Quantitative and qualitative mutational impact of ionizing radiation on normal cells.

The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.

Low Dose Rate Radiation Regulates M2-like Macrophages in an Allergic Airway Inflammation Mouse Model.

We investigated the effects of low dose rate radiation (LDR) on M1 and M2 macrophages in an ovalbumin-induced mouse model of allergic airway inflammation and asthma. After exposure to LDR (1 Gy, 1.818 mGy/h) for 24 days, mice were euthanized and the changes in the number of M1 and M2 macrophages in the bronchoalveolar lavage fluid and lung, and M2-associated cytokine levels, were assessed. LDR treatment not only restored the M2-rich microenvironment but also ameliorated asthma-related progression in a macrophage-dependent manner. In an ovalbumin-induced mouse model, LDR treatment significantly inhibited M2, but not M1, macrophage infiltration. M2-specific changes in macrophage polarization during chronic lung disease reversed the positive effects of LDR. Moreover, the levels of cytokines, including chemokine (C-C motif) ligand (CCL) 24, CCL17, transforming growth factor beta 1, and matrix metalloproteinase-9, decreased in ovalbumin-sensitized/challenged mice upon exposure to LDR. Collectively, our results indicate that LDR exposure suppressed asthmatic progression, including mucin accumulation, inflammation, and Type 2 T helper (Th2) cytokine (interleukin (IL)-4 and IL-13) production. In conclusion, LDR exposure decreased Th2 cytokine secretion in M2 macrophages, resulting in a reduction in eosinophilic inflammation in ovalbumin-sensitized/challenged mice.

Differential Effects of Low and High Radiation Dose Rates on Mouse Spermatogenesis.

The adverse effects of radiation are proportional to the total dose and dose rate. We aimed to investigate the effects of radiation dose rate on different organs in mice. The mice were subjected to low dose rate (LDR, ~3.4 mGy/h) and high dose rate (HDR, ~51 Gy/h) radiation. LDR radiation caused severe tissue toxicity, as observed in the histological analysis of testis. It adversely influenced sperm production, including sperm count and motility, and induced greater sperm abnormalities. The expression of markers of early stage spermatogonial stem cells, such as Plzf, c-Kit, and Oct4, decreased significantly after LDR irradiation, compared to that following exposure of HDR radiation, in qPCR analysis. The compositional ratios of all stages of spermatogonia and meiotic cells, except round spermatid, were considerably reduced by LDR in FACS analysis. Therefore, LDR radiation caused more adverse testicular damage than that by HDR radiation, contrary to the response observed in other organs. Therefore, the dose rate of radiation may have differential effects, depending on the organ; it is necessary to evaluate the effect of radiation in terms of radiation dose, dose rate, organ type, and other conditions.

Hypoxia induces immunogenic cell death of cancer cells by enhancing the exposure of cell surface calreticulin in an endoplasmic reticulum stress-dependent manner.

Hypoxia is associated with resistance to anticancer therapies. Additionally, it is involved in the immune evasion of cancer cells by inducing an immunosuppressive microenvironment. However, the role of hypoxia in modulating the immunogenicity of cancer cells remains unknown. Hypoxia is known to induce endoplasmic reticulum (ER) stress, which serves a key role in inducing the cell surface exposure of calreticulin, a marker of immunogenic cell death. The present study investigated whether hypoxia influenced the immunogenicity of cancer cells using FACS, western blot analysis and syngenic mouse tumor model. The results revealed that hypoxia induced the cell surface exposure of calreticulin in human and mouse breast cancer cell lines depending on ER stress. Enhanced cell surface exposure of calreticulin induced by hypoxia resulted in an increase in anticancer immunity in a mouse model, which suggested that hypoxia induced immunogenic cell death. Notably, hypoxia did not significantly modulate the cell surface exposure of CD47, an antagonist of calreticulin function in cancer immunogenicity. These results suggest that hypoxia may enhance the immunogenicity of cancer cells themselves, in addition to its role in inducing an immunosuppressive cancer microenvironment.

High-Dose Radiation-Induced Changes in Murine Small Intestinal Motility: Are the Changes in the Interstitial Cells of Cajal or in the Enteric Nervous System?

Murine small intestinal motility consists of phasic contraction from interstitial cells of Cajal (ICC) and migrating motor complexes (MMCs) from the enteric nervous system. The number of ICC is reduced in various gastrointestinal disorders, and this effect can be reversed once the disorder is resolved through cellular and tissue remodelling. Exposure to high-dose radiation can induce inflammation and alter intestinal motility. In this study, we investigated the changes in the small intestinal motility of 8- to 10-week-old male C3H/HeN mice after high-dose (13 Gy) irradiation. The aim of this study was to determine whether those changes are caused by changes in the ICC or enteric nervous system. After irradiation, the small intestine was dissected and stored in oxygenated Krebs-Ringer bicarbonate solution. The tension of contractions and intracellular membrane potentials were recorded at day 0, 1, 3 and 5 after irradiation and compared with those of sham-irradiated mice. Histological evaluation was performed by immunohistochemistry and apoptosis was evaluated. Quantitative real-time polymerase chain reaction (qPCR) for c-kit mRNA was also performed. Phasic contractions were not changed at day 0, 1, 3 and 5 after irradiation and did not significantly differ from those in the control mice. Slow waves were also sustained after irradiation. However, the frequency of migrating motor complexes (MMCs) was significantly higher at day 0 and 1 after exposure and the amplitude and area under the curve were significantly lower at day 3 after exposure compared with control mice. MMCs were recovered at day 5 with no difference from those of the control mice. ICC were detected after irradiation by immunohistochemistry for c-kit, and c-kit mRNA levels did not differ between sham-irradiated and irradiated mice. Histological evaluation showed that the most severe inflammation was detected at day 3 after irradiation, and apoptosis was detected only in the mucosa. Acetylcholine increased the contractility after irradiation, and tetrodotoxin decreased the number of MMCs in sham-irradiated and irradiated mice. N(w)-oxide-l-arginine (L-NA) increased the number of MMCs. MMCs were recovered after L-NA treatment at day 3 after irradiation. Sodium nitroprusside decreased the MMCs in sham-irradiated and irradiated mice. Exposure to high-dose radiation did not alter phasic contractions and slow waves in the small intestine of mice, which suggests that ICC and their functions may be sustained after high-dose irradiation. Mucosal inflammation was severe after irradiation and there were some changes in MMCs related to the enteric nervous system.

Continuous Exposure to Low-Dose-Rate Gamma Irradiation Reduces Airway Inflammation in Ovalbumin-Induced Asthma.

Although safe doses of radiation have been determined, concerns about the harmful effects of low-dose radiation persist. In particular, to date, few studies have investigated the correlation between low-dose radiation and disease development. Asthma is a common chronic inflammatory airway disease that is recognized as a major public health problem. In this study, we evaluated the effects of low-dose-rate chronic irradiation on allergic asthma in a murine model. Mice were sensitized and airway-challenged with ovalbumin (OVA) and were exposed to continuous low-dose-rate irradiation (0.554 or 1.818 mGy/h) for 24 days after initial sensitization. The effects of chronic radiation on proinflammatory cytokines and the activity of matrix metalloproteinase-9 (MMP-9) were investigated. Exposure to low-dose-rate chronic irradiation significantly decreased the number of inflammatory cells, methylcholine responsiveness (PenH value), and the levels of OVA-specific immunoglobulin E, interleukin (IL)-4, and IL-5. Furthermore, airway inflammation and the mucus production in lung tissue were attenuated and elevated MMP-9 expression and activity induced by OVA challenge were significantly suppressed. These results indicate that low-dose-rate chronic irradiation suppresses allergic asthma induced by OVA challenge and does not exert any adverse effects on asthma development. Our findings can potentially provide toxicological guidance for the safe use of radiation and relieve the general anxiety about exposure to low-dose radiation.

Administration of granulocyte colony-stimulating factor with radiotherapy promotes tumor growth by stimulating vascularization in tumor-bearing mice.

Although granulocyte-colony stimulating factor (G-CSF) is commonly used to support recovery from radiation-induced side-effects, the precise effects of G-CSF on colon cancer under radiotherapy remain poorly understood. In the present study, to investigate the effects of tumor growth following radiotherapy and G-CSF administration in a murine xenograft model of colon cancer, female BALB/c mice were injected with cells of a colon carcinoma cell line (CT26) with irradiation and G-CSF, alone or in combination. Mice received 2 Gy of focal radiation daily for 5 days and intraperitoneal injection of G-CSF (100 µg/kg/day) after irradiation for 7 days. Changes in the levels of myeloperoxidase (MPO), vascular endothelial growth factor (VEGF), matrix metalloproteinase type 9 (MMP-9) and CD31 were assessed in the mouse cancer induced by injection of colon cancer cells. We observed that G-CSF increased the number of circulating neutrophils, but facilitated tumor growth. However, G-CSF treatment did not affect radiation-induced cytotoxicity and cell viability in CT26 cells in vitro. Increased levels of myeloperoxidase, a neutrophil marker and those of vascular endothelial growth factor were observed in tumors with G-CSF supplementation. In addition, we found that increased levels of CD31 and matrix metalloproteinase-9 were correlated with the enhanced tumor growth after G-CSF treatment. Therefore, these data suggest that G-CSF may contribute to tumor growth and decrease the antitumor effect of radiotherapy, possibly by promoting vascularization in cancer lesions.

Protective Effect of Administered Rolipram against Radiation-Induced Testicular Injury in Mice.

PURPOSE: Pelvic irradiation for the treatment of cancer can affect normal cells, such as the rapidly proliferating spermatogenic cells of the testis, leading to infertility, a common post-irradiation problem. The present study investigated the radioprotective effect of rolipram, a specific phosphodiesterase type-IV inhibitor known to increase the expression and phosphorylation of the cyclic adenosine monophosphate response element-binding protein (CREB), a key factor for spermatogenesis, with the testicular system against pelvic irradiation. MATERIALS AND METHODS: Male C57BL/6 mice were treated with pelvic irradiation (2 Gy) and rolipram, alone or in combination, and were sacrificed at 12 hours and 35 days after irradiation. RESULTS: Rolipram protected germ cells from radiation-induced apoptosis at 12 hours after irradiation and significantly increased testis weight compared with irradiation controls at 35 days. Rolipram also ameliorated radiation-induced testicular morphological changes, such as changes in seminiferous tubular diameter and epithelial height. Additionally, seminiferous tubule repopulation and stem cell survival indices were higher in the rolipram-treated group than in the radiation group. Moreover, rolipram treatment counteracted the radiation-mediated decrease in the sperm count and mobility in the epididymis. CONCLUSIONS: These protective effects of rolipram treatment prior to irradiation may be mediated by the increase in pCREB levels at 12 hours post-irradiation and the attenuated decrease in pCREB levels in the testis at 35 days post-irradiation in the rolipram-treated group. These findings suggest that activation of CREB signaling by rolipram treatment ameliorates the detrimental effects of acute irradiation on testicular dysfunction and the related male reproductive functions in mice.

Radiation-induced eosinophilic, polymorphic, and pruritic eruption in a pig skin model.

Eosinophilic, polymorphic and pruritic eruption associated with radiotherapy (EPPER) can occur in cancer patients after irradiation. In this study, we characterized the clinical and histopathological features of pig skin that developed widespread polymorphic and pruritic skin lesions following localized 50 Gy gamma-irradiation. The pigs developed pruritus 5-7 weeks after irradiation, and infiltration of the dermis by eosinophils was detected 4-7 weeks after irradiation. The irradiated animals also showed transiently increased numbers of peripheral eosinophils 5-7 weeks after treatment. Irradiation induced desquamation after 2-4 weeks, which and the desquamation gradually resolved after 7 weeks. These pathological changes correspond to those seen in irradiated human skin, indicating that this model could be useful for elucidating the pathogenesis of EPPER and for developing therapeutic and prophylactic methods.

Molecular characterization of the aryl hydrocarbon receptor (AhR) pathway in goldfish (Carassius auratus) exposure to TCDD: the mRNA and protein levels.

In bony fish or other aquatic vertebrates, the aryl hydrocarbon receptor (AhR) signaling pathway is initiated by exposure to polycyclic (or/and halogenated) aromatic hydrocarbons (PAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), which subsequently induces the up-regulated expression of a series of related genes (such as cytochrome P4501A (CYP1A)). However, a lack of applicable protein reagents hinders our further understanding of the AhR signaling pathway, which focuses only on gene-based investigations. The goldfish (Carassius auratus) is an ideal model for a study of environmental pollution in whole-Asian fresh water. Here, three sensitive and specific polyclonal antisera against goldfish AhR1, AhR2, and CYP1A proteins were developed. These antisera not only bound the in-vitro synthesized target proteins, but recognized the real proteins expressed in goldfish tissues, with minimal cross-reactivity to non-specific proteins. Together with the analysis of semi-quantitative RT-PCR and polyclonal-antibody-based sandwich ELISA, we confirmed that goldfish AhRs differed in the expression (mRNA and protein levels) patterns among test tissues. Importantly, the relative abundance of each AhR mRNA levels from the different tissues showed no obvious consistency with their protein levels. After exposure to TCDD, goldfish AhR2 showed a more sensitivity than AhR1, and stimulated CYP1A expression directly, similar with the other reported fish models. Overall, development of these antibodies in this study will allow valuable and versatile investigations to further understand the AhR signaling pathway, and different expression (mRNA and protein) patterns represent the first step in determining the regulatory mechanisms underlying the TCDD-exposed aquatic environment.

Protective effect of genistein on radiation-induced intestinal injury in tumor bearing mice.

BACKGROUND: Radiation therapy is the most widely used treatment for cancer, but it causes the side effect of mucositis due to intestinal damage. We examined the protective effect of genistein in tumor-bearing mice after abdominal irradiation by evaluation of apoptosis and intestinal morphological changes. METHODS: Mouse colon cancer CT26 cells were subcutaneously injected at the flank of BALB/c mice to generate tumors. The tumor-bearing mice were treated with abdominal radiation at 5 and 10 Gy, and with genistein at 200 mg/kg body weight per day for 1 d before radiation. The changes in intestinal histology were evaluated 12 h and 3.5 d after irradiation. To assess the effect of the combination treatment on the cancer growth, the tumor volume was determined at sacrifice before tumor overgrowth occurred. RESULTS: Genistein significantly decreased the number of apoptotic nuclei compared with that in the irradiation group 12 h after 5 Gy irradiation. Evaluation of histological changes showed that genistein ameliorated intestinal morphological changes such as decreased crypt survival, villus shortening, and increased length of the basal lamina 3.5 d after 10 Gy irradiation. Moreover, the genistein-treated group exhibited more Ki-67-positive proliferating cells in the jejunum than the irradiated control group, and crypt depths were greater in the genistein-treated group than in the irradiated control group. The mean weight of the CT26 tumors was reduced in the group treated with genistein and radiation compared with the control group. CONCLUSION: Genistein had a protective effect on intestinal damage induced by irradiation and delayed tumor growth. These results suggest that genistein is a useful candidate for preventing radiotherapy-induced intestinal damage in cancer patients.

Influence of divalent metal ions on E2-induced ER pathway in goldfish (Carassius auratus) hepatocytes.

Metal ions existing in the environment could influence the estrogen pathway in aquatic animal, but the detailed mechanism is still delusive. We here showed that in male Carassius auratus hepatocytes, copper (Cu) or cadmium (Cd), did not directly induce vitellogenin (VTG) expression. Interestingly, co-exposure with Cd²âº (or Cu²âº) and 17-ß-estradiol (E2) greatly increased the VTG level, comparing with single treatment of E2. Meanwhile, Cd²âº or Cu²âº (but not E2) triggers HSP70 expression. But, mixture of Cd²âº or Cu²âº with E2 did not obviously raise HSP70 level. E2 also had no obvious effect on reactive oxygen species. Co-treatment of Cd²âº and E2 showed no obvious increase compared to single treatment with Cd²âº. We further assume that Cd²âº-involved oxidative stress generates misfolded proteins, resulting in the competition of HSP70 proteins from a heterocomplex (with estrogen receptor). Thus, dissociation of the heterocomplex actives the receptor-ligand binding activity and promotes the E2-induced VTG expression.

Sequence conservation in the internal transcribed spacers and 5.8S ribosomal RNA of parasitic scuticociliates Miamiensis avidus (Ciliophora, Scuticociliatia).

The scuticociliate Miamiensis avidus is a histophagous parasite that causes high mortality in cultured marine fishes, with clinical signs of severe ulcers and hemorrhages in the skeletal muscle. The internal transcribed spacer (ITS) region, which is widely used in taxonomy and molecular phylogeny because of a high degree of variation, was compared for 21 cloned strains of M. avidus (Ciliophora, Scuticociliatia). These strains were isolated from olive flounder Paralichthys olivaceus, ridged-eye flounder Pleuronichthys cornutus and spotted knifejaw Oplegnathus fasciatus in Korea and Japan. The ITS1 (140 bp), ITS2 (236 bp) and 5.8S (119 bp) regions from 21 strains were identical, indicating that these regions are highly conserved in M. avidus. Phylogenic analysis of ITS2 shows that the ciliate should be included in the Philasterida with a close relationship to Pseudocohnilembus hargisi. This study exhibits the first detailed analysis of the ITS1, 5.8 S and ITS2 rRNA regions of M. avidus.