Additive effects of simulated microgravity and ionizing radiation in cell death, induction of ROS and expression of RAC2 in human bronchial epithelial cells

Abstract

Radiation and microgravity are undoubtedly two major factors in space environment that pose a health threat to astronauts. However, the mechanistic study of their interactive biological effects is lacking. In this study, human lung bronchial epithelial Beas-2B cells were used to study the regulation of radiobiological effects by simulated microgravity (using a three-dimensional clinostat). It was found that simulated microgravity together with radiation induced drop of survival fraction, proliferation inhibition, apoptosis, and DNA double-strand break formation of Beas-2B cells additively. They also additively induced Ras-related C3 botulinum toxin substrate 2 (RAC2) upregulation, leading to increased NADPH oxidase activity and increased intracellular reactive oxygen species (ROS) yield. The findings indicated that simulated microgravity and ionizing radiation presented an additive effect on cell death of human bronchial epithelial cells, which was mediated by RAC2 to some extent. The study provides a new perspective for the better understanding of the compound biological effects of the space environmental factors.

Fig. 1. Both simulated microgravity and X-ray irradiation induced cell survival inhibition.

Beas-2B cells were treated by simulated microgravity for 48 h first and then exposed to X-ray irradiation at indicated doses. a Cell survival fraction was determined by colony formation assay. b Cell survival curves were drawn for cells subjected to irradiation and/or simulated microgravity. The experiments were done in triplicate and error bars indicate SEM. IR, irradiation group; SMG, simulated microgravity group; SMG + IR, simulated microgravity combined with irradiation group. For the compounding treatment, cells were treated by simulated microgravity for 48 h first and then exposed to X-ray irradiation. Cells were cultured under 1 g during and after exposure (the same below). *Compared with control group. #Compared with IR group, */^#p < 0.05, **/##p < 0.01, ***/###p < 0.001.

Fig. 2. Both simulated microgravity and radiation induced cell proliferation inhibition.

Beas-2B cells were treated by simulated microgravity for 48 h first and then exposed to 2 Gy X-ray irradiation. Cell proliferation curves were drawn from 0 to 96 h post irradiation. The experiments were done in triplicate and error bars indicate SEM. Ctrl, control group; IR, irradiation group; SMG, simulated microgravity group; SMG + IR, simulated microgravity combined with irradiation group.

Fig. 3. The γH2AX foci levels in the Beas-2B cells subjected to different treatments.

Beas-2B cells were treated by simulated microgravity for 48 h first and then exposed to 0.5 Gy X-ray irradiation. Irradiated cells were fixed at 1 h or 24 h post irradiation for γH2AX foci assay. a Immunofluorescent staining of the γH2AX in the cells exposed to simulated microgravity and/or 0.5 Gy X-rays. b The γH2AX foci yields in the cells exposed to simulated microgravity and/or 0.5 Gy X-rays. The experiments were done in triplicate and error bars indicate SEM. Ctrl, control group; IR, irradiation group; SMG, simulated microgravity group; SMG + IR, simulated microgravity combined with irradiation group. Scale bar represents 50 μm. *Compared with control group. #Compared with IR group. &Compared with SMG group. */#/&: p < 0.05.

Fig. 4. Simulated microgravity and radiation induced apoptosis additively.

Beas-2B cells were treated by simulated microgravity for 48 h first and then exposed to 2 Gy X-ray irradiation. Samples were collected at 48 h post irradiation. a Representative images of the cytometric analysis of the apoptosis. b The apoptosis rates of the cells exposed to simulated microgravity and/or 2 Gy X-rays. The experiments were done in triplicate and error bars indicate SEM. Ctrl, control group; IR, irradiation group; SMG, simulated microgravity group; SMG + IR, simulated microgravity combined with irradiation group. *Compared with control group. #Compared with IR group. &Compared with SMG group. */#/&: p < 0.05, **: p < 0.01.

Fig. 5. Simulated microgravity and radiation increased RAC2 expression, NADPH oxidase activity, and ROS yield additively.

Beas-2B cells were treated by simulated microgravity for 48 h first and then exposed to 2 Gy X-ray irradiation. Samples for qRT-PCR, western blotting, NADPH consumption assay, as well as ROS production assay were all collected at 2 h post irradiation. a Transcriptional level of RAC2 was determined in Beas-2B cells exposed to simulated microgravity and/or 2 Gy X-rays. b Protein level of RAC2 as determined by western blotting. c NADPH consumption in Beas-2B cells exposed to simulated microgravity and/or 2 Gy X-rays. d ROS yields in the treated Beas-2B cells. All experiments were done in triplicate and error bars indicate SEM. Ctrl, control group; IR, irradiation group; SMG, simulated microgravity group; SMG + IR, simulated microgravity combined with irradiation group. *Compared with control group. #Compared with IR group. &Compared with SMG group. */#/&: p < 0.05.