It has been reported that DNA double‐strand breaks (DSB) can be induced by cytoplasm irradiation, and that both reactive free radicals and mitochondria are involved in DSB formation. However, the ...cellular antioxidative responses that are stimulated and the biological consequences of cytoplasmic irradiation remain unknown. Using the Single Particle Irradiation system to Cell (SPICE) proton microbeam facility at the National Institute of Radiological Sciences (NIRS Japan), the response of nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) antioxidative signaling to cytoplasmic irradiation was studied in normal human lung fibroblast WI‐38 cells. Cytoplasmic irradiation stimulated the localization of NRF2 to the nucleus and the expression of its target protein, heme oxygenase 1. Activation of NRF2 by tert‐butylhydroquinone mitigated the levels of DSB induced by cytoplasmic irradiation. Mitochondrial fragmentation was also promoted by cytoplasmic irradiation, and treatment with mitochondrial division inhibitor 1 (Mdivi‐1) suppressed cytoplasmic irradiation‐induced NRF2 activation and aggravated DSB formation. Furthermore, p53 contributed to the induction of mitochondrial fragmentation and activation of NRF2, although the expression of p53 was significantly downregulated by cytoplasmic irradiation. Finally, mitochondrial superoxide (MitoSOX) production was enhanced under cytoplasmic irradiation, and use of the MitoSOX scavenger mitoTEMPOL indicated that MitoSOX caused alterations in p53 expression, mitochondrial dynamics, and NRF2 activation. Overall, NRF2 antioxidative response is suggested to play a key role against genomic DNA damage under cytoplasmic irradiation. Additionally, the upstream regulators of NRF2 provide new clues on cytoplasmic irradiation‐induced biological processes and prevention of radiation risks.
Only the cytoplasm was targeted by microbeam, and cytoplasmic irradiation stimulated NRF2 antioxidative response for protection against cytoplasmic irradiation‐induced DNA damage. Also, p53 was found to promote mitochondrial fragmentation, which led to activating the NRF2 antioxidative response. Elevation of mitochondrial superoxide was an elicitor for NRF2 activation, changes in p53 level, and mitochondrial morphology.
Abstract
Radiation cancer therapy with ultra-high dose rate exposure, so called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response. The aim of this study ...was to clarify whether FLASH exposure of proton beam would be effective in reducing the DNA strand break induction. We applied a simple model system, pBR322 plasmid DNA in aqueous 1 × TE solution, where DNA single strand breaks (SSBs) and double strand breaks (DSBs) can be precisely quantified by gel electrophoresis. Plasmid DNA were exposed to 27.5 MeV protons in the conventional dose rate of 0.05 Gy/s (CONV) and ultra-high dose rate of 40 Gy/s (FLASH). With both dose rate, the kinetics of the SSB and DSB induction were proportional to absorbed dose. The SSB induction of FLASH was significantly less than CONV, which were 8.79 ± 0.14 (10−3 SSB per Gy per molecule) and 10.8 ± 0.68 (10−3 SSB per Gy per molecule), respectively. The DSB induction of FLASH was also slightly less than CONV, but difference was not significant. Altogether, 27.5 MeV proton beam at 40 Gy/s reduced SSB and not DSB, thus its effect may not be significant in reducing lethal DNA damage that become apparent in acute radiation effect.
FLASH radiotherapy appears to kill off tumor cells while sparing healthy tissues, by irradiation at ultra high dose rate (>40 Gy s
−1
). The present study aims to clarify the mechanism of the sparing ...effect by proton irradiation under the FLASH conditions from a viewpoint of radiation chemistry. To do so, we evaluate radiation chemical yields (
G
values) of 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA), which is produced by water radiolysis using coumarin-3-carboxylic acid (C3CA) solution as a radical scavenger of hydroxyl radicals. We shoot 27.5 MeV protons in the dose rate ranging from 0.05 to 160 Gy s
−1
. The recombination process of hydroxyl radicals produced is followed by varying the concentration of C3CA from 0.2 to 20 mM, which corresponds to the scavenging time scale from 7.1 to 714 ns. The
G
value of 7OH-C3CA produced decreases with increasing dose rate on the same scavenging time scale. Additionally, the trend of the relative
G
value normalized at a scavenging time scale of 100 ns, where radical-radical reaction subsides, is consistent in the examined dose rate range. This finding implies that
G
values of 7OH-C3CA produced reduce with increasing dose rate due to the oxygen depletion. We experimentally present that the sparing effect for healthy tissues would be seen even with a proton beam under the FLASH conditions due to the depletion of oxygen.
Yield of 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA) significantly decreases at FLASH condition with the dose rate of >40 Gy s
−1
, compared to that at conventional condition of 0.05 Gy s
−1
, due to the oxygen depletion in the solution.
It is well known that mitochondria and the endoplasmic reticulum (ER) play important roles in radiation response, but their functions in radiation-induced bystander effect (RIBE) are largely unclear. ...In this study, we found that when a small portion of cells in a population of human lung fibroblast MRC-5 cells were precisely irradiated through either the nuclei or cytoplasm with counted microbeam protons, the yield of micronuclei (MN) and the levels of intracellular reactive oxygen species (ROS) in nonirradiated cells neighboring irradiated cells were significantly increased. Mito/ER-tracker staining demonstrated that the mitochondria were clearly activated after nuclear irradiation and ER mass approached a higher level after cytoplasmic irradiation. Moreover, the radiation-induced ROS was diminished by rotenone, an inhibitor of mitochondria activation, but it was not influenced by siRNA interference of
, an ER regulation protein. While for nuclear irradiation, rotenone-enhanced radiation-induced ER expression, and
siRNA eliminated radiation-induced activation of mitochondria, these phenomena were not observed for cytoplasmic irradiation. Bystander MN was reduced by rotenone but enhanced by
siRNA. When the cells were treated with both rotenone and
siRNA, the MN yield was reduced for nuclear irradiation but was enhanced for cytoplasmic irradiation. Our results suggest that the organelles of mitochondria and ER have different roles in RIBE with respect to nuclear and cytoplasmic irradiation, and the function of ER is a prerequisite for mitochondrial activation.
We have been studying the effectiveness of direct action, which induces clustered DNA damage leading to cell killing, relative to indirect action. Here a new criterion Direct Ation-Based Biological ...Effectiveness (DABBLE) is proposed to understand the contribution of direct action for cell killing induced by C ions. DABBLE is defined as the ratio of direct action to indirect action. To derive this ratio, we describe survival curves of mammalian cells as a function of the number of OH radicals produced 1 ps and 100 ns after irradiation, instead of the absorbed dose. By comparing values on the vertical axis of the survival curves at a certain number of OH radicals produced, we successfully discriminate the contribution of direct action induced by C ions from that of indirect action. DABBLE increases monotonically with increasing linear energy transfer (LET) up to 140 keV/μm and then drops, when the survival curves are described by the number of OH radicals 1 ps after irradiation. The trend of DABBLE is in agreement with that of relative biological effectiveness (RBE) of indirect action. In comparison, the value of DABBLE increases monotonically with LET, when the survival curves are described by the number of OH radicals 100 ns after irradiation. This finding implies that the effectiveness of C ion therapy for cancer depends on the contribution of direct action and we can follow the contribution of direct action over time in the chemical phase.
This study aimed to determine whether the radiation-induced bystander effect (RIBE) is affected by radiation quality. To mimic the different radiation qualities of the direct action (D)/indirect ...action (ID) ratio, A549 cells were exposed to X-rays, with either 100 mM of the radical scavenger, thio-urea (TU+), or null (TU-). Biological responses in irradiated and bystander cells were compared at equal lethal effects of a 6% survival dose, which was estimated from the survival curves to be 8 Gy and 5 Gy for TU+ and TU-, respectively. Cyclooxygenase-2 (COX-2) expression in TU- irradiated cells increased up to 8 h post-irradiation, before decreasing towards 24 h. The concentration of prostaglandin E2 (PGE2), a primary product of COX-2 and known as a secreted inducible factor in RIBE, increased over 3-fold compared with that in the control at 8 h post-irradiation. Conversely, COX-2 expression and PGE2 production of TU+ irradiated cells were drastically suppressed. These results show that the larger D/ID suppressed COX-2 expression and PGE2 production in irradiated cells. However, in contrast to the case in the irradiated cells, COX-2 expression was equally observed in the TU- and TU+ co-cultured bystander cells, which showed the highest expression levels at 24 h post-irradiation. Taken together, these findings demonstrate that radiation quality, such as the D/ID ratio, may be an important factor in the alteration of signalling pathways involved in RIBE.
Radiation cancer therapy with ultra-high dose rate (UHDR) exposure, so-called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response to therapy. The aim of ...this study was to clarify whether a 59.5 MeV proton beam at an UHDR of 48.6 Gy/s could effectively reduce the DNA damage of pBR322 plasmid DNA in solution compared to the conventional dose rate (CONV) of 0.057 Gy/s.
A simple system, consisting of pBR322 plasmid DNA in 1× Tris-EDTA buffer, was initially employed for proton beam exposure. We then used formamidopyrimidine-DNA glycosylase (Fpg) enzymes. which convert oxidative base damages of oxidized purines to DNA strand breaks, to quantify DNA single strand breaks (SSBs) and double strand breaks (DSBs) by agarose gel electrophoresis.
Our findings showed that the SSB induction rate (SSB per plasmid DNA/Gy) at UHDR and the induction of Fpg enzyme sensitive sites (ESS) were significantly reduced in UHDR compared to CONV. However, there was no significant difference in DSB induction and non-DSB cluster damages.
UHDR of a 59.5 MeV proton beam could reduce non-clustered, non-DSB damages, such as SSB and sparsely distributed ESS. However, this effect may not be significant in reducing lethal DNA damage that becomes apparent only in acute radiation effects of mammalian cells and in vivo studies.
It has been reported that in cells exposed to low-dose radiation, radio-adaptive responses can be induced which make irradiated cells refractory to subsequent high-dose irradiation. However, whether ...adaptive responses are possible when only the cytoplasm, not the nucleus, of the cell is exposed to radiation is still unclear. In this study, using the proton microbeam facility at the National Institute of Radiological Sciences (Japan), we found that cytoplasmic irradiation activates radio-adaptive responses in normal human lung fibroblast WI-38 cells. Our results showed that when cells received cytoplasmic irradiation with 500 protons prior to 2 Gy or 6 Gy X-ray broad-beam irradiation, the DNA double-strand break levels were significantly reduced. In contrast, at cytoplasmic irradiation with less than 100 protons, the radio-adaptive response was not detected. Moreover, the time interval between cytoplasmic irradiation and whole-cell X-ray irradiation should be longer than 6 h for the induction of adaptive responses. In addition, cytoplasmic irradiation elevated the level of cellular mitochondrial superoxide, which enhanced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2) and its mediated nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (NRF2). This signaling pathway contributed to cytoplasmic irradiation-induced adaptive response as supported by the observations that treatment with the mitochondrial superoxide scavenger mito-tempol, ERK 1/2 inhibitor U0126 or NRF2 inhibitor ML385 could repress the adaptive response. Overall, we showed that cytoplasmic irradiation induces radio-adaptive responses and that mitochondrial superoxide/ERK 1/2/NRF2 signaling is a mechanism. Our results provide new information on the biological effects induced by cytoplasm-targeted irradiation.
Nuclear factor (erythroid-derived 2)-like 2 (NRF2), well-known as a master antioxidative response regulator in mammalian cells, is considered as a potential target for radiation protection and cancer ...therapy sensitization. We examined the response of NRF2 signaling in normal human lung fibroblast WI-38 cells to nucleus targeted irradiation by 3.4 MeV proton microbeam. Nucleus targeted irradiation stimulated the nucleus accumulation of NRF2 and the expression of its target gene, heme oxygenase 1 (HO-1). The nucleus accumulation of NRF2 increased from 3 h to 12 h post 500 proton irradiation. In the 500 protons range, higher number of protons resulted in increased NRF2 nucleus accumulation. Activating NRF2 with
-butylhydroquinone reduced DNA double-strand break (DSB) formation in nucleus targeted irradiation by 15%. Moreover, ATM phosphorylation was found in nucleus targeted irradiation. Inhibiting ATM with ku55933 prevented NRF2 nucleus accumulation. Furthermore, nucleus targeted irradiation activated ERK 1/2, and ROS-ERK 1/2 signaling regulated NRF2 nucleus accumulation. Taken together, NRF2 signaling was activated by nucleus targeted irradiation and mitigated DNA DSB. The discovery of ATM and ERK 1/2 as upstream regulators of NRF2 signaling in nucleus targeted cells revealed new information regarding radiation protection.
We proposed a depth-intensity curve discrimination (DCD) method for assessing absorbed dose contributions of hydroxyl radicals (HO·) due to thermal neutrons, fast neutrons, and gamma rays in ...accelerator-based mixed radiation fields, that are relevant to boron neutron capture therapy (BNCT). The HO· yields of thermal neutrons, fast neutrons, and gamma-rays measured by coumarin-3-carboxylic acid solutions were discriminately evaluated using depth-intensity curves of their radiations and maximum likelihood-expectation maximization algorithm. The discrimination accuracies of the three types of radiation were 2.8%, 4.2%, and 1.0%, respectively. We demonstrated that the DCD method is appropriate to determine absorbed dose contributions of HO· in complex accelerator-based neutron fields for BNCT.
•We developed the depth-intensity curve discrimination (DCD) method.•HO· yields of thermal neutrons, fast neutrons, and gamma rays were evaluated with DCD method.•The discrimination accuracies were more than 95% in each radiation.•DCD method is useful for assessing HO· based absorbed dose in accelerator-based neutron field.
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