Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.     

Redox-dependent modulation of aconitase activity in intact mitochondria

It has previously been reported that exposure of purified mitochondrial or cytoplasmic aconitase to superoxide (O(2)(-)(*) or hydrogen peroxide (H(2)O(2)) leads to release of the Fe-alpha from the enzyme's [4Fe-4S](2+) cluster and to inactivation. Nevertheless, little is known regarding the response of aconitase to pro-oxidants within intact mitochondria. In the present study, we provide evidence that aconitase is rapidly inactivated and subsequently reactivated when isolated cardiac mitochondria are treated with H(2)O(2). Reactivation of the enzyme is dependent on the presence of the enzyme's substrate, citrate. EPR spectroscopic analysis indicates that enzyme inactivation precedes release of the labile Fe-alpha from the enzyme's [4Fe-4S](2+) cluster. In addition, as judged by isoelectric focusing gel electrophoresis, the relative level of Fe-alpha release and cluster disassembly does not reflect the magnitude of enzyme inactivation. These observations suggest that some form of posttranslational modification of aconitase other than release of iron is responsible for enzyme inactivation. In support of this conclusion, H(2)O(2) does not exert its inhibitory effects by acting directly on the enzyme, rather inactivation appears to result from interaction(s) between aconitase and a mitochondrial membrane component responsive to H(2)O(2). Nevertheless, prolonged exposure of mitochondria to steady-state levels of H(2)O(2) or O(2)(-)(*) results in disassembly of the [4Fe-4S](2+) cluster, carbonylation, and protein degradation. Thus, depending on the pro-oxidant species, the level and duration of the oxidative stress, and the metabolic state of the mitochondria, aconitase may undergo reversible modulation in activity or progress to [4Fe-4S](2+) cluster disassembly and proteolytic degradation.     

Rotenone-like action of the branched-chain phytanic acid induces oxidative stress in mitochondria

Phytanic acid (Phyt) increase is associated with the hereditary neurodegenerative Refsum disease. To elucidate the still unclear toxicity of Phyt, mitochondria from brain and heart of adult rats were exposed to free Phyt. Phyt at low micromolar concentrations (maximally: 100 nmol/mg of protein) enhances superoxide (O(2)(.))(2) generation. Phyt induces O(2)(.) in state 3 (phosphorylating), as well as in state 4 (resting). Phyt stimulates O(2)(.) generation when the respiratory chain is fed with electrons derived from oxidation of glutamate/malate, pyruvate/malate, or succinate in the presence of rotenone. With succinate alone, Phyt suppresses O(2)(.) generation caused by reverse electron transport from succinate to complex I. The enhanced O(2)(.) generation by Phyt in state 4 is in contrast to the mild uncoupling concept. In this concept uncoupling by nonesterified fatty acids should abolish O(2)(.) generation. Stimulation of O(2)(.) generation by Phyt is paralleled by inhibition of the electron transport within the respiratory chain or electron leakage from the respiratory chain. The interference of Phyt with the electron transport was demonstrated by inhibition of state 3- and p-trifluoromethoxyphenylhydrazone (FCCP)-dependent respiration, inactivation of the NADH-ubiquinone oxidoreductase complex in permeabilized mitochondria, decrease in reduction of the synthetic electron acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in state 4, and increase of the mitochondrial NAD(P)H level in FCCP-uncoupled mitochondria. Thus, we suggest that complex I is the main site of Phyt-stimulated O(2)(.) generation. Furthermore, inactivation of aconitase and oxidation of the mitochondrial glutathione pool show that enhanced O(2)(.) generation with chronic exposure to Phyt causes oxidative damage.     

Production of reactive oxygen species by mitochondria: central role of complex III

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.     

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.     

Accumulation of long-chain acylcarnitine and 3-hydroxy acylcarnitine molecular species in diabetic myocardium: identification of alterations in mitochondrial fatty acid processing in diabetic myocardium by shotgun lipidomics

Diabetic cardiomyopathy is the result of maladaptive changes in energy homeostasis. However, the biochemical mechanisms underlying dysfunctional lipid metabolism in diabetic myocardium are incompletely understood. Herein, we exploit shotgun lipidomics to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which was reversible upon insulin treatment. Analysis of acylcarnitine molecular species in myocardium unexpectedly identified acylcarnitine molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species. Synthesis of 3-hydroxy acylcarnitine identified the natural products as the 3-hydroxylated acylcarnitines through comparisons of diagnostic fragmentation patterns of synthetic and naturally occurring constituents using tandem mass spectrometry. Diabetes induced an increase of both calcium-independent phospholipase A(2) (iPLA(2)) mRNA and iPLA(2) activity in rat myocardium. Cardiac ischemia in myocardium genetically engineered to overexpress iPLA(2) dramatically increased the amount of acylcarnitine present in myocardium. Moreover, mechanism-based inactivation of iPLA(2) in either wild-type or transgenic myocardium ablated a substantial portion of the acylcarnitine increase. Collectively, these results identify discrete insulin remediable abnormalities in mitochondrial fatty acid processing in diabetic myocardium and identify iPLA(2) as an important enzymatic contributor to the pool of fatty acids that can be used for acylcarnitine synthesis and energy production in myocardium.     

Mitochondrial fatty acid oxidation and oxidative stress: Lack of reverse electron transfer-associated production of reactive oxygen species

Reverse electron transfer (RET) from succinate to NAD⁺ is known to be accompanied by high generation of reactive oxygen species (ROS). In contrast, oxidation of fatty acids by mitochondria, despite being a powerful source of FADH₂, does not lead to RET-associated high ROS generation. Here we show that oxidation of carnitine esters of medium- and long-chain fatty acids by rat heart mitochondria is accompanied by neither high level of NADH/NAD⁺ nor intramitochondrial reduction of acetoacetate to β-hydroxybutyrate, comparable to those accompanying succinate oxidation, although it produces the same or higher energization of mitochondria as evidenced by high transmembrane potential. Also in contrast to the oxidation of succinate, where conversion of the pH difference between the mitochondrial matrix and the medium into the transmembrane electric potential by addition of nigericin results in a decrease of ROS generation, the same treatment during oxidation of octanoylcarnitine produces a large increase of ROS. Analysis of respiratory chain complexes by Blue Native polyacrylamide gel electrophoresis revealed bands that could tentatively point to supercomplex formation between complexes II and I and complexes II and III. However, no such association could be found between complex I and the electron transferring flavoprotein that participates in fatty acid oxidation. It is speculated that structural association between respective respiratory chain components may facilitate effective reverse electron transfer.     

Redox regulation of endogenous substrate oxidation by cardiac mitochondria

Reactive oxygen species (ROS) play important roles in regulating mitochondrial function, as well as in ischemia-reperfusion injury and cardioprotection. Here we show that, in the absence of exogenous substrates, cardiac mitochondria have a surprisingly large capacity to phosphorylate ADP by oxidizing endogenous substrates, provided that H2O2 is removed from the extramitochondrial environment and a reduced environment is maintained in the matrix. In isolated mitochondria without exogenous substrates, addition of catalase and the membrane-permeant reducing agent N-acetylcysteine (Nac) or the ROS scavenger mercaptopropionyl glycine significantly increased the ability to phosphorylate added ADP, as demonstrated by 1) full recovery of membrane potential (Deltapsi) and matrix volume from ADP-induced dissipation and shrinkage, 2) ADP-dependent increase in O2 consumption, and 3) enhanced rate of ATP synthesis. Removal of extramitochondrial H2O2 by catalase was required to stimulate endogenous substrate oxidation, as shown by the increase in O2 consumption and Deltapsi. This effect was greatly enhanced by addition of Nac or mercaptopropionyl glycine to suppress oxidation-induced ROS increases in the matrix. Theoretical considerations, as well as reversible inhibition of O2 consumption with 3-mercaptopropionic acid and pyruvate in state 3, indicate that these substrates are fatty acids. Under in vivo conditions in which powerful antioxidant conditions are maintained, this mechanism may be important in stimulation of beta-oxidation and ATP production at low levels of extramitochondrial fatty acids. Incapacitation of this mechanism may potentially contribute to mitochondrial dysfunction during oxidative stress.     

Comparative biochemistry of beta-oxidation. An investigation into the abilities of isolated heart mitochondria of various animal species to oxidize long-chain fatty acids, including the C22:1 monoenes.

Rates of acylcarnitine oxidation by isolated heart mitochondria from various animal species were measured polarographically, and by using a spectrophotometric assay [see Osmundsen & Bremer (1977) Biochem. J. 164, 621-633]. Polarographic measurements do not give a correct guide to abilities to beta-oxidize very-long-chain acylcarnitines, in particular C22:1 fatty acylcarnitines. 2. No significant species differences were detected in the abilities to beta-oxidize various C22:1 fatty acylcarnitines. Significant species differences were, however, detected when rates of beta-oxidation were correlated with rates of respiration brought about by very-long-chain acylcarnitines. We concluded that some aspects of oxidative metabolism (possibly the oxidation of tricarboxylic acid-cycle intermediates) are inhibited by very-long-chain fatty acids in some species (e.g. the rat and the cat but not in others (e.g. the pig and the rabbit). 3. It is proposed that the pattern of variation of rates of oxidation of various acylcarnitines (as measured spectrophotometrically) of various chain lengths can be used as a guide to the chain-length specificities of the acyl-CoA dehydrogenases of beta-oxidation (EC 1.3.99.3).     

Sensitized photooxygenation and peroxidase-catalyzed inactivation of xanthine oxidase--evidence of cysteine damage by singlet oxygen

Xanthine oxidase (XO) has been investigated for its decreased activity in several cancerous tissues and constitutive generation of reactive oxygen species (ROS) in vivo seems to contribute significantly to its inactivation. Singlet oxygen (1O2) production has been suggested to be relevant when considering folic acid metabolism by cancer cells. Thus, the susceptibility of XO to inactivation by 1O2 generated either by the bioenergized systems folic acid/peroxidase/GSH/Mn2+/O2 and malonaldehyde/peroxidase/Mn2+/O2 or by methylene blue (MB) or eosin-sensitized photooxygenation was studied. Our results showed that other ROS were also responsible for XO inactivation when MB was used. In contrast, eosin produced almost exclusively 1O2. Kinetic studies of XO oxidation in the malonaldehyde/peroxidase system showed that histidine (His) is a competitive inhibitor with respect to XO. A similar result was observed in the eosin-photosensitized process, suggesting the involvement of 1O2 in both processes. In addition, an efficient quenching of XO oxidation by guanosine in the folic acid/peroxidase system was observed. Amino acid analysis revealed that cysteine (Cys) is more affected than other XO amino acids also prone to oxidation such as tyrosine (Tyr), methionine (Met) and His. These results indicate that 1O2 may cause oxidative damage to the Cys residues of XO, with loss of enzyme activity. Alteration of the flavin prosthetic site is hypothesized.     

Double mode of inhibition-inducing interactions of 1,4-naphthoquinone with urease: arylation versus oxidation of enzyme thiols

In their inhibition-inducing interactions with enzymes, quinones primarily utilize two mechanisms, arylation and oxidation of enzyme thiol groups. In this work, we investigated the interactions of 1,4-naphthoquinone with urease in an effort to estimate the contribution of the two mechanisms in the enzyme inhibition. Jack bean urease, a homohexamer, contains 15 thiols per enzyme subunit, six accessible under non-denaturing conditions, of which Cys592 proximal to the active site indirectly participates in the enzyme catalysis. Unlike by 1,4-benzoquinone, a thiol arylator, the inactivation of urease by 1,4-naphthoquinone under aerobic conditions was found to be biphasic, time- and concentration-dependent with a non-linear residual activity-modified thiols dependence. DTT protection studies and thiol titration with DTNB suggest that thiols are the sites of enzyme interactions with the quinone. The inactivated enzyme had approximately 40% of its activity restored by excess DTT supporting the presence of sulfenic acid resulting from the oxidation of enzyme thiols by ROS. Furthermore, the aerobic inactivation was prevented in approximately 30% by catalase, proving the involvement of hydrogen peroxide in the process. When H2O2 was directly applied to urease, the enzyme showed susceptibility to this inactivation in a time- and concentration-dependent manner with the inhibition constant of H2O2 Ki = 3.24 mM. Additionally, anaerobic inactivation of urease was performed and was found to be weaker than aerobic. The results obtained are consistent with a double mode of 1,4-naphthoquinone inhibitory action on urease, namely through the arylation of the enzyme thiol groups and ROS generation, notably H2O2, resulting in the oxidation of the groups.     

Long-chain acylcarnitine content determines the pattern of energy metabolism in cardiac mitochondria

In the heart, a nutritional state (fed or fasted) is characterized by a unique energy metabolism pattern determined by the availability of substrates. Increased availability of acylcarnitines has been associated with decreased glucose utilization; however, the effects of long-chain acylcarnitines on glucose metabolism have not been previously studied. We tested how changes in long-chain acylcarnitine content regulate the metabolism of glucose and long-chain fatty acids in cardiac mitochondria in fed and fasted states. We examined the concentrations of metabolic intermediates in plasma and cardiac tissues under fed and fasted states. The effects of substrate availability and their competition for energy production at the mitochondrial level were studied in isolated rat cardiac mitochondria. The availability of long-chain acylcarnitines in plasma reflected their content in cardiac tissue in the fed and fasted states, and acylcarnitine content in the heart was fivefold higher in fasted state compared to the fed state. In substrate competition experiments, pyruvate and fatty acid metabolites effectively competed for the energy production pathway; however, only the physiological content of acylcarnitine significantly reduced pyruvate and lactate oxidation in mitochondria. The increased availability of long-chain acylcarnitine significantly reduced glucose utilization in isolated rat heart model and in vivo. Our results demonstrate that changes in long-chain acylcarnitine contents could orchestrate the interplay between the metabolism of pyruvate–lactate and long-chain fatty acids, and thus determine the pattern of energy metabolism in cardiac mitochondria.     

Activation of aconitase in mouse fast-twitch skeletal muscle during contraction-mediated oxidative stress

Aconitase is a mitochondrial enzyme that converts citrate to isocitrate in the tricarboxylic acid cycle and is inactivated by reactive oxygen species (ROS). We investigated the effect of exercise/contraction, which is associated with elevated ROS production, on aconitase activity in skeletal muscle. Humans cycled at 75% of maximal workload, followed by six 60-s bouts at 125% of maximum workload. Biopsies were taken from the thigh muscle at rest and after the submaximal and supramaximal workloads. Isolated mouse extensor digitorum longus (EDL; fast twitch) and soleus (slow twitch) muscles were stimulated to perform repeated contractions for 10 min. Muscles were analyzed for enzyme activities and glutathione status. Exercise did not affect aconitase activity in human muscle despite increased oxidative stress, as judged by elevated levels of oxidized glutathione. Similarly, repeated contractions did not alter aconitase activity in soleus muscle. In contrast, repeated contractions significantly increased aconitase activity in EDL muscle by 50%, despite increased ROS production. This increase was not associated with a change in the amount of immunoreactive aconitase (Western blot) but was markedly inhibited by cyclosporin A, an inhibitor of the protein phosphatase calcineurin. Immunoprecipitation experiments demonstrated that aconitase was phosphorylated on serine residues. Aconitase in cell-free extracts was inactivated by the addition of the ROS hydrogen peroxide. In conclusion, the results suggest that aconitase activity can be regulated by at least two mechanisms: oxidation/reduction and phosphorylation/dephosphorylation. During contraction, a ROS-mediated inactivation of aconitase can be overcome, possibly by dephosphorylation of the enzyme. The dual-control system may be important in maintaining aerobic ATP production during muscle contraction. glutathione; reactive oxygen species     

Fatty acids as modulators of the cellular production of reactive oxygen species

Long-chain nonesterified ("free") fatty acids (FFA) and some of their derivatives and metabolites can modify intracellular production of reactive oxygen species (ROS), in particular O(2)(-) and H(2)O(2). In mitochondria, FFA exert a dual effect on ROS production. Because of slowing down the rate of electron flow through Complexes I and III of the respiratory chain due to interaction within the complex subunit structure, and between Complexes III and IV due to release of cytochrome c from the inner membrane, FFA increase the rate of ROS generation in the forward mode of electron transport. On the other hand, due to their protonophoric action on the inner mitochondrial membrane ("mild uncoupling effect"), FFA strongly decrease ROS generation in the reverse mode of electron transport. In the plasma membrane of phagocytic neutrophils and a number of other types of cells, polyunsaturated FFA stimulate O(2)(-) generation by NADPH oxidase. These effects of FFA can modulate signaling functions of ROS and be, at least partly, responsible for their proapoptotic effects in several types of cells.     

Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase 11 and hydrogen peroxide

Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis. Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H2O2 directly in the lungs. We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H2O2, as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite. The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore. Oxidation of pyocyanin was observed also when exogenous H2O2 was replaced by a H2O2-generating system consisting of NADH and the pigment itself. That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H2O2. In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation. This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H2O2 removal. This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest.     

Molecular mechanisms of 6-hydroxydopamine-induced cytotoxicity in PC12 cells: involvement of hydrogen peroxide-dependent and -independent action

The neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to generate an experimental model of Parkinson's disease. It has been reported that reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide (H2O2), generated from 6-OHDA are involved in its cytotoxicity; however, the contribution and role of ROS in 6-OHDA-induced cell death have not been fully elucidated. In the present study using PC12 cells, we observed the generation of 50 microM H2O2 from a lethal concentration of 100 microM 6-OHDA within a few minutes, and compared the sole effect of H2O2 with 6-OHDA. Catalase, an H2O2-removing enzyme, completely abolished the cytotoxic effect of H2O2, while a significant but partial protective effect was observed against 6-OHDA. 6-OHDA induced peroxiredoxin oxidation, cytochrome c release, and caspase-3 activation. Catalase exhibited a strong inhibitory effect against the peroxiredoxin oxidation, and cytochrome c release induced by 6-OHDA; however, caspase-3 activation was not effectively inhibited by catalase. On the other hand, 6-OHDA-induced caspase-3 activation was inhibited in the presence of caspase-8, caspase-9, and calpain inhibitors. These results suggest that the H2O2 generated from 6-OHDA plays a pivotal role in 6-OHDA-induced peroxiredoxin oxidation, and cytochrome c release, while H2O2- and cytochrome c-independent caspase activation pathways are involved in 6-OHDA-induced neurotoxicity. These findings may contribute to explain the importance of generated H2O2 and secondary products as a second messenger of 6-OHDA-induced cell death signal linked to Parkinson's disease.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

Inactivation of alcohol dehydrogenase in rice seedlings is related to a microsomal fatty acid alpha-oxidation system

The selective inactivation of alcohol dehydrogenase by the inactivator found in the microsomal fraction of rice (Oryza sativa) seedlings growing in air (Shimomura, S. & Beevers, H. (1983) Plant Physiol. 71, 736-741; 742-746) was further studied. This inactivation was found to be essentially dependent on the presence of free fatty acids. The specificity for fatty acids and the inhibitory effects of imidazole, 2-hydroxyfatty acids and dithiothreitol on the inactivation were all consistent with the properties of the fatty acid alpha-oxidation system in plants. Both O2 consumption and decarboxylation of fatty acid due to alpha-oxidation were also demonstrated in rice microsomes. When purified rice alcohol dehydrogenase was added to the alpha-oxidation system in rice microsomes, the decarboxylation of fatty acid was inhibited, and the cysteinyl residues of alcohol dehydrogenase were oxidized. The oxidation of two cysteinyl residues per monomer resulted in the complete inactivation of the enzyme. The activity of the inactivator in the isolated microsomes was gradually lost during storage and was rapidly lost upon heating. The inactivation of alcohol dehydrogenase was observed even when the enzyme was separated from microsomes by a dialysis membrane. These results indicate that the inactivation of alcohol dehydrogenase is closely related to fatty acid alpha-oxidation. We postulate that an intermediate of alpha-oxidation is the inactivator.     

Stanozolol treatment decreases the mitochondrial ROS generation and oxidative stress induced by acute exercise in rat skeletal muscle

Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.