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.

Ascorbic Acid 2-Glucoside Pretreatment Protects Cells from Ionizing Radiation, UVC, and Short Wavelength of UVB.

Ascorbic acid 2-glucoside (AA2G), glucosylated ascorbic acid (AA), has superior properties for bioavailability and stability compared to AA. Although AA2G has shown radioprotective properties similar to AA, effects for UV light, especially UVC and UVB, are not studied. AA2G was tested for cytotoxicity and protective effects against ionizing radiation, UVC, and broadband and narrowband UVB in Chinese hamster ovary (CHO) cells and compared to AA and dimethyl sulfoxide (DMSO). Pretreatment with DMSO, AA, and AA2G showed comparative protective effects in CHO wild type and radiosensitive xrs5 cells for cell death against ionizing radiation with reducing the number of radiation-induced DNA damages. Pretreatment with AA and AA2G protected CHO wild type and UV sensitive UV135 cells from UVC and broadband UV, but not from narrowband UVB. DMSO showed no protective effects against tested UV. The UV filtration effects of AA and AA2G were analyzed with a spectrometer and spectroradiometer. AA and AA2G blocked UVC and reduced short wavelengths of UVB, but had no effect on wavelengths above 300nm. These results suggest that AA2G protects cells from radiation by acting as a radical scavenger to reduce initial DNA damage, as well as protecting cells from certain UVB wavelengths by filtration.

Intrinsic Radiosensitivity and Cellular Characterization of 27 Canine Cancer Cell Lines.

Canine cancer cell lines have progressively been developed, but are still underused resources for radiation biology research. Measurement of the cellular intrinsic radiosensitivity is important because understanding the difference may provide a framework for further elucidating profiles for prediction of radiation therapy response. Our studies have focused on characterizing diverse canine cancer cell lines in vitro and understanding parameters that might contribute to intrinsic radiosensitivity. First, intrinsic radiosensitivity of 27 canine cancer cell lines derived from ten tumor types was determined using a clonogenic assay. The 27 cell lines had varying radiosensitivities regardless tumor type (survival fraction at 2 Gy, SF2 = 0.19-0.93). In order to understand parameters that might contribute to intrinsic radiosensitivity, we evaluated the relationships of cellular radiosensitivity with basic cellular characteristics of the cell lines. There was no significant correlation of SF2 with S-phase fraction, doubling time, chromosome number, ploidy, or number of metacentric chromosomes, while there was a statistically significant correlation between SF2 and plating efficiency. Next, we selected the five most radiosensitive cell lines as the radiosensitive group and the five most radioresistant cell lines as the radioresistant group. Then, we evaluated known parameters for cell killing by ionizing radiation, including radiation-induced DNA double strand break (DSB) repair and apoptosis, in the radiosensitive group as compared to the radioresistant group. High levels of residual γ-H2AX foci at the sites of DSBs were present in the four out of the five radiosensitive canine cancer cell lines. Our studies suggested that substantial differences in intrinsic radiosensitivity exist in canine cancer cell lines, and radiation-induced DSB repair was related to radiosensitivity, which is consistent with previous human studies. These data may assist further investigations focusing on the detection of DSB for predicting individual response to radiation therapy for dogs, regardless of tumor type.