Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis.heterochromatin | high linear energy transfer | high charge and energy particles | 53BP1 | ionizing radiation induced foci I onizing radiation (IR) may induce cancer and loss of neural function or death in humans. Low (e.g., γ-and X-rays) and high [i.e., high charge and energy (HZE)] linear energy transfer (LET) IR induces a plethora of DNA damage, and the damage complexity increases with an increase in the LET of the radiation (1-3). Isolated DNA lesions (mainly induced by low-LET radiation), including double-strand breaks (DSBs), single-strand breaks (SSBs), and damaged bases located at a distance from other damage, are generally repaired efficiently. Substantial evidence indicates that high-LET radiation induces complex DNA damage, a unique class of DNA lesions that includes two or more individual lesions within one or two helical turns of the DNA (4). These lesions can be abasic sites (apurinic/apyrimidinic sites or APs), damaged bases (oxidized purines or pyrimidines), SSBs, or DSBs (5). Convincing evidence indicates that complex DNA lesions are more difficult to repair than isolated lesions and in some instances are irreparable; however, it is unclear why clustered lesions are difficult to repair.The biological consequences of complex DNA damage range from point mutations and loss of genetic material to cellular lethality due to repair impairment and lesion or repair-intermediate persistency. Clustered lesions induce intra-and interchromosomal insertions, and inversions often in association with large deletions (6). FISH-painting methodologies were used to show that high-LET IR induces a high fraction of chromosome rearrangements (7). Recently, it has been suggested that non-DSB clusters, if unrepaired, can lead to the formation of mutations and chromosome abnormalities (8). After exposure to high-LET radiation, immortalized...

Bystander effects induced by low dose of ionizing radiation have been shown to widely exist in many cell types and may have a significant impact on radiation risk assessment. Though many studies have been reported on this phenomenological observation, the mechanisms underlying this process are not clear, especially on the questions of how soon after irradiation the bystander effects can be initiated and how far this bystander signal can be propagated once it is started. DNA double-strand breaks (DSBs) induced by ionizing radiation or carcinogenic chemicals can be visualized in situ using gamma-H2AX immunofluorescent staining. Our previous studies have shown that in situ visualization of DSBs could be used to assess irradiation-induced extranuclear/extracellular (bystander) effect at an early stage after irradiation. In the present studies, we used this method to investigate the time and spatial effects of damage signals on unirradiated bystander cells. The results showed that increased DSBs in irradiated and unirradiated bystander areas could be visualized 2 min after radiation and reached its maximum 30 min after radiation. The average levels of DSB formation at 30 min post-1cGy irradiation in the irradiated and unirradiated bystander areas were 3-fold and 2-fold higher than those of the sham-irradiated control cells, respectively. Afterwards, the formation of DSBs declined with incubation time and remained steady for at least 6 h at a level that was statistically higher than their controls. The results also showed that the bystander signal derived from irradiated cells could be transferred to anywhere in the dish and the percentage of DSBs in the unirradiated bystander cells was not dependent on the dose delivered. Moreover, the fraction of DSB positive cells in unirradiated bystander areas showed a time-dependent increase based on its distance to the irradiated area at very early stage post-irradiation. Both lindane and DMSO significantly suppressed the yield of DSBs in the cells of unirradiated bystander areas, which suggest that gap junctional intercellular communication and reactive oxygen species played important roles in the induction of the bystander effects, both in irradiated and unirradiated bystander areas.

Little is known about the effects of space radiation on the human body. There are a number of potential chronic and acute effects, and one major target for noncarcinogenic effects is the human vasculature. Cellular stress, inflammatory response, and other radiation effects on endothelial cells may affect vascular function. This study was aimed at understanding the effects of space ionizing radiation on the formation and maintenance of capillary-like blood vessels. We used a 3D human vessel model created with human endothelial cells in a gel matrix to assess the effects of low-LET protons and high-LET iron ions. Iron ions were more damaging and caused significant reduction in the length of intact vessels in both developing and mature vessels at a dose of 80 cGy. Protons had no effect on mature vessels up to a dose of 3.2 Gy but did inhibit vessel formation at 80 cGy. Comparison with γ radiation showed that photons had even less effect, although, as with protons, developing vessels were more sensitive. Apoptosis assays showed that inhibition of vessel development or deterioration of mature vessels was not due to cell death by apoptosis even in the case of iron ions. These are the first data to show the effects of radiation with varying linear energy transfer on a human vessel model.

No abstract