Biomedical subjects
Publications and source records attributed to G Obe.
Chinese hamster ovary (CHO) cells were treated with bovine pancreatic DNase I using the method of electroporation. The enzyme induced chromosomal aberrations in a S-phase independent manner. The frequencies of polycentric chromosomes induced in the G1 phase of the cell cycle are positively correlated with the dose of DNase I. The distributions of DNase I-induced polycentric chromosomes were overdispersed.
Chinese hamster ovary (CHO) cells were treated with the restriction endonuclease AluI using various methods; namely, treatment in the presence of hypertonic concentrations of glycerol or sorbitol and electroporation. The frequencies of chromosomal aberrations induced were scored in first post-treatment metaphases. For all treatment schedules linear dose-effect relationships of polycentric chromosomes were found. The distribution of polycentric chromosomes induced by AluI was overdispersed. Inhibition of cellular energy metabolism with sodium azide and 2-deoxyglucose led to a strong reduction of the frequencies of chromosomal aberrations when the treatment with AluI was carried out in the presence of glycerol or sorbitol, but not when carried out by electroporation. This is interpreted to mean that glycerol- or sorbitol-mediated cellular uptake of AluI, but not the uptake via electroporation, is an energy-dependent mechanism.
Human peripheral lymphocytes were isolated from whole blood and exposed to culture medium of reduced osmolality. This hypotonic treatment led to a significant increase in the frequencies of chromosomal aberrations when the osmolality was reduced to 60 mOsm/kg H2O and below. Maximum damage occurred when the hypotonic treatment was done 27 or 30 h after starting the cultures. We also looked for the induction of sister-chromatid exchanges (SCE) by hypotonic culture conditions, but the SCE frequencies were not influenced.
Gaseous nitric oxide (NO) and nitrogen dioxide (NO2) were tested for their potential to induce DNA single-strand breaks (SSBs) in Chinese hamster cells (V79 cells). The alkaline elution assay was used for the detection of SSBs. V79 cells were exposed to NO and NO2 in N2 in varying concentrations (0-500 p.p.m.) and over varying periods (5-30 min). NO treatment did not result in any detectable DNA damage. NO2 led to a dose- and time-dependent increase of the rate of SSBs and the amount was dependent on concentration of NO2 and the length of exposure. The lowest observable effective concentration which was statistically different from control values was 10 p.p.m. exposed for 20 min. The metabolites of both gases, nitrite (NO2-) and nitrate (NO3-), had no DNA-damaging activity up to a concentration of 1 mM. The mechanism by which NO2 may generate SSBs is discussed.
The radioactive fallout of the Chernobyl accident caused an increase in radiation dose of 20 to 110 per cent over the normal environmental burden to the inhabitans of Salzburg City in Austria (in a distance of about 1300 km from the accident). The structural chromosome aberration in the lymphocytes of the peripheral blood of 15 test-persons have been investigated one year after the accident. From two of these we know also the aberration frequencies before the accident which were significantly lower. The results from all test persons were pooled according to their Cs137 and Cs134 content, measured by whole body counter. Their mean additional blood doses from the incorporated caesium plus the external fallout radiation were 0.23, 0.36 and 0.55 mGy/yr. The aberration frequencies increased with dose. The slope of the best straight-line through the points was 2.0 +/- 0.7 total chromosome type aberrations in 100 metaphases per mGy/yr. This result fits in well with former investigations of persons with individually calculated radiation burden from the environment. The sharp increase with dose at this low level is not compatible with values extrapolated from high doses. The usual dose assessment based on chromosome aberrations extrapolated from high to low doses is therefore not possible in the range considered in this investigation.
Cultivation of human peripheral lymphocytes (HPL) in the presence of 50-Hz electromagnetic fields (EMFs) does not alter the spontaneous frequencies of sister-chromatid exchanges (SCE) and of chromosomal aberrations (CA), but leads to an enhancement of the cell cycle progression of HPLs in vitro. Pretreatment of HPLs with trenimon (TRN), diepoxybutane (DEB), or methylnitrosourea (MNU) in the G0 phase of the cell cycle results in dose-dependent elevations of the SCE frequencies. In some cases culturing of HPLs pretreated with MNU or TRN in the presence of EMFs led to significantly higher frequencies of SCEs when compared to cells cultivated in the absence of EMFs. Since we did not use multiple fixation times these data may rather result from differential influences on HPL subsets than from EMF exposure.
Pretreatment of Chinese hamster ovary cells with proteases or with NaN3 leads to less chromosomal aberrations when the cells are posttreated with Alu I compared to the treatment of cells with Alu I alone. The same result is obtained when the cells are treated with Alu I at 0 degree C instead of 37 degrees C. The cells recover from the protease treatment when they are kept in medium before treatment with Alu I. These results are interpreted to mean that Alu I is bound by surface receptors and that the Alu I-receptor complexes are internalized by an energy-dependent endocytotic process.
Cell-free extracts of Lactobacillus bifidus have been reported as possessing DNA-repair-eliciting properties in UV-irradiated human cells, and suggestions have been made that these extracts could be used to protect human skin cells from DNA-damaging effects induced by natural UV light. In view of the importance of these findings, and because extracts of other bifidobacteriae had previously been shown to possess genotoxic activity in bacterial systems, it seemed appropriate to perform some experiments aimed at evaluating the ability of cell-free extracts of L. bifidus, as well as the bacterial suspension medium, to modulate DNA repair and/or to exert potentially adverse genotoxic effects in a variety of mammalian cells in culture. Chinese hamster cells, human fibroblasts, and human lymphocytes were used to evaluate the influence of the extract on the repair of UV-damaged DNA and on several biological effects (cell cycle progression, cell killing, chromosomal aberrations, and sister-chromatid exchanges) induced by DNA-damaging agents. The results show that neither the extract nor the suspension have any influence on DNA repair or other biological endpoints induced by UV and other DNA-damaging agents. In conclusion, the present findings do not indicate that cell-free extracts of L. bifidus specifically promote the repair of UV-damaged DNA in human cells. Neither do they indicate that these extracts have a promoting activity on UV-induced (misrepair) mutagenesis in mammalian cells. Finally, the present experiments indicate that the L. bifidus extracts used in our experiments are devoid of any direct mutagenic and/or genotoxic activity in mammalian cells.
Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the G0 (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sister-chromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in G0 cells and that G1 cells can repair both DEB and MNU induced lesions.
The restriction endonuclease Alu I induces chromosome-type aberrations in Chinese hamster ovary cells whose frequencies are considerably elevated in the presence of high concentrations of MgCl2, (NH4)2SO4, CaCl2 or NaCl. The most plausible explanation for these findings is that salt leads to partial dehistonization of the chromatin which makes more recognition sites available for Alu I.
The dose-response for radiation-induced chromosome aberrations in human lymphocytes is usually fitted to the quadratic model. This assumes that the slope is essentially linear at low doses. Empirical observations of linearity at less than 200 mGy are, however, sparse. Some data have been published indicating a non-linear (threshold) response and these are reviewed. In particular one study with X-rays showed a plateau in response up to 50 mGy and with a significant dip below the control level at 4 mGy. The mechanism proposed to explain non-linearity is that low doses stimulate the enzymic repair capability of lymphocytes. Preliminary data are presented from a large experiment by six laboratories in which the low dose-response for X-rays has been re-examined. The plateau in the dose-response relationship, if it exists, does not extend to doses above approximately 10 mGy. No irradiated cells yielded aberration levels significantly below the control. Over the range 0-300 mGy the response can be fitted to a linear regression. There are, however, variations in sensitivity between cells from different donors. An unexpected finding was that some lymphocytes contained greater than 1 exchange aberrations. This may indicate a small subset of cells that are especially susceptible to the induction of aberrations by low doses.
Treatment of yeast cells proficient in the repair of radiation damage (Saccharomyces cerevisiae) with the restriction endonuclease Alu I leads to a positive dose-effect relationship between inactivation level and enzyme concentration. The data suggest an uptake of the active restriction enzyme into the cells and a relationship between induction of DNA double-strand breaks and cell killing.
The formation of mutagenic compounds from cyclophosphamide (CPA) by S9-fractions from liver (S9L) or kidney (S9K) of pregnant CBA and C 57 BL mice was investigated, using point mutations in Salmonella typhimurium (TA 1535) and the induction of sister chromatid exchanges (SCE) in human peripheral lymphocytes (HPL) or Chinese hamster ovary (CHO) cells as end points. In addition, the teratological response of CBA and C 57 BL mice to CPA on day 11 of pregnancy was analysed in vivo. The results are as follows: (1) S9L from CBA mice was more effective than S9L from C 57 BL mice in metabolizing CPA to products inducing mutations in Salmonella and SCEs in HPL and CHO cells. (2) S9L was more effective than S9K from both strains of mice. (3) In vivo pretreatment of mice with a single dose of CPA (20 mg/kg) reduced the in vitro metabolizing capacity of S9L and S9K significantly and led to the disappearance of the interstrain difference. (4) The embryolethal and teratogenic effects of CPA were stronger in C 57 BL than in CBA mice; the types of teratological effects were partially different in the two strains.
Heat treatment of CHO cells in the G1-phase of the cell cycle leads to chromatid-type aberrations in first posttreatment metaphases. Posttreatment of heat-treated cells with the alkylating agent trenimon leads to a synergistic effect on the production of chromatid-type exchanges. These results indicate that heat induces lesions which like the lesions produced by trenimon give rise to chromatid-type aberrations during the first posttreatment S-phase, and that these lesions can interact with each other to produce chromatid-type exchanges. Treatment of CHO cells in the G1-phase of the cell cycle with the restriction endonuclease Alu I induces chromosomal aberrations. Pretreatment of cells with heat leads to a reduction of Alu I induced chromosome-type aberrations. When cells are allowed to recover after heat treatment for 22 h, the aberration frequencies produced by Alu I are the same as in cells not treated with heat. These findings can be explained by assuming that heat-induced accumulation of accessory proteins in the chromatin protects the DNA from being cut by Alu I, and that the cells recovered from the heat-induced protein accumulation after 22 h.
Alcoholics have a higher frequency of chromosomal aberrations and sister-chromatid exchanges (SCEs) in their peripheral lymphocytes. In human and mammalian cells in vitro, ethanol generally does not induce genetic damage, but it induces SCEs in the presence of an exogenous metabolic system. In human lymphocytes in vitro, ethanol induces SCEs in the presence of alcohol dehydrogenase. In animals in vivo, ethanol induces a variety of genetic effects, including SCEs, micronuclei, dominant lethal mutations and aneuploidy in mouse eggs. There is some indication that ethanol may lead to genetic damage in sperm. In bacteria, ethanol is at best marginally active. Ethanol leads to anomalous chromosome segregation in Aspergillus, to mutations in yeast, to chromosomal aberrations and SCEs in plant root tips and to disturbances of meiosis and micronuclei in tetrads in Zea and Tradescantia respectively. The first metabolite of ethanol, acetaldehyde is mutagenic in a variety of test systems. The mutagenic activity of acetaldehyde in bacteria is questionable, but there is no doubt of its mutagenic activity in a variety of eukaryotic test systems in vitro as well as in vivo.
Treatment of G1-phase Chinese hamster ovary (CHO) cells with the restriction endonuclease Dra I (recognition site TTT/AAA) leads to the induction of chromosome-type aberrations. The dose-effect relationships or the frequencies of polycentric chromosomes have a strong linear component. Prelabelling of the cells with bromodeoxyuridine (B) leads to a strong suppression by the chromosome breaking activity of Dra I. This may be explained by assuming that substitution of T by B renders the recognition site of Dra I resistant to being cut by the enzyme.
Bleomycin induced DNA single-strand breaks in Chinese hamster V 79 cells which were detected by the alkaline filter elution assay. In the presence of propylgallate, an antioxidant, the amount of DNA single-strand breaks was significantly reduced. The production of DNA strand breaks by methylnitronitrosoguanidine used as a positive control was not influenced by propylgallate. It is suggested that propylgallate inhibits the generation of DNA single-strand breaks by trapping reactive oxygen species produced by the bleomycin-iron(II) complex.