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International audience; The temperature dependence and the ouabain sensitivity of Na,K-ATPase was examined in the nephron of normal, cold-exposed, and hibernating jerboas. The transport and hydrolytic activity of renal Na,K-ATPase displayed similar temperature dependence in rats and normal jerboas. Cold-resistance of Na,K-ATPase appeared in cold-exposed jerboas and further increased during hibernation. Three subpopulations of Na,K-ATPase displaying very high (Ki approximately 10(-13) M), high (Ki approximately 10(-9) M) and low sensitivity to ouabain (Ki approximately 10(-6) M) were detected in the thick ascending limb and collecting duct of jerboas. In thick ascending limbs, the subpopulation of very high sensitivity to ouabain disappeared in cold-exposed animals, which accounted for the previously reported decrease in Na,K-ATPase activity. In collecting ducts of cold-exposed animals, the subpopulation of very high sensitivity to ouabain also disappeared, but the resulting decrease in activity was overbalanced by the appearance of the subpopulation of high sensitivity.

The variables affecting the frequency of ouabain-resistant mutant clones have been studied in a strain of foetal lung fibroblasts. Optimum mutant recovery was obtained when cells were selected in 10 ′-6 M ouabain at a cell density of 2 × 10 4 cells/cm 2 (10 6 cells per 100-mm dish). The spontaneous mutation rate was estimated to be 4 × 10 −8 per cell generation. Treatment with the mutagens ethyl methanesulfonate (EMS), N -methyl- N ′-nitro- N -nitrosoguanidine, and UV light increased the frequency of mutant colonies by an order of magnitude. The maximum number of mutants after mutagenesis with EMS occurred after two population doublings of growth in non-selective medium prior to selection and depended on the dose of EMS. Ouabain-resistance is a useful marker for studies of quantitative mutagenesis in human cells.

The interaction between ultraviolet light (UV-C) from germicidal lamps (254 nm) and near-ultraviolet light (UV-B) from Westinghouse Sun Lamps (290-345 nm) was studied in Chinese hamster V79 cells by measuring the effectiveness of combined exposures to induce the resistance to 6-thioguanine or to ouabain. Exposure of cells to a conditioning dose of UV-B (approximately 70% survival) results in significant inhibition of the induction by UV-C of cells resistant to ouabain. The inhibition is lost, however, if cells are incubated for 12 h at 37 degrees C between exposures. Inhibition is also observed when cells are preirradiated with a dose of UV-B filtered with polystyrene (300-345 nm) which, in itself, has no effect on cell killing. Conditioning exposures of unfiltered or filtered UV-B light do not inhibit the induction of 6-thioguanine-resistant cells by UV-C light, and the effects of UV-B and UV-C light are largely independent.

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.

The mutagenicity of vinyl chloride, vinylidene chloride (1,1-dichloroethylene) and chloroprene (2-chloro-1,3-butadiene) was tested in V79 Chinese hamster cells in the presence of a 15 000 × g liver supernatant from phenobarbitone-pre-treated rats and mice. Mutations in terms of 8-azaguanine and ouabain resistance were induced in a dose-related fashion by exposure to vapour of vinyl chloride in the presence of liver supernatant from phenobarbitone-pretreated rats. Vapours of vinylidene chloride and chloroprene induced a doserelated toxicity in the presence of liver supernatant from phenobarbitone-pretreated rats, but these two compounds were not mutagenic in V79 Chinese hamster cells under the present assay conditions. The results are discussed with regard to the metabolic activation of the compounds and to the correlation with their carcinogenicity in man and experimental animals.

Following treatment with ultraviolet light or ethyl methane-sulphonate mouse lymphoma L5178Y cells were cultured for different periods in the presence of non-toxic levels of the tumour promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA). There were no significant changes in either the spontaneous or induced frequencies of ouabain-resistant variants recovered after TPA treatment. These results confirm that any effect of TPA on mutagenesis may be by modifying metabolic cooperation and the recovery of resistant cells.

GEM 199 cells derived from an erythrophoroma of goldfish ( Carassius auratus ), which had a high plating efficiency, were used to investigate the lethal and mutational effects of radiations (UV and γ-rays) and chemicals (4NQPO and MNNG). The cells were more resistant to γ-rays than mammalian cells and CAF-MM1 cells derived from the normal fin tissue of goldfish. They were also more resistant to UV-irradiation than CAF-MM1 cells. Photoreactivation after UV-irradiation was present in GEM 199 cells for both survival and mutation. The initial shoulder of the survival curve of UV-irradiated cells was reduced greatly by caffeine, suggesting a high activity of the post-replication repair. The spontaneous mutation frequency to ouabain resistance was 1−5 × 10 −6 clones per viable cell. MNNG was effective in inducing ouabain-resistant mutation, while 4NQO and γ-rays did not induce mutation.

— We compared mutagenesis and sister chromatid exchange (SCE) induction by 193 nm and 308 nm pulsed excimer laser radiation with 254 nm low intensity continuous wave UV light in Chinese hamster ovary (CHO) cells in culture. The 254 nm radiation was most mutagenic of the radiations, in accordance with expectation, and also was most effective in increasing the level of SCEs. The 193 nm radiation was mutagenic at the ouabain resistance locus, but not at the HGPRT locus. However, 193 nm radiation was also strongly cytotoxic at energies producing measurable mutations. This radiation also caused a dose-related increase in SCEs. Pulsed excimer radiation at 308 nm was mutagenic at both loci, and also increased the incidence of SCEs. Comparison of the ratio of mutants/surviving cells at the D37 after radiation showed similar values for 254 nm and 308 nm at the HGPRT locus, but at the ouabain resistance locus, the ratio for the 308 nm radiation was about 5 times that for 254 nm radiation. These results indicate that some risk for mutagenesis may accompany the use of excimer radiation in the UVA region in therapeutic applications.

The present paper deals with effects of 12- O -tetradecanoylphorbol-13-acetate (TPA) on the frequency of induced mutations to 6-mercaptopurine (6MP) and ouabain resistance in Chinese hamster and mouse cells. UV light, bovine adenovirus 3(BAV-3) and 5-bromodeoxyuridine (BrdU) were used as mutagens. TPA was shown to raise the frequency of gene mutations induced by UV light and BAV-3 but it did not enhance the mutagenic effect of BrdU. We also examined the ability of BAV-3 and BrdU to induced tumours in mice. BrdU was shown to have no carcinogenic effect. The results suggest that TPA enhances the mutagenic effect only for carcinogenic mutagens.

The biological activity of potassium dichromate (K2Cr2O7) was assayed in V79/AP4 Chinese hamster cells by measuring two mutational end points, thioguanine (TG) resistance and ouabain (OUA) resistance, and two non-mutational end points, cytotoxicity and sister chromatid exchanges (SCE). By exposing the cells for 1 h to the chemical, all biological end points examined were affected by the treatment in a dose-dependent manner. Moreover the combined use of the two selective systems indicated that chromium induces base-pair substitutions in mammalian cells in culture.

Summary The spontaneous frequency of mutants resistant to growth inhibition by ouabain (OUAR mutants) was found to be about 5·10−5 per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1·10−6 in 1 mM ouabain. No increase in the frequency of OUAR mutants was found in cultures treated with γ-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than γ-rays for the induction of thioguanine-resistant mutants in these cells. Some evidence was found for an interaction between cellular radiation damage and ouabain-resistance, which may lead to a reduction in the survival of OUAR mutants in irradiated populations, but this damage seemed insufficient to account for inability to detect radiation-induced OUAR mutants. Reproducibly large increases in the frequency of OUAR mutants were found in cultures treated with various concentrations of ethyl methanesulphonate (EMS) by respreading cells in 1 mM ouabain for up to 8 days after EMS treatment. The concentration-OUAR mutant induction curve was approximately linear with low EMS concentrations. Recent evidence is reviewed in support of the suggestion, made in earlier studies, that ionising radiation is unable to induce OUAR mutants because of the severity of the genetic damage it causes.