Hydrogen peroxide lethality is associated with a decreased ability to maintain positive DNA supercoiling

Hydrogen peroxide is more toxic to mammalian cells at 37 degrees C than 0 degree C at all concentrations studied. Histone-free nuclei (nucleoids) extracted from treated cells have a reduced ability to maintain positive DNA supercoiling, with the maximum effect at the higher temperature. Prior exposure of cells to sodium ascorbate at 0 degree C increased both toxicity and the inhibition of nuclear supercoil rewinding. After exposure at 0 degrees C, normal levels of supercoiling returned with both a fast and a slow component, kinetics characteristic of DNA single-strand break repair; the fast component was eliminated when cells were exposed at 37 degrees C due to in situ rejoining. At least a portion of the lethal lesions induced by hydrogen peroxide are DNA double-strand breaks (dsb) because the dsb repair-deficient mutant, xrs-5, is approximately two to three times more sensitive than wild-type cells over the initial portion of the survival curve. However, the increased toxicity found after exposure at 37 degrees C is observed equally in both cell lines, indicating that temperature-dependent cell killing is not directly linked to DNA dsb. It is suggested that cell killing at 37 degrees C is mediated through two linked processes. First, hydrogen peroxide may disrupt cation-stabilized nuclear supercoiling by direct ion oxidation. Second, as a part of the oxidation process, hydrogen peroxide will produce potentially cytotoxic free radicals close to the DNA-linked metal site, limited in extent only by the presence of chemicals capable of reducing metal ions prior to reoxidation.     

DNA strand break and rejoining in cultured human fibroblasts exposed to fast neutrons or gamma rays

The production and rejoining of DNA single-strand and double-strand breaks have been monitored in monolayer cultures of proliferating human skin fibroblasts by means of sensitive techniques. Cells were irradiated with low doses of either 60Co gamma-rays or 14.6 MeV neutrons at 0 degrees C (0-5 Gy for measurement of single-strand breaks by alkaline elution and 0-50 Gy for double-strand breaks measured by neutral elution). The yield of single-strand breaks induced by neutrons was 30 per cent of that produced by the same dose of gamma-rays; whilst in the induction of double-strand breaks neutrons were 1.6 times as effective as gamma-rays. Upon post-irradiation incubation of cells at 37 degrees C, neutron-induced single-strand and double-strand breaks were rejoined with a similar time-course to gamma-induced breaks. Rejoining followed biphasic kinetics; of the single-strand breaks, 50 per cent disappeared within 2 min after gamma-rays and 6-10 min after neutrons. Fifty per cent of the double-strand breaks disappeared within 10 min, after gamma-rays and neutrons. Cells derived from patients suffering from ataxia-telangiectasia showed the same capacity for repair of single- and double-strand breaks induced by 14.6 MeV neutrons, as cells established from normal donors. The comparison of neutrons and gamma-rays in the induction of DNA breaks did not explain the elevated r.b.e. on high LET radiation. However, a study of the variation in the spectrum of lesions induced by different radiation sources will probably contribute to the clarification of the relative importance of other radio products.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.     

Effect of glutathione depletion on the cytotoxicity of xenobiotics and induction of single-strand DNA breaks by ionizing radiation in isolated hamster round spermatids

The role of glutathione (GSH) in cellular protection mechanisms in round spermatids from hamsters was studied. Isolated spermatids were largely depleted of GSH by treating the cells for 2 h with the GSH conjugating agent diethyl maleate (DEM). This treatment resulted in a 90% decrease of the cellular GSH content, but did not affect the ATP content. Exposure of isolated spermatids to cumene hydroperoxide (CHP), a compound which is detoxicated by the GSH redox cycle, showed that the cytotoxicity of the peroxide was markedly potentiated by GSH depletion of the cells. The cytotoxicity was reflected by the cellular ATP content. A decrease of the ATP content of the GSH-depleted spermatids was observed at 5-6-fold lower CHP concentrations, as compared to control cells. An increased cytotoxicity in GSH-depleted cells was also observed using 1-chloro-2,4-dinitrobenzene (CDNB), which is a reactive compound that is detoxicated by glutathione conjugation. The induction of single-strand DNA breaks by gamma radiation was 3-5-fold higher in GSH-depleted spermatids as compared to control cells. This radiation-induced damage was estimated under hypoxic conditions (500 p.p.m. O2 in N2). GSH depletion did not affect the repair of single-strand DNA breaks following the irradiation. The present results indicate that cellular GSH has an important function in the defence mechanisms of round spermatids against peroxides, electrophilic xenobiotics and radiation-induced DNA damage.     

Antioxidant effect of dipyridamole (DIP) and its derivative RA 25 upon lipid peroxidation and hemolysis in red blood cells

The antioxidant effects of dipyridamol (DIP), a coronary vasodilator, and its derivative RA-25 were compared in intact red blood cells (RBC) and in isolated ghost membranes. Both compounds are quite effective antioxidants in cumene hydroperoxide-induced lipid peroxidation of RBC, showing a much smaller effect for hydrogen peroxide oxidation. The antioxidant effect of DIP was considerably higher than that of RA25. For isolated ghost membranes, the apparent IC50 (the drug concentration that produces 50% inhibition of lipid peroxidation) in cumene hydroperoxide-induced peroxidation was 25 microM, while the maximum protective effect of RA-25 was around 30% in the drug concentration range of 50-100 microM. The drugs can protect the oxidative hemolysis induced by cumene hydroperoxide with a lower effect when the hemolysis is induced by H2O2. The significant antioxidant effect against damages induced by cumene hydroperoxide suggests that DIP, due to its lipophilic character, can interact with RBC membranes, and the protective effect is associated with the binding of the drug to the membrane. On the other hand, RA-25 is more hydrophilic than DIP, binds to the membrane to a smaller extent, and, for this reason, has a lower antioxidant effect.     

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.     

Oxidative Stress Responses in the Unicellular Cyanobacterium Synechococcus Pcc 7942

Oxidative stress responses were tested in the unicellular cyanobacterium synechococcus PCC 7942 (R-2). Cells were exposed to hydrogen peroxide, cumene hydroperoxide and high light intensities. The extent and time course of oxidative stress were related to the activities of ascorbate peroxidase and catalase. Ascorbate peroxidase was found to be the major enzyme involved in the removal of hydrogen peroxide under the tested oxidative stresse. Catalase activity was inhibited in cells, treated with high H₂O₂ concentrations, and was not induced under photooxidative stress. Catalase was specifically induced in cells treated with cumene hydroperoxide.

Superoxide dismutase activity increased under conditions generating superoxide, such as high light intensities. The induction of the antioxidative enzymes was light dependent and was inhibited by chloramphenicol.     

Effect of 3-aminobenzamide on DNA strand-break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide

The influence of the nuclear ADP-ribosyltransferase inhibitor 3-aminobenzamide on the DNA strand-break rejoining kinetics and cytotoxicity in Chinese hamster ovary cells following H2O2 treatment was investigated. For the DNA damage studies, cells were treated on ice with H2O2 (0-20 microM) for 1 h in serum-free medium, after which the H2O2 was removed and the cells were allowed to repair their damage in complete medium at 37 degrees C in the presence or absence of 3-aminobenzamide (5 mM) for periods up to 2 h. The DNA strand breaks remaining as a function of time were then estimated by alkaline elution. A linear relationship between the H2O2 concentration and the initial level of DNA single-strand breaks (zero time allowed for repair) was observed. No double-strand breaks or DNA-protein cross-links were detected at these doses. The rejoining of single-strand breaks after H2O2 (20 microM) alone was characterized by a single exponential process with a t1/2 of approx. 5 min. However, in the presence of 3-aminobenzamide, rejoining was much slower and biphasic, with t1/2 of approx. 10 and 36 min. The inhibitory action of 3-aminobenzamide was concentration-dependent and completely reversible in that, when the 3-aminobenzamide was removed from the treated cultures, the strand-break rejoining kinetics rapidly returned to the t1/2 of 5 min typical of H2O2 alone. Considerably higher concentrations of H2O2 (up to 600 microM) were required for cell killing compared to the DNA damage studies. Cell killing by H2O2 alone was characterized by a shoulderless, exponential survival curve (D0 = 880 microM). The cytotoxicity was potentiated when the cells were treated with 3-aminobenzamide (5 mM) for 1 h after the H2O2 treatment; the survival curve with 3-aminobenzamide also assumed a biphasic character (D0 of 212 microM and 520 microM). These results are consistent with the theory that OH.-induced single-strand breaks do not normally represent lethal lesions to the cell because of their rapid, efficient repair. However, interference with these repair processes (in this case by 3-aminobenzamide) can alter this relationship, possibly allowing lesion fixation.     

Finger temperatures during military field training at 0 to −29 °C

1. Skin and rectal temperatures were recorded continuously in 70 measurements during typical tasks of infantry and artillery training at 0 to −29 °C. The duration of the measurements varied from 55 min to 9.5 h.

2. The distribution of finger skin temperatures was quite similar at ambient temperature ranges 0 to −10 °C and −10 to −20 °C, while at −20 to −30 °C the finger temperatures were clearly lower.

3. At different ambient temperature ranges, 20–69% of finger temperatures were low enough to cause cold thermal sensations.

4. Sensation of cold was experienced at a finger temperature of 11.6±3.7 °C (mean±SD).     

Improved Control of Otiorhynchus sulcatus at 9°C by Cold-stored Heterorhabditis megidis UK211

The effect of storage temperature (9 and 20°C) on North West European Heterorhabditis megidis isolate UK211 for control of Otiorhynchus sulcatus larvae at 9°C is assessed. O. sulcatus mortality increased from -5.3% (corrected mortality) using freshly produced nematodes, to 27.1% using nematodes that had been cold-stored for 12 weeks. The number of nematodes invading the insect larvae increased almost 27-fold. Nematode storage at 9°C for 11 to 12 weeks weeks resulted in significantly higher O. sulcatus mortality (41%) than storage at 20°C for 2 to 3 weeks (12%). Thus, cold storage does enhance nematode infectivity for O. sulcatus larvae.     

Primary DNA damage in human blood lymphocytes exposed in vitro to 2450 MHz radiofrequency radiation

Human peripheral blood samples collected from three healthy human volunteers were exposed in vitro to pulsed-wave 2450 MHz radiofrequency (RF) radiation for 2 h. The RF radiation was generated with a net forward power of 21 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The average power density at the position of the cells in the flask was 5 mW/cm(2). The mean specific absorption rate, calculated by finite difference time domain analysis, was 2.135 (+/-0.005 SE) W/kg. Aliquots of whole blood that were sham-exposed or exposed in vitro to 50 cGy of ionizing radiation from a (137)Cs gamma-ray source were used as controls. The lymphocytes were examined to determine the extent of primary DNA damage (single-strand breaks and alkali-labile lesions) using the alkaline comet assay with three different slide-processing schedules. The assay was performed on the cells immediately after the exposures and at 4 h after incubation of the exposed blood at 37 +/- 1 degrees C to allow time for rejoining of any strand breaks present immediately after exposure, i.e. to assess the capacity of the lymphocytes to repair this type of DNA damage. At either time, the data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to the comet tail length, fluorescence intensity of the migrated DNA in the tail, and tail moment. The conclusions were similar for each of the three different comet assay slide-processing schedules examined. In contrast, the response of lymphocytes exposed to ionizing radiation was significantly different from RF-radiation- and sham-exposed cells. Thus, under the experimental conditions tested, there is no evidence for induction of DNA single-strand breaks and alkali-labile lesions in human blood lymphocytes exposed in vitro to pulsed-wave 2450 MHz radiofrequency radiation, either immediately or at 4 h after exposure.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 microM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and alpha-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 microM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C0t of -3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 microM alpha-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to cross-linking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or alpha-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 microM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 microM ouabain. We confirmed that cumene hydroperoxide at greater than 10 microM lowers cloning efficiency and that this is largely prevented by selenite or alpha-tocopherol. Additionally, selenite or alpha-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or alpha-tocopherol. Selenium increased replicative lifespan by 10-20% and alpha-tocopherol by 22-30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and alpha-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.     

Comparisons of blood viscosity between amphibians and mammals at 3°C and 38°C

(1) The range of temperature exposure of endotherms is narrow compared to ectotherms that can experience daily and seasonal temperature fluxes. (2) Comparison of the blood viscosity of amphibians (bullfrog, Woodhouse's toad, and marine toad) and mammals (horse, dog, and rat) at 3°C and 38°C was undertaken to determine if the effect of temperature on blood viscosity was diminished in amphibians relative to mammals. (3) Mammals did not consistently show greater changes in blood viscosity, plasma viscosity, or relative viscosity with decreasing temperatures relative to the amphibians in this study. (4) These data do not support our hypothesis that blood viscosity of amphibians is less affected by temperature than mammalian blood.     

The formation of DNA single-strand breaks and alkali-labile sites in human blood lymphocytes exposed to 365-nm UVA radiation

The potency of UVA radiation, representing 90% of solar UV light reaching the earth׳s surface, to induce human skin cancer is the subject of continuing controversy. This study was undertaken to investigate the role of reactive oxygen species in DNA damage produced by the exposure of human cells to UVA radiation. This knowledge is important for better understanding of UV-induced carcinogenesis. We measured DNA single-strand breaks and alkali-labile sites in human lymphocytes exposed ex vivo to various doses of 365-nm UV photons compared to X-rays and hydrogen peroxide using the comet assay. We demonstrated that the UVA-induced DNA damage increased in a linear dose-dependent manner. The rate of DNA single-strand breaks and alkali-labile sites after exposure to 1 J/cm² was similar to the rate induced by exposure to 1 Gy of X-rays or 25 μM hydrogen peroxide. The presence of either the hydroxyl radical scavenger dimethyl sulfoxide or the singlet oxygen quencher sodium azide resulted in a significant reduction in the UVA-induced DNA damage, suggesting a role for these reactive oxygen species in mediating UVA-induced DNA single-strand breaks and alkali-labile sites. We also showed that chromatin relaxation due to hypertonic conditions resulted in increased damage in both untreated and UVA-treated cells. The effect was the most significant in the presence of 0.5 M Na⁺, implying a role for histone H1. Our data suggest that the majority of DNA single-strand breaks and alkali-labile sites after exposure of human lymphocytes to UVA are produced by reactive oxygen species (the hydroxyl radical and singlet oxygen) and that the state of chromatin may substantially contribute to the outcome of such exposures.     

Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measured by the comet assay

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrheal illness in immunocompromised individuals. This organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irradiation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral comet assay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25 kGy) and were incubated for various periods (6-96 h) after exposure to 10 kGy. The comet tail moment showed that the number of DNA double-strand breaks increased concomitantly with the gamma irradiation dose. When investigating rejoining after irradiation at 10 kGy, double-strand breaks peaked at 6 h postirradiation, and rejoining was highest at 72 h postirradiation. The observed rejoining pattern suggests that repair process occurs slowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradiation, 10 kGy.     

Exposure of cells to nonlethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization

The cytotoxicity of hydrogen peroxide is, at least partly, mediated by the induction of intralysosomal iron-catalyzed oxidative reactions with damage to lysosomal membranes and leakage of destructive contents. We hypothesize that minor such leakage may be nonlethal, and the ensuing cellular degeneration repairable. Consequently, we investigated, using a model system of cultured J-774 cells, the effects of hydrogen peroxide in moderate concentrations on cellular viability, lysosomal membrane integrity, morphology, and ATP and reduced glutathione concentrations. These parameters were initially estimated directly after a 30 min exposure to a bolus dose of hydrogen peroxide in phosphate buffered saline at 37°C, and then again following subsequent recovery periods of different lengths under ordinary culture conditions. All cells survived an exposure to 250 μM hydrogen peroxide for 30 min, whereas 350 and 500 μM exposure was lethal to a small fraction of cells. The oxidative stress caused early, time- and dose-dependent, partial relocalization of the lysosomotropic weak base acridine orange from the lysosomal compartment to the cytosol. This phenomenon is known to parallel leakage of damaging lysosomal contents such as hydrolytic enzymes. There were also signs of cellular damage in the form of surface blebbing and increased autophagocytosis, more marked with the higher doses of hydrogen peroxide. Also found was a rapid depletion of ATP and GSH. These alterations were all reversible, as long as cells were exposed to nonlethal amounts of hydrogen peroxide. Based on these and previous findings, we suggest that lysosomes are less stable organelles than has hitherto been assumed. Restricted lysosomal leakage might be a common event, for example, during sublethal oxidative stress, causing reversible, degenerative alterations, which are repaired by autophagocytosis.     

Purification and properties of a glutathione peroxidase from Southern bluefin tuna (Thunnus maccoyii) liver

A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 micromol NADPH oxidised min(-1) mg(-1) protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 microM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX.     

Effects of variation in glutathione peroxidase activity on DNA damage and cell survival in human cells exposed to hydrogen peroxide and t-butyl hydroperoxide.

The selenium-dependent glutathione peroxidase activities of two human cell lines, the colon carcinoma HT29 and the mesothelioma P31, cultured in medium containing 2% serum, increased from 195 to 541 and from 94 to 361 units/mg of protein respectively after supplementation with 100 nM-selenite. The catalase activity remained unchanged by this treatment. The effects of the obtained variation in glutathione peroxidase activities were investigated by exposing cells to H2O2 and t-butyl hydroperoxide. Selenite supplementation resulted in a decrease in H2O2-induced DNA single-strand breaks in both HT29 and P31 cells. A small, but significant, decrease in the number of DNA single-strand breaks for low doses (10-50 microM) of t-butyl hydroperoxide was found only in P31 cells and not in HT29 cells. We could detect neither induction of double-strand breaks (detection limit approx. 1000 breaks per cell) nor DNA-protein cross-links after exposing the cells to the two peroxides. In spite of the apparent protective effect of increased glutathione peroxidase activity on DNA single-strand break formation, there were no differences between selenite-supplemented and non-supplemented cells in cell survival after exposure to peroxide.     

Isolation and partial characterisation of a mammalian cell mutant hypersensitive to topoisomerase II inhibitors and X-rays

We have isolated, following one-step mutagenesis, a Chinese hamster ovary cell mutant hypersensitive to the intercalating agent, adriamycin (4-fold compared to parental CHO-K1 cells). This agent exerts at least part of its cytotoxic action via inhibition of the nuclear enzyme, topoisomerase II. The mutant, designated ADR-3, showed hypersensitivity to all classes of topoisomerase II inhibitors, including actinomycin D, amsacrine (m-AMSA), etoposide (VP16) and mitoxantrone. ADR-3 cells also showed cross-sensitivity to ionizing radiation, but not to UV light. Cellular accumulation of radiolabeled actinomycin D was similar in parental and mutant cells. At equimolar doses, adriamycin induced more protein-associated DNA single- and double-strand breaks in ADR-3 cells than in CHO-K1 cells. Topoisomerase II activity was elevated to a small but significant degree in ADR-3 cells, and this was reflected in a 1.5-fold higher level of topoisomerase II protein in ADR-3 than in CHO-K1 cells, as judged by Western blotting. ADR-3 cells were hypersensitive to cumene hydroperoxide but cross-resistant to hydrogen peroxide, suggesting possible abnormality in the detoxification of peroxides by glutathione peroxidase or catalase. Glutathione peroxidase activity against hydrogen peroxide was similar in CHO-K1 and ADR-3 cell extracts, but activity against cumene hydroperoxide was evaluated to a small but significant extent in mutant cells. Catalase levels were not significantly different in ADR-3 and CHO-K1 cells. ADR-3 cells were recessive in hybrids with parental CHO-K1 cells with respect to sensitivity to topoisomerase II inhibitors and X-rays, and represent a different genetic complementation group from the previously reported adriamycin-sensitive mutant, ADR-1 [Davies et al., J. Biol. Chem., 263 (1988) 17724-17729].