Relationship between serum reactive oxidative metabolite level and skin reaction in an irradiated rat model

Abstract

Purpose. Ionizing radiation generates free radicals and reactive oxygen species that induce DNA damage in vivo. This study aimed to determine the relationship between serum reactive oxygen metabolite (ROM) levels and skin reaction after irradiation in a rat model. Methods and materials. I. Female Wistar rats were classified into 0 Gy (control), 2 Gy, and 30 Gy groups; serum ROM levels were measured in the very acute phase. II. Other female Wistar rats were classified into 0 Gy (control), 30 Gy, 50 Gy, and 70 Gy groups; serum ROM levels were measured before and 3, 7, 16, 24, 31, and 38 days after irradiation. Skin reaction was evaluated according to the SRS (0–5) twice every week. Results. Serum ROM levels in the subacute phase were significantly higher in the 50 and 70 Gy groups than in the 0 and 30 Gy groups [p = 0.029, repeated-measure analysis of variance (ANOVA)]. As expected, SRSs increased in the order of the 0 Gy, 30 Gy, 50 Gy, and 70 Gy groups and differed significantly among these groups (p < 0.001, repeated-measure ANOVA). Peak serum ROM levels were observed 16 days after irradiation in all irradiated groups and corresponded with the appearance of visible skin reaction after irradiation. Conclusions. Serum ROM levels may be useful for evaluating radiation damage in mammals. Further investigations are required to investigate changes in intracellular metabolism after irradiation at gene and protein levels.     

The effects of high-power microwaves on the ultrastructure of Bacillus subtilis

Aims:  To investigate the microbicidal mechanisms of high-power microwave (2·0 kW) irradiation on Bacillus subtilis and to determine the effect of this procedure on the ultrastructure of the cell wall.
Methods and Results:  We performed viability test, examined cells using transmission electron microscopy (TEM), and measured the release of intracellular proteins and nucleic acids. The inactivation rate of B. subtilis by 2·0-kW microwave irradiation was higher than that of a domestic microwave (0·5 kW). Few proteins were released from either microwaved or boiled cells. However, the leakage of nucleic acids from 2·0-kW-microwaved cells was significantly higher than that of 0·5-kW-microwaved or boiled cells. Therefore, we examined ultrastructural alterations of microwaved or boiled cells to analyse the pattern of release of cytoplasmic contents. Although boiled cells did not show any ultrastructural changes on TEM, 2·0-kW-microwaved cells showed disruption of the cell wall.
Conclusion:  The microbicidal mechanisms of 2·0-kW microwave irradiation include damage to the microbial cell wall, breakage of the genomic DNA, and thermal coagulation of cytoplasmic proteins.
Significance and Impact of the Study:  TEM images showed that the cytoplasmic protein aggregation and cell envelope damage by microwave irradiation were different from the ultrastructural changes observed after boiling.     

Morphometry of the FRTL-5 cells after irradiation

The morphological changes of in vitro irradiated FRTL-5 cells and their ability to grow in semi-solid medium were studied morphometrically. FRTL-5 cells were grown in medium with 4 different concentrations of TSH (0, 0.1, 1, 10 mU/ml). After irradiation with 0 Gy, 2 Gy and 4 Gy, the cells were seeded on glass cover-slips and in methocel. Fourteen days after irradiation, the morphometric analysis of FRTL-5 cells and their nuclei was performed. The results showed that irradiation and different doses of TSH have influence on FRTL-5 cell size, more on their nuclei than on the cells as a whole. Growing of FRTL-5 cells in the methocel indicates the possible transformation of these cells after long-culturing in the TSH medium and after irradiation.     

The effect of amphotericin B on the ultrastructure of Prototheca species

Prototheca zopfii and Prototheca wickerhamii strains were exposed to subinhibitory concentrations of the antimycotic amphotericin B, and the effect of the treatment on their ultrastructure was assessed. The results revealed ultrastructural changes in the treated cells, expressed by swelling of mitochondria, degradation of cell organelles, accumulation of microbody like structures, lipid droplets and starch granules in the cytoplasm, and changes in the inner layer of the cell wall.     

Destruction of Pigments and Ultrastructural Changes in Cyanobacteria during Photodamage

Sequential stages of pigment degradation and ultrastructural changes were examined in cyanobacteria Anabaena variabilis ATCC 29413, Synechococcus sp. PCC 6301 (Anacystis nidulans) and S. elongatus B-267 during irradiation of cell suspensions with high-intensity light. Early manifestations of photooxidative destruction were evident as profound changes in ultrastructure of thylakoids; in A. variabilis these changes appeared even before bleaching of pigments. Concomitant to these alterations, the cytoplasmic matrix turned homogenous and the nucleoid was subject to degradation, while ultrastructural changes of cytoplasmic membrane and cell walls became evident in some species. In A. variabilis these changes were related to a subsequent autolysis of cells. Synechococcus strains demonstrated comparatively high resistance to irradiation. The experimental data were compared with previously described behavior of the same species of cyanobacteria cultured under photooxidative conditions. This comparison revealed principal similarity and species-specific features in the destructive changes of thylakoids and other cell components of cyanobacterial cells.     

Astereological analysis of FRTL-5 cells as an effect of thyrotropin and irradiation

FRTL-5 cell line is a cloned epithelial follicular cell line from Fischer rat thyroids. This cell line expresses many of the thyroid differentiated markers in vitro. Their growth and function depend on thyrotropin (TSH) as the main regulatory hormone. In this astereological analysis, the authors concentrate on FRTL-5 nuclei as the most vulnerable part of the cell. Using morphometrical variables, they wished to discover the morphologically identifiable sign of transformation of FRTL-5 cells after irradiation and to study the effect of different TSH concentrations. FRTL-5 cells were grown in a medium of 4 different concentrations of TSH (0, 0.1, 1, 10 mU/ml) and irradiated with 0 Gy, 2 Gy, and 4 Gy. The results showed that the nuclear-cytoplasmic ratio decreases after irradiation with doses of 4 Gy or if TSH was included in the medium. The nuclear maximum diameter of FRTL-5 cells increased with higher concentrations of TSH more obviously after irradiation with 4 Gy than with 2 Gy. On the basis of astereological analysis, it was concluded that different concentrations of TSH and irradiation exert an effect especially upon FRTL-5 cell nuclei. The possible transformation of FRTL-5 cells after culturing in TSH medium and after irradiation could be confirmed by injection into an animal of the Fischer strain.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Proton irradiation induced reactive oxygen species promote morphological and functional changes in HepG2 cells

The increased use of proton therapy has led to the need of better understanding the cellular mechanisms involved. The aim of this study was to investigate the effects induced by the accelerated proton beam in hepatocarcinoma cells. An existing facility in IFIN-HH, a 3 MV Tandetron? accelerator, was used to irradiate HepG2 human hepatocarcinoma cells with doses between 0 and 3 Gy. Colony formation was used to assess the influence of radiation on cell long-term replication. Also, the changes induced at the mitochondrial level were shown by increased ROS and ATP levels as well as a decrease in the mitochondrial membrane potential. An increased dose has induced DNA damages and G₂/M cell cycle arrest which leads to caspase 3/7 mediated apoptosis and senescence induction. Finally, the morphological and ultrastructural changes were observed at the membrane level and the nucleus of the irradiated cells. Thus, proton irradiation induces both morphological and functional changes in HepG2 cells.     

Increased G2 delay in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc

Cell cycle perturbation after irradiation was studied in five cell lines transfected with oncogenes. Two immortalized, radio-sensitive cell lines with D0s of 1.06 and 1.08 Gy were compared to three radioresistant cell lines with D0s of 1.68-2.17 Gy. The sensitive cell lines were transfected with the v-myc or c-myc oncogenes, the resistant cell lines with the v-myc plus H-ras oncogenes. Exponentially growing populations were exposed to 5, 10, or 15 Gy of orthovoltage radiation. The percentage of cells in each phase of the cell cycle was determined at various times after irradiation using flow cytometry. All cell lines underwent a dose-dependent arrest in G2 phase after irradiation, but the resistant cell lines underwent a significantly longer arrest in G2 phase after irradiation than did the sensitive cell lines. In conjunction with other results from our laboratories, we suggest that this difference in G2 arrest may be the basis for the increased resistance of cells transfected with oncogenes to irradiation.     

The effect of He-Ne laser radiation on the survival of HeLa cells subjected to ionizing radiation]

A monolayer of HeLa cells, at the stationary phase of growth, exposed to He-Ne laser radiation (632.8 nm; 100 J/m2) either 5 min or 60 min prior to gamma irradiation (0.1-10 Gy; 6.75 Gy/min), or 5 min after irradiation has been investigated. With a 5-min interval between irradiation sessions (both sequences) the survival curves are virtually the same as those for gamma-irradiated cells only. With He-Ne laser radiation delivered 60 min before gamma irradiation with doses exceeding 5 Gy, a fraction of radioresistant cells is identified whose D0 is almost twice as high as D0 of basic cell mass (3.6 and 1.7 Gy respectively. The survival curve becomes a two-component one. A hypothesis is proposed that He-Ne laser radiation activates, in some cells, the processes that promote the repair of radiation damages.     

Low-Dose Electron-Beam Irradiation for the Improvement of Biofilm Formation by Probiotic Lactobacilli

The effects of 50–150 gray electron-beam irradiation on the biofilm-formation ability and cell surface hydrophobicity of the commercial strain, Lactobacillus acidophilus DDS®-1, from Lacto-G (a marketed synbiotic formulation) and the putative probiotic, L. rhamnosus Vahe, were evaluated. No significant changes in cell surface hydrophobicity were found after irradiation, while increases in biofilm-formation abilities were documented for both investigated microorganisms 0.22 ± 0.03 vs. 0.149 ± 0.02 (L. rhamnosus Vahe, 150 Gy) and 0.218 ± 0.021 vs. 0.17 ± 0.012 (L. acidophilus DDS®-1, 150 Gy). Given this, the use of electron-beam irradiation (50–100 Gy) for the treatment of L. rhamnosus Vahe and L. acidophilus DDS®-1 cells may be considered in product sterilization, quality improvement, and packaging practices.

     

Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

氧离子辐射对体外人精子自发化学发光、运动性、顶体反应和存活率的影响(英文)

我们研究了不同剂量(0,0.25,0.5,1,2,4,8,16,32,64Gy)3.17MeV/u氧离子对体外人精子自发化学发光(SCL)、运动性,顶体反应(AR)和存活率的影响。结果表明,精子SCL随辐射剂量明显增强,最低有效剂量为0.5Gy;精子运动性在0.5—2Gy,AR在0.5—4Gy辐射组均明显高于其对照组,但均随辐射剂量的进一步增加而明显降低;在0.25—8Gy剂量之间,精子存活率与其对照组相比无明显变化,但当剂量进一步增加至16—64Gy时,存活率急剧下降。这些结果提示,低剂量重离子辐射能对精子运动性及AR等功能产生兴奋效应。而大剂量则明显抑制这些功能,甚至造成精子死亡。其作用机制可能与重离子辐射诱导的自由基反应有关。     

Langerhans cell number and morphology in mouse footpad epidermis after X irradiation

Effects of 250-kV X rays on epidermal Langerhans cells were studied in CBA/CaH mice. One group received 20 Gy to the feet, another 8 Gy to the whole body, and a third both the 8 Gy whole-body and a 12 Gy local dose to the feet. Mice from each group and controls were sacrificed at intervals from 1 to 64 days later. ATPase-positive cells in sheets of footpad epidermis were counted by light microscopy. The density of Langerhans cells in controls was 1515 +/- 36/mm2 (mean +/- SE; n = 34). By 3 days after irradiation they became rounded and less dendritic and numbers gradually reached a nadir by 10 days, at 18% of controls after 20 Gy and 57% of controls after 8 Gy. Some of the remainder exhibited bizarre morphology and ultrastructural abnormalities. After local irradiation of the feet Langerhans cell numbers recovered rapidly between 14 and 16 days, although their distribution was uneven until 30 days after irradiation. Repopulation was delayed after an 8 Gy whole-body dose by at least 3 weeks. These results demonstrate that high local doses of X rays substantially but transiently deplete the epidermal Langerhans cell population and support the hypothesis that functional hemopoietic tissue is required for extensive Langerhans cell replenishment.     

The effect of graded doses of fission neutrons or X rays on the lymphoid compartment of the thymus in mice

Young adult CBA/H mice were exposed to graded doses of whole-body irradiation with either fast fission neutrons or 300 kVp X rays at center-line dose rates of 0.1 and 0.3 Gy/min, respectively. Dose-response curves were determined at Days 2 and 5 after irradiation for the total thymic cell survival and for the survival of thymocytes defined by monoclonal anti-Thy-1, -Lyt-1, -Lyt-2, and -T-200 antibodies as measured by flow cytofluorometric analysis. Cell dose-response curves of thymocytes show, 2 days after irradiation, a two-component curve with a radiosensitive part and a part refractory to irradiation. The radiosensitive part of the dose survival curve of the Lyt-2+ cells, i.e., mainly cortical cells, has a D0 value of about 0.26 and 0.60 Gy for neutrons and X rays, respectively, whereas that of the other cell types has corresponding D0 values of about 0.30 and 0.70 Gy. The radiorefractory part of the dose-response curves cannot be detected beyond 5 days after irradiation. At that time, the Lyt-2+ cells are again most radiosensitive with a D0 value of 0.37 and 0.99 Gy for neutrons and X rays, respectively. The other measured cell types have corresponding D0 values of about 0.47 Gy. The fission neutron RBE values for the reduction in the thymocyte populations defined by either monoclonal anti-Thy-1, -Lyt-1, -Lyt-2, or -T-200 antibodies to 1.0% vary from 2.6 to 2.8. Furthermore, the estimated D0 values of the Thy-1-, T-200- intrathymic precursor cells which repopulate the thymus during the bone marrow independent phase of the biphasic thymus regeneration after whole-body irradiation are 0.64-0.79 Gy for fission neutrons and 1.32-1.55 Gy for X rays.     

Effect of 12C+5 ion beam irradiation on cell viability and plant regeneration in callus,protoplasts and cell suspensions of Lavatera thuringiaca

Summary The biological effects of irradiation with¹²C⁺⁵ ion beam on plant cells have been analyzed. Protoplasts and cell suspensions ofLavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis +Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with¹²C⁺⁵ ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50 %, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the¹²C⁺⁵ ion beam in asymmetric protoplast fusion experiments is discussed.     

Effect of γ-radiation on development,yield and quality of microtubers <Emphasis Type="Italic">in vitro</Emphasis> in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Explants obtained from in vitro-propagated plantlets of two potato cultivars, Shepody and Atlantic, were treated with five doses of γ-radiation (0, 2, 4, 6 and 8 Gy) to investigate the stimulating effects of low irradiation on the production and quality of microtubers in vitro. Microtubers of both cultivars treated with γ-radiation initiated 5 d earlier than in the non-irradiated control. The whole period of microtuberization was prolonged by 10 – 15 d with 4, 6 and 8 Gy irradiation treatment for cv. Atlantic. Irradiation of the plantlets (4 Gy) led to a significant increase not only in the microtuber number (116.7 and 34.5 % over the control) but also in the fresh mass (77.6 and 23.2 % in Shepody and Atlantic, respectively). Low dose irradiation (2 – 4 Gy) increased the starch content of microtubers. High doses (6 – 8 Gy) enhanced ascorbic acid and reducing sugar contents. 4 – 6 Gy doses also effectively increased the protein contents of microtubers.     

A morphometric study on the effects of ethylene treatment in promoting abscission of tobacco flower pedicels

The ultrastructural changes observed in ethylene-induced abscission of tobacco flower pedicels (Nicotiana tabacum L. `Little Turkish') were studied by the techniques of morphometric analysis. The surface area of the membranes, relative volume of the organelles, and the number of organelles were determined for both ethylene-treated and control cells. In pedicels exposed to ethylene for 4.5 to 5 hours, abscission was evident within the separation zone. The most significant change in cell structure was observed in the surface area of the rough endoplasmic reticulum which more than doubled with ethylene treatment of the tissue.     

mRNA Expression Profiles for Prostate Cancer following Fractionated Irradiation Are Influenced by p53 Status

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy × 10, 1 Gy × 10, and 2 Gy × 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 × 10^-73, 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 × 10^-30) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.     

ULTRASTRUCTURAL ALTERATIONS IN OAK LEAVES PARASITIZED BY TAPHRINA CAERULESCENS

Leaves of Quercus velutina Lam. parasitized by Taphrina caerulescens (Desm.) Tulanse were examined with the electron microscope to ascertain cellular aberrations that were induced by the fungus. Major changes were most apparent in the lower epidermis, where cellular divisions and enlargement were common, the cell walls becoming irregularly thickened. Many of the ultrastructural features of the diseased cells resembled those of meristematic cells; the absence of a large central vacuole, a disproportionately large nucleus, and an abundance of cytoplasmic organelles. Chloroplasts with large amounts of starch were also routinely observed.