Molecular alterations produced in bacterial cells by ionizing radiation

Biochemical effects of high doses of 0.8 Mev electrons onEscherichia coli B were studied using infrared spectroscopy (IR). Aqueous suspensions of the bacterial cells were irradiated in open petri dishes. After exposure, films of these cells were examined for absorption of light between 4000 cm^–1 to 600 cm^–1. The qualitative aspects of the changes in the absorption spectra indicative of molecular alteration were noted and attempts were made to interpret them. The damage is selective in that some molecular groups are affected more than others. In general the changes indicate breakup of biopolymers and overall oxidation. All exposure doses given were above 1.0×10⁶ Roentgen.     

DNA repair deficiency and BPDE-induced chromosomal alterations in CHO cells

The induction of chromosomal aberrations and sister chromatid exchanges by BPDE was evaluated in parental and different DNA repair deficient Chinese hamster ovary cell lines in order to elucidate the mechanisms involved in their induction. These included the parental line (AA8), nucleotide excision repair (UV4, UV5, UV61), base excision repair (EM9), homologous recombination repair (Irs1SF) and non-homologous end joining (V3-3) deficient ones. The ranking of different cell lines for BPDE-induced chromosome aberrations was: UV4, Irs1SF, UV5, UV 61, EM9, V3-3, and AA8 in a descending order. Cells deficient in NER and HRR were found to be very sensitive, indicating the importance of these pathways in the repair of lesions induced by BPDE. For induction of SCEs, HRR and BER deficient cells were refractory, whereas the other cell lines responded with a dose-dependent increase. The possible mechanisms involved in BPDE-induced chromosomal alterations are discussed.     

The induction by ionizing radiation of chromosomal aberrations in rhesus monkey pre-meiotic germ cells: effects of dose rate and radiation quality

The induction of reciprocal translocations in rhesus monkey stem-cell spermatogonia was studied using multivalent analysis at metaphase of primary spermatocytes. Animals were exposed to 1 Gy gamma-rays at dose rates of 140 and 0.2 mGy/min or to 0.25 Gy acute 2 MeV neutrons. Reduction of the dose rate from 140 mGy/min to 0.2 mGy/min did not result in a lowering of the frequencies of recovered translocations of 0.43%. The neutron data indicated an RBE (neutrons vs. X-rays) of 2.1, which is clearly lower than the value of 4 obtained in the mouse. It is made plausible that in general mammalian species with high sensitivities for the cytotoxic effects of ionizing radiation, such as the rhesus monkey, will exhibit relatively high threshold dose rates below which no further reduction in aberration yield occurs, whereas in more resistant species, such as the mouse, the threshold dose rate will be at a very low level. Similarly, resistant species will show relatively high RBE values for neutron irradiation and sensitive species low ones.     

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     

Critical target and dose and dose-rate responses for the induction of chromosomal instability by ionizing radiation.

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min(-1)). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around approximately 30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the gamma-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min(-1)) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.     

Mutational alterations induced in yeast by ionizing radiation

The cyc1-9 ochre (UAA) mutant and the cyc1-179 amber (UAG) mutant of the yeast Saccharomyces cerevisiae were reverted with X-rays and -particles. The amino acid sequence changes of iso-1-cytochromes c from 36 of the intragenic revertants were determined by amino acid analysis and peptide mapping, aided by partial amino acid sequencing of 4 revertants. In addition, the DNA segments encompassing 3 unusual mutations with complex changes were cloned and sequenced. This study and previous studies of 16 other revertants of cyc1-9 and cyc1-179 revealed that ionizing radiation primarily induces single base-pair substitutions; 47 of the 52 revertants arose by transversions and transitions without any apparent preference. However, the A·T→T·A substitution at the first base pair for the cyc1-179 UAG codon, leading to the normal protein, was not detected, nor was it found previously in 32 revertants of cycl-179 obtained spontaneously or induced with various other mutagens; apparently, there is a prohibition of certain base-pair substitutions at certain sites in DNA. In addition, 5 of the 52 revertants arose by multiple changes within a short region of 11 base pairs. These consisted of the deletion of 6 base pairs, the substitution of 3 base pairs, and 3 different kinds of substitutions of two base pairs. Compared to other mutagens previously tested with the cyc1 system, ionizing radiation produces the most random types of base-pair substitutions.     

Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells

Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively.     

The influence of incorporated bromodeoxyuridine on mutagenicity testing by sister chromatid exchange induction inVicia faba root tip cells

The induction of sister chromatid exchanges (SCEs) inVicia faba root-tip cells after short-term (2 h) and long-term (24 h) treatments with alkylating agents (N-methyl-N-nitrosourea, ethyl methanesulphonate) and maleic hydrazide was studied. The primary roots were treated with mutagens before or after 5-bromodeoxyuridine (BrdU) incorporation into DNA and the influence of mutagen application on SCE induction in the cells with non- and BrdU-substituted chromosomal DNA. On the contrary, application of maleic hydrazide after the incorporation of BrdU into DNA strongly increased the rate of SCEs. The lowest limit concentrations of mutagens capable of significantly increasing SCE frequency in the cells with non-substituted DNA after the long-term treatment were estimated.     

The induction of chromosome damage in CHO cells by beryllium and radiation given alone and in combination

Studies were conducted to determine the effects of BeSO4 or X rays, alone and in combination, on cell cycle kinetics, cell killing, and the production of chromosome aberrations in Chinese hamster ovary (CHO) cells. The concentration of BeSO4 required to kill 50% of CHO cells exposed to BeSO4 for 20 h was determined to be 1.1 mM with 95% confidence intervals of 0.72 to 1.8 mM. During the last 2 h of the 20-h beryllium treatment (0.2 and 1.0 mM), cells were exposed to 0.0, 1.0, or 2.0 Gy of X rays. Exposure to either BeSO4 or X rays produced a change in cell cycle kinetics which resulted in an accumulation of cells in the G2/M stage of the cell cycle. However, combined exposure to both agents resulted in a block similar to that observed following exposure to X rays only. The background level of chromosome damage was 0.05 +/- 0.015 aberrations/cell in the CHO cells. Seven hours after the end of exposure to 0.2 and 1.0 mM beryllium, 0.03 +/- 0.003 and 0.09 +/- 0.02 aberrations/cell, respectively, were observed. The data for chromosome aberrations following X-ray exposure were fitted to a linear model with a coefficient of 0.14 +/- 0.01 aberrations/cell/Gy. When beryllium was combined with the X-ray exposure the interactive response was predicted by a multiplicative model and was significantly higher (P less than 0.05) than predicted by an additive model. The influence of time after radiation exposure on the interaction between beryllium and X rays was also determined. No interaction between beryllium and X-ray exposure in the induction of chromosome-type aberrations (P greater than 0.05) was detected. The frequency of chromatid-type exchanges and total aberrations was significantly higher (P less than 0.05) in the radiation plus beryllium-exposed cells when compared to cells exposed to X rays only, at both 9 and 12 h after X-ray exposure. These data suggest that the multiplicative interaction may be limited to cells in the S and G2 stages of the cell cycle.     

Influence of sodium butyrate on the induction of radiation-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells.

CHO cells were pre-treated with sodium butyrate (SB) for 24 h and then X-irradiated in G1. Metaphases were scored for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs). The data were compared with those obtained after irradiation of cells not pre-treated with SB and showed that SB has different effects on the endpoints examined. The frequencies of dicentric chromosomes were elevated and of small acentric rings (double minutes, DMs) reduced. These results are discussed to be a consequence of conformational changes in hyperacetylated chromatin which could lead to more interchromosomal and to less intrachromosomal exchanges. SB itself induces a few SCEs but suppresses the induction of SCEs by X-rays. We assume that a minor part of radiation induced SCEs are 'false' resulting from structural chromosomal aberrations, such as inversions, induced in G1. Inversions are the symmetrical counterparts of DMs. If inversions are suppressed by SB treatment to a similar extent as DMs a small reduction of SCEs by SB can be expected.     

Cell-type-dependent induction of eotaxin and CCR3 by ionizing radiation

Eotaxin is an eosinophil-specific C-C chemokine that is implicated in the pathogenesis of eosinophilic inflammatory diseases, such as asthma and atopic dermatitis, by acting specifically on its receptor CCR3. Using RT-PCR analysis, we show that the expression of eotaxin is upregulated upon treatment with ionizing radiation (IR) in human dermal fibroblasts, but not in the bronchial epithelial cell line A549. In contrast, the gene encoding CCR3 is markedly induced in both cell types. None of the genes coding for other CCR3 ligands are significantly induced by IR in these cell types. cDNA array analysis of irradiated versus nonirradiated A549 cells and human dermal fibroblasts confirm and extend these results, and support the observation that regulation of eotaxin/CCR3-induction by IR occurs in a selective and cell-type-dependent manner. They further suggest that the induction of signaling via eotaxin and CCR3 may be an important step leading to eosinophilia in patients with radiation exposure.     

Exogenous catalase introduced in CHO cells by electroporation does not protect against chromosome damage induced by ionizing radiation.

The relative importance of hydrogen peroxide generated as a consequence of irradiation with X-rays for the production of chromosomal aberrations has been studied in cultured CHO cells. Catalase introduced into cells by electroporation protected DNA from strand breakage induced by hydrogen peroxide given 4h later, and the yield of chromosome aberrations was also reduced. Nevertheless, when the cells were irradiated after treatment with catalase following a similar protocol and the yield of chromosomal aberrations analyzed at metaphase, no protective effect was observed as compared with cells treated with X-rays alone. These observations seem to support the hypothesis that hydroxyl radicals generated from hydrogen peroxide are not a major factor responsible for chromosome damage induced by ionizing radiation.     

Different induction of adaptive response to ionizing radiation in normal and neoplastic cells

Since the beneficial effects of low-dose radiation (0.01 Gy) are usually observed in normal cells, we investigated whether the adaptive response was induced by low-dose radiation in neoplastic cells of different origin as well as in normal cells. Cell lines used in this experiment were as follows: mouse lymphocytes (NL); L929 cells established from mouse connective tissue; primary mouse keratinocytes (PK); line 308 from mouse papilloma; X-ray sensitive lymphoma cells, L5178Y-S and EL-4 cells from mouse lymphoma. The adaptive response was determined by cell survival and apoptosis. The involvement of apoptosis in the adaptive response was examined by ELISA and TUNEL assay. Adaptive response was induced by pretreatment with low-dose radiation of 0.01 Gy in normal cells such as NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. In addition, the reduction of apoptosis by pretreatment with low-dose radiation was observed in NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. These results suggested that the adaptive response could be induced by pretreatment with low-dose radiation and the phenomena were observed in normal cells, not in neoplastic cells. In addition, pretreatment with low-dose radiation reduced apoptosis, suggesting that an anti-apoptotic pathway may be involved in the adaptive response. This revised version was published online in July 2006 with corrections to the Cover Date.     

High frequency induction of mitotic recombination by ionizing radiation in Mlh1 null mouse cells

Mitotic recombination in somatic cells involves crossover events between homologous autosomal chromosomes. This process can convert a cell with a heterozygous deficiency to one with a homozygous deficiency if a mutant allele is present on one of the two homologous autosomes. Thus mitotic recombination often represents the second mutational step in tumor suppressor gene inactivation. In this study we examined the frequency and spectrum of ionizing radiation (IR)-induced autosomal mutations affecting Aprt expression in a mouse kidney cell line null for the Mlh1 mismatch repair (MMR) gene. The mutant frequency results demonstrated high frequency induction of mutations by IR exposure and the spectral analysis revealed that most of this response was due to the induction of mitotic recombinational events. High frequency induction of mitotic recombination was not observed in a DNA repair-proficient cell line or in a cell line with an MMR-independent mutator phenotype. These results demonstrate that IR exposure can initiate a process leading to mitotic recombinational events and that MMR function suppresses these events from occurring.     

Proximity effects in chromosome aberration induction by low-LET ionizing radiation

Although chromosome aberrations are known to derive from distance-dependent mis-rejoining of chromosome fragments, evaluating whether a certain model describes such “proximity effects” better than another one is complicated by the fact that different approaches have often been tested under different conditions. Herein, a biophysical model (“BIANCA”, i.e. BIophysical ANalysis of Cell death and chromosome Aberrations) was upgraded, implementing explicit chromosome-arm domains and two new models for the dependence of the rejoining probability on the fragment initial distance, r. Such probability was described either by an exponential function like exp(−r/r₀), or by a Gaussian function like exp(−r²/2σ²), where r₀ and σ were adjustable parameters. The second, and last, parameters was the yield of “Cluster Lesions” (CL), where “Cluster Lesion” defines a critical DNA damage producing two independent chromosome fragments. The model was applied to low-LET-irradiated lymphocytes (doses: 1–4 Gy) and fibroblasts (1–6.1 Gy). Good agreement with experimental yields of dicentrics and centric rings, and thus their ratio (“F-ratio”), was found by both the exponential model (with r₀ = 0.8 μm for lymphocytes and 0.7 μm for fibroblasts) and the Gaussian model (with σ = 1.1 μm for lymphocytes and 1.3 μm for fibroblasts). While the former also allowed reproducing dose-responses for excess acentric fragments, the latter substantially underestimated the experimental curves. Both models provided G-ratios (ratio of acentric to centric rings) higher than those expected from randomness, although the values calculated by the Gaussian model were lower than those calculated by the exponential one. For lymphocytes the calculated G-ratios were in good agreement with the experimental ones, whereas for fibroblasts both models substantially underestimated the experimental results, which deserves further investigation. This work suggested that, although both models performed better than a step model (which previously allowed reproducing the F-ratio but underestimated the G-ratio), an exponential function describes proximity effects better than a Gaussian one.     

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [³H]DNA in alkaline sucrose gradients or by direct assay of the amount of [³H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.     

Actin paracrystal induction by forskolin and by db-cAMP in CHO cells

Forskolin, a hypotensive diterpine, is assumed to be a potent activator of adenylate cyclase leading to increased levels of cAMP. When this drug is used at 10(-5) M on CHO-C14 cells in culture, it induces within 15 min actin paracrystals in all cells. At this time the paracrystals are mostly situated close to the cell periphery. Electron microscopy (EM) shows structures typical of actin paracrystals. Scanning electron microscopy (SEM) reveals a reduction in surface microvilli and blebs. Identical results can be obtained by adding 1 mM db-cAMP to the culture medium directly. The paracrystals are observed within 15 min and thus represent one of the earliest ultrastructural changes so far described for reverse transformation of CHO cells by db-cAMP. The microtubular and vimentin profiles appear unchanged by forskolin treatment of CHO-K1 cells. Out of currently unknown reasons forskolin does not induce the actin transformation in several other commonly used cell lines.