Free Radical Biology & Medicine, Vol. 28, No. 10, pp. 1456  1462, 2000
Copyright © 2000 Elsevier Science Inc.
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Forum: Therapeutic Applications of Reactive Oxygen and Nitrogen Species
in 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
(Received 23 December 1999; Accepted 4 January 2000)
Abstract Oxidative stress has traditionally been viewed as a stochastic process of cell damage resulting from aerobic
metabolism, and antioxidants have been viewed simply as free radical scavengers. Only recently has it been recognized
that 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 antioxidant
defense systems while antagonizing redox-sensitive signal transduction may allow improved clinical management of
chronic inflammatory or degenerative conditions, including Alzheimer s disease. Introduction of antioxidant therapies
into 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
detection or manipulation of free radicals in vitro, let
alone in vivo. Moreover, the techniques brought to bear
During the past 10 15 years, the field of  free radical
on free radical chemistry were esoteric, largely limited to
research has risen from relative obscurity to become a
spin trapping methods, and required expensive and often
mainstream element of biomedical science, and for good
inaccessible instrumentation. Most importantly, the
reason. Since Commoner s first detection of free radicals
pathophysiological sequelae of oxidative stress have
in a biological system (germinating barley seeds) in 1954
been notoriously difficult to quantify. Despite these im-
[1], free radical biology had mostly been the proprietary
pediments, the medical significance of oxidative stress
domain of physical chemists. The chemical entities stud-
has become increasingly recognized to the point that it is
ied by these scientists were ephemeral, almost to the
now considered to be a component of virtually every
point of abstraction. Very few techniques existed for the
disease process. The ascendancy of free radical biology
is attributable to several major factors. First, new tech-
Kenneth Hensley holds a Ph.D. in Physical Chemistry from the niques have been invented (and are still being invented)
University of Kentucky. He has served as a research scientist at the
to quantify oxidative stress in vivo, although the existing
Oklahoma Medical Research Foundation for the past four years. His
technology is poorly suited for routine clinical applica-
current research investigates the relationship between oxidative stress
and neuroinflammation in the aging human brain, with special empha- tions. Second, the inseparable relationship of oxidative
sis on basic mechanisms of neurodegeneration in Alzheimer s disease.
stress to inflammation has become incontrovertible along
Dr. Robinson, Dr. Gabbita, and Mr. Salsman currently pursue studies of
with the recognition that certain reactive ROS function
oxidative injury at the Oklahoma Medical Research Foundation with
special emphasis on Alzheimer s disease. Dr. Floyd is head of the Free as messenger molecules to propagate inflammatory sig-
Radical Biology and Aging Research Program at the Oklahoma Med-
nals. Third, the discovery of nitric oxide (NO) as a
ical Research Foundation. His current research interests center on the
vasodilator and immune mediator has stimulated the
biology of aging and the role of nitric oxide in age-related pathologies
of the central nervous system. interest of mainstream biologists and clinicians to an
Address correspondence to: Kenneth Hensley, Ph.D., Free Radical
almost unprecedented degree. As free radical/oxidative
Biology and Aging Research Program, Oklahoma Medical Research
stress research enters the 21st century, we face the chal-
Foundation, Oklahoma City, OK 73104, USA; Tel: [phone];
Fax: [phone]; E-Mail: [e-mail]. lenge of transferring our nascent (but burgeoning)
1456
ROS and cell signaling 1457
knowledge of oxidative pathology from the laboratory oxidative stress, it seems reasonable that dietary or phar-
into the clinic and the pharmacy. New therapeutic strat- macological practices that bolster the ROS scavenging
egies can, and will be developed which rationally incor- capacity should somehow improve health. Considerable
porate antioxidants into the management of chronic dis- epidemiological and clinical data, and huge amounts of
ease. The purpose of this review is to highlight promising animal data, corroborate this hypothesis. While a com-
new developments in antioxidant therapy, particularly plete review is outside the scope of this discussion, it is
with respect to strategies aimed at uncoupling oxidative worth noting that natural variation in antioxidant levels
stress from redox-sensitive signal transduction. A final correlate negatively with certain pathologies, particularly
section of the review summarizes current challenges in of the cardiovascular system. Most famously, plasma
the practical assessment of oxidative stress, which must -tocopherol correlates negatively with risk of ischemic
be overcome before antioxidant therapy can achieve its heart disease in several large, cross-sectional studies
clinical potential. [14 16]. The usual explanation for this phenomenon is
that -toc inhibits low density lipoprotein oxidation, an
etiological factor in atherosclerotic plaque development
ROS AS TOXINS: ANTIOXIDANTS AS SCAVENGERS OF
[reviewed in 17]. Clinical studies designed to supplement
REACTIVE INTERMEDIATES
antioxidant defenses, particularly by dietary administra-
Until relatively recently, oxidative stress was consid- tion of -toc (50 1000 mg/day) have shown some mar-
ered purely from the toxicological perspective. A rela- ginal benefit but not as much as might be expected based
tively small number of free radicals such as the super- on epidemiological statistics. For instance, a 40% in-
oxide anion (O2" ) and the hydroxyl radical (HO" ) were crease in plasma -toc is epidemiologically correlated
recognized as minor by-products of oxidative phosphor- with a 60 80% reduced risk of ischemic heart disease
ylation. By 1973, Britton Chance and colleagues [2] had [14]. Paradoxically, clinical augmentation of plasma
determined that approximately 2% of the oxygen re- -tocopherol by the same amount confers only small
duced by the mitochondrion forms O2" or the dismuta- cardiovascular benefit in heart disease patients [18] with
tion product H2O2. This estimate has been confirmed no effect, or even a marginal increase, in all-cause mor-
repeatedly [3]. Superoxide and peroxide react with metal tality [19]. Even more disconcerting, supplementation
ions to promote additional radical generation, with the with the lipophilic antioxidant -carotene actually exac-
release of the particularly reactive hydroxyl [4]. Hy- erbates cancer risk among smokers [20]. Thus, while
droxyl radicals react at nearly diffusion-limited rates antioxidant levels are clearly important in promoting
with any component of the cell, including lipids, DNA health, the supplementation of antioxidant defenses in
and proteins. The net result of this nonspecific free human subjects will prove much more complicated than
radical attack is a loss of cell integrity, enzyme function, the simple, casual administration of presumptively ben-
and genomic stability [5 8]. Consequently, numerous eficial free radical scavengers.
detoxification mechanisms have evolved to deal with The main problem faced by clinicians and basic sci-
oxyradical stress. Superoxide dismutase (SOD) converts entists is that  antioxidant function is much more com-
O2" to H2O2, which is subsequently reduced to water by plex than simple free radical scavenging, and dietary
catalase or otherwise decomposed by glutathione-depen- supplementation with a particular antioxidant is likely to
dent peroxidases. Small-molecule reducing agents such perturb the natural balance of other antioxidants. As a
as glutathione thereby buffer the intracellular environ- case in point, dietary supplementation with -toc causes
ment against ROS. In synergy with the aqueous defense a profound and immediate decrease in plasma concen-
mechanisms, lipid-phase antioxidants exist naturally to tration of -tocopherol ( -toc), a minor unmethylated
scavenge radical intermediates. -tocopherol ( -toc, vi- tocopherol [21 23]. -Tocopherol has been virtually un-
tamin E) is the principle lipid-phase antioxidant [9 11]. studied, but some reports indicate that -toc may scav-
Hydroxyl (or alkoxyl) radical attack on tocopherol forms enge reactive nitrogen species (RNS) in a way that -toc
a stabilized phenolic radical which is reduced back to the cannot, forming the nitration product 5-nitro- -tocoph-
phenol by ascorbate and NADH/NADPH-dependent re- erol as a reaction product [21,22]. A very recent cardio-
ductase enzymes [9]. Over the past decade, the menag- vascular study reports that dietary -toc is much more
erie of ROS has been expanded to include reactive ni- efficacious than -toc at decreasing susceptibility to oc-
trogen species (RNS) derived from NO reaction with clusive thrombus, with plasma concentration-normalized
superoxide or peroxide [12,13]. Specific defense mech- efficacy of -toc exceeding that of -toc by a factor of 20
anisms evolved to counteract RNS stress will probably or more [24]. Clearly, much more basic research is
be identified in coming years. needed to understand the interplay among natural anti-
Given the extreme reactivity of most oxyradicals and oxidant systems and the synergies inherent to these sys-
the number of defense mechanisms evolved to counteract tems.
1458 K. HENSLEY et al.
Fig. 1. Western blots demonstrating synchronous phospho-activation of four distinct protein kinase cascades in primary rat astrocytes
initiated by addition of exogenous H2O2. Stat-3 Signal Transducer and Activator of Transcription-3 (target residue: pSer727); JNK
c-Jun amino terminal kinase (target residues: pThr183-pro184-pTyr185); AKT protein kinase B or RAC (target residue : pSer183);
p38 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 on
12% 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 chemiluminescent
substrates.
ROS AS SIGNALING MOLECULES: POTENTIAL
kinases which are activated by phosphorylation of spe-
TARGETS FOR ANTI-INFLAMMATORY THERAPEUTICS
cific regulatory domains. For example, NF- B is acti-
vated upon phosphorylation of an inhibitory subunit
As discussed previously, oxidative stress has long
(I B). Conveniently, specific antibodies are now avail-
been considered an  accident of aerobic metabolism; a
able against the phosphorylated activation sites of many
stochastic process of free radical production and nonspe-
protein kinases so that activation of a particular enzyme
cific tissue damage which is fundamentally unregulated
can be assessed by standard immunoblot techniques.
aside from the normal phalanx of antioxidant defense
Figure 1 illustrates the phosphoactivation of several ma-
mechanisms. In recent years, a paradigm shift has been
jor protein kinase pathways in cultured primary rat as-
occurring wherein certain ROS and RNS have become
trocytes exposed to low concentrations of exogenous
appreciated as signaling molecules whose production
H2O2.
may be regulated as a part of routine cellular signal
Work from our group and others indicates that H2O2
transduction [reviewed in 25]. The seminal work by
may be synthesized endogenously in certain cell types as
Baeurle and colleagues first showed that certain tran-
a response to activation by specific cytokines or growth
scription factors of the NF B/rel family can be activated
factors [28 30]. This endogenous H2O2 then acts as a
not only by receptor-targeted ligands but also by direct
second messenger to stimulate protein kinase cascades
application of oxidizing agents (particularly H2O2) or
coupled to inflammatory gene expression, or in control of
ionizing radiation [26,27]. Subsequently, several other
the cell cycle. The earliest convincing studies that impli-
protein kinase cascades and transcription factors have
cated H2O2 as an endogenous messenger were performed
been discovered to possess redox-sensitive elements. The
common paradigm in all redox-sensitive signal transduc- by Sunderesan et al. [31] using, as a model system,
tion pathways is the presence of intermediate protein vascular smooth muscle cells (VSMCs) stimulated with
ROS and cell signaling 1459
platelet-derived growth factor (PDGF). PDGF receptor transducer, and illustrates possible targets for pharmaco-
binding caused peroxide formation which could be in- logical antagonism.
hibited by intracellular expression of catalase. Catalase The p38MAPK pathway is a particularly relevant target
expression inhibited PDGF signal transduction by sup- for antioxidant antagonism in chronic inflammatory dis-
pressing protein tyrosine phosphorylation [31]. Antioxi- ease. p38MAPK regulates expression of inflammatory cy-
tokines including IL1 [36] and largely regulates expres-
dants, particularly thiol-reducing agents such as
sion of iNOS and COX-II [37,38]. We have observed
N-acetyl-cysteine, could mimic the inhibitory effects of
p38MAPK hyperphosphorylation in Alzheimer s diseased
catalase and prevent redox activation of ligand-coupled
(AD) brain tissue, in plaques and neurons where protein
protein kinase cascades [31].
nitration is also evident [39,40]. In separate work, Wal-
Subsequent studies by a number of groups, particu-
ton and colleagues [41] have observed similar p38MAPK
larly that of Sue Goo Rhee and colleagues [29], have led
phosphorylation in microglia of postischemic rodent
to the hypothesis that H2O2 acts through the transient
brain, where protein oxidation and nitration are salient
oxidative inactivation of protein tyrosine phosphatases
pathological phenomena [42,43]. Brain-accessible anti-
(PTPs) which contain a nucleophilic cysteine as a cata-
oxidants and antagonists of redox signaling may, there-
lytic element of the active site. Rhee has shown that
fore, have wide utility in the therapeutic interdiction of
epidermal growth factor (EGF) binding to epidermoid
neuroinflammatory events occurring in AD, stroke, and
cells induces rapid loss of PTP reactivity that can be
other neurodegenerative disease.
restored by glutathione-dependent reductive pathways
The recognition that ROS may stimulate inflamma-
[29]. As in the case of PDGF, EGF receptor binding
tory signaling pathways comes with considerable clinical
causes intracellular production of H2O2 within the time-
ramifications. Once we can identify the sources and
frame of PTP inactivation [28,29]. In separate but con-
targets of  second-messenger ROS, new avenues will
temporaneous work, Denu and Tanner demonstrated that
be open for the development of novel pharmacophores
H2O2 reacts with PTPs in vitro to convert the active-site
that function both as antioxidants and nonsteroidal anti-
cysteine into a metastable sulfinic acid [32]. Subsequent
inflammatory agents. PBN, for instance, decreases brain
reduction by glutathione restores the enzyme to its active
protein oxidation during ischemia/reperfusion injury or
form. Alternatively, phosphatase reaction with oxidized
normal aging [44,45]. Additionally, PBN can protect
glutathione could transiently inactivate a PTP during a
animals from systemic inflammation induced by bacte-
redox signaling event [33].
rial endotoxin [46]. We have shown that inflammatory
We have observed strong evidence for peroxide-me-
gene transcription and iNOS expression are simulta-
diated, phosphatase-dependent signal transduction using
neously suppressed by the nitrone within the same ani-
a cytokine stimulus directed against primary rat astro-
mal models [47 49]. Moreover, the transcription of pro-
cytes [30,34]. We find that both interleukin-1 (IL1 )
apoptotic elements such as caspase 3 and Fas antigen are
and H2O2 will promote phospho-activation of the p38-
suppressed by PBN in rats subjected to experimental
mitogen activated protein kinase (p38MAPK) in a manner
septic shock [49]. These diverse actions can be explained
that can be antagonized with submillimolar quantities of
by nitrone antagonism of redox-sensitive signal trans-
NAC or the nitrone-based antioxidant phenyl-N-tert-bu-
duction pathways including, but not limited to, the
tylnitrone (PBN) [30]. Interestingly, PBN has been found
p38MAPK cascade. Unfortunately, the precise site of ac-
efficacious in preventing ischemia/reperfusion injury,
tion of PBN has not been elucidated. Future research will
septic shock, and other trauma, though the mechanism of
need to identify the exact source of second-messenger
action has been indeterminate [reviewed in 35]. In IL1 -
ROS, better pinpoint the targets of this ROS, and identify
treated astrocytes, total phosphatase activity decreases
regulatory elements against which novel pharmaco-
simultaneously with p38MAPK phospho-activation, and
phores may be designed.
returns to baseline as p38MAPK becomes dephosphory-
lated (inactivated). Both NAC and PBN maintain phos-
MONITORING OXIDATIVE STRESS: A BIOANALYTICAL
phatase activity at or above baseline values [30] while
CHALLENGE AND A BIOMEDICAL NECESSITY
promoting global protein dephosphorylation [34]. Fi-
nally, we were able to measure H2O2 biosynthesis in
Despite widespread scientific and public perception
IL1 -treated astrocytes and found this to be inhibited by
that antioxidants are  good, and the incontrovertible
1 mM PBN [30]. Thus, several lines of evidence argue
evidence that oxidative damage is deleterious in chronic
that H2O2 is used as a ubiquitous messenger substance to
disease, serious barriers exist to the introduction of an-
inactivate regulatory phosphatase enzymes and promote tioxidant therapies into clinical medicine. The greatest of
inflammatory signal transduction. Figure 2 schematically these barriers is the fact that we cannot currently deter-
summarizes the probable function of H2O2 as a signal mine which individuals might benefit from which anti-
1460 K. HENSLEY et al.
Fig. 2. Postulated mechanism of peroxide-mediated redox signaling. Arrows indicate stimulatory pathways; indicate inhibitory
pressures. Signaling is initiated by specific ligand-receptor interactions. Typically, a series of protein kinase intermediates propagate
the signal toward nuclear transcription factors. Other signaling pathways must exist to facilitate the H2O2 production observed by
several 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 directly
through 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 will
allow maximal signal output through the protein kinase cascade. The oxidized, inactive protein phosphatase can be regenerated into
the active form by further reduction by GSH in a reaction catalyzed by thioredoxin (Trdx). Reactivated phosphatase activity will cause
dephosphorylation of intermediate protein kinases and transcription factors, thereby terminating the redox-sensitive signal. Potential
sites of pharmacological action would include the putative peroxide-generator, as well as various intermediate kinase enzymes such
as p38MAPK. Agents that maintain phosphatase activity in the face of an oxidative challenge would, in general, be expected to
antagonize the redox signaling process.
oxidant therapy. The optimum daily dose of even com- The onus is upon free radical researchers to develop
mon antioxidants such as -tocopherol and vitamin C are sensitive, facile, and accurate assays for oxidative stress
subject to some debate, while no guidelines have ever that predict the type of antioxidant supplementation that
been considered for less-common, but possibly no less might be appropriate to a specific individual. Moreover,
significant antioxidants such as -tocopherol. While we such bioanalytical tools must allow a clinician to monitor
have a poor idea of the biological effects inherent to a patient s response to treatment, in much the same way
supplementation with natural antioxidants, we have no as the physician would monitor cholesterol or blood
idea whatsoever of the effects of synthetic antioxidants glucose or any other clinically-relevant parameter. Our
in the human subject. As alluded to previously, certain group has been active in the development of high per-
subgroups might even react negatively to antioxidants, as formance liquid chromatography with electrochemical
evidenced by the apparent exacerbation of lung cancer detection (HPLC-ECD) as a tool for the routine assess-
among patients taking -carotene [20]. Beyond the de- ment of oxidative stress [50 52]. Specific, discreet ana-
termination of therapeutic strategy, a clinician should lytes can be selectively measured by HPLC-ECD, and
have some means of determining the responsiveness of these analytes may indicate something of the nature of an
his patient to the prescribed treatment. How can one oxidative insult. For example, HPLC-ECD can detect
monitor antioxidant status in a clinical setting? Cur- nitrated tyrosines (3-nitrotyrosine) and 5-nitro- -tocoph-
rently, there is no satisfying answer to such a question. erol as indicators of NO involvement in a disease process
ROS and cell signaling 1461
[10] Burton, G. W.; Joyce, A.; Ingold, K. U. First proof that vitamin E
[50]. Nonspecific oxidation might be indicated by in-
is major lipid-soluble, chain-breaking antioxidant in human blood
creases in the hydroxyl reaction products o-tyrosine or
plasma. Lancet 2:27; 1982.
m-tyrosine or by tyrosine dimers; or, alternatively, by
[11] Ingold, K. U.; Webb, A. C.; Witter, D.; Burton, G. W.; Metcalfe,
T. A.; Muller, D. P. Vitamin E remains the major lipid-soluble,
increased conversion of -toc to the corresponding p-
chain-breaking antioxidant in human plasma even in individuals
quinone [50 52].
suffering severe vitamin E deficiency. Arch. Biochem. Biophys.
Other researchers have successfully indexed oxidative
259:224 225; 1987.
[12] Squadrito, G. L.; Pryor, W. A. Oxidative chemistry of nitric
stress by gas chromatography in combination with mass
oxide: the roles of superoxide, peroxynitrite, and carbon dioxide.
spectrometry (GC-MS). GC-MS analysis of low molec-
Free Radic. Biol. Med. 25:392 403; 1998.
ular weight hydrocarbons in breath [53], or specific ara-
[13] Koppenol, W. H. The basic chemistry of nitrogen monoxide and
chidonic acid peroxidation products (isoprostanes) in peroxynitrite. Free Radic. Biol. Med. 25:385 391; 1998
[14] Gey, K. F.; Puska, P.; Moser, U. K. Inverse correlation between
fluids [54,55], may prove amenable to clinical medicine.
plasma vitamin E and mortality from ischemic heart disease in
Morrow, Montine and colleagues [55], for instance, have
cross-cultural epidemiology. Am. J. Clin. Nutr. 53(Suppl. 1):
measured increased F1-isoprostanes in cerebrospinal 326S 334S; 1991.
[15] Stampfer, M. J.; Hennekens, C. H.; Manson, J. E.; Coldizt, G. A.;
fluid of patients with Alzheimer s disease. AD is one
Rosner, B.; Willett, W. C. Vitamin E consumption and the risk of
illness with a clear oxidative stress component wherein
coronary artery disease in women. N. Engl. J. Med. 328:1444
antioxidant supplementation (specifically, with -to-
1449; 1993.
[16] Rimm, E. B.; Stampfer, M. J.; Ascherio, A.; Giovannucci, E.;
copherol) confers a small, but significant clinical benefit
Colditz, G. A.; Willett, W. C. Vitamin E consumption and the risk
manifest by delays in primary outcome indicators (e.g.,
of coronary heart disease in men. N. Engl. J. Med. 328:1450
time of entry into a nursing home or loss of ability to
1456; 1993.
[17] Esterbauer, H.; Gebicki, J.; Puhl, H.; Jurgens, G. The role of lipid
perform routine daily function) [56]. Before antioxidant
peroxidation and antioxidants in modification of LDL. Free
therapy becomes accepted, detailed longitudinal studies
Radic. Biol. Med. 13:341 390; 1992.
will need to be conducted which evaluate panels of
[18] Stephens, N. G.; Parsons, A.; Schofield, P. M.; Kelly, F.; Cheese-
man, K.; Mitchinson, M. J. Randomised controlled trial of vitamin
oxidative biomarkers along with traditional clinical end-
E in patients with coronary disease: Cambridge Heart Antioxidant
points in patients undergoing treatment for diverse
Study (CHAOS). Lancet 347:781 786; 1996.
chronic illnesses. The publication of such studies will
[19] Rapola, J. M.; Virtamo, J.; Ripatti, S.; Huttumen, J. K.; Albanes,
D.; Taylor, P. R.; Heinonen, O. P. Randomised trial of alpha
usher in a new and exciting period in the history of
tocopherol and beta carotene supplements on incidence of major
oxidative stress research and will signal the final matu-
coronary events in men with previous myocardial infarction.
ration of the discipline.
Lancet 349:1715 1720; 1997.
[20] The Alpha-Tocopherol Beta Carotene Cancer Prevention Study
Group. The effect of vitamin E and beta carotene on the incidence
Acknowledgements  This work was supported in part by the National
of lung cancer and other cancers in male smokers. N. Engl.
Institutes of Health (NS35747) and the Oklahoma Center for the
J. Med. 330:1029 1035; 1994.
Advancement of Science and Technology (OCAST H67-097).
[21] Cooney, R. V.; Franke, A. A.; Harwood, P. J.; Hatch-Pigott, V.;
Custer, L. J.; Mordan, L. J. -Tocopherol detoxification of nitro-
REFERENCES gen dioxide: superiority to -tocopherol. Proc. Natl. Acad. Sci.
USA 90:1771 1775; 1993.
[1] Commoner, B.; Townsend, J.; Pake, G. E. Free radicals in bio-
[22] Christen, S.; Woodall, A. A.; Shigenaga, M. K.; Southwell-Keely,
logical materials. Nature 174:689 691; 1954.
P. T.; Duncan, M. W.; Ames, B. N. -Tocopherol traps mutagenic
[2] Boveris, A.; Chance, B. The mitochondrial generation of hydro-
electrophiles such as NOx and complements -tocopherol: phys-
gen peroxide: general properties and effect of hyperbaric oxygen.
iological implications. Proc. Natl. Acad. Sci. USA 94:3217 3222;
Biochem. J. 134:707 716; 1973.
1997.
[3] Hensley, K.; Pye, Q. N.; Maidt, M. L.; Stewart, C. A.; Robinson,
[23] Goss, S. P. A.; Hogg, N.; Kalyanaraman, B. The effect of -to-
K. A.; Jaffrey, F.; Floyd, R. A. Interaction of -phenyl-N-tert-
copherol on the nitration of -tocopherol by peroxynitrite. Arch.
butyl nitrone and alternative electron acceptors with complex I
Biochem. Biophys. 363:333 340; 1999.
indicates a substrate reduction site upstream from the rotenone
[24] Saldeen, T.; Li, D.; Mehta, J. L. Differential effects of alpha- and
binding site. J. Neurochem. 71:2549 2557; 1998.
gamma-tocopherol on low-density lipoprotein oxidation, super-
[4] Stadtman, E. R. Metal ion-catalyzed oxidation of proteins: bio-
oxide activity, platelet aggregation and arterial thrombogenesis.
chemical mechanism and biological consequences. Free Radic.
J. Am. Coll. Cardiol. 34:1208 1215; 1999.
Biol. Med. 9:315 325; 1990.
[25] Suzuki, Y. J.; Forman, H. J.; Sevanian, A. Oxidants as stimulators
[5] Stadtman, E. R.; Berlett, B. S. Fenton chemistry. Amino acid
of signal transduction. Free Radic. Biol. Med. 22:269  285; 1997.
oxidation. J. Biol. Chem. 266:17201 17211; 1991.
[26] Schreck, R.; Rieber, P.; Baeuerle, P. A. Reactive oxygen inter-
[6] Floyd, R. A. The role of 8-hydroxyguanine in carcinogenesis.
mediates as apparently widely used messengers in the activation
Carcinogenesis 11:1447 1450; 1990.
of the NF-kappa B transcription factor and HIV-1. EMBO J.
[7] Gille, J. J.; van Berkel, C. G.; Joenje, H. Mutagenicity of meta-
10:2247 2258; 1991.
bolic oxygen radicals in mammalian cell cultures. Carcinogenesis
[27] Schreck, R.; Albermann, K.; Baeuerle, P. A. Nuclear factor kappa
15:2695 2699; 1994.
B: an oxidative stress-responsive transcription factor of eukary-
[8] Halliwell, B. Oxygen and nitrogen are pro-carcinogens. Damage
otic cells (a review). Free Radic. Res. Commun. 17:221 237;
to DNA by reactive oxygen, chlorine and nitrogen species: mea-
surement, mechanism and the effects of nutrition. Mutat. Res. 1992.
443:37 52; 1999. [28] Bae, Y. S.; Kang, S. W.; Seo, M. S.; Baines, I. C.; Tekle, E.;
[9] Buettner, G. R. The pecking order of free radicals and antioxi- Chock, P. B.; Rhee, S. G. Epidermal growth factor (EGF)-induced
dants: lipid peroxidation, alpha tocopherol and ascorbate. Arch. generation of hydrogen peroxide. Role in EGF receptor-mediated
Biochem. Biophys. 300:535 543; 1993. tyrosine phosphorylation. J. Biol. Chem. 272:217 221; 1997.
1462 K. HENSLEY et al.
[29] Lee, S. R.; Kwon, K. S.; Kim, S. R.; Rhee, S. G. Reversible [46] Pogrebniak, H. W.; Merino, M. J.; Hahn, S. M.; Mitchell, J. B.;
inactivation of protein-tyrosine phosphatase 1B in A431 cells Pass, H. I. Spin trap salvage from endotoxemia: the role of
stimulated with epidermal growth factor. J. Biol. Chem. 273: cytokine down-regulation. Surgery 112:130 139; 1992.
15366 15372; 1998. [47] Miyajima, T.; Kotake, Y. Spin trapping agent, phenyl N-tert-butyl
[30] Robinson, K.; Stewart, C. A.; Pye, Q. N.; Nguyen, X.; Kenney, nitrone, inhibits induction of nitric oxide synthase in endotoxin-
L.; Salsman, S.; Floyd, R. A.; Hensley, K. Redox sensitive protein induced shock in mice. Biochem. Biophys. Res. Commun. 215:
phosphatase activity regulates the phosphorylation state of p38 114 121; 1995.
protein kinase in primary astrocyte culture. J. Neurosci Res. [48] Sang, H.; Wallis, G. L.; Stewart, C. A.; Kotake, Y. Expression of
55:724 732; 1999. cytokines and activation of transcription factors in lipopolysac-
[31] Sundaresan, M.; Yu, Z. X.; Ferrans, V. J.; Irani, K.; Finkel, T. charide-administered rats and their inhibition by phenyl N-tert-
Requirement for generation of H2O2 for platelet-derived growth
butylnitrone (PBN). Arch. Biochem. Biophys. 363:341 348;
factor signal transduction. Science 270:296 299; 1995.
1999.
[32] Denu, J. M.; Tanner, K. G. Specific and reversible inactivation of
[49] Stewart, C. A.; Hyam, K.; Wallis, G.; Sang, H.; Robinson, K. A.;
protein tyrosine phosphatases by hydrogen peroxide: evidence for
Floyd, R. A.; Kotake, Y.; Hensley, K. Phenyl-N-tert-butylnitrone
a sulfenic acid intermediate and implications for redox regulation.
demonstrates broad-spectrum inhibition of apoptosis-associated
Biochemistry 37:5633 5642; 1998.
gene expression in endotoxin-treated rats. Arch. Biochem. Bio-
[33] Barrett, W. C.; DeGnore, J. P.; Konig, S.; Fales, H. M.; Keng,
phys. 365:71 74; 1999.
Y. F.; Zhang, Z. Y.; Yim, M. B.; Chock, P. B. Regulation of
[50] Hensley, K.; Williamson, K.; Gabbita, S. P.; Grammas, P.; Floyd,
PTP1B via glutathionylation of the active site cysteine 215. Bio-
R. A. Determination of biological oxidative stress using high
chemistry 18:6699 6705; 1999.
performance liquid chromatography with electrochemical detec-
[34] Robinson, K. A.; Stewart, C. A.; Pye, Q. N.; Floyd, R. A.;
tion (HPLC-ECD). J. High Res. Chromatogr. 22:429 437; 1999.
Hensley, K. Basal protein phosphorylation is decreased and phos- [51] Hensley, K.; Maidt, M. L.; Yu, Z. Q.; Sang, H.; Markesbery,
phatase activity increased by an antioxidant and a free radical trap
W. R.; Floyd, R. A. Electrochemical analysis of protein nitroty-
in primary rat glia. Arch. Biochem. Biophys. 365:211 215; 1999.
rosine and dityrosine in the Alzheimer brain indicates region-
[35] Hensley, K.; Carney, J. M.; Stewart, C. A.; Tabatabaie, T.; Pye,
specific accumulation. J. Neurosci. 18:8126 8132; 1998.
Q. N.; Floyd, R. A. Nitrone-based free radical traps as neuropro-
[52] Hensley, K.; Maidt, M. L.; Pye, Q. N.; Stewart, C. A.; Wack, M.;
tective agents in cerebral ischemia and other pathologies. Int. Rev.
Tabatabaie, T.; Floyd, R. A. Quantitation of protein-bound 3-ni-
Neurobiol. 40:299 317; 1997.
trotyrosine and 3,4-dihydroxyphenylalanine by high-performance
[36] Baldassare, J. J.; Bi, Y.; Bellone, C. J. The role of p38 mitogen-
liquid chromatography with electrochemical array detection.
activated protein kinase in IL-1 beta transcription. J. Immunol.
Anal. Biochem. 251:187 195; 1997.
162:5367 5673; 1999.
[53] Arterbery, V. E.; Pryor, W. A.; Jiang, L.; Sehnert, S. S.; Foster,
[37] Bhat, N. R.; Zhang, P.; Lee, J. C.; Hogan, E. L. Extracellular
W. M.; Abrams, R. A.; Williams, J. R.; Wharam, M. D. Jr.; Risby,
signal-regulated kinase and p38 subgroups of mitogen-activated
T. H. Breath ethane generation during clinical total body irradi-
protein kinases regulate inducible nitric oxide synthase and tumor
ation as a marker of oxygen-free-radical-mediated lipid peroxi-
necrosis factor gene expression in endotoxin-stiumulated pri-
dation: a case study. Free Radic. Biol. Med. 17:569 576; 1994.
mary glial cultures. J. Neurosci. 18:1633 1641; 1998.
[54] Montine, T. J.; Beal, M. F.; Cudkowicz, M. E.; O Donnell, H.;
[38] Da Silva, J.; Pierrat, B.; Mary, J. L.; Lesslauer, W. Blockade of
Margolin, R. A.; McFarland, L.; Bachrach, A. F.; Zackert, W. E.;
p38 mitogen-activated protein kinase pathway inhibits inducible
Roberts, L. J.; Morrow, J. D. Increased CSF F2-isoprostane
nitric oxide synthase expression in mouse astrocytes. J. Biol.
concentration in probable AD. Neurology 52:562 565; 1999.
Chem. 272:28373 28380; 1997.
[55] Roberts, L. J. II; Montine, T. J.; Markesbery, W. R.; Tapper,
[39] Hensley, K.; Floyd, R. A.; Zheng, N.-Y.; Nael, R.; Robinson,
A. R.; Hardy, P.; Chemtob, S.; Dettbarn, W. D.; Morrow, J. D.
K. A.; Nguyen, X.; Pye, Q. N.; Stewart, C. A.; Geddes, J.;
Formation of isoprostane-like compounds (neuroprostanes) in
Markesbery, W. R.; Patel, E.; Johnson, G. V. W.; Bing, G. p38
vivo from docosahexaenoic acid. J. Biol. Chem. 273:13605
kinase is activated in Alzheimer disease brain. J. Neurochem.
13612; 1998.
72:2053 2058; 1999.
[56] Sano, M.; Ernesto, C.; Thomas, R. G.; Klauber, M. R.; Schafer,
[40] Smith, M. A.; Harris, P. L. R.; Sayre, L. M.; Beckman, J. S.;
K.; Grundman, M.; Woodbury, P.; Growdon, J.; Cotman, C. W.;
Perry, G. Widespread peroxynitrite-mediated damage in Alzhei-
Pfeiffer, E.; Schneider, L. S.; Thal, L. J. A controlled trial of
mer s disease. J. Neurosci. 17:2653 2657; 1997.
selegiline, alpha-tocopherol, or both as treatment for Alzheimer s
[41] Walton, K. M.; DiRocco, R.; Bartlett, B. A.; Koury, E.; Marcy,
disease. The Alzheimer s Disease Cooperative Study. N. Engl.
V. R.; Jarvis, B.; Shaefer, E. M.; Bhat, R. V. Activation of p38
J. Med. 336:1216 1222; 1997.
MAPK in microglia after ischemia. J. Neurochem. 70:1764
1767; 1998.
[42] Tanaka, K.; Shirai, T.; Nagata, E.; Dembo, T.; Fukuuchi, Y.
ABBREVIATIONS
Immunohistochemical detection of nitrotyrosine in postischemic
cortex in gerbil. Neurosci. Lett. 235:85 88; 1997.
[43] Eliasson, M. J.; Huang, Z.; Ferrante, R. J.; Sasamata, M.; Mol-
RNS reactive nitrogen species
liver, M. E.; Snyder, S. H.; Moskowitz, M. A. Neuronal nitric
ROS reactive oxygen species
oxide synthase activation and peroxynitrite formation in ischemic
stroke linked to neural damage. J. Neurosci. 19:5910 5918; PBN phenyl-tert-butylnitrone
1999.
NAC N-acetyl cysteine
[44] Oliver, C. N.; Starke-Reed, P. E.; Stadtman, E. R.; Liu, G. J.;
-toc alpha tocopherol
Carney, J. M.; Floyd, R. A. Oxidative damage to brain proteins,
loss of glutamine synthetase activity, and production of free -toc gamma;-tocopherol
radicals during ischemia/reperfusion-induced injury to gerbil
IL1  interleukin-1
brain. Proc. Natl. Acad. Sci. USA 87:5144 5147; 1990.
p38MAPK p38-mitogen activated protein kinase
[45] Carney, J. M.; Starke-Reed, P. E.; Oliver, C. N.; Landum, R. W.;
Cheng, M. S.; Wu, J. F.; Floyd, R. A. Reversal of age-related PTP protein tyrosine phosphatase
increase in brain protein oxidation, decrease in enzyme activity,
HPLC-ECD high performance liquid chromatography
and loss in temporal and spatial memory by chronic administra-
with electrochemical detection
tion of the spin-trapping compound N-tert-butyl-alpha-phenylni-
trone. Proc. Natl. Acad. Sci. USA 88:3633 3636; 1991. AD Alzheimer s disease