Volume 383, Issue 3-4

Present knowledge on peroxiredoxins is reviewed with special emphasis on catalytic principles, specificities and biological function. Peroxiredoxins are low efficiency peroxidases using thiols as reductants. They appear to be fairly promiscuous with respect to the hydroperoxide substrate; the specificities for the donor substrate vary considerably between the subfamilies, comprising GSH, thioredoxin, tryparedoxin and the analogous CXXC motifs in bacterial AhpF proteins. Peroxiredoxins are definitely responsible for antioxidant defense in bacteria (AhpC), yeast (thioredoxin peroxidase) and trypanosomatids (tryparedoxin peroxidase). They are considered to determine virulence of mycobacteria and trypanosomatids. In higher plants they are involved in balancing hydroperoxide production during photosynthesis. In higher animals peroxiredoxins appear to be involved in the redoxregulation of cellular signaling and differentiation, displaying in part opposite effects.

Human and rabbit reticulocyte 15-lipoxygenase (15-lipoxygenase-1) and the leukocytetype 12-lipoxygenases (12/15-lipoxygenases) of pig, beef, mouse and rat constitute a particular subfamily of mammalian lipoxygenases (reticulocytetype lipoxygenases) with unique properties and functions. They catalyze enzymatic lipid peroxidation in complex biological structures via direct dioxygenation of phospholipids and cholesterol esters of biomembranes and plasma lipoproteins. Moreover, they are a source of free radicals initiating nonenzymatic lipid peroxidation and other oxidative processes. Expression and activity of reticulocytetype lipoxygenases are highly regulated. Moreover, the susceptibility of intracellular membranes toward these lipoxygenases is controlled and may be increased together with lipoxygenase activity under conditions of oxidative stress. Thus, oxidative stress may favor a concerted package of lipoxygenase mediated enzymatic and nonenzymatic lipid peroxidation and cooxidative processes. Reaction of reticulocytetype lipoxygenases with lowdensity lipoprotein renders the latter atherogenic and appears to be involved in the formation of atherosclerotic lesions.

The ingredients of oxidative stress include a variety of reactive species such as reactive oxygen and reactive nitrogen species (ROS, RNS). While sulfur is usually considered as part of cellular antioxidant systems there is mounting evidence that reactive sulfur species (RSS) with stressor properties similar to the ones found in ROS are formed under conditions of oxidative stress. Thiols as well as disulfides are easily oxidised to sulfur species with sulfur in higher oxidation states. Such agents include thiyl radicals, disulfides, sulfenic acids and disulfideSoxides. They rapidly oxidise and subsequently inhibit thiolproteins and enzymes and can be considered as a separate class of oxidative stressors providing new antioxidant drug targets.

Nitrogen dioxide (NO2) is an oxidizing free radical which can initiate a variety of destructive pathways in living systems, and several diseases are suspected to be connected with both exogenously and endogenously formed NO2. Peroxynitrite (ONOO/ONOOH) is believed to be an important endogenous source of NO2 radicals, but other sources, among them enzymatically ones, have been identified recently. It also became clear during the last few years that in vivo formation of 3-nitrotyrosine strictly depends on the availability of NO2 radicals. Since nitrogen dioxide is a very toxic compound an arsenal of antioxidants (e.g. vitamin C, glutathione, vitamin E, and βcarotene) must eliminate this harmful radical in vivo. Here the recently identified superoxide (O2)dependent formation of peroxynitrate (O2NOO) and the central role of vitamin C are of special importance.

Nitric oxide (NO) and peroxynitrite (ONOO) avidly interact with mitochondrial components, leading to a range of biological responses spanning from the modulation of mitochondrial respiration, mitochondrial dysfunction to the signaling of apoptotic cell death. Physiological levels of NO primarily interact with cytochrome c oxidase, leading to a competitive and reversible inhibition of mitochondrial oxygen uptake. In turn, this leads to alterations in electrochemical gradients, which affect calcium uptake and may regulate processes such as mitochondrial transition pore (MTP) opening and the release of proapoptotic proteins. Large or persistent levels of NO in mitochondria promote mitochondrial oxidant formation. Peroxynitrite formed either extra or intramitochondrially leads to oxidative damage, most notably at complexes I and II of the electron transport chain, ATPase, aconitase and Mnsuperoxide dismutase. Mitochondrial scavenging systems for peroxynitrite and peroxynitritederived radicals such as carbonate (CO3.) and nitrogen dioxide radicals (NO2) include cytochrome c oxidase, glutathione and ubiquinol and serve to partially attenuate the reactions of these oxidants with critical mitochondrial targets. Detection of nitrated mitochondrial proteins in vivo supports the concept that mitochondria constitute central loci of the toxic effects of excess reactive nitrogen species. In this review we will provide an overview of the biochemical mechanisms by which NO and ONOO regulate or alter mitochondrial functions.

Nitric oxide, generated by endogenous nitric oxide synthases or nitric oxide donors, can promote or prevent apoptosis induced by diverse proapoptotic stimuli in cell culture models. Both mitochondrialdependent and independent apoptotic signaling pathways mediate this dichotomous cellular response to nitric oxide. The molecular mechanisms behind these effects are complex and involve a number of nitrogen oxiderelated species that are more reactive than nitric oxide itself. The local cellular environment plays a dynamic role in determining the nature and concentration of these species. Important components of the microenvironment include: the cellular redox state, glutathione, transition metals and the presence of other oxygen and nitrogencentered radicals. In particular, redoxsensitive nitrosating species are favorably generated under physiological conditions and capable of modifying multiple cell signaling pathways through reversible Snitrosation reactions. Cytochrome c release from mitochondria is an important mechanism for the activation of caspase-3 and the initiation of cell death in response to intrinsic proapoptotic stimuli, including oxidative and nitrosative stress. In turn, caspases and mitogen associated protein kinases may modulate cytochrome c release through their effects on the Bcl-2 family of proteins. This review will focus on (i) the importance of the cellular environment in determining the fate of nitric oxide and (ii) the ability of Snitrosation to regulate mitochondrialdependent apoptosis at the level of mitochondrial bioenergetics, cytochrome c release, caspases, mitogen associated protein kinases, and the Bcl-2 family of proteins.

It is well established that fatty acid metabolites of cyclooxygenase, lipoxygenase (LOX), and cytochrome P450 are implicated in essential aspects of cellular signaling including the induction of programmed cell death. Here we review the roles of enzymatic and nonenzymatic products of polyunsaturated fatty acids in controlling cell growth and apoptosis. Also, the spontaneous oxidation of polyunsaturated fatty acids yields reactive aldehydes and other products of lipid peroxidation that are potentially toxic to cells and may also signal apoptosis. Significant conflicting data in terms of the role of LOX enzymes are highlighted, prompting a reevaluation of the relationship between LOX and prostate cancer cell survival. We include new data showing that LNCaP, PC3, and Du145 cells express much lower levels of 5-LOX mRNA and protein compared with normal prostate epithelial cells (NHP2) and primary prostate carcinoma cells (TP1). Although the 5-LOX activating protein inhibitor MK886 killed these cells, another 5-LOX inhibitor AA861 hardly showed any effect. These observations suggest that 5-LOX is unlikely to be a prostate cancer cell survival factor, implying that the mechanisms by which LOX inhibitors induce apoptosis are more complex than expected. This review also suggests several mechanisms involving peroxisome proliferator activated receptor activation, BCL proteins, thiol regulation, and mitochondrial and kinase signaling by which cell death may be produced in response to changes in nonesterified and nonprotein bound fatty acid levels. Overall, this review provides a context within which the effects of fatty acids and fatty acid oxidation products on signal transduction pathways, particularly those involved in apoptosis, can be considered in terms of their overall importance relative to the much better studied protein or peptide signaling factors.

Following the requirement for cells to cope with oxidative stress, there are cellular adaptation mechanisms at the level of gene expression. Much of what is known about oxidantinduced signaling in mammalian cells was found in experiments using hydrogen peroxide as an oxidant. However, since the biochemical reactivities of various oxidants significantly differ, oxidative stress is not necessarily identical independent of the oxidant employed to bring it about. Here, the biological actions of peroxynitrite and singlet oxygen are presented, focusing on signaling effects. Peroxynitrite is generated in biological systems in the diffusioncontrolled reaction of superoxide with nitrogen monoxide and is thus likely to be produced in the vicinity of activated macrophages. Singlet oxygen is generated by stimulated neutrophils in vivo and may further be generated photochemically, e.g. upon exposure of cells to ultraviolet A radiation. Exposure of cells to either of these oxidants elicits a cellular stress response, entailing the activation of signaling cascades that regulate proliferative and apoptotic responses, such as mitogenactivated protein kinase cascades or the phosphoinositide 3-kinase/Akt cascade. Two mechanisms for the oxidantinduced activation of a signaling cascade may be envisaged: (i) the indirect targeting of the cascade by interrupting negative regulation, and (ii) an activating oxidation of one of the constituting components of the cascade. Examples for both mechanisms in relation to peroxynitrite and singlet oxygen are discussed.

Molecules provided with an antioxidant function may have additional properties, the latter being sometimes of greater importance than the former. In the last ten years, αtocopherol has revealed precise cellular functions, some of which are independent of its antioxidant/radical scavenging ability. At the posttranslational level, αtocopherol inhibits protein kinase C and 5-lipoxygenase and activates protein phosphatase 2A and diacylglycerol kinase. Some genes (CD36, αTTP, αtropomyosin, and collagenase) are affected by αtocopherol at the transcriptional level. αTocopherol also induces inhibition of cell proliferation, platelet aggregation and monocyte adhesion. These effects are unrelated to the antioxidant activity of vitamin E, but rather are believed to be a result of specific interactions of vitamin E with components of the cell, e. g. proteins, enzymes and membranes. This review focuses on novel nonantioxidant functions of αtocopherol and discusses the possibility that many of the effects previously attributed to the antioxidant functions can also be explained by nonantioxidant mechanisms.

Basal steadystate levels of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2deoxyguanosine (8-oxoG) are observed in all types of cells, most probably due to a continuous generation of reactive oxygen species (ROS) in the cellular oxygen metabolism, and it has long been suspected that they might play an important role in the initiation of carcinogenesis. Experimental evidence for this assumption can be obtained by studying the effects of a modulation the steadystate levels, either by in or decreasing the generation of oxidative DNA damage, on spontaneous mutation rates and cancer incidence. However, clear answers have not yet been obtained by these strategies. It is still doubtful whether an efficient reduction of the in vivo steadystate levels can achieved by application of antioxidants, and effects observed under oxidative stress conditions (i.e. increased oxidative DNA damage) are inconclusive due to the pronounced epigenetic effects of ROS on signal transduction and gene expression (tumor promotion). In addition, the reliable quantification of the basal levels of oxidative DNA modifications is still major problem. Recently, the generation of mice deficient in the repair 8-oxoG (ogg1/ mice) has opened the door for an alternative approach. Results obtained so far indicate that an increase by less than five 8-oxoG residues per 106 bp in the liver of the knockout animals is associated with a two to threefold higher spontaneous mutation frequency in transgenic genes. However, the increase in the ogg1/ mice of the steadystate level of 8-oxoG and the spontaneous mutation frequency was only observed in the liver and apparently too small to enhance the spontaneous cancer incidence significantly. The limited effect seems to be due to a backup repair system for 8-oxoG in the ogg1/ mice, and it can be expected that the inactivation of this pathway doubleknockout mice will lead to higher effects and a better assessment of the risk associated with endogenous oxidative DNA damage.

Hypothermia is a wellknown strategem to protect biological material against injurious or degradative processes and is widely used in experimental and especially in clinical applications. However, hypothermia has also proved to be strongly injurious to a variety of cell types. Hypothermic injury to mammalian cells has long been attributed predominantly to disturbances of cellular ion homeostasis, especially of sodium homeostasis. For many years, reactive oxygen species have hardly been considered in the pathogenesis of hypothermic injury to mammalian cells. In recent years, however, increasing evidence for a role of reactive oxygen species in hypothermic injury to these cells has accumulated. Today there seems to be little doubt that reactive oxygen species decisively contribute to hypothermic injury in diverse mammalian cells. In some cell types, such as liver and kidney cells, they even appear to play the central role in hypothermic injury, outruling by far a contribution of the cellular ion homeostasis. In these cells, the cellular chelatable, redoxactive iron pool appears to be decisively involved in the pathogenesis of hypothermic injury and of coldinduced apoptosis that occurs upon rewarming of the cells after a (sublethal) period of cold incubation.

A very small, predominantly cytosolic pool of iron ions plays the central role in the cellular iron metabolism. It links the cellular iron uptake with the insertion of the metal in iron storage proteins and other essential ironcontaining molecules. Furthermore, this transit (labile) pool is essentially involved in the pathogenesis of a number of diseases. Due to its high physiological and pathophysiological significance, numerous methods for its characterization have been developed during the last five decades. Most of these methods, however, influence the size and nature of the transit iron pool artificially, as they are not applicable to viable biological material. Recently, fluorescence spectroscopic methods for measurements within viable cells have become available. Although these methods avoid the artifacts of previous methods, studies using fluorescent iron indicators revealed that the intracellular transit iron pool, which is methodically assessed as chelatable iron, is substantially defined by the method and/or the ironchelating indicator applied for its detection, since the iron ions are bound to a large number of different ligands in different metabolic compartments. A more comprehensive characterization of the nature and the role of the thus not uniform cellular transit iron pool therefore requires parallel employment of different indicator molecules, which clearly differ in their intracellular distribution and their physicochemical characteristics.

There is an increasing awareness of the role of certain nutritional components, including dietary flavonoids found in fruit, vegetables and beverages, in the maintenance of health and prevention of chronic diseases. In this regard, recent studies highlight an exciting role with respect to their potential neuroprotective actions, in particular towards deficits commonly observed with aging, such as reduced performance of cognitive, memory and learning tasks. These neurological functions, and possible mechanisms involved in controlling them, can be influenced by supplementation of single dietary flavonoids, or as part of a flavonoidrich preparation. With this, a renewed emphasis is aimed at further understanding their modes and sites of action. Moreover a common theme among many in vitro studies examining mechanisms of neuroprotection is the failure to include biologically relevant metabolites of the flavonoids known to enter the circulation, and thus most likely to be bioavailable to cells and tissues. This oversight will ultimately influence the mechanisms of action proposed to explain the neuroprotection observed in animals and human studies. As such, emerging findings suggest a variety of potential mechanisms of action of flavonoids and their bioavailable metabolites in cytoprotection against oxidative stress, which may be independent of conventional antioxidant reducing activities. Such mechanisms might involve their interaction with cell signalling cascades, their influence on gene expression and the down regulation of pathways leading to cell death.

Many neurodegenerative disorders and syndromes are associated with an excessive generation of reactive oxygen species (ROS) and oxidative stress. The pathways to nerve cell death induced by diverse potential neurotoxins such as peptides, excitatory amino acids, cytokines or synthetic drugs commonly share oxidative downstream processes, which can cause either an acute oxidative destruction or activate secondary events leading to apoptosis. The pathophysiological role of ROS has been intensively studied in in vitro and in vivo models of chronic neurodegenerative diseases such as Alzheimers disease (AD) and of syndromes associated with rapid nerve cell loss as occuring in stroke. In AD, oxidative neuronal cell dysfunction and cell death caused by protofibrils and aggregates of the ADassociated amyloid β protein (Aβ) may causally contribute to pathogenesis and progression. ROS and reactive nitrogen species also take part in the complex cascade of events and the detrimental effects occuring during ischemia and reperfusion in stroke. Direct antioxidants such as chainbreaking free radical scavengers can prevent oxidative nerve cell death. Although there is ample experimental evidence demonstrating neuroprotective activities of direct antioxidants in vitro, the clinical evidence for antioxidant compounds to act as protective drugs is relatively scarce. Here, the neuroprotective potential of antioxidant phenolic structures including αtocopherol (vitamin E) and 17βestradiol (estrogen) in vitro is summarized. In addition, the antioxidant and cytoprotective activities of lipophilic tyrosine and tryptophancontaining structures are discussed. Finally, an outlook is given on the neuroprotective potential of aromatic amines and imines, which may comprise novel lead structures for antioxidant drug design.

The photoreceptors in the retina, designed to initiate the cascade of events which link the incoming light to the sensation of vision, are susceptible to damage by light, particularly blue light. The damage can lead to cell death and diseases. The turnover of retinal, an essential element of the visual process, is the basis of the events that lead to damage. Free retinal, absorbing in the blue region of the visible spectrum, is phototoxic, and is a precursor of the (photo)toxic compound A2E, which specifically targets cytochrome oxidase and thereby induces cell death by apoptosis. Cell death induced by A2E in the dark is prevented by negatively charged phospholipids. The blue lightfiltering molecules lutein and zeaxanthin are tailormade substances protecting the retina. In vitro, they protect cytochrome oxidase against the permanent damage caused by A2E in combination with light. These novel findings should enable us to prevent or cure the dry form of agerelated macular degeneration, the leading cause of severe visual impairment in humans living in developed countries.

Nitric oxide (NO) is a free radical species that diffuses and concentrates in the hydrophobic core of lowdensity lipoprotein (LDL) to serve as a potent inhibitor of lipid oxidation processes. Peroxynitrite (PN), the product of the diffusionlimited reaction between NO and superoxide (O2), represents a relevant mediator of oxidative modifications in LDL. The focus of this review is the analysis of interactions between NO and PN and its secondary reactions with oxygen radicals on LDL oxidation, which are relevant in the development of the early steps as well as progression of atherosclerosis. We propose that the balance between rates of PN and NO production, which greatly depends on oxidative stress processes within the vascular wall, will critically determine the final extent of oxidative LDL modifications leading or not to scavenger receptormediated LDL uptake and foam cell formation.

Dietary antioxidants such as carotenoids, tocopherols, vitamin C or flavonoids exhibit biological activities that are not directly related to their antioxidant properties. The parent compounds and/or their metabolites have impact on cellular signaling pathways, influence the expression of certain genes or act as inhibitors of regulatory enzymes. Thus, they reveal additional biological effects which might be of importance in context with the prevention of degenerative diseases related to the consumption of a diet rich in antioxidants. This review focuses on known nonantioxidant properties of carotenoids, including retinoiddependent signaling, stimulation of gap junctional communications, impact on the regulation of cell growth and induction of detoxifying enzymes, such as cytochrome P450-dependent monooxygenases.

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. The production of reactive oxygen and nitric oxide species causes oxidative modifications of proteins often combined with a loss of their biological function. Like most partially denatured proteins, moderately oxidized proteins are more sensitive to proteolytic attack by proteases. The diverse cellular proteolytic systems are an important secondary defense against oxidative stress by degrading oxidized and damaged proteins, thereby preventing their intracellular accumulation. In mammalian cells, a range of proteases exists which are distributed throughout the cell. In this review we summarize the function of the cytosolic (proteasome and calpains), the lysosomal, the mitochondrial and the nuclear proteolytic pathways in response to oxidative stress. Particular emphasis is given to the proteasomal system, since this pathway appears to be the most important proteolytic system involved in the removal of oxidatively modified or damaged proteins.

The morphogenetic transitions of the N. crassa asexual life cycle are responses to a hyperoxidant state in which probably singlet oxygen is generated. Induction of catalase activity and catalase oxidation by singlet oxygen are consequences of this recurrent hyperoxidant state. Here the biochemical properties and regulation of two large monofunctional catalases are reviewed, and a new catalaseperoxidase gene and activity is described. Catalase-3 is associated to growing and Catalase-1 to nongrowing cells. Under stressful conditions one of these catalases is synthesized, depending on whether growth can be continued or a resistant cell has to be made. The catalaseperoxidase Catalase-2 was possibly derived from a bacterial enzyme. In contrast to the other catalases, Catalase-2 had catalase and peroxidase activity. Catalase-2 was expressed under conditions in which vacuolization of hyphae is observed. All three enzymes have a chlorin in its active site instead of ferroprotoheme IX and are resistant to molar concentrations of hydrogen peroxide. These and all other catalases tested so far are oxidized by singlet oxygen, probably at the heme moiety. The catalase activity is virtually unaffected by oxidation, but the enzymes are probably degraded more rapidly than the unmodified ones.

Alterations in cellular hydration not only contribute to metabolic regulation, but also critically determine the cellular response to different kinds of stress. Whereas cell swelling triggers anabolic pathways and protects cells from heat and oxidative challenge, cellular dehydration contributes to insulin resistance and catabolism and increases the cellular susceptibility to stressinduced damage. Intracellular accumulation of organic osmolytes, cell cycle delay and the expression of heat shock proteins provide cellular tolerance to hyperosmolarity and protect against stressors under dehydrating conditions. This article discusses some mechanisms by which alterations in cell hydration contribute to cytoprotection or cell damage. In addition, the close relationship between osmotic and oxidative stress and the contribution of isoosmotic shrinkage to apoptotic cell death are considered.

Thioredoxin (TRX) is a 12 kDa protein with redoxactive dithiol (CysGlyProCys) in the active site. TRX is induced by a variety of stresses including viral infection and inflammation. The promoter sequences of the TRX gene contain a series of stressresponsive elements including ORE, ARE, XRE, CRE and SP-1. TRX promotes DNA binding of transcription factors such as NFκB, AP-1 and p53. TRX interacts with target proteins modulating the activity of those proteins. We have identified TRX binding protein-2 (TBP-2), which was identical to vitamin D3 upregulated protein 1 (VDUP1). Potential action of TBP-2/VDUP1 as a redoxsensitive tumor suppressor will be discussed. There is accumulating evidence for the involvement of TRX in the protection against infectious and inflammatory disorders. We will discuss the role of TRXdependent redox regulation of the host defense mechanism, in particular its relation to the emerging concept of constitutive and/or inducible TRX on special cell types with dendritic and stellate morphology in the immune, endocrine and nervous systems, which we provisionally designate as dendritic stellate TRX producer cells (DST cell types).

Reactive oxygen species production and glutathione depletion in mammalian male germ cells are physiological events that are requisite to the functional maturation and capacitation of spermatozoa. In relation to this oxidative stress, an oxidation of the bulk of protein sulfydryl groups takes place during the final phases of male germ cell maturation. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase catalyzes this reaction, and accounts for both the assembly of the midpiece of spermatozoa and chromatin condensation. This process highlights the role of H2O2 and selenium in spermatogenesis and provides a mechanism for coupling a physiologically controlled oxidative stress to a specialized phenotypic function.

Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma concentration of lipid hydroperoxides, detectable by a sensitive chemiluminescence procedure. This is associated with increased susceptibility of LDL to oxidation, apparently due a structural perturbation at the particle surface brought about by lipid oxidation products. The postprandial modification of LDL is at least partially accounted for by an increase of LDL, a subfraction containing lipid oxidation products where apoproteinB-100 (apoB-100) is denatured. Consuming the meal with a suitable source of antioxidants, such as those found in red wine, minimizes this postprandial oxidative stress. The inhibition of peroxidation of lipids present in the meal during digestion is a possible mechanism for the observed protection of LDL. The in vivo oxidatively modified LDL has numerous features that correspond to the atherogenic minimally modified LDL produced in vitro. These modified particles could account for a relevant link between nutrition and early biological processes that foster the development of atherosclerosis.

A watersoluble [18O]labeled endoperoxide derived from N,Ndi(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide (DHPN18O2) has been shown to act as a clean chemical source of [18O]labeled molecular singlet oxygen. This allows the assessment of the singlet oxygen (1O2) reactivity toward biological targets such as DNA. The present work focuses on the qualitative identification of the main 1O2-oxidation products of 8-oxo-7,8-dihydro-2deoxyguanosine, which was achieved using high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLCESIMS/MS). Thus, the [18O]labeled and unlabeled imidazolone and oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features as those of the oxidized guanidinohydantoin molecule was detected. Our data strongly suggest that the imidazolone and oxazolone nucleosides are generated via the rearrangement of an unstable 5-hydroperoxide intermediate. Interestingly, the combined use of appropriate tools, including isotopically labeled singlet oxygen and the high resolution HPLCESIMS/MS technique, has allowed to shed new light on the 1O2- mediated oxidation reactions of guanine DNA components.

The oxidation of low density lipoprotein (LDL) by lipoxygenase has been implicated in the pathogenesis of atherosclerosis. It has been known that lipoxygenasemediated lipid peroxidation proceeds in general via regio, stereo and enantiospecific mechanisms, but that it is sometimes accompanied by a share of random hydroperoxides as side reaction products. In this study we investigated the oxidation of various substrates (linoleic acid, methyl linoleate, phosphatidylcholine, isolated LDL, and human plasma) by the arachidonate 15-lipoxygenases from rabbit reticulocytes and soybeans aiming at elucidating the effects of substrate, lipoxygenase and reaction milieu on the contribution and mechanism of random oxidation and also the effect of antioxidant. The specific character of the rabbit 15-lipoxygenase reaction was confirmed under all conditions employed here. However, the specificity by soybean lipoxygenase was markedly dependent on the conditions. When phosphatidylcholine liposomes and LDL were oxygenated by soybean lipoxygenase, the product pattern was found to be exclusively regio, stereo, and enantiorandom. When free linoleic acid was incorporated into PC liposomes and oxidized by soybean lipoxygenase, the free acid was specifically oxygenated, whereas esterified linoleate gave random oxidation products exclusively. Radicalscavenging antioxidants such as αtocopherol, ascorbic acid and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol selectively inhibited the random oxidation but did not influence specific product formation. It is assumed that the random reaction products originate from free radical intermediates, which have escaped the active site of the enzyme and thus may be accessible to radical scavengers. These data indicate that the specificity of lipoxygenasecatalyzed lipid oxidation and the inhibitory effects of antioxidants depend on the physicochemical state of the substrate and type of lipoxygenase and that they may change completely depending on the conditions.

Oxidatively modified lowdensity lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redoxactive transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidasederived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophilmediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5106 cells/ml) incubated with human LDL (0.25 mg protein/ml) timedependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating longlived oxidants such as Nchloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 M) protected against neutrophilmediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, preloading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl and Nchloramine dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.

Dietary hydroperoxides are being discussed as potential health hazards contributing to oxidative stressrelated diseases. However, how foodborn hydroperoxides could exert systemic effects remains elusive in view of the limited chances to be absorbed. Therefore, the metabolic fate of 13-HPODE (13-hydroperoxy octadecadienoic acid), 13-HODE (13-hydroxy octadecadienoic acid) and linoleic acid (LA) was investigated in a CaCo-2 cell monolayer as a model of the intestinal epithelium. [1-14C]-13-HPODE, up to a noncytotoxic concentration of 100 M, did not cross the CaCo-2 cell monolayer unreduced if applied to the luminal side. The [1-14C]HPODEderived radioactivity was preferentially recovered from intracellular and released diacylglycerols (DG), phospholipids (PL) and cholesterol esterified with oxidized fatty acids (oxCE). A similar distribution pattern was obtained with 13- HODE. In contrast, LA is preferentially incorporated into triacylglycerols (TG), cholesteryl esters (CE) and PL (but mainly released as TG). 13-HPODE dosedependently decreased the incorporation of LA into released TG, while LA accumulated in cellular and released DGs, effects similarily exerted by 13-HODE. We concluded that foodborn hydroperoxy fatty acids are instantly reduced by the gastrointestinal glutathione peroxidase, which was previously shown to persist in selenium deficiency. Accordingly, modulation of the glutathione peroxidases by selenium deprivation/repletion did not modify the disturbance of the lipid metabolism by 13-HPODE. Thus, hydroperoxy fatty acids disturb intestinal lipid metabolism by being esterified as hydroxy fatty acids into complex lipids, and may render lipoproteins synthesized thereof susceptible to further oxidative modifications.

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H2O2 and peroxyl radicals both generated Sglutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause Sglutathiolation. Thus, oxidative damage from H2O2 or AAPH was prevented by protein Sglutathiolation, while a direct reaction between GSH and oxidant likely prevents HOClmediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly Sglutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.

Oxidants have been shown to be involved in alcoholinduced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcoholinduced liver injury in rats. Male Wistar rats were fed highfat liquid diets with or without ethanol (10 14 g kg 1 day 1) and green tea (300 mg kg 1 day 1) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (~4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0 550 mg dl 1 day 1). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35±3 IU/l) by enteral ethanol (114±18); inclusion of green tea extract in the diet significantly blunted this increase (65±10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFa protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcoholinduced liver injury, most likely by preventing oxidative stress.

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three smallmolecule membrane antioxidants (βcarotene, αtocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that βcarotene provides protection against singlet oxygenmediated lipid peroxidation, but does not slow free radicalmediated lipid peroxidation. α Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chainbreaking antioxidant as evident from its ability to blunt oxygen consumption during free radical mediated lipid peroxidation. These three smallmolecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.

Thioredoxin reductase (TR), a flavoprotein, catalyzes the reduction of oxidized thioredoxin in a NADPHdependent manner, and contains a selenocysteine residue near the Cterminus. TR plays an important role in protecting against oxidative stress and in regulating cell growth and cell death. Constitutive TR expression has been observed in several cell types of the mammalian body, including endothelial cells. The latter are continually exposed to both exogenous and endogenous sources of nitric oxide (NO) and NOderived species. Reactive nitrogen species (RNS) are associated with pathological events, contributing to the cell and tissue damage accompanying inflammation, atherogenesis and autoimmune diseases. In this study, we report on the effect of peroxynitrite on TR in human umbilical vein endothelial cells (HUVECs). Exposure to the peroxynitrite donor SIN-1 for 1 h resulted in a decrease in TR activity. Interestingly, the activity was completely restored within 24 h. To further examine this mechanism, the expression of TR at the mRNA and protein level was examined. TR mRNA levels were markedly increased by treatment of SIN-1 within 6 h, and TR protein level was also increased after the treatment in HUVECs. These results suggest that the inactivation of TR by peroxynitrite might be involved in the upregulation of the TR gene in HUVECs. Therefore, HUVECs have a unique protective mechanism that allows the maintenance of balance in intracellular redox status via TR induction as an adaptive response to nitrooxidative stress.

Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy 1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steadystate concentrations (0.1 0.5 nmolmin 1106 cells) of H2O2 generated intracellularly activate cJun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both cJun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Poly (ADPribose) (PAR) is formed upon activation of the DNA repair enzyme poly(ADPribose) polymerase (PARP), and therefore was suggested as a new marker of apoptosis. Since DNA of epidermal cells represents a wellknown chromophore for UVB irradiation, and UVB is known to generate H2O2 in keratinocytes, we hypothesized that PAR is a very sensitive marker of UVB and H2O2-induced apoptosis in keratinocytes. In order to test this hypothesis, human immortalized keratinocytes (HaCaT) were UVBirradiated or treated with H2O2, and subsequently apoptosis was identified by comparing conventional parameters such as morphological analysis, DNA laddering, and TUNEL assay, with PAR formation. Both, UVB and H2O2 treatment induced PAR formation in HaCaT cells in a dosedependent manner, and its formation was detected as early as 4 h after irradiation, and at lower UVB doses (10 mJ/cm2) than observed by DNA laddering and the TUNEL assay. In conclusion, the detection of PAR formation is a very sensitive and early method for the identification of apoptotic cells in UVBinduced apoptosis of human keratinocytes.

Hypochlorous acid (HOCl) is a bactericidal compound formed by activated neutrophils during inflammation. Overproduction of HOCl causes damage to tissues at the site of neutrophil accumulation. The deleterious effects of excessive HOCl formation can be attenuated using antioxidants. Thiols and thioethers are known to be very effective HOCl scavengers. In the present study, the potency of several sulfurcontaining compounds to protect acetylcholinesterase, glutathione Stransferase P1-1 (GST P1-1) and α1-antiproteinease against inactivation by HOCl was determined. Surprisingly, glutathione disulfide was an effective protector of acetylcholinesterase against hypochlorous acid. Glutathione disulfide did not provide protection for GST P1-1 and α1-antiproteinease against oxidative inactivation by HOCl. The implications of this finding are discussed.

We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitindependent proteolytic pathway [Ikemoto et al., FASEB J. 15 (2001), 1279 1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acidreactive substance (TBARS) and glutathione disulfide (GSSG), in gastrocnemius muscle of tailsuspended rats. Glutathione (GSH) concentration in the muscle significantly decreased from day 5 and reached a minimum value on day 10. Tail suspension reciprocally increased concentrations of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail suspension. To prevent ubiquitination associated with oxidative stress, we also administered an antioxidative nutrient, cysteine, to tailsuspended rats. Intragastric supplementation of 140 mg/rat of cysteine for 2 weeks or longer normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, compared with supplementation of the equimolar amount of alanine. The cysteine supplementation significantly suppressed the loss of hindlimb muscle mass. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by unweighting.