Alkaline Comet Assay to Detect DNA Damage.

The comet assay is an effective method for identifying DNA breaks and alkali-labile sites induced by genotoxins. Performed as a single-cell electrophoresis, this assay is especially simplistic, and the results are easily reproducible. DNA breakage can be quantitatively assessed by the induced comet tail regions, which can be measured using a variety of comet software. This protocol will finish within approximately two hours with adequate preparation, and digitized images can be taken using a confocal or standard fluorescence microscopes after staining the cell nucleus with a DNA dye.

Biological Effects of Monoenergetic Carbon Ions and Their Associated Secondary Particles.

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.

Cytotoxicity and genotoxicity of blue LED light and protective effects of AA2G in mammalian cells and associated DNA repair deficient cell lines.

Light emitting diode (LED) devices emit narrow bands of the blue, green, and red light spectrum rather than the continuous spectrum emitted from sunlight and fluorescent light bulbs. LED devices have become considerably common in society, and the fluence of blue light from LED devices is more intense than other light sources. Previous studies presented that the blue light spectrum may harness potentially inimical genotoxicity. Therefore, the aim of this study was to investigate this potential cytotoxicity and genotoxicity, as well as identify the mechanism of the cellular effects induced by blue LED light exposure in mammalian cell lines with their DNA repair deficient mutants. Our results demonstrated that blue LED light induced both oxidative stress to cells and cytotoxic and genotoxic effects including reduction of clonogenicity, cell cycle arrest, induction of sister chromatid exchanges, endoreduplicated chromosomes, and increased frequency of HPRT locus mutations. In DNA repair deficient cells, particularly those involving double strand break repair deficiency, cells presented hypersensitivity to blue LED light exposure. Blue LED light also induced chromosome aberrations more in DNA repair deficient cells than wild type cells. The cytotoxicity of blue LED light was reduced by an effective antioxidant, ascorbic acid 2-glucoside, which can suppress blue LED light induced oxidative stress. These results indicated that prolonged, high intensity exposure to blue LED light induces genotoxic stress to cells, and oxidative stress induced by blue LED light is targeting DNA to induce these biological effects.

High LET-Like Radiation Tracks at the Distal Side of Accelerated Proton Bragg Peak.

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.

Evaluating the Genotoxic and Cytotoxic Effects of Thymidine Analogs, 5-Ethynyl-2'-Deoxyuridine and 5-Bromo-2'-Deoxyurdine to Mammalian Cells.

BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces gene and chromosomal mutations and induces sensitization to photons, EdU's effects have not been extensively studied yet. Therefore, we investigated EdU's potential cytotoxic and mutagenic effects and its related underlying mechanisms when administered to Chinese hamster ovary (CHO) wild type and DNA repair-deficient cells. EdU treatment displayed a higher cytotoxicity and genotoxicity than BrdU treatment. Cells with defective homologous recombination repair displayed a greater growth delay and severe inhibition of clonogenicity with EdU compared to wild type and other DNA repair-deficient cells. Inductions of sister chromatid exchange and hypoxanthine phosphorybosyl transferase (HPRT) mutation were observed in EdU-incorporated cells as well. Interestingly, on the other hand, EdU did not induce sensitization to photons to the same degree as BrdU. Our results demonstrate that elevated concentrations (similar to manufacturers suggested concentration; >5-10 µM) of EdU treatment were toxic to the cell cultures, particularly in cells with a defect in homologous recombination repair. Therefore, EdU should be administered with additional precautions.