Radiation Induced Non-Targeted Response: Mechanism and Potential Clinical Implications

Generations of students in radiation biology have been taught that heritable biological effects require direct damage to DNA. Radiation-induced non-targeted/bystander effects represent a paradigm shift in our understanding of the radiobiological effects of ionizing radiation in that extranuclear and extracellular effects may also contribute to the biological consequences of exposure to low doses of radiation. Although radiation induced bystander effects have been well documented in a variety of biological systems, including 3D human tissue samples and whole organisms, the mechanism is not known. There is recent evidence that the NF-κB-dependent gene expression of interleukin 8, interleukin 6, cyclooxygenase- 2, tumor necrosis factor and interleukin 33 in directly irradiated cells produced the cytokines and prostaglandin E2 with autocrine/paracrine functions, which further activated signaling pathways and induced NF-κB-dependent gene expression in bystander cells. The observations that heritable DNA alterations can be propagated to cells many generations after radiation exposure and that bystander cells exhibit genomic instability in ways similar to directly hit cells indicate that the low dose radiation response is a complex interplay of various modulating factors. The potential implication of the non-targeted response in radiation induced secondary cancer is discussed. A better understanding of the mechanism of the non-targeted effects will be invaluable to assess its clinical relevance and ways in which the bystander phenomenon can be manipulated to increase therapeutic gain in radiotherapy

207-nm UV Light—A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. II: In-Vivo Safety Studies

Background UVC light generated by conventional germicidal lamps is a well-established anti-microbial modality, effective against both bacteria and viruses. However, it is a human health hazard, being both carcinogenic and cataractogenic. Earlier studies showed that single-wavelength far-UVC light (207 nm) generated by excimer lamps kills bacteria without apparent harm to human skin tissue in vitro. The biophysical explanation is that, due to its extremely short range in biological material, 207 nm UV light cannot penetrate the human stratum corneum (the outer dead-cell skin layer, thickness 5–20 μm) nor even the cytoplasm of individual human cells. By contrast, 207 nm UV light can penetrate bacteria and viruses because these cells are physically much smaller. Aims To test the biophysically-based hypothesis that 207 nm UV light is not cytotoxic to exposed mammalian skin in vivo. Methods Hairless mice were exposed to a bactericidal UV fluence of 157 mJ/cm2 delivered by a filtered Kr-Br excimer lamp producing monoenergetic 207-nm UV light, or delivered by a conventional 254-nm UV germicidal lamp. Sham irradiations constituted the negative control. Eight relevant cellular and molecular damage endpoints including epidermal hyperplasia, pre-mutagenic UV-associated DNA lesions, skin inflammation, and normal cell proliferation and differentiation were evaluated in mice dorsal skin harvested 48 h after UV exposure. Results While conventional germicidal UV (254 nm) exposure produced significant effects for all the studied skin damage endpoints, the same fluence of 207 nm UV light produced results that were not statistically distinguishable from the zero exposure controls. Conclusions As predicted by biophysical considerations and in agreement with earlier in vitro studies, 207-nm light does not appear to be significantly cytotoxic to mouse skin. These results suggest that excimer-based far-UVC light could potentially be used for its anti-microbial properties, but without the associated hazards to skin of conventional germicidal UV lamps

RAD9 deficiency enhances radiation induced bystander DNA damage and transcriptomal response

Background Radiation induced bystander effects are an important component of the overall response of cells to irradiation and are associated with human health risks. The mechanism responsible includes intra-cellular and inter-cellular signaling by which the bystander response is propagated. However, details of the signaling mechanism are not well defined. Methods We measured the bystander response of Mrad9 +/+ and Mrad9 −/− mouse embryonic stem cells, as well as human H1299 cells with inherent or RNA interference-mediated reduced RAD9 levels after exposure to 1 Gy α particles, by scoring chromosomal aberrations and micronuclei formation, respectively. In addition, we used microarray gene expression analyses to profile the transcriptome of directly irradiated and bystander H1299 cells. Results We demonstrated that Mrad9 null enhances chromatid aberration frequency induced by radiation in bystander mouse embryonic stem cells. In addition, we found that H1299 cells with reduced RAD9 protein levels showed a higher frequency of radiation induced bystander micronuclei formation, compared with parental cells containing inherent levels of RAD9. The enhanced bystander response in human cells was associated with a unique transcriptomic profile. In unirradiated cells, RAD9 reduction broadly affected stress response pathways at the mRNA level; there was reduction in transcript levels corresponding to genes encoding multiple members of the UVA-MAPK and p38MAPK families, such as STAT1 and PARP1, suggesting that these signaling mechanisms may not function optimally when RAD9 is reduced. Using network analysis, we found that differential activation of the SP1 and NUPR1 transcriptional regulators was predicted in directly irradiated and bystander H1299 cells. Transcription factor prediction analysis also implied that HIF1α (Hypoxia induced factor 1 alpha) activation by protein stabilization in irradiated cells could be a negative predictor of the bystander response, suggesting that local hypoxic stress experienced by cells directly exposed to radiation may influence whether or not they will elicit a bystander response in neighboring cells

Detection of Chromosomal Instability in Bystander Cells after Si490-Ion Irradiation

There is increasing evidence that two of the biological effects associated with low-dose ionizing radiation, genomic instability and bystander responses, may be linked. To verify and validate the link between the two phenomena, the ability of Si490 ions (high-energy particles associated with radiation risk in space) to induce bystander responses and chromosomal instability in human bronchial epithelial (HBEC-3kt) cells was investigated. These studies were conducted at both the population and single cell level in irradiated and nonirradiated bystander cells receiving medium from the irradiated cultures. At the general population level, transfer of medium from silicon-ion (Si490)-irradiated cultures (at doses of 0.073 Gy, 1.2 Gy and 2 Gy) to nonirradiated bystander cells resulted in small increases in the levels of chromosomal aberrations at the first division. Subsequently, single cell clones isolated from irradiated and bystander populations were analyzed for the appearance of de novo chromosome-type aberrations after 50 population doublings using mFISH. Both irradiated and bystander clones demonstrated chromosomal instability (as seen by the de novo appearance of translocations and chromosomal fragments), albeit to different degrees, whereas sham-treated controls showed relatively stable chromosomal patterns. The results presented here highlight the importance of nontargeted effects of radiation on chromosomal instability in human epithelial cells and their potential relevance to human health

Correction: High Throughput Measurement of γH2AX DSB Repair Kinetics in a Healthy Human Population.

The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay

Far-UVC light prevents MRSA infection of superficial wounds in vivo.

Prevention of superficial surgical wound infections from drug-resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA) currently present major health care challenges. The majority of surgical site infections (SSI) are believed to be caused by airborne transmission of bacteria alighting onto the wound during surgical procedures. We have previously shown that far-ultraviolet C light in the wavelength range of 207-222 nm is significantly harmful to bacteria, but without damaging mammalian cells and tissues. It is important that the lamp be fitted with a filter to remove light emitted at wavelengths longer than 230 nm which are harmful.Using a hairless mouse model of infection of superficial wounds, here we tested the hypothesis that 222-nm light kills MRSA alighting onto a superficial skin incisions as efficiently as typical germicidal light (254 nm), but without inducing skin damage.To simulate the scenario wherein incisions are infected during surgical procedures as pathogens in the room alight on a wound, MRSA was spread on a defined area of the mouse dorsal skin; the infected skin was then exposed to UVC light (222 nm or 254 nm) followed by a superficial incision within the defined area, which was immediately sutured. Two and seven days post procedure, bactericidal efficacy was measured as MRSA colony formation unit (CFU) per gram of harvested skin whereas fixed samples were used to assess skin damage measured in terms of epidermal thickness and DNA photodamage.In the circumstance of superficial incisions infected with bacteria alighting onto the wound, 222-nm light showed the same bactericidal properties of 254-nm light but without the associated skin damage.Being safe for patient and hospital staff, our results suggested that far-UVC light (222 nm) might be a convenient approach to prevent transmission of drug-resistant infectious agents in the clinical setting