Narongchai Autsavapromporn

The role of gap junction communication and oxidative stress in the propagation of toxic effects among high-dose α-particle-irradiated human cells.

Abstract We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.

Gap Junction Communication and the Propagation of Bystander Effects Induced by Microbeam Irradiation in Human Fibroblast Cultures: The Impact of Radiation Quality

Understanding the mechanisms underlying the bystander effects of low doses/low fluences of low- or high-linear energy transfer (LET) radiation is relevant to radiotherapy and radiation protection. Here, we investigated the role of gap-junction intercellular communication (GJIC) in the propagation of stressful effects in confluent normal human fibroblast cultures wherein only 0.036–0.144% of cells in the population were traversed by primary radiation tracks. Confluent cells were exposed to graded doses from monochromatic 5.35 keV X ray (LET ∼6 keV/μm), 18.3 MeV/u carbon ion (LET ∼103 keV/μm), 13 MeV/u neon ion (LET ∼380 keV/μm) or 11.5 MeV/u argon ion (LET ∼1,260 keV/μm) microbeams in the presence or absence of 18-α-glycyrrhetinic acid (AGA), an inhibitor of GJIC. After 4 h incubation at 37°C, the cells were subcultured and assayed for micronucleus (MN) formation. Micronuclei were induced in a greater fraction of cells than expected based on the fraction of cells targeted by primary radiation, and the effect occurred in a dose-dependent manner with any of the radiation sources. Interestingly, MN formation for the heavy-ion microbeam irradiation in the absence of AGA was higher than in its presence at high mean absorbed doses. In contrast, there were no significant differences in cell cultures exposed to X-ray microbeam irradiation in presence or absence of AGA. This showed that the inhibition of GJIC depressed the enhancement of MN formation in bystander cells from cultures exposed to high-LET radiation but not low-LET radiation. Bystander cells recipient of growth medium harvested from 5.35 keV X-irradiated cultures experienced stress manifested in the form of excess micronucleus formation. Together, the results support the involvement of both junctional communication and secreted factor(s) in the propagation of radiation-induced stress to bystander cells. They highlight the important role of radiation quality and dose in the observed effects.

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.

Human cell responses to ionizing radiation are differentially affected by the expressed connexins

In multicellular organisms, intercellular communication is essential for homeostatic functions and has a major role in tissue responses to stress. Here, we describe the effects of expression of different connexins, which form gap junction channels with different permeabilities, on the responses of human cells to ionizing radiation. Exposure of confluent HeLa cell cultures to 137Cs γ rays, 3.7 MeV α particles, 1000 MeV protons or 1000 MeV/u iron ions resulted in distinct effects when the cells expressed gap junction channels composed of either connexin26 (Cx26) or connexin32 (Cx32). Irradiated HeLa cells expressing Cx26 generally showed decreased clonogenic survival and reduced metabolic activity relative to parental cells lacking gap junction communication. In contrast, irradiated HeLa cells expressing Cx32 generally showed enhanced survival and greater metabolic activity relative to the control cells. The effects on clonogenic survival correlated more strongly with effects on metabolic activity than with DNA damage as assessed by micronucleus formation. The data also showed that the ability of a connexin to affect clonogenic survival following ionizing radiation can depend on the specific type of radiation. Together, these findings show that specific types of connexin channels are targets that may be exploited to enhance radiotherapeutic efficacy and to formulate countermeasures to the harmful effects of specific types of ionizing radiation.

Genetic changes in progeny of bystander human fibroblasts after microbeam irradiation with X-rays, protons or carbon ions: The relevance to cancer risk

Abstract Purpose: Radiation-induced bystander effects have important implications in radiotherapy. Their persistence in normal cells may contribute to risk of health hazards, including cancer. This study investigates the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of harmful effects in progeny of bystander cells. Materials and methods: Confluent human skin fibroblasts were exposed to microbeam radiations with different linear energy transfer (LET) at mean absorbed doses of 0.4 Gy by which 0.036–0.4% of the cells were directly targeted by radiation. Following 20 population doublings, the cells were harvested and assayed for micronucleus formation, gene mutation and protein oxidation. Results: Our results showed that expression of stressful effects in the progeny of bystander cells is dependent on LET. The progeny of bystander cells exposed to X-rays (LET ∼6 keV/μm) or protons (LET ∼11 keV/μm) showed persistent oxidative stress, which correlated with increased micronucleus formation and mutation at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus. Such effects were not observed after irradiation by carbon ions (LET ∼103 keV/μm). Interestingly, progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultures were exposed to X-rays. Conclusions: These findings suggest that cellular exposure to proton and heavy charged particle with LET properties similar to those used here can reduce the risk of lesions associated with cancer. The ability of cells to communicate via gap junctions at the time of irradiation appears to impact residual damage in progeny of bystander cells.

Participation of gap junction communication in potentially lethal damage repair and DNA damage in human fibroblasts exposed to low- or high-LET radiation.

Existing research has not fully explained how different types of ionizing radiation (IR) modulate the responses of cell populations or tissues. In our previous work, we showed that gap junction intercellular communication (GJIC) mediates the propagation of stressful effects among irradiated cells exposed to high linear energy transfer (LET) radiations, in which almost every cells is traversed by an IR track. In the present study, we conducted an in-depth study of the role of GJIC in modulating the repair of potentially lethal damage (PLDR) and micronuclei formation in cells exposed to low- or high-LET IR. Confluent human fibroblasts were exposed in the presence or absence of a gap junction inhibitor to 200kV X rays (LET∼1.7keV/μm), carbon ions (LET∼76keV/μm), silicon ions (LET∼113keV/μm) or iron ions (LET∼400keV/μm) that resulted in isosurvival levels. The fibroblasts were incubated for various times at 37°C. As expected, high-LET IR were more effective than were low-LET X rays at killing cells and damaging DNA shortly after irradiation. However, when cells were held in a confluent state for several hours, PLDR associated with a reduction in DNA damage, occurred only in cells exposed to X rays. Interestingly, inhibition of GJIC eliminated the enhancement of toxic effects, which resulted in an increase of cell survival and reduction in the level of micronucleus formation in cells exposed to high, but not in those exposed to low-LET IR. The experiment shows that gap-junction communication plays an important role in the propagation of stressful effects among irradiated cells exposed to high-LET IR while GJIC has only a minimal effect on PLDR and DNA damage following low-LET irradiation. Together, our results show that PLDR and induction of DNA damage clearly depend on gap-junction communication and radiation quality.

Impact of Co-Culturing with Fractionated Carbon-Ion-Irradiated Cancer Cells on Bystander Normal Cells and Their Progeny

The purpose of this study was to compare the biological effects of fractionated doses versus a single dose of high-LET carbon ions in bystander normal cells, and determine the effect on their progeny using the layered tissue co-culture system. Briefly, confluent human glioblastoma (T98G) cells received a single dose of 6 Gy or three daily doses of 2 Gy carbon ions, which were then seeded on top of an insert with bystander normal skin fibroblasts (NB1RGB) growing underneath. Cells were co-cultured for 6 h or allowed to grow for 20 population doublings, then harvested and assayed for different end points. A single dose of carbon ions resulted in less damage in bystander normal NB1RGB cells than the fractionated doses. In contrast, the progeny of bystander NB1RGB cells co-cultured with T98G cells exposed to fractionated doses showed less damage than progeny from bystander cells co-cultured with single dose glioblastoma cells. Furthermore, inhibition of gap junction communication demonstrated its involvement in the stressful effects in bystander cells and their progeny. These results indicate that dose fractionation reduced the late effect of carbon-ion exposure in the progeny of bystander cells compared to the effect in the initial bystander cells.

Bystander effect and genomic instability in human cells and their progeny after irradiation with X rays, protons or carbon ions: role of gap junction communication

Purpose: Ionizing Radiation (IR)-induced bystander effects and genomic instability have important implication in radiotherapy and radioprotection. Their persistence in the progeny of normal cells may contribute to risk of long-term radiation-related health effect in human, including cancer. Hence, this study investigates the role of gap junction intercellular communication (GJIC) and the quality of radiation in the propagation of stressful effects in the unirradiated bystander cells and their progeny. Material and methods: Human skin fibroblasts in the confluent state were exposed to microbeam irradiations with different linear energy transfer (LET) at mean absorbed dose of 0.4 Gy, in the presence or absence of GJIC inhibitor (AGA) by which 0.036-0.4% of cells were directly targeted by IR. After 4 h irradiation or following 20 population doublings, the cells were harvested and assayed for micronucleus (MN) formation, gene mutation and protein oxidation. Results: Our results showed that high-LET carbon microbeams (LET ∼103 keV/μm) and high-LET proton microbeams (LET ∼11 keV/μm) were more effective than low-LET X ray microbeam (LET ∼6 keV/μm) in the induction of DNA damage (MN formation) in bystander cells. Interestingly, significant attenuation of MN formation occurred in bystander cells in the presence of AGA after proton and carbon microbeams. In contrast, incubation of the cells with AGA did not significantly affect the induction of MN formation in bystander cells during confluent holding after X irradiation. Further, the progeny of bystander cells exposed to X rays or protons showed persistent oxidative stress which correlated MN formation and mutation frequency. Such effects were not observed after irradiation by carbon ions. Importantly, the progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultured were exposed to X rays. Taken together, the overall results show the expression of stressful effects in the bystander cells and their progeny are dependent on the radiation quality or LET. Conclusions: Our findings suggest that the involvement of GJIC-dependent of radiation quality in the propagation of radiation induces stressful effects to bystander cells and their progeny. In addition, this work provides a strong support to the fact that carbon ions can significantly reduce the risk of cancer and have potential implications in the therapeutic outcome of radiotherapy compared to X rays or protons. Citation Format: Narongchai Autsavapromporn, Ianik Plante, Cuihua Liu, Teruaki Konishi, Noriko Usami, Tomoo Funayama, Yukio Uchihori, Tom K. Hei, Edouard I. Azzam, Sirikan Yamada, Takeshi Murakami, Masao Suzuki. Bystander effect and genomic instability in human cells and their progeny after irradiation with X rays, protons or carbon ions: role of gap junction communication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2015-1815

Radiation-quality-dependent bystander effects induced by the microbeams with different radiation sources

A central paradigm in radiation biology has been that only cells ‘hit’ by a track of radiation would be affected to induce radiobiological consequences, and cells ‘not hit’ should not be. This is the basis of the current system for risk estimation of radiobiological effects. However, it has recently been challenged by so-called non-targeted effects, such as bystander effect, and such radiation-induced cellular responses may have important implications for risk evaluation of low-dose-rate radiations as well as in tumor radiotherapy. Our group has been studying radiation-quality bystander cellular effects using the microbeams with different radiation sources. It is essentially important for evaluating risk such a low-dose-rate exposure as the accident of Fukushima Daiichi Nuclear Power Plants to examine bystander effects induced by low-LET electromagnetic radiations, such as X or gamma rays. We have been studying the cellular responses in normal human fibroblasts by targeted cell nucleus irradiations with monochromatic X-ray microbeams (5.35 keV) produced by Photon Factory in High Energy Accelerator Research Organization. The results indicated that the bystander effect in cell- killing effect was observed in the targeted cell nucleus irradiation, not in the random irradiation containing both cell nucleus and cytoplasm by Poisson distribution. The results suggest that energy deposition in cytoplasm is an important role of inducing bystander effects in case of low-LET radiations. We have also been investigating high-LET-radiation induced bystander effects using the heavy-ion microbeams at Takasaki Ion Accelerators for Advanced Radiation Application in Japan Atomic Energy Agency. Only 0.04% of the total numbers of normal human fibroblasts were irradiated with C-ion (220 MeV), Ne-ion (260 MeV) and Ar-ion (460 MeV) microbeams collimated at 20 μm in diameter. Cell-killing effect and gene mutation at HPRT locus in the cells irradiated with C ions were higher beyond our expectations and returned the estimated values that only 0.04% of the total cells were irradiated when using the specific inhibitor of gap junctions. On the other hand, no induced biological effects were observed in Ne and Ar ions whether the inhibitor was applied or not. The result suggested that the C-ion microbeam was capable of inducing bystander cellular effects via gap junction-mediated cell-cell communication. There is clear evidence that bystander cellular effects are dependent on radiation quality. It is also important for highly developed heavy-ion radiotherapy to identify bystander effects induced by spatially low-fluence irradiations with heavy-ion beams. We have been investigating the biological effects using human tumor cell lines. The results clearly showed that bystander effects were observed in the carbon-ion irradiation but not in other ions as well as the effects in normal fibroblasts. Furthermore, the bystander cell-killing effect in tumor cell lines was strongly induced in the cells harboring wild-type P53 not in mutated-type P53 cells. The results provide the important implication for a tailor-made therapy using carbon ions.