Differentiation and delayed cell death in embryonal stem cells exposed to low doses of ionising radiation

Embryonal stem cells have been used to study the effects of environmentally relevant doses of radiation on cell death and differentation. The ES cells were found to have a greater than 60% chance of surviving the traversal of a single alpha-particle, the lowest possible dose of high linear energy transfer radiation a cell may receive. The ES cells appeared to possess the cell cycle checkpoints believed to prevent the transmission of the radiation damage. However, delayed effects were observed in the progeny. An increased incidence of apoptosis and haempoietic differentiation capacity was found to persist in the ES cell population over many cell divisions. Since both cell death and differentiation are known to play a key role in tissue kinetics, an ES cell model will provide a valuable and versatile cell system for studying the role of cell death and differentiation in the pathology of radiogenic diseases.     

G2 chromatid damage and repair kinetics in normal human fibroblast cells exposed to low- or high-LET radiation

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high-LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle.     

Sister-chromatid exchange and cell kinetics in CHO-K1 cells, human fibroblasts and lymphoblastoid cells exposed in vitro to asbestos and glass fibre

Possible mutagenic activity of the asbestos dusts crocidolite and chrysotile, and fine and coarse glass, was assessed in CHO-K1 cells, human fibroblasts and human lymphoblastoid cells using the sister-chromatid exchange assay and by examining the effects on cell kinetics. Asbestos caused no dose-related increase in sister-chromatid exchange levels in any of the cell types. However, mitotic delay was induced in CHO-K1 cells and human fibroblasts. The order of magnitude of induced delay in CHO-K1 cells was chrysotile greater than fine glass greater than crocidolite greater than coarse glass. Mitotic inhibition was more pronounced in these cells if they were still in suspension when initially exposed to the dusts compared with 1 h after plating.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to low doses of heavy-ion radiation

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1-4Gy) of high-LET IR (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.     

Sister chromatid exchange frequency in human lymphocytes exposed to ionizing radiation in vivo and in vitro

This paper reports statistically significant elevations in peripheral blood lymphocyte sister chromatid exchange frequencies in persons occupationally exposed to low levels of ionizing radiation when compared with unexposed persons. Low doses of X or gamma rays administered in vitro also produce significant elevations in sister chromatid exchange frequencies, though the magnitude of the increases is dependent upon culture medium and other factors.     

The genetic basis of resistance to ionising radiation damage in cultured mammalian cells

To test the genetic similarity of independently-isolated hamster cell mutants sensitive to ionising radiation, these were fused in pairs and the hybrids exposed to X-rays. Some mutants (irs1, irs3, xrs-1, XR-1, BLM2) were found to complement all others tested for radiosensitivity in hybrids, and are therefore in separate genetic groups. The mutants irs2 and V-E5, both isolated from V79 cells, did not complement and therefore belong to the same group. Another pair, EM7 and irs1SF, formed hybrids with intermediate levels of survival between mutant and wild-type. However, the parental cells fused to irs1SF also showed intermediate sensitivity, suggesting a semi-dominant mutant phenotype rather than a lack of complementation. Crosses of some of these hamster mutants to the radiosensitive mouse mutant M10 showed clear complementation (irs1 x M10, irs2 x M10) but for others the complementation did not greatly exceed the sensitivity of one (irs3 x M10) or both mutants (XR-1 x M10). Taken with our previously-published data, these results show that there are at least 8 genetic groups determining resistance to ionising radiation damage in rodent cells.     

DNA damage in cells exposed to ionizing radiation

Ionizing radiation induces variety of structural lesions in DNA of irradiated organisms. Their formation depends largely on the degree of cell oxygenation, the level of endogenous antioxidants, on DNA-protein complexes and compactization of DNA in the chromatin and activity of DNA repair systems. All ionizing radiation-induced DNA lesions can arbitrarily be divided into two groups. Group 1 includes singly damaged sites (single-sites): base modification, single-strand breaks, alkaline-labile sites (including a basic sites). Group 2 contains: locally multiply damaged sites (clustered lesions), double-strand breaks, intermolecular cross-links. The yields of lesions of group 2 increases with high linear energy transfer of radiation and these lesions play a dominant role in the radiation death, formation of chromosome and gene mutations, cell transformation.     

mFISH analysis of chromosome aberrations in workers occupationally exposed to mixed radiation

We performed a study on the presence of chromosome aberrations in a cohort of plutonium workers of the Mayak production association (PA) with a mean age of 73.3 ± 7.2 years to see whether by multi-color fluorescence in situ hybridization (mFISH) translocation analysis can discriminate individuals who underwent occupational exposure with internal and/or external exposure to ionizing radiation 40 years ago. All Mayak PA workers were occupationally exposed to chronic internal alpha-radiation due to incorporated plutonium-239 and/or to external gamma-rays. First, we obtained the translocation yield in control individuals by mFISH to chromosome spreads of age-matched individuals and obtained background values that are similar to previously published values of an international study (Sigurdson et al. in Mutat Res 652:112–121, 2008). Workers who had absorbed a total dose of >0.5 Gy external gamma-rays to the red bone marrow (RBM) displayed a significantly higher frequency of stable chromosome aberrations relative to a group of workers exposed to <0.5 Gy gamma-rays total absorbed RBM dose. Thus, the translocation frequency may be considered to be a biological marker of external radiation exposure even years after the exposure. In a group of workers who were internally exposed and had incorporated plutonium-239 at a body burden >1.48 kBq, mFISH revealed a considerable number of cells with complex chromosomal rearrangements. Linear associations were observed for translocation yield with the absorbed RBM dose from external gamma-rays as well as for complex chromosomal rearrangements with the plutonium-239 body burden.     

Cytogenetic analysis of peripheral blood lymphocytes of occupational workers exposed to low levels of ionising radiation.

The incidence of chromosomal aberrations was analysed in peripheral blood lymphocytes of occupationally exposed people having cumulative doses of 500 mSv. The exposed individuals showed higher frequencies of dicentrics as well as acentrics than normal controls. Absorbed radiation dose was calculated by using in vitro dose response curve established for Cobalt-60 gamma rays. In the control constituting 17 healthy individuals, two dicentrics were detected among 3700 metaphases analysed. In the exposed group 27 dicentrics and one centric ring was detected among 8400 metaphases analysed. Due to small number of dicentrics scored in each individual, the dose estimate suffers from a large statistical uncertainty. The collective dose was found to be 1.89 Gy. This is in good agreement with the corrected physical doses, assuming a mean life of 10 years for the disappearance of lymphocytes. The physical doses accumulated during the last 10 years of occupation were also in good agreement with the biological dose estimate.     

Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.     

Ultrastructural alterations in ciliary cells exposed to ionizing radiation

Summary Early effects of ionizing radiation were investigated in an experimental in vitro system using the ciliary cells of the tracheal mucous membrane of the rabbit, irradiated at 30° C and at more than 90% humidity. The changes in physiological activities of the ciliary cells caused by irradiation were continuously registered during the irradiation. The specimens were examined immediately after irradiation electron microscopically. The morphological changes in irradiated material after 10–70 Gy are compared with normal material. After 40–70 Gy, scanning electron microscopy revealed the formation of vesicles on cilia, and club-like protrusions and adhesion of their tips. After 30–70 Gy, a swelling of mitochondrial membranes and cristae was apparent transmission electron microscopically. The membrane alterations caused by irradiation are assumed to disturb the permeability and flow of ATP from the mitochondria, which in turn leads to the recorded changes in the activity of the ciliated cells.This investigation was supported by grants from Konung Gustaf V:s Jubileumsfond, John and Augusta Perssons Stiftelse, B. Kamprads Fond, the Faculty of Medicine, University of Lund, Sweden and the Swedish Medical Research Council (No. B77-17X-03897-05)The authors are greatly indebted to Miss Inger Norling, Miss Marianne Palmegren and Miss Birgitta Sandström for their excellent technical assistance     

Comparison of UV-induced unscheduled DNA synthesis in lymphocytes exposed to low doses of ionising radiation in vivo and in vitro

In an attempt to understand and ascertain the stimulatory effects of low-dose ionising radiation, a study was conducted to compare the changes in the UV-induced repair capacity of human blood cells exposed to low conditioning doses of ionising radiation under in vivo and in vitro conditions. A significant increase in the rate of UV induced Unscheduled DNA synthesis (UDS) in lymphocytes pre-exposed to low doses of ionising radiation was observed both under in vitro and in vivo conditions. There was also a significant correlation between the adapting dose and net UDS in lymphocytes of radiation workers implying that the triggering action of the adaptation process is dose dependent. However, on comparing the extent of UV-induced UDS of the in vivo and in vitro exposures, significantly higher rates of UDS were observed in the lymphocytes of radiation workers when compared to a corresponding in vitro adapting dose. We postulate that the response in vivo is much more pronounced due to cell repopulating events and extra cellular secretory factors like hormones etc,.