Iron catalysis of lipid peroxidation in ferroptosis: Regulated enzymatic or random free radical reaction?

Duality of iron as an essential cofactor of many enzymatic metabolic processes and as a catalyst of poorly controlled redox-cycling reactions defines its possible biological beneficial and hazardous role in the body. In this review, we discuss these two "faces" of iron in a newly conceptualized program of regulated cell death, ferroptosis. Ferroptosis is a genetically programmed iron-dependent form of regulated cell death driven by enhanced lipid peroxidation and insufficient capacity of thiol-dependent mechanisms (glutathione peroxidase 4, GPX4) to eliminate hydroperoxy-lipids. We present arguments favoring the enzymatic mechanisms of ferroptotically engaged non-heme iron of 15-lipoxygenases (15-LOX) in complexes with phosphatidylethanolamine binding protein 1 (PEBP1) as a catalyst of highly selective and specific oxidation reactions of arachidonoyl- (AA) and adrenoyl-phosphatidylethanolamines (PE). We discuss possible role of iron chaperons as control mechanisms for guided iron delivery directly to their "protein clients" thus limiting non-enzymatic redox-cycling reactions. We also consider opportunities of loosely-bound iron to contribute to the production of pro-ferroptotic lipid oxidation products. Finally, we propose a two-stage iron-dependent mechanism for iron in ferroptosis by combining its catalytic role in the 15-LOX-driven production of 15-hydroperoxy-AA-PE (HOO-AA-PE) as well as possible involvement of loosely-bound iron in oxidative cleavage of HOO-AA-PE to oxidatively truncated electrophiles capable of attacking nucleophilic targets in yet to be identified proteins leading to cell demise.

Improved spatial resolution of matrix-assisted laser desorption/ionization imaging of lipids in the brain by alkylated derivatives of 2,5-dihydroxybenzoic acid.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) is one of the major techniques for mass spectrometry imaging (MSI) of biological systems along with secondary-ion mass spectrometry (SIMS) and desorption electrospray mass spectrometry (DESI). The inherent variability of MALDI-MSI signals within intact tissues is related to the heterogeneity of both the sample surface and the matrix crystallization. To circumvent some of these limitations of MALDI-MSI, we have developed improved matrices for lipid analysis based on structural modification of the commonly used matrix 2,5-dihydroxybenzoic acid (DHB). METHODS: We have synthesized DHB containing -C6H13 and -C12H25 alkyl chains and applied these matrices to rat brain using a capillary sprayer. We utilized a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer to analyze lipid extracts and tissue sections, and examined these sections with polarized light microscopy and differential interference contrast microscopy. RESULTS: O-alkylation of DHB yields matrices, which, when applied to brain sections, follow a trend of phase transition from crystals to an oily layer in the sequence DHB → DHB-C6H13 → DHB-C12H25 . MALDI-MSI images acquired with DHB-C12H25 exhibited a considerably higher density of lipids than DHB. CONCLUSIONS: Comparative experiments with DHB and DHB-C12H25 are presented, which indicate that the latter matrix affords higher lateral resolution than the former.

Molecular design of new inhibitors of peroxidase activity of cytochrome c/cardiolipin complexes: fluorescent oxadiazole-derivatized cardiolipin.

Interaction of a mitochondria-specific anionic phospholipid, cardiolipin (CL), with an intermembrane protein, cytochrome c (cyt c), yields a peroxidase complex. During apoptosis, the complex induces accumulation of CL oxidation products that are essential for detachment of cyt c from the mitochondrial membrane, induction of permeability transition, and release of proapoptotic factors into the cytosol. Therefore, suppression of the peroxidase activity and prevention of CL oxidation may lead to discovery of new antiapoptotic drugs. Here, we report a new approach to regulate the cyt c peroxidase activity by using modified CL with an oxidizable and fluorescent 7-nitro-2,1,3-benzoxadiazole (NBD) moiety (NBD-CL). We demonstrate that NBD-CL forms high-affinity complexes with cyt c and blocks cyt c-catalyzed oxidation of several peroxidase substrates, cyt c self-oxidation, and, most importantly, inhibits cyt c-dependent oxidation of polyunsaturated tetralinoleoyl CL (TLCL) and accumulation of TLCL hydroperoxides. Electrospray ionization mass spectrometry and fluorescence analysis revealed that oxidation and cleavage of the NBD moiety of NBD-CL underlie the inhibition mechanism. We conclude that modified CL combining a nonoxidizable monounsaturated trioleoyl CL with a C(12)-NBD fragment undergoes a regiospecific oxidation thereby representing a novel inhibitor of cyt c peroxidase activity.

An ESR and HPLC-EC assay for the detection of alkyl radicals.

The correlation of lipid peroxidation with release of alkanes (RH) is considered a noninvasive method for the in vivo evaluation of oxidative stress. The formation of RH is believed to reflect a lipid hydroperoxide (LOOH)-dependent generation of alkoxyl radicals (LO*) that undergo beta-scission with release of alkyl radicals (R*). Alternatively, R* could be spin-trapped with a nitrone before the formation of RH and analyzed by ESR. Extracts from the liver and lung of CCl(4)- and asbestos-treated rats that were previously loaded with nitrones exhibited ESR spectra suggesting the formation of iso-propyl, n-butyl, ethyl, and pentyl radical-derived nitroxides. In biological systems, various nitroxides with indistinguishable ESR spectra could be formed. Hence, experiments with N-tert-butyl-alpha-phenylnitrone (PBN) for spin trapping of R* were carried out in which the nitroxides formed were separated and analyzed by HPLC with electrochemical detection (EC). The C(1-5) homologous series of PBN nitroxides and hydroxylamines were synthesized, characterized by ESR, GC-MS, and HPLC-EC, and used as HPLC standards. For in vivo generation and spin trapping of R*, rats were loaded with CCl(4) and PBN. The HPLC-EC chromatograms of liver extracts from CCl(4)-treated rats demonstrated the formation of both the nitroxide and hydroxylamine forms of PBN/*CCl(3), as well as the formation of a series of unidentified PBN nitroxides and hydroxylamines. However, formation of PBN adducts with retention times similar to these of the PBN/C(2-5) derivatives was not observed. In conclusion, we could not correlate the production of PBN-detectable alkyl radicals with the reported CCl(4)-dependent production of C(1-5) alkanes. We speculate that the major reason for this is the low steady-state concentrations of R* produced because only a small fraction of LO* undergo beta-scission to release R*.

Oxidation of the ketoxime acetoxime to nitric oxide by oxygen radical-generating systems.

Ketoximes undergo a cytochrome P450-catalyzed oxidation to nitric oxide and ketones in liver microsomes. In addition, nitric oxide synthase (NOS) can catalyze the oxidative denitration of the >C=N-OH group of amidoximes. The objective of this work was to characterize the oxidation of a ketoxime (acetoxime) and to assess the ability of NOS to catalyze the generation of nitric oxide/nitrogen monoxide (*NO) from acetoxime. Acetoxime was oxidized to NO2- (and NO3-) by microsomes enriched with several P450 isoforms, including CYP2E1, CYP1A1, and CYP2B1. Nitric oxide was identified as an intermediate in the overall reaction. Superoxide dismutase and catalase significantly inhibited the reaction. Exogenous iron increased the microsomal generation of NO2- from acetoxime, while metal chelators (desferrioxamine, EDTA, DTPA) inhibited it. A Fenton-like system (Fe2+ plus H2O2, pH 7.4) consumed acetoxime with production of NO2- and NO3-, whereas oxidation by superoxide or by H2O2 was inefficient. The results presented suggest a role for hydroxyl radical-like oxidants in the oxidation of acetoxime to nitric oxide. O-Acetylacetoxime and O-tert-butylacetoxime were not oxidized by a Fenton system or by liver microsomes to any significant extent. Formation of the 5,5'-dimethyl-1-pyrroline-N-oxide/. OH adduct by a Fenton system was significantly inhibited by acetoxime, while O-acetylacetoxime and O-tert-butylacetoxime were inactive. These results suggest that the. OH-dependent oxidation of acetoxime initially proceeds via abstraction of a hydrogen atom from its hydroxyl group, as opposed to the oxidation of its >C=N- function. HepG2 cells with low levels of expression of P450 did not significantly produce NO2- from acetoxime, while HepG2 cells expressing CYP2E1 did, and this generation was blocked by a CYP2E1 inhibitor. Acetoxime was inactive either as a substrate or as an inhibitor of iNOS activity. These results indicate that reactive oxygen species play a key role in the oxidation of acetoxime to. NO by liver microsomes by a mechanism involving H abstraction from the OH moiety by hydroxyl radical-like oxidants and suggest the possibility that acetoxime may be an effective producer of. NO primarily in the liver by a pathway independent of NOS.

Preparation and properties of S-nitroso-L-cysteine ethyl ester, an intracellular nitrosating agent.

In this report, a protocol for the preparation of the hydrochloride of S-nitroso-L-cysteine ethyl ester (SNCEE.HCl; 2) is presented. The synthesis of 2 has been targeted because S-nitroso-L-cysteine (SNC; 2b), which is extensively used for trans-S-nitrosation of thiol-containing proteins, has a limited ability of crossing cellular membranes. The nitrosothiol 2 was prepared via direct S-nitrosation of the hydrochloride of L-cysteine ethyl ester (CEE.HCl; 1a) with ethyl nitrite. 2 is relatively stable in crystal form and when neutralized to SNCEE (2a) in aqueous solutions treated with chelators of metal ions. Traces of metal ions, however, triggered the decomposition of 2a to nitric oxide and a S-centered radical, which were detected by ESR spectrometry. In contrast to 2b, 2a is a lipophilic compound that was taken up by human neutrophils. The latter process was paralleled by inhibition of the NADPH oxidase-dependent generation of superoxide anion radicals, presumably via reaction(s) of intracellular trans-S-nitrosation. Intracellular accumulation of S-nitrosothiols was observed with 2a but not with 2b. It is expected that the use of 2a will be advantageous when intracellular reactions of trans-S-nitrosation are to be studied.

Mitochondrial permeability transition induced by 1-hydroxyethyl radical.

Impairment of mitochondrial functions has been found in ethanol-induced liver injury. Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat liver microsomal systems. Experiments were carried out to evaluate the ability of HER to cause mitochondrial swelling as an indicator of the mitochondrial permeability transition (MPT). Electron spin resonance (ESR) spectroscopy was used to detect HER and to study its interaction with mitochondria. The ESR signal intensity of the spin adduct formed from alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone (POBN) and HER generated from either a thermic decomposition of 1,1'-dihydroxyazoethane (DHAE) or a Fenton reaction system containing ethanol was markedly diminished by the addition of mitochondria, indicating an interaction between HER and mitochondria. Exposure of rat liver mitochondria to HER generated from either system caused swelling, as reflected by a decrease in absorbance at 540 nm, in a HER concentration-dependent and a cyclosporin A-sensitive manner. Mitochondrial swelling was also induced in the Fenton reaction system without ethanol. The DHAE-dependent generation of HER in mitochondrial suspension resulted in a decrease of membrane protein thiols and collapse of the membrane potential (delta psi). The swelling induced by HER was prevented by glutathione and vitamin E, but not by superoxide dismutase. Catalase did not prevent the swelling caused by the acetaldehyde/hydroxylamine O-sulfonate (HOS) system, but was inhibitory in the Fenton reaction system with or without ethanol. These results indicate that HER, as well as hydroxyl radical, can induce the MPT, and suggest the possibility that the collapse of delta psi caused by HER may, at least in part, contribute to impairment of mitochondrial function caused by ethanol and in ethanol-induced liver injury.

Interaction of 1-hydroxyethyl radical with antioxidant enzymes.

There is considerable interest in the role of the 1-hydroxyethyl radical (HER) in the toxic effects of ethanol. The goal of this study was to evaluate the effects of HER on classical antioxidant enzymes. The interaction of acetaldehyde with hydroxylamine-o-sulfonic acid has been shown to produce 1, 1'-dihydroxyazoethane (DHAE); this compound appears to be highly unstable, and its decomposition leads to the generation of HER. Addition of DHAE into a solution of PBN led to the appearance of the typical EPR spectra of PBN/HER adduct. No PBN/HER spin adduct was detected when DHAE was incubated with 0.1 M PBN in the presence of GSH. In the absence of PBN, DHAE oxidized ascorbic acid to semidehydroascorbyl radical, presumably via an ascorbate-dependent one-electron reduction of HER back to ethanol. Catalase was progressively inactivated by exposure to DHAE-generated HER in a time and HER concentration-dependent manner. Ascorbic acid and PBN gave full protection to catalase against HER-dependent inactivation. The antioxidants 2-tert-butyl-4-methylphenol, propylgallate, and alpha-tocopherol-protected catalase against inactivation by 84, 88, and 39%, respectively. Other antioxidant enzymes were also sensitive to exposure to HER. Glutathione reductase, glutathione peroxidase, and superoxide dismutase were inactivated by 46, 36, and 39%, respectively, by HER. The results reported here plus previous results showing HER interacts with GSH, ascorbate, and alpha-tocopherol suggest that prolonged generation of HER in cells from animals chronically exposed to ethanol may lower the antioxidant defense status, thereby contributing to mechanisms by which ethanol produces a state of oxidative stress and produces toxicity.

Metabolism of carbon tetrachloride to trichloromethyl radical: An ESR and HPLC-EC study.

Extensive ESR spin-trapping studies with alpha-phenyl-N-tert-butylnitrone (PBN) have shown that carbon tetrachloride (CCl(4)) is metabolized to trichloromethyl radical ((*)CCl(3)). However, the ESR analysis of alpha-phenyl-N-tert-butylnitrone (PBN)-spin trapped (*)CCl(3) in biological systems appears to be complicated. It has been reported that after in vivo administration of PBN and CCl(4) to rats, most of the PBN-CCl(3) adduct collected in the bile was ESR silent, suggesting reduction of the nitroxide to its hydroxylamine form. The PBN-CCl(3) nitroxide was also shown to undergo a NADPH-dependent reduction in the presence of liver microsomes. Thus, it appears that the variability (or the absence) of the ESR signal of PBN-CCl(3) nitroxide in biological systems reflects, at least in part, the fluctuations in the equilibrium between the nitroxide and hydroxylamine forms of this adduct. To test this possibility, ESR and HPLC experiments with electrochemical detection (EC) were conducted for analysis of the major redox form of the PBN-CCl(3) adduct in vivo. Standard procedures for the in vitro preparation of both redox forms of PBN-CCl(3) and for their HPLC-EC analysis and electrochemical profiles were established. The intensity of the initially observed ESR spectrum of PBN-CCl(3) nitroxide of the liver extract from a CCl(4)- and PBN-treated rat was relatively constant; after an addition of K(3)[Fe(CN)(6)] to the extract, the intensity of the ESR spectrum increased by 1 order of magnitude, most likely due to the co-oxidation of ESR silent PBN-derived hydroxylamines. The addition of PBN-CCl(3) nitroxide to the liver homogenate resulted in the rapid loss of the ESR signal. The HPLC-EC analysis of the liver extract revealed that the in vivo spin trapping of (*)CCl(3) with PBN leads to a preferential formation of the ESR silent PBN-CCl(3) hydroxylamine. The predominant presence of the hydroxylamine derivative was also detected in the blood of a CCl(4)-treated rat. The results of this work are discussed in terms of combination of the ESR spin trapping and HPLC-EC techniques for the detection of ESR silent radical adducts in biological systems.

Metabolites of acetaminophen trigger Ca2+ release from liver microsomes.

Release of mitochondrial calcium is believed to play a key role in the toxicity of acetaminophen in biological systems. Elevated cytosolic Ca2+ may also result from activation of calcium releasing channels. The major metabolites of acetaminophen, benzoquinone imine and 1,4-benzoquinone, induced Ca2+ release in isolated rat liver microsomes. The 1,4-benzoquinone-induced release of calcium was suppressed by ryanodine and fully inhibited by reduced glutathione. Concentrations of 1,4-benzoquinone that induced Ca2+ release did not affect the activity of the microsomal Ca2+, Mg2+-APTase. The binding of [3H]ryanodine to liver microsomes, however, was significantly decreased by 1,4-benzoquinone, suggesting a direct interaction of this metabolite with the ryanodine-binding protein (ryanodine receptor). These results suggest that cellular Ca2+ levels may be elevated by acetaminophen by pathways involving, in part, activation of Ca2+ releasing channels such as the ryanodine receptor.

ESR and HPLC-EC analysis of the interaction of hydroxyl radical with DMSO: rapid reduction and quantification of POBN and PBN nitroxides.

The low stability of hydroxyl radical (OH.)-derived nitroxides is a limiting factor for direct spin-trapping of OH. in biological systems. The latter experimental difficulty is partly solved with the introduction of dimethyl sulfoxide (DMSO) into the studied systems. Hydroxyl radical oxidizes DMSO to methyl radical, which forms relatively stable nitroxides. The results of the present work provide evidence that in alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and alpha-phenyl-N-tert-butylnitrone (PBN) spin-trapping experiments aimed to detect methyl radical in biological systems, the nitroxides formed can be reduced to their ESR-"silent" hydroxylamine derivatives. The nitroxides and their hydroxylamine derivatives were successfully analyzed by HPLC with electrochemical (EC) and UV detection. The lowest limits of UV and EC detection of POBN/CH3 hydroxylamine was evaluated to be in the micro- and nanomolar range, respectively. In parallel ESR and HPLC-EC analysis of the metabolism of menadione by either HepG2 cells or isolated rat hepatocytes in the presence of DMSO, the HPLC-EC method has proven to be more sensitive in detecting the production of methyl radical. The use of the HPLC-EC detection of POBN/CH3 and PBN/CH3 is expected to be advantageous in detection of hydroxyl radical in biological systems in the presence of DMSO.

ESR and HPLC-EC analysis of ethanol oxidation to 1-hydroxyethyl radical: rapid reduction and quantification of POBN and PBN nitroxides.

Extensive ESR spin-trapping studies have shown that ethanol is oxidized to 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. The ESR detection of POBN/HER nitroxide in bile, and formation of antibodies, which recognize HER adducts in alcoholics, suggest that HER is produced in vivo. In liver, where ethanol is primarily metabolized, only traces of PBN/HER nitroxide are documented. One limitation of the ESR spin-trapping technique, however, is that the nitroxides formed in the presence of cellular reductants can be metabolized to the corresponding ESR "silent" hydroxylamines. Ascorbate and NADPH plus liver microsomes were found to reduce rapidly both POBN/HER and PBN/HER nitroxides to their ESR "silent" hydroxylamine derivatives. An HPLC method with electrochemical detection was developed for the detection and quantification of both POBN/HER and PBN/HER nitroxides, as well as their hydroxylamines. Both the diastereomers of the POBN/HER nitroxide and hydroxylamine can be detected, as can both isomers of the PBN/HER nitroxide, and it is estimated that the sensitivity of the HPLC procedure is in the nM range when using EC detection. The hydroxylamines are stable in ethanol, while pH-dependent auto-oxidation occurs in aqueous buffers. Some of the characteristics associated with HER formation by microsomes as detected with ESR (e.g., sensitivity to SOD and catalase, increase after induction of CYP2E1) are reproduced with the HPLC method. By quantification of the POBN/HER hydroxylamine, the NADPH-dependent rates of HER formation by microsomes from pyrazole-treated rats are estimated to be about 1-1.5 nmol HER per min per mg microsomal protein. This rate is less, as compared to the two electron-dependent rate of acetaldehyde formation by these microsomes, about 10-15 nmol per min per mg protein. Thus, at first approximation, the one electron-dependent rate of ethanol oxidation is about 10% the two electron-dependent rate by isolated microsomes. The HPLC procedure can readily detect the POBN/HER and PBN/HER nitroxides and their hydroxylamine derivatives in the same sample and may be of value in detecting HER spin-trapped adducts under biological reducing conditions.

Redox-cycling of iron ions triggers calcium release from liver microsomes.

Elevation of cytosolic calcium levels has been shown to occur via oxidation of critical protein thiols in liver microsomes. Elevated cytosolic Ca2+ may also result from activation of calcium releasing channels. In the presence of NADPH or ascorbic acid, iron ions produced a concentration-dependent release of calcium from liver microsomes. Under anaerobic conditions, the iron-induced release of calcium was inhibited, suggesting that a reaction of oxidation triggers the releasing process. The calcium releasing process at pH 7.0 appears to be highly sensitive to activation by iron ions, as effective concentrations (e.g., 2-5 microM) did not alter the Ca2+, Mg2+-ATPase or the phospholipid component of the microsomal membranes. Iron-induced Ca2+-release could occur under conditions in which there was no iron-induced microsomal lipid peroxidation. Under conditions of intense lipid peroxidation, PBN fully prevented the iron-induced accumulation of thiobarbituric reactive reagents without affecting the release of Ca2+, suggesting that lipid peroxidation is not the mechanism by which iron causes release of calcium. Trolox, GSH and high concentrations of ascorbate, however, strongly inhibited the iron-induced calcium release, most likely due to modulation of the Fe2+/Fe3+ ratio. While the IP3 receptor system is considered to be the main regulator of calcium release, liver also contains a ryanodine-sensitive calcium releasing store. The iron-induced calcium release at pH 7.0 was blocked by ruthenium red, a specific inhibitor of the ryanodine receptor, and Fe2+ (but not Fe3+) decreased the binding of ryanodine, a specific ligand for the ryanodine-sensitive calcium channel. These results suggest that redox-cycling of iron ions results in an activation of a ryanodine-sensitive calcium channel. Activation of calcium releasing channels by iron may play a role in the evolution of various hepatic disorders that are associated with chronic iron overload in humans.

Interaction of 1-hydroxyethyl radical with glutathione, ascorbic acid and alpha-tocopherol.

Ethanol has been shown to be oxidized to a free radical metabolite, the 1-hydroxyethyl radical (HER). Interaction of HER with cellular antioxidants may contribute to the known ability of ethanol administration to lower levels of GSH and alpha-tocopherol. Experiments were carried out to establish a model system for the generation of HER and to study its interaction with GSH, ascorbic acid and alpha-tocopherol. A standard reaction for formation of azo-compounds using acetaldehyde and hydroxylamine-O-sulfonic acid was applied for the synthesis of 1,1'-dihydroxyazoethane (CH3CH(OH)-N=N-CH(OH)CH3). Although stable at -70 degrees C, thermal decomposition of this compound at room temperature was shown to produce HER, detected by EPR spectrometry as the PBN/HER or DMPO/HER spin adducts, and validated by computer simulation. GSH, present at the beginning of the experiment, inhibited formation of the PBN/HER signal. However, GSH did not cause any decay of pre-formed PBN/HER spin adduct. GSH was consumed in the presence of the HER-generating system in a reaction largely reversed by addition of NADPH plus glutathione reductase. Ascorbate also inhibited formation of the PBN/HER spin adduct and rapidly reduced the pre-formed adduct. HER amplified the oxidation of ascorbate, which was associated with the formation of the semidehydroascorbyl radical. Alpha-tocopherol was also consumed in the presence of HER. Production of HER in intact HepG2 cells by the redox cycling of 2,3-dimethoxy-1,4-naphthoquinone was associated with consumption of GSH. These data demonstrate the use of a simple chemical system for the controlled, continuous formation of HER and indicate that cellular antioxidants such as GSH, ascorbate, and alpha-tocopherol, interact with HER. The ability of agents such as ascorbate to reduce the PBN/HER spin adduct to EPR-silent product(s) may mask the quantitative detection of HER in biological systems.

Thiol oxidation and cytochrome P450-dependent metabolism of CCl4 triggers Ca2+ release from liver microsomes.

Elevation of cytosolic calcium levels has been shown to occur after exposure to hepatotoxins such as CCl4. This has been associated with inhibition of the Ca2+, Mg(2+)-ATPase which pumps calcium into the endoplasmic reticulum. Elevated cytosolic Ca2+ may also result from activation of calcium releasing channels. In the presence of NADPH, CCl4 produced a concentration-dependent release of calcium from liver microsomes after a lag period. The lag period was shorter with microsomes from pyrazole-treated rats in which CYP2E1 is induced, as compared to saline microsomes. The calcium releasing process appears to be very sensitive to activation by CCl4 as effective concentrations (e.g., 50 microM) did not affect the Ca2+, Mg(2+)-ATPase or produce lipid peroxidation. Inhibition of the CCl4-induced release of calcium by 4-methylpyrazole and by anti-CYP2E1 IgG, and the requirement for NADPH, indicates that CCl4 metabolism is required for the activation of calcium release. The lag period for CCl4-induced release of calcium was associated with the time required to deplete alpha-tocopherol from the microsomal membranes; however, lipid peroxidation was not observed at these levels of CCl4, and the lag period for CCl4-induced release of calcium was shorter under anaerobic than aerobic conditions, suggesting a possible role for CCl3 in the mechanism of activation. Production of CCl3 was observed by ESR spin-trapping experiments with PBN; PBN prevented the CCl4-induced calcium release, presumably by interacting with CCl3 and other reactive species. Calcium release was produced by thiol oxidants such as 2,2'-dithiodipyridine. Lipophilic thiols such as mercaptoethanol or cysteamine could partially reverse the CCl4-induced calcium release, whereas GSH was ineffective. While the IP3 receptor system is considered as the main regulator of calcium release, liver also contains ryanodine-sensitive calcium releasing stores. The CCl4-induced calcium release was blocked by ruthenium red, a specific inhibitor of the ryanodine receptor; ruthenium red did not block CCl4 metabolism to CCl3. CCl4 increased the binding of ryanodine, a specific ligand for the ryanodine-sensitive calcium channel. These results suggest that metabolism of CCl4 to reactive species by cytochrome P450 results in an activation of a ryanodine-sensitive calcium channel, perhaps due to oxidation of lipophilic thiols of the channel. Activation of calcium releasing channels may play a role in the elevated cytosolic calcium levels found in the liver after treatment with hepatotoxins.

Ubiquinone-dependent recycling of vitamin E radicals by superoxide.

Superoxide-driven reduction of alpha-tocopheroxyl radical in the presence of ubiquinone-10 has been demonstrated in dimethylsulfoxide. Our HPLC measurements showed that ubiquinone-10 protected alpha-tocopherol against oxidation by KO2 in a concentration-dependent manner. alpha-Tocopherol was oxidized by KO2 to form ESR-detectable radicals of tocopherol ubisemiquinone. In the presence of ubiquinone-10, neither these radicals nor alpha-tocopheroxyl radicals (generated by uv light or PbO2) could be detected in ESR spectra. Instead, ESR signals of ubisemiquinone-10 radicals were observed. Formation of ubisemiquinone-10 radicals from ubiquinone-10 upon addition of KO2 was ascertained by their characteristic ESR and uv-vis spectra. alpha-Tocopherol caused a concentration-dependent decrease of the ubisemiquinone-10 radical steady-state concentration. We conclude that one-electron reduction of ubiquinone-10 by superoxide ion resulting in the formation of ubiquinone-10 radicals caused redox-cycling of alpha-tocopherol from its phenoxyl radical, thus preventing loss of alpha-tocopherol. This suggests that coenzyme Q may have another physiological function, i.e., protection of alpha-tocopherol against superoxide-driven oxidation.

Antioxidant paradoxes of phenolic compounds: peroxyl radical scavenger and lipid antioxidant, etoposide (VP-16), inhibits sarcoplasmic reticulum Ca(2+)-ATPase via thiol oxidation by its phenoxyl radical.

The effectiveness of a phenolic antioxidant as a radical scavenger is determined by its reactivity toward peroxyl radicals and also by the reactivity of the anti-oxidant phenoxyl radical toward oxidation substrate. If the phenoxyl radical efficiently interacts with vitally important biomolecules, this interaction may result in oxidative damage rather than antioxidant protection. In the present work, we studied effects of phenoxyl radicals generated from a phenolic antitumor drug, Etoposide (VP-16), on oxidation of thiols and activity of Ca(2+)-ATPase in sarcoplasmic reticulum (SR) membranes from skeletal muscles. We found that VP-16 is an effective scavenger of peroxyl radicals as judged by its ability to inhibit a water-soluble azo-initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced (i) chemiluminescence (oxidation) of luminol, (ii) fluorescence decay (oxidation) of cis-parinaric acid incorporated in SR membranes, and (iii) peroxidation of SR membrane lipids. VP-16 did not prevent AAPH-induced oxidation of sulfhydryl groups and inhibition of Ca(2+)-ATPase in SR membranes. Electron spin resonance measurements showed that AAPH-induced VP-16 phenoxyl radicals were reduced by interaction with SR thiols. By using tyrosinase to generate VP-16 phenoxyl radicals as the only source of free radicals in the model system, we found that inhibition of Ca(2+)-ATPase was accompanied by oxidation of about 5 mol of Ca(2+)-ATPase SH groups per 1 mol of oxidized VP-16. Secondary products of VP-16 oxidation (including VP-16 o-quinone) were not efficient in inhibiting SR Ca(2+)-ATPase. Reduction of VP-16 phenoxyl radicals by ascorbate protected against AAPH- and tyrosinase-induced thiol oxidation and Ca(2+)-ATPase inhibition. The results suggest that efficient phenolic scavengers of peroxyl radicals such as VP-16--which are commonly considered as potent antioxidants--may themselves produce oxidative stress due to secondary reactions of their phenoxyl radicals with thiols.

Reduction of phenoxyl radicals by thioredoxin results in selective oxidation of its SH-groups to disulfides. An antioxidant function of thioredoxin.

Thioredoxin is an important cellular redox buffer. In this report, we describe the reaction of thioredoxin with phenoxyl radicals. The vicinal sulfhydryls of the bis(cysteinyl) active site sequence reduced phenoxyl radicals released in horseradish peroxidase-catalyzed oxidation of phenol. Redox cycling of phenol was accompanied by selective oxidation of thioredoxin sulfhydryls to disulfides. HPLC/UV-vis measurements showed that the SH:phenol oxidation ratio was 15:1 under the conditions used. At the end of the reaction, oxidized thioredoxin was quantitatively recovered in the reduced form with dithiothreitol. Oxidation of sulfhydryls to sulfoxy derivatives, oxidation of other amino acid residues, and formation of covalent adducts with phenolic metabolites (quinones) were not detected by LC-MS. While the thiyl radical of glutathione was readily detected with the spin trap 5,5-dimethyl-1-pyrroline N-oxide, no ESR-detectable DMPO-thiyl adducts formed during the oxidation of thioredoxin. Similarly, oxidation of vicinal sulfhydryls of dihydrolipoic acid did not produce DMPO-thiyl spin adducts, indicating that fast intramolecular cyclization to disulfide occurred with thioredoxin. Measurements of the superoxide dismutase-sensitive chemiluminescence response of lucigenin demonstrated that thioredoxin oxidation was accompanied by release of superoxide, most likely via disulfide radical anion-mediated one-electron reduction of oxygen. We propose that formation of disulfides is characteristic of the phenoxyl radical-catalyzed oxidation of vicinal sulfhydryls in both small thiols and disulfide-forming oxidoreductases. Reversibility of the phenoxyl radical-catalyzed modification of thioredoxin may be responsible for its function as an efficient cytosolic antioxidant.

Endogenous ascorbate regenerates vitamin E in the retina directly and in combination with exogenous dihydrolipoic acid.

Vitamin E (alpha-tocopherol) is the major lipid-soluble antioxidant of retinal membranes whose deficiency causes retinal degeneration. Its antioxidant function is realized via scavenging peroxyl radicals as a result of which phenoxyl radicals of alpha-tocopherol are formed. Our hypothesis is that alpha-tocopherol phenoxyl radicals can be reduced by endogenous reductants in the retina, providing for alpha-tocopherol recycling. The results of this study demonstrate for the first time that: (i) endogenous ascorbate (vitamin C) in retinal homogenates and in rod outer segments is able to protect endogenous alpha-tocopherol against oxidation induced by UV-irradiation by reducing the phenoxyl radical of alpha-tocopherol, (ii) in the absence of ascorbate, neither endogenous nor exogenously added glutathione (GSH) is efficient in protecting alpha-tocopherol against oxidation; (iii) GSH does not substantially enhance the protective effect of ascorbate against alpha-tocopherol oxidation; (iv) exogenous dihydrolipoic acid (DHLA), although inefficient in direct reduction of the alpha-tocopherol phenoxyl radical, is able to enhance the protective effect of ascorbate by regenerating it from dehydroascorbate. Thus, regeneration of alpha-tocopherol from its phenoxyl radical can enhance its antioxidant effectiveness in the retina. The recycling of alpha-tocopherol opens new avenues for pharmacological approaches to enhance antioxidants of the retina.

Phenoxyl radical-induced thiol-dependent generation of reactive oxygen species: implications for benzene toxicity.

Mechanisms of phenoxyl radical-induced generation of oxygen radicals potentially involved in toxicity of benzene were studied. We hypothesized that phenoxyl radical intermediates formed from phenolic metabolites of benzene by oxidative enzymes (e.g., peroxidases, tyrosinase) are able to damage biomolecules via (i) oxidation of low-molecular-weight thiols and protein thiols and (ii) thiol-dependent generation of oxygen radicals and subsequent oxidation of DNA. Phenoxyl radicals were generated by the oxidation of phenol by myeloperoxidase+H2O2, horseradish peroxidase+H2O2, or tyrosinase. The reaction of phenolphenoxyl radicals with GSH and dihydrolipoic acid was studied. Our HPLC measurements showed that both thiols reduced the phenoxyl radical back to phenol. This reaction was accompanied by the formation of thiyl radicals (detected by ESR as 5,5-dimethyl-1-pyrroline-N-oxide/glutathione thiyl radical spin adducts) and of superoxide radicals (measured by their chemiluminescence response in the presence of lucigenin). Hydroxylation of 2'-deoxyguanosine to 8-oxo-7,8-dihydro-2'-deoxyguanosine was demonstrated in the course of the tyrosinase-catalyzed oxidation of phenol in the presence of dihydrolipoic acid and Fe(III)-EDTA. Redox-cycling of phenoxyl radicals by thiols produces oxygen radicals which can be responsible for the oxidative damage of DNA by radical intermediates of benzene metabolism.