Electrostatic contribution in the catalysis of O2*- dismutation by superoxide dismutase mimics. MnIIITE-2-PyP5+ versus MnIIIBr8T-2-PyP+

The Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) is a potent superoxide dismutase (SOD) mimic in vitro and was beneficial in rodent models of oxidative stress pathologies. Its high activity has been ascribed to both the favorable redox potential of its metal center and to the electrostatic facilitation assured by the four positive charges encircling the metal center. Its comparison with the non-alkylated, singly charged analogue Mn(III) beta-octabromo meso-tetrakis(2-pyridyl)porphyrin (Mn(III)Br(8)T-2-PyP(+)) enabled us to evaluate the electrostatic contribution to the catalysis of O(2)() dismutation. Both compounds exhibit nearly identical metal-centered redox potential for Mn(III)/Mn(II) redox couple: +228 mV for Mn(III)TE-2-PyP(5+) and +219 mV versus NHE for Mn(III)Br(8)T-2-PyP(+). The eight electron-withdrawing beta pyrrolic bromines contribute equally to the redox properties of the parent Mn(III)T-2-PyP(+) as do four quaternized cationic meso ortho pyridyl nitrogens. However, the SOD-like activity of the highly charged Mn(III)TE-2-PyP(5+) is >100-fold higher (log k(cat) = 7.76) than that of the singly charged Mn(III)Br(8)T-2-PyP(+) (log k(cat) = 5.63). The kinetic salt effect showed that the catalytic rate constants of the Mn(III)TE-2-PyP(5+) and of its methyl analogue, Mn(III)TM-2-PyP(5+), are exactly 5-fold more sensitive to ionic strength than is the k(cat) of Mn(III)Br(8)T-2-PyP(+), which parallels the charge ratio of these compounds. Interestingly, only a small effect of ionic strength on the rate constant was found in the case of penta-charged para (Mn(III)TM-4-PyP(5+)) and meta isomers (Mn(III)TM-3-PyP(5+)), indicating that the placement of the positive charges in the close proximity of the metal center (ortho position) is essential for the electrostatic facilitation of O(2)() dismutation.     

Nitrosylation of manganese(II) tetrakis(N-ethylpyridinium-2-yl)porphyrin: a simple and sensitive spectrophotometric assay for nitric oxide.

Reaction between NO(*) and manganese tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) was investigated at 25 degrees C. At high excess of NO(*) (1.5 mM) the reaction with the oxidized, air-stable form Mn(III)TE-2-PyP(5+) (5 microM), proceeds very slowly (t(1/2) congruent with 60 min). The presence of excess ascorbate (1 mM) produces the reduced form, Mn(II)TE-2-PyP(4+), which reacts with NO(*) stoichiometrically and in the time of mixing (k congruent with 1 x 10(6) M(-1) s(-1)). The high rate of formation and the stability of the product, Mn(II)TE-2-PyP(NO)(4+) (?Mn(NO)?(6)), make the reaction outcompete the reaction of NO(*) with O(2). Our in vitro measurements show a linear absorbance response upon addition of NO to a PBS, pH 7.4, solution containing an excess of ascorbate over Mn(III)TE-2-PyP(5+). Thus, the observed interactions can be the basis of a convenient and sensitive spectrophotometric assay for NO(*). Also, it may have important implications for the in vivo behavior of Mn(III)TE-2-PyP(5+) which is currently exploited as a possible therapeutic agent for various oxygen-radical related disorders.     

Only one of a wide assortment of manganese-containing SOD mimicking compounds rescues the slow aerobic growth phenotypes of both Escherichia coli and Saccharomyces cerevisiae strains lacking superoxide dismutase enzymes

A variety of manganese-containing coordination compounds, frequently termed superoxide dismutase (SOD) mimics, have been reported to have SOD activity in vitro and to be effective at improving conditions related to increased oxidative stress in multicellular organisms. We tested the effectiveness of several of these compounds in substituting for authentic SOD enzymes in two simple systems--the prokaryote Escherichia coli and the single-celled eukaryote, Saccharomyces cerevisiae--where strains are available that completely lack cytoplasmic SOD activity and are thus significantly impaired in their ability to grow aerobically. Most of the compounds tested, including Euk-8 and Euk-134, manganese salen derivatives developed by Eukarion; M40403, a manganese complex of a bis(cyclohexylpyridine)-substituted macrocyclic ligand developed by Metaphore; and several manganese porphyrin derivatives, were ineffective in both systems. Only the manganese tetrapyridyl porphyrin complex MnTM-2-PyP and two close relatives were effective in rescuing aerobic growth of E. coli lacking SOD, and, in the case of sod1Delta yeast, only MnTM-2-PyP itself was fully effective. Surprisingly, several compounds reported to be beneficial in other in vivo model systems (Euk-8, Euk-134, M40403) were actually toxic to these organisms lacking SOD, although they had no effect on the wild-type parent strains. Our results suggest the possibility that the beneficial effects of some of the so-called "SOD mimic drugs" may be due to some property other than in vivo superoxide dismutase activity.     

Tetrahydrobiopterin rapidly reduces the SOD mimic Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin

Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) effectively scavenges reactive oxygen and nitrogen species in vitro, and protects in vivo, in different rodent models of oxidative stress injuries. Further, Mn(III)TE-2-PyP(5+) was shown to be readily reduced by cellular reductants such as ascorbic acid and glutathione. We now show that tetrahydrobiopterin (BH(4)) is also able to reduce the metal center. Under anaerobic conditions, in phosphate-buffered saline (pH 7.4) at 25 +/- 0.1 degrees C, reduction of Mn(III)TE-2-PyP(5+) occurs through two reaction steps with rate constants k(1) = 1.0 x 10(4) M(-1) s(-1) and k(2) = 1.5 x 10(3) M(-1) s(-1). We ascribe these steps to the formation of tetrahydrobiopterin radical (BH(4)(.+)) (k(1)) that then undergoes oxidation to 6,7-dihydro-8H-biopterin (k(2)), which upon rearrangement gives rise to 7,8-dihydrobiopterin (7,8-BH(2)). Under aerobic conditions, Mn(III)TE-2-PyP(5+) catalytically oxidizes BH(4). This is also true for its longer chain alkyl analog, Mn(III) ortho-tetrakis(N-n-octylpyridinium-2-yl)porphyrin. The reduced Mn(II) porphyrin cannot be oxidized by 7,8-BH(2) or by l-sepiapterin. The data are discussed with regard to the possible impact of the interaction of Mn(III)TE-2-PyP(5+) with BH(4) on endothelial cell proliferation and hence on tumor antiangiogenesis via inhibition of nitric oxide synthase.     

Metalloporphyrins improve the survival of Sod2-deficient neurons

The objective of this study was to determine whether metalloporphyrin catalytic antioxidants influence the survival of neuronal cultures in an in vitro model of age-related mitochondrial oxidative stress. Neuronal cultures were prepared from cerebral cortices of manganese superoxide dismutase (MnSOD or Sod2) knockout (homozygous -/-, heterozygous -/+ or wild-type +/+) mice. The ability of catalytic antioxidants, manganese tetrakis-(4-benzoic acid) porphyrin (MnTBAP) and manganese tetrakis-(N-ethyl-2-pyridyl) porphyrin (MnTE-2-PyP) to influence the survival of cultured cerebrocortical neurones from Sod2-replete (+/+) and Sod2-deficient (+/- or -/-) mice was assessed. Sod2-/- cultures showed accelerated cell death in serum-free conditions when grown in ambient oxygen. MnTBAP and MnTE-2-PyP delayed the death of Sod2-/- cultures and improved the survival of Sod2+/+ and Sod2+/- cultures in serum-free conditions. The results suggest that metalloporphyrin antioxidants can delay neuronal death resulting specifically from increased mitochondrial oxidative stress. Furthermore, Sod2-deficient neuronal cultures provide a simple model system to screen the biological efficacy of mitochondrial antioxidants.     

Dependence of excitotoxic neurodegeneration on mitochondrial aconitase inactivation

Using the inactivation of mitochondrial and cytosolic aconitases as markers of compartment-specific superoxide (O2(-)) production, we show that oxygen-glucose deprivation (OGD) or excitotoxin exposure produce a time-dependent inactivation of mitochondrial, but not cytosolic, aconitase in cortical cultures. To determine if mitochondrial O2(-) production was an important determinant in neuronal death resulting from OGD, metalloporphyrins with varying superoxide dismutase (SOD) activity were tested for their ability to protect against mitochondrial aconitase inactivation and cell death. OGD-induced mitochondrial aconitase inactivation and cell death was inhibited by manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP), manganese tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) and NMDA receptor antagonists. By contrast, NMDA- or kainate (KA)-induced mitochondrial aconitase inactivation and cell death was inhibited by MnTBAP, but not MnTE-2-PyP. Moreover, both MnTBAP and MnTE-2-PyP penetrated mitochondrial fractions of cortical cells. These data suggest that mitochondrial aconitase inactivation closely correlates with subsequent neuronal death following excitotoxicity produced by OGD or NMDA/KA exposure. Assessment of biological rather biochemical antioxidant activities better predicted neuroprotection by metalloporphyrins. Moreover, antioxidants that protect oxidant-sensitive mitochondrial targets such as aconitase may be useful as therapies for disease states involving excitotoxicity.     

Bioconjugates of cyclodextrins of manganese salen-type ligand with superoxide dismutase activity

6A,6B-Dideoxy-6A,6B-di[(N-salicylidene)amino]-beta-cyclodextrin was synthesized and characterized by NMR, UV and CD spectroscopy in order to prepare a N,N(')-bis-(salicylidene)ethane-1,2-diamine (SalenH(2)) type ligand. The manganese(III) complex was synthesized and characterized by UV and cyclic voltammetry and compared to EUK-8. The superoxide dismutase (SOD)-like and catalase-like activities were tested by indirect assay. The cyclodextrin complex shows a larger solubility than EUK-8 and good SOD-like activity. Catalase activity is also shown.     

Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion

Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP(5+) in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 microg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-beta subunit as a key protein induced by MnTnHex-2-PyP(5+) treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP(5+) protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.     

Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode Caenorhabditis elegans

According to the oxidative damage theory a primary cause of aging is the accrual of molecular damage from reactive oxygen species (ROS), particularly superoxide and its derivatives. This predicts that treatments that reduce ROS levels should retard aging. Using the nematode Caenorhabditis elegans, we tested the effects on stress resistance and life span of treatment with EUK-8 and EUK-134, synthetic mimetics of the antioxidant enzyme superoxide dismutase (SOD), which neutralises superoxide. Treatment with SOD mimetics elevated in vivo SOD activity levels, particularly in mitochondria, where up to 5-fold increases in SOD activity were recorded. Treatment with exogenous SOD mimetics did not affect endogenous protein SOD levels. Where life span was reduced by the superoxide generators paraquat and plumbagin, EUK-8 treatment increased life span in a dose-dependent fashion. Yet in the absence of a superoxide generator, treatment with EUK-8 or EUK-134 did not increase life span, even at doses that were optimal for protection against pro-oxidants. Thus, an elevation of SOD activity levels sufficient to increase life span when it is limited by superoxide generators does not retard aging in the absence of superoxide generators. This suggests that C. elegans life span is not normally limited by levels of superoxide and its derivatives.     

A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrin, combines superb potency and lipophilicity with low toxicity

The Mn porphyrins of k(cat)(O(2)(.-)) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP(5+) is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP(5+) has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH(2) group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP(5+). The high SOD-like activity is preserved and even enhanced: log k(cat)(O(2)(.-))=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP(5+) and MnTnHep-2-PyP(5+), respectively. MnTnBuOE-2-PyP(5+) was tested in an O(2)(.-) -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5-30 μM. MnTnHep-2-PyP(5+) was already toxic at 5 μM, and MnTnHex-2-PyP(5+) became toxic at 30 μM. In a mouse toxicity study, MnTnBuOE-2-PyP(5+) was several-fold less toxic than either MnTnHex-2-PyP(5+) or MnTnHep-2-PyP(5+).     

The overlapping roles of manganese and Cu/Zn SOD in oxidative stress protection

In various organisms, high intracellular manganese provides protection against oxidative damage through unknown pathways. Herein we use a genetic approach in Saccharomyces cerevisiae to analyze factors that promote manganese as an antioxidant in cells lacking Cu/Zn superoxide dismutase (sod1 Delta). Unlike certain bacterial systems, oxygen resistance in yeast correlates with high intracellular manganese without a lowering of iron. This manganese for antioxidant protection is provided by the Nramp transporters Smf1p and Smf2p, with Smf1p playing a major role. In fact, loss of manganese transport by Smf1p together with loss of the Pmr1p manganese pump is lethal to sod1 Delta cells despite normal manganese SOD2 activity. Manganese-phosphate complexes are excellent superoxide dismutase mimics in vitro, yet through genetic disruption of phosphate transport and storage, we observed no requirement for phosphate in manganese suppression of oxidative damage. If anything, elevated phosphate correlated with profound oxidative stress in sod1 Delta mutants. The efficacy of manganese as an antioxidant was drastically reduced in cells that hyperaccumulate phosphate without effects on Mn SOD activity. Non-SOD manganese can provide a critical backup for Cu/Zn SOD1, but only under appropriate physiologic conditions.     

Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are known to play an important role in the proliferation and viability of vascular smooth muscle cells. In this study, we determined the effects of increased superoxide dismutase and catalase activity on fetal pulmonary arterial smooth muscle cell (FPASMC) proliferation and viability using EUK-134, a superoxide dismutase/catalase mimetic. Treatment of FPASMC with EUK-134 or with a combination of superoxide dismutase and catalase enzymes decreased superoxide and hydrogen peroxide levels as detected by the fluorescent dyes dihydroethidium and dichlorodihydrofluorescein diacetate, respectively. EUK-134 (5 microM) attenuated serum-induced FPASMC proliferation, whereas 50 microM EUK-134 decreased the number of viable cells, suggesting cell death. Conversely, combined superoxide dismutase and catalase enzyme activity equivalent to 50 microM EUK-134 prevented proliferation but did not reduce the number of viable FPASMC. The loss of mitochondrial membrane potential after 18 h, an increase in caspase-9 and caspase-3 activity after 24 h, and the subsequent appearance of TdT-mediated dUTP nick end labeling-positive nuclei were detected in FPASMC after treatment with 50 microM EUK-134. This indicates an induction of programmed rather than necrotic cell death and suggests that prolonged removal of ROS is required to stimulate apoptosis. Compounds such as EUK-134 may, therefore, prove more effective than enzymic antioxidants over longer periods, especially when the aim is to decrease the number of smooth muscle cells in diseases resulting from excessive muscularization.     

Overexpression of Mn-containing superoxide dismutase in transgenic Drosophila melanogaster.

The general objective of this study was to examine the role of mitochondria in the aging process. Two alternative hypotheses were tested: (i) that overexpression of Mn superoxide dismutase (Mn SOD) in the mitochondria of Drosophila melanogaster would slow the accrual of oxidative damage and prolong survival or (ii) that there is an evolved optimum level of superoxide anion radical, such that overexpression of Mn SOD would have deleterious or neutral effects. Microinjection and mobilization of a transgene, which contained a 9-kb genomic sequence encoding Mn SOD, produced 15 experimental lines overexpressing Mn SOD by 5-116% relative to the parental y w strain. Comparisons between these lines and control lines containing inserted vector sequences alone indicated that the mean longevity of the experimental lines was decreased by 4-5% relative to controls. There were no compensatory changes in the metabolic rate, level of physical activity, or the levels of other antioxidants, namely Cu-Zn SOD, catalase, and glutathione. There were no differences between groups in rates of mitochondrial hydrogen peroxide release, protein oxidative damage, or resistance to 100% oxygen or starvation conditions. The experimental lines had a marginally increased resistance to moderate heat stress. These results are consistent with the existence of an optimum level of Mn SOD activity which minimizes oxidative stress. The naturally evolved level of Mn SOD activity in Drosophila appears to be near the optimum required under normal conditions, although the optimum may be shifted to a higher level under more stressful conditions.     

Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae

Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import.     

Reduction of manganese porphyrins by flavoenzymes and submitochondrial particles: a catalytic cycle for the reduction of peroxynitrite

The reduction of manganese(III) meso-tetrakis((N-ethyl)pyridinium-2-yl)porphyrin (MnTE-2-PyP) to manganese(II) was catalyzed by flavoenzymes such as xanthine oxidase and glucose oxidase, and by Complex I and Complex II of the mitochondrial electron transport chain. The reduced manganese porphyrin has been previously shown to react rapidly with superoxide and carbonate radical anion. Herein, we describe the reaction of a reduced manganese porphyrin with peroxynitrite that proceeds as a two-electron process, has a rate constant greater than 7 x 10(6) M(-1) s(-1) (at pH 7.25 and 37 degrees C), and produces nitrite and the Mn(IV)Porphyrin. The Mn(II)/Mn(IV) redox cycle was used to divert peroxynitrite from the inactivation of succinate dehydrogenase. In a typical experiment, 5 microM MnTE-2-PyP in the presence of excess succinate was able to protect the succinate dehydrogenase and succinate oxidase activities of submitochondrial particles challenged with a cumulative dose of 140 microM peroxynitrite infused in the course of 2 h. Other MnPorphyrins that are reduced more slowly do not provide as much protection underscoring the rate limiting character of the reduction step. The data presented here serve to rationalize the pharmacological action of MnPorphyrins as peroxynitrite reduction catalysts in vivo and opens avenues for the development of MnPorphyrins to protect mitochondria from oxidative damage.     

Manganese-porphyrin reactions with lipids and lipoproteins

Manganese porphyrin complexes serve to catalytically scavenge superoxide, hydrogen peroxide, and peroxynitrite. Herein, reactions of manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) with lipids and lipid hydroperoxides (LOOH) are examined. In linoleic acid and human low-density lipoprotein (LDL), MnTE-2-PyP(5+) promotes oxidative reactions when biological reductants are not present. By redox cycling between Mn(+3) and Mn(+4) forms, MnTE-2-PyP(5+) initiates lipid peroxidation via decomposition of 13(S)hydroperoxyoctadecadienoic acid [13(S)HPODE], with a second-order rate constant of 8.9 x 10(3) M(-1)s(-1)and k(cat) = 0.32 s(-1). Studies of LDL oxidation demonstrate that: (i) MnTE-2-PyP(5+) can directly oxidize LDL, (ii) MnTE-2-PyP(5+) does not inhibit Cu-induced LDL oxidation, and (iii) MnTE-2-PyP(5+) plus a reductant partially inhibit lipid peroxidation. MnTE-2-PyP(5+) (1-5 microM) also significantly inhibits FeCl(3) plus ascorbate-induced lipid peroxidation of rat brain homogenate. In summary, MnTE-2-PyP(5+) initiates membrane lipid and lipoprotein oxidation in the absence of biological reductants, while MnTE-2-PyP(5+) inhibits lipid oxidation reactions initiated by other oxidants when reductants are present. It is proposed that, as the Mn(+3) resting redox state of MnTE-2-PyP(5+) becomes oxidized to the Mn(+4) redox state, LOOH is decomposed to byproducts that propagate lipid oxidation reactions. When the manganese of MnTE-2-PyP(5+) is reduced to the +2 state by biological reductants, antioxidant reactions of the metalloporphyrin are favored.     

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100mM ethanol for 3days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis(N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N'-monomethyl-l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.     

Differential responses of stress proteins, antioxidant enzymes, and photosynthetic efficiency to physiological stresses in the Florida red tide dinoflagellate, Karenia brevis

This study identifies stress proteins and antioxidant enzymes that may play a role in the survival strategies of the Florida red tide dinoflagellate, Karenia brevis. Heat shock protein 60 (Hsp 60), mitochondrial small heat shock protein (mitosHsp), chloroplastic small heat shock protein (chlsHsp), Mn superoxide dismutase (SOD), and Fe SOD were first identified by Western blotting. The induction of these proteins in laboratory cultures in response to elevated temperatures, hydrogen peroxide, lead, or elevated light intensities was next assessed. In parallel, F(V)/F(M), a measurement of photosynthetic efficiency and common proxy of cellular stress, was determined. Hsp 60, Fe SOD, and Mn SOD were induced following exposure to elevated temperatures, hydrogen peroxide, or lead. MitosHsp responded only to heat, whereas chlsHsp responded only to H(2)O(2)-induced stress. The expression of stress proteins and antioxidant enzymes appears to be a more sensitive indicator of heat or chemically induced stresses than F(V)/F(M). However, F(V)/F(M) decreased significantly in response to elevated light intensities that did not induce the expression of stress proteins. These results identify for the first time stress proteins and antioxidant enzymes in K. brevis, provide evidence for differential sensitivity of cellular organelles to various sources of stress, and confirm the presence of conserved stress responses observed across phyla in a dinoflagellate.     

The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2

Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.