Non-targeted effects of radiation: a personal perspective on the role of exosomes in an evolving paradigm.

PURPOSE: Radiation-induced non-targeted effects (NTE) have implications in a variety of areas relevant to radiation biology. Here we evaluate the various cargo associated with exosomal signaling and how they work synergistically to initiate and propagate the non-targeted effects including genomic instability and bystander effects. CONCLUSIONS: Extra cellular vesicles, in particular exosomes, have been shown to carry bystander signals. Exosome cargo may contain nucleic acids, both DNA and RNA, as well as proteins, lipids, and metabolites. These cargo molecules have all been considered as potential mediators of NTE. A review of current literature shows mounting evidence of a role for ionizing radiation in modulating both the numbers of exosomes released from affected cells as well as the content of their cargo, and that these exosomes can instigate functional changes in recipient cells. However, there are significant gaps in our understanding, particularly regarding modified exosome cargo after radiation exposure and the functional changes induced in recipient cells.

Induction of Genomic Instability in a Primary Human Fibroblast Cell Line Following Low-Dose Alpha-Particle Exposure and the Potential Role of Exosomes.

PURPOSE: To study the induction of genomic instability (GI) in the progeny of cell populations irradiated with low doses of alpha-particles and the potential role of exosome-encapsulated bystander signalling. METHODS: The induction of GI in HF19 normal fibroblast cells was assessed by determining the formation of micronuclei (MN) in binucleate cells along with using the alkaline comet assay to assess DNA damage. RESULTS: Low dose alpha-particle exposure (0.0001-1 Gy) was observed to produce a significant induction of micronuclei and DNA damage shortly after irradiation (assays performed at 5 and 1 h post exposure, respectively). This damage was not only still evident and statistically significant in all irradiated groups after 10 population doublings, but similar trends were observed after 20 population doublings. Exosomes from irradiated cells were also observed to enhance the level of DNA damage in non-irradiated bystander cells at early times. CONCLUSION: very low doses of alpha-particles are capable of inducing GI in the progeny of irradiated cells even at doses where <1% of the cells are traversed, where the level of response was similar to that observed at doses where 100% of the cells were traversed. This may have important implications with respect to the evaluation of cancer risk associated with very low-dose alpha-particle exposure and deviation from a linear dose response.

Radiation-Induced Senescence Bystander Effect: The Role of Exosomes.

Ionizing Radiation (IR), especially at high doses, induces cellular senescence in exposed cultures. IR also induces "bystander effects" through signals released from irradiated cells, and these effects include many of the same outcomes observed following direct exposure. Here, we investigate if radiation can cause senescence through a bystander mechanism. Control cultures were exposed directly to 0, 0.1, 2, and 10 Gy. Unirradiated cells were treated with medium from irradiated cultures or with exosomes extracted from irradiated medium. The level of senescence was determined post-treatment (24 h, 15 days, 30 days, and 45 days) by ß-galactosidase staining. Media from cultures exposed to all four doses, and exosomes from these cultures, induced significant senescence in recipient cultures. Senescence levels were initially low at the earliest timepoint, and peaked at 15 days, and then decreased with further passaging. These results demonstrate that senescence is inducible through a bystander mechanism. As with other bystander effects, bystander senescence was induced by a low radiation dose. However, unlike other bystander effects, cultures recovered from bystander senescence after repeated passaging. Bystander senescence may be a potentially significant effect of exposure to IR, and may have both beneficial and harmful effects in the context of radiotherapy.

Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

Radiation-induced Chromosome Instability: The Role of Dose and Dose Rate.

Nontargeted effects include radiation-induced genomic instability (RIGI) which is observed in the progeny of cells exposed to ionizing radiation and can be manifested in different ways, including chromosomal instability and micronucleus (MN) formation. Since genomic instability is commonly observed in tumors and has a role in tumor progression, RIGI has the potential of being an important mechanism for radiation-induced cancer. The work presented explores the role of dose and dose rate on RIGI, determined using a MN assay, in normal primary human fibroblast (HF19) cells exposed to either 0.1 Gy or 1 Gy of X-rays delivered either as an acute (0.42 Gy/min) or protracted (0.0031 Gy/min) exposure. While the expected increase in MN was observed following the first mitosis of the irradiated cells compared to unirradiated controls, the results also demonstrate a significant increase in MN yields in the progeny of these cells at 10 and 20 population doublings following irradiation. Minimal difference was observed between the two doses used (0.1 and 1 Gy) and the dose rates (acute and protracted). Therefore, these nontargeted effects have the potential to be important for the low-dose and dose-rate exposure. The results also show an enhancement of the cellular levels of reactive oxygen species after 20 population doublings, which suggests that ionising radiation (IR) could potentially perturb the homeostasis of oxidative stress and so modify the background rate of endogenous DNA damage induction. In conclusion, the investigations have demonstrated that normal primary human fibroblast (HF19) cells are susceptible to the induction of early DNA damage and RIGI, not only after a high dose and high dose rate exposure to low linear energy transfer, but also following low dose, low dose rate exposures. The results suggest that the mechanism of radiation induced RIGI in HF19 cells can be correlated with the induction of reactive oxygen species levels following exposure to 0.1 and 1 Gy low-dose rate and high-dose rate x-ray irradiation.

Electrical behavior of stored erythrocytes after exposure to gamma radiation and the role of alpha-lipoic acid as radioprotector.

The effects of gamma rays (25, 50 and 100 Gy) on stored erythrocytes were studied by measuring their dielectric properties and observing their morphology under scanning electron microscopy. Alpha lipoic acid (a potent natural antioxidant) was introduced prior to irradiation for radioprotection. It can be concluded that the dose level of 25 Gy can be considered a safe sterile dose; however, irradiation doses of 50 and 100 Gy should be applied with the addition of alpha-acid to preserve the cell viability.