Manuela Buonanno

Long-term consequences of radiation-induced bystander effects depend on radiation quality and dose and correlate with oxidative stress.

Widespread evidence indicates that exposure of cell populations to ionizing radiation results in significant biological changes in both the irradiated and nonirradiated bystander cells in the population. We investigated the role of radiation quality, or linear energy transfer (LET), and radiation dose in the propagation of stressful effects in the progeny of bystander cells. Confluent normal human cell cultures were exposed to low or high doses of 1GeV/u iron ions (LET ∼ 151 keV/µm), 600 MeV/u silicon ions (LET ∼ 51 keV/µm), or 1 GeV protons (LET ∼ 0.2 keV/µm). Within minutes after irradiation, the cells were trypsinized and co-cultured with nonirradiated cells for 5 h. During this time, irradiated and nonirradiated cells were grown on either side of an insert with 3-µm pores. Nonirradiated cells were then harvested and allowed to grow for 20 generations. Relative to controls, the progeny of bystander cells that were co-cultured with cells irradiated with iron or silicon ions, but not protons, exhibited reduced cloning efficiency and harbored higher levels of chromosomal damage, protein oxidation and lipid peroxidation. This correlated with decreased activity of antioxidant enzymes, inactivation of the redox-sensitive metabolic enzyme aconitase, and altered translation of proteins encoded by mitochondrial DNA. Together, the results demonstrate that the long-term consequences of the induced nontargeted effects greatly depend on the quality and dose of the radiation and involve persistent oxidative stress due to induced perturbations in oxidative metabolism. They are relevant to estimates of health risks from exposures to space radiation and the emergence of second malignancies after radiotherapy.

Increased frequency of spontaneous neoplastic transformation in progeny of bystander cells from cultures exposed to densely ionizing radiation.

An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10T½ mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P<0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in co-culture with cells irradiated with iron ions, but not with protons.

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.

Intercellular communication amplifies stressful effects in high-charge, high-energy (HZE) particle-irradiated human cells.

Gap junction intercellular communication/Cell killing/Potentially lethal damage repair/DNA damage/ Linear energy transfer of space radiation. Understanding the mechanisms that underlay the biological effects of particulate radiations is essential for space exploration and for radiotherap y. Here, we investigated the role of gap junction intercellular communication (GJIC) in modulating harmful effects induced in confluent cultures wherein most cells are traversed by one or more radiation tracks. We focused on the effect of radiation quality (linear energy transfer; LET) on junctional propagation of DNA damage and cell death among the irradiated cells. Confluent normal human fibroblasts were exposed to gr aded doses of 1 GeV protons (LET ~0.2 keV/μm) or 1 GeV/u iron ions (LET ~151 keV/μm) and were assayed for clonogenic survival and for micronucleus formation, a reflection of DNA damage, shortly after irradiation and following longer incubation periods. Iron ions were ~2.7 fold more effective than protons at killing 90% of the cells in the exposed cultures when assayed within 5–10 minutes after irradiation. When cells were held in the confluent state for several hours after irradiation, substantial potentially lethal damage repair (PLDR), coupled with a reduction in micronucleus formation, occurred in cells exposed to protons, but not in those exposed to iron ions. In fact, such confluent holding after exposure to a similarly toxic dose of iron ions enhanced the induced toxic effect. However, following iron ion irradiation, inhibition of GJIC by 18-α-glycyrrhetinic acid eliminated the enhanced toxicity and red uced micronucleus formation to levels below those detected in cells assayed shortly after irradiation. The data show that low-LET radiation induces strong PLDR within hours, but that high-LET radiation with similar immediate toxicity does not induce PLDR and its toxicity increases with time following irradiation. The results also show that GJIC among irradiated cells amplifies stressful effects following exposure to high-, but not low-LET radiation, and that GJIC has only minimal effect on cellular recovery following low-LET irradiation.

Health risks of space exploration: targeted and nontargeted oxidative injury by high-charge and high-energy particles.

SIGNIFICANCE During deep space travel, astronauts are often exposed to high atomic number (Z) and high-energy (E) (high charge and high energy [HZE]) particles. On interaction with cells, these particles cause severe oxidative injury and result in unique biological responses. When cell populations are exposed to low fluences of HZE particles, a significant fraction of the cells are not traversed by a primary radiation track, and yet, oxidative stress induced in the targeted cells may spread to nearby bystander cells. The long-term effects are more complex because the oxidative effects persist in progeny of the targeted and affected bystander cells, which promote genomic instability and may increase the risk of age-related cancer and degenerative diseases. RECENT ADVANCES Greater understanding of the spatial and temporal features of reactive oxygen species bursts along the tracks of HZE particles, and the availability of facilities that can simulate exposure to space radiations have supported the characterization of oxidative stress from targeted and nontargeted effects. CRITICAL ISSUES The significance of secondary radiations generated from the interaction of the primary HZE particles with biological material and the mitigating effects of antioxidants on various cellular injuries are central to understanding nontargeted effects and alleviating tissue injury. FUTURE DIRECTIONS Elucidation of the mechanisms underlying the cellular responses to HZE particles, particularly under reduced gravity and situations of exposure to additional radiations, such as protons, should be useful in reducing the uncertainty associated with current models for predicting long-term health risks of space radiation. These studies are also relevant to hadron therapy of cancer.

Germicidal Efficacy and Mammalian Skin Safety of 222-nm UV Light

We have previously shown that 207-nm ultraviolet (UV) light has similar antimicrobial properties as typical germicidal UV light (254 nm), but without inducing mammalian skin damage. The biophysical rationale is based on the limited penetration distance of 207-nm light in biological samples (e.g. stratum corneum) compared with that of 254-nm light. Here we extended our previous studies to 222-nm light and tested the hypothesis that there exists a narrow wavelength window in the far-UVC region, from around 200–222 nm, which is significantly harmful to bacteria, but without damaging cells in tissues. We used a krypton-chlorine (Kr-Cl) excimer lamp that produces 222-nm UV light with a bandpass filter to remove the lower- and higher-wavelength components. Relative to respective controls, we measured: 1. in vitro killing of methicillin-resistant Staphylococcus aureus (MRSA) as a function of UV fluence; 2. yields of the main UV-associated premutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) in a 3D human skin tissue model in vitro; 3. eight cellular and molecular skin damage endpoints in exposed hairless mice in vivo. Comparisons were made with results from a conventional 254-nm UV germicidal lamp used as positive control. We found that 222-nm light kills MRSA efficiently but, unlike conventional germicidal UV lamps (254 nm), it produces almost no premutagenic UV-associated DNA lesions in a 3D human skin model and it is not cytotoxic to exposed mammalian skin. As predicted by biophysical considerations and in agreement with our previous findings, far-UVC light in the range of 200–222 nm kills bacteria efficiently regardless of their drug-resistant proficiency, but without the skin damaging effects associated with conventional germicidal UV exposure.

A Mouse Ear Model for Bystander Studies Induced by Microbeam Irradiation.

Radiation-induced bystander effects have been observed in vitro and in cell and tissue culture models, however, there are few reported studies showing these effects in vivo. To our knowledge, this is the first reported study on bystander effects induced by microbeam irradiation in an intact living mammal. The mouse ear was used to investigate radiation-induced bystander effects in keratinocytes, utilizing a 3 MeV proton microbeam (LET 13.1 keV/μm) with a range in skin of about 135 μm. Using a custom-designed holder, the ear of an anesthetized C57BL/6J mouse was flattened by gentle suction and placed over the microbeam port to irradiate cells along a 35 μm wide, 6 mm long path. Immunohistochemical analysis of γ-H2AX foci formation in tissue sections revealed, compared to control tissue, proton-induced γ-H2AX foci formation in one of the two epidermal layers of the mouse ear. Strikingly, a higher number of cells than expected showed foci from direct irradiation effects. Although the proton-irradiated line was ~35 μm wide, the average width spanned by γ-H2AX-positive cells exceeded 150 μm. Cells adjacent to or in the epidermal layer opposite the γ-H2AX-positive region did not exhibit foci. These findings validate this mammalian model as a viable system for investigating radiation-induced bystander effects in an intact living organism.

Microbeam irradiation of C. elegans nematode in microfluidic channels

To perform high-throughput studies on the biological effects of ionizing radiation in vivo, we have implemented a microfluidic tool for microbeam irradiation of Caenorhabditis elegans. The device allows the immobilization of worms with minimal stress for a rapid and controlled microbeam irradiation of multiple samples in parallel. Adapted from an established design, our microfluidic clamp consists of 16 tapered channels with 10-μm-thin bottoms to ensure charged particle traversal. Worms are introduced into the microfluidic device through liquid flow between an inlet and an outlet, and the size of each microchannel guarantees that young adult worms are immobilized within minutes without the use of anesthesia. After site-specific irradiation with the microbeam, the worms can be released by reversing the flow direction in the clamp and collected for analysis of biological endpoints such as repair of radiation-induced DNA damage. For such studies, minimal sample manipulation and reduced use of drugs such as anesthetics that might interfere with normal physiological processes are preferable. By using our microfluidic device that allows simultaneous immobilization and imaging for irradiation of several whole living samples on a single clamp, here we show that 4.5-MeV proton microbeam irradiation induced DNA damage in wild-type C. elegans, as assessed by the formation of Rad51 foci that are essential for homologous repair of radiation-induced DNA damage.

Single-cell responses to ionizing radiation

While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of “housekeeping” genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories—those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.

Integrated interdisciplinary training in the radiological sciences

The radiation sciences are increasingly interdisciplinary, both from the research and the clinical perspectives. Beyond clinical and research issues, there are very real issues of communication between scientists from different disciplines. It follows that there is an increasing need for interdisciplinary training courses in the radiological sciences. Training courses are common in biomedical academic and clinical environments, but are typically targeted to scientists in specific technical fields. In the era of multidisciplinary biomedical science, there is a need for highly integrated multidisciplinary training courses that are designed for, and are useful to, scientists who are from a mix of very different academic fields and backgrounds. We briefly describe our experiences running such an integrated training course for researchers in the field of biomedical radiation microbeams, and draw some conclusions about how such interdisciplinary training courses can best function. These conclusions should be applicable to many other areas of the radiological sciences. In summary, we found that it is highly beneficial to keep the scientists from the different disciplines together. In practice, this means not segregating the training course into sections specifically for biologists and sections specifically for physicists and engineers, but rather keeping the students together to attend the same lectures and hands-on studies throughout the course. This structure added value to the learning experience not only in terms of the cross fertilization of information and ideas between scientists from the different disciplines, but also in terms of reinforcing some basic concepts for scientists in their own discipline.