Heat shock inactivates cellular antioxidant defenses against hydrogen peroxide: protection by glucose

Hyperthermia is used in cancer treatment and potentiates the cytotoxicity of radiation and certain chemotherapy drugs. The mechanism(s) of heat killing and those involved in heat potentiation of cytotoxic modalities are not understood. This study examines whether heat shock causes a redox imbalance, leading to oxidative changes in Chinese hamster ovary cells. Decreases in the GSH/GSSG ratio reflected an oxidative imbalance in heated (42 degrees C) and in H(2)O(2)-challenged cells. Glucose provided protection against these changes. Glucose also protected cells against cytotoxicity of H(2)O(2) and/or hyperthermia (42 to 43 degrees C). Glucose appears to protect cells against H(2)O(2) and heat shock by providing NADPH through its metabolism via the pentose phosphate cycle (PC). When cells were deprived of glucose, there was a marked decrease in the GSH/GSSG ratio and in NADPH levels, indicating a severe redox imbalance. Glucose deprivation caused cell death, which was consistent with increased accumulation of H(2)O(2), since three distinct H(2)O(2)-detoxifying systems (N-acetyl-L-cysteine, sodium pyruvate, and catalase) rescued cells against cytotoxicity. Nontoxic levels of H(2)O(2) stimulated a corresponding increase in both PC activity and NADPH levels. NADPH levels and basal activity of the PC increased at 42 degrees C. However, the oxidant-stimulated increases in PC activity and NADPH levels were lost in heated cells. Therefore, heat shock inactivates an important cellular defense mechanism against oxidants. These findings suggest that heat shock may enhance the cytotoxicity of oxidants by inhibiting increases in PC activity following oxidative stress. These data are potentially relevant to understanding the potentiation of cytotoxicity of radiation and oxidant-generating drugs by heat shock, used in combined modality cancer treatment.     

Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells.

Multidrug resistance is a major obstacle for the successful use of chemotherapy. The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump. We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine. The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay. We examined the ability of hyperthermia (42 degrees C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products. Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid. CH(R)C5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells. Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity. Hyperthermia (42 degrees C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CH(R)C5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CH(R)C5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time). Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CH(R)C5 and AuxB1 cells. Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.     

Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts

Survival after H2O2 exposure or heat shock of asynchronous Chinese hamster ovary cells (HA-1) was assayed following pretreatment with mildly toxic doses of either H2O2 or hyperthermia. H2O2 cytotoxicity at 37 degrees C, expressed as a function of mM H2O2 was found to be dependent on cell density at the time of treatment. The density dependence reflected the ability of cells to reduce the effectiveness of H2O2 as a cytotoxic agent. When the survival data were plotted as a function of mumoles H2O2/cell at the beginning of the treatment, survival was independent of cell density. Cells pretreated with 0.1 mM (3-5 mumoles/cell X 10(-7)) H2O2 for 1 hr at 37 degrees C (30-50% survival) became resistant to a subsequent H2O2 treatment 16-36 hr after pretreatment [dose modifying factor (DMF) at 1% isosurvival = 4-6]. Their resistance to 43 degrees C heating, however, was only slightly increased over controls 16-36 hr following pretreatment (DMF at 1% isosurvival = 1.2). During this same interval, the synthesis of protein migrating in the 70 kD region of a one-dimensional SDS-polyacrylamide gel was enhanced twofold in the H2O2-pretreated cells. When the cells were heated for 15 min at 45 degrees C (40-60% survival), the survivors became extremely resistant to 43 degrees C heating and somewhat resistant to H2O2 (DMF at 1% isosurvival = 2). The heat-induced resistance to heat developed much more rapidly (reached a maximum between 6 and 13 hr) following pretreatment than the heat-induced resistance to H2O2 (16-36 hr). The enhanced synthesis of 70 kD protein after heat shock was greater in magnitude and occurred more rapidly following preheating than following H2O2 pretreatment. The cells that became resistant to H2O2 by either pretreatment (H2O2 or heat shock) also increased their ability to reduce the H2O2 cytotoxicity from the treatment medium beyond that of the untreated HA-1 cells. This may be one of the mechanisms involved in the increased resistance and a common adaptive mechanism induced by both stresses. These data indicate that mammalian cells develop resistance to H2O2 following mild pretreatment with H2O2 or heat shock. The cross-resistance induced by H2O2 and heat shock reinforce the hypothesis that some overlap in mechanisms exist between the cellular responses to these two stresses. However, the failure of H2O2 pretreatment to induce much resistance to heat indicates that there are also differences in the actions of the two agents.     

Oxidative damage to fibronectin. II. The effect of H2O2 and the hydroxyl radical.

The effect of H2O2 and the hydroxyl radical (.OH) on fibronectin was investigated. .OH was generated in three ways: (i) by radiolysis with 60Co under N2O, or by the Fenton system using either (ii) equimolar Fe(2+)-EDTA and H2O2 or (iii) H2O2 and catalytic amounts of Fe(2+)-EDTA recycled with ascorbate. Each system had a different effect. H2O2 alone caused no changes, even at an 800-fold molar excess. Radiolytic .OH caused a rapid loss of tryptophan fluorescence, an increase in bityrosine fluorescence, and extensive crosslinking. The Fenton system using Fe-EDTA, H2O2, and ascorbate caused a loss in tryptophan fluorescence, a smaller increase in bityrosine than was seen with radiolytic .OH, and a threefold increase in carbonyl groups. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis fragmentation of fibronectin was seen. In contrast, when .OH was generated with equimolar Fe-EDTA and H2O2, the only change was a small increase in bityrosine fluorescence at the highest dose of oxidant. None of the systems used affected cysteine. All the changes except the loss of tryptophan by radiolytic .OH were completely inhibited with mannitol. The differences seen with radiolytic .OH and the Fe-EDTA, H2O2, ascorbate system were not solely due to O2 in the latter system since similar results were obtained under N2. The differences between radiolytic .OH and the Fenton systems could be partly due to the components of the latter systems reacting with .OH and thus competing with fibronectin. Our results demonstrate that the extent and type of fibronectin damage by .OH is dependent on the mode of radical generation.     

Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide

The combination of ascorbate, transition metal ions, and hydrogen peroxide (H(2)O(2)) is an efficient hydroxyl radical generating system called "the Udenfriend system." Although the pro-oxidant role of ascorbate in this system has been well characterized in vitro, it is uncertain whether ascorbate also acts as a pro-oxidant under physiological conditions. To address this question, human plasma, used as a representative biological fluid, was either depleted of endogenous ascorbate with ascorbate oxidase, left untreated, or supplemented with 25 microM-1 mM ascorbate. Subsequently, the plasma samples were incubated at 37 degrees C with 50 microM-1 mM iron (from ferrous ammonium sulfate), 60 or 100 microM copper (from cupric sulfate), and/or 200 microM or 1 mM H(2)O(2). Although endogenous and added ascorbate was depleted rapidly in the presence of transition metal ions and H(2)O(2), no cholesterol ester hydroperoxides or malondialdehyde were formed, i.e., ascorbate protected against, rather than promoted, lipid peroxidation. Conversely, depletion of endogenous ascorbate was sufficient to cause lipid peroxidation, the rate and extent of which were enhanced by the addition of metal ions but not H(2)O(2). Ascorbate also did not enhance protein oxidation in plasma exposed to metal ions and H(2)O(2), as assessed by protein carbonyl formation and depletion of reduced thiols. Interestingly, neither the rate nor the extent of endogenous alpha-tocopherol oxidation in plasma was affected by any of the treatments. Our data show that even in the presence of redox-active iron or copper and H(2)O(2), ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in human plasma in vitro.     

Hypothermia injury/cold-induced apoptosis--evidence of an increase in chelatable iron causing oxidative injury in spite of low O2-/H2O2 formation.

When incubated at 4 degrees C, cultured rat hepatocytes or liver endothelial cells exhibit pronounced injury and, during earlier rewarming, marked apoptosis. Both processes are mediated by reactive oxygen species, and marked protective effects of iron chelators as well as the protection provided by various other antioxidants suggest that hydroxyl radicals, formed by classical Fenton chemistry, are involved. However, when we measured the Fenton chemistry educt hydrogen peroxide and its precursor, the superoxide anion radical, formation of both had markedly decreased and steady-state levels of hydrogen peroxide did not alter during cold incubation of either liver endothelial cells or hepatocytes. Similarly, there was no evidence of an increase in O2-/H2O2 release contributing to cold-induced apoptosis occurring on rewarming. In contrast to the release/level of O2- and H2O2, cellular homeostasis of the transition metal iron is likely to play a key role during cold incubation of cultured hepatocytes: the hepatocellular pool of chelatable iron, measured on a single-cell level using laser scanning microscopy and the fluorescent indicator phen green, increased from 3.1 +/- 2.3 microM (before cold incubation) to 7.7 +/- 2.4 microM within 90 min after initiation of cold incubation. This increase in the cellular chelatable iron pool was reversible on rewarming after short periods of cold incubation. The cold-induced increase in the hepatocellular chelatable iron pool was confirmed using the calcein method. These data suggest that free radical-mediated hypothermia injury/cold-induced apoptosis is primarily evoked by alterations in the cellular iron homeostasis/a rapid increase in the cellular chelatable iron pool and not by increased formation of O2-/H2O2.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

Enhancement of cell killing by induction of apoptosis after treatment with mild hyperthermia at 42 degrees C and cisplatin.

Ohtsubo, T., Igawa, H., Saito, T., Matsumoto, H., Park, H. J., Song, C. W., Kano, E. and Saito, H. Enhancement of Cell Killing by Induction of Apoptosis after Treatment with Mild Hyperthermia at 42 degrees C and Cisplatin. Radiat. Res. 156, 103-109 (2001).We examined the interactive effects of cisplatin (1.0 microg/ml) combined with hyperthermia on cell killing and on the induction of apoptosis in IMC-3 human maxillary carcinoma cells. The cytotoxic effects of hyperthermia on IMC-3 cells at 44 degrees C were greater than at 42 degrees C, as has been reported for many other cells. The induction of apoptosis, DNA fragmentation and poly(ADP-ribose) polymerase cleavage were greater after hyperthermia at 44 degrees C for 30 min compared with treatment at 42 degrees C for 105 min, even though both of these heat doses were isoeffective in reducing cell survival to 50%. Treatment with cisplatin at 37 degrees C for up to 120 min did not result in cytotoxicity or the induction of apoptosis. The enhancement ratio for treatment with cisplatin at 42 degrees C was greater than that at 44 degrees C. More apoptosis was induced after the treatment with cisplatin at 42 degrees C compared to treatment with cisplatin at 44 degrees C. Taking these findings together, the combination of cisplatin and hyperthermia at 42 degrees C appeared to be more effective than cisplatin with hyperthermia at 44 degrees C for the induction of apoptosis in IMC-3 cells.     

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.     

Cytotoxicity of quinolones toward eukaryotic cells. Identification of topoisomerase II as the primary cellular target for the quinolone CP-115,953 in yeast.

The quinolone CP-115,953 (6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid) represents a novel mechanistic class of drugs with potent activity against eukaryotic topoisomerase II in vitro (Robinson, M. J., Martin, B. A., Gootz, T. D., McGuirk, P. R., Moynihan, M., Sutcliffe, J. A., and Osheroff, N. (1991) J. Biol. Chem. 266, 14585-14592). Although the quinolone is highly toxic to mammalian cells in culture, its mechanism of cytotoxic action is not known. Therefore, yeast was used as a model system to determine whether topoisomerase II is the primary target responsible for the in vivo effects of CP-115,953. The quinolone was equipotent to etoposide at enhancing DNA breakage mediated by the Saccharomyces cerevisiae type II enzyme. Moreover, at concentrations as low as 5 microM, CP-115,953 was cytotoxic to yeast cells that carried wild type topoisomerase II (TOP2+). By utilizing a yeast strain that expressed the top2-1 temperature-sensitive mutant, the effect of topoisomerase II activity on quinolone cytotoxicity was determined. At the permissive temperature of 25 degrees C, cells were highly sensitive to CP-115,953. However, at the semipermissive temperature of 30 degrees C (where in vivo enzyme activity is present but is greatly diminished), cells displayed only marginal sensitivity to the quinolone at concentrations as high as 50 microM. These results strongly suggest that topoisomerase II is the primary physiological target responsible for quinolone cytotoxicity and that CP-115,953 kills cells by converting the type II enzyme into a cellular poison.     

Oxygen- and temperature-dependent cytotoxic and radiosensitizing effects of cis-dichlorodiammineplatinum(II) on human NHIK 3025 cells in vitro

The radiosensitizing effect of the chemotherapeutic drug cis-dichlorodiammineplatinum(II) (cis-DDP) was tested on human NHIK 3025 cells cultivated in vitro. cis-DDP was found to exert a radiomodifying effect under hypoxic but not under aerobic conditions. These results confirm that cis-DDP may act as a radiosensitizer of hypoxic cells; however, the radiosensitizing effect was seen only at concentrations of cis-DDP having a considerable cytotoxic activity, and for practical reasons concerning survival level the highest drug concentration that was investigated was 15 microM at 37 degrees C. The radiosensitizing effect was of a dose-modifying type and with a dose-modifying factor (DMF) of 1.2 at 15 microM in hypoxic cells. The radiosensitizing as well as the cytotoxic effect of cis-DDP was found to be strongly temperature dependent. Isoeffect doses of cis-DDP was reduced with a factor of 3 at 22 as compared to 37 degrees C. We also found that hypoxic cells were less sensitive to cis-DDP than cells treated in the presence of oxygen. To test the correlation between cytotoxicity and radiosensitization on the one hand and cellular uptake of cis-DDP on the other, cell-associated Pt was measured by atomic absorption spectroscopy. From these studies the cytotoxicity of cis-DDP at 22 and 37 degrees C under aerobic conditions was found to be the same as long as the amount of cell-associated Pt (i.e., the cellular uptake) was the same. However, whether the cells were treated under hypoxic or aerobic conditions, the cellular uptake of Pt was the same. While the radiosensitizing effect was present at 37 and at 40 degrees C, no such effect could be found at 22 degrees C. Since the cytotoxicity of cis-DDP as well as the drug uptake was reduced about three times at 22 as compared to 37 degrees C, we increased the concentration threefold, to 50 microM at 22 degrees C. Still no radiosensitizing effect was found at this temperature.     

Nitroxides block DNA scission and protect cells from oxidative damage.

The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H2O2. Oxidative damage induced by H2O2 was assayed by monitoring cell survival. The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated the H2O2-induced damage. The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability. The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H2O2 cytotoxicity. The xthA cells were protected by catalase but not by superoxide dismutase (SOD). Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H2O2 toxicity, without lowering H2O2 concentration. To check whether nitroxides protect against O2.(-)-independent injury also, experiments were repeated under hypoxia. These nitroxides also protected hypoxic cells against H2O2, suggesting alternative modes of protection. Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.     

Hydrogen peroxide metabolism in alveolar macrophages after exposure to hypoxia and heat

High temperature can change the effects of intra- and intercellular regulators and therefore modify the cellular response to hypoxia. We investigated H(2)O(2) production by alveolar macrophages, isolated from adult male rats, which were incubated under conditions of oxygen deficiency and high temperature (experiment in vitro). The incubation of these cells for 2 hours at 10 % or 5 % oxygen led only to slight fluctuations in the H(2)O(2) level, while the rise of temperature from 37 degrees C up to 42 degrees C significantly increased its generation. Level of thiobarbituric acid-reactive substances (TBARS) underwent similar changes. Under these conditions the accumulation of H(2)O(2) was found to be caused mainly by its decreased cleavage rather than its enhanced production. This is indicated by decreased catalase and glutathione peroxidase activity together with a parallel absence of significant changes in superoxide dismutase (SOD) activity. Slight fluctuation of reduced glutathione level and the pronounced increase of glucose-6-phosphate dehydrogenase (G6PD) activity were detected. Strong (5 %) but not moderate (10 %) lack of oxygen led to a sharp increase in formation of cellular nitrite ions by alveolar macrophages. In general, our data showed that high temperature did not lead to any qualitative shifts of defined hypoxia-derived changes in oxidant/antioxidant balance in alveolar macrophages, but promoted sensitivity of cells to oxygen shortage.     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).     

Calcitriol sensitizes colon cancer cells to H2O2-induced cytotoxicity while inhibiting caspase activation

The anti-cancer activity of calcitriol, the active metabolite of Vitamin D, in the colon is usually attributed to its anti-proliferative and pro-differentiative actions. The levels of reactive oxygen species (ROS) are high in colon carcinomas due to increased aerobic metabolism and exposure to various anti-cancer modalities. We examined whether calcitriol modulates the response of colon cancer cells to the cytotoxic action of the common mediator of ROS injury, H2O2. Pretreatment with calcitriol (100 nM, 48 h) sensitized HT-29 colon cancer cells to cell death induced by acute exposure to H2O2 or chronic exposure to the H2O2 generating system, glucose/glucose-oxidase. Although the morphological features of H2O2-induced HT-29 cell death are consistent with apoptosis, we detected no executioner caspase activation in response to cytotoxic concentrations of H2O2 and treatment with a pan-caspase inhibitor did not affect H2O2-induced cytotoxicity nor its enhancement by calcitriol. Conversely, exposure of HT-29 cells to sub-toxic concentrations of H2O2 resulted in low executioner caspase activation that was inhibited by pretreatment with calcitriol. The sensitization of colon cancer cells to ROS-induced cytotoxicity may contribute to its assumed action as a chemopreventive agent and to its therapeutic potential alone or in combination with other anti-cancer modalities.     

Photo-oxidation of cells generates long-lived intracellular protein peroxides

Singlet oxygen is generated by several cellular, enzymatic, and chemical reactions as well as by exposure to UV or visible light in the presence of a sensitizer. Consequently, this oxidant has been proposed to be a damaging agent many pathologies. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study, we show that illumination of viable rose bengal-loaded THP-1 (human monocyte-like) cells with visible light gives rise to intracellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of rose bengal, is enhanced in D(2)O, and is decreased by azide, consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to about 1.5 nmoles peroxide per 10(6) cells, or 10 nmoles/mg cell protein, and account for up to approximately 15% of the O(2) consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, cellular protein peroxide levels decrease with t(1/2) about 4 h at 37 degrees C. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions gives rise to radicals as detected by EPR spin trapping. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer leads to novel long-lived, but reactive, intracellular protein peroxides via singlet oxygen-mediated reactions.     

Resistance to alloxan of tumoral insulin-producing cells

Rat pancreatic islets and insulin-producing cells of the RINm5F line were incubated for 5 min at 7 or 23 degrees C in media containing 3H2O and either L-[1-14C]glucose or [2-14C]alloxan. In the islets the intracellular distribution space of [2-14C]alloxan represented, at 7 and 23 degrees C respectively, 11.4 +/- 1.0 and 25.5 +/- 2.3% of the intracellular 3H2O space. In the RINm5F cells, the distribution space of [2-14C]alloxan failed to be affected by the ambient temperature and represented, after correction for extracellular contamination, no more than 5.2 +/- 0.5% of the intracellular 3H2O space. Preincubation for 30 min at 7 degrees C in the presence of alloxan (10 mM) failed to affect subsequent D-[U-14C]glucose oxidation in the tumoral cells, whilst causing a 70% inhibition of glucose oxidation in the islets. It is proposed that RINm5F cells are resistant to the cytotoxic action of alloxan, this being attributable, in part at least, to poor uptake of the diabetogenic agent.     

Hypothermia enhances bcl-2 expression and protects against oxidative stress-induced cell death in Chinese hamster ovary cells.

Oxidative stress is one of the major causes of cellular injury. Various reactive oxygen (ROS) and nitrogen (RNS) species such as superoxide, hydroxyl radical, peroxynitrite, and nitric oxide are involved in the manifestations of different types of organ toxicity and the resultant syndromes, symptoms, or diseases. Hypothermic conditions have been reported to reduce the oxidative stress in various in vitro and in vivo studies. In the present study, we sought to determine the effect of lowered temperatures on oxidative stress-induced cell death in Chinese hamster ovary (CHO) cells. We also investigated the oxidative stress-induced alterations in the expression of anti-apoptotic protein, bcl-2, in CHO cells at lowered temperatures. CHO cells were incubated at four different temperatures of 30, 32, 35, and 37 degrees C (control temperature) from 1 to 4 d. In another set, the cells were incubated with 100 microM hydrogen peroxide (H(2)O(2)) for 30 min before harvesting at different time points. The cells were harvested at 1, 2, 3, and 4 d. Cell survival was significantly higher at 30 degrees C as compared to 37 degrees C over 4 d of incubation. In cells incubated with H(2)O(2), significantly higher cell viability was observed at lower temperatures as compared to the cells incubated at 37 degrees C. The activity of glutathione peroxidase (GSH-Px) also increased significantly at lower temperatures. Lowered temperature also provided a significant increase in the expression of anti-apoptotic protein, bcl-2 after 4 d of incubation. These data suggest that hypothermic conditions lowers the risk of oxidative stress-induced cellular damage and programmed cell death by increasing the activity of GSH-Px and by the induction in the expression of the anti-apoptotic protein, bcl-2.     

Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss reaction

Cultured hepatocytes pretreated with the ferric iron chelator deferoxamine were resistant to the toxicity of H2O2 generated by either glucose oxidase or by the metabolism of menadione (2-methyl-1,4-naphthoquinone). Ferric, ferrous, or cupric ions restored the sensitivity of the cells to H2O2. Deferoxamine added to hepatocytes previously treated with this chelator prevented the restoration of cell killing by only ferric iron. The free radical scavengers mannitol, thiourea, benzoate, and 4-methylmercapto-2-oxobutyrate protected either native cells exposed to H2O2 or pretreated hepatocytes exposed to H2O2 and given ferric or ferrous iron. Superoxide dismutase prevented the killing of native hepatocytes by either glucose oxidase or menadione. With deferoxamine-pretreated hepatocytes, superoxide dismutase prevented the cell killing dependent upon the addition of ferric but not ferrous iron. Catalase prevented the killing by menadione of deferoxamine-pretreated hepatocytes given either ferric or ferrous iron. Deferoxamine pretreatment did not prevent the toxicity of t-butyl hydroperoxide but did, however, prevent that of cumene hydroperoxide. It is concluded that both ferric iron and superoxide ions are required for the killing of cultured hepatocytes by H2O2. The toxicity of H2O2 is also dependent upon its reaction with ferrous iron to form hydroxyl radicals by the Fenton reaction. The ferrous iron needed for this reaction is formed by the reduction of cellular ferric iron by superoxide ions. Such a sequence corresponds to the so-called iron-catalyzed Haber-Weiss reaction, and the present report documents its participation in the killing of intact hepatocytes by H2O2. Cumene hydroperoxide but not t-butyl hydroperoxide closely models the toxicity of hydrogen peroxide.