Alterations in cardiac contractile proteins due to oxygen free radicals.

In view of the potential role of free radicals in the genesis of cardiac abnormalities under different pathophysiological conditions and the importance of contractile proteins in determining heart function, this study was undertaken to examine the effects of oxygen free radicals on the rat heart myofibrils. Xanthine plus xanthine oxidase (X + XO) which is known to generate superoxide anions (O2-) and hydrogen peroxide (H2O2), an activated species of oxygen, was found to decrease Ca(2+)-stimulated ATPase activity, increase Mg(2+)-ATPase activity and reduce sulfhydryl (SH) group contents in myofibrils; these effects were completely prevented by superoxide dismutase (SOD) plus catalase (CAT). Both H2O2 and hypochlorous acid (HOCl), an oxidant, produced actions on cardiac myofibrils similar to those observed by X + XO. The effects of H2O2 and HOCl were prevented by CAT and L-methionine, respectively. N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), inhibitors of SH groups, also produced effects similar to those seen with X + XO. Dithiothreitol (DTT), a well known sulfhydryl-reducing agent, prevented the actions of X + XO, H2O2, HOCl, NEM and DTNB. These results suggest that marked changes in myofibrillar ATPase activities by different species of oxygen free radicals may be mediated by the oxidation of SH groups.     

Hypochlorite- and hypobromite-mediated radical formation and its role in cell lysis.

Activated leukocytes generate the potent oxidants HOCl and HOBr via the formation of H(2)O(2) and the release of peroxidase enzymes (myeloperoxidase, eosinophil peroxidase). HOCl and HOBr are potent microbiocidal agents, but excessive or misplaced production can cause tissue damage and cell lysis. In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently. These radicals are nitrogen-centered, cell-protein-derived species and have parameters identical to those detected with red blood cells and HOBr. Exposure of these cells to HOBr did not give detectable radicals. Overall these experiments demonstrate that HOCl and HOBr react with different selectivity with cellular targets, and that this can result in radical formation. This radical generation can precede, and may play a role in, cell lysis.     

In vitro production of free oxygen radicals induced by pulsed ultrasounds in whole blood exposed to diagnostical frequencies and intensities.

DNA alkalinization experiments on lymphocytes from sonicated whole blood and on in vitro cultured lymphocytes in presence of free radical scavengers (superoxide dismutase, catalase and mannitol) showed that lesions inflicted upon DNA by pulsed ultrasounds could be ascribed to production of free radicals (O2-, OH.) and H2O2, which could mediate the production of still unidentified organic radicals, likely to be responsible for DNA damage.     

Interaction of tert-butyl hydroperoxide with hypochlorite induces peroxyl radicals. A chemiluminescence study

The interaction of hypochlorite (HOCl/OCl-) with tert-butyl hydroperoxide ((CH3)3COOH) was investigated by chemiluminescence. It was shown that the addition of HOCl/OCl- to (CH3)3COOH induces a fast chemiluminescent flash. The intensity of this flash increases with the increase in both HOCl/OCl- and (CH3)3COOH concentration. The chemiluminescence is quenched in a concentration-dependent manner in the presence of free radical spin traps N-tert-butyl nitrone and alpha-(4-pyridyl-1-oxyl)-N-tert-butyl nitrone. This fact proves that free radicals take part in the interaction of HOCl/OCl- and (CH3)3COOH. Hypochlorite yielded a very similar chemiluminescence spectrum in its reaction with (CH3)3COOH as Ce4+. It differed considerably from the spectrum in the system H2O2 and HOCl/OCl-. It is well known that the interaction of Ce4+ and (CH3)3COOH produces peroxyl radicals. These results confirm the hyothesis that the interaction of HOCl/OCl- and (CH3)3COOH is mediated by peroxyl radicals. Thus, organic hydroperoxides always present in unsaturated lipids can induce lipid peroxidation processes in the reaction with HOCl/OCl-.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Molecular chlorine generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes produces 5-chlorocytosine in bacterial RNA

Myeloperoxidase, a heme enzyme secreted by activated phagocytes, uses H(2)O(2) and Cl(-) to generate the chlorinating intermediate hypochlorous acid (HOCl). This potent cytotoxic oxidant plays a critical role in host defenses against invading pathogens. In this study, we explore the possibility that myeloperoxidase-derived HOCl might oxidize nucleic acids. When we exposed 2'-deoxycytidine to the myeloperoxidase-H(2)O(2)-Cl(-) system, we obtained a single major product that was identified as 5-chloro-2'-deoxycytidine using mass spectrometry, high performance liquid chromatography, UV-visible spectroscopy, and NMR spectroscopy. 5-Chloro-2'-deoxycytidine production by myeloperoxidase required H(2)O(2) and Cl(-), suggesting that HOCl is an intermediate in the reaction. However, reagent HOCl failed to generate 5-chloro-2'-deoxycytidine in the absence of Cl(-). Moreover, chlorination of 2'-deoxycytidine was optimal under acidic conditions in the presence of Cl(-). These results implicate molecular chlorine (Cl(2)), which is in equilibrium with HOCl through a reaction requiring Cl(-) and H(+), in the generation of 5-chloro-2'-deoxycytidine. Activated human neutrophils were able to generate 5-chloro-2'-deoxycytidine. Cellular chlorination was blocked by catalase and heme poisons, consistent with a myeloperoxidase-catalyzed reaction. The myeloperoxidase-H(2)O(2)-Cl(-) system generated similar levels of 5-chlorocytosine in RNA and DNA in vitro. In striking contrast, only cell-associated RNA acquired detectable levels of 5-chlorocytosine when intact Escherichia coli was exposed to the myeloperoxidase system. This observation suggests that oxidizing intermediates generated by myeloperoxidase selectively target intracellular RNA for chlorination. Collectively, these results indicate that Cl(2) derived from HOCl generates 5-chloro-2'-deoxycytidine during the myeloperoxidase-catalyzed oxidation of 2'-deoxycytidine. Phagocytic generation of Cl(2) therefore may constitute one mechanism for oxidizing nucleic acids at sites of inflammation.     

纤维二糖脱氢酶生成羟自由基和还原各种自由基的研究

利用电子顺磁共振（ＥＳＲ）技术和硫代巴比妥酸（ＴＢＡ）反应研究了纤维二糖脱氢酶（ＣＤＨ）生成·ＯＨ和还原各种自由基的能力．以纤维二糖为电子供体时,ＣＤＨ可以生成·ＯＨ．·ＯＨ生成量与ＣＤＨ、Ｆｅ３＋和Ｏ２的浓度有关．加入过氧化氢酶可使·ＯＨ的生成明显减少．ＣＤＨ可以还原自旋加合物［ＰＢＮ－ＯＨ］·、氮氧自由基和天然木素分子中的自由基．结果表明,ＣＤＨ具有生成·ＯＨ和还原各种自由基的能力．对该酶在木质纤维素降解中的作用进行了探讨     

Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence

Stimulated neutrophils produce several potent oxidants including H2O2, O2- and HOCl. Previous studies have revealed all of these compounds to be capable of oxidizing luminol, a reagent often used to indicate, by its chemiluminescence, the oxidative burst of neutrophils. Data presented in this paper indicate that H2O2 and HOCl spontaneously react at physiologic pH to produce luminol-dependent chemiluminescence 100 times the sum of the chemiluminescence of either reagent alone. This enhancement is due to a co-oxidation by HOCl and H2O2, or to a novel oxidant generated by the interaction of HOCl and H2O2. The HOCl scavenger, taurine, inhibits the chemiluminescence. Evidence is presented against the participation of hydroxyl radical, O2- or singlet oxygen in the oxidation of luminol by HOCl and H2O2. These findings have implications for potential anti-inflammatory compounds.     

Protein kinase C involvement in lipid peroxidation and cell membrane damage induced by oxygen-based radicals in hepatocytes

We investigated the effects of oxygen-based radicals induced by t-butyl hydroperoxide or H2O2/Cu2+ on cultured hepatocytes. Radical exposure caused membrane lesions (blebs), lactate dehydrogenase release and lipid peroxidation (i.e. formation of malondialdehyde) in cells. As expected, radical scavengers (catalase, alpha-tocopherol) strongly inhibited these phenomena. A similar or even superior inhibitory effect was achieved by the protein kinase C (PKC) inhibitors H-7 and phloretin. These agents did not reveal notable radical scavenging properties as assessed by their ability to break down H2O2. The PKC stimulators 4 beta-phorbol-12-myristate-13 and 1-olyeoyl-2-acetyl-sn-glycerol intensified the detrimental actions of the radical-inducing agents. [3H]Phorbol-12,13-dibutyrate-binding studies showed that membrane association of PKC is markedly increased in hepatocytes after exposure to H2O2/Cu2+ or t-butyl hydroperoxide. These results suggest that PKC membrane translocation and activation may be important for mediating membrane damage and lipid peroxidation after cells are exposed to oxygen-based radicals.     

Hypochlorite-induced oxidation of thiols: Formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.

We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca(2+) handling.     

Role of monochloramine in the oxidation of erythrocyte hemoglobin by stimulated neutrophils

Stimulation of the oxygen (O2) metabolism of isolated human neutrophilic leukocytes resulted in oxidation of hemoglobin of autologous erythrocytes without erythrocyte lysis. Hb oxidation could be accounted for by reduction of O2 to superoxide (O-2) by the neutrophils, dismutation of O-2 to yield hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (Cl-) by H2O2 to yield hypochlorous acid (HOCl), the reaction of HOCl with endogenous ammonia (NH+4) to yield monochloramine ( NH2Cl ), and the oxidative attack of NH2Cl on erythrocytes. NH2Cl was detected when HOCl reacted with the NH+4 and other substances released into the medium by neutrophils. The amount of NH+4 released was sufficient to form the amount of NH2Cl required for the observed Hb oxidation. Oxidation was increased by adding myeloperoxidase or NH+4 to increase NH2Cl formation. Due to the volatility of NH2Cl , Hb was oxidized when neutrophils and erythrocytes were incubated separately in a closed container. Oxidation was decreased by adding catalase to eliminate H2O2, dithiothreitol to reduce HOCl and NH2Cl , or taurine to react with HOCl or NH2Cl to yield taurine monochloramine . NH2Cl was up to 50 times more effective than H2O2, HOCl, or taurine monochloramine as an oxidant for erythrocyte Hb, whereas HOCl was up to 10 times more effective than NH2Cl as a lytic agent. NH2Cl contributes to oxidation of erythrocyte components by stimulated neutrophils and may contribute to other forms of neutrophil oxidative cytotoxicity.     

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.     

The antioxidant action of taurine, hypotaurine and their metabolic precursors.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.     

Hypochlorous acid is a potent inhibitor of GST P1-1.

Glutathione S-transferase is a phase II detoxification enzyme that can be inactivated by H(2)O(2). During oxidative stress various other reactive oxygen species are generated that are more reactive than the relatively stable H(2)O(2). Hypochlorous acid (HOCl) is a powerful oxidant which is highly reactive towards a range of biological substrates. We studied the influence of HOCl on the activity of GST P1-1. HOCl inhibits purified glutathione S-transferase P1-1 in a concentration dependent manner with an IC(50)-value of 0.6 microM, which is more than 1000 times as low as IC(50) reported for H(2)O(2). HOCl lowered the V(max) value, but did not affect the K(m) for CDNB. Our results show that HOCl is a potent, non-competitive inhibitor of GST P1-1. The relevance of this effect is discussed.     

Role of hydroxyl radicals in the iron-ethylenediaminetetraacetic acid mediated stimulation of microsomal oxidation of ethanol

The microsomal oxidation of ethanol or 1-butanol was increased by ferrous ammonium sulfate-ethylenediaminetetraacetic acid (1:2) (Fe-EDTA) (3.4-50 microM). The increase was blocked by hydroxyl radical scavenging agents such as dimethyl sulfoxide or mannitol. The activities of aminopyrine demethylase or aniline hydroxylase were not affected by Fe-EDTA. The accumulation of H2O2 was decreased in the presence of Fe-EDTA, consistent with an increased utilization of H2O2. Other investigators have shown that Fe-EDTA increases the formation of hydroxyl radicals in systems where superoxide radicals are generated. The stimulation by Fe-EDTA appears to represent a pathway involving hydroxyl radicals rather than catalase because (1) stimulation occurred in the presence of azide, which inhibits catalase, (2) stimulation occurred in the presence of 1-butanol, which is not an effective substrate for catalase, and (3) stimulation was blocked by hydroxyl radical scavenging agents, which do not affect catalase-mediated oxidation of ethanol. A possible role for contaminating iron in the H2O or buffers could be ruled out since similar results were obtained with or without chelex-100 treatment of these solutions. The stimulatory effect by Fe-EDTA required microsomal electron transfer with NADPH, and H2O2 could not replace the NADPH-generating system. In the absence of microsomes or catalase, Fe-EDTA also stimulated the coupled oxidation of ethanol during the oxidation of xanthine by xanthine oxidase. These results suggest that during microsomal electrom transfer, conditions may be appropriate for a Fenton type or a modified Haber-Weiss type of reaction to occur, leading to the production of hydroxyl radicals.     

Distinct modes of cell death induced by different reactive oxygen species: amino acyl chloramines mediate hypochlorous acid-induced apoptosis

Oxidants derived from inflammatory phagocytes compose a key element of the host immune defense system and can kill mammalian cells by one of several different mechanisms. In this report, we compare mechanisms of cell death induced in human B lymphoma cells by the inflammatory oxidants superoxide, H(2)O(2), and HOCl. The results indicate that the mode of cell death induced depends on the nature of the oxidant involved and the medium in which the cells are treated. When human Burkitt's lymphoma cells are exposed to superoxide anion, generated as a flux from xanthine and xanthine oxidase, the cells die by a non-apoptotic mechanism (pyknosis/necrosis) identical to that seen when cells are treated with a bolus of reagent H(2)O(2). Addition of superoxide dismutase has no effect, whereas catalase is completely protective, indicating that exogenously generated superoxide kills cells entirely through its dismutation into H(2)O(2). In contrast, cells treated in culture media with reagent HOCl die largely by apoptosis. HOCl-induced apoptosis is mediated by aminoacyl chloramines generated in the culture media and can be mimicked by treatment of cells with taurine chloramine or with long lived chloramines generated from modified Lys or Arg. The results suggest that in a physiological milieu in which O(2)(-) and H(2)O(2) are the main oxidants being formed, the principal form of cell death may be necrotic, and under inflammatory conditions in which HOCl is generated, apoptotic cell death may predominate.     

Effect of hydrogen peroxide on intracellular pH in the human atrial myocardium

The cardiac injury observed during myocardial ischemia and reperfusion has been shown to be a consequence of a complex mechanism in which the accumulation of hydrogen peroxide (H2O2) and other oxygen free radicals (OFRs), and intracellular pH (pHi) are believed to play a major role. However, the effect of H2O2 on pHi has not been well characterized in the human atrial myocardium. In the present study, we superfused hydrogen peroxide into the human atrial tissue in order to assess the effects of oxygen free radicals on the pHi, and, furthermore, to test the ability of certain potential cardioprotective agents, including scavengers of the *OH free radical (N-(mercaptopropionyl)-glycine; N-MPG) and the HOCl free radical (L-methionine), to protect against oxidative-induced pHi challenge. The human atrial tissues were obtained from patients undergoing corrective open-heart surgery. The ratiometric recordings of pHi were measured using the pH-sensitive, dual-excitation and dual-emission fluorescent dye BCECF (2', 7'-bis(carboxyethyl)-5, 6-carboxyfluorescein acetoxymethyl ester). By continuously monitoring pHi changes in human atrial myocardium, we have found, for the first time, that (a) H2O2 (30 microM-3 mM) induced a significant dose-dependent intracellular acidosis, (b) N-MPG caused a significant block on the intracellular acidosis induced by 3 mM H2O2, whereas L-methionine did not, and (c) Hoe 694, a specific Na+/H+ exchanger (NHE) inhibitor, caused a similar extents like that induced by 3 mM H2O2. Our data suggest that the effects of H2O2 are caused mainly through the generation of *OH, which is attributed to the intracellular acidosis seen in the human atrial trabecular muscle. The possible underlying mechanism for H2O2-induced acidosis is likely due to its inhibition on the activity of NHE and other acid extruders, as the pHi changes after H2O2 exposure could be detected even though the activity of NHE was completely blocked by 30 mM Hoe 694.