Protection against oxidative protein damage induced by metal-catalyzed reaction or alkylperoxyl radicals: comparative effects of melatonin and other antioxidants

Melatonin is a well-known hydroxyl radical (*OH) scavenger that protects DNA and lipids from free radical attack. In this paper, we studied the ability of melatonin to prevent oxidative damage to bovine serum albumin (BSA) induced by two different paradigms: the metal-catalyzed oxidation (MCO) induced by Cu(2+)/H(2)O(2) and the alkoxyl and alkylperoxyl radicals formed by the azo initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH, 40 mM). The protective effects of melatonin were compared with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), glutathione (GSH), ascorbate, 3,4',5-trihydroxy-trans-stilbene (resveratrol, 0.1 microM-4 mM) and mannitol (50 microM-100 mM). Melatonin efficiently prevented protein modification induced by both models, as assayed by polyacrylamide gel electrophoresis and carbonyl content. Both trolox and ascorbate had an obvious pro-oxidant effect in the Cu(2+)/H(2)O(2) model, whereas both prevented BSA damage induced by AAPH. In the MCO model, the efficacy of GSH in terms of protein protection was higher than melatonin at relatively high concentrations (250 microM-4 mM); however, at lower concentrations (50-250 microM), the efficacy of melatonin was superior to GSH. D-Mannitol (50 microM-100 mM) and resveratrol did not protect BSA from the site-specific damage induced by Cu(2+)/H(2)O(2). On the other hand, the relative protective efficiency in the AAPH model was melatonin approximately trolox>GSH>ascorbate.     

Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes

The metabolism and toxicity of formaldehyde (CH2O) in isolated rat hepatocytes was found to be dependent upon the intracellular concentration of glutathione (GSH). Using hepatocytes depleted of GSH by treatment with diethyl maleate (DEM), the rate of CH2O (5.0 mM) disappearance was significantly decreased. Formaldehyde decreased the concentration of GSH in hepatocytes, probably by the extrusion of the CH2O-GSH adduct, S-hydroxymethylglutathione. Formaldehyde toxicity was potentiated in cells pretreated with 1.0 mM DEM as measured by the loss of membrane integrity (NADH stimulation of lactate dehydrogenase (LDH) activity) and an increase in lipid peroxidation (formation of thiobarbituric acid-reactive compounds). This potentiation of toxicity was both CH2O concentration-dependent and time-dependent. There was an excellent correlation between the increase in lipid peroxidation and the decrease in cell viability. L-Methionine (1.0 mM) both protected the cells from toxicity caused by the combination of 8.0 mM CH2O and 1.0 mM DEM and increased the cellular GSH concentration. The antioxidants, ascorbate, butylated hydroxytoluene (BHT) and alpha-tocopherol (10, 25 and 125 microM), all exhibited dose-dependent protection against toxicity produced by 8.0 mM CH2O and 1.0 mM DEM. At toxic concentrations of CH2O (10.0-13.0 mM), administered by itself, lipid peroxidation did not increase concomitantly with the decrease in cell viability and the addition of antioxidants (125 microM) did not influence CH2O toxicity. These results suggest that CH2O toxicity in GSH-depleted hepatocytes may be mediated by free radicals as a result of the effect of CH2O on a critical cellular pool of GSH. However, cells with normal concentrations of GSH are damaged by CH2O by a different mechanism.     

Mitochondrial function and reactive oxygen species action in relation to boar motility

Flow cytometric assays of viable boar sperm were developed to measure reactive oxygen species (ROS) formation (oxidization of hydroethidine to ethidium), membrane lipid peroxidation (oxidation of lipophilic probe C(11)-BODIPY(581/591)), and mitochondrial inner transmembrane potential (DeltaPsi(m); aggregation of mitochondrial probe JC-1) during hypothermic liquid storage and freeze-thawing of boar semen and to investigate relationships among ROS, motility, DeltaPsi(m), and ATP production. Basal ROS formation and membrane lipid peroxidation were low in viable sperm of both fresh and frozen-thawed semen, affecting < or =4%. Sperm in fresh, liquid-stored and frozen-thawed semen appeared to be equally susceptible to the activity ROS generators xanthine/xanthine oxidase, FeSO(4)/ascorbate, and hydrogen peroxide (H(2)O(2)). Of the ROS generators tested, FeSO(4)/ascorbate was specific for membrane lipid peroxidation, whereas menadione, xanthine/xanthine oxidase, and H(2)O(2) were specific for oxidization of hydroethidine. Menadione (30microM) and H(2)O(2) (300microM) decreased (P<0.05) motility by 90% during 60min of incubation. Menadione decreased (P<0.05) the incidence of sperm with high DeltaPsi(m) by 95% during 60min of the incubation, although ATP content was not decreased (P>0.05) until 120min. In contrast, H(2)O(2) did not affect DeltaPsi(m) or ATP at any time. The formation of ROS was not associated with any change in viability (90%) for either menadione or H(2)O(2) through 120min. Overall, the inhibitory affects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to an ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.     

Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells

We have compared the abilities of ascorbate and reduced glutathione (GSH) to act as intracellular free radical scavengers and protect cells against radical-mediated lipid peroxidation. Phenoxyl radicals were generated in HL60 cells, through the action of their myeloperoxidase, by adding H2O2 and phenol. Normally cultured cells, which contain no ascorbate; cells that had been preloaded with ascorbate; and those that had been depleted of GSH with buthionine sulfoximine were investigated. Generation of phenoxyl radicals resulted in the oxidation of ascorbate and GSH. Ascorbate loss was much greater in the absence of GSH, and adding glucose gave GSH-dependent protection against ascorbate loss. Ascorbate, or glucose metabolism, had little effect on the GSH loss. Glutathionyl radical formation was detected by spin trapping with DMPO in cells lacking ascorbate, and the signal was suppressed by ascorbate loading. Addition of phenol plus H2O2 to the cells caused lipid peroxidation, as measured with C11-BODIPY. Peroxidation was greatest in cells that lacked both ascorbate and GSH. Either scavenger alone gave substantial inhibition but optimal protection was seen with both present. These results indicate that GSH and ascorbate can each act as an intracellular radical scavenger and protect against lipid peroxidation. With both present, ascorbate is preferred and acts as the ultimate radical sink for phenoxyl or glutathionyl radicals. However, GSH is still consumed by metabolically recycling dehydroascorbate. Thus, recycling scavenging by ascorbate does not spare GSH, but it does enable the two antioxidants to provide more protection against lipid peroxidation than either alone.     

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated with Cu(2+) ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu(2+) to Cu(+) and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu(+) oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.     

Zinc in the prevention of Fe2+-initiated lipid and protein oxidation

In the present study we characterized the capacity of zinc to protect lipids and proteins from Fe2+-initiated oxidative damage. The effects of zinc on lipid oxidation were investigated in liposomes composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS) at a molar relationship of 60:40 (PC:PS, 60:40). Lipid oxidation was evaluated as the oxidation of cis-parinaric acid or as the formation of 2-thiobarbituric acid-reactive substances (TBARS). Zinc protected liposomes from Fe2+ (2.5-50 microM)-supported lipid oxidation. However, zinc (50 microM) did not prevent the oxidative inactivation of glutamine synthetase and glucose 6-phosphate dehydrogenase when rat brain supernatants were oxidized in the presence of 5 microM Fe2+ and 0.5 mM H2O2. We also studied the interactions of zinc with epicatechin in the prevention of lipid oxidation in liposomes. The simultaneous addition of 0.5 microM epicatechin (EC) and 50 microM zinc increased the protection of liposomes from oxidation compared to that observed in the presence of zinc or EC separately. Zinc (50 microM) also protected liposomes from the stimulatory effect of aluminum on Fe2+-initiated lipid oxidation. Zinc could play an important role as an antioxidant in biological systems, replacing iron and other metals with pro-oxidant activity from binding sites and interacting with other components of the oxidant defense system.     

Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.     

Generation of Hydroxyl Radicals and Its Relation to Cellular Oxidative Damage in Plants Subjected to Water Stress

The hydroxyl radical ('OH) is one of the roost reactive mdieales known to chemistry and is believed to be a major active free radicle responsible for modifications of macmmolecules and cellular damage. Two lines of evidence strongly indicate that 'OH radicals are generated in a Fenton-type Haber-Weiss reactions in plants subjected to water stress. Firstly, water stress causes an increase in the concentration of catalytic metals, which are critical for Fenton-like reactions to proceed in vivo. Furthermore, subrmillimolar concentrations of H2O2 and ascorbic acid(or O2－ ) in the drought-stressed plants are large enough to support the Fentontype Haber-Weiss reactions. Secondly, there is oxidation of proteins and lipids in the drought-stressed plants; a process that requires a catalytic metal and that, at least for protein oxidation, is mediated by the 'OH radicals. Protein oxidation is thought to involve binding of metal ions to the proteins and subsequent site-specific attack by the 'OH radicals arising from the roetal-catalysed decomposition of H2O2. It has been proposed that protein oxidation may be a better index than lipid peroxidation because the latter fields many different products and these only appear after a lag period. The validity of malondialdehyde (MDA), an early product of lipid peroxidation, as an index of lipid peroxidation has been argued by the non-specific method of its measurement. The 'OH radicals are not the only necessary initiator for lipid peroxidation and lipid peroxidation is not usually involved in plants exposed to water stress.     

Relevance of apple polyphenols as antioxidants in human plasma: contrasting in vitro and in vivo effects

Apples are a major source of flavonoids in the Western diet, and flavonoid-rich foods may help protect against chronic diseases by antioxidant mechanisms. In the present study we investigated: (1) the antioxidant capacity of representative apple polyphenols and their contribution to the total antioxidant capacity of apple extracts; (2) the effects of adding apple extract to human plasma in vitro on oxidation of endogenous antioxidants and lipids; and (3) the effects of apple consumption by humans on ex vivo oxidation of plasma antioxidants and lipids. We found that the apple-contained flavonols and flavanols, quercetin, rutin, (-)-epicatechin, and (+)-catechin, had a higher antioxidant capacity than the dihydrochalcones, phloridzin and phloretin, and the hydroxycinnamate, chlorogenic acid. However, together these apple polyphenols contributed less than 20% to the total antioxidant capacity of aqueous apple extracts. When human plasma was exposed to a constant flux of aqueous peroxyl radicals, endogenous ascorbate (70.0 +/- 10.3 microM) was oxidized within 45 min of incubation, while endogenous urate (375 +/- 40 microM) and alpha-tocopherol (24.7 +/- 1.2 microM) were oxidized after ascorbate. Addition of 7.1 or 14.3 micrograms/ml total phenols of apple extract did not protect ascorbate from oxidation, but increased the half-life (t1/2) of urate from 136 +/- 15 to 192 +/- 16 and 208 +/- 23 min, respectively (p < 0.05 each), and t1/2 of alpha-tocopherol from 141 +/- 18 to 164 +/- 8 min (p = ns) and 188 +/- 8 min (p < 0.05). Lipid peroxidation started after ascorbate depletion, and addition of apple extract increased the lag time preceding detectable lipid peroxidation from 36.3 +/- 3.7 to 50.9 +/- 2.7 min (p < 0.05) and 70.4 +/- 4.2 min (p < 0.001). However, when six healthy volunteers ate five apples and plasma was obtained up to 4 h after apple consumption, no significant increases in the resistance to oxidation of endogenous urate, alpha-tocopherol, and lipids were found. Thus, despite the high antioxidant capacity of individual apple polyphenols and apple extracts and the significant antioxidant effects of apple extract added to human plasma in vitro, ingestion of large amounts of apples by humans does not appear to result in equivalent in vivo antioxidant effects of apple polyphenols.     

Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage

The present study describes the effects of exposure of bovine sperm to mild and more intense ROS generating conditions. The membrane integrity of the incubated sperm was assessed and the incubated sperm were used for IVF after which the percentages of cleavage and blastocyst formation were determined for a period up to 9 days. The incubated sperm samples showed increased levels of molecular oxidation in the plasma membrane, the mitochondria, the cytosol and to a lesser extent in the sperm's DNA. The sperm membrane integrity as well as the first cleavage rates obtained with sperm from mild ROS generating conditions (100 microM H2O2) were not different from sperm incubated without pro-oxidants. However, exposure of sperm to more severe oxidative stress (500 mM H2O2 or a combination of 100 microM ascorbic acid, 20 microM FeSO4 and 500 microM H2O2) led to plasma membrane oxidation, reduced percentages of cleaved embryos and a reduction in the percentages of cleaved embryos that developed to the blastocyst stage. From these results, we conclude that the impact of oxidative stress to sperm becomes primarily manifest after the first cleavage of the formed zygote. Importantly, the level of lipid peroxidation in the sperm plasma membrane significantly correlates with blastocyst formation when the corresponding sperm is used for in vitro fertilization of oocytes.     

Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings.

Leaves of maize (Zea mays L.) seedlings were supplied with different concentrations of abscisic acid (ABA). Its effects on the levels of superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and the content of catalytic Fe, the activities of several antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), the contents of several non-enzymatic antioxidants such as ascorbate (ASC), reduced glutathione (GSH), alpha-tocopherol (alpha-TOC) and carotenoid (CAR), and the degrees of the oxidative damage to the membrane lipids and proteins were examined. Treatment with 10 and 100 microM ABA significantly increased the levels of O(2)(-) and H(2)O(2), followed by an increase in activities of SOD, CAT, APX and GR, and the contents of ASC, GSH, alpha-TOC and CAR in a dose- and time-dependent pattern in leaves of maize seedlings. An oxidative damage expressed as lipid peroxidation, protein oxidation, and plasma membrane leakage did not occur except for a slight increase with 100 microM ABA treatment for 24 h. Treatment with 1,000 microM ABA led to a more abundant generation of O(2)(-) and H(2)O(2) and a significant increase in the content of catalytic Fe, which is critical for H(2)O(2)-dependent hydroxyl radical production. The activities of these antioxidative enzymes and the contents of alpha-TOC and CAR were still maintained at a higher level, but no longer further enhanced when compared with the treatment of 100 microM ABA. The contents of ASC and GSH had no changes in leaves treated with 1,000 microM ABA. These results indicate that treatment with low concentrations of ABA (10 to 100 microM) induced an antioxidative defence response against oxidative damage, but a high concentration of ABA (1,000 microM) induced an excessive generation of AOS and led to an oxidative damage in plant cells.     

Lipid peroxidation causes an increase of lipid order and a decrease of 5'-nucleotidase activity in the liver plasma membrane.

The effect of peroxidation on 5'-nucleotidase activity as well as on membrane microviscosity has been investigated in liver plasma membranes from Wistar rats. The peroxidation was performed with 100 microM H2O2 and 200 microM FeSO4 and/or with 5 mM t-butylhydroperoxide. Treatment of the membranes with these oxidizing agents resulted in an elevation of the transition temperatures of the polarization of the lipid fluorescent probes 1,6 diphenyl-1,3,5 hexatriene (DPH), 3-p-(6-phenyl) 1,3,5 hexatriene phenylpropionic acid (PA-DPH) as well as of the fluorescent thiol reagent N-(1-pyrene) maleimide (1-PM). The peroxidation resulted in a decrease of the activity of 5'nucleotidase. Our data support that the increase of membrane microviscosity of the lipid domain regulates the activity of 5'-nucleotidase.     

Silicon-mediated alleviation of Mn toxicity in Cucumis sativus in relation to activities of superoxide dismutase and ascorbate peroxidase

The effects of exogenous silicon (Si) on plant growth, activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase, and concentrations of ascorbate and glutathione were investigated in cucumber (Cucumis sativus L.) plants treated with excess manganese (Mn) (600 microM). Compared with the treatment of normal Mn (10 microM), excess Mn significantly increased H2O2 concentration and lipid peroxidation indicated by accumulation of thiobarbituric acid reactive substances. The leaves showed apparent symptoms of Mn toxicity and the plant growth was significantly inhibited by excess Mn. The addition of Si significantly decreased lipid peroxidation caused by excess Mn, inhibited the appearance of Mn toxicity symptoms, and improved plant growth. This alleviation of Mn toxicity by Si was related to a significant increase in the activities of SOD, APX, DHAR and GR and the concentrations of ascorbate and glutathione.     

Time-course of oxidation of lipids in human cerebrospinal fluid in vitro

Oxidative mechanisms play an important role in the pathogenesis of Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. To assess whether the oxidation of brain lipoproteins plays a role in the development of these pathologies, we investigated whether the lipoproteins of human cerebrospinal fluid (CSF) are susceptible to oxidative modification in vitro. We studied oxidation time-course for up to 100 h of human CSF in the absence (autooxidation) or presence of exogenous oxidants. Autooxidation of diluted CSF was found to result in a slow accumulation of lipid peroxidation products. The time-course of lipid hydroperoxide accumulation revealed three consecutive phases, lag-phase, propagation phase and plateau phase. Qualitatively similar time-course has been typically found in human plasma and plasma lipoproteins. Autooxidation of CSF was accelerated by adding exogenous oxidants, delayed by adding antioxidants and completely inhibited by adding a chelator of transition metal ions. Autooxidation of CSF also resulted in the consumption of endogenous ascorbate, alpha-tocopherol, urate and linoleic and arachidonic acids. Taking into account that (i) lipid peroxidation products measured in our study are known to be derived from fatty acids, and (ii) lipophilic antioxidants and fatty acids present in CSF are likely to be located in CSF lipoproteins, we conclude that lipoproteins of human CSF are modified in vitro during its autooxidation. This autooxidation appears to be catalyzed by transition metal ions, such as Cu(II) and Fe(III), which are present in native CSF. These data suggest that the oxidation of CSF lipoproteins might occur in vivo and play a role in the pathogenesis of neurodegenerative diseases.     

Oxidation of ferrous iron during peroxidation of lipid substrates

Oxidation of Fe2+ in solution was dependent upon medium composition and the presence of lipid. The complete oxidation of Fe2+ in 0.9% saline was markedly accelerated in the presence of phosphate or EDTA and the ferrous oxidation product formed was readily recoverable as Fe2+ by ascorbate reduction. In contrast, in the presence of either brain synaptosomal membranes, phospholipid liposomes, fatty acid micelles or H2O2, less than 50% of the Fe2+ oxidized during an incubation could be recovered as Fe2+ via reduction with ascorbate. In the presence of unsaturated lipid, oxidation of Fe2+ was associated with peroxidation of lipid, as assessed by the uptake of O2 and formation of thiobarbituric acid-reactive products during incubations. Although relatively little Fe2+ oxidation or lipid peroxidation occurred in saline with synaptosomes or linoleic acid micelles during an incubation with Fe2+ alone, significant Fe2+ oxidation and lipid peroxidation occurred in incubations containing a 1:1 ratio of Fe2+ and Fe3+. Extensive Fe2+ oxidation and lipid peroxidation also occurred with Fe2+ alone in saline incubations with either linolenic or arachidonic acid acid micelles or liposomes prepared from dilinoleoylphosphatidylcholine. While a 1:1 ratio of Fe2+ and Fe3+ enhanced thiobarbituric acid-reactive product formation in incubations containing linolenic or arachidonic micelles, it reduced the rate of O2 consumption as compared with Fe2+ alone. The results demonstrate that oxidation of Fe2+ in incubations containing lipid substrates is linked to and accelerated by peroxidation of those substrates. Furthermore, the results suggest that oxidation of Fe2+ in the presence of lipid or H2O2 creates forms of iron which differ from those formed during simple Fe2+ autoxidation.     

Interaction of PGBx and peroxides with cytochrome c and inhibition of lipid peroxidation

PGBx, a derivative of prostaglandin B1, stimulated the oxidation of cytochrome c in the presence of H2O2. Although the reaction was nonenzymatic, the apparent activation energies of 12 and 4.9 kcal above and below the transition at 21.5 degrees C were similar to those for oxidation by cytochrome oxidase. Depletion of H2O2 and oxidation of cytochrome c followed similar time courses, suggesting that H2O2 was consumed in the reaction. PGBx was a specific requirement, but organic hydroperoxides (ethyl and T-butyl) could replace H2O2. Low concentrations of ethyl or t-butyl hydroperoxide initially stimulated the oxidation of cytochrome c; this stimulation disappeared before completion of the oxidation, but was restored when the hydroperoxide concentration was renewed, suggesting that these hydroperoxides were probably also consumed in the reaction. The concentration of PGBx (8.9 microM) required for half-maximum stimulation of the oxidation was similar to the apparent Kd for its dissociation from oxidized cytochrome c (6.8 microM). Binding data and CD spectra suggested that a 1:1 complex between cytochrome c and PGBx was formed, altering the conformation of the heme region. This conformational change caused a shift of the Soret absorption peak from 410 to 406 nm and may be responsible for the enhanced oxidizability of the cytochrome c by H2O2. Cytochrome c inhibited lipid peroxidation in microsomes, an effect enhanced by the addition of PGBx. In the absence of lipid peroxidation, cytochrome c and PGBx stimulated NADPH oxidation via NADPH-cytochrome c reductase. Thus the inhibition of lipid peroxidation by cytochrome c and PGBx may involve either the removal of hydroperoxides or deviation of electron transfer away from the pathway for lipid peroxidation.     

The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress

Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.     

Cellular signalling and free-radical modulating activities of the novel peptidomimetic L-glutamyl-histamine.

A novel histamine-containing peptidomimetic, L-glutamyl-histamine (L-Glu-Hist), has been synthesized and characterized as a possible cytokine mimic which might lead to cellular responses of improved specificity. The energy-minimized 3-D conformations of L-Glu-Hist derived from its chemical structure stabilize Fe2+-chelating complexes. L-Glu-Hist concentration-dependently accelerates a decrease in ferrous iron in ferrous sulfate solution and shows ferroxidase-like activity at concentrations less than 3 mM in the phenanthroline assay, whereas in the concentration range 3-20 mM it restricts the availability of Fe2+ to phenanthroline by chelation of iron ions. At low concentrations (less than or about 1 mM), L-Glu-Hist stimulates peroxidation of phosphatidylcholine in liposomes catalyzed by a superoxide anion radical (O2)-generating system (Fe2+ + ascorbate) and, at high concentrations (*10 mM), it suppresses lipid peroxidation (LPO) in liposomes. The stimulation of LPO by L-Glu-Hist is related to its ability at low concentrations (*0.05 mM) to release O2 free radicals as determined by the superoxide dismutase-inhibitable reduction of cytochrome c. The release of O2 by L-Glu-Hist might result from its ferroxidase-like activity, while its inhibition of LPO is due to chelation of Fe2+, prevention of the formation of free radicals, and degradation of lipid hydroperoxides at 5-20 mM L-Glu-Hist concentrations. L-Glu-Hist releases O2 at concentrations which stimulate [3H]thymidine incorporation into DNA and proliferation of mouse spleen lymphocytes and also of mononuclear cells from human blood. The induction of lymphocyte proliferation by L-Glu-Hist is dose-dependent in the 0.01-0.05 mM concentration range, although the maximal stimulation of LPO in the O2-dependent system is observed at higher L-Glu-Hist concentrations (*1 mM). Thus, low concentrations of oxygen free radicals released by L-Glu-Hist may provide a very fast, specific, and sensitive trigger for lymphocyte proliferation and immunoregulation.     

Ferric(III) ions inhibits copper(II)/hydrogen peroxide-catalyzing lipid peroxidation in human erythrocyte membranes.

1. Effect of ferric ions (Fe3+) on the lipid peroxidation catalyzed by copper ions (Cu2+) and hydrogen peroxide (H2O2) was studied in human erythrocyte membranes. 2. The formation of thiobarbituric acid-reactive products elicited by CuCl2/H2O2 was inhibited by FeCl3 in a concentration-dependent manner; 0.25 mM FeCl3 were enough to cause 50% inhibition of the formation of peroxides. 3. The inhibitory effect of FeCl3 is not due to competition against Cu2+. 4. FeCl3 inhibited the initiation, but did not inhibit the propagation of Cu2+/H2O2-catalyzing lipid peroxidation. 5. In the heat- or trypsin-treated erythrocyte membranes, FeCl3 had no inhibitory effect on Cu2+/H2O2-catalyzing lipid peroxidation. 6. Sodium azide, an inhibitor of catalase, had no effect on the inhibitory effect of FeCl3. 7. These results suggest that a protein factor(s), which is not catalase, is involved in the inhibition of Cu2+/H2O2-catalyzing lipid peroxidation by Fe3+.