Cytochrome c-catalyzed oxidation of organic molecules by hydrogen peroxide.

Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.     

Variables in xanthine oxidase-initiated luminol chemiluminescence: Implications for chemiluminescence measurements in biological systems

We tested the effects of generally used chemiluminescence inhibitors on an example of luminol chemiluminescence elicited by xanthine oxidase/hypoxanthine system, and attempted to assess their capabilities in discovering the reaction pathways leading to chemiluminescence. Luminol itself is a xanthine oxidase inhibitor and its concentration affects the reaction mechanism. Maximal chemiluminescence response was observed at luminol concentration inhibiting urate production. Chemiluminescence was totally inhibited by superoxide dismutase, the inhibition by catalase depended on luminol concentration. Ferricytochrome c, a detector of superoxide, either stimulated or inhibited chemiluminescence in a concentration-dependent manner. Chemiluminescence was highly stimulated by peroxidases. A pronounced inhibition of chemiluminescence was caused by chelators; 1 mm desferal and 0.01 mm diethyldithiocarbamate. It is suggested that measurement of luminol chemiluminescence is not a suitable method for discrimination among individual reactive oxygen species and their quantitative determination in biological systems.     

Luminol chemiluminescence using xanthine and hypoxanthine as xanthine oxidase substrates

Luminol chemiluminescence induced by the xanthine or hypoxanthine-O2-xanthine oxidase system is analyzed and compared. Characteristics of the light emission curves were examined considering the conventional reaction scheme for the oxidation of both substrates in the presence of xanthine oxidase. The ratio of the areas of the rate of superoxide production during substrate oxidation to uric acid. The O2-. to uric acid ratio for each substrate can account for differences in xanthine and hypoxanthine-supported light emission, since uric acid is a strong inhibitor of O2-.-dependent luminol chemiluminescence. These results are consistent with a free radical scavenging role for uric acid. A similar but weaker scavenging effect of xanthine may also contribute to the observed differences in chemiluminescent yields between both substrates.     

Metabolic activation of 1-naphthol and phenol by a simple superoxide-generating system and human leukocytes

Phenol and 1-naphthol, products of benzene and naphthalene biotransformation, are metabolized during O2- generation by xanthine oxidase/hypoxanthine and phorbol myristate acetate (PMA)-stimulated human neutrophils. The addition of 1-naphthol to xanthine oxidase/hypoxanthine incubations resulted in the formation of 1,4-naphthoquinone (1,4-NQ) whereas phenol addition yielded only small quantities of hydroquinone, catechol and a unidentified reducible product but not 1,4-benzoquinone. This formation of 1,4-NQ was dependent upon hypoxanthine, xanthine oxidase, and 1-naphthol and was inhibited by the addition of superoxide dismutase (SOD) demonstrating that the conversion was O2-mediated. During O2- generation by PMA-stimulated neutrophils, the addition of phenol interfered with luminol-dependent chemiluminescence and resulted in covalent binding of phenol to protein. Protein binding was 80% inhibited by the addition of azide or catalase to the incubations indicating that bioactivation was peroxidase-mediated. In contrast, the addition of 1-naphthol to PMA-stimulated neutrophils interfered with superoxide-dependent cytochrome c reduction as well as luminol-dependent chemiluminescence and also resulted in protein binding. Protein binding was only partially inhibited by azide or catalase. The addition of SOD in combination with catalase resulted in a significantly greater inhibition of binding when compared to that of catalase alone. The results of these experiments indicate that phenol and 1-naphthol are converted to reactive metabolites during superoxide generating conditions but by different mechanisms. The formation of reactive metabolites from phenol was almost exclusively peroxidase-mediated whereas the bioactivation of 1-naphthol could occur by two different mechanisms, a peroxidase-dependent and a direct superoxide-dependent mechanism.     

Detection of activated O2 species in vitro and in rat lungs by chemiluminescence

This study used chemiluminescence, an "on-line" photon-counting technique, to detect and characterize activated O2 species in vitro and in isolated rat lungs. The sensitivity and specificity of enhanced chemiluminescence for superoxide anion (O2-.) and hydrogen peroxide (H2O2) was evaluated in vitro. The effect of media conditions (such as O2 tension, albumin concentration, and sulfhydryl group availability) on luminescence was assessed in vitro. Xanthine-xanthine oxidase (X-XO) primarily produced superoxide anion in vitro. Enhanced chemiluminescence varied directly with the dose of luminescent probe used and the quantity of activated O2 species administered. The strength of the luminescent signal was also dependent on the concentration of albumin and O2 in the media. Lucigenin was more sensitive than luminol to the presence of O2-. and, unlike luminol, lucigenin did not alter radical production by XO. However, neither luminescent probe was specific for O2-., as both detected H2O2 and O2 in vitro. H2O2-induced chemiluminescence was inhibited by catalase but not superoxide dismutase (SOD), while X-XO-induced luminescence was inhibited by SOD but not catalase. SOD-inhibitable chemiluminescence was a sensitive and specific marker for O2-. production in vitro. Once the sensitivity-specificity of enhanced chemiluminescence was defined in vitro, this technique was used to explore the mechanism by which exogenous X-XO reduced hypoxic vasoconstriction in isolated rat lungs. The vascular paresis, caused by administration of X-XO to the rat lung, resulted from a brief burst of O2-. production rather than a sustained alteration of lung radical levels.     

Oxidative stress induced by ciprofloxacin in Staphylococcus aureus

Staphylococcus aureus with multiple sensitivity to ciprofloxacin, was investigated to detect alterations in the production of superoxide anion (O(2)(-)), other reactive oxidant species (ROS), and superoxide dismutase (SOD), and to relate them with ciprofloxacin accumulation and sensitivity. Oxidative stress was studied by means of Nitroblue Tetrazolium reaction (NBT) and chemiluminescence (CL); lucigenin was employed to detect O(2)(-), and luminol was used to measure other ROS. Sensitive strains exhibited higher intracellular O(2)(-) increase than resistant ones when incubated with ciprofloxacin. SOD was determined in normal conditions and induction was investigated in the presence of ciprofloxacin. These assays demonstrated that resistant and sensitive strains exported a great amount of SOD and that the induction of SOD intracellular was insufficient to counteract the augment of O(2)(-) in the cytoplasm of sensitive strains. Accumulation of ciprofloxacin, researched by spectrofluorometry, showed high levels of antibiotic in sensitive strains which increased the O(2)(-) causing more oxidative stress than in resistant S. aureus.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase. A requiem for H2O4.

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O2- during the first few minutes of the reaction. H2O2 decreases this accumulation of O2- presumably because of the Haber-Weiss reaction (H2O2+O2- leads to OH- +OH+O2) and very small amounts of superoxide dismutase eliminate it. This accumulation of O2- was demonstrated in terms of a burst of reduction of cytochrome c, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H2O4-, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

Characteristics of luminol chemiluminescence induced by the catalytic action of myeloperoxidase

The effects of pH, luminol myeloperoxidase and hydrogen peroxide concentrations on the intensity of luminol chemiluminescence induced by myeloperoxidase catalysis were investigated. It was found that the intensity of luminescence is proportional to the enzyme concentration (up to 8.10(-8) M) and reaches the saturation level at higher enzyme concentrations. The dependence of chemiluminescence intensity on [H2O2] is bell-shaped: at H2O2 concentrations above 1.10(-4) M the luminescence is inhibited with a maximum at neutral values of pH. Luminol at concentrations above 5.10(-5) M inhibits this process. It was demonstrated that the effects of singlet oxygen, superoxide and hydroxyl radicals on the chemiluminescence reaction are insignificant. Luminol oxidation in the course of the myeloperoxidase reaction is induced by hypochlorite.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation

The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.     

Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.     

Inhibition of myeloperoxidase by salicylhydroxamic acid.

Salicylhydroxamic acid inhibited the luminol-dependent chemiluminescence of human neutrophils stimulated by phorbol 12-myristate 13-acetate or the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). This compound had no inhibitory effect on the kinetics of O2.- generation or O2 uptake during the respiratory burst, but inhibited both the peroxidative activity of purified myeloperoxidase and the chemiluminescence generated by a cell-free myeloperoxidase/H2O2 system. The concentration of salicylhydroxamic acid necessary for complete inhibition of myeloperoxidase activity was 30-50 microM (I50 values of 3-5 microM) compared with the non-specific inhibitor NaN3, which exhibited maximal inhibition at 100-200 microM (I50 values of 30-50 microM). Whereas taurine inhibited the luminol chemiluminescence of an H2O2/HOC1 system by HOC1 scavenging, this compound had little effect on myeloperoxidase/H2O2-dependent luminol chemiluminescence; in contrast, 10 microM-salicylhydroxamic acid did not quench HOC1 significantly but greatly diminished myeloperoxidase/H2O2-dependent luminol chemiluminescence, indicating that its effects on myeloperoxidase chemiluminescence were largely due to peroxidase inhibition rather than non-specific HOC1 scavenging. Salicylhydroxamic acid prevented the formation of myeloperoxidase Compound II, but only at low H2O2 concentrations, suggesting that it may compete for the H2O2-binding site on the enzyme. These data suggest that salicylhydroxamic acid may be used as a potent inhibitor to delineate the function of myeloperoxidase in neutrophil-mediated inflammatory events.     

Measurement of superoxide radicals produced by human activated neutrophils by accumulation of stable nitroxide radicals detected by MR spectroscopy

An important index of neutrophil function is the production of superoxide radicals (O2-) upon activation. Thus a development of a new adequate assay of O2- generation measurement is of great interest for phagocyte researchers. The present article considers the quantitative determination of O2- generation based on the interaction of O2- with 1-oxy-2,2,6,6-tetramethyl-4-oxypiperidine producing 4-oxo-2,2,6,6-piperidine-1-oxyl, detected by ESR. The kinetic curve of nitroxyl radical (NR) formation has a linear character. The NR formation rate after a short induction period (appr. 2 min.) approaches 3.3 X 10(-3) M/s, where cell concentration was 4 X 10(5) per ml. Hydroxylamine (3.8 mM) auto-oxidation rate is negligible as compared with activated neutrophils and is equal to 2 X 10(-9) M/s. Sensitivity NR to the presence of superoxide dismutase (SOD) came as evidence that NR formation is due O2- radicals. SOD (10(-7) M) inhibits NR formation by 90%. Hydroxylamine oxidation by O2- is an irreversible reaction--20-min incubation of activated neutrophils with NR do not influence NR concentration. The NR generation rate dependence upon the neutrophil concentration is linear in the cell concentration range from 4 X 10(5 up to 6 X 10(6) per ml. In this range a quantitative measurement of O2- production is suitable. The sensitivity of hydroxylamine assay is close to the sensitivity of chemiluminescent method, but specificity is higher, as SOD inhibits chemiluminescence only by 50%.     

The effect of some tannins on trout erythrocytes exposed to oxidative stress

In order to gain more knowledge on the role of tannins as antioxidants, their ability to protect (Salmo irideus) erythrocytes against oxidative stress was investigated. Antioxidant activity of different tannins (tannic, gallic and ellagic acid) was evaluated by chemiluminescence (CL) techniques using lucigenin and luminol as chemiluminogenic probes for the superoxide radical generated by the xanthine/xanthine oxidase system and hydrogen peroxide, respectively. The superoxide-scavenging activity of these tannins was shown for all the compounds; however, it is not clear if this is due to their ability of scavenging the superoxide radical or to their inhibitory activity on xanthine oxidase. Tannic and ellagic acid showed a marked effect on the reduction of H2O2-luminol chemiluminescence. The influence of these tannins on the rate of hemolysis in stressed trout erythrocytes was investigated and the results indicate that tannic acid accelerates the hemolytic event while gallic and ellagic acid have no significant effect. The possible protective action of these compounds against oxidative DNA damage was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique, used to detect primary DNA damage in individual cells. The results here reported show that tannins under study are capable at low concentrations of protecting DNA breakage, while at high concentrations they can be genotoxic.     

Calmodulin participation in oxygen radical-induced cardiac sarcoplasmic reticulum calcium uptake reduction

The effect of scavengers of oxygen radicals on canine cardiac sarcoplasmic reticulum (SR) Ca2+ uptake velocity was investigated at pH 6.4, the intracellular pH of the ischemic myocardium. With the generation of oxygen radicals from a xanthine-xanthine oxidase reaction, there was a significant depression of SR Ca2+ uptake velocity. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD), a scavenger of .O2-, or denatured SOD had no effect on the depression of Ca2+ uptake velocity induced by the xanthine-xanthine oxidase reaction. However, catalase, which can impair hydroxyl radical (.OH) formation by destroying the precursor H2O2, significantly inhibited the effect of the xanthine-xanthine oxidase reaction. This effect of catalase was enhanced by SOD, but not by denatured SOD. Dimethyl sulfoxide (Me2SO), a known .OH scavenger, completely inhibited the effect of the xanthine-xanthine oxidase reaction. The observed effect of oxygen radicals and radical scavengers was not seen in the calmodulin-depleted SR vesicles. Addition of exogenous calmodulin, however, reproduced the effect of oxygen radicals and the scavengers. The effect of oxygen radicals was enhanced by the calmodulin antagonists (compounds 48/80 and W-7) at concentrations which showed no effect alone on Ca2+ uptake velocity. Taken together, these findings strongly suggest that .OH, but not .O2-, is involved in a mechanism that may cause SR dysfunction, and that the effect of oxygen radicals is calmodulin dependent.     

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.     

Experimental study on photodynamic diagnosis of cancer mediated by chemiluminescence probe

A novel method of photodynamic diagnosis of cancer mediated by chemiluminescence probe is presented. The mechanism for photodynamic therapy involves singlet oxygen ((1)O(2)) generated by energy transfer from photosensitizers. (1)O(2) can react with 3,7-dihydro-6-[4-[2-(N'-(5-fluoresceinyl)thioureido)ethoxy]phenyl]-2-methylimidazo[1,2-a]pyrazin-3-one sodium salt (FCLA), which is a Cypridina luciferin analog and a specific chemiluminescence probe for detecting (1)O(2) and superoxide (O(2)(-)). The reaction of FCLA and (1)O(2) can give emission with peak wavelength at about 532 nm. In the present study, FCLA was chosen as an optical reporter of (1)O(2) produced from the photosensitization reaction of hematoporphyrin derivative in model solution and in nude mice with transplanted mammary cancer. Photosensitized chemiluminescence from the reaction of FCLA with (1)O(2) was detected by a highly sensitive Intensified Charge-Coupled Device detector. The chemiluminescence was markedly inhibited by the addition of 10 mmol/l sodium azide (NaN(3)) to the model solution and minor effects were observed at the addition of 10 micromol/l superoxide dismutase, 20 mmol/l mannitol and 100 microg/ml catalase, respectively, thus indicating that (1)O(2) generation from photosensitization reaction mainly results in light emission. Experiments in vivo with tumor-bearing mice showed a clear chemiluminescence image of tumor. The study suggests that this novel method may be applicable to the diagnosis of superficial tumors.     

Phosphate inhibition of the copper- and zinc-containing superoxide dismutase: a reexamination

Phosphate was reported to be an inhibitor of copper- and zinc-containing superoxide dismutase (SOD) [de Freitas, D.M., & Valentine, J.S. (1984) Biochemistry 23, 2079-2082]. Thus SOD activity, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), was decreased by approximately 50% when the assay was made 10 mM in phosphate, and the ionic strength was adjusted with sodium fluoride. The inhibitory effect of phosphate was attributed to the neutralization of the positive charge on the guanidino residue of Arg-141. We have reexamined the effects of phosphate inhibition of SOD and found that the enzyme has identical activity in phosphate or HEPES buffer when the ionic strength is adjusted with NaBr. The putative inhibitory effect of phosphate appears to have been due to fluoride inhibition of the superoxide generating system of xanthine/xanthine oxidase. We have confirmed this result by using a photochemical generation of O2- in addition to the enzymatic generation of O2-. Chemical modification of the lysine residues to homoarginines does not affect the activity of the enzyme and does not impart a phosphate sensitivity. Chemical modification with phenylglyoxal caused approximately 80% inactivation of the native enzyme and 90% inactivation of the O-methylisourea-modified enzyme. Our results suggest that phosphate does not inhibit the copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) beyond the expectations of its effect on ionic strength.     

Cell death by reactive oxygen species generated from water-soluble cationic metalloporphyrins as superoxide dismutase mimics.

We investigated the effect on cell death of reactive oxygen species induced by water-soluble cationic metalloporphyrins with superoxide dismutase (SOD) activity. The SOD activity of 5,10,15,20-tetrakis(4-N-methylpyridyl)]porphine (MPy(4)P) containing Fe, Mn or Cu was measured using a cytochrome c assay by the xanthine/xanthine oxidase system and stopped-flow kinetic analysis. Cell viability of four cell lines treated with metalloporphyrins, mitomycin c (MMC), or cisplatin was estimated by a trypan blue exclusion assay. FeMPy(4)P with a high SOD activity showed a significant cytotoxicity compared with MMC and cisplatin, while CuMPy(4)P without SOD activity exhibited no cytotoxicity. However, MnMPy(4)P showing an SOD activity as high as that of FeMPy(4)P did not indicate cytotoxicity. These findings suggest that FeMPy(4)P as SOD mimic converts intracellular O2(*-) to H(2)O(2) and that it rapidly reacts with H(2)O(2) to form *OH, causing DNA damage and inducing cell death. On the other hand, MnMPy(4)P did not participate in the Fenton reaction, so that DNA damage in the cells treated with MnMPy(4)P was not observed. In addition, the cytotoxicity by the metalloporphyrin was inversely correlated with the SOD activity of the cells and the selective damage at cellular and DNA levels was confirmed. We believe that for an anticancer drug with antioxidant ability O(2)(*-) is useful as a target molecule to induce selective cell death between cancer and normal cells and that metalloporphyrins showing SOD activity and Fenton-like reaction are a new class of anticancer agents.