Golden Hamster Embryo Cells Research Articles

Relief of delayed oxidative stress by ascorbic acid can suppress radiation-induced cellular senescence in mammalian fibroblast cells

Ionizing radiation-induced cellular senescence is thought to be caused by nuclear DNA damage that cannot be repaired. However, here we found that radiation induces delayed increase of intracellular oxidative stress after irradiation. We investigated whether the relief of delayed oxidative stress by ascorbic acid would suppress the radiation-induced cellular senescence in Syrian golden hamster embryo (SHE) cells. We observed that the level of oxidative stress was drastically increased soon after irradiation, then declined to the level in non-irradiated cells, and increased again with a peak on day 3 after irradiation. We found that the inductions of cellular senescence after X-irradiation were reduced along with suppression of the delayed induction of oxidative stress by treatment with ascorbic acid, but not when oxidative stress occurred immediately after irradiation. Moreover, treatment of ascorbic acid inhibited p53 accumulation at 3 days after irradiation. Our data suggested a delayed increase of intracellular oxidative stress levels plays an important role in the process of radiation-induced cellular senescence by p53 accumulation.

Discrimination of a transformation phenotype in Syrian golden hamster embryo (SHE) cells using ATR-FTIR spectroscopy

Primary Syrian hamster embryo (SHE) cells might be used to assess morphological transformation following treatment with chemical carcinogens. We employed attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to interrogate SHE colonies, as complex biomolecules absorb in the mid-infrared (IR; λ = 2–20 μm) giving vibrational spectra associated with structure and function. Early-passage SHE cells were cultured (pH 6.7) in the presence or absence of benzo[ a]pyrene (B[ a]P; 5.0 μg/ml). Unstained colonies were applied to an ATR crystal, and vibrational spectra were obtained in the ATR mode using a Bruker Vector 22 FTIR spectrometer with Helios ATR attachment. These were individually baseline-corrected and normalised. Spectra were then analysed using principal component analysis (PCA) plus linear discriminant analysis (LDA). PCA was used to reduce the dataset dimensions before LDA was employed to reveal clustering. This determined whether wavenumber–absorbance relationships expressed as single points (scores) in ‘hyperspace’ might on the basis of multivariate distance reveal biophysical differences associated with morphologies in vehicle control (non-transformed or transformed) or carcinogen-treated (non-transformed or transformed) cells. Retrospectively designated SHE colonies (following staining and microscopic analysis) clustered according to whether they were vehicle control (non-transformed), B[ a]P-treated (non-transformed) or transformed (control and B[ a]P-treated). Scores plots pointed to a B[ a]P-treated phenotype and derived loadings plots highlighted distinguishing markers in control transformed vs. B[ a]P-treated transformed; these were mostly associated with Amide I, Amide II and phosphate stretching (asymmetric and symmetric) vibrations. Combined application of ATR-FTIR spectroscopy and unsupervised (PCA)/supervised (LDA) may be a novel approach to scoring SHE colonies for morphological transformation.

Test for antioxidant ability by scavenging long-lived mutagenic radicals in mammalian cells and by blood test with intentional radicals: an application of gallic acid

Antioxidant ability of gallic acid (GA) are determined both by electron spin resonance measurement of long-lived radicals produced in γ-ray irradiated Syrian golden hamster embryo cells with GA and by hemolysis measurement with GA when blood cells are submitted to radicals. Scavenging properties of GA are determined by the reaction rate constant with long-lived mutagenic radicals in the cells while the blood test allows to analyze the global effects of this compound: radical scavenger+metal ion chelator+regeneration of intra- and extra-cellular antioxidant.

Scavenging of long-lived radicals by (−)-epigallocatechin-3- O-gallate and simultaneous suppression of mutation in irradiated mammalian cells

Effect of (−)-epigallocatechin-3- O-gallate (EGCg) on scavenging long-lived radicals and its biological significance were investigated using electron-spin-resonance spectroscopy and mutation assay in cultured human embryo cells. EGCg scavenged long-lived radicals in irradiated golden hamster embryo cells and albumin solution, and simultaneously reduced mutation frequency in the irradiated human embryo cells. These results indicate that long-lived radials are involved in the induction of mutation by radiation.

Reaction of long-lived radicals and vitamin C in γ-irradiated mammalian cells and their model system at 295 K. Tunneling reaction in biological system

When golden hamster embryo (GHE) cells or concentrated albumin solution (0.1 kg dm −3) that is a model system of cells is irradiated with γ-rays at 295 K, organic radicals produced can be observed by ESR. The organic radicals survive at both 295 and 310 K for such a long time as 20 h. The long-lived radicals in GHE cells and the albumin solution react with vitamin C by the rate constants of 0.007 dm 3 mol −1 s −1 and 0.014 dm 3 mol −1 s −1, respectively. The long-lived radicals in human cells cause gene mutation, which is suppressed by addition of vitamin C. The isotope effect on the rate constant ( k) for the reaction of the long-lived radicals and vitamin C has been studied in the albumin solution by use of protonated vitamin C and deuterated vitamin C. The isotope effect ( k H/ k D) was more than 20 ≈ 50 and was interpreted in terms of tunneling reaction.

Radiation-induced emission from golden hamster embryo cells

Emission from high-energy-electron-irradiated golden hamster embryo (GHE) cells has been studied over the temperature range 12–300 K both by a one-shot-single-photon-counting method and by photocurrent measurements with an oscilloscope. Emission from the irradiated phosphate buffered saline (PBS) also has been studied. The emission spectra from PBS at 12 and 77 K show a maximum around 330 and 380 nm, respectively, which are the same spectra as those from irradiated pure H 2O. The emission from irradiated GHE consists of the new band at 480 nm in addition to the emission from H 2O. The 480 nm emission is observed at the temperature range of 12–300 K, though the emission at 300 K is much lower than that at low temperature. The 480 nm emission is ascribed to the transition from excited organic substances in GHE cells. The intensity of 480 nm emission at 300 K increases linearly with increasing irradiation-dose in the range of 11–600 Gy.

Mutations in ras genes in cells cultured from mouse skin tumors induced by ultraviolet irradiation.

Mutations in ras oncogenes were detected in cultured cells of mouse skin tumors induced by near-UV irradiation. DNA extracted from the UV-induced tumor cells was transfected to golden hamster embryo cells, and focus-forming ability was confirmed in 22 of 26 cell strains, 15 of which had the repetitive mouse sequence. Mouse ras genes were detected in 10 of these 22 cell strains. Point mutations in the ras genes were at Ha-ras codon 13 (GGC-->GTC in two strains, GGC-->AGC in one strain), Ki-ras codon 61 (CAA-->GAA in two strains), and N-ras codon 61 (CAA-->CAT in two strains, CAA-->AAA in two strains). In one tumor cell strain no base change was directed. Most mutations occurred at dipyrimidine sites. Pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproducts are the likely cause of the skin cancers. The base change occurred preferentially at G.C base pairs, and transversions predominated.

Studies on Mechanisms and Blockade of Carcinogenic Action of Female Sex Hormones

Tumours of mice are induced by administration of Inj. Hydroxyprogesteroni Caproatis Co. (EP) in a practical subthreshold dose of carcinogenesis or 2. 5-5 times the human contraceptive dose (simply referred to as 2. 5- to 5-fold dose) combined with whole-body 0. 5 Gy gamma-ray irradiation. Malignant transformation of Syrian golden hamster embryo (SHE) cells is also induced by 5-fold dose of EP combined with 0. 3 Gy gamma-ray irradiation in vitro, thereby indicating that synergistic carcinogenesis can be obtained by combined use of physical and chemical carcinogens.The mechanisms of synergistic carcinogenesis have been further explained by cytogenetics, damage extent of the target cell DNA and production of free radicals. The Chinese traditional medicine with antioxidat-ing effect (Sulekang Capsule, SC), food additive--butylated hydroxyanisole (BHA) and green tea can effectively inhibit the carcinogenic effect of EP or EP combined with gamma rays in mice. They all have marked ability to scavenge or remove the free radicals and thereby reduce the DNA damage.

Electron Spin Resonance Studies of Free Radicals in Gamma-Irradiated Golden Hamster Embryo Cells: Radical Formation at 77 and 295 K, and Radioprotective Effects of Vitamin C at 295 K

Formation of free radicals in golden hamster embryo (GHE) cells produced by gamma irradiation at 77 and 295 K has been studied by electron spin resonance (ESR) spectroscopy. The yields of free radicals in the gamma-irradiated frozen cells at 77 K increase linearly with increasing dose in the range from 8 Gy to 1.3 kGy, suggesting that the mechanism of radical formation at a dose of 8 Gy is the same as that at 1.3 kGy. When GHE cells are irradiated with 5 kGy gamma rays at 295 K and then the ESR spectrum is measured at 77 K, a part of the organic radicals produced can be observed clearly. The organic radicals survive in GHE cells for more than 24 h at room temperature. The formation of the organic radicals by gamma irradiation is suppressed upon the addition of vitamin C to the GHE cells. When an aqueous solution of albumin (0.1 kg dm-3) is gamma-irradiated with 5 kGy at 295 K, albumin radicals are observed by ESR. The formation of the albumin radicals is suppressed drastically upon the addition of vitamin C. The efficient radioprotection of vitamin C against radical formation has been interpreted in terms of the scavenging of albumin radicals by vitamin C.

Do OH radicals react with organic substances in gamma-irradiated frozen cells of golden hamster embryo?

Reactivity of OH radicals, produced by γ-irradiation, in golden hamster embryo (GHE) cells and in phosphate-buffered saline (PBS) solutions containing nuclei of the GHE cells was studied at 77 and 111 K by ESR spectroscopy. Since OH radicals do not diffuse in frozen cells at 77 K, they do not react with organic substances, such as protein and DNA, in the cells at 77 K. The efficiency of production and trapping of OH radicals at high concentration of organic substances in cells are the same as that at low concentration. Thus OH radicals produced near the organic substances do not react with them, being trapped at 77 K. When GHE cells or PBS-nuclei solutions are irradiated by γ-rays at 77 K and then warmed to 111 K, OH radicals decay fast, while the amounts of organic radicals remain constant at this temperature. These results indicate that OH radicals do not react with organic substances in γ-irradiated cells during warming of the irradiated cells. Therefore it was concluded that OH radicals are not the main reactive species responsible for biological effects in γ-irradiated frozen cells.

Mutagenic and transforming effects of soft-X-rays with resonance energy of phosphorus K-absorption edge.

Syrian golden hamster embryo (SHE) cells were exposed to synchrotron-produced monochromatic X-rays at 5.747 (2.159 keV), 5.763 (2.153 keV) and 5.779 A (2.147 keV). Although X-rays of all wavelengths induced mutations and chromatid aberrations in a dose-dependent manner, when cells were irradiated with 2.153 keV X-rays, which correspond to the resonance energy of the phosphorus K-absorption edge, the frequencies of mutation and chromatid aberration at equal dose levels were higher than for X-rays of the other wavelengths. At equal survival levels, however, there was no difference in the frequencies of mutations and chromatid aberrations in cells irradiated with soft X-rays. On the other hand, the frequency of morphological transformation in cells irradiated with 2.147 keV X-rays was higher than those irradiated with 2.153 keV and 2.159 keV X-rays. The relative biological effectiveness compared to cobalt-60 gamma-rays in morphological transformation was 2.8 for 2.147 keV, 1.1 for 2.159 keV and 1.0 for 2.153 keV at a 37% survival level.

Radiation-induced chromosome damage in G1 phase cells as breaks in premature chromosome condensation (PCC) and its biological meaning.

We studied radiation-induced chromosome fragmentation in the G1-phase of Syrian golden hamster embryo (SHE) cells by premature chromosome condensation (PCC). SHE cells were irradiated with 14N ions (95 MeV) and 4He ions (22 MeV) generated by the cyclotron and 137Cs gamma rays. The number of chromosome breaks in PCC (PCC break) induced by 14N ions was higher than in those by the others. High linear energy transfer (LET) radiation (heavy ions) is more effective in the induction of PCC breaks than low LET radiation (gamma-rays). 530 kev/microns 14N induced PCC 2.4 times more than 137Cs gamma rays. There is an inverse relationship between the number of chromatid deletions and the incidence of mutation or transformation. There is, however, a good correlation between the incidence of PCC breaks and frequencies of mutation and/or morphological transformation. Although over 95% of the PCC breaks induced by gamma-rays were rejoined within 8 hours of post-irradiation incubation, only 35 to 45% of PCC breaks induced by heavy ions were rejoined. These results suggest that there is a qualitative difference in the initial chromosome damage caused by heavy ions and gamma rays.

Heavy ion-induced chromosome breakage studied by premature chromosome condensation (PCC) in Syrian hamster embryo cells.

We have studied induction and repair of chromosome damage induced by high linear energy transfer (LET) heavy ions in G1/G0 interphase Syrian golden hamster embryo (SHE) cells as revealed by the premature chromosome condensation (PCC) technique. The number of chromosome breaks in condensed chromosomes induced by high LET heavy ions was higher than those induced by 137Cs gamma-rays. Compared with 137Cs gamma rays, the relative biological effectiveness (RBE) for PCC breaks was 1.5 for 35 keV/microns 4He ions, 1.9 for 77keV/microns 4He ions, and 2.5 for 530keV/microns 14N ions. Although 95% of the PCC breaks induced by gamma-rays rejoined during 8 h post-irradiation incubation, only 35-45% of fragments induced by high LET radiations rejoined in the same time. These results suggest that there is a difference, spatial or qualitative, in the initial chromosome damage produced by high LET radiations and low LET radiations.

Expression dynamics of transforming phenotypes in X-irradiated Syrian golden hamster embryo cells

We investigated the dynamics of expression for morphological transformation, for a1025horage-independently growing (A1025h −) cells and for mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in X-irradiated Syrian golden hamster embryo (SHE) cells. No A1025h − cells were detected with 0–14 days of post-irradiation i1025ubation before selection. No mutants at the HGPRT locus were detected with 0–5 days of post-irradiation incubation before selection. The maximum number of mutants for all doses was found after post-irradiation incubation for 8 days. On the other hand, the highest frequency of morphological transformants for all doses was detected with 0 days of post-irradiation incubation. The frequency of induction of morphological transformants increased with increasing dose. Then morphological transformants abruptly decreased with increasing lengths of post-irradiation incubation and no morphological transformants were detected with 14 days of post-irradiation incubation before selection (< 10 −4). A large fraction of morphological transformants (more than 86%) was cloned with feeder cells and expressed more extensive phenotypes of malignant transformation, such as the acquisition of anchorage-independent growth, immortality in vitro and tumorigenicity during further subculturing.

Differential heat sensitivity of normal and transformed Syrian golden hamster embryo cells in confluence

The heat sensitivities of normal and transformed cells derived from Syrian golden hamster embryo (SHE) cells were investigated. There were no differences in heat sensitivities between normal and transformed cells when they were exponentially growing. When confluent cultures were heated in situ, only normal cells showed a reduced heat sensitivity, 4-5 times less than that of exponentially growing cells. However, when these confluent cultures were trypsinized, diluted in medium and heated in suspension, the difference disappeared. When mixed cultures of normal and transformed S11 cells in confluence were heated at 43 degrees C, transformed S11 cells were selectively killed. The induction of HSP70 synthesis by heating in normal SHE cells and transformed counterparts were investigated in confluence using monoclonal antibody. The rate of enhanced HSP70 synthesis after heat treatment did not correlate with the difference in heat sensitivities between normal and transformed SHE cells in confluence as measured by cell survival. The results that normal SHE cells become more resistant to heat than their transformed counterparts only in confluence suggest that cell-to-cell contact plays an important role in the heat resistance of normal SHE cells in vivo.

Radioprotective Effects of Dimethyl Sulfoxide in Golden Hamster Embryo Cells Exposed to γ Rays at 77 K: I. Radical Formation as Studied by Electron Spin Resonance

Formation of free radicals in golden hamster embryo (GHE) cells in the frozen living state by gamma irradiation has been studied by electron spin resonance spectroscopy at 4.2 and 77 K. The relative yields of H atoms, OH radicals, and organic radicals trapped in the irradiated GHE cells are 12, 72, and 16%, respectively, of total radical yields. When dimethylsulfoxide (DMSO) is added to GHE cells at 77 K, a large quantity of CH2SOCH3 radicals (DMSO radicals) are formed after gamma irradiation. The yields of OH radicals are not affected by the addition of DMSO. When the GHE cell-DMSO mixtures are irradiated with gamma rays at 77 K and then warmed to 111 K, the OH radicals decay, whereas the DMSO radicals do not increase complementarily. Moreover, the decay rates of the OH radicals at 111 K do not depend upon the concentration of DMSO. Thus OH radicals do not react with DMSO during warming of the irradiated sample. When H atoms are produced by gamma irradiation of acid ice at 60 K, the decay rates of the H atoms at 77 K increase with increasing DMSO concentration, indicating that DMSO reacts with H atoms (CH3SOCH3 + H----.CH2SOCH3 + H2) at 77 K by quantum-mechanical tunneling. When the GHE cell-DMSO mixture is irradiated with gamma rays at 77 or 4.2 K in the dark, DMSO ions are produced in addition to DMSO radicals. Therefore it is concluded that DMSO does not scavenge OH radicals, but does capture H atoms, holes and/or electrons in the gamma-irradiated cells, resulting in the remarkable formation of DMSO radicals. This scavenger effect of DMSO may be related to the radioprotection of DMSO against cell killing described in the companion paper (Watanabe et al., Radiat. Res., this issue).

Radioprotective Effects of Dimethyl Sulfoxide in Golden Hamster Embryo Cells Exposed to γ Rays at 77 K: II. Protection from Lethal, Chromosomal, and DNA Damage

Golden hamster embryo cells were exposed to 137Cs gamma rays in the presence or absence of dimethyl sulfoxide at both 310 and 77 K. Dimethyl sulfoxide gave significant protection against cell killing at both 310 and 77 K. The extent of radioprotection with 1.28 M dimethyl sulfoxide at 77 K was 85-89% of the lethal effects observed in the absence of dimethyl sulfoxide at 310 K; the dose-modifying factor was 5.7. Dimethyl sulfoxide also exerted protected against gamma-ray-induced DNA single-strand breaks and chromosomal aberrations with a maximum protection of 80-100% at a dimethyl sulfoxide concentration of 1.28 M at 77 K. At 77 K, H atoms, ion holes, and electrons can migrate through frozen cells but OH radicals cannot diffuse. Thus the protective effects of dimethyl sulfoxide against cell killing, chromosomal aberrations, and DNA single-strand breaks at 77 K may be due to the scavenging of H atoms or other ions, rather than OH radicals.

Application of Transmission Electron Microscope and Scanning Electron Microscope in experimental tumor studies

Transmission electron microscope(TEM) and scanning electron microscope(SEM) were widely used in experimental tumor studies. They are useful for evaluation of cellular transformation in vitro, classification of histological types of tumors and treating effect of tumors. We have obtained some results as follows:1. Studies on the malignant transformation of mammalian cells in vitro. Syrian golden hamster embryo cells(SGHEC) were transformed in vitro by ThO2 and/or ore dust. In a few days after dust added into medium, some dust crystals were phagocytized. Two weeks later, malignant transformation took place. These cells were of different size, nuclear pleomorphism, numerous ribosomes, increasing of microvilli on cell surface with various length and thickness, and blebs and ruffles(Figs. 1,2). Myelomonocytic leukemic transformation of mouse embryo cells(MEC) was induced in vitro by 3H-TdR. Transformed cells were become round from fusiform. The number of mitochondria and endoplasmic reticulum was reduced, ribosomes and nucleoli increased, shape of nuclei irregular, microvilli increased, and blebs and ruffles appeared(Fig. 3).

Analysis of Chromatid Damage in G2Phase Induced by Heavy Ions and X-rays

The induction of chromatid aberrations was examined in Golden hamster embryo (GHE) cells 2 h after exposure to X-rays or heavy ions. Compared with X-rays the frequencies of gaps and deletions were almost one-half in the case of He ions, and much lower in cells irradiated with N ions. The induction of exchanges by He ions and N ions was also less than that by X-rays. The relative number of gaps, deletions and exchanges were 0.4, 0.5 and 0.4 for He ions, and 0.2, 0.2 and 0.4 for N ions. The mitotic indices were almost the same in X-irradiated and heavy-ion-exposed cells. Chromatid aberrations induced by heavy ions tended to persist longer than those induced by X-rays during successive culture passages after exposure. The present results suggest a difference in the quality of chromatid damage induced by X-rays and heavy ions.

Trisomy of chromosome 9q: specific chromosome change associated with tumorigenicity during the process of X-ray-induced neoplastic transformation in golden hamster embryo cells.

We have reported that trisomy of chromosome 7 is commonly observed in anchorage-independent clones isolated from X-irradiated golden hamster embryo cells. All 10 clones derived from different irradiated populations showed tumorigenicity when 1 x 10(7) cells were injected s.c. into nude mice (BALB/c, nu/nu). From karyotypic analysis, we found that 8 of 10 cells showed trisomy of chromosome 9. One cell line had a translocation between chromosomes 9q and 19q and trisomy of chromosome 7. The other cell line contained trisomy of chromosome 9 and a translocation between chromosomes 7q and 8q. Using Southern blot analysis, we observed no amplification of v-myc, v-Ha-ras, v-Ki-ras or N-ras-related oncogenes. Furthermore, we could not detect either an increase in expression of v-myc- and v-Ha-ras-related genes or the activation of any oncogene, by the NIH 3T3 transfection assay. Our results suggest that trisomy of chromosome 7 is insufficient for the expression of tumorigenicity and that increased dosage of chromosome 9q may play an important role in the malignant progression of X-ray-induced neoplastic transformation.