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Following carbon ion beam irradiation in mammalian cells, such as used in carbon ion radiotherapy (CIRT), it has been suggested that the balance between whether nonhomologous end joining (NHEJ) or homologous recombination (HR) is utilized depends on the DNA double-strand break (DSB) complexity. Here, we quantified DSB distribution and identified the importance of each DSB repair pathway at increasing depths within the carbon ion spread-out Bragg peak (SOBP) beam range. Chinese hamster ovary (CHO) cell lines were irradiated in a single biological system capable of incorporating the full carbon ion SOBP beam range. Cytotoxicity and DSB distribution/repair kinetics were examined at increasing beam depths using cell survival as an endpoint and γ-H2AX as a surrogate marker for DSBs. We observed that proximal SOBP had the highest number of total foci/cell and lowest survival, while distal SOBP had the most dense tracks. Both NHEJ- and HR-deficient CHO cells portrayed an increase in radiosensitivity throughout the full carbon beam range, although NHEJ-deficient cells were the most radiosensitive cell line from beam entrance up to proximal SOBP and demonstrated a dose-dependent decrease in ability to repair DSBs. In contrast, HR-deficient cells had the greatest ratio of survival fraction at entrance depth to the lowest survival fraction within the SOBP and demonstrated a linear energy transfer (LET)-dependent decrease in ability to repair DSBs. Collectively, our results provide insight into treatment planning and potential targets to inhibit, as HR was a more beneficial pathway to inhibit than NHEJ to enhance the cell killing effect of CIRT in targeted tumor cells within the SOBP while maintaining limited unwanted damage to surrounding healthy cells.

(1) Background: Hyperbaric oxygen (HBO) exposure induces oxidative stress that may lead to DNA damage, which has been observed in human peripheral blood lymphocytes or non-human cells. Here, we investigated the impact of hyperbaric conditions on two human osteoblastic cell lines: primary human osteoblasts, HOBs, and the osteogenic tumor cell line SAOS-2. (2) Methods: Cells were exposed to HBO in an experimental hyperbaric chamber (4 ATA, 100% oxygen, 37 °C, and 4 h) or sham-exposed (1 ATA, air, 37 °C, and 4 h). DNA damage was examined before, directly after, and 24 h after exposure with an alkaline comet assay and detection of γH2AX+53BP1 colocalizing double-strand break (DSB) foci and apoptosis. The gene expression of TGFß-1, HO-1, and NQO1, involved in antioxidative functions, was measured with qRT-PCR. (3) Results: The alkaline comet assay showed significantly elevated levels of DNA damage in both cell lines after 4 h of HBO, while the DSB foci were similar to sham. γH2AX analysis indicated a slight increase in apoptosis in both cell lines. The increased expression of HO-1 in HOB and SAOS-2 directly after exposure suggested the induction of an antioxidative response in these cells. Additionally, the expression of TGF-ß1 was negatively affected in HOB cells 4 h after exposure. (4) Conclusions: in summary, this study indicates that osteoblastic cells are sensitive to the DNA-damaging effects of hyperbaric hyperoxia, with the HBO-induced DNA damage consisting largely of single-strand DNA breaks that are rapidly repaired.

DNA double-strand breaks (DSBs) are the main factor behind carbon-ion radiation therapy (CIRT)-induced cell death. Nuclear interactions along the beam path between the primary carbon ions and targets result in nuclear fragmentation of carbon ions and recoiled particles. These secondary particles travel further distances past the Bragg peak to the tail region, leading to unwanted biological effects that may result in cytotoxicity in critical organs and secondary induced tumors following CIRT. Here, we confirmed that the density of the DSB distributions increases as the cell survival decreases at the Bragg peak and demonstrated that by visualizing DSBs, the various LET fragmentation ions and recoiled particles produced differences in their biological effects in the post-Bragg peak tail regions. This suggests that the density of the DSBs within the high-LET track structures, rather than only their presence, is important for inducing cell death. These results are essential for CIRT treatment planning to limit the amount of healthy cell damage and reducing both the late effect and the secondary tumor-associated risk.

BACKGROUND: Nuclear medicine patients are isolated in a room after the injection of a radiopharmaceutical. They may be active Wi-Fi option of its smartphone mobile or other environmental radiofrequency waves. The hypothesis of this study was the evaluation of increased biological effects of the simultaneous exposure to gamma-ray and the Wi-Fi waves by measuring the level of the increased double strand-breaks DNA in peripheral blood lymphocyte in the rat.
METHODS: Fifty male Wistar rats were exposed for 2, 24, and 72 h only by Wi-Fi, 99m Tc, and simultaneously by Wi-Fi and 99m Tc. The power density levels of Wi-Fi emitter at 15 cm was 4.2nW/ c m 2 . An activity of 100 μCi of 99m Tc was injected intraperitoneally. Blood samples were taken by cardiac puncture following general anesthesia. Mononuclear cells are extraction by Ficoll-Hypaque density gradient centrifugation. The number of gamma-H2AX foci per nucleus was counted by flow cytometry. The statistical differences between experimental groups at 2, 24, and 72 h were determined with a repeated measure\'s analysis of variance. The significant difference between groups at the same time was analyzed with the Kruskal-Wallis Test.
RESULTS: The manner of gamma-H2AX expression was not the same for three groups in time. The number of gamma-H2AX foci between the three groups was a significant difference after 72 h.
CONCLUSIONS: Simultaneous Wi-Fi and gamma-ray exposures can increase the number of double-strand break DNA in peripheral blood lymphocytes to exposure of gamma-ray to 72 h after technetium injection in the rat.

Lung cancer has the highest mortality rate worldwide and is often diagnosed at late stages, requiring genotoxic chemotherapy with significant side effects. Cancer prevention has become a major focus, including the use of dietary and supplemental antioxidants. Thus, we investigated the ability of an antioxidant formulation (AOX1) to reduce DNA damage in human bronchial epithelial cells (BEAS-2B) with and without the combination of apple peel flavonoid fraction (AF4), or its major constituent quercetin (Q), or Q-3-O-d-glucoside (Q3G) in vitro. To model smoke-related genotoxicity, we used cigarette-smoke hydrocarbon 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) as well as methotrexate (MTX) to induce DNA damage in BEAS-2B cells. DNA fragmentation, γ-H2AX immunofluorescence, and comet assays were used as indicators of DNA damage. Pre-exposure to AOX1 alone or in combination with AF4, Q, or Q3G before challenging with NNKOAc and MTX significantly reduced intracellular reactive oxygen species (ROS) levels and DNA damage in BEAS-2B cells. Although NNKOAc-induced DNA damage activated ATM-Rad3-related (ATR) and Chk1 kinase in BEAS-2B cells, pre-exposure of the cells with tested antioxidants prior to carcinogen challenge significantly reduced their activation and levels of γ-H2AX (p ≤ 0.05). Therefore, AOX1 alone or combined with flavonoids holds promise as a chemoprotectant by reducing ROS and DNA damage to attenuate activation of ATR kinase following carcinogen exposure.

Proton therapy is a type of hadron radiotherapy used for treating solid tumors. Unlike heavy charged elements, proton radiation is considered to be low LET (Linear Energy Transfer) radiation, like X-rays. However, the clinical SOBP (Spread Out Bragg Peak) proton radiation is considered to be higher in relative biological effectiveness (RBE) than both X-ray and their own entrance region. The RBE is estimated to be 1.1-1.2, which can be attributed to the higher LET at the SOBP region than at the entrance region. In order to clarify the nature of higher LET near the Bragg peak of proton radiation and its potential cytotoxic effects, we utilized a horizontal irradiation system with CHO cells. Additionally, we examined DNA repair mutants, analyzed cytotoxicity with colony formation, and assessed DNA damage and its repair with γ-H2AX foci assay in a high-resolution microscopic scale analysis along with the Bragg peak. Besides confirming that the most cytotoxic effects occurred at the Bragg peak, extended cytotoxicity was observed a few millimeters after the Bragg peak. γ-H2AX foci numbers reached a maximum at the Bragg peak and reduced dramatically after the Bragg peak. However, in the post-Bragg peak region, particle track-like structures were sporadically observed. This region contains foci that are more difficult to repair. The peak and post-Bragg peak regions contain rare high LET-like radiation tracks and can cause cellular lethality. This may have caused unwanted side effects and complexities of outputs for the proton therapy treatment.

Among heavy metals, cadmium is considered one of the most toxic and dangerous environmental factors, contributing to stress by disturbing the delicate balance between production and scavenging of reactive oxygen species (ROS). To explore possible relationships and linkages between Cd(II)-induced oxidative stress and the consequent damage at the genomic level (followed by DNA replication stress), root apical meristem (RAM) cells in broad bean (V. faba) seedlings exposed to CdCl2 treatment and to post-cadmium recovery water incubations were tested with respect to H2O2 production, DNA double-strand breaks (γ-phosphorylation of H2AX histones), chromatin morphology, histone H3S10 phosphorylation on serine (a marker of chromatin condensation), mitotic activity, and EdU staining (to quantify cells typical of different stages of nuclear DNA replication). In order to evaluate Cd(II)-mediated epigenetic changes involved in transcription and in the assembly of nucleosomes during the S-phase of the cell cycle, the acetylation of histone H3 on lysine 5 (H3K56Ac) was investigated by immunofluorescence. Cellular responses to cadmium (II) toxicity seem to be composed of a series of interlinked biochemical reactions, which, via generation of ROS and DNA damage-induced replication stress, ultimately activate signal factors engaged in cell cycle control pathways, DNA repair systems, and epigenetic adaptations.

In stomach, metaplasia can arise from differentiated chief cells that become mitotic via paligenosis, a stepwise program. In paligenosis, mitosis initiation requires reactivation of the cellular energy hub mTORC1 after initial mTORC1 suppression by DNA damage induced transcript 4 (DDIT4 aka REDD1). Here, we use DDIT4-deficient mice and human cells to study how metaplasia increases tumorigenesis risk.
A tissue microarray of human gastric tissue specimens was analyzed by immunohistochemistry for DDIT4. C57BL/6 mice were administered combinations of intraperitoneal injections of high-dose tamoxifen (TAM) to induce spasmolytic polypeptide-expressing metaplasia (SPEM) and rapamycin to block mTORC1 activity, and N-methyl-N-nitrosourea (MNU) in drinking water to induce spontaneous gastric tumors. Stomachs were analyzed for proliferation, DNA damage, and tumor formation. CRISPR/Cas9-generated DDIT4-/- and control human gastric cells were analyzed for growth in vitro and in xenografts with and without 5-fluorouracil (5-FU) treatment.
DDIT4 was expressed in normal gastric chief cells in mice and humans and decreased as chief cells became metaplastic. Paligenotic Ddit4-/- chief cells maintained constitutively high mTORC1, causing increased mitosis of metaplastic cells despite DNA damage. Lower DDIT4 expression correlated with longer survival of patients with gastric cancer. 5-FU-treated DDIT4-/- human gastric epithelial cells had significantly increased cells entering mitosis despite DNA damage and increased proliferation in vitro and in xenografts. MNU-treated Ddit4-/- mice had increased spontaneous tumorigenesis after multiple rounds of paligenosis induced by TAM.
During injury-induced metaplastic proliferation, failure of licensing mTORC1 reactivation correlates with increased proliferation of cells harboring DNA damage, as well as increased tumor formation and growth in mice and humans.

Advanced techniques allow investigating cellular DNA damage measurements. Ionizing radiation produces multiple DNA damages. Among them, DNA double strand breaks are most toxic to cells. DSBs can form mutations, chromosome aberrations, and cell killing. Although DSBs in cells can be detected directly by neutral elution, pulse field gel electrophoresis, and premature chromosome condensation, recent technologies like cellular immunocytochemistry-based fluorescence detection allow us to visualize the DSBs in cells. Here, we describe gamma-H2AX and Rad51 focus formation assay, which play an important role in DNA damage responses.