Investigation On Progeny Genomic Instability In NL20 Human Bronchial Epithelial Cells Induced By Low Dose Irradiation With Different Radiation Quality

Objectives:Low dose irradiation-induced genomic instability is defined as the biological phenomenon that delayed effects, such as chromosomal translocations, deletions, increased micronuclei rate, etc. occur in the progeny of irradiated cells via transmission of ionizing radiation "memory" to the progeny through cell replication process. Currently non-targeted effects, including progeny genomic instability, bystander effects, etc., have brought a huge impact on radiation-induced carcinogenesis and traditional radiation risk assessment. Thus it has become one of the hot topics in the field of radiation biology. The study of low dose irradiation-induced progeny genomic instability has now been focusing on the underlying molecular mechanisms and the influence on the organisms,In this study, NL20 human bronchial epithelial cells were used to explore progeny genomic instability and the relevant mechanisms induced by low dose irradiation with different radiation quality. First, whether low dose of X-rays or a particles could induce DNA damage in the progeny of NL20 cells was verified; The damage in the progeny of NL20 cells caused by high-energy Fe ions was further explored to pave the way for the detailed investigation on the biological consequences of low dose of high LET ionizing radiation.Methods:In the first part of this study, micronucleus formation and immunofluorescence of 53BP1 foci (DNA damage surrogate) were used as the biological endpoints to demonstrate that low dose X-rays could induce DNA damage in irradiated NL20 cells, and this damage effect could transmit to the progeny cells. Then an user-friendly α-irradiation equipment which was designed and built by ourselves was used to study the changes of NL20 cells and their progeny at different times. Finally, we explored the radiation quality dependence of DNA damage in irradiated NL20 cells and their progeny cells.In the second part of this study, iron ion beam irradiation using Lanzhou heavy ion accelerator was used to investigate the effects on the progeny of irradiated NL20 cells. Firstly micronucleus formation and 53BP1 foci fluorescence were used to detect DNA damages in the progeny of NL20 cells. Then, the proliferation assay and cell cycle assay were used to evaluate cell growth in the progeny of irradiated cell. Intracellular ROS detection kit was used to measure ROS level in the progeny of irradiated NL20 cells with iron ions. Finally, the expression levels of SOD1, SOD2 in the progeny of irradiated NL20 cells were detected by western blotting.Results:1. After fractionated irradiation with X-rays or a particles (3x0.3 Gy of X-rays or 3×0.1Gy of a particles), the percentages of 53BP1 foci positive cells and nuclei with micronucleus in the 3rd、6th progeny of NL20 cells are much higher than that in the control group, while with a single irradiation (0.9 Gy of X-rays or 0.3 Gy of a particles), the 3rd progeny cells show no statistical difference from the control group in terms of DNA damage. Compared with X-irradiation, a particles cause a bigger increase trend in the percentage of 53BP1 foci positive cells and micronucleus formation in 3rd、 6th、18th progeny cells of NL20 cells, although not statistically significant.2.0.1 Gy iron ion irradiation causes an significant increase in the nuclei with micronucleus in the 6th、12th progeny of irradiated NL20 cells. However, there is no difference in 18th progeny cells of NL20 cells compared with the relative control. In addition, the percentage of 53BP1 foci positive cells in the6th、12th、18th progeny cells of irradiated NL20 cells is statistically significantly higher than the control group, suggesting that DNA damage caused by 0.1 Gy of Fe ions persists in NL20 progeny cells. Data of cell cycle distribution display that compared with the relative control, there is a significantly G1 phase delay in irradiated group, which is consistent with proliferation data. Next, ROS level changes in the progeny of Fe-irradiated NL20 cells were explored with ROS detection kit. The 19-22th progeny cells of NL20 cells have a significantly higher intracellular ROS levels than the control groups. Finally, the Western blot results show that the levels of SOD1 and SOD2 in 22th progeny cells of NL20 cells markedly decline, suggesting that the antioxidant system in the progeny of irradiated NL20 cells is obviously weakened.Conclusions:1. Low doses of X-rays and a-irradiation can induce DNA damage in irradiated NL20 cells. Also, this damage effect can transmit to its progeny cells. However, the damage in the progeny of irradiated cells does not appear with single irradiation mode.2. DNA damage induced by iron ion irradiation in NL20 cells can transmit to and persist in their progeny cells. Also, after iron ion irradiation the proliferation of the progeny cells is markedly slowed down and there is a significantly G1 phase arrest. Moreover, the intracellular ROS levels, the expression levels of SOD 1 and SOD2 are abnormal.3. Radiation quality plays an important role in low dose irradiation-induced progeny genomic instability. However, its specific mechanism needs further study.