Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Does hydrogen peroxide exist “free” in biological systems?

Hydrogen peroxide (H₂O₂) can diffuse far from the site of production to intracellular locations where biological effects may be greater. The diffusion range is extended by H₂O₂ carriers formed spontaneously by hydrogen bonding with monomeric and polymeric compounds, including amino and dicarboxylic acids, peptides, proteins, nucleic acid bases, and nucleosides. Hydrogen peroxide adducts (HPAs) are readily synthesized, e.g., crystalline histidine (His)-H₂O₂ adducts. An equilibrium exists between an adduct-forming compound and H₂O₂. The detection and relative stabilities of HPAs are measured by the degree of decomposition of H₂O₂ as influenced by test compounds in buffered solution competing with glucose or fructose for H₂O₂. The HPAs delay decomposition of H₂O₂ up to several hundredfold. The overall charge on an HPA, i.e., its ability to penetrate cell membranes, influences the cytotoxic and clastogenic effects of H₂O₂. Growth inhibition of Salmonella typhimurium LT₂ by H₂O₂ is enhanced by neutral HPAs but decreased by anionic HPAs. Addition of catalase 1, 10, or 30 min after inoculation of S. typhimurium LT₂ reduces or nearly eliminates partial growth inhibition by H₂O₂, but a neutral HPA, expecially his-H₂O₂, transported H₂O₂ into the cells within 1 min, and in about 10 min completely inhibited growth. The stability of HPAs decreases with increasing pH or increasing temperature, while added Fe(II) in the presence and absence of EDTA accelerates H₂O₂ and HPA decomposition. Calculations indicate H₂O₂ hydrogen bonds with nucleic acid-base pairs with no apparent bond strain and energy stabilization comparable to normal hydrogen bonding.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (·MbFe(IV)=O) and/or ferrylmyoglobin (MbFe(IV)=O). Reduction of MbFe(IV)=O showed first-order kinetics for a 1-2 times stoichiometric excess of H₂O₂ to MMb while a 3-10 times stoichiometric excess of H₂O₂ resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H₂O₂ and BSA were influenced by [H₂O₂] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H₂O₂. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H₂O₂ to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H₂O₂ and the hyper-valent iron-center of H₂O₂-activated MMb.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

Redox Cycling of Human Methaemoglobin by H2O2 Yields Persistent Ferryl Iron and Protein Based Radicals

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H₂O₂, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H₂O₂ resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H₂O₂ showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H₂O₂. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalatic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10 fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: ∼20 mol H₂O₂ and abolished the protein radical steady state. The biological implications for these results are discussed.     

Hydroxylamine potentiates the effect of low dose hydrogen peroxide in glioma cells independent of p53

We had earlier shown that higher concentration of hydrogen peroxide (H₂O₂) induced p53-dependent apoptosis in glioma cell line with wild type p53 but had minimal effect on cells with mutated p53. Here we show a potentiating effect of hydroxylamine (HA), an inhibitor of catalase, on a nontoxic dose of H₂O₂ in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H₂O₂. Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H₂O₂ as compared to cells treated with each component alone in both the cell lines. Dimethyl sulfoxide (DMSO) protected cells. Cytosolic cytochrome c and activated caspase 3 were detected at 4 h. The results suggest that higher levels of intracellular ROS, generated by HA+H₂O₂ act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway.     

ESR Spin Trapping Detection of Hydroxyl Radicals in the Reactions of Cr(V) Complexes with Hydrogen Peroxide

Electron spin resonance (ESR) measurments provide direct evidence for the involvement of Cr(V) in the reduction of Cr(VI) by NAD(P)H. Addition of hydrogen peroxide (H₂O₂) to NAD(P)H-Cr(VI) reaction mixtures suppresses the Cr(V) signal and generates hydroxyl (OH) radicals (as detected via spin trapping), suggesting that Cr(V) reacts with H₂O₂ to generate the OH radicals. Reaction between H₂O₂ and a Cr(V)-glutathione complex. and between H₂O₂ and several Cr(V)-cdrboxylato complexes also produces OH radicals. These results suggest that Cr(V) complexes catalyze the generation of OH radicals from H₂O₂, and that OH radicals might play a significant role in the mechanism of Cr(VI) cytotoxicity.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

Coffee drinking increases levels of urinary hydrogen peroxide detected in healthy human volunteers

Freshly-voided human urine contains significant concentrations of hydrogen peroxide (H₂O₂). This H₂O₂ appears to arise in whole or in part by superoxide-dependent autoxidation of urinary biomolecules. Since instant coffee also contains high levels of H₂O₂, we examined the effect of coffee drinking on urinary levels of H₂O₂. Studies on healthy human volunteers showed that coffee drinking is rapidly and reproducibly followed by increased levels of H₂O₂ detectable in the urine for up to 2 h after drinking the coffee. The levels of H₂O₂ detected in urine suggest that exposure of human tissues to H₂O₂ may be greater than is commonly supposed. It is possible that H₂O₂ in urine could act as an antibacterial agent, and that H₂O₂ is involved in the regulation of glomerular function.     

Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by myeloperoxidase-generated "reactive species"

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. With MPO/H₂O₂/NaCl, LADH lipoamide reductase and diaphorase activities significantly decreased as a function of incubation time. Iodide, bromide, thiocyanide and chloride effectively supplemented the MPO/H₂O₂ system, KI and NaCl being the most and the least effective supplements, respectively. LADH inactivation by MPO/H₂O₂/NaCl and by NaOCl was similarly prevented by thiol compounds such as GSH, L-cysteine, N-acetylcysteine, penicillamine and N-(2-mercaptopropionyl-glycine) in agreement with the role of HOCl in LADH inactivation by MPO/H₂O₂/NaCl. LADH was also inactivated by MPO/NADH/halide, MPO/H₂O₂/NaNO₂ and MPO/NADH/NaNO₂ systems. Catalase prevented the action of the NADH-dependent systems, thus supporting H₂O₂ production by NADH-supplemented LADH. MPO inhibitors (4-aminobenzoic acid hydrazide, and isoniazid), GSH, L-cysteine, L-methionine and L-tryptophan prevented LADH inactivation by MPO/H₂O₂/NaNO₂. Other MPO systems inactivating LADH were (a) MPO/H₂O₂/chlorpromazine; (b) MPO/H₂O₂/monophenolic systems, including L-tyrosine, serotonin and acetaminophen and (c) MPO/H₂O₂/di- and polyphenolic systems, including norepinephrine, catechol, nordihydroguaiaretic acid, caffeic acid, quercetin and catechin. Comparison of the above effects and those previously reported with pig myocardial LADH indicates that both enzymes were similarly affected by the MPO-dependent systems, allowance being made for T. cruzi LADH diaphorase inactivation and the greater sensitivity of its LADH lipoamide reductase activity towards the MPO/H₂O₂/NaCl system and NaOCl.     

Effect of hydrogen peroxide in redox status estimation using nitroxyl spin probe

A procedure for estimating in vivo redox status using EPR and a hydrogen peroxide (H₂O₂)-dependent spin probe method is described. The mechanism of decreasing spin clearance in the selenium-deficient (SeD) rat is discussed. The in vivo decay constant of the nitroxyl spin probe in the liver region of SeD rats appeared to be slightly lower that of the selenium-adequate control (SeC) group, and was significantly smaller than that of normal rats. Bile H₂O₂ levels in normal rats were significantly lower than those in SeD rats. The in vivo decay constant of the spin probe in SeD rats depended on the bile H₂O₂ level. Furthermore, H₂O₂ was detected in the bile in all SeD rats, whereas bile H₂O₂ could be detected in only half of the normal rats. It was found that the in vivo decay constant of the spin probe in normal rats also depended on whether bile H₂O₂ was detected or not. In vivo decay constants were smaller in rats subjected to the surgical operation than in the nonoperated groups. The EPR signal of the nitroxyl radical in the liver homogenate was increased by addition of H₂O₂, which was administered 30 min before the rat was killed. It appears that H₂O₂ can oxidize the hydroxylamine formed following reduction of the spin probe in the liver.     

Role of Hydroxyl Radicals in Escherzchza Colz Killing Induced by Hydrogen Peroxide

Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH -) generated by H₂O₂ and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H₂O₂ lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxarnine, did not affect the survival of cells exposed to 2.5mM H₂O₂. In addition cell vulnerability to the same H₂O₂ concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H₂O₂ higher than 10mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content.

It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.     

Enzymic Pathways Involved in Cell Response to H2O2

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H₂O₂ was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H₂O₂ in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H₂O₂ corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H₂O₂, resulting in the overall cell survival different from that obtained after inhibition of either.     

Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H₂O₂/halide, MPO/NADH/halide and MPO/H₂O₂/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H₂O₂ system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H₂O₂/NaCl system. NaOCl and the MPO/H₂O₂/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/ NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H₂O₂ production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horseradish peroxidase suplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H₂O₂/NaNO₂ system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated “reactive species” oxidize essential thiol groups at LADH catalytic site.     

Purification of cytochrome c peroxidase for monitoring H2O2 production.

One of the most precise methods of determining hydrogen peroxide (H₂O₂) formation by biological systems is based on measuring the rate of enzyme-substrate complex formation between H₂O₂ and cytochrome c peroxidase (CCP). The main problem with this method is that CCP is not commercially available and has to be prepared in the laboratory. We have modified some currently available methods for purifying a highly active preparation of CCP in about 4 d. It includes a batch extraction of protein using DEAE-sepharose followed by concentration either by lyophilization or by passing the extract through a small DEAE-sepharose column instead of by ultrafiltration. The concentrated preparation is passed through a Sephadex G-75 column and the final CCP crystallized against water. The final preparations had a purity index (PI, ratio of absorbance at 408 nm/280 nm, equivalent to heme/protein ratio) above 1.2. These changes make the overall procedure very simple, preserving enzyme activity and spectral properties. In addition, we point out that special care has to be taken to eliminate cytochrome c from crude CCP extracts. Cytochrome c not only introduces an artifact when determining PI, but is also may act as a hydrogen donor for CCP when monitoring H₂O₂ formation, thus decreasing the sensitivity of this method.     

Why does SOD overexpression sometimes enhance,sometimes decrease,hydrogen peroxide production? A minimalist explanation

Toxic effects of superoxide dismutase (SOD) overexpression are commonly attributed to increased hydrogen peroxide (H₂O₂) production. Still, published experiments yield contradictory evidence on whether SOD overexpression increases or decreases H₂O₂ production. We analyzed this issue using a minimal mathematical model. The most relevant mechanisms of superoxide consumption are treated as pseudo first-order processes, and both superoxide production and the activity of enzymes other than SOD were considered constant. Even within this simple framework, SOD overexpression may increase, hold constant, or decrease H₂O₂ production. At normal SOD levels, the outcome depends on the ratio between the rate of processes that consume superoxide without forming H₂O₂ and the rate of processes that consume superoxide with high (≥ 1) H₂O₂ yield. In cells or cellular compartments where this ratio is exceptionally low (< 1), a modest decrease in H₂O₂ production upon SOD overexpression is expected. Where the ratio is higher than unity, H₂O₂ production should increase, but at most linearly, with SOD activity. The results are consistent with the available experimental observations. According to the minimal model, only where most superoxide is eliminated through H₂O₂-free processes does SOD activity have the moderately large influence on H₂O₂ production observed in some experiments.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.