reactive oxygen species, cell signaling, and cell injury

Forum: Therapeutic Applications of Reactive Oxygen and Nitrogen Speciesin Human Disease

REACTIVE OXYGEN SPECIES, CELL SIGNALING, AND CELL INJURY

KENNETH HENSLEY, KENT A. ROBINSON, S. PRASAD GABBITA , SCOTT SALSMAN, and ROBERT A. FLOYD

Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

(Received23 December1999;Accepted4 January2000)

Abstract—Oxidative stress has traditionally been viewed as a stochastic process of cell damage resulting from aerobicmetabolism, and antioxidants have been viewed simply as free radical scavengers. Only recently has it been recognizedthat reactive oxygen species (ROS) are widely used as second messengers to propagate proinflammatory or growth-stimulatory signals. With this knowledge has come the corollary realization that oxidative stress and chronic inflam-mation are related, perhaps inseparable phenomena. New pharmacological strategies aimed at supplementing antioxidantdefense systems while antagonizing redox-sensitive signal transduction may allow improved clinical management ofchronic inflammatory or degenerative conditions, including Alzheimer’s disease. Introduction of antioxidant therapiesinto mainstream medicine is possible and promising, but will require significant advances in basic cell biology,pharmacology, and clinical bioanalysis. © 2000 Elsevier Science Inc.

Keywords—Inflammation, Antioxidant, Phosphatase, Nitric oxide, Nitrone, Free radical

INTRODUCTION

During the past 10–15 years, the field of “free radicalresearch” has risen from relative obscurity to become amainstream element of biomedical science, and for goodreason. Since Commoner’s first detection of free radicalsin a biological system (germinating barley seeds) in 1954[1], free radical biology had mostly been the proprietarydomain of physical chemists. The chemical entities stud-ied by these scientists were ephemeral, almost to thepoint of abstraction. Very few techniques existed for the

detection or manipulation of free radicals in vitro, letalone in vivo. Moreover, the techniques brought to bearon free radical chemistry were esoteric, largely limited tospin trapping methods, and required expensive and ofteninaccessible instrumentation. Most importantly, thepathophysiological sequelae of oxidative stress havebeen notoriously difficult to quantify. Despite these im-pediments, the medical significance of oxidative stresshas become increasingly recognized to the point that it isnow considered to be a component of virtually everydisease process. The ascendancy of free radical biologyis attributable to several major factors. First, new tech-niques have been invented (and are still being invented)to quantify oxidative stress in vivo, although the existingtechnology is poorly suited for routine clinical applica-tions. Second, the inseparable relationship of oxidativestress to inflammation has become incontrovertible alongwith the recognition that certain reactive ROS functionas messenger molecules to propagate inflammatory sig-nals. Third, the discovery of nitric oxide (NO) as avasodilator and immune mediator has stimulated theinterest of mainstream biologists and clinicians to analmost unprecedented degree. As free radical/oxidativestress research enters the 21st century, we face the chal-lenge of transferring our nascent (but burgeoning)

Kenneth Hensley holds a Ph.D. in Physical Chemistry from theUniversity of Kentucky. He has served as a research scientist at theOklahoma Medical Research Foundation for the past four years. Hiscurrent research investigates the relationship between oxidative stressand neuroinflammation in the aging human brain, with special empha-sis on basic mechanisms of neurodegeneration in Alzheimer’s disease.Dr. Robinson, Dr. Gabbita, and Mr. Salsman currently pursue studies ofoxidative injury at the Oklahoma Medical Research Foundation withspecial emphasis on Alzheimer’s disease. Dr. Floyd is head of the FreeRadical Biology and Aging Research Program at the Oklahoma Med-ical Research Foundation. His current research interests center on thebiology of aging and the role of nitric oxide in age-related pathologiesof the central nervous system.

Address correspondence to: Kenneth Hensley, Ph.D., Free RadicalBiology and Aging Research Program, Oklahoma Medical ResearchFoundation, Oklahoma City, OK 73104, USA; Tel: (405) 271-7569;Fax: (405) 271-1795; E-Mail: [email protected].

Free Radical Biology & Medicine, Vol. 28, No. 10, pp. 1456–1462, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

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knowledge of oxidative pathology from the laboratoryinto the clinic and the pharmacy. New therapeutic strat-egies can, and will be developed which rationally incor-porate antioxidants into the management of chronic dis-ease. The purpose of this review is to highlight promisingnew developments in antioxidant therapy, particularlywith respect to strategies aimed at uncoupling oxidativestress from redox-sensitive signal transduction. A finalsection of the review summarizes current challenges inthe practical assessment of oxidative stress, which mustbe overcome before antioxidant therapy can achieve itsclinical potential.

ROS AS TOXINS: ANTIOXIDANTS AS SCAVENGERS OF

REACTIVE INTERMEDIATES

Until relatively recently, oxidative stress was consid-ered purely from the toxicological perspective. A rela-tively small number of free radicals such as the super-oxide anion (O2

•2) and the hydroxyl radical (HO•) wererecognized as minor by-products of oxidative phosphor-ylation. By 1973, Britton Chance and colleagues [2] haddetermined that approximately 2% of the oxygen re-duced by the mitochondrion forms O2

•2 or the dismuta-tion product H2O2. This estimate has been confirmedrepeatedly [3]. Superoxide and peroxide react with metalions to promote additional radical generation, with therelease of the particularly reactive hydroxyl [4]. Hy-droxyl radicals react at nearly diffusion-limited rateswith any component of the cell, including lipids, DNAand proteins. The net result of this nonspecific freeradical attack is a loss of cell integrity, enzyme function,and genomic stability [5–8]. Consequently, numerousdetoxification mechanisms have evolved to deal withoxyradical stress. Superoxide dismutase (SOD) convertsO2

•2 to H2O2, which is subsequently reduced to water bycatalase or otherwise decomposed by glutathione-depen-dent peroxidases. Small-molecule reducing agents suchas glutathione thereby buffer the intracellular environ-ment against ROS. In synergy with the aqueous defensemechanisms, lipid-phase antioxidants exist naturally toscavenge radical intermediates.A-tocopherol (a-toc, vi-tamin E) is the principle lipid-phase antioxidant [9–11].Hydroxyl (or alkoxyl) radical attack on tocopherol formsa stabilized phenolic radical which is reduced back to thephenol by ascorbate and NADH/NADPH-dependent re-ductase enzymes [9]. Over the past decade, the menag-erie of ROS has been expanded to include reactive ni-trogen species (RNS) derived from NO reaction withsuperoxide or peroxide [12,13]. Specific defense mech-anisms evolved to counteract RNS stress will probablybe identified in coming years.

Given the extreme reactivity of most oxyradicals andthe number of defense mechanisms evolved to counteract

oxidative stress, it seems reasonable that dietary or phar-macological practices that bolster the ROS scavengingcapacity should somehow improve health. Considerableepidemiological and clinical data, and huge amounts ofanimal data, corroborate this hypothesis. While a com-plete review is outside the scope of this discussion, it isworth noting that natural variation in antioxidant levelscorrelate negatively with certain pathologies, particularlyof the cardiovascular system. Most famously, plasmaa-tocopherol correlates negatively with risk of ischemicheart disease in several large, cross-sectional studies[14–16]. The usual explanation for this phenomenon isthat a-toc inhibits low density lipoprotein oxidation, anetiological factor in atherosclerotic plaque development[reviewed in 17]. Clinical studies designed to supplementantioxidant defenses, particularly by dietary administra-tion of a-toc (50–1000 mg/day) have shown some mar-ginal benefit but not as much as might be expected basedon epidemiological statistics. For instance, a 40% in-crease in plasmaa-toc is epidemiologically correlatedwith a 60–80% reduced risk of ischemic heart disease[14]. Paradoxically, clinical augmentation of plasmaa-tocopherol by the same amount confers only smallcardiovascular benefit in heart disease patients [18] withno effect, or even a marginal increase, in all-cause mor-tality [19]. Even more disconcerting, supplementationwith the lipophilic antioxidantb-carotene actually exac-erbates cancer risk among smokers [20]. Thus, whileantioxidant levels are clearly important in promotinghealth, the supplementation of antioxidant defenses inhuman subjects will prove much more complicated thanthe simple, casual administration of presumptively ben-eficial free radical scavengers.

The main problem faced by clinicians and basic sci-entists is that “antioxidant” function is much more com-plex than simple free radical scavenging, and dietarysupplementation with a particular antioxidant is likely toperturb the natural balance of other antioxidants. As acase in point, dietary supplementation witha-toc causesa profound and immediate decrease in plasma concen-tration of g-tocopherol (g-toc), a minor unmethylatedtocopherol [21–23].g-Tocopherol has been virtually un-studied, but some reports indicate thata-toc may scav-enge reactive nitrogen species (RNS) in a way thata-toccannot, forming the nitration product 5-nitro-a-tocoph-erol as a reaction product [21,22]. A very recent cardio-vascular study reports that dietaryg-toc is much moreefficacious thana-toc at decreasing susceptibility to oc-clusive thrombus, with plasma concentration-normalizedefficacy ofg-toc exceeding that ofa-toc by a factor of 20or more [24]. Clearly, much more basic research isneeded to understand the interplay among natural anti-oxidant systems and the synergies inherent to these sys-tems.

1457ROS and cell signaling

ROS AS SIGNALING MOLECULES: POTENTIAL

TARGETS FOR ANTI-INFLAMMATORY THERAPEUTICS

As discussed previously, oxidative stress has longbeen considered an “accident” of aerobic metabolism; astochastic process of free radical production and nonspe-cific tissue damage which is fundamentally unregulatedaside from the normal phalanx of antioxidant defensemechanisms. In recent years, a paradigm shift has beenoccurring wherein certain ROS and RNS have becomeappreciated as signaling molecules whose productionmay be regulated as a part of routine cellular signaltransduction [reviewed in 25]. The seminal work byBaeurle and colleagues first showed that certain tran-scription factors of the NFkB/rel family can be activatednot only by receptor-targeted ligands but also by directapplication of oxidizing agents (particularly H2O2) orionizing radiation [26,27]. Subsequently, several otherprotein kinase cascades and transcription factors havebeen discovered to possess redox-sensitive elements. Thecommon paradigm in all redox-sensitive signal transduc-tion pathways is the presence of intermediate protein

kinases which are activated by phosphorylation of spe-cific regulatory domains. For example, NF-kB is acti-vated upon phosphorylation of an inhibitory subunit(IkB). Conveniently, specific antibodies are now avail-able against the phosphorylated activation sites of manyprotein kinases so that activation of a particular enzymecan be assessed by standard immunoblot techniques.Figure 1 illustrates the phosphoactivation of several ma-jor protein kinase pathways in cultured primary rat as-trocytes exposed to low concentrations of exogenousH2O2.

Work from our group and others indicates that H2O2

may be synthesized endogenously in certain cell types asa response to activation by specific cytokines or growthfactors [28–30]. This endogenous H2O2 then acts as asecond messenger to stimulate protein kinase cascadescoupled to inflammatory gene expression, or in control ofthe cell cycle. The earliest convincing studies that impli-cated H2O2 as an endogenous messenger were performedby Sunderesan et al. [31] using, as a model system,vascular smooth muscle cells (VSMCs) stimulated with

Fig. 1. Western blots demonstrating synchronous phospho-activation of four distinct protein kinase cascades in primary rat astrocytesinitiated by addition of exogenous H2O2. Stat-35 Signal Transducer and Activator of Transcription-3 (target residue: pSer727); JNK 5c-Jun amino terminal kinase (target residues: pThr183-pro184-pTyr185); AKT 5 protein kinase B or RAC (target residue : pSer183);p38 5 p38MAPK (target residues: pThr180-Gly181-pTyr182). Antibodies recognize phosphorylated residues and other epitopic compo-nents near the phosphorylation sites. Cells were stimulated with the indicated bolus of peroxide for 5 min, lysed, electrophoresed on12% polyacrylamide gels, and probed with the appropriate phosphorylation-state specific primary antibody (New England Biolabs,Beverly, MA, USA). Blots were developed using horseradish peroxidase-conjugated secondary antibodies and chemiluminescentsubstrates.

1458 K. HENSLEY et al.

platelet-derived growth factor (PDGF). PDGF receptorbinding caused peroxide formation which could be in-hibited by intracellular expression of catalase. Catalaseexpression inhibited PDGF signal transduction by sup-pressing protein tyrosine phosphorylation [31]. Antioxi-dants, particularly thiol-reducing agents such asN-acetyl-cysteine, could mimic the inhibitory effects ofcatalase and prevent redox activation of ligand-coupledprotein kinase cascades [31].

Subsequent studies by a number of groups, particu-larly that of Sue Goo Rhee and colleagues [29], have ledto the hypothesis that H2O2 acts through the transientoxidative inactivation of protein tyrosine phosphatases(PTPs) which contain a nucleophilic cysteine as a cata-lytic element of the active site. Rhee has shown thatepidermal growth factor (EGF) binding to epidermoidcells induces rapid loss of PTP reactivity that can berestored by glutathione-dependent reductive pathways[29]. As in the case of PDGF, EGF receptor bindingcauses intracellular production of H2O2 within the time-frame of PTP inactivation [28,29]. In separate but con-temporaneous work, Denu and Tanner demonstrated thatH2O2 reacts with PTPs in vitro to convert the active-sitecysteine into a metastable sulfinic acid [32]. Subsequentreduction by glutathione restores the enzyme to its activeform. Alternatively, phosphatase reaction with oxidizedglutathione could transiently inactivate a PTP during aredox signaling event [33].

We have observed strong evidence for peroxide-me-diated, phosphatase-dependent signal transduction usinga cytokine stimulus directed against primary rat astro-cytes [30,34]. We find that both interleukin-1b (IL1b)and H2O2 will promote phospho-activation of the p38-mitogen activated protein kinase (p38MAPK) in a mannerthat can be antagonized with submillimolar quantities ofNAC or the nitrone-based antioxidant phenyl-N-tert-bu-tylnitrone (PBN) [30]. Interestingly, PBN has been foundefficacious in preventing ischemia/reperfusion injury,septic shock, and other trauma, though the mechanism ofaction has been indeterminate [reviewed in 35]. In IL1b-treated astrocytes, total phosphatase activity decreasessimultaneously with p38MAPK phospho-activation, andreturns to baseline as p38MAPK becomes dephosphory-lated (inactivated). Both NAC and PBN maintain phos-phatase activity at or above baseline values [30] whilepromoting global protein dephosphorylation [34]. Fi-nally, we were able to measure H2O2 biosynthesis inIL1b-treated astrocytes and found this to be inhibited by1 mM PBN [30]. Thus, several lines of evidence arguethat H2O2 is used as a ubiquitous messenger substance toinactivate regulatory phosphatase enzymes and promoteinflammatory signal transduction. Figure 2 schematicallysummarizes the probable function of H2O2 as a signal

transducer, and illustrates possible targets for pharmaco-logical antagonism.

The p38MAPK pathway is a particularly relevant targetfor antioxidant antagonism in chronic inflammatory dis-ease. p38MAPK regulates expression of inflammatory cy-tokines including IL1b [36] and largely regulates expres-sion of iNOS and COX-II [37,38]. We have observedp38MAPK hyperphosphorylation in Alzheimer’s diseased(AD) brain tissue, in plaques and neurons where proteinnitration is also evident [39,40]. In separate work, Wal-ton and colleagues [41] have observed similar p38MAPK

phosphorylation in microglia of postischemic rodentbrain, where protein oxidation and nitration are salientpathological phenomena [42,43]. Brain-accessible anti-oxidants and antagonists of redox signaling may, there-fore, have wide utility in the therapeutic interdiction ofneuroinflammatory events occurring in AD, stroke, andother neurodegenerative disease.

The recognition that ROS may stimulate inflamma-tory signaling pathways comes with considerable clinicalramifications. Once we can identify the sources andtargets of “second-messenger” ROS, new avenues willbe open for the development of novel pharmacophoresthat function both as antioxidants and nonsteroidal anti-inflammatory agents. PBN, for instance, decreases brainprotein oxidation during ischemia/reperfusion injury ornormal aging [44,45]. Additionally, PBN can protectanimals from systemic inflammation induced by bacte-rial endotoxin [46]. We have shown that inflammatorygene transcription and iNOS expression are simulta-neously suppressed by the nitrone within the same ani-mal models [47–49]. Moreover, the transcription of pro-apoptotic elements such as caspase 3 and Fas antigen aresuppressed by PBN in rats subjected to experimentalseptic shock [49]. These diverse actions can be explainedby nitrone antagonism of redox-sensitive signal trans-duction pathways including, but not limited to, thep38MAPK cascade. Unfortunately, the precise site of ac-tion of PBN has not been elucidated. Future research willneed to identify the exact source of second-messengerROS, better pinpoint the targets of this ROS, and identifyregulatory elements against which novel pharmaco-phores may be designed.

MONITORING OXIDATIVE STRESS: A BIOANALYTICAL

CHALLENGE AND A BIOMEDICAL NECESSITY

Despite widespread scientific and public perceptionthat antioxidants are “good,” and the incontrovertibleevidence that oxidative damage is deleterious in chronicdisease, serious barriers exist to the introduction of an-tioxidant therapies into clinical medicine. The greatest ofthese barriers is the fact that we cannot currently deter-mine which individuals might benefit from which anti-


oxidant therapy. The optimum daily dose of even com-mon antioxidants such asa-tocopherol and vitamin C aresubject to some debate, while no guidelines have everbeen considered for less-common, but possibly no lesssignificant antioxidants such asa-tocopherol. While wehave a poor idea of the biological effects inherent tosupplementation with natural antioxidants, we have noidea whatsoever of the effects of synthetic antioxidantsin the human subject. As alluded to previously, certainsubgroups might even react negatively to antioxidants, asevidenced by the apparent exacerbation of lung canceramong patients takingb-carotene [20]. Beyond the de-termination of therapeutic strategy, a clinician shouldhave some means of determining the responsiveness ofhis patient to the prescribed treatment. How can onemonitor antioxidant status in a clinical setting? Cur-rently, there is no satisfying answer to such a question.

The onus is upon free radical researchers to developsensitive, facile, and accurate assays for oxidative stressthat predict the type of antioxidant supplementation thatmight be appropriate to a specific individual. Moreover,such bioanalytical tools must allow a clinician to monitora patient’s response to treatment, in much the same wayas the physician would monitor cholesterol or bloodglucose or any other clinically-relevant parameter. Ourgroup has been active in the development of high per-formance liquid chromatography with electrochemicaldetection (HPLC-ECD) as a tool for the routine assess-ment of oxidative stress [50–52]. Specific, discreet ana-lytes can be selectively measured by HPLC-ECD, andthese analytes may indicate something of the nature of anoxidative insult. For example, HPLC-ECD can detectnitrated tyrosines (3-nitrotyrosine) and 5-nitro-g-tocoph-erol as indicators of NO involvement in a disease process

Fig. 2. Postulated mechanism of peroxide-mediated redox signaling. Arrows indicate stimulatory pathways;¢ indicate inhibitorypressures. Signaling is initiated by specific ligand-receptor interactions. Typically, a series of protein kinase intermediates propagatethe signal toward nuclear transcription factors. Other signaling pathways must exist to facilitate the H2O2 production observed byseveral labs [e.g., references 28,30]. The sites of intracellular peroxide generation are currently subject to some debate; however,mitochondria and plasma membrane-bound oxidoreductase enzymes have been postulated to serve this function. Endogenously-generated H2O2 causes transient inactivation of sensitive protein tyrosine phosphatases (PTP-SH); this reaction may occur directlythrough a sulfenic acid intermediate (PTP-SOH) or indirectly via formation of a mixed glutathione intermediate (PTP-S-SG).Glutathione oxidation by peroxide is readily catalyzed by glutathione peroxidase (GSH-Px). Removal of phosphatase inhibition willallow maximal signal output through the protein kinase cascade. The oxidized, inactive protein phosphatase can be regenerated intothe active form by further reduction by GSH in a reaction catalyzed by thioredoxin (Trdx). Reactivated phosphatase activity will causedephosphorylation of intermediate protein kinases and transcription factors, thereby terminating the redox-sensitive signal. Potentialsites of pharmacological action would include the putative peroxide-generator, as well as various intermediate kinase enzymes suchas p38MAPK. Agents that maintain phosphatase activity in the face of an oxidative challenge would, in general, be expected toantagonize the redox signaling process.


[50]. Nonspecific oxidation might be indicated by in-creases in the hydroxyl reaction products o-tyrosine orm-tyrosine or by tyrosine dimers; or, alternatively, byincreased conversion ofa-toc to the corresponding p-quinone [50–52].

Other researchers have successfully indexed oxidativestress by gas chromatography in combination with massspectrometry (GC-MS). GC-MS analysis of low molec-ular weight hydrocarbons in breath [53], or specific ara-chidonic acid peroxidation products (isoprostanes) influids [54,55], may prove amenable to clinical medicine.Morrow, Montine and colleagues [55], for instance, havemeasured increased F1-isoprostanes in cerebrospinalfluid of patients with Alzheimer’s disease. AD is oneillness with a clear oxidative stress component whereinantioxidant supplementation (specifically, witha-to-copherol) confers a small, but significant clinical benefitmanifest by delays in primary outcome indicators (e.g.,time of entry into a nursing home or loss of ability toperform routine daily function) [56]. Before antioxidanttherapy becomes accepted, detailed longitudinal studieswill need to be conducted which evaluate panels ofoxidative biomarkers along with traditional clinical end-points in patients undergoing treatment for diversechronic illnesses. The publication of such studies willusher in a new and exciting period in the history ofoxidative stress research and will signal the final matu-ration of the discipline.

Acknowledgements— This work was supported in part by the NationalInstitutes of Health (NS35747) and the Oklahoma Center for theAdvancement of Science and Technology (OCAST H67-097).

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ABBREVIATIONS

RNS—reactive nitrogen speciesROS—reactive oxygen speciesPBN—phenyl-tert-butylnitroneNAC—N-acetyl cysteinea-toc—alpha tocopherolg-toc—gamma;-tocopherolIL1b—interleukin-1bp38MAPK—p38-mitogen activated protein kinasePTP—protein tyrosine phosphataseHPLC-ECD—high performance liquid chromatography

with electrochemical detectionAD—Alzheimer’s disease


reactive oxygen species, cell signaling, and cell injury

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