Opposite Roles for p38MAPK-Driven Responses and Reactive Oxygen Species in the Persistence and Resolution of Radiation-Induced Genomic Instability

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as ⁵⁶Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a threshold-like response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.     

The effect of carbon irradiation is associated with greater oxidative stress in mouse intestine and colon relative to γ-rays

Carbon irradiation due to its higher biological effectiveness relative to photon radiation is a concern for toxicity to proliferative normal gastrointestinal (GI) tissue after radiotherapy and long-duration space missions such as mission to Mars. Although radiation-induced oxidative stress is linked to chronic diseases such as cancer, effects of carbon irradiation on normal GI tissue have not been fully understood. This study assessed and compared chronic oxidative stress in mouse intestine and colon after different doses of carbon and γ radiation, which are qualitatively different. Mice (C57BL/6J) were exposed to 0.5 or 1.3?Gy of γ or carbon irradiation, and intestinal and colonic tissues were collected 2 months after irradiation. While part of the tissues was used for isolating epithelial cells, tissue samples were also fixed and paraffin embedded for 4 µm thick sections as well as frozen for biochemical assays. In isolated epithelial cells, reactive oxygen species and mitochondrial status were studied using fluorescent probes and flow cytometry. We assessed antioxidant enzymes and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in tissues and formalin-fixed tissue sections were stained for 4-hydroxynonenal, a lipid peroxidation marker. Data show that mitochondrial deregulation, increased NADPH oxidase activity, and decreased antioxidant activity were major contributors to carbon radiation-induced oxidative stress in mouse intestinal and colonic cells. When considered along with higher lipid peroxidation after carbon irradiation relative to γ-rays, our data have implications for functional changes in intestine and carcinogenesis in colon after carbon radiotherapy as well as space travel.     

The effect of alpha-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. alpha-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of gamma-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP+ +NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.     

Induction and processing of oxidative clustered DNA lesions in 56Fe-ion-irradiated human monocytes

Space and cosmic radiation is characterized by energetic heavy ions of high linear energy transfer (LET). Although both low- and high-LET radiations can create oxidative clustered DNA lesions and double-strand breaks (DSBs), the local complexity of oxidative clustered DNA lesions tends to increase with increasing LET. We irradiated 28SC human monocytes with doses from 0-10 Gy of (56)Fe ions (1.046 GeV/ nucleon, LET = 148 keV/microm) and determined the induction and processing of prompt DSBs and oxidative clustered DNA lesions using pulsed-field gel electrophoresis (PFGE) and Number Average Length Analysis (NALA). The (56)Fe ions produced decreased yields of DSBs (10.9 DSB Gy(-1) Gbp(-1)) and clusters (1 DSB: approximately 0.8 Fpg clusters: approximately 0.7 Endo III clusters: approximately 0.5 Endo IV clusters) compared to previous results with (137)Cs gamma rays. The difference in the relative biological effectiveness (RBE) of the measured and predicted DSB yields may be due to the formation of spatially correlated DSBs (regionally multiply damaged sites) which result in small DNA fragments that are difficult to detect with the PFGE assay. The processing data suggest enhanced difficulty compared with gamma rays in the processing of DSBs but not clusters. At the same time, apoptosis is increased compared to that seen with gamma rays. The enhanced levels of apoptosis observed after exposure to (56)Fe ions may be due to the elimination of cells carrying high levels of persistent DNA clusters that are removed only by cell death and/or "splitting" during DNA replication.     

The effect of α-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP⁺+NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.     

Total-body irradiation with high-LET particles: acute and chronic effects on the immune system

Although the immune system is highly susceptible to radiation-induced damage, consequences of high linear energy transfer (LET) radiation remain unclear. This study evaluated the effects of 0.1 gray (Gy), 0.5 Gy, and 2.0 Gy iron ion (56Fe(26)) radiation on lymphoid cells and organs of C57BL/6 mice on days 4 and 113 after whole body exposure; a group irradiated with 2.0 Gy silicon ions (28Si) was euthanized on day 113. On day 4 after 56Fe irradiation, dose-dependent decreases were noted in spleen and thymus masses and all major leukocyte populations in blood and spleen. The CD19(+) B lymphocytes were most radiosensitive and NK1.1(+) natural killer (NK) cells were most resistant. CD3(+) T cells were moderately radiosensitive and a greater loss of CD3(+)/CD8(+) T(C) cells than CD3(+)/CD4(+) T(H) cells was noted. Basal DNA synthesis was elevated on day 4, but response to mitogens and secretion of interleukin-2 and tumor necrosis factor-alpha were unaffected. Signs of anemia were noted. By day 113, high B cell numbers and low T(C) cell and monocyte percents were found in the 2.0 Gy 56Fe group; the 2.0 Gy 2)Si mice had low NK cells, decreased basal DNA synthesis, and a somewhat increased response to two mitogens. Collectively, the data show that lymphoid cells and tissues are markedly affected by high linear energy transfer (LET) radiation at relatively low doses, that some aberrations persist long after exposure, and that different consequences may be induced by various densely ionizing particles. Thus simultaneous exposure to multiple radiation sources could lead to a broader spectrum of immune dysfunction than currently anticipated.     

Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability.     

Heavy Ion Radiation Exposure Triggered Higher Intestinal Tumor Frequency and Greater β-Catenin Activation than γ Radiation in APCMin/+ Mice

Risk of colorectal cancer (CRC) after exposure to low linear energy transfer (low-LET) radiation such as γ-ray is highlighted by the studies in atom bomb survivors. On the contrary, CRC risk prediction after exposure to high-LET cosmic heavy ion radiation exposure is hindered due to scarcity of in vivo data. Therefore, intestinal tumor frequency, size, cluster, and grade were studied in APC^Min/+ mice (n = 20 per group; 6 to 8 wks old; female) 100 to 110 days after exposure to 1.6 or 4 Gy of heavy ion ⁵⁶Fe radiation (energy: 1000 MeV/nucleon) and results were compared to γ radiation doses of 2 or 5 Gy, which are equitoxic to 1.6 and 4 Gy ⁵⁶Fe respectively. Due to relevance of lower doses to radiotherapy treatment fractions and space exploration, we followed 2 Gy γ and equitoxic 1.6 Gy ⁵⁶Fe for comparative analysis of intestinal epithelial cell (IEC) proliferation, differentiation, and β-catenin signaling pathway alterations between the two radiation types using immunoblot, and immunohistochemistry. Relative to controls and γ-ray, intestinal tumor frequency and grade was significantly higher after ⁵⁶Fe radiation. Additionally, tumor incidence per unit of radiation (per cGy) was also higher after ⁵⁶Fe radiation relative to γ radiation. Staining for phospho-histone H3, indicative of IEC proliferation, was more and alcian blue staining, indicative of IEC differentiation, was less in ⁵⁶Fe than γ irradiated samples. Activation of β-catenin was more in ⁵⁶Fe-irradiated tumor-free and tumor-bearing areas of the intestinal tissues. When considered along with higher levels of cyclin D1, we infer that relative to γ radiation exposure to ⁵⁶Fe radiation induced markedly reduced differentiation, and increased proliferative index in IEC resulting in increased intestinal tumors of larger size and grade due to preferentially greater activation of β-catenin and its downstream effectors.     

MiR-21 is continually elevated long-term in the brain after exposure to ionizing radiation

Ionizing radiation stimulates miR-21 expression in different types of mammalian cells in culture. However, it remains unclear whether radiation could stimulate miR-21 expression in brain cells and tissue and, if so, how long the upregulation of miR-21 would be maintained after exposure to different types of radiation. To answer these questions, we examined the miR-21 levels in irradiated mouse hippocampal cells and brain tissue from mice at different times up to 1 year after whole-body exposure to 0.5 Gy of X rays [low linear energy transfer (LET)] or (56)Fe ions (high LET). The results showed that radiation stimulated miR-21 expression in mouse hippocampal cells and upregulation of EGFR, which is similar to that in human hepatocytes, as we reported previously. Interestingly, the miR-21 levels gradually increased within 1 year after irradiation, although there was no significant difference in the miR-21 low- and high-LET irradiated mice. The high expression of miR-21 in the brain was also associated with high expression of EGFR in irradiated mice; thus our data strongly support that EGFR and miR-21 are in a positive regulatory loop, because it is known that radiation stimulates miR-21 through the EGFR/Stat3 pathway and miR-21 activates the EGFR pathway. Since the brain is relatively resistant to radiation-induced histomorphological changes, our findings may provide a new way to explore radiation-induced pathological changes in the brain by investigating miR-21 and its multiple targets.     

Redox changes induced in hippocampal precursor cells by heavy ion irradiation

Hippocampal precursors retain the capacity to proliferate and differentiate throughout life, and their progeny, immature neurons, can undergo neurogenesis, a process believed to be important in maintaining the cognitive health of an organism. A variety of stresses including irradiation have been shown to deplete neural precursor cells, an effect that inhibits neurogenesis and is associated with the onset of cognitive impairments. Our past work has shown that neural precursor cells exposed to X-rays or protons exhibit a prolonged increase in oxidative stress, a factor we hypothesize to be critical in regulating the function of these cells after irradiation and other stresses. Here we report that irradiation of hippocampal precursor cells with high-linear energy transfer (LET) 1 GeV/nucleon ⁵⁶Fe ions leads to significantly higher levels of oxidative stress when compared to lower LET radiations (X-rays, protons). Irradiation with 1 Gy of ⁵⁶Fe ions elicits twofold to fivefold higher levels of reactive oxygen species (ROS) compared to unirradiated controls, and at lower doses (≤1 Gy) neural precursors exhibit a linear dose response 6 h after heavy ion exposure. The use of the antioxidant lipoic acid (LA) was able to reduce ROS levels below background levels when added before or after ⁵⁶Fe ion irradiation. These results conclusively show that low doses of ⁵⁶Fe ions can elicit significant levels of oxidative stress in neural precursor cells. Given the prevalence of heavy ions in space and the duration of interplanetary travel, these data suggest that astronauts are at risk for developing cognitive decrements. However, our results also indicate that antioxidants delivered before as radioprotective agents or after as mitigating agents hold promise as effective countermeasures for ameliorating certain adverse effects of heavy ion exposure to the CNS.     

Effects of radiation quality on interactions between oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation damages DNA and also induces oxidative stress, which can affect the function of proteins involved in DNA repair, thereby causing repair of DNA damage to become less efficient. We previously developed a mathematical model of this potentially synergistic relationship and applied it to γ-ray exposure data on the radiation-resistant prokaryote Deinococcus radiodurans. Here, we investigate the effects of radiation quality on these processes by applying the model to data on exposures of D. radiodurans to heavy ions with linear energy transfer (LET) of 18.5–11,300 keV/μm. The model adequately describes these data using three parameters combinations: radiogenic DNA damage induction, repair protein inactivation and cellular repair capacity. Although statistical uncertainties around best-fit parameter estimates are substantial, the behaviors of model parameters are consistent with current knowledge of LET effects: inactivation cross-sections for both DNA and proteins increase with increasing LET; DNA damage yield per unit of radiation dose also increases with LET; protein damage per unit dose tends to decrease with LET; DNA and especially protein damage yields are reduced when cells are irradiated in the dry state. These results suggest that synergism between oxidative stress and DNA damage may play an important role not only during γ-ray exposure, but during high-LET radiation exposure as well.     

Detection of oxidative stress induced by low- and high-linear energy transfer radiation in cultured human epithelial cells

A standardized dichlorofluorescin (DCF) fluorometric assay capable of measuring radiation-induced oxidative stress was used to determine the effectiveness of protons and high-mass, high-atomic number (Z) and high-energy (HZE) particles to produce oxidative stress in vitro. Protons were found to be about equally as effective as X rays in the generation of oxidative stress in cultured cells. However, 56Fe-ion beams with energies of 1 GeV/nucleon and 5 GeV/nucleon were less effective than X rays or gamma rays in inducing dichlorofluorescin (DCFH) oxidation. The relatively lower slope values for the dose responses of HZE-particle radiation-induced DCFH oxidation indicate that the sensitivity of the DCF fluorometric assay is probably dependent on the linear energy transfer (LET) of the radiation beam.     

A comparison of radiation-induced mitochondrial damage between neural progenitor stem cells and differentiated cells

Mitochondria play a key role in maintaining cellular homeostasis during stress responses, and mitochondrial dysfunction contributes to carcinogenesis, aging, and neurologic disease. We here investigated ionizing radiation (IR)-induced mitochondrial damage in human neural progenitor stem cells (NSCs), their differentiated counterparts and human normal fibroblasts. Long-term fractionated radiation (FR) with low doses of X-rays for 31 d enhanced mitochondrial activity as evident by elevated mitochondrial membrane potential (ΔΨm) and mitochondrial complex IV (cytochrome c oxidase) activity to fill the energy demands for the chronic DNA damage response in differentiated cells. Subsequent reduction of the antioxidant glutathione via continuous activation of mitochondrial oxidative phosphorylation caused oxidative stress and genomic instability in differentiated cells exposed to long-term FR. In contrast, long-term FR had no effect on the mitochondrial activity in NSCs. This cell type showed efficient DNA repair, no mitochondrial damage, and resistance to long-term FR. After high doses of acute single radiation (SR) (> 5 Gy), cell cycle arrest at the G2 phase was observed in NSCs and human fibroblasts. Under this condition, increase in mitochondria mass, mitochondrial DNA, and intracellular reactive oxygen species (ROS) levels were observed in the absence of enhanced mitochondrial activity. Consequently, cellular senescence was induced by high doses of SR in differentiated cells.

In conclusion, we demonstrated that mitochondrial radiation responses differ according to the extent of DNA damage, duration of radiation exposure, and cell differentiation.     

Effects of 1 GeV/nucleon ⁵⁶Fe particles on longevity, carcinogenesis and neuromotor ability in Atm-deficient mice

As therapeutic uses of high-LET radiation become more prevalent and human space exploration continues to be a focus of NASA, it is important to understand the biological effects of high-LET radiation and the role of genetics in sensitivity to high-LET radiation. To study genetic susceptibility to radiation, we used mice deficient in Atm activity (AtmΔSRI). ATM is important in DNA repair, apoptosis and cell cycle regulation. Although homozygous mutations in ATM are rare, the prevalence of ATM heterozygosity is estimated to be 1% and results in an increased cancer risk. We found that the effects of 1 Gy 1 GeV/nucleon ??Fe particles on life span and tumorigenesis are genotype- and sex-specific. Significant effects of 1 Gy 1 GeV/nucleon ??Fe particles on incidence of non-cancer end points were seen; however, 2 Gy 1 GeV/nucleon ??Fe particles significantly affected neuromotor ability. Our results represent an extensive investigation into the late effects of high-LET radiation exposure in a sex- and genotype-dependent manner and provide a baseline for understanding the long-term risks of high-LET radiation.     

Acute effects of iron-particle radiation on immunity. Part II: Leukocyte activation, cytokines and adhesion

The effects of high-linear energy transfer (LET) radiation on immune function have not been clearly established. The major goal of this study was to evaluate leukocyte responses after whole-body exposure to high-LET radiation. C57BL/6 mice were exposed to 0, 0.5, 2 and 3 Gy (56)Fe(26+) particles (1055 MeV/nucleon, 148.2 keV/microm) and killed humanely 4 days after exposure. Spontaneous synthesis of DNA in blood and spleen cells was increased significantly in groups receiving either 2 or 3 Gy (P < 0.001). In contrast, a significant depression in the response of T lymphocytes to phytohemagglutinin (PHA) and concanavalin A (ConA) was noted (P < 0.005); the response to lipopolysaccharide (LPS), a B-cell mitogen, was similar among groups. A cytometric bead array assay revealed that the level of tumor necrosis factor alpha (Tnfa) secreted by splenocytes increased significantly with increasing (56)Fe-particle dose (P < 0.05); interferon gamma, interleukin2 (Il2), Il4 and Il5 were unaffected. Flow cytometry analysis showed that 2 and 3 Gy markedly reduced splenic mononuclear cells expressing the activation markers CD25 and CD71, both with and without the T-cell marker CD3 (P < 0.05); proportions also varied significantly. Similar patterns were noted in mononuclear and granular cells with adhesion markers CD11b and, to a lesser extent, CD54 (P < 0.05). The results show that a single, acute exposure to high-LET radiation induced changes that can profoundly alter leukocyte functions. The implications of the data are discussed in relation to low-LET radiation, altered gravity, and space flight.     

Redox signaling (cross-talk) from and to mitochondria involves mitochondrial pores and reactive oxygen species

This review highlights the important role of redox signaling between mitochondria and NADPH oxidases. Besides the definition and general importance of redox signaling, the cross-talk between mitochondrial and Nox-derived reactive oxygen species (ROS) is discussed on the basis of 4 different examples. In the first model, angiotensin-II is discussed as a trigger for NADPH oxidase activation with subsequent ROS-dependent opening of mitochondrial ATP-sensitive potassium channels leading to depolarization of mitochondrial membrane potential followed by mitochondrial ROS formation and respiratory dysfunction. This concept was supported by observations that ethidium bromide-induced mitochondrial damage suppressed angiotensin-II-dependent increase in Nox1 and oxidative stress. In another example hypoxia was used as a stimulator of mitochondrial ROS formation and by using pharmacological and genetic inhibitors, a role of mitochondrial ROS for the induction of NADPH oxidase via PKC? was demonstrated. The third model was based on cell death by serum withdrawal that promotes the production of ROS in human 293T cells by stimulating both the mitochondria and Nox1. By superior molecular biological methods the authors showed that mitochondria were responsible for the fast onset of ROS formation followed by a slower but long-lasting oxidative stress condition based on the activation of an NADPH oxidase (Nox1) in response to the fast mitochondrial ROS formation. Finally, a cross-talk between mitochondria and NADPH oxidases (Nox2) was shown in nitroglycerin-induced tolerance involving the mitochondrial permeability transition pore and ATP-sensitive potassium channels. The use of these redox signaling pathways as pharmacological targets is briefly discussed.     

p53-independent downregulation of histone gene expression in human cell lines by high- and low-let radiation

Using microarrays to analyze differential gene expression as a function of p53 status and radiation quality, we observed downregulation of a large set of histone genes in p53 wild-type TK6 cells 24 h after exposure to equitoxic doses of high-LET (1.67 Gy 1 GeV/amu (56)Fe ions) or low-LET (2.5 Gy γ rays) radiation. Quantitative real-time PCR of specific subtypes of core (H2A, H2B, H3 and H4) and linker (H1) histones confirmed this result. DNA synthesis and histone gene expression are tightly coordinated during the S phase of the cell cycle, and both processes are regulated by cell cycle checkpoints in response to DNA damage caused by ionizing radiation. However, we observed similar repression of histone gene expression in both TK6 cells and their p53-null derivative NH32 after radiation exposure, although the histone gene expression was not decreased to the same extent in NH32 cells as it was in TK6 cells. We also found decreased histone gene expression that was dose- and time-dependent in the colon cancer cell line HCT116 and its p53-null derivative. These results show that both high- and low-LET radiation exposure negatively regulate histone gene expression in human lymphoblastoid and colon cancer cell lines independent of p53 status.     

Relative biological effectiveness of 12C and 28Si radiation in C57BL/6J mice

Study of heavy ion radiation-induced effects on mice could provide insight into the human health risks of space radiation exposure. The purpose of the present study is to assess the relative biological effectiveness (RBE) of (12)C and (28)Si ion radiation, which has not been reported previously in the literature. Female C57BL/6J mice (n = 15) were irradiated using 4-8 Gy of (28)Si (300 MeV/nucleon energy; LET 70 keV/μm) and 5-8 Gy of (12)C (290 MeV/nucleon energy; LET 13 keV/μm) ions. Post-exposure, mice were monitored regularly, and their survival observed for 30 days. The LD(50/30) dose (the dose at which 50 % lethality occurred by 30-day post-exposure) was calculated from the survival curve and was used to determine the RBE of (28)Si and (12)C in relation to γ radiation. The LD(50/30) for (28)Si and (12)C ion is 5.17 and 7.34 Gy, respectively, and the RBE in relation to γ radiation (LD(50/30)-7.25 Gy) is 1.4 for (28)Si and 0.99 for (12)C. Determination of RBE of (28)Si and (12)C for survival in mice is not only important for space radiation risk estimate studies, but it also has implications for HZE radiation in cancer therapy.     

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.     

A manganese porphyrin complex is a novel radiation protector

Exposure of cells to ionizing radiation leads to formation of reactive oxygen species, which are associated with radiation-induced cytotoxicity. Therefore, compounds that scavenge reactive oxygen species may confer radioprotective effects. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. The objective of this study was to investigate the effects of manganese(III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on radiation-dependent toxicity. We investigated the protective role of MnTMPyP against ionizing radiation in U937 cells and mice. On exposure to ionizing radiation, there was a distinct difference between control cells and cells pretreated with MnTMPyP with respect to viability, cellular redox status, and oxidative damage to cells. Lipid peroxidation, oxidative DNA damage, and protein oxidation were significantly lower in the cells treated with MnTMPyP when the cells were exposed to ionizing radiation. The [GSSG]/[GSH + GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP+ + NADPH] ratio was lower in control cells compared with MnTMPyP-treated cells. Ionizing radiation-induced mitochondrial damage, as reflected by the altered mitochondrial permeability transition, increase in accumulation of reactive oxygen species, reduction of ATP production, and morphological change, was significantly higher in control cells than in MnTMPyP-treated cells. MnTMPyP administration for 14 days at a daily dosage of 5 mg/kg provided substantial protection against killing and oxidative damage in mice exposed to whole-body irradiation. These data indicate that MnTMPyP may have great application potential as a new class of in vivo, non-sulfur-containing radiation protectors.