Selenite-induced variation in glutathione peroxidase activity of three mammalian cell lines: no effect on radiation-induced cell killing or DNA strand breakage

The selenium-dependent glutathione peroxidase activities of three mammalian cell lines, HT29, P31, and N-18, cultured in medium with low serum content, increased about 2-, 5-, and 40-fold, respectively, after supplementation with 100 nM selenite. Catalase, CuZn superoxide dismutase, and Mn superoxide dismutase activities were not generally influenced by selenite supplementation, and there was only a minor nonselenium-dependent glutathione peroxidase activity in the investigated cell lines. Gamma-irradiated control and selenite-supplemented cells showed no changes in the surviving fractions, as estimated by clonogenic survival or [3H]-thymidine uptake, nor were there any significant differences between the two groups in the induction of DNA strand breaks after gamma irradiation under repairing (37 degrees C) or nonrepairing (0 degrees C) conditions. The results suggest that selenium-dependent glutathione peroxidase does not contribute significantly to the radiation resistance of cultured mammalian cells.     

Induction and Rejoining of DNA Single-Strand Breaks in Relation to Cellular Growth in Human Cells Exposed to Three Hydroperoxides at 0°C and 37°C

There was a 5-fold increase in cytotoxicity for cumene hydroperoxide, 10-fold for tert-butyl hydroperoxide and 25-fold for hydrogen peroxide, under metabolizing conditions (37°C) in comparison to nonmetabolizing conditions (0°C), when human P31 cells were exposed for 60 min. The induction of DNA single-strand breaks correlated poorly with cytotoxicity. Hydrogen peroxide was by far the most effective agent inducing single-strand breaks irrespective of temperature. Cumene hydroperoxide produced fewer strand breaks than tert-butyl hydroperoxide despite its greater cytotoxicity at either 37°C or at 0°C. The pattern of single-strand break induction did not change with temperature. The number of breaks, however, increased when the cells were exposed at 37°C. The pattern of rejoining was similar for hydrogen peroxide- and tert-butyl hydroperoxide-induced breaks at both temperatures whereas the rejoining of cumene hydroperoxide-induced breaks deviated somewhat from this pattern. The results indicate that there is no clear-cut relationship between induction of DNA single-strand breaks and cytotoxicity after hydroperoxide exposure.     

Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi.

The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals.     

Cytotoxic and DNA-damaging effects of 1,2-bis(sulfonyl)hydrazines on human cells of the Mer+ and Mer- phenotype

A series of 1,2-bis(sulfonyl)hydrazines with the capacity to function as alkylating agents have been evaluated for their toxicity towards Mer- HT29 and Mer- BE cells, and for their ability to produce DNA damage expressed as single-strand breaks and DNA interstrand cross-links. Compounds of this class with methylating potential showed a marked difference in their capacity to inhibit the growth of Mer- and Mer+ cells, being considerably more toxic to BE Mer- cells. Dose-dependent DNA single-strand breaks were induced by these agents, with the quantity of breaks produced in Mer- and Mer+ cells being essentially the same. Maintenance of these lesions did not appear to explain the differential in toxicity to BE and HT29 cells. A chloroethylating compound of this class was also more toxic to Mer- BE cells than to Mer+ HT29 cells, but the differential toxicity was considerably less than that of the methylating agents of the series. The chloroethylating agent did not produce measurable single-strand breaks of the DNA of treated cells, but caused more DNA interstrand cross-links in Mer- cells than in Mer+ cells. Thus, DNA interstrand cross-links may be at least in part responsible for the cell kill produced by this agent. The findings suggest that methylating and chloroethylating derivatives of the 1,2-bis(sulfonyl)hydrazine family have different biochemical determinants of their cytodestructive actions.     

Oxygen toxicity in the perfused rat liver and lung under hyperbaric conditions.

1. In the lung and liver of tocopherol-deficient rats, the activities of glutathione peroxidase and glucose 6-phosphate dehydrogenase were increased substantially, suggesting an important role for both enzymes in protecting the organ against the deleterious effects of lipid peroxides. 2. Facilitation of the glutathione peroxidase reaction by infusing t-butyl hydroperoxide caused the oxidation of nicotinamide nucleotides and glutathione, resulting in a concomitant increase in the rate of release of oxidized glutathione into the perfusate. Thus the rate of production of lipid peroxide and H2O2 in the perfused organ could be compared by simultaneous measurement of the rate of glutathione release and the turnover number of the catalase reaction. 3. On hyperbaric oxygenation at 4 X 10(5)Pa, H2O2 production, estimated from the turnover of the catalase reaction, was increased slightly in the liver, and glutathione release was increased slightly, in both lung and liver. 4. Tocopherol deficiency caused a marked increase in lipid-peroxide formation as indicated by a corresponding increase in glutathione release under hyperbaric oxygenation, with a further enhancement when the tocopherol-deficient rats were also starved. 5. The study demonstrates that the primary response to hyperbaric oxygenation is an elevation of the rate of lipid peroxidation rather than of the rate of formation of H2O2 or superoxide.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

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.     

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.     

Responses of glutathione system and antioxidant enzymes to exhaustive exercise and hydroperoxide.

Glutathione (gamma-glutamylcysteinylglycine) is one of the major antioxidants in the body. The present study investigated the changes of glutathione status, oxidative injury, and antioxidant enzyme systems after an exhaustive bout of treadmill running and/or hydroperoxide injection in male Sprague-Dawley rats. Concentrations of total and reduced glutathione in deep vastus lateralis muscle were significantly increased (P less than 0.01) after exhaustive exercise with either hydroperoxide (t-butyl hydroperoxide) or saline injection, whereas hydroperoxide alone had no significant effect. Exhaustive exercise increased muscle glutathione disulfide content by 75 and 60% (P less than 0.05), respectively, in hydroperoxide and saline groups. Concentrations of glutathione-related amino acids glutamate, cysteine, and aspartate were significantly increased in the same muscle after exhaustion. Hepatic glutathione status was not affected by either hydroperoxide injection or exercise. Glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase activities were significantly elevated after exhaustive exercise with or without hydroperoxide injection in muscle but not in liver. Hydroperoxide and exhaustive exercise enhanced lipid peroxidation in muscle and liver, respectively. It is concluded that exhaustive exercise can impose a severe oxidative stress on skeletal muscle and that glutathione systems as well as antioxidant enzymes are important in coping with free radical-mediated muscle injury.     

Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence.

Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.     

Hydrogen peroxide-induced DNA damage in bovine lens epithelial cells

The present investigation was undertaken to determine the types and extent of DNA damage resulting from incubation of primary cultures of bovine lens epithelial cells with hydrogen peroxide. Significant numbers of DNA single-strand breaks were detected by alkaline elution after exposure to as little as 25 microM H2O2 for 5 min at 37 degrees C. The extent of single-strand breakage was concentration dependent and linear from 25 to 200 microM H2O2. The observed single-strand breaks appear primarily due to the action of the hydroxyl radical via a Fenton reaction as both an iron chelator, 1,10-phenanthroline and OH. scavengers, including DMSO, KI and glycerol, significantly inhibited the DNA-damaging effect of H2O2. Diethyldithiocarbamate, an inhibitor of superoxide dismutase, further potentiated the DNA-damaging effects of H2O2, presumably by increasing the steady-state concentration of Fe2+. DNA-protein cross-linking was not observed. In addition, significant levels of 5,6-saturated thymine residues or pyrimidine dimers were not detected after modification of the alkaline elution methodology to allow the use of either E. coli endonuclease III or bacteriophage T4 endonuclease V, respectively. No double-strand breaks were detected after incubation of epithelial cell cultures with H2O2 concentrations of up to 400 microM for 10 min and subsequent neutral filter elution. Since, in vivo, the lens epithelium contains populations of both quiescent and dividing cells, the degree of susceptibility to oxidative damage was also studied in actively growing and plateau-phase cultures. Reduced levels of single-strand breakage were observed when plateau-phase cultures were compared to actively growing cells. In contrast, essentially no differences in repair rates were noted at equitoxic doses of H2O2. The above results suggest that lens epithelial cells may be particularly sensitive to oxidative damage and thus are a good model system in which to study the effects of oxidative stress.     

GSH-dependent peroxidase activity of the rice (Oryza sativa) glutaredoxin,a thioltransferase

Glutaredoxin (Grx) is a 12-kDa thioltransferase that reduces disulfide bonds of other proteins and maintains the redox potential of cells. In addition to its oxidoreductase activity, we report here that a rice Grx (OsGrx) can also function as a GSH-dependent peroxidase. Because of this antioxidant activity, OsGrx protects glutamine synthetase from oxidative damage. Individually replacing the conserved Cys residues in OsGrx with Ser shows that Cys(23), but not Cys(26), is essential for the thioltransferase and GSH-dependent peroxidase activities. Kinetic characterization of OsGrx reveals that the maximal catalytic efficiency (V(max)/K(m)) is obtained with cumene hydroperoxide rather than H(2)O(2) or t-butyl hydroperoxide.     

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.     

Properties of glutathione release observed during reduction of organic hydroperoxide, demethylation of aminopyrine and oxidation of some substances in perfused rat liver, and their implications for the physiological function of catalase.

The enhanced reduction of t-butyl hydroperoxide by glutathione peroxidase is accompanied by a decrease in the cellular concentration of both glutathione and NADPH in isolated liver cells, resulting in the release of GSSG (oxidized glutathione) from the perfused rat liver. This phenomenon, first reported by H. Sies, C. Gerstenecker, H. Menzel & L. Flohé (1972) (FEBS Lett. 27, 171-175), can be observed under a variety of conditions, not only with the acceleration of the glutathione peroxidase reaction by organic peroxides, but also during the oxidation of glycollate and benzylamine, during demethylation of aminopyrine in the liver of the phenobarbital-pretreated rat and during oxidation of uric acid in the liver of the starved rat pretreated with 3-amino-1,2,4-triazole. The rate of release of GSSG is altered markedly by changes in the metabolic conditions which affect the rate of hepatic NADPH generation. Thus, regardless of whether achieved by enhanced oxidation of glutathione by glutathione peroxidase or by oxidation of NADPH through other metabolic pathways, an increase in the cellular concentration of GSSG appears to facilitate its release. It has been found that, in addition to the hexose monophosphate shunt, the mitochondrial NADH-NADP+ transhydrogenase reaction plays an important role in supplying reducing equivalents to the glutathione peroxidase reaction and in maintaining the cellular oxidation-reduction state of the nicotinamide nucleotides. Spectrophotometric analysis of the steady-state concentration of the catalase-H2O2 intermediate with simultaneous measurement of the rate of release of GSSG leads to the conclusion that intracellular compartmentation of catalase in the peroxisomes and glutathione peroxidase in the cytosol and mitochondria distinguishes the reactivities of these enzymes one from the other, and facilitates their effective cooperation in hydroperoxide metabolism in the liver.     

Effects of metal ion chelators on DNA strand breaks and inactivation produced by hydrogen peroxide in Escherichia coli: detection of iron-independent lesions.

In order to study the role of metallic ions in the H2O2 inactivation of Escherichia coli cells, H2O2-sensitive mutants were treated with metal ion chelators and then submitted to H2O2 treatment. o-Phenanthroline, dipyridyl, desferrioxamine, and neocuproine were used as metal chelators. Cell sensitivity to H2O2 treatment was not modified by neocuproine, suggesting that copper has a minor role in OH production in E. coli. On the other hand, prior treatment with iron chelators protected the cells against the H2O2 lethal effect, indicating that iron participates in the production of OH. However, analysis of DNA sedimentation profiles and DNA degradation studies indicated that these chelators did not completely block the formation of DNA single-strand breaks by H2O2 treatment. Thiourea, a scavenger of OH, caused a reduction in both H2O2 sensitivity and DNA single-strand break production. The breaks observed after treatment with metal chelators and H2O2 were repaired 60 min after H2O2 elimination in xthA but not polA mutant cells. Therefore, we propose that there are at least two pathways for H2O2-induced DNA lesions: one produced by H2O2 through iron oxidation and OH production, in which lesions are repaired by the products of the xthA and polA genes, and the other produced by an iron-independent pathway in which DNA repair requires polA gene products but not those of the xthA gene.     

Ebselen augments its peroxidase activity by inducing nrf-2-dependent transcription

Ebselen is an organoselenium compound that acts as a glutathione peroxidase mimic. Since ebselen is a hydrophobic, thio-reactive compound capable of interacting with Keap-1, we tested its ability to activate nrf-2-dependent responses in the human hepatocarcinoma derived cell line, HepG2. Ebselen (25 microM) increased expression of an nrf-2 response element reporter in transient transfection experiments by 4-fold. Although, the induction was lower than that observed with classic nrf-2 inducer, sulforaphane (10 microL; 7-fold), ebselen also induced expression of native NAD(P)H:quinone oxidoreductase (1.6-fold) activity; induction of this protein is known to be dependent on nrf-2 action. Treatment of HepG2 cells with ebselen increased glutathione levels after 12 (1.5-fold) or 24 (1.9-fold)h of treatment. Treatment of the cells with either sulforaphane or ebselen 24 h prior to treatment with varying concentrations of t-butyl hydroperoxide increased the half maximal lethal dose from 28 to 42 microM and 58 microM for sulforaphane and ebselen, respectively. The protective effects of ebselen treatment were greater with pretreatment (IC50=58 microM) than simultaneous addition (IC50=45 microM). The protein synthesis inhibitor cycloheximide blocked increases in intracellular glutathione synthesis and partially blocked the protective effects of this regimen on increasing cell survival following t-butyl hydroperoxide treatment. Likewise co-treatment with the MEK 1 inhibitor, PD98059, which has been shown to inhibit nrf-2-dependent gene activation, partially inhibited the ebselen-dependent increases in IC50 while not affecting the control cells. We conclude that nrf-2 activation augments the role of ebselen as an antioxidant or by indirect induction of cellular antioxidant defences.     

DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2)

Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2). As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis.     

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells

Pluripotent mouse embryonic stem cells (mES cells) exhibit ∼ 100 large γH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (> 10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PK_cs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous γH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of γH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, γH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive γH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).