Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant, ascorbate

Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O(2)(·-), reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP(5+), MnTnHex-2-PyP(5+), and a meta isomer MnTnHex-3-PyP(5+), which differ greatly with regard to their metal-centered reduction potential, E(1/2) (Mn(III)P/Mn(II)P) and lipophilicity, were explored. Employing Mn(III)P/Mn(II)P redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP(5+) was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP(5+) is most prone to oxidative degradation with H(2) , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.     

Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics,MnTE-2-PyP5+ and MnTnHex-2-PyP5+

The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP⁵⁺ (AEOL10113, FBC-007) and MnTnHex-2-PyP⁵⁺ have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O₂^•− and ONOO⁻ and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood–brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP⁵⁺ is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP⁵⁺, which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24 h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10 mg/kg MnTE-2-PyP⁵⁺ and 0.5 or 2 mg/kg MnTnHex-2-PyP⁵⁺. Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (C_max, C_24 h, t_max, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUC_oral/AUC_iv) is 23% for MnTE-2-PyP⁵⁺ and 21% for MnTnHex-2-PyP⁵⁺. Despite the fivefold lower dose administered, the AUC values for liver, heart, and spleen are higher for MnTnHex-2-PyP⁵⁺ than for MnTE-2-PyP⁵⁺ (and comparable for other organs), clearly demonstrating the better tissue penetration and tissue retention of the more lipophilic MnTnHex-2-PyP⁵⁺.     

Protective role of ortho-substituted Mn(III) N-alkylpyridylporphyrins against the oxidative injury induced by tert-butylhydroperoxide

Abstract

The present work addresses the role of two ortho-substituted Mn(III) N-alkylpyridylporphyrins, alkyl being ethyl in MnTE-2-PyP⁵⁺ and n-hexyl in MnTnHex-2-PyP⁵⁺, on the protection against the oxidant tert-butylhydroperoxide (TBHP). Their protective role was studied in V79 cells using endpoints of cell viability (MTT and crystal violet assays), intracellular O₂?– generation (dihydroethidium assay) and glutathione status (DTNB and monochlorobimane assays). MnPs per se did not show cytotoxicity (up to 25 μM, 24 h). The exposure to TBHP resulted in a significant decrease in cell viability and in an increase in the intracellular O₂^?– levels. Also, TBHP depleted total and reduced glutathione and increased GSSG. The two MnPs counteracted remarkably the effects of TBHP. Even at low concentrations, both MnPs were protective in terms of cell viability and abrogated the intracellular O₂^?– increase in a significant way. Also, they augmented markedly the total and reduced glutathione contents in TBHP-treated cells, highlighting the multiple mechanisms of protection of these SOD mimics, which at least in part may be ascribed to their electron-donating ability.     

Mn porphyrin-based SOD mimic,MnTnHex-2-PyP5+, and non-SOD mimic,MnTBAP3−, suppressed rat spinal cord ischemia/reperfusion injury via NF-κB pathways

Abstract

Herein we have demonstrated that both superoxide dismutase (SOD) mimic, cationic Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP⁵⁺), and non-SOD mimic, anionic Mn(III) meso-tetrakis(4-carboxylatophenyl)porphyrin (MnTBAP^3?), protect against oxidative stress caused by spinal cord ischemia/reperfusion via suppression of nuclear factor kappa B (NF-κB) pro-inflammatory pathways. Earlier reports showed that Mn(III) N-alkylpyridylporphyrins were able to prevent the DNA binding of NF-κB in an aqueous system, whereas MnTBAP^3? was not. Here, for the first time, in a complex in vivo system—animal model of spinal cord injury—a similar impact of MnTBAP^3?, at a dose identical to that of MnTnHex-2-PyP⁵⁺, was demonstrated in NF-κB downregulation. Rats were treated subcutaneously at 1.5 mg/kg starting at 30 min before ischemia/reperfusion, and then every 12 h afterward for either 48 h or 7 days. The anti-inflammatory effects of both Mn porphyrins (MnPs) were demonstrated in the spinal cord tissue at both 48 h and 7 days. The downregulation of NF-κB, a major pro-inflammatory signaling protein regulating astrocyte activation, was detected and found to correlate well with the suppression of astrogliosis (as glial fibrillary acidic protein) by both MnPs. The markers of oxidative stress, lipid peroxidation and protein carbonyl formation, were significantly reduced by MnPs. The favorable impact of both MnPs on motor neurons (Tarlov score and inclined plane test) was assessed. No major changes in glutathione peroxidase- and SOD-like activities were demonstrated, which implies that none of the MnPs acted as SOD mimic. Increasing amount of data on the reactivity of MnTBAP^3? with reactive nitrogen species (RNS) (.NO/HNO/ONOO^?) suggests that RNS/MnTBAP^3?-driven modification of NF-κB protein cysteines may be involved in its therapeutic effects. This differs from the therapeutic efficacy of MnTnHex-2-PyP⁵⁺ which presumably occurs via reactive oxygen species and relates to NF-κB thiol oxidation; the role of RNS cannot be excluded.     

Effect of lipophilicity of Mn (III) ortho N-alkylpyridyl- and diortho N,N′-diethylimidazolylporphyrins in two in-vitro models of oxygen and glucose deprivation-induced neuronal death

In vivo investigations have confirmed the beneficial effects of hydrophilic, cationic Mn(III) porphyrin-based catalytic antioxidants in different models of oxidative stress. Using a cell culture model of rat mixed neuronal/glial cells, this study investigated the effect of MnTnOct-2-PyP⁵⁺ on oxygen and glucose deprivation (OGD)-induced cell death as compared to the effects of widely studied hydrophilic analogues MnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺ and a standard compound, dizocilpine (MK-801). It was hypothesized that the octylpyridylporphyrin, MnTnOct-2-PyP⁵⁺, a lipophilic but equally potent antioxidant as the other two porphyrins, would be more efficacious in reducing OGD-induced cell death due to its higher bioavailability. Cell death was evaluated at 24 h using lactate dehydrogenase (LDH) release and propidium iodide staining. At concentrations from 3–100 µm, all three porphyrins reduced cell death as compared to cultures exposed to OGD alone, the effects depending upon the concentrations and type of treatment. To assess the effect of lipophilicity the additional experiments were performed using submicromolar concentrations of MnTnOct-2-PyP⁵⁺ in an organotypic hippocampal slice model of OGD with propidium iodide and Sytox staining. When compared to oxygen and glucose deprivation alone, concentrations of MnTnOct-2-PyP⁵⁺ as low as 0.01 µm significantly (p<0.001; power 1.0) reduced neuronal cells similar to control. This is the first in vitro study on the mammalian cells which indicates that MnTnOct-2-PyP⁵⁺ is up to 3000-fold more efficacious than equally potent hydrophilic analogues, due entirely to its increased bioavailability. Such remarkable increase in efficacy parallels 5.7-orders of magnitude increase in lipophilicity of MnTnOct-2-PyP⁵⁺ (log P=?0.77) when compared to MnTE-2-PyP⁵⁺ (log P_ow=?6.43), P_ow being partition coefficient between n-octanol and water.     

Bioavailability of metalloporphyrin-based SOD mimics is greatly influenced by a single charge residing on a Mn site

Abstract

In the cell Mn porphyrins (MnPs) likely couple with cellular reductants which results in a drop of total charge from 5+ to 4+ and dramatically increases their lipophilicity by up to three orders of magnitude depending upon the length of alkylpyridyl chains and type of isomer. The effects result from the interplay of solvation, lipophilicit and stericity. Impact of ascorbate on accumulation of MnPs was measured in E. coli and in Balb/C mouse tumours and muscle; for the latter measurements, the LC/ESI-MS/MS method was developed. Accumulation was significantly enhanced when MnPs were co-administered with ascorbate in both prokaryotic and eukaryotic systems. Further, MnTnHex-2-PyP⁵⁺ accumulates 5-fold more in the tumour than in a muscle. Such data increase our understanding of MnPs cellular and sub-cellular accumulation and remarkable in vivo effects. The work is in progress to understand how coupling of MnPs with ascorbate affects their mechanism of action, in particular with respect to cancer therapy.     

Manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin acts as a pro-oxidant to inhibit electron transport chain proteins,modulate bioenergetics,and enhance the response to chemotherapy in lymphoma cells

The manganese porphyrin, manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin (MnTE-2-PyP⁵⁺), acts as a pro-oxidant in the presence of intracellular H₂O₂. Mitochondria are the most prominent source of intracellular ROS and important regulators of the intrinsic apoptotic pathway. Due to the increased oxidants near and within the mitochondria, we hypothesized that the mitochondria are a target of the pro-oxidative activity of MnTE-2-PyP⁵⁺ and that we could exploit this effect to enhance the chemotherapeutic response in lymphoma. In this study, we demonstrate that MnTE-2-PyP⁵⁺ modulates the mitochondrial redox environment and sensitizes lymphoma cells to antilymphoma chemotherapeutics. MnTE-2-PyP⁵⁺ increased dexamethasone-induced mitochondrial ROS and oxidation of the mitochondrial glutathione pool in lymphoma cells. The combination treatment induced glutathionylation of Complexes I, III, and IV in the electron transport chain, and decreased the activity of Complexes I and III, but not the activity of Complex IV. Treatment with the porphyrin and dexamethasone also decreased cellular ATP levels. Rho(0) malignant T-cells with impaired mitochondrial electron transport chain function were less sensitive to the combination treatment than wild-type cells. These findings suggest that mitochondria are important for the porphyrin’s ability to enhance cell death. MnTE-2-PyP⁵⁺ also augmented the effects of 2-deoxy-D-glucose (2DG), an antiglycolytic agent. In combination with 2DG, MnTE-2-PyP⁵⁺ increased protein glutathionylation, decreased ATP levels more than 2DG treatment alone, and enhanced 2DG-induced cell death in primary B-ALL cells. MnTE-2-PyP⁵⁺ did not enhance dexamethasone- or 2DG-induced cell death in normal cells. Our findings suggest that MnTE-2-PyP⁵⁺ has potential as an adjuvant for the treatment of hematologic malignancies.     

Mn porphyrin in combination with ascorbate acts as a pro-oxidant and mediates caspase-independent cancer cell death

Resistance to therapy-mediated apoptosis in inflammatory breast cancer, an aggressive and distinct subtype of breast cancer, was recently attributed to increased superoxide dismutase (SOD) expression, glutathione (GSH) content, and decreased accumulation of reactive species. In this study, we demonstrate the unique ability of two Mn(III) N-substituted pyridylporphyrin (MnP)-based SOD mimics (MnTE-2-PyP⁵⁺ and MnTnBuOE-2-PyP⁵⁺) to catalyze oxidation of ascorbate, leading to the production of excessive levels of peroxide, and in turn cell death. The accumulation of peroxide, as a consequence of MnP+ascorbate treatment, was fully reversed by the administration of exogenous catalase, showing that hydrogen peroxide is essential for cell death. Cell death as a consequence of the action of MnP+ascorbate corresponded to decreases in GSH levels, prosurvival signaling (p-NF-κB, p-ERK1/2), and in expression of X-linked inhibitor of apoptosis protein, the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and annexin V staining, administration of a pan-caspase inhibitor, Q-VD-OPh, did not reverse the observed cytotoxicity. MnP+ascorbate-treated cells showed nuclear translocation of apoptosis-inducing factor, suggesting the possibility of a mechanism of caspase-independent cell death. Pharmacological ascorbate has already shown promise in recently completed phase I clinical trials, in which its oxidation and subsequent peroxide formation was catalyzed by endogenous metalloproteins. The catalysis of ascorbate oxidation by an optimized metal-based catalyst (such as MnP) carries a large therapeutic potential as an anticancer agent by itself or in combination with other modalities such as radio- and chemotherapy.     

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

A novel heterobinuclear mixed valence complex [Fe^IIICu^II(BPBPMP)(OAc)₂]ClO₄, 1, with the unsymmetrical N₅O₂ donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H₂BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe^III–()OH–Cu^IIOH₂]⁺ species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe^III-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe^IIIM^II, where M^II is Zn^II, Mn^II, Ni^II, or Fe^II). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 M), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7×10⁷ over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.Electronic Supplementary Material Supplementary material is available for this article at .     

Manganoporphyrins and ascorbate enhance gemcitabine cytotoxicity in pancreatic cancer

Pharmacological ascorbate (AscH⁻) selectively induces cytotoxicity in pancreatic cancer cells vs normal cells via the generation of extracellular hydrogen peroxide (H₂O₂), producing double-stranded DNA breaks and ultimately cell death. Catalytic manganoporphyrins (MnPs) can enhance ascorbate-induced cytotoxicity by increasing the rate of AscH⁻ oxidation and therefore the rate of generation of H₂O₂. We hypothesized that combining MnPs and AscH⁻ with the chemotherapeutic agent gemcitabine would further enhance pancreatic cancer cell cytotoxicity without increasing toxicity in normal pancreatic cells or other organs. Redox-active MnPs were combined with AscH⁻ and administered with or without gemcitabine to human pancreatic cancer cell lines, as well as immortalized normal pancreatic ductal epithelial cells. The MnPs MnT2EPyP (Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin pentachloride) and MnT4MPyP (Mn(III)tetrakis(N-methylpyridinium-4-yl) porphyrin pentachloride) were investigated. Clonogenic survival was significantly decreased in all pancreatic cancer cell lines studied when treated with MnP + AscH⁻ + gemcitabine, whereas nontumorigenic cells were resistant. The concentration of ascorbate radical (Asc^•−, an indicator of oxidative flux) was significantly increased in treatment groups containing MnP and AscH⁻. Furthermore, MnP + AscH⁻ increased double-stranded DNA breaks in gemcitabine-treated cells. These results were abrogated by extracellular catalase, further supporting the role of the flux of H₂O₂. In vivo growth was inhibited and survival increased in mice treated with MnT2EPyP, AscH⁻, and gemcitabine without a concomitant increase in systemic oxidative stress. These data suggest a promising role for the use of MnPs in combination with pharmacologic AscH⁻ and chemotherapeutics in pancreatic cancer.     

Spectral and kinetic investigation on oxidation and reduction of water soluble porphyrin-manganese(III) complex

In this work the oxidation and reduction reactions of Mn^III-Coproporphyrin-I (Mn^III-CPI) have been studied and four forms of manganese-CPI complexes have been characterized. This complex was observed to be highly reactive (at basic pH) towards Mn(II), hypochlorite, hydrogen peroxide and oxone, forming [Mn^IV(O)CPI(OH)]⁻ that was unstable and, after a short time, formed again [Mn^IIICPI(OH)₂]⁻. With an excess of NaClO, a further oxidation of the complex [Mn^IV(O)CPI(OH)]⁻, provoked a significant spectral change for the [Mn^V(O)CPI(OH)] formation that showed, in the time, a partial polymerization. [Mn^IIICPI(OH)₂]⁻ was reduced by sodium dithionite to form the very unstable complex of [Mn^IICPI(OH)]⁻ that successively degraded with Mn(II) release.     

Endothelial cell phagokinesis in response to specific metal ions

Salts of Cu^{I, II}, Ni^II, Sn^II, In^III and a sub-fraction of the cupoprotein ceruloplasmin induced phagokinesis of cultured aortal endothelial cells. A variant aortal endothelial cell line was highly sensitive; cells travelled up to 1000 μm in 24 h in response to 2× 10^?6 M SnCl₂. Other metal ions tested (Zn^II, Co^II, Mn^II, Cr^II, Fe^III, Al^III, Sb^III and Mo^II) were not active. The motility response of endothelial cells to Cu ions in vitro is proposed as a model system for studying early events in neovascularization and as a sensitive assay for detecting angiogenic activity in fractions from cells and tissues.     

Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus

Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn²⁺, veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn²⁺ and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.     

Manganese(III) complexes of N2O2 donor 5-bromosalicylideneimine ligands: Combined effects of electron withdrawing substituents and chelate ring size variations on electrochemical properties

A series of mononuclear manganese(III) complexes of formulae [Mn(L)(X)(H₂O)] (1-13) and [Mn(L)(X)] (14-17) (X = ClO₄, F, Cl, Br, I, NCS, N₃), derived from the Schiff bases of 5-bromosalicylaldehyde and different types of diamine (1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopropane and 1,4-diaminobutane), have been synthesized and characterized by the combination of IR, UV-Vis spectroscopies, cyclic voltammetry and by X-ray crystallography. The redox properties of all the manganese(III) complexes show grossly identical features consisting of a reversible or quasireversible Mn^III/Mn^II reduction. Besides Mn^III/Mn^II reduction, the complexes 4, 5, 10, 13 and 16 also show reversible or quasireversible Mn^III/Mn^IV oxidation. A linear correlation has been found for the complexes 5, 7, 11 and 13 [Mn(L²)(X)(H₂O)] (X = F, Cl, Br, I) when E_1/2 [Mn^III/Mn^II] is plotted against Mulliken electronegativities (χ_M). The effect of the flexibility of the ligand on redox potential has been studied. It has been observed that the manganese(II) state is stabilized with increasing flexibility of the ligand environment. The crystal structure of 6 shows an octahedral geometry.     

Comparison of two Mn porphyrin-based mimics of superoxide dismutase in pulmonary radioprotection

Development of radiation therapy (RT)-induced lung injury is associated with chronic production of reactive oxygen and nitrogen species (ROS/RNS). MnTE-2-PyP5+ is a catalytic Mn porphyrin mimic of SOD, already shown to protect lungs from RT-induced injury by scavenging ROS/RNS. The purpose of this study was to compare MnTE-2-PyP5+ with a newly introduced analogue MnTnHex-2-PyP5+, which is expected to be a more effective radioprotector due to its lipophilic properties. This study shows that Fischer rats which were irradiated to their right hemithorax (28 Gy) have less pulmonary injury as measured using breathing frequencies when treated with daily subcutaneous injections of MnTE-2-PyP5+ (3 and 6 mg/kg) or MnTnHex-2-PyP5+ (0.3, 0.6, or 1.0 mg/kg) for 2 weeks after RT. However, at 16 weeks post-RT, only MnTE-2-PyP5+ at a dose of 6 mg/kg is able to ameliorate oxidative damage, block activation of HIF-1alpha and TGF-beta, and impair upregulation of CA-IX and VEGF. MnTnHex-2-PyP5+ at a dose of 0.3 mg/kg is effective only in reducing RT-induced TGF-beta and CA-IX expression. Significant loss of body weight was observed in animals receiving MnTnHex-2-PyP5+ (0.3 and 0.6 mg/kg). MnTnHex-2-PyP5+ has the ability to dissolve lipid membranes, causing local irritation/necrosis at injection sites if given at doses of 1 mg/kg or higher. In conclusion, both compounds show an ability to ameliorate lung damage as measured using breathing frequencies and histopathologic evaluation. However, MnTE-2-PyP5+ at 6 mg/kg proved to be more effective in reducing expression of key molecular factors known to play an important role in radiation-induced lung injury.     

Towards the mechanisms involved in the antioxidant action of Mn<Superscript>III</Superscript> [<Emphasis Type="Italic">meso</Emphasis>-tetrakis(4-<Emphasis Type="Italic">N</Emphasis>-methyl pyridinium) porphyrin] in mitochondria

Aerobic organisms are afforded with an antioxidant enzymatic apparatus that more recently has been recognized to include cytochrome c, as it is able to prevent hydrogen peroxide generation by returning electrons from the superoxide ion back to the respiratory chain. The present study investigated the glutathione peroxidase (GPx), superoxide dismutase (SOD) and cytochrome c-like antioxidant activities of para Mn(III)TMPyP in isolated rat liver mitochondria (RLM) and mitoplasts. In RLM, Mn^IIITMPyP decreased the lipid-peroxide content associated with glutathione (GSH) depletion consistent with the use of GSH as a reducing agent for high valence states of Mn^IIITMPyP. SOD and cytochrome c antioxidant activities were also investigated. Mn^IITMPyP was able to reduce ferric cytochrome c, indicating the potential to remove a superoxide ion by returning electrons back to the respiratory chain. In antimicyn A-poisoned mitoplasts, Mn^IIITMPyP efficiently decreased the EPR signal of DMPO-OH adduct concomitant with GSH depletion. The present results are consistent with SOD and GPx activities for Mn^IIITMPyP and do not exclude cytochrome c-like activity. However, considering that para Mn^IIITMPyP more efficiently reduces, rather than oxidizes, superoxide ion; electron transfer from the Mn^IITMPyP to the respiratory chain might not significantly contribute to the superoxide ion removal, since most of Mn^IITMPyP is expected to be produced at the expense of NADPH/GSH oxidation. The present results suggest GPx-like activity to be the principal antioxidant mechanism of Mn^IIITMPyP, whose efficiency is dependent on the NADPH/GSH content in cells.