Studies on hydroperoxide-dependent folic acid degradation by hemin

Hemin (ferric protoporphyrin IX chloride) in the presence of hydrogen peroxide or tert-butyl hydroperoxide was found to cleave folic acid at the C9-N10 bond. The ferrous form of hemin was not involved in hydroperoxide-dependent folic acid degradation, as indicated by the lack of inhibition by carbon monoxide. Molecular oxygen was not required for the degradation. GSH-Mn(II) or NAD(P)H in the presence of molecular oxygen did not support hemin-mediated folic acid degradation. The degradation increased as the temperature was elevated from 10 to 70 degrees C. Ascorbic acid and azide were potent inhibitors. Superoxide dismutase and hydroxyl radical quenchers, such as ethanol, mannitol, benzoate, and dimethyl sulfoxide did not inhibit the reaction. Catalase inhibited hydrogen peroxide-supported degradation but not the tert-butyl hydroperoxide-dependent one. Thiol compounds, such as thioglycolic acid, thiourea, glutathione, cysteine, and 2-mercaptoethanol, inhibited the hydrogen peroxide-dependent degradation but supported the tert-butyl hydroperoxide-mediated one. N5-formyl tetrahydrofolic acid, but not N10-formyl folic acid, was degraded by hemin in the presence of H2O2 or TBHP. The data obtained are suggestive of a mechanism similar to N-demethylation reactions catalyzed by cytochrome P-450 and some peroxidases.     

Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase

The lipid-soluble peroxides, tert-butyl hydroperoxide and peroxidized cardiolipin, each react with bovine cytochrome c oxidase and cause a loss of electron-transport activity. Coinciding with loss of activity is oxidation of Trp19 and Trp48 within subunits VIIc and IV, and partial dissociation of subunits VIa and VIIa. tert-Butyl hydroperoxide initiates these structural and functional changes of cytochrome c oxidase by three mechanisms: (1) radical generation at the binuclear center; (2) direct oxidation of Trp19 and Trp48; and (3) peroxidation of bound cardiolipin. All three mechanisms contribute to inactivation since blocking a single mechanism only partially prevents oxidative damage. The first mechanism is similar to that described for hydrogen peroxide [Biochemistry43:1003-1009; 2004], while the second and third mechanism are unique to organic hydroperoxides. Peroxidized cardiolipin inactivates cytochrome c oxidase in the absence of tert-butyl hydroperoxide and oxidizes the same tryptophans within the nuclear-encoded subunits. Peroxidized cardiolipin also inactivates cardiolipin-free cytochrome c oxidase rather than restoring full activity. Cardiolipin-free cytochrome c oxidase, although it does not contain cardiolipin, is still inactivated by tert-butyl hydroperoxide, indicating that the other oxidation products contribute to the inactivation of cytochrome c oxidase. We conclude that both peroxidized cardiolipin and tert-butyl hydroperoxide react with and triggers a cascade of structural alterations within cytochrome c oxidase. The summation of these events leads to cytochrome c oxidase inactivation.     

Peroxidatic oxidation of benzo[a]pyrene and prostaglandin biosynthesis.

The arachidonic acid dependent oxidation of benzo[a]pyrene to a mixture of 3,6-, 1,6-, and 6,12-quinones has been studied by using enzyme preparations from sheep seminal vesicles. Maximal oxidation is observed at 100 microM benzo[a]pyrene and 150 microM arachidonic acid. The arachidonic acid dependent oxidation is peroxidatic and utilizes prostaglandin G2 (PGG2), generated in situ from arachidonate, as the hydroperoxide substrate. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid is equivalent to PGG2 as a hydroperoxide substrate, but hydrogen peroxide, cumene hydroperoxide, and tert-butyl hydroperoxide are much poorer substrates. Arachidonic acid dependent benzo[a]pyrene oxidation by microsomal and solubilized enzyme preparations is markedly.     

Lipid peroxide makes rabbit platelet hyperaggregable to agonists through phospholipase A2 activation

Treatment of rabbit platelets with tert-butyl hydroperoxide and Fe2+ caused increasing arachidonic acid release, lysophosphatidylcholine formation, and aggregation with increasing concentrations of Fe2+. A combination of tert-butyl hydroperoxide and a low concentration of Fe2+, which by itself causes slight or no such activation, elicited synergistic release of arachidonic acid and aggregation under stimulation with a suboptimal concentration of collagen or arachidonic acid as an agonist. These responses were inhibited by pretreatment of the platelets with vitamin E or mepacrine in a concentration-dependent manner, but not by uric acid. The arachidonic acid release was dependent on the presence of Ca2+ in the medium. Synergistic formation of lysophosphatidylcholine, but not diacylglycerol, was also observed under this condition. The aggregation was also inhibited by indomethacin, a cyclooxygenase inhibitor. Cyclooxygenase activity was not affected by the oxidative treatment. These results suggest that lipid peroxide formed in membranes causes phospholipase A2 to become hypersusceptible to the agonist used, making the platelets hyperaggregable.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Hydroperoxides selectively inhibit human erythrocyte membrane enzymes

Treatment of washed erythrocytes with tert-butyl hydroperoxide (0.5 mM, 10 min) inhibited basal Ca2+ + Mg2+-ATPase activity by 40% and calmodulin-stimulated activity by 54%. The inhibition was accompanied by the formation of methemoglobin and the aggregation of some membrane proteins into a high-molecular-weight polymer. Membranes, isolated from washed erythrocytes, showed a similar pattern of inhibition. Basal Ca2+ + Mg2+-ATPase activity was inhibited 50% at 10 min and 70% at 30 min while calmodulin-stimulated activity was inhibited 70% at 10 min and 84% at 30 min. Thiobarbituric acid-reactive products formed slowly during the first 10 min and then increased sharply between 10 and 30 min. The polymerization of membrane proteins was also observed during the tert-butyl hydroperoxide exposure. Inhibition of erythrocyte membrane enzymes was selective. The Na+ + K+-stimulated Mg2+ ATPase, like the Ca2+ + Mg2+-ATPase, was sensitive to membrane oxidation but the activities of Mg2+-ATPase and acetylcholinesterase were less inhibited by tert-butyl hydroperoxide. Acetylcholinterase was found to be very resistant to hydroperoxide treatment with less than 10% loss of activity. The effects of two other hyproperoxides on enzyme inhibition were studied also. Cumene hydroperoxide (0.5 mM) was found to be as potent as tert-butyl hydroperoxide but hydrogen peroxide at 10 mM did not produce thiobarbituric acid-reactive products or inhibit Ca2+ + Mg2+-ATPase activity until after 20 min. The selective effects of peroxides on these enzyme activities are discussed.     

Low concentration of oxidant and nitric oxide donors stimulate proliferation of human endothelial cells in vitro

Proliferation of human umbilical vein endothelial cells in vitro was inhibited by high concentrations of oxidants and nitric oxide donors but stimulated by low (micromolar or submicromolar) concentrations of hydrogen peroxide, menadione, tert-butyl hydroperoxide, AAPH, nitroglycerin, SIN-1 and sodium nitroprusside. The stimulation seems to be dependent upon generation of secondary reactive oxygen species as inferred from attenuation of cell proliferation by superoxide dismutase and catalase. These results point to another type of possible artefact of cell culture, viz. stimulation of cell proliferation by low concentrations of oxidants.     

Alterations in inositol phosphate production during oxidative stress in isolated hepatocytes

The level of inositol phosphates was measured in rat hepatocytes treated with 2-methyl-1,4-naphthoquinone (menadione) or tert-butyl hydroperoxide, which cause Ca2+ mobilization from intracellular stores and an increase in cytosolic free Ca2+ concentration. Although neither agent produced any apparent changes in the resting level of inositol phosphates, pretreatment of hepatocytes with either menadione or tert-butyl hydroperoxide, as well as with several sulfhydryl reagents, markedly inhibited the increase in inositol phosphates induced by both hormonal and nonhormonal stimuli. Addition of dithiothreitol to menadione- or tert-butyl hydroperoxide-treated hepatocytes reversed this inhibition and reestablished responsiveness to extracellular stimuli. Our findings suggest that the inhibition of the inositol phosphate response by menadione and tert-butyl hydroperoxide occurs through the modification of critical sulfhydryl group(s) and that the alterations in intracellular Ca2+ homeostasis occurring during the metabolism of menadione and tert-butyl hydroperoxide in hepatocytes are not mediated by inositol phosphates.     

Induction of vascular relaxation by hydroperoxides

Hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, and 3-chloroperoxybenzoic acid (CPB) and 15-HPETE relaxed, in a concentration dependent manner rat aortic rings contracted with PGF2 alpha (1 X 10(-5)). Relaxation is not inhibited by either indomethacin (2 X 10(-5) M), a cyclo-oxygenase inhibitor or eicosatetraynoic acid (1 X 10(-5) M), a dual cyclo-oxygenase and lipoxygenase inhibitor. Rings with intact endothelium relaxed to a greater degree on exposure to CPB and 15-HPETE. Methylene blue, a soluble guanylate cyclase inhibitor (1 X 10(-5) M) blocked the relaxation elicited by the five peroxides, whereas both superoxide dismutase (scavenger of superoxide anion) and mannitol (scavenger of hydroxyl radical) have no effect. We conclude that relaxation of vascular smooth muscle is a general property of peroxides and that the endothelium may in some instances facilitate this effect.     

HeLp, a heme-transporting lipoprotein with an antioxidant role

Plasma lipoproteins involved in lipid transport are target for free radical-evoked pathological conditions in several mammalian models. The main hemolymphatic protein of Boophilus microplus is a heme-binding lipoprotein (HeLp, for Heme LipoProtein) that carries dietary heme produced from degradation of vertebrate hemoglobin to tissues of the tick. Addition of heme to phospholipid liposomes resulted in intense lipid peroxidation, which was inhibited by addition of HeLp. HeLp prevented lysis of red blood cells by heme. HeLp also inhibited reactions of heme with tert-butyl hydroperoxide (t-BOOH) or hydrogen peroxide. HeLp, quite differently from other lipoproteins, presents a protective intrinsic mechanism to counteract heme toxicity, while preserving the heme molecule to be reused by the tick. This is the first report of a lipoprotein acting as an antioxidant particle against heme-induced radical damage.     

Pro-oxidative effects of Tempo in systems containing oxidants

2,2,6,6-Tetramethylpiperidine-1-oxyl (Tempo), previously reported by us to augment oxidation of glutathione induced by peroxynitrite (Glebska J, Skolimowski J, Kudzin Z, Gwozdzinski K, Grzelak A, Bartosz G. Pro-oxidative activity of nitroxides in their reactions with glutathione. Free Radic Biol Med 2003; 35: 310-316) was found to increase oxidation of glutathione induced by various oxidants, including persulfate, tert-butyl hydroperoxide and hydrogen peroxide. Tempo augmented also the inactivation and thiol loss of alcohol dehydrogenase induced by 2,2'-azobis(2-amidinopropane) (AAPH) and oxidative degradation of deoxyribose induced by ammonium persulfate and tert-butyl hydroperoxide. These results point to a pro-oxidative effect of nitroxides on a range of biomolecules subjected to the action of various oxidants.     

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.     

Interaction of tert-butyl hydroperoxide with hypochlorous acid. A spin trapping and chemiluminescence study

The formation of radical species during the reaction of ter-tbutyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (aN = 1.537 mT, aH beta = 0.148 mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (aN = 1.484 mT, aH beta = 0.233 mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl-alpha-phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe2+ or Ce4+ in the presence of POBN. Using Fe2+ a spin adduct with a N = 1.633 mT and aH beta = 0.276 mT was observed. The major spin adduct formed with Ce4+ was characterised by a N = 1.480 mT and aH beta = 0.233 mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce4+. The addition of Fe2+ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce4+ the same spin adduct and the same luminescence profile. Because Ce4+ is known to oxidize organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.     

Identification in Saccharomyces cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress.

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.     

Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite.

Peroxiredoxins are antioxidant enzymes whose peroxidase activity depends on a redox-sensitive cysteine residue at the active center. In this study we investigated properties of the active center cysteine of bovine 1-Cys peroxiredoxin using a recombinant protein (BRPrx). The only cysteine residue of the BRPrx molecule was oxidized rapidly by an equimolar peroxide or peroxynitrite to the cysteine sulfenic acid. Approximate rates of oxidation of BRPrx by different peroxides were estimated using selenium glutathione peroxidase as a competitor. Oxidation of the active center cysteine of BRPrx by H2O2 proceeded only several times slowly than that of the selenocysteine of glutathione peroxidase. The rate of oxidation varied depending on peroxides tested, with H2O2 being about 7 and 80 times faster than tert-butyl hydroperoxide and cumene hydroperoxide, respectively. Peroxynitrite oxidized BRPrx slower than H2O2 but faster than tert-butyl hydroperoxide. Further oxidation of the cysteine sulfenic acid of BRPrx to higher oxidation states proceeded slowly. Oxidized BRPrx was reduced by dithiothreitol, dihydrolipoic acid, and hydrogen sulfide, and demonstrated peroxidase activity (about 30 nmol/mg/min) with these reductants as electron donors. beta-Mercaptoethanol formed a mixed disulfide and did not support peroxidase activity. Oxidized BRPrx did not react with glutathione, cysteine, homocysteine, N-acetyl-cysteine, and mercaptosuccinic acid.     

Oxidation of cytochrome b5 by hydroperoxides in rat liver.

1. Spectral changes following the addition of hydroperoxides to isolated hepatocytes and to perfused rat liver were observed. Cytochrome b5 is the major, if not the only, hemoprotein exhibiting redox changes under these conditions; cytochrome b5 is oxidized by added hydroperoxides, e.g. tert-butyl or cumene hydroperoxides. No spectral changes attributable to cytochrome b5 were observed with tert-butanol. 2. The effect is present also when the mitochondrial respiratory chain is inhibited by antimycin A, and it is not observable with isolated mitochondria. On the other hand, the oxidation of cytochrome b5 by hydroperoxides is readily demonstrable in microsomal fractions in presence of NADH. 3. Spectral evidence for a participation of the other microsomal hemoprotein, cytochrome P-450, in the hydroperoxide-linked effects was not obtained. Thus, in hepatocytes from phenobarbital-pretreated rats, no formation of cytochrome P-420, no displacement of a type I substrate, hexobarbital, and no major steady state redox change of cytochrome P-450 was detectable. However, when cytochrome P-450 was dithionite-reduced, an oxidation of this cytochrome occurred upon subsequent hydroperoxide addition. 4. Hydrogen peroxide addition to hepatocytes also leads to a lower steady-state degree of reduction of cytochrome b5. Evidence is provided with hepatocytes from rats pretreated with 3-amino-1,2,4-triazole that H2O2 generated intracellularly, e.g. from added glycolate, also causes a detectable oxidation of cytochrome b5. 5. The mechanism of these hydroperoxide effects remains to be established, and it is not clear whether cytochrome b5 reacts directly or indirectly. However, it is suggested that these effects may be of significance for the further study of cytochrome-b5-linked metabolic pathways.     

Enhancement by nitrite of peroxide-induced degradation of uric acid and 3-N-ribosyluric acid

The oxidation of uric acid and 3-N-ribosyluric acid by hydrogen peroxide and methemoglobin was stimulated by the addition of sodium nitrite, which alone has no effect on the urates. The urates were not oxidized by either hydrogen peroxide alone or hydrogen peroxide and sodium nitrite unless methemoglobin was present. t-Butyl hydroperoxide also oxidized the urates in the presence of methemoglobin, but the reaction was not stimulated by sodium nitrite. The addition of either sodium azide or potassium cyanide reduced the rate of the reaction with either hydrogen peroxide or t-butyl hydroperoxide both in the presence and absence of sodium nitrite. Possible explanations for the stimulation by nitrite of peroxide-induced degradation of urates are presented.     

Further structural studies of zosterine

Traces of either ferrous or ferric salts greatly increase the rate of the stepwise degradation of reducing sugars by alkaline hydrogen peroxide, as measured by formation of formic acid; addition of larger proportions of iron salts causes relatively smaller effects. The results showed that, unless unusually strict precautions are taken to exclude traces of iron, the free-radical cleavage of the hydroperoxide adducts of reducing sugars is far more rapid than the ionic cleavage. The catalytic effect of iron salts is counteracted by addition of magnesium salts. With d-glucose, inhibition of the catalytic effect of iron by magnesium depends on both the magnesium-iron ratio and the concentration at a given ratio. Measurements with various molar proportions of the salts indicated that a magnesium-iron complex, containing six atoms of magnesium to one of iron, is formed. Presumably, removal of iron by formation of this complex inhibits the free-radical degradation of hydroperoxide adducts. In marked contrast to the results obtained with reducing sugars, the degradation of potassium glyoxylate and of glyoxal by alkaline hydrogen peroxide is extremely rapid, and not catalyzed by iron or inhibited by magnesium. The results are in accord with an ionic, rather than a free-radical, cleavage of the hydroperoxide adducts of these compounds. The rapidity of the ionic reaction may be attributed to the ready availability of an electron pair from the adjoining carbon atom.     

Cytochrome P-450 deficiency and resistance to t-butyl hydroperoxide of hepatoma microsomal lipid peroxidation

Lipid peroxidation of microsomes from rat liver and Morris hepatoma 9618A was induced by means of tert-butyl hydroperoxide (t-BuOOH). In rat liver microsomes t-BuOOH stimulated an early formation of lipid hydroperoxides (LOOH) and an increasing accumulation of malondialdehyde; t-BuOOH was completely consumed and cytochrome P-450 was rapidly destroyed. In hepatoma microsomes (60% deficiency of cytochrome P-450) a remarkable inhibition of both malondialdehyde and LOOH was observed; t-BuOOH was consumed only partially and cytochrome P-450 was destroyed slowly. In the presence of aminopyrine, malondialdehyde production was inhibited to the same extent (about 70%) in normal and tumour microsomes. The concentration of t-BuOOH required to achieve half-maximal velocity of malondialdehyde accumulation was comparable in the two microsome types. It is proposed that the deficiency of cytochrome P-450 limits the activation of t-BuOOH to the free radical species which initiate lipid peroxidation. Low cytochrome P-450 content would also affect the LOOH-dependent propagation of lipid peroxidation.     

Ferrous ion-mediated cytochrome P-450 degradation and lipid peroxidation in adrenal cortex mitochondria.

The relationship between the degradation reaction of cytochrome P-450 and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe2+, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H2O2, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe2+-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome P-450 is closely related to lipid peroxidation.