradiation. That is the largest decrease in viability compared with 6, 8 and 10 Gy that led to viability decrease to 24±3.4, 12±0.7 and 9±0.7% respectively.