A rapidly growing body of experimental evidence indicates that ionizing radiation induces biological effects in non-irradiated bystander cells that have received signals from adjacent or distant irradiated cells. This phenomenon, which has been termed the ionizing radiation-induced bystander effect, challenges the long-standing paradigm that radiation traversal through the nucleus of a cell is a prerequisite to elicit genetic damage or a biological response. Bystander effects have been observed in a number of experimental systems, and cells whose nucleus or cytoplasm is irradiated exert bystander responses. Bystander cells manifest a multitude of biological consequences, such as genetic and epigenetic changes, alterations in gene expression, activation of signal transduction pathways, and delayed effects in their progeny. Several mediating mechanisms have been proposed. These involve gap junction-mediated intercellular communication, secreted soluble factors, oxidative metabolism, plasma membrane-bound lipid rafts, and calcium fluxes. This paper reviews briefly the current knowledge of the bystander effect with a focus on proposed mechanisms. The potential benefit of bystander effects to cancer radiotherapy will also be discussed.

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The risks of exposure to low dose ionizing radiation (below 100 mSv) are estimated by extrapolating from data obtained after exposure to high dose radiation, using a linear no-threshold model (LNT model). However, the validity of using this dose-response model is controversial because evidence accumulated over the past decade has indicated that living organisms, including humans, respond differently to low dose/low dose-rate radiation than they do to high dose/high dose-rate radiation. In other words, there are accumulated findings which cannot be explained by the classical "target theory" of radiation biology. The radioadaptive response, radiation-induced bystander effects, low-dose radio-hypersensitivity, and genomic instability are specifically observed in response to low dose/low dose-rate radiation, and the mechanisms underlying these responses often involve biochemical/molecular signals that respond to targeted and non-targeted events. Recently, correlations between the radioadaptive and bystander responses have been increasingly reported. The present review focuses on the latter two phenomena by summarizing observations supporting their existence, and discussing the linkage between them from the aspect of production of reactive oxygen and nitrogen species.

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Irradiation of murine subcutaneous stroma before implantation of tumor cells leads to retarded tumor growth. This effect is called Tumor Bed Effect (TBE) and can be used to assess the sensitivity of stromal tissue to radiation. We tested the ability of stromal tissue to recover from X-ray-induced damage as a function of the time interval between X-irradiation and implantation of tumor cells over a period of 195 days. We also assessed the effects of a second test treatment of X-irradiation before implantation to assess residual damage by the first radiation treatment. The tumor bed effect in C57Bl10×DBA2 mice observed after X-ray treatment and implantation of M8013 cells (from a transplantable mouse mammary carcinoma) declines with the time that elapses between X-rays and implantation. Implantation of tumor cells 195 days after initial irradiation of 10 or 20 Gy resulted in a considerably smaller TBE. The half-time of the decay is estimated as about 50 days. The extent of the recovery was then tested in two-fraction experiments, with radiation fractions separated by intervals of 30 or 180 days. In the experiment with re-irradiation at an interval of 30 days after the first radiation dose of 20 Gy hardly any recovery was observed, whereas at an interval of 180 days a considerable recovery was observed. We presume that the recovery in TBE that was observed a long time after the irradiation results from a proliferative stimulus to endothelial cells which takes place during the post-irradiation period. The proliferative response leads to cell death of the X-ray damaged endothelial cells and thereafter these are replaced by healthy cells.

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We examined the effect of whole body low-dose γ-irradiation on the status of the antioxidant defense system in the rodent kidneys at different time intervals. Young male Balb/c mice were exposed to whole body radiation from a ⁶⁰Co source at doses of 10, 25 and 50 cGy (48.78 cGy/min). Antioxidant status and lipid peroxidation were estimated in the kidneys at 4, 12 and 24 h after irradiation. Lipid peroxidation increased between 33% and 49% and reduced glutathione between 12% and 47% at 12 h at different radiation doses. Reduced glutathione level remained significantly (p < 0.05) elevated even at 24 h after irradiation to 25 cGy. Superoxide dismutase activity also increased by 37% at 12 h on exposure of animals to all the doses up to 50 cGy. Catalase activity increased significantly at 12 h on exposure to 10 cGy and 50 cGy. Interestingly, glutathione peroxidase activity increased by 31% at 4 h and subsequently returned to control levels at 24 h after exposure to 50 cGy. Glutathione reductase activity increased by 10-12% at 12 h after exposure to 25 cGy and 50 cGy. The results suggest that the whole body exposure of animals to gamma radiation stimulates the antioxidant defense system in the kidneys within 4 to 24 h after irradiation, at doses of 25 cGy and 50 cGy.

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The radiation-sensitive mutant "ric1" has a defect in the repair mechanism of DNA double strand breaks induced by γ-rays in early embryogenesis. In this study, the new radiation-sensitive Medaka (Oryzias latipes) strain, ric1olvas-GFP was established to monitor the development of germ cells in vivo. The development of germ cells was normal in ric1olvas-GFP, but embryonic germ cells at Stage 7 (32-cell stage) and Stage 33 (extensive proliferating stage of PGCs) showed higher radiosensitivity. There was no sex difference in germ cell radiosensitivity at Stage 7, but female embryos showed higher radiosensitivity than male at Stage 33. In embryos obtained by crossing ric1 female with olvas-GFP male, germ cells showed similar radiosensitivity to ric1olvas-GFP and increased sensitivity compared to embryos obtained from crossing wild-type female with olvas-GFP male at Stage 7. These results suggest that germ cells have the ric1 dependent DNA repair system during embryogenesis and the maternal ric1gene factor may play a critical role in radiosensitivity at an early developmental stage.

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Ionizing radiation induces a variety of lesions in DNA, each of which can be used as a bio-indicator for biological dosimetry or the study of the radioprotective effects of substances. To assess gamma ray-induced DNA damage in vivo in mouse leukocytes at various doses and the effect of famotidine, blood was collected from Balb/c male mice after irradiation with 4 Gy γ-rays at different time intervals post-irradiation. To assess the response, mice were irradiated with doses of gamma-rays at 1 to 4 Grays. Famotidine was injected intra-peritoneally (i.p) at a dose of 5 mg/kg at various time intervals before irradiation. Four slides were prepared from each sample and alkaline comet assay was performed using standard protocols. Results obtained show that radiation significantly increases DNA damage in leukocytes in a dose dependent manner (p < 0.01) when using appropriate sampling time after irradiation, because increasing sampling time after irradiation resulted in a time dependent disappearance of DNA damage. Treatment with only 5 mg/kg famotidine before 4 Gy irradiation led to almost 50% reduction in DNA damage when compared with those animals which received radiation alone. The radioprotective capability of famotidine might be attributed to radical scavenging properties and an anti-oxidation mechanism.

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Nutmeg, the dried seed kernel of Myristica fragrans, MF (Family: Myristicaceae) possesses antifungal, hepatoprotective and antioxidant properties. Its radioprotective effect against 6, 8 and10 Gy gamma radiation was evaluated by 30 day survival assay. Regression analysis yielded LD_50/30 as 6.83 Gy and 8.89 Gy for irradiated only and (MF + radiation) groups, respectively. The dose reduction factor was computed as 1.3. Administration of MF significantly enhanced hepatic glutathione (GSH) and decreased testicular lipid peroxidation (LPO) level whereas acid phosphatase (ACP) and alkaline phosphatase (ALP) activity did not show any significant alteration. Irradiation resulted in significant elevation in LPO level and ACP activity, and decreased the GSH content and ALP activity. MF pretreatment effectively protected against radiation induced biochemical alteration as reflected by a decrease in LPO level and ACP activity, and an increase in GSH and ALP activity. The present study has implications for the potential use of MF as a radioprotector.

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p53 is one of the most frequently mutated genes in mammary carcinomas (MCs). To detect tumor suppressor genes cooperating with a hetero-deficient p53 gene in mammary carcinogenesis, we first examined allelotypes in MCs from (BALB/cHeA × MSM/Ms) F₁- p53^+/- and (BALB/cHeA × 129/SvEv) F₁- p53^+/- female mice, and then surveyed down-regulated genes in the allelic loss regions. Genome-wide screening at 42 loci identified frequent (more than 30%) loss of heterozygosity (LOH) on chromosomes 5, 8, 11, 12, 14 and 18 in the MCs from either of the F₁ mice. The MCs in the p53^+/- mice indicated highly frequent LOH, especially on chromosomes 8, 11 and 12, distinct from other mouse tumors. More than 60% of the 38 MCs from (BALB/cHeA × MSM/Ms) F₁- p53 ^+/- mice showed LOH in a region ranging from D8Mit85 (105.0 Mb from centromere) to D8Mit113 (111.8 Mb) on chromosome 8, a region syntenic to human chromosome 16q22.1, on which LOH has been found in breast cancers. RT-PCR analyses revealed that the LOH of chromosome 8 was associated with the reduced and/or complete loss of expression of Cdh1 and Cdh5 genes in 15 (58%) and 8 (31%) of 26 MCs derived from the F₁ mice, respectively. Thus, inactivation of Cdh1 and Cdh5 is likely to cooperate with the loss of p53, suggesting a possible tumor suppressive function of these genes in mammary carcinogenesis.

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Thermionic emission (TE)-type X-ray generators have been exclusively used in medicine, but there are many difficulties in making these X-ray sources compact. A field emission (FE)-type X-ray generator using carbon nanotubes is a newly-developed compact system that can be as small as several cm in length. Considering the compactness of the equipment, the FE-type X-ray generator may become a useful tool for endoscopic, intracavitary or intraoperative radiotherapy in the future. The aim of this study was to investigate the biological effects of X-rays generated by the FE-type X-ray source in comparison with those of conventional TE-type X-rays. Mouse thymic lymphoma 3SB cells were irradiated by an FE-type X-ray generator developed by our group and a conventional TE-type X-ray source under identical conditions. DNA damage after radiation was detected by foci formation of phospho-H2AX (γ-H2AX). Effect on the cell cycle was analyzed by flow cytometry. Activation of the DNA damage checkpoint was analyzed by immunoblotting. Induction of apoptosis was studied using the TUNEL assay. In terms of induction of DNA damage (DNA double-strand breaks), activation of cell cycle checkpoints (p53 stabilization, p21 induction, Chk1 and Chk2 phosphorylations), and induction of apoptotic cell death, FE-type X-rays were as effective as TE-type X-rays, and FE-type X-rays appeared to be applicable to radiation therapy.

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The purpose of the present work was to examine gene expression patterns in a rat keratinocyte line exposed to a ⁵⁶Fe ion beam. The cells were exposed to 1.01 geV/nucleon ⁵⁶Fe ions generated by the NASA Space Radiation Laboratory facility. Data from Affymetrix rat microarrays (RAT 230_2) were processed by BRB ArrayTools 3.3.0 software, and the Gene Ontogeny (GO) database was utilized to categorize significantly responding genes. Cell cycle distribution was analyzed by flow cytometry, and cell survival was based on the colony survival assay. At 24 h after 3.0 Gy of ⁵⁶Fe ion radiation, 69 known genes were significantly (p ≤ 0.001) altered (88% upregulated) and 16 of 97 categories of genes at level 7 in the GO hierarchy were significantly altered (p ≤ 0.01) including "mitosis", "DNA repair" and "positive regulation of cell cycle" in comparison to control. Flow cytometry revealed that 60% of irradiated cells versus 10% of control cells were in G₂/M phase of the cell cycle at 24 h after ⁵⁶Fe ion radiation. Colony survival rate for rat keratinocytes irradiated with 3.0 Gy of ⁵⁶Fe ion was 6.6% versus control at 24 h after irradiation. The results in the present study suggest a link between the increased expression of cell cycle genes and cell death.

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Interleukin-11 (IL-11) is a well known anti-inflammatory cytokine that is associated with cell growth, and also participates in limiting X-ray irradiation induced intestinal mucosal injury. The aim of this study was to evaluate the protective effect of IL-11 on the cell injury induced by X-ray irradiation in rat intestinal epithelial IEC-18 cells. Recombinant human IL-11 (rhIL-11) treated cells were irradiated and then examined for cell viability. To evaluate irradiation injury, trypan blue staining was used to detect the dead cells. The viability of irradiated cells was up-regulated by rhIL-11 treatment and also resulted in the activation of p90 ribosomal S6 kinase (p90RSK) and S6 ribosomal protein (S6Rp). Wortmannin, a specific inhibitor of PI3K, suppressed the activation of S6Rp in rhIL-11 treated cells, and decreased the up-regulation of viability by rhIL-11 treatment in irradiated cells. The TUNEL assay was also perfomed to estimate the rate of apoptosis in X-ray induced cell death. There was no difference in the results between trypan blue staining and the TUNEL assay. Further, rhIL-11 down-regulated the expression of cleaved caspase-3 in irradiated cells. These results suggest that rhIL-11 may play an important role in protection from radiation injury.

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