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Vol. 53, Issue 1, 135-159, March 2001
Institute of Pharmacology, University of Messina, Messina, Italy (S.C., A.P.C.); and Metaphore Pharmaceuticals, St. Louis, Missouri (D.P.R., D.S.)
Abstract
I. Introduction
A. Oxygen Radical
B. Nitrogen Species
II. DNA Damage
III. Poly(ADP-Ribose) Synthetase
IV. Relative Importance of Reactions of Glutathione with Nitric Oxide, Oxyradicals, and Peroxynitrite in Endotoxic Shock and Inflammation
V. Superoxide Dismutase
VI. Radical Generation
A. In Ischemia/Reperfusion
B. In Shock and Inflammation
VII. Pharmacological Intervention to Reduce Reactive Oxygen Species Generation in Shock, Inflammation, and Ischemia/Reperfusion
A. Peroxynitrite Decomposition Catalysts as Anti-inflammatory Agents
B. Catalytic Antioxidants
C. Metalloporphyrins
1. Effects of Metalloporphyrins in Inflammation.
2. Effect of Metalloporphyrins in Endotoxic and Hemorrhagic Shock.
3. Limitations of Metalloporphyrins.
D. New Rational Synthetic Enzymes: Manganese(II)-Based Superoxide Dismutase Mimics
1. Characterization of Superoxide Dismutase Activity.
2. Catalyst/Drug Design.
3. Anti-Inflammatory Activity of Superoxide Dismutase Mimics.
4. Attenuation of Myocardial Ischemia/Reperfusion Injury by Superoxide Dismutase Mimics.
VIII. Conclusions and Future Directions
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A vast amount of circumstantial evidence implicates oxygen-derived free radicals (especially superoxide and hydroxyl radical) and high-energy oxidants (such as peroxynitrite) as mediators of inflammation, shock, and ischemia/reperfusion injury. The aim of this review is to describe recent developments in the field of oxidative stress research. The first part of the review focuses on the roles of reactive oxygen species (ROS) in shock, inflammation, and ischemia/reperfusion injury. The second part of the review deals with the novel findings using recently identified pharmacological tools (e.g., peroxynitrite decomposition catalysts and selective superoxide dismutase mimetics (SODm) in shock, ischemia/reperfusion, and inflammation. 1) The role of ROS consists of immunohistochemical and biochemical evidence that demonstrates the production of ROS in shock, inflammation, and ischemia/reperfusion injury. ROS can initiate a wide range of toxic oxidative reactions. These include initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane sodium/potassium ATPase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation, and ischemia/reperfusion. 2) Treatment with either peroxynitrite decomposition catalysts, which selectively inhibit peroxynitrite, or with SODm, which selectively mimic the catalytic activity of the human superoxide dismutase enzymes, have been shown to prevent in vivo the delayed vascular decompensation and the cellular energetic failure associated with shock, inflammation, and ischemia/reperfusion injury. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical, and hydrogen peroxide) are all potential reactants capable of initiating DNA single-strand breakage, with subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase, leading to eventual severe energy depletion of the cells and necrotic-type cell death. Antioxidant treatment inhibits the activation of poly(ADP-ribose) synthetase and prevents the organ injury associated with shock, inflammation, and ischemia/reperfusion.
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I. Introduction |
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A. Oxygen Radical
A free radical is defined as any atom or molecule possessing
unpaired electrons. Molecular oxygen, O2, is a
biradical with two such unpaired electrons. The biologically relevant
free radicals derived from oxygen are the superoxide anion
(O
2), the perhydroxyl radical (protonated superoxide,
HO2), the hydroxyl radical (HO·), and free radical
nitric oxide (NO·). The one electron reduction of oxygen (i.e.,
the addition of one electron to an oxygen molecule) results in
formation of O2 (also known as the
superoxide radical), whereas the two-electron reduction product of
oxygen, when fully protonated, forms hydrogen peroxide
(H2O2). A third species of
activated oxygen, known as singlet oxygen, is recognized as a possible
contributor to oxidative stress in living systems. Singlet oxygen is a
high-energy, electron-spin paired state of dioxygen that is
approximately 1 eV higher in energy than ground-state triplet oxygen
and is capable of oxidizing a number of biological molecules, including
lipid- and olefinic-containing molecules. Finally, an additional
reduction product of oxygen, HO·, is the most reactive and least
selective of all the oxy radical oxidizing agents.
It was believed initially that the toxicity ascribed to the
superoxide radical was caused by superoxide's direct interaction with
biological targets. It is now clear that many tissue effects of
O2 result from the secondary formation of other oxygen
radicals in addition to direct reactions of superoxide (or its
conjugate acid) with biological targets, such as lipids (Aikens and
Dix, 1991; Dix and Aikens, 1993
), catecholamines (Misra and Fridovich, 1972; Heikkila and Cohen, 1973; Rao and Hayon, 1975; Macarthur et al.,
2000), and DNA (Dix et al., 1996). Superoxide in aqueous media
undergoes a spontaneous second-order reaction with itself, a
dismutation reaction that yields one molecule each of
H2O2 and oxygen (see
reaction 1, Table 1) in a relatively slow
reaction at pH 7.4 (the second-order rate constant is of the order of
104.5) when compared with the rate at which
superoxide or HO2 can abstract an H-atom from such key
biological targets as catecholamines or the allylic CH in lipid where
the second-order rate constant exceeds 107.
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Although the dismutation would be spontaneous at physiological pH at
high superoxide concentrations, the concentration of superoxide
approaches 10 µM (physiological) as the self-reaction slows down
considerably and its lifetime becomes extended to many seconds.
Consequently, nature has evolved a class of superoxide dismutase
(SOD2) enzymes to
remove this deleterious free radical byproduct of oxygen metabolism.
These enzymes can react rapidly with superoxide (rates approaching or
exceeding 109) and dismutate the radical to the
nonradical products, O2 and H2O2, faster than
superoxide can react with other potential biological targets. The short
half-life should not be misinterpreted as mitigating the potential
reactivity of O2 because the half-life is actually quite long
in relation to the phenomenal diffusion coefficient of the radical.
Given that superoxide can interact with a variety of biological target
molecules, the reaction with the enzyme literally can shunt the
superoxide production into
H2O2 and oxygen. Thus, it
is conceivable that, in vivo, the presence of the highly active SOD
enzymes will lead to an increase in the local concentration of
H2O2.
The most reactive oxy radical is HO2. It was proposed many
years ago that it could be produced from the interaction of O2 and H2O2 by a chemical
process known as the Haber-Weiss reaction (reaction 2, Table 1).
However, detailed studies of the rate of this reaction have shown that
it could not take place under physiological conditions. An alternative
explanation, which is now widely accepted, is that trace amounts of
metal ions, primarily ferrous ion, react with
H2O2 in what is known as
the iron-catalyzed Fenton reaction to produce the hydroxyl radical.
Normally, ferrous ion is not present in vivo, but it is produced by the
action of superoxide on ferric ion (reactions 3 and 4, Table 1) present in iron storage proteins; thus, liberating soluble ferrous ion. There
is considerable debate as to whether protein-bound metal ions (e.g.,
lactoferrin, hemoglobin, etc.) catalyze this reaction to any great
degree. Sensitive measurements of the free (unbound) iron concentration
in tissues, such as synovial fluid, have been reported to show a
sufficient concentration to catalyze reactions (3) and (4) in an
inflamed joint. Finally, recent attention has been drawn to what is
called "site-specific" hydroxyl radical formation (Czapski et al.,
1983), wherein an iron ion bound to a macromolecule catalyzes
HO· generation at the actual site on the substrate where
cleavage eventually ensues.
In summary, tissue toxicity from extracellular superoxide generation seems to be based on its direct reactivity with numerous types of biological molecules (lipid, DNA, RNA, catecholamines, steroids, etc.) and from its dismutation to form H2O2 and the concomitant reduction of ferric ion to ferrous ion; reaction of these two products yields the highly toxic hydroxyl radical that may cleave covalent bonds in proteins and carbohydrates, cause lipid peroxidation, and destroy cell membranes. There are three strategies available to "detoxify" or prevent formation of locally produced oxygen radicals: 1) deliver SOD or an SODm to the area; 2) deliver catalase or a related peroxide scavenger, or 3) chelate (and thereby inactivate) the trace iron that catalyzes the reaction.
B. Nitrogen Species
NO· is synthesized from the guanidino group of
L-arginine by a family of enzymes termed NO·
synthases (NOSs). Three isoforms have been described and cloned: endothelial cell NOS (ecNOS or type 3), brain NOS (bNOS, nNOS, or type 1), and inducible macrophage-type NOS (iNOS or type 2). All of
the NOS isoforms can be inhibited to varying degrees with N-substituted L-arginine analogs
(e.g., N-methyl-L-arginine). The
formation of NO· is linked to the incorporation of
O2 into the molecule. All NOS isoforms are
dependent on NADPH and calmodulin. In iNOS, calmodulin is present in a
tightly bound form; thus iNOS produces NO· in a sustained manner
in the presence of adequate substrate (Geller and Billiar, 1998;
Marletta, 1993; Stuehr, 1997). Many of the biological actions of
NO· are mediated through the guanylyl cyclase/cyclic GMP (cGMP)
system. NO·, a lipophilic small molecule, diffuses to adjacent
cells and readily enters the cytosol, where it activates soluble
guanylyl cyclase by binding to the iron on its heme component, thereby moving the iron out of the plane of the porphyrin ring. Increased levels of cGMP trigger a reduction of calcium concentration by enhancing extrusion of calcium and its sequestration into intracellular stores. The decrease in intracellular calcium concentration is responsible for the NO·-mediated relaxation of vascular and
nonvascular smooth muscle, inhibition of platelet adherence and
aggregation, inhibition of neutrophil chemotaxis, and signal
transduction in the central and peripheral nervous systems (Ignarro,
1991; Moncada et al., 1991; Dusting, 1995). It is now well established
that NO· also has several cGMP-independent actions. The
cytotoxic effects of NO· (in high local concentrations) involves
the inhibition of key mitochondrial iron-sulfur enzymes, including
NADH:ubiquinone oxidoreductase, NADH:succinate oxidoreductase, and
aconitase (Nathan, 1992). cGMP-independent activation by NO of other
enzymes, such as cyclooxygenase, has also been described. This action
may be related to the reaction of NO· with the iron-heme center
at the active site of the enzyme (Salvemini and Masferrer, 1996).
NO· inhibits the activity of cytochrome P-450 enzymes (Khatsenko et al., 1993). NO· may modulate gene transcription and
translation: in endothelial cells, it activates c-fos (Felley-Bosco et
al., 1994), whereas in neurons it potentiates the effect of calcium on
gene expression linked to the c-fos promoter (Peunova and Enikolopov,
1993). Many inflammatory conditions are associated with production of
comparatively large amounts of NO·, produced by iNOS, with
consequent cytotoxic effects. iNOS, first identified in macrophages,
can be expressed in essentially any cell type. Although constitutive
expression of iNOS has been localized to the kidney, the intestine, and
the bronchial epithelia, iNOS is expressed typically in response to
immunological stimuli and produces nanomoles, rather than picomoles, of
NO·. Once produced in high local concentrations, NO· may
act as cytostatic and cytotoxic molecules for fungal, bacterial, helminthic, and protozoal organisms, as well as tumor cells. Bacterial lipopolysaccharide and a variety of proinflammatory cytokines also
induce the expression of iNOS in a number of nonhematopoietic cells,
including fibroblasts, glial cells, and cardiac myocytes, as well as
vascular and nonvascular smooth muscle cells (Nathan, 1992). iNOS
produces large amounts of NO· for prolonged periods. The
expression of iNOS is regulated both at the level of transcription and
at the level of iNOS mRNA stability. The mechanism of iNOS induction
involves de novo transcription and the biosynthesis of new protein.
Induction of iNOS can be inhibited by numerous agents, including
glucocorticoids, thrombin, macrophage deactivation factor, tumor growth
factor-
, platelet-derived growth factor, interleukin (IL)-4, IL-8,
IL-10, and IL-13. Induction of iNOS may have either toxic or protective
effects. Factors that seem to dictate the consequences of iNOS
expression include the type of insult, the tissue type, the level and
duration of iNOS expression, and probably the redox status of the
tissue. Much attention has focused on the toxicity of iNOS. For
example, induction of iNOS in endothelial cells produces endothelial
injury (Palmer et al., 1992). Induction of iNOS has been shown to
inhibit cellular respiration in macrophages and vascular smooth muscle
cells; these processes can lead to cell dysfunction and cell death.
Such processes, when occurring within vascular smooth muscle cells,
play a key role in the pathogenesis of the vascular hyporeactivity and
progressive vascular decompensation associated with various forms of
circulatory shock (Szabó, 1995). In clear contrast, expression of
iNOS in liver cells suppressed endotoxin and tumor growth
factor-
-induced toxicity (Kim et al., 1997; Ou et al., 1997).
Overexpression of iNOS by gene transfer also limits lipopolysaccharide
(LPS)-induced toxicity in endothelial cells (Tzeng et al., 1997).
Simultaneous generation of NO· and O2 favors the
production of a toxic reaction product, peroxynitrite anion
(ONOO
) (Beckman et al., 1990), and this product
may account for some of the deleterious effects associated with
NO· production. This peroxynitrite-forming reaction has since
been shown to be diffusion controlled
(kobs, = 6.7 × 109 M1
s1), indicating that competition of NO·
with SOD for superoxide is feasible (Huie and Padmaja, 1993).
Beckman noted that peroxynitrite production increases as the square of
the fluxes of these precursors. Moreover, certain forms of SOD enzymes
are inactivated by reaction with peroxynitrite, and this can create
positive feedback for ONOO formation
(Ischiropoulos et al., 1992a; Beckman et al., 1994a). Hence, it is
reasonable to conclude that peroxynitrite overproduction may occur
readily in vivo. Once near or inside a cell,
ONOO can damage or deplete a number of vital
components [e.g., DNA by strand scission (King et al., 1992; Groves
and Marla, 1995; Groves et al., 1996), lipids by peroxidation (Radi et
al., 1991a; Rubbo et al., 1994), aconitase (Castro et al., 1994;
Hausladen and Fridovich, 1994), and antioxidant availability (Van der
Vliet et al., 1994; Vasquez-Vivar et al., 1996)].
A considerable portion of the toxic effects previously attributed to
NO· or O2 alone may in fact be modulated by
peroxynitrite (Table 2). The resulting
oxidative stress may cause cell death and tissue damage that
characterize a number of human disease states, among them neurological
disorders and stroke, inflammatory bowel disease, arthritis, toxic
shock, and acute reperfusion injuries. In fact, recent studies suggest
that peroxynitrite, and not NO·, may be the ultimate cytotoxic
species in many conditions (Castro et al., 1994; Hausladen and
Fridovich, 1994; Szabó et al., 1996a).
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In cells exposed to exogenous peroxynitrite or to compounds that
simultaneously generate NO· and superoxide, marked changes in
the level of cellular energetics and DNA integrity occur. For instance,
in pulmonary type II cells, inhibition by peroxynitrite of sodium
uptake has been reported (Hu et al., 1994). Mitochondrial respiration
is profoundly inhibited by peroxynitrite in neurons and glial cells
(Bolanos et al., 1995), cultured monocytic macrophages (Szabó and
Salzman, 1995; Szabó et al., 1996b), and cultured rat aortic
smooth muscle cells (Szabó et al., 1996b). Although a decrease in
the activity of succinate-cytochrome c reductase and
cytochrome c oxidase was found in neurons exposed to
peroxynitrite, only cytochrome c oxidase was affected in
isolated mitochondria exposed to peroxynitrite. These findings suggest the contribution of secondary cellular pathways to the toxicity of
peroxynitrite (Bolanos et al., 1995). Inactivation of mitochondrial enzymes increases the amounts of
H2O2 generated by the
mitochondria (Radi et al., 1994), which may further contribute to
cellular injury, in an additive or synergistic fashion.
Similarly, in macrophages (Szabó and Salzman, 1995), smooth
muscle cells (Szabó et al., 1996b), and neurons (Heales et al., 1994), immunostimulation leads to the inhibition of mitochondrial respiration. This inhibition is due to peroxynitrite, rather than "pure" NO· formation, because the suppression of cell
respiration can be restored by both NOS inhibitors and by superoxide or
peroxynitrite scavengers.
Although exposure to high concentrations of peroxynitrite leads
to rapid cell death associated with rapid energetic derangements, lower
concentrations of peroxynitrite can, after several hours, lead to
apoptotic cell death (Bonfoco et al., 1995; Estevez et al., 1995; Salgo
et al., 1995). In isolated tissues and organs, peroxynitrite elicits a
variety of alterations. Peroxynitrite infusion causes a reduction in
myocardial contractility in isolated perfused hearts (Schulz et al.,
1995) and induces an impairment of the endothelium-dependent relaxant
ability (Villa et al., 1994). The finding that the development of this
endothelial dysfunction can be prevented by NO· donors (Villa et
al., 1994) supports the notion that toxic acute effects are due to
OONO formation (Moro et al., 1994, 1995).
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II. DNA Damage |
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Free radical-mediated reactions can cause structural alterations
in DNA (e.g., nicking, base-pair mutations, rearrangements, deletions,
insertions, and sequence amplification). The endogenous reactions that
are likely to contribute to ongoing DNA damage are oxidation,
methylation, depurination, and deamination (Totter, 1980; Ames, 1989).
NO or, more likely, reactive products derived from it, such as
NO2, ONOO,
N2O3 and
HNO2, are mutagenic agents, with the potential to
produce nitration, nitrosation, and deamination reactions on DNA bases (Routledge et al., 1994). Methylation of cytosines in DNA is important for the regulation of gene expression, and normal methylation patterns
can be altered during carcinogenesis (Weitzman et al., 1994).
Conversion of guanine to 8-hydroxyguanine, a frequent result of
reactive oxygen species (ROS) attack (Halliwell and Aruoma, 1991;
Dizdaroglu, 1993; Box et al., 1995), has been found to alter the
enzyme-catalyzed methylation of adjacent cytosines (Weitzman et al.,
1994)
thus providing a link between oxidative DNA damage and altered
methylation patterns.
The chemistry of DNA damage by several ROS has been well characterized
in vitro (Steenken, 1989; Dizdaroglu, 1993; Epe, 1993; Box et al.,
1995), although specific information is needed about the changes
produced by peroxyl (RO2), alkoxyl (RO·), ozone
(O3), and several of the reactive nitrogen
species (RNS) (e.g., ONOO) is lacking.
Different ROS affect DNA in different ways [e.g., H2O2 does not react with
DNA bases at all (Halliwell and Aruoma, 1991; Dizdaroglu, 1993)],
whereas HO· generates a multiplicity of products from all four
DNA bases, and this pattern seems to be a diagnostic "fingerprint"
of HO· attack (Halliwell and Aruoma, 1991). By contrast
O2 selectively attacks guanine (Epe, 1993; Van den Akker et
al., 1994). The most commonly produced base lesion, and the one most
often measured as an index of oxidative DNA damage, is
8-hydroxyguanine. It is sometimes measured as the nucleoside,
8-hydroxydeoxyguanosine (Floyd et al., 1986; Ames, 1989). These assay
methods have been reviewed in detail (Floyd et al., 1986; Halliwell and
Aruoma, 1991; Halliwell and Dizdaroglu, 1992; Dizdaroglu, 1993).
Damage to DNA by ROS/RNS seems to occur naturally, in that low
steady-state levels of base damage products have been detected in
nuclear DNA from human cells and tissues (Floyd et al., 1986; Ames,
1989; Halliwell and Dizdaroglu, 1992; Richter, 1992; Musarrat and Wani,
1994). The pattern of damage to the purine and pyrimidine bases
suggests that at least some of the damage occurs by HO· attack,
suggesting that HO· is formed in the nucleus in vitro (Halliwell
and Dizdaroglu, 1992). ROS/RNS can also damage mitochondrial DNA, and
such damage has been suggested to be important in several human
diseases and in the aging process (Harman, 1992; Shigenaga et al.,
1994). Mitochondria are often said to be the most important
intracellular source of ROS, but it is difficult to unambiguously
confirm this postulate (Halliwell and Gutteridge, 1985). However, it
seems very likely that the mitochondrial electron transport chain
generates ROS in vivo (Ambrosio et al., 1993; Guidot et al., 1993) and
that mitochondrial DNA is damaged by them. The roles that ROS or RNS play in the DNA damage have not yet been completely elucidated. This
apparent increased net oxidative damage in mitochondrial DNA compared
with nuclear DNA could be because of the proximity of mitochondrial DNA
to ROS generated during electron transport, the lack of histone
proteins to protect the DNA against attack, or inefficient repair, so
that base damage accumulates to higher levels.
DNA damage can be repaired by the action of a series of enzymes
(Demple and Harrison, 1994). However, DNA from human cells and tissues
contains low levels of DNA base damage products (Ames, 1989; Malins and
Haimanot, 1991; Halliwell and Dizdaroglu, 1992; Bashir et al., 1993;
Jaruga et al., 1994; Adachi et al., 1995), suggesting that these
enzymes do not achieve complete removal of modified bases, perhaps
because they operate at close to maximum capacity in vivo. DNA damage
by ROS/RNS can cause multiple lesions, including single and double
strand breaks, apurinic/apyrimidinic sites and modified pyrimidines and
purines. Repair of these lesions occurs primarily by base excision
repair, although nucleotide excision repair may also be involved. A
repair system for the abasic apurinic/apyrimidinic sites produced by
spontaneous depurination also exists. Areas of current interest include
the role of poly(ADP-ribose) polymerase (PARP) in the rejoining of DNA
strand breaks, including those induced by ROS (Satoh et al., 1993;
Satoh and Lindahl, 1994).
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III. Poly(ADP-Ribose) Synthetase |
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Poly(ADP) synthetase (PARS) [also known as PARP or
poly(ADP-ribose) transferase] is a protein modifying and
nucleotide-polymerizing enzyme that is present abundantly in the
nucleus (Althaus and Richter, 1987; De Murcia and Menissier-De Murcia,
1994). The obligatory trigger of PARS activation is the nicks and
breaks in the DNA strand, which can be induced by a variety of
environmental stimuli and free radical (or oxidant) attacks; these
include the oxidants HO2, HO·, and
ONOO, ionizing radiation, and genotoxic agents,
such as
N-methyl-N'-nitro-N-nitrosoguanidine. The physiological function of PARS and poly(ADP-ribosylation) is still
under much debate. From studies using pharmacological inhibitors of
PARS, poly(ADP-ribosylation) has been suggested to regulate gene
expression and gene amplification, cellular differentiation and
malignant transformation, cellular division, and DNA replication, as
well as apoptotic cell death (Althaus and Richter, 1987; Lautier et
al., 1993; De Murcia and Menissier-De Murcia, 1994; Lindahl et al.,
1995; Wang et al., 1995; Simbulan-Rosenthal et al., 1996). However,
recent studies using cells from PARS(-/-) mice have failed to
demonstrate a role for PARS in the process of apoptosis induced by
various apoptotic signals, such as the Fas ligand or dexamethasone (De
Murcia et al., 1997; Morrison et al., 1997; Wang et al., 1995, 1997).
In the 1980s, Berger and Okamoto have observed rapid depletion of NAD+ due to PARS activation, leading to cellular ATP depletion, and functional alterations of the cell, with eventual necrotic-type cell death. The main cytotoxic triggers used in these studies in vitro were alkylating agents, radiation, and H2O2, whereas the most frequently used PARS inhibitors were nicotinamide, 3-aminobenzamide, and benzamide.
Research into the "suicidal" role of PARS gained new momentum in
the mid-1990s because of the observations in vitro that NO· or
peroxynitrite can trigger DNA single-strand breakage and PARS activation (Radons et al., 1994; Eliasson et al., 1997; Szabó et
al., 1996b). NO· and peroxynitrite can also inhibit
mitochondrial respiration and exert other cytotoxic effects on their
own. Thus, it is likely that a synergistic relationship exists between
the PARS-mediated pathways and PARS-independent pathways of cellular
metabolic suppression (Fig. 1).
Furthermore, the observations that NO· and peroxynitrite are
important mediators of the cellular damage in various forms of
inflammation and reperfusion injury suggest that the PARS-related
suicide pathway might play a role in various pathophysiological
conditions in vivo (Fig. 2).
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IV. Relative Importance of Reactions of Glutathione with Nitric Oxide, Oxyradicals, and Peroxynitrite in Endotoxic Shock and Inflammation |
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Glutathione is a known oxyradical scavenger (Darley-Usmar and
Halliwell, 1996). Moreover, glutathione can react with NO· to
form S-nitrosoglutathione, a vasodilator compound (Simon et al., 1993). Thus, theoretically, the mechanism of the observed vascular alterations in the BSO-pretreated cells, and tissues may be
related to peroxynitrite, oxyradicals, NO·, or the combination
of these. From data in the literature, it seems unlikely that a
glutathione-NO· reaction plays a major role in the observed
changes. This conclusion was based on the following considerations: 1)
in accordance with previous studies (Jia and Furchgott, 1993; Stamler,
1995), we found no differences in the endothelium-independent relaxant
effect of
S-nitroso-N-acetyl-DL-penicillamine
in control and BSO-treated animals, suggesting that the reduction in
endogenous glutathione does not affect NO-induced relaxant responses;
and 2) in vitro studies in macrophages and other cell types have
established that endogenous glutathione only protects against very high
(pharmacologically relevant) fluxes of NO·, but not against
lower levels of NO· production, such as the ones that are
relevant to the in vitro or in vivo conditions in our experiments
(Sakanashi et al., 1991; Walker et al., 1995). On the other hand, the
possibility that the enhancement of cytotoxicity in BSO-treated cells
or tissues after LPS treatment is related, in part, to increased
oxyradical-induced cytotoxic effects is a good possibility. Data
supports that depletion of endogenous glutathione enhances the
cytotoxic effects of H2O2 and oxyradicals, and we have also observed an enhancement of
H2O2 toxicity in
endothelial cells and smooth muscle cells (Cuzzocrea et al., 1998c). In
the experiments that involve LPS stimulation, it is conceivable that a
more pronounced inhibition of mitochondrial respiration by
oxygen-derived free radicals and oxidants can lead to a dysfunctional
electron transfer, with more superoxide production from the
mitochondria. This positive feedback cycle would also lead to an
enhancement of peroxynitrite production, with subsequent increased
cytotoxicity. It is noteworthy in this context that it is the
production of superoxide, not the production of NO·, that
represents the rate-limiting factor in the production of peroxynitrite
during endotoxemia (Szabó and Salzman, 1995).
It must be kept in mind that, in immunostimulated cells, the production
of various oxygen- and nitrogen-derived free radicals and oxidants
occurs in a simultaneous fashion. Therefore, it is conceivable that
important interactions exist between these various species in terms of
oxidative potential and cytotoxicity. For instance, in respect to
peroxynitrite-induced oxidative injury, it is well established that the
ratio of NO· and superoxide determines the oxidant capacity, and
excess NO· reduces peroxynitrite-induced oxidative processes
(Rubbo et al., 1994; Szabó and Salzman, 1995; Petit et al.,
1996). H2O2, on the other
hand, prolongs the half-life of peroxynitrite (Miles et al., 1996), and
synergizes with peroxynitrite in terms of cytotoxicity. Thus, it is
possible that the cytotoxic effects we observed in response to
immunostimulation represent the sum of a complex interaction between
various oxygen- and nitrogen-derived radicals and oxidants. Nevertheless, based on the similarities between the effects of exogenously added peroxynitrite and LPS treatment, and considering the
simultaneous protective effects of
N-methyl-L-arginine and tetrakis-(4-benzoic acid) porphyrin (MnTBAP) against the
vascular failure in response to LPS challenge (see above), we propose
that peroxynitrite, or a peroxynitrite-derived oxidant, contributes to
protein oxidation in response to immunostimulation.
Recent studies demonstrate that endogenous glutathione plays an
important role in reducing vascular hyporeactivity and endothelial dysfunction in response to peroxynitrite and endotoxic shock, as well
as in acute inflammation. In fact we have shown that BSO-treated rats
developed a significant inflammatory response, as compared with the
animals that have a normal glutathione system. These findings are in
agreement with previous suggestions that glutathione plays an important
role in blocking the oxidant-induced injury and, specifically, in
blocking the peroxynitrite-induced injury (Karoui et al., 1996;
Cuzzocrea et al., 1998c). A variety of additive or synergistic
cytotoxic processes triggered by peroxynitrite may contribute to acute
and delayed cytotoxicity, and depletion of glutathione may also
interfere with these pathways. This points out the importance of intact
glutathione pools, as protective mechanisms against the vascular
failure under conditions of oxidant stress, shock, and inflammation.
There are several ways to improve glutathione status and/or replenish
cellular glutathione stores. For instance, cell-permeable glutathione
analogs have been described (Morris et al., 1995). These strategies may
represent alternative or additional approaches to other approaches
directed toward the prevention of the loss of vascular patency in shock
and inflammation.
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V. Superoxide Dismutase |
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Under normal circumstances, formation of O2 (the
one-electron reduction product of oxygen) is kept under
tight control by SOD enzymes. These include the Mn enzyme in
mitochondria (SOD2) and Cu/Zn enzyme present in the cytosol (SOD1) or
extracellular surfaces (SOD3). The importance of SOD2 is highlighted by
the findings that, in contrast to SOD1 (Reaume et al., 1996) and SOD3 (Carlsson et al., 1995), SOD2 knockout is lethal to mice
(Lebovitz et al., 1996; Melov et al., 1999). In acute and
chronic inflammation, the production of O2 is increased at a
rate that overwhelms the capacity of the endogenous SOD enzyme defense
system to remove them. The result of such imbalance results in
O2-mediated damage. A proposal that O2 was intimately
involved with the inflammatory response was raised as early as the
1970s through the pioneering work of McCord and Fridovich (McCord and
Fridovich, 1969). Some important proinflammatory roles for O2
(Fig. 3) include endothelial cell damage
and increased microvascular permeability (Droy-Lefaix et al., 1991;
Haglind et al., 1994), formation of chemotactic factors such as
leukotriene B4 (Fantone and Ward, 1982; Deitch et
al., 1990), recruitment of neutrophils at sites of inflammation (Boughton-Smith et al., 1993; Salvemini et al., 1996a, 1999), lipid
peroxidation and oxidation, DNA single-strand damage (Dix et al.,
1996), and formation of ONOO (Beckman et al., 1990;
Ischropoulos et al., 1992a; Crow and Beckman, 1995; Salvemini et al.,
1998, 1999). Most of the knowledge gathered about the roles of
superoxide in disease has been collected by the use of the native SOD
enzyme and, more recently, by data generated in transgenic animals that
overexpress the human enzyme (Huber et al., 1980; Uematsu et al., 1994;
Fridovich, 1995).
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VI. Radical Generation |
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A. In Ischemia/Reperfusion
Following ischemia, superoxide is produced during the reperfusion
phase, and it rapidly reacts with NO· and forms
ONOO. This has been demonstrated in the heart
(Matheis et al., 1992; Naseem et al., 1995; Schulz and Warnbolt, 1995),
liver (Ma et al., 1995), kidney (Yu et al., 1994), intestine
(Szabó et al., 1995a), brain (Cazevielle et al., 1993; Fagni et
al., 1994; Gunasekar et al., 1995), and lung (Ischiropoulos et al.,
1995; Kooy et al., 1995). Under these conditions, prevention of
ONOO generation by inhibition of NO·
biosynthesis markedly reduces reperfusion injury, as shown by reduced
pulmonary lipid peroxidation (Ischiropoulos et al., 1995) or improved
myocardial mechanical performance (Schulz and Warnbolt, 1995).
A growing body of evidence supports a role for
ONOO and other reactive species in neuronal
injury associated with ischemia/reperfusion injury in the central
nervous system. The original proposition (Beckman, 1991), that
ONOO (and not NO· or O2
independently) is a major cytotoxic mediator in the neuronal injury
during stroke and N-methyl-D-aspartic
acid (NMDA) receptor activation, was based on theoretical
considerations and previous evidence showing that reperfusion injury in
the central nervous system is associated with activation of NMDA
receptors, which then triggers the production of O2 and
NO·. There is now indirect evidence to show that NMDA receptor
activation is associated with a marked increase in HO·-like
activity in the brain (blocked by inhibition of NOS), which is
presumably due to ONOO generation (Hammer et
al., 1993). The involvement of O2 and the protective effect of
O2 neutralizing strategies (Cazevielle et al., 1993; Dawson et
al., 1993; Lafon-Cazal et al., 1993; Fagni et al., 1994; Beal et al.,
1995; Crow and Beckman, 1995; Dawson, 1995; Gunasekar et al., 1995), as
well as the involvement of NO· and the protective effect of NOS
inhibition (Huang et al., 1994; Smith et al., 1994; Schulz et al.,
1995; Zielasek et al., 1995), has been well established in various
forms of central nervous system injury.
Similar to inflammation and shock, the mechanism of
ONOO-induced cellular damage in the
ischemia/reperfusion remains a subject for future investigations, but
presumably involves multiple mechanisms. Both in vivo and in vitro
evidence clearly suggest the involvement of PARS in the neuronal damage
associated with NO· (or ONOO) production
in response to NMDA receptor activation (Wallis et al., 1993; Cosi et
al., 1994; Zhang et al., 1994, 1995).
Endothelial cells appear to be major regulators of neutrophil traffic,
regulating the process of neutrophil chemoattraction, adhesion, and
migration from the vasculature to the tissue. During the early phase of
reperfusion, P-selectin is rapidly released to the cell surface
from preformed storage pools after exposure to certain stimulisuch as
H2O2, histamine, or
complementand allows the leukocytes to roll along the endothelium
(Geng et al., 1990). ICAM-1, constitutively expressed on the surface of
endothelial cells, is then involved in neutrophil adhesion (Geng et
al., 1990; Farhood et al., 1995). Hypoxic endothelial cells synthesize
proinflammatory cytokines, which can up-regulate endothelial expression
of the constitutive adhesion molecule ICAM-1 in autocrine fashion
(Shreeniwas et al., 1992). The expression of P-selectin and ICAM-1
corresponds with the induction of neutrophil recruitment, which is
maximal within the first hour of reperfusion, and persists at a lower rate in the late phase of reperfusion (Clark et al., 1995). In accordance with these findings, it has been demonstrated that ischemia
and reperfusion induced the appearance of P-selectin on the endothelial
vascular wall and up-regulated the surface expression of ICAM-1 on
endothelial cells. Thus, it has been hypothesized that oxidative and
nitrosative damage in ischemia/reperfusion and shock requires the
presence of ROS, which are mainly produced from the massive neutrophil
infiltration (Cuzzocrea et al., 1998c).
B. In Shock and Inflammation
Important cardiovascular consequences of circulatory shock include
reduced responsiveness of arteries and veins to exogenous or endogenous
vasoconstrictor agents (vascular hyporeactivity), myocardial
dysfunction, and disrupted intracellular energetic processes. Recent
studies prompted these conclusions based mainly on results obtained
with the use of NOS inhibitors, but they did not or could not
distinguish between the effects of NO· versus
ONOO. Recent data demonstrate that authentic
ONOO is capable of mimicking many of the
cardiovascular alterations associated with shock (endothelial
dysfunction, vascular hyporeactivity, myocardial failure, and cellular
energetic failure) (see above). In circulatory shock, proinflammatory
cytokines invoke a pleiotropic cellular response, including the
stimulation of oxygen-centered free radicals, such as O2. The
majority of NO· produced by macrophages is converted to
ONOO (Ischiropoulos et al., 1992b). The
production of ONOO (evidenced by increased
nitrotyrosine immunoreactivity or increased oxidation of the
fluorescent probe dihydrorhodamine 123 to rhodamine 123) has recently
been demonstrated in endotoxic shock and in hemorrhagic shock (Wizemann
et al., 1994; Szabó et al., 1995b).
A large number of studies demonstrate the protective effect of SOD in
various models of endotoxic and hemorrhagic shock and splanchnic artery
occlusion/reperfusion injury (McKechnie et al., 1986; Wang et al.,
1990; Rhee et al., 1991; Youn et al., 1991; McCord, 1993; Kapoor and
Prasad, 1995; Salvemini et al., 1999). Furthermore, there is a large
amount of evidence to show that the production of reactive species
such as O2, H2O2,
and HO· occurs at the site of inflammation and contributes
to tissue damage (Salvemini et al., 1996a; Cuzzocrea et al., 1997).
Inhibitors of NOS activity reduce the severity of inflammation and
support a role for NO· in the pathophysiology associated with
various models of inflammation (Tracey et al., 1995; Wei et al., 1995;
Salvemini et al., 1996b; Cuzzocrea et al., 1997). In addition to
NO·, ONOO is also generated during
inflammation damage (Salvemini et al., 1996b; Cuzzocrea et al., 1997).
The involvement of ONOO in these conditions is
strongly supported by direct measurements. For example, in arthritis,
nitrotyrosine levels increase in plasma and synovial fluid (Kaur and
Halliwell, 1994). In ileitis (Miller et al., 1995) and in
endotoxin-induced intestinal inflammation (Chamulitat et al., 1996),
there is immunocytochemical documentation (increased nitrotyrosine
immunoreactivity in the inflamed tissues) of augmented
ONOO production (Fig.
4).
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The pathophysiological role of NO· and
ONOO in the gastrointestinal damage elicited by
endotoxin or chronic inflammation has been the subject of a variety of
detailed investigations. The ability of authentic
ONOO to cause severe colonic inflammation has
been documented (Rachmilewitz et al., 1993). The production of
ONOO in colitis may be even more pronounced
because of the parallel down-regulation of SOD (Seo et al., 1995),
which makes the O2 available for coupling with NO·.
Desferrioxamine, a putative peroxynitrite scavenger (Denicola et al.,
1995), or SOD protects against the gastric damage elicited by
NO· donors, supporting the view that peroxynitrite (and not
NO· per se) is the cytotoxic species in these models (Lamarque
and Whittle, 1995). Recent investigations have also concluded that inhibition of PARS exerts beneficial effects in shock (Szabó et
al., 1997), reperfusion injury (Zhang et al., 1994; Zingarelli et al.,
1996; Cuzzocrea et al., 1997; Thiemermann et al., 1997), and
inflammation (Szabó et al., 1997, 1998; Cuzzocrea et al., 1998a,b).
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VII. Pharmacological Intervention to Reduce Reactive Oxygen Species Generation in Shock, Inflammation, and Ischemia/Reperfusion |
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Interventions, which reduce the generation or the effects of ROS
exert beneficial effects in a variety of models of inflammation and
shock. These therapeutic interventions include a vitamin E-like antioxidant (Cuzzocrea et al., 1999a), an SODm (Wang et al., 1990; Cuzzocrea et al., 1999b), and a ONOO
decomposition catalyst (Salvemini et al., 1998). The therapeutic efficacy of SOD itself in animals with systemic inflammation, hemorrhage, or shock is controversial. The following reasons may explain the lack of effect of SOD against the tissue injury associated with local or systemic inflammation: 1) SOD metabolized O2 to H2O2. Without efficient
removal of H2O2, however,
H2O2 is converted to the
highly toxic HO· (Goode and Webster, 1993). Indeed, SOD may
function as a pro-oxidant by catalyzing the conversion of
H2O2 to HO· (Yim et
al., 1990), such as is believed to be the case in Down's syndrome. 2)
Neither SOD nor O2 easily cross biological membranes. Thus, an
increase in the amounts of extracellular SOD does not attenuate the
effects of the O2 generated by intracellular sources (Meister,
1992). In contrast to SOD, spin-trapping nitrones, such as
phenyl-N-tert-butyl nitrone, consistently improve
outcome in rat models of endotoxic (McKechnie et al., 1986; Hamburger
and McCay, 1989) and traumatic shock (Novelli et al., 1986; Novelli, 1992). The early phase of the inflammatory process is related to the
production of histamine, leukotrienes, platelet-activating factor, and
possibly cyclo-oxygenase products, whereas the delayed phase of
the inflammatory response has been linked to neutrophil infiltration
and the production of neutrophil-derived free radicals and oxidants,
such as H2O2, O2,
and HO·, as well as the release of other neutrophil-derived
mediators (Ohishi et al., 1989; Salvemini et al., 1996b).
A. Peroxynitrite Decomposition Catalysts as Anti-inflammatory Agents
Pathologies driven by the formation of peroxynitrite are amenable
to pharmacological intervention at the level of the reactant (NO· and O2) or the product
(ONOO). Indeed, SOD and/or inhibitors of NOS
have been effective in attenuating both acute and chronic inflammatory
responses in animal models of human diseases. We have recently
identified a novel class of anti-inflammatory agents:
peroxynitrite decomposition catalysts.
The peroxynitrite anion is formed and can be prepared via by a number
of pathways (Fig. 5), particularly
through various oxidations of nitrogen oxides and photolysis and
radiolysis of solid nitrate salts. As previously noted, formation of
peroxynitrite by the combination of NO· and O2
radicals is quite favorable (Huie and Padmaja, 1993), as is the
combination of NO2 and HO·
(kobs = 4.5 × 109 M1
s1) (Logager and Sehested, 1993). Four
practical laboratory syntheses of peroxynitrite are known, including:
1) the original route, in which intermediate NO+
from dehydration of nitrous acid is trapped with peroxide (Beckman et
al., 1994a); 2) nucleophilic attack of peroxide on alkyl nitrites (Moncada and Higgs, 1993); 3) ozonolysis of aqueous azide solutions (Gleu and Hubold, 1935; Pryor et al., 1995); and 4) photolysis and
radiolysis of nitrate salts (King et al., 1992). Yields are readily
observed and quantified spectrophotometrically by the characteristic
yellow color of the anion (
max = 302 nm,
max = 1670 ± 50 M1 cm1) (Beckman et
al., 1994b).
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Peroxynitrite is stable in alkaline solution, but the conjugate acid
(pKa = 6.80) (Logager and Sehested,
1993) is colorless and unstable, and isomerizes rapidly to nitrate,
which is considerably more stable (
H° = 40 kcal/mol) (Squadrito et
al., 1995). An additional acid-catalyzed pathway is observed at extreme
acidity, which apparently corresponds to decomposition of
[H2OONO]+ (Benton and
Moore, 1970).
The rearrangement of peroxynitrite to nitrate is coupled intimately
with the oxidation chemistry of this species, and both reactions have
been the subject of recent investigations and intense debate.
Peroxynitrite and its conjugate acid are strong oxidants, capable of
effecting one- and two-electron reactions akin to those of HO·,
nitrogen dioxide (NO2), and nitrosoniun cation. Oxidations of
thiols (Radi et al., 1991b; Van der Vliet et al., 1994), sulfides (Pryor et al.; 1994; Padmaja et al., 1996), transition metal complexes (Goldstein and Czapski, 1995; Groves and Marla, 1995), deoxyribose (Beckman et al., 1990), halide ions (Hughes et al., 1971; Goldstein and
Czapski, 1995), ascorbate (Barlett et al., 1995; Squadrito et al.,
1995), olefins (Halfpenny and Robinson, 1992), benzenes (Halfpenny and
Robinson, 1992), phenols (Halfpenny and Robinson, 1992, 1996; Beckman
et al., 1992; Ischiropoulos et al., 1992a; Van der Vliet et al.,
1994a,b; Groves and Marla, 1995; Ischiropoulos et al., 1996; Ramezanian
et al., 1996), and other aromatics (Halfpenny and Robinson, 1996; Van
der Vliet et al., 1994b; Alvarez et al., 1996) by peroxynitrite have
been described.
Peroxynitrite is a particularly effective oxidant of aromatic molecules
and organosulfur compounds that include free amino acids and peptide
residues. Cysteine and glutathione, which are significant components of
antioxidant reservoirs, are converted to disulfides (Radi et al.,
1991b; Van der Vliet et al., 1994b). Methionine is converted to
sulfoxide or is fragmented to ethylene and dimethyldisulfide (Pryor et
al., 1994; Padmaja et al., 1996). Dimethyl sulfoxide is oxidized to
formaldehyde (Beckman et al., 1990). Methyl acrylate is polymerized
(Halfpenny and Robinson, 1992). Tyrosine and tryptophan undergo
one-electron oxidations to radical cations, which are competitively
hydroxylated, nitrated, and dimerized (Ischiropoulos et al., 1992b; Van
der Vliet et al., 1994b; Alvarez et al., 1996; Ramezanian et al.,
1996). The formation of nitrotyrosine is particularly favorable, and
the appearance of this product in biological samples is taken as
diagnostic of exposure to peroxynitrite (Salvemini et al., 1996b).
Purine nucleotides are vulnerable to oxidation (Douki and Cadet, 1996;
Uppu et al., 1996) and to adduct formation (Douki et al., 1996).
Peroxynitrite reacts with a number of metal-containing enzymes. It can
inactivate aconitase as can superoxide (Keyer and Imlay, 1996) by
oxidative cleavage of the labile iron site from the parent cluster
(Hausladen and Fridovich, 1996), and certain SOD enzymes by nitration
of critical residues (Ischiropoulos et al., 1992b). Peroxynitrite is
proposed to down-regulate neuronal NOS by oxidation of a requisite
cofactor (Huhmer et al., 1996). Heme prosthetic groups of cytochromes
(Thomson et al., 1995) and peroxidases (Floris et al., 1993) are
oxidized reversibly by peroxynitrite. Furthermore, peroxynitrite can
release metal ions from the active sites of other enzymes (Swain et
al., 1994; Crow et al., 1995).
The kinetics of substrate oxidation by peroxynitrite is extremely
complex (Pryor and Squadrito, 1995). When the solution pH is fixed near
physiologically relevant values, so that peroxynitrite and its
conjugate acid are present in fixed ratios, observed rates of substrate
oxidation and peroxynitrite loss are first-order in peroxynitrite.
These rates typically increase from the peroxynitrite isomerization
limit in proportion to increasing substrate concentrations; however, a
significant zero-order limit is frequently encountered. Fairly high
substrate concentrations, for example, several millimolar ascorbate
(Barlett et al., 1995), are often required to attain first-order
behavior. This behavior has been interpreted to involve competition
between direct, first-order reaction of peroxynitrite with substrate
and quenching by substrate of any activated form of peroxynitrite
complex (Pryor and Squadrito, 1995).
The reaction of peroxynitrite with methionine at pH 7.4, 25°C, is the
prototypical example (Pryor et al., 1994), although similar kinetics is
observed with ascorbate (Squadrito et al., 1995). At high methionine
concentrations, the sulfoxide is formed from a two-electron oxidation
by peroxynitrite; observed peroxynitrite decay rates are first-order in
methionine, but the extrapolated intercept with no added methionine is
several times faster than the intrinsic isomerization rate of
peroxynitrite. At lower methionine concentrations, the observed rates
drop below the first-order limit, and an increasing mole fraction of
the observed products is ethylene produced by decomposition of a
sulfur-centered radical cation that results from a one-electron
oxidation. The one-electron and two-electron oxidation products are
proposed to arise from discrete reactions with activated and
ground-state peroxynitrite, respectively.
The approach to a zero-order limit with increasing substrate indicates that the activated form is formed in a rapidly reversible equilibrium between peroxynitrite/peroxynitrous acid, which is a stronger oxidant. Furthermore, the rates of isomerization and zero-order oxidation are typically similar, which suggests that these processes follow coincident paths through the intermediate.
The nature of the activated state is the subject of considerable
debate. Some researchers favor the formulation as a pair of radicals,
HO· and NO2, which results from reversible peroxide
bond homolysis and can proceed either to oxidations or to recombination
as nitrate (Halfpenny and Robinson, 1996; Beckman et al., 1990; King et
al., 1992; Van der Vliet et al., 1994a; Alvarez et al., 1996). It has also been suggested that the intermediate is a trans-isomer
that is formed via isomerization of the thermodynamically favored
cis geometry by hindered rotation about the nitrogen
peroxide bond (Hughes and Nicklin, 1968; Koppenol et al., 1992;
Goldstein and Czapski, 1995; Pryor et al., 1994, 1996; Squadrito
et al., 1995; Tsai et al., 1996a,b). The discrepancy in viewpoints is
of more than academic interest, due to the extremely rapid and general oxidative reactivity of the product: HO·.
Interest in peroxynitrite chemistry recently has grown in significance,
but experimental evidence on this point is limited and not completely
definitive. Certain results seem to favor the radical hypothesis. For
example, HO· and NO2 radicals recombine to form
peroxynitrite as a major product (Logager and Sehested, 1993), which
provides reversibility. The bond dissociation enthalpies can be
calculated with some assumptions and coincide closely with experimental
activation enthalpies for isomerization (e.g., 17 versus 18 ± 1 kcal/mol) (Mahoney, 1970; Koppenol et al., 1992). However, other
possible processes are calculated to have similar activation
enthalpies, and it has been argued that the experimental entropy is too
small to accommodate homolysis (Koppenol et al., 1992). Radical
trapping products, particularly hydroxylated aromatics and oxidized
spin traps, are reported to form (Halfpenny and Robinson, 1996; Beckman
et al., 1990; Van der Vliet et al., 1994b), but yields relative to
charged peroxynitrite are invariably very low (Shi et al., 1994;
Richeson and Ingold, 1996). Finally, the rate of isomerization to
nitrate shows none of the dependence on solution viscosity that is
expected for diffusive radical recombination (Pryor et al., 1996). A
detour around such objections is an efficient cage recombination
process, but the distinction between tightly caged radicals and a
geometric isomerization becomes an issue more of semantics than of physics.
Formulation of the activated state as the trans-isomer seems
to be reasonable from a structural and theoretical standpoint. High-level calculations find that the cis-isomer is more
stable (McGrath et al., 1988; Tsai et al., 1996a,b), and predictions for this isomer match experimental spectra (Tsai et al., 1996a,b). Isomerization of the trans-isomer to nitrate is predicted to
be more facile than that of the cis-isomer, the terminal
peroxide oxygen atom being more favorably disposed geometrically and
electronically for intramolecular addition to nitrogen in the former
(Tsai et al., 1996a). Furthermore, interconversion of the isomers by
hindered rotation about the partial bond between nitrogen and the
peroxide is predicted to be markedly more facile for the protonated
acid than for the anion (McGrath and Rowland, 1994; Tsai et al., 1996a) that neatly explains the acid requirement of the nitrate isomerization reaction. However, reports of direct experimental observation of the
trans-isomer are notably absent from the literature. In addition, yields of oxidized product versus peroxynitrite loading from
some substrates that react only with the activated state (McGrath et
al., 1988; Yang et al., 1992; Crow et al., 1994) and ferricyanide
([Fe(CN)6]4) (Goldstein
and Czapski, 1995) are reported to be substoichiometric and independent
of substrate concentration. This result implies that substrate
quenching of the intermediate is in competition with nitrate formation
or some other decay reaction (e.g., to nitrite), directly from the
ground state (Goldstein and Czapski, 1995).
Other activation mechanisms, such as heterolysis to HO· and
NO2+, are ruled out by the
experimental observation that double labeling of peroxynitrite with
18O across the peroxide bond produces
double-labeled nitrate upon isomerization (Anbar and Taube, 1954).
Wider use of isotope labels as tracers (18O) or
as magnetic probes (15N) may assist future
clarification of the nature of the activated state in the oxidation and
isomerization reactions.
It should be noted that a few examples of alternative routes to
peroxynitrite-like oxidative biochemistry have been suggested. One such
possibility is a redox cycle that forms toxic hydroxyl radicals from
adventitious ferrous ions and
H2O2, which is closed by
NO· reduction of derived ferric ions (Farias-Eisner et al.,
1996). Alternatively, the oxidation of nitrite to
ClNO2 by hypochlorite, which is formed in vivo
from the action of peroxidase enzymes on chloride, provides another
biologically derived radical precursor and nitration agent (Eiserich et
al., 1996). Nitryl chloride has been shown to effect nitration of
tyrosine in vitro, which is often taken as a biomarker for
peroxynitrite exposure. Finally, peroxynitrite reacts rapidly with
carbon dioxide, yielding an adduct that is a stronger oxidant than
peroxynitrite alone (Lymar and Hurst, 1995). Because carbon dioxide is
present in vivo in equilibrium with bicarbonate, the reactivity of
peroxynitrite with various target molecules can be amplified (Lymar et
al., 1996); however, bicarbonate actually seems to moderate the
bacteriocidal properties of peroxynitrite (Zhu et al., 1992; Lymar and
Hurst, 1996), perhaps by opening up benign decomposition pathways such as hydrolysis (Lymar and Hurst, 1996).
Known and possible reactions of peroxynitrite are summarized in Fig. 5. Two important notions emerge from these mechanistic investigations. First, formation of a highly oxidizing (possibly a radical) intermediate can be preempted by a competitive, direct reaction with ground-state peroxynitrite, especially as the unprotonated anion. Second, the kinetic barriers forming this oxidizing species, and nitrate-forming isomerization, are considerable; such reactivity may therefore be amenable to selective catalysis. These points lead the discussion to strategies for blocking the deleterious biochemistry of peroxynitrite.
Such strategies must aim to decrease either the flux or the intrinsic
lifetime of the peroxynitrite. Three particular tactics would
accomplish such purposes: 1) blockage of peroxynitrite formation by
limiting the availability of NO· and O2, either through
inhibition of NOS or acceleration of superoxide dismutation; 2)
competitive stoichiometric trapping of peroxynitrite; or 3) catalysis
of peroxynitrite decomposition to benign products (e.g., isomerization
to nitrate). All three approaches afford possibilities for
pharmacological intervention, but the last one is particularly
attractive. Identification of a highly active catalytic peroxynitrite
isomerase would facilitate destruction of many equivalents of
peroxynitrite formed over an extended time interval from a single,
substoichiometric drug dose. As previously noted, the possibility of
such catalysis is real and has now been demonstrated by highly stable
molecules in vitro.
The initial experimental demonstration of peroxynitrite isomerization
catalysis was reported recently (Stern et al., 1996). Addition of iron
porphyrin complexes
(H2O)FeIII(L) (L = 5,10,15,20-tetrakis-(N-methyl-4'pyridyl)
porphyrinato; 5,10,15,20-tetrakis-(2',4',6'-trimethyl-3',5'-disulfonatophenyl) porphyrinato) to solutions measurably reduced the lifetime of peroxynitrite under physiologically relevant conditions (pH 7.4, 37°C). Furthermore, the product ion distribution shifted markedly toward innocuous nitrate at the expense of nitrite. The porphyrin complexes were extremely robust and attained high peroxynitrite isomerization rates at micromolar concentrations, even at peroxynitrite concentrations in excess of 100 µM. These qualities are quite remarkable, and the potential efficacy of these catalysts as drugs can
be predicted from a purely chemical standpoint. For this reason, the
search for other redox-active complexes that will accomplish catalysis
of the peroxynitrite isomerization to nitrate continues.
The catalytic reactions are governed by Michaelis-Menten kinetics,
typical of enzymes, and involve formation of an oxidized intermediate
complex, O = FeIV(L), which was observed by
use of time-resolved stopped-flow UV-visible spectrophotometry.
Therefore, irreversible turnover of peroxynitrite to nitrate results
from a reversible formation of the catalytic intermediate, O = FeIV(L), and NO2 radical. The
intermediate was generated independently and was shown to quench
rapidly upon addition of the NO2 gas or nitrite ion.
The proposed catalytic cycle is illustrated in Fig.
6 (Stern et al., 1996). In keeping with
this scheme, yields of oxidized catalyst intermediate and observed
peroxynitrite lifetimes were nonlinearly dependent on peroxynitrite
concentration; higher loadings saturated the catalyst in the oxidized
state and raised the lifetime of the peroxynitrite. Addition of
antioxidants, such as ascorbate, competitively quenches the
intermediate and accelerates catalysis (Jensen et al., submitted for
publication). Peroxynitrite decomposition becomes more efficient simply
by increasing the concentration of either the complex or antioxidant,
and harmful oxidations become less competitive.
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As previously noted, heme prosthetic groups are oxidized by
peroxynitrite (Floris et al., 1993); for example, reaction of myeloperoxidase was reported to be as rapid as that observed for the
synthetic catalysts. It is likely that peroxidase enzymes, in addition
to their previously recognized biochemical role as peroxide scavengers,
might function additionally as endogenous peroxynitrite isomerases
(Lymar and Hurst, 1996). Myeloperoxidase is, for example, excreted by
neutrophils, and may use OONO in the
inflammatory immune response. This hypothesis was supported by further
experiments that demonstrated that mammalian heme haloperoxidases are
as active as the synthetic porphyrins (Jensen et al., submitted for
publication). Nature already practices such catalysis! Hence, an active
drug will duplicate peroxidase reactivity at critical sites where these
enzymes are not present in optimal quantities. The synthetic porphyrin
complexes also have an advantage over the endogenous enzymes in higher
reactivity with antioxidants, which permits reductase activity to
compete with the isomerization reaction and further decreases
peroxynitrite lifetimes (Jensen et al., submitted for publication).
Identification of the peroxynitrite decomposition catalysts offers the scientific community the exciting opportunity to elucidate and further our understanding of the roles of peroxynitrite in animal models of diseases. This may lead to breakthroughs in understanding the pathophysiological importance of this molecule. Therefore, direct pharmacological intervention with unique peroxynitrite decomposition catalysts specifically designed to decompose peroxynitrite to innocuous nitrate represents a novel strategy to tackle a wide range of disease states that are potentially governed and driven by the overt production of this cytotoxic molecule.
B. Catalytic Antioxidants
SOD and catalase are metalloproteins that use efficient
dismutation reactions in their mechanisms to detoxify ROS. A
dismutation reaction involves a series of one- or two-electron
transfers, where the electrons are accepted from one O2 or
H2O2 and then donated to
another (Klug-Roth et al., 1973; Waldo and Penner-Hahn, 1995).
These efficient reactions do not require reducing equivalents and thus
do not require energy from the cell to operate. The overexpression of
these enzymes in cell culture and in whole animals has provided protection against the deleterious effects of a wide range of oxidative
stress paradigms (Krall et al., 1988; Sohal et al., 1995; Ho et al.,
1998). The use of SOD and catalase as therapeutic agents to attenuate
ROS-induced injury responses has had mixed success (Shaffer et al.,
1987; Thibeault et al., 1991; Wispe et al., 1992; Lardot et al., 1996;
Simonson et al., 1997). The main limitations of these natural products
are their large size, which limits cell permeability, short circulating
half-life, antigenicity, and expense. An increasing number of
low-molecular weight SODm have been developed to overcome some of these limitations.
C. Metalloporphyrins
Manganese-based metalloporphyrin complexes have been shown to
possess at least four distinct antioxidant properties (Lawrence and
Sawyer, 1979; Faulkner et al., 1994; Day et al., 1995, 1997, 1999; Szabó et al., 1996a; Batinic-Haberle et al., 1998). These include scavenging O2,
H2O2,
ONOO, and lipid peroxyl radicals. The manganese
moiety of the SOD mimetics functions in the dismutation reaction with
O2 by alternate reduction and oxidation changing in its
valence between Mn(III) and Mn(II), much like native SODs. The catalase
activity of metalloporphyrins could be attributed to their extensive
conjugated ring system that undergoes reversible one-electron
oxidations, much like the heme prosthetic groups of endogenous
catalases and peroxidases (Dolphin et al., 1971). In general,
metalloporphyrins with higher SOD activity possessed greater catalase
activity. It is noteworthy that the catalase activity of such complexes
is less than 1% that of native catalases. However, despite this,
manganese porphyrins are still able to protect cells from a toxic
amount of H2O2 (Day et al.,
1997). The mechanism by which metalloporphyrins scavenge ONOO is thought to involve the formation of an
oxo-Mn(IV) complex that can be readily reduced to the Mn(III) oxidation
state by a wide variety of endogenous antioxidants (i.e., ascorbate and glutathione) and even by O2. The exact mechanism by which
metalloporphyrins inhibit lipid peroxidation is not known, but is
thought to be similar to that described for
ONOO scavenging.
1. Effects of Metalloporphyrins in Inflammation.
In vitro
models of oxidative stress have been useful both in terms of confirming
the antioxidant activities of metalloporphyrins obtained in cell-free
systems and predicting their use as antioxidants in more complex in
vivo models of human disease. Metalloporphyrins have been shown to be
protective in a wide variety of in vitro oxidative stress models
involving the generation of O2,
H2O2, and
ONOO alone or in concert. At micromolar levels,
they seems to be nontoxic and protect cultured cells against the
toxicity of O2 generators [paraquat (Day et al., 1995) and
pyocyanine (Gardner et al., 1996)], H2O2 generator [glucose
oxidase (Day et al., 1997)], and ONOO injury
produced by endotoxin (Szabó et al., 1996a) or
ONOO itself (Misko et al., 1998).
Metalloporphyrins are also potent inhibitors of lipid peroxidation (Day
et al., 1999).
).
Carrageenan-induced paw edema is a model of acute inflammation. Both
ironIII
tetrakis-(2,4,6-trimethyl-3,5-disulfonatophenyl) porphyrin
and ironIII
tetrakis-(N-methyl pyridinium-4-yl) porphyrin
caused a dose-dependent reduction in swelling in paw tissue (Salvemini
et al., 1998). In a rat model of lung pleurisy, the intraperitoneal
treatment with MnTBAP before carrageenan administration was found to
suppress inflammatory responses in a dose-dependent manner (Cuzzocrea
et al., 1999b). The most profound effects of MnTBAP were on depressing the neutrophil influx and reducing nitrotyrosine formation, a marker of
ONOO formation in inflammation.
2. Effect of Metalloporphyrins in Endotoxic and Hemorrhagic Shock.
A common complication of bacterial sepsis is the phenomenon
referred to as endotoxic shock that results in oxidative tissue damage
partially resulting from the formation of ONOO
(Kilbourn and Griffith, 1992). MnTBAP protected against some of the
detrimental effects associated with endotoxic shock, including vessel
contractility and cellular energy deficit (Zingarelli et al., 1997).
MnTBAP also provided similar protective effects in a hemorrhagic shock
model in the rat (Szabó, 1998). The efficacy of MnTBAP in these
models probably relates to its ONOO-scavenging
activity in addition to its O2-scavenging activity.
3. Limitations of Metalloporphyrins.
Although MnTBAP
has proven to be a very effective compound in a wide range of
oxidative stress paradigms, we have found that its potency and efficacy
can be quite variable. One general limitation of metalloporphyrins is
their poor blood-brain permeability that complicates their use in
neurodegenerative diseases and will require development of new
compounds to overcome this obstacle. A general problem for these SOD
mimetics is that they can react with a wide variety ROS. This creates
some confusion in the literature when used to demonstrate a specific
role for O2. Finally, because ROS/RNS have roles in cell
signaling and, as a consequence, roles in controlling gene expression
(Simon et al., 1998; Duranteau et al., 1998) and in host defense,
antioxidant therapies may have an impact on these physiological processes.
D. New Rational Synthetic Enzymes: Manganese(II)-Based Superoxide Dismutase Mimics
Protective and beneficial roles of SOD have been demonstrated in a
broad range of disease, both preclinically and clinically (Babior,
1982; Halliwell and Gutteridge, 1985; Maxwell, 1995). Orgotein (bovine
Cu,ZnSOD) showed promising results as a human therapeutic in acute and
chronic conditions, including rheumatoid arthritis and osteoarthritis,
as well as side effects associated with chemotherapy and radiation
therapy (Niwa et al., 1985). There are drawbacks and issues associated
with the use of the native enzymes as therapeutic agents (e.g.,
solution instability, immunogenicity of nonhuman enzymes, bell-shaped
dose response curves, high susceptibility to proteolytic digestion) and
as pharmacological tools (e.g., they do not penetrate cells or cross
the blood-brain barrier, thus limiting the dismutation of superoxide
only to the extracellular space or compartments).
To overcome the limitations associated with native enzyme therapy the
Salvemini group has developed a series of SOD mimetics that selectively
catalyze the dismutation of O2 (Fig.
7). An important and unique property of
these SODm is that they are stable to dissociation and oxidation in the
Mn(II) oxidation state and that the rate-determining step in the
catalytic cycle for dismutation of superoxide is oxidation of the
Mn(II) oxidation state not reduction of Mn(III) as occurs with the
MnIII porphyrin complexes. Those relevant
biological oxidants (ROS) that have been demonstrated not to react with
these Mn(II) complexes, I, include NO·,
OONO,
H2O2,
O2, and OCl (Riley et
al., 1996). This property is not shared by other classes of SODm or
scavengers, including several metalloporphyrins such as
tetrakis-(N-ethyl-2-pyridyl) porphyrin and
MnTBAP, since they have been shown to interact with a variety of
oxidants, including ONOO and
H2O2 (Patel and Day, 1999).
Although these agents are anti-inflammatory (Patel and Day, 1999), it
is not clear at this stage how important the removal of superoxide is
in the context of the inflammatory response because of their lack of
selectivity for superoxide. Furthermore, this new class of SOD mimetics
is not deactivated by ONOO or
H2O2 (Riley, unpublished
results). This is an added advantage over the native manganese SOD
(MnSOD) enzyme, since the native enzyme is nitrated and deactivated by
ONOO (Macmillan-Crow and Thompson, 1999).
|
1. Characterization of Superoxide Dismutase Activity.
The
discovery and activity of the SOD enzymes were first reported by
Fridovich and McCord using a cytochrome c assay (McCord and
Fridovich, 1969). In this assay, the ferricytochrome c is reduced by superoxide to give the reduced form of cytochrome
c, which gives a spectrophotometric change. Inhibition of
this reduction of cytochrome c was taken as a measure of SOD
activity. Since then, the cytochrome c assay and other
indirect assays have been used by investigators to assess the SOD
activity of enzymes and putative SOD mimetics. Difficulty with these
indirect assays can arise from several sources. For example, an agent
with putative SOD activity can oxidize the reduced cytochrome (possibly
resulting in a false-positive for SOD activity), reduce ferricytochrome c (potentially leading to a false-negative), or react
stoichiometrically, not catalytically, with superoxide (i.e., a
scavenger of superoxide) (Weiss et al., 1993). The indirect assays do
not discriminate among these processes and additionally do not provide
information regarding the mechanism of action of putative SODm.
). From this type
of analysis, an uncatalyzed decay of superoxide (second-order kinetics)
can be distinguished from a catalyzed decay of superoxide (first-order
kinetics) in the presence of a large excess of superoxide over the
complex being screened. A second-order catalytic rate constant
(kcat) can be obtained for an agent
with true catalytic SOD activity. This direct determination of a true
kcat can be used to directly compare
and quantitate the SOD activities of enzymes and/or mimetics under a
given set of conditions (e.g., defined pH and temperature). No direct
comparisons can be made between the
kcat value and activity obtained from
the cytochrome c assay or other indirect assays.
2. Catalyst/Drug Design.
Initial efforts focused on the
synthesis of Mn-based complexes as low molecular weight SOD mimics that
could function as selective and active SOD catalysts. This
decision to pursue Mn complexes was based largely on considerations of
toxicity. Of the three metals known (Fe, Mn, and Cu) to catalyze
O2 to H2O2 and
oxygen, manganese is the least toxic to mammalian systems as the free aquated metal ion and is also the least likely of the three
M2+ ions to react with
H2O2 to generate HO·
(Fenton chemistry). Although Mn-based complexes of desferal (Darr et
al., 1987; Faulkner et al., 1994), quinolol (Howie and Sawyer, 1976),
cyclam (Rush et al., 1991), and salen (Baudry et al., 1993) have been
reported to have SOD activity based on indirect methodologies previously described, analysis of these complexes by the stopped-flow kinetic methodology (Riley et al., 1991) demonstrated that these complexes have no detectable catalytic SOD activity. More
likely, these Mn complexes react stoichiometrically with superoxide
(Weiss et al., 1993), resulting in their apparent activity in the
indirect assays.
1
s1 at pH 7.4 and 21°C for 1)
(Riley et al., 1997). From structure-activity studies, we find that
increasing the complex ring size to a 16-membered ring (complex
2) results in a substantial loss of SOD activity with a
kcat of 1.0 × 106 M1
s1 (Riley et al., 1997). Further increasing the
macrocycle size to a 17-membered ring (complex 3 or
4) gives complexes with no detectable SOD activity
(kcat < 5 × 105 M1
s1) as shown by stopped-flow analysis.
Importantly, it was found that, in general, increasing the substitution
on the carbon backbone of the macrocyclic ring resulted in Mn(II)
complexes with greatly enhanced stability toward dissociation while
generally retaining SOD activity (Riley et al., 1996). Additionally, a
large number of complexes were prepared with ligands possessing methyl
substituents in various combinations of position and stereochemistry (Riley et al., 1996) to probe how position, stereochemistry, and number
of substituents affect the catalytic SOD activity. Kinetic studies have
also revealed that certain structural elements act synergistically
giving complexes with substantially enhanced SOD activity and enhanced
stability [e.g., Mn(II) complexes containing trans-cyclohexano groups (complexes 5 and
6)]. Complex 5 with one
trans-cyclohexano group has a
kcat value of 9.09 × 107 M1
s1 at pH 
7.4 and complex 6 with
two trans-cyclohexano (both R,R)
groups has a kcat value of 1.21 × 108 M1
s1 at pH 7.4 (kcat for the all S mirror
image isomer is identical to 6, whereas the R, R,S,
S-isomer, 7, has no catalytic activity). By electron
spin resonance (ESR) analysis, we have shown that complex 8 has high in vivo stability and is greater than 90% intact in the liver
of rats 30 min after intravenous injection.
3. Anti-Inflammatory Activity of Superoxide Dismutase Mimics.
The SOD mimetics 1 and 5 were tested for
their ability to inhibit human neutrophil-mediated injury to human aortic endothelial cells in vitro (Hardy et al., 1994). Human neutrophils were activated to produce superoxide from exposure to tumor
necrosis factor- and the complement component C5a. The extent of
injury to the endothelial cells, which was measured by the release of
51Cr-labeled chromate from prelabeled cells, was
linearly dependent on the amount of superoxide produced. Many of our
SOD mimics have been shown to attenuate the neutrophil-mediated injury
to the endothelial cells. Fluorescent studies of the neutrophil
respiratory burst indicated that the SOD mimic does not prevent the
generation of superoxide by the activated neutrophils. The SOD mimics
also protected the endothelial cells against injury caused by
xanthine/xanthine oxidase, a biochemical system that produces
superoxide. The catalytic nature of the dismutation of superoxide by
the SOD mimics is evident from comparing the protective effects of the
SOD mimics with the lack of protectiveness by the structurally related
Mn(II)-based complex 9 that has no detectable SOD activity.
In addition, the H2O2
scavenger catalase, the iron chelator desferrioxamine, and serine
protease inhibitors did not protect against the neutrophil-mediated injury. These results were consistent with superoxide mediating the
human neutrophil-induced injury to human aortic endothelial cells;
thus, the Mn(II)-based SOD mimics may be viable agents to prevent
oxidative injury due to neutrophils. Bovine erythrocyte Cu,ZnSOD
protected the endothelial cells in a concentration-dependent manner
from neutrophil-dependent injury, but the results were inconsistent
(Hardy et al., 1994). On a SOD activity basis, as assessed by
stopped-flow, the SOD enzyme was much less effective at protecting the
endothelial cells than the SOD mimics. The efficacy of the SOD mimics
in attenuating the neutrophil-mediated injury may be due to the
compound's ability to gain access to the intracellular space. We have
synthesized Mn(II)-based SOD mimics with a broad range of
lipophilicities (log P values of 4 to +2) that may further allow control of the degree of intracellular penetration (Riley et al.,
1996). At high doses, the SOD enzyme lost some of its efficiency to
block the neutrophil-dependent injury to the endothelial cells (Hardy
et al., 1994). This bell-shaped dose-response curve is a common
characteristic of the SOD enzymes, and high doses of the SOD enzymes
exhibit proinflammatory effects (Dowling et al., 1993). The
proinflammatory effects of the SOD enzyme is not well understood, but
it is speculated to be due to its reaction with the dismutation product
H2O2 to generate HO·
radicals via Fenton chemistry (Mao et al., 1993). The lack of a
bell-shaped dose-response curve with the SOD mimetics may be related to
the selective reactivity of the SOD mimics with superoxide and the
complexes' inability to react with
H2O2.
1
s1 at pH 8.1 and 21°C), whereas the E. coli MnSOD that we tested had no detectable SOD activity
(kcat < 5 × 105 M1
s1) (Furchgott and Vanhoutte, 1989). Again, it
is important to assay putative SOD mimics and the SOD enzymes by a
direct method of analysis for SOD activity as the indirect methods
(namely, the cytochrome c assay) indicated that both of
these enzymes had comparable SOD activity.
When administered intravenously, the SOD mimic 1 attenuated
the dilute aqueous acetic acid-induced inflammation of the colon in
mice with an ED50 of 10 mg/kg, which is
equivalent to the ED50 of the compound when it
was administered intracolonically (Weiss et al., 1996). In contrast,
the bovine erythrocyte Cu,ZnSOD failed to protect against the
inflammatory injury when administered intravenously, presumably due to
the enzyme's short in vivo half-life. The SOD mimic 1 was
also effective at inhibiting the inflammation and neutrophil
infiltration induced by the intradermal administration of the
neutrophil chemoattractant leukotriene. These results are consistent with a role for superoxide in the mediation of oxidative tissue injury and the infiltration of neutrophils. These results indicate that the SOD mimetics may prove useful as novel
anti-inflammatory agents for the treatment of conditions mediated, in
part, by superoxide produced by activated polymorphonuclear leukocytes (neutrophils).
4. Attenuation of Myocardial Ischemia/Reperfusion Injury by
Superoxide Dismutase Mimics.
Reperfusion of the ischemic
myocardium can result in a burst of superoxide production as shown by
ESR spin-trapping studies (Zweier, 1988). The superoxide produced as a
result of reperfusion of the ischemic tissue has been proposed to be a
mediator of the reperfusion injury to the myocardium. We evaluated the
cardioprotective effects of the Mn(II)-based SOD mimetics in isolated
heart preparations and in the in vivo models of myocardial
ischemia/reperfusion injury. When perfused at a concentration of 20 µM, the SODm 1 inhibited the release of creatine kinase
and intracellular potassium in Langendorff-perfused rabbit isolated
hearts subjected to 30 min of global ischemia followed by 45 min
reperfusion (Kilgore et al., 1994). In the same model, complex
1 also decreased irreversible damage in the reperfused
ischemic hearts as indicated by results obtained with a radiolabeled
monoclonal antibody to the intracellular protein myosin. The SOD mimic
1 also protected against myocardial ischemia-reperfusion
injury to the isolated primate heart.
). Myocardial infarct size was reduced from 44.2 ± 5.6% to 25.7 ± 4.3% by the SOD mimic when
administered intravenously at a total dose of 16 mg/kg. Hemodynamic
parameters were unaffected, except for a transient hypotensive effect
that will be commented on in the following section on NO potentiation. At an identical dose, complex 9, which had no detectable SOD
activity failed to protect against the reperfusion injury of the
ischemic myocardium in the dog, indicating that the injury was mediated
by superoxide. Further myocardial ischemia/reperfusion studies were
conducted in the cat to evaluate the mechanism of protection of the SOD
mimetics (Venturini et al., 1994). The SOD mimic 5 protected
the feline myocardium from necrosis as a result of a period of ischemia
of 75 min followed by a 4.5-h period of reperfusion. The protection was
dose-dependent and a statistically significant reduction in necrosis as
a percentage of the area at risk (AAR) (9.9 ± 2.8% with
5 versus 29.0 ± 3.8% in controls) was observed at an
intravenous dose of 5 µmol/kg. Creatine kinase release and the
infiltration of neutrophils into the myocardium were also inhibited by
complex 5.
A dose-dependent, not bell-shaped, dose-response curve was observed
with the SOD mimic 5 in the feline myocardial
ischemia/reperfusion model (Venturini et al., 1994). With the SOD mimic
5, the same amount of protection against necrosis was
observed at 50 µmol/kg (9.3 ± 5.6% necrosis in AAR) as at 5 µmol/kg (9.9 ± 2.8% necrosis in AAR). However, as noted
previously, the SOD enzyme had a bell-shaped dose-response curve in the
feline model of the ischemia/reperfusion injury (Venturini et al.,
1994). The SOD mimic 6, which has a higher
kcat (more SOD activity) and is more
lipophilic than complex 5, significantly inhibited the
myocardial necrosis at a dose of 2 µmol/kg (Venturini et al., 1994).
Complex 6 also attenuated creatine kinase release and the
infiltration of neutrophils in a concentration-dependent manner. ESR
stability studies in the isolated rabbit heart indicated that the SOD
mimic 6 has a high degree of stability in the myocardium
under ischemic conditions. ESR studies also showed that complex
6 could partition into the myocardium and could dramatically
attenuate the burst of superoxide-derived radicals at the time of
reperfusion. As in the dog studies, the inactive complex 9 did not inhibit the necrosis arising from reperfusion of the feline
ischemic myocardium, indicating that superoxide is a mediator of the
injury (Venturini et al., 1994). Mn(II) chloride did not protect
against the reperfusion injury, indicating that complex 1 was not attenuating reperfusion injury due to the release of Mn(II)
(Venturini et al., 1994). Based on these in vitro and in vivo studies
with the SOD mimics, we have established an important role for
superoxide in reperfusion injury to the ischemic myocardium. By
catalyzing the dismutation of superoxide, the SOD mimetics may prove
useful as therapeutic agents to attenuate myocardial
ischemia-reperfusion injury following myocardial infarction.
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VIII. Conclusions and Future Directions |
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TopPreviousNextReferences
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In initial studies, it was proposed that superoxide acts as an inactivator of NO·, since SOD prolongs the biological half-life of NO·. Along the lines of this concept, it has been suggested that NO· can limit the cytotoxicity of superoxide. On the other hand the reaction of NO· and superoxide has been shown to yield peroxynitrite, a reactive oxidant species, and an important mediator of cell damage under conditions of inflammation and oxidant stress. The evidence presented herein favors the view that the reaction of NO· and superoxide yields peroxynitrite, which under many conditions enhances the cytotoxic potential of its "precursors". Clearly, the ratio of NO· and superoxide is very important since NO· can act as an inactivator of the biological activity of peroxynitrite. The finding that peroxynitrite can be formed by the combination of superoxide with NO produced by ecNOS in pathophysiological conditions, such as the early phases of shock and reperfusion injury, deserves further discussion. Previous studies, demonstrating protective effects of inhibition of the constitutive isoforms of NOS in the endothelium (ecNOS) and in the central nervous system (bNOS), have proposed that the toxicity is due to enhanced NO formation by these constitutive enzymes. Although this is certainly one possibility, another explanation should also be considered, namely that simultaneous generation of superoxide enhances the toxic potential of NO and inhibition of constitutive NOS activity prevents the formation of peroxynitrite. By recognizing that the formation of peroxynitrite can occur from superoxide and NO produced by ecNOS, the conventional wisdom of "small amounts of NO are beneficial, large amounts of NO are toxic" needs to be revised. In fact, as discussed previously, large amounts of NO may suppress the oxidant reactivity of peroxynitrite. Whether such an action plays an important role in the protective effect of NO donors in various pathophysiological conditions (such as ischemia/reperfusion injury and various forms of shock needs to be further investigated). In addition, it is clear that the cytotoxicity of peroxynitrite and ROS in various pathophysiological conditions will depend on the endogenous antioxidant status (glutathione levels, vitamin E, vitamin C, SOD, etc.). It is conceivable that small amounts of peroxynitrite are produced under basal, physiological conditions (since, in many cell types, NO from the constitutive NOS isoforms and superoxide from mitochondria and other cellular sources are always produced). It is also probable that the endogenous antioxidant systems are sufficient to neutralize such low-level peroxynitrite production. Selective pharmacological inhibition of iNOS in shock and inflammation is expected to be of significant therapeutic benefit, since it would maintain the physiological functions of ecNOS (such as inhibition of platelet and white cell adhesion, maintenance of vasodilatory tone, etc.), while inhibiting the generation of cytotoxic concentrations of NO. In view of our current knowledge, however, such an approach would not completely eliminate the formation of peroxynitrite, especially during the phases of reperfusion or fluid resuscitation. On the other hand, nonisoform-selective inhibition of NOS, while having the potential of completely eliminating peroxynitrite generation, would have deleterious side effects on its own, due to the absence of ecNOS.
Understanding the signal transduction mechanisms used by free radicals
to modify the course of disease will undoubtedly elucidate important
molecular targets for future pharmacological intervention. It is clear
that low molecular weight SOD "synzymes" can serve as
powerful tools to pharmacologically dissect the mechanism(s) by which
O2 exerts its effects. The Mn(II)-based SOD mimetics I described herein provide an example of an unique approach for the development of artificial enzymes as future drugs. In certain
cases where an enzyme of potential therapeutic benefit does not have
the appropriate properties for a drug, a synthetic, small molecule
enzyme mimetic can conceivably be designed with chemical and physical
characteristics suitable for a therapeutic. The SOD mimics are
catalytic drugs (i.e., the compounds do not involve a
stoichiometric interaction with a biological target, such as a
receptor, but instead enhance the rate of conversion of superoxide to
O2 and
H2O2 without the complex
itself being consumed). The SOD mimics have been rigorously
characterized for SOD activity by stopped-flow kinetic analysis, for in
vitro stability by kinetic and thermodynamic assays, and for in vivo
stability by ESR and radiochemical studies. The ability of the SOD
mimics to scavenge superoxide in vivo has also been demonstrated by ESR studies. In vitro and in vivo studies have demonstrated that the SOD
mimetics have potent anti-inflammatory properties, attenuate myocardial
ischemia/reperfusion injury, and prolong the half-life of NO·,
an antithrombotic and vascular relaxant. Therefore, the SOD mimetics
may find clinical utility in diseases mediated, in part, by superoxide
(Fig. 8). Previous negative clinical
studies with the SOD enzymes should not preclude clinical demonstration
of utility by the SOD mimics because the SOD mimics have numerous advantages over the enzymes, including a normal dose-response curve,
membrane permeability, stability, cost, and lack of immunogenicity, as
well as potential oral activity. In light of the critical roles of
superoxide in disease and cellular signaling, these new, highly potent
"synzymes" have tremendous potential in the treatment of numerous
diseases, ranging from acute and chronic inflammation to shock and
ischemia/reperfusion injury.
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Footnotes |
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1 Address for correspondence: Salvatore Cuzzocrea, Ph.D., Institute of Pharmacology, School of Medicine, University of Messina, Torre Biologica-Policlinico Universitario Via C. Valeria-Gazzi, 98100 Messina, Italy. E-mail: salvator{at}www.unime.it
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Abbreviations |
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SOD, superoxide dismutase; SODm, SOD mimetics; NOS, nitric-oxide synthase; AAR, area at risk; bNOS, brain NOS; BSO, L-buthionine-(S,R)-sulfoximine; ecNOS, endothelial cell NOS; ESR, electron spin resonance; ICAM, intercellular adhesion molecule; iNOS, inducible macrophage-type NOS; LPS, lipopolysaccharide; MnSOD, manganese superoxide dismutase; MnTBAP, tetrakis-(4-benzoic acid) porphyrin; NMDA, N-methyl-D-aspartic acid; PARP, poly(ADP-ribose) polymerase; PARS, poly(ADP-ribose) synthetase; RNS, reactive nitrogen species; ROS, reactive oxygen species.
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References |
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2 production by human neutrophils.
Can J Physiol Pharmacol
60:
1353-1358[Medline].
0031-6997/01/5301-135-159$3.00
PHARMACOLOGICAL REVIEWS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
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 M. M. Berger Antioxidant Micronutrients in Major Trauma and Burns: Evidence and Practice Nutr Clin Pract, October 1, 2006; 21(5): 438 - 449. [Abstract] [Full Text] [PDF]
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 Z. Cao, H. Zhu, L. Zhang, X. Zhao, J. L. Zweier, and Y. Li Antioxidants and Phase 2 Enzymes in Cardiomyocytes: Chemical Inducibility and Chemoprotection Against Oxidant and Simulated Ischemia-Reperfusion Injury Experimental Biology and Medicine, September 1, 2006; 231(8): 1353 - 1364. [Abstract] [Full Text] [PDF]
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T. Ohashi, Y. Ito, T. Matsuno, S. Sato, K. Shimokata, and H. Kume Paradoxical Effects of Hydrogen Peroxide on Human Airway Anion Secretion J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 296 - 303. [Abstract] [Full Text] [PDF]
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L. F. Andersen, D. R Jacobs Jr, M. H Carlsen, and R. Blomhoff Consumption of coffee is associated with reduced risk of death attributed to inflammatory and cardiovascular diseases in the Iowa Women's Health Study Am. J. Clinical Nutrition, May 1, 2006; 83(5): 1039 - 1046. [Abstract] [Full Text] [PDF]
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T. Genovese, E. Mazzon, C. Crisafulli, R. Di Paola, C. Muia, P. Bramanti, and S. Cuzzocrea Immunomodulatory Effects of Etanercept in an Experimental Model of Spinal Cord Injury J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1006 - 1016. [Abstract] [Full Text] [PDF]
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 R. Zamora, Y. Vodovotz, B. Betten, C. Wong, B. Zuckerbraun, K. F. Gibson, and H. R. Ford Intestinal and hepatic expression of BNIP3 in necrotizing enterocolitis: regulation by nitric oxide and peroxynitrite Am J Physiol Gastrointest Liver Physiol, November 1, 2005; 289(5): G822 - G830. [Abstract] [Full Text] [PDF]
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 P. J. Barnes New approaches to COPD Eur. Respir. Rev., September 1, 2005; 14(94): 2 - 11. [Abstract] [Full Text] [PDF]
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 T. B. Mysore, T. A. Shinkel, J. Collins, E. J. Salvaris, N. Fisicaro, L. J. Murray-Segal, L. E.A. Johnson, D. A. Lepore, S. N. Walters, R. Stokes, et al. Overexpression of Glutathione Peroxidase With Two Isoforms of Superoxide Dismutase Protects Mouse Islets From Oxidative Injury and Improves Islet Graft Function Diabetes, July 1, 2005; 54(7): 2109 - 2116. [Abstract] [Full Text] [PDF]
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T. Genovese, E. Mazzon, R. D. Paola, C. Muia, M. D. Threadgill, A. P. Caputi, C. Thiemermann, and S. Cuzzocrea Inhibitors of Poly(ADP-Ribose) Polymerase Modulate Signal Transduction Pathways and the Development of Bleomycin-Induced Lung Injury J. Pharmacol. Exp. Ther., May 1, 2005; 313(2): 529 - 538. [Abstract] [Full Text] [PDF]
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S. Cuzzocrea, R. Di Paola, E. Mazzon, U. Cortes, T. Genovese, C. Muia, W. Li, W. Xu, J.-H. Li, J. Zhang, et al. PARG activity mediates intestinal injury induced by splanchnic artery occlusion and reperfusion FASEB J, April 1, 2005; 19(6): 558 - 566. [Abstract] [Full Text] [PDF]
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 P. J. Barnes, I. M. Adcock, and K. Ito Histone acetylation and deacetylation: importance in inflammatory lung diseases Eur. Respir. J., March 1, 2005; 25(3): 552 - 563. [Abstract] [Full Text] [PDF]
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 P. J. Barnes Mediators of Chronic Obstructive Pulmonary Disease Pharmacol. Rev., December 1, 2004; 56(4): 515 - 548. [Abstract] [Full Text] [PDF]
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 X. Cui, C. Parent, H. Macarthur, S. D. Ochs, E. Gerstenberg, S. Solomon, Y. Fitz, R. L. Danner, S. M. Banks, C. Natanson, et al. Severity of sepsis alters the effects of superoxide anion inhibition in a rat sepsis model J Appl Physiol, October 1, 2004; 97(4): 1349 - 1357. [Abstract] [Full Text] [PDF]
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C. Di Filippo, F. Rossi, E. Ongini, P. Del Soldato, M. Perretti, and M. D'Amico The Distinct Alterations Produced in Cardiovascular Functions by Prednisolone and Nitro-prednisolone (NCX-1015) in the Rat Highlight a Causal Role for Endothelin-1 J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 1133 - 1141. [Abstract] [Full Text] [PDF]
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 A. Sarkar, Y. Sreenivasan, G. T. Ramesh, and S. K. Manna {beta}-D-Glucoside Suppresses Tumor Necrosis Factor-induced Activation of Nuclear Transcription Factor {kappa}B but Potentiates Apoptosis J. Biol. Chem., August 6, 2004; 279(32): 33768 - 33781. [Abstract] [Full Text] [PDF]
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 A. Ceriello and E. Motz Is Oxidative Stress the Pathogenic Mechanism Underlying Insulin Resistance, Diabetes, and Cardiovascular Disease? The Common Soil Hypothesis Revisited Arterioscler. Thromb. Vasc. Biol., May 1, 2004; 24(5): 816 - 823. [Abstract] [Full Text]
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S. CUZZOCREA, E. MAZZON, L. DUGO, R. DI PAOLA, A. P. CAPUTI, and D. SALVEMINI Superoxide: a key player in hypertension FASEB J, January 1, 2004; 18(1): 94 - 101. [Abstract] [Full Text] [PDF]
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 J. Pleiner, F. Mittermayer, G. Schaller, C. Marsik, R. J. MacAllister, and M. Wolzt Inflammation-induced vasoconstrictorhyporeactivity is caused by oxidative stress J. Am. Coll. Cardiol., November 5, 2003; 42(9): 1656 - 1662. [Abstract] [Full Text] [PDF]
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 A. Yildirim, M. Gumus, S. Dalga, Y. N. Sahin, and F. Akcay Dehydroepiandrosterone Improves Hepatic Antioxidant Systems after Renal Ischemia-Reperfusion Injury in Rabbits Ann. Clin. Lab. Sci., October 1, 2003; 33(4): 459 - 464. [Abstract] [Full Text] [PDF]
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 G. E. Callera, R. M. Touyz, S. A. Teixeira, M. N. Muscara, M. H. C. Carvalho, Z. B. Fortes, D. Nigro, E. L. Schiffrin, and R. C. Tostes ETA Receptor Blockade Decreases Vascular Superoxide Generation in DOCA-Salt Hypertension Hypertension, October 1, 2003; 42(4): 811 - 817. [Abstract] [Full Text] [PDF]
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 L. Prokai, K. Prokai-Tatrai, P. Perjesi, A. D. Zharikova, E. J. Perez, R. Liu, and J. W. Simpkins Quinol-based cyclic antioxidant mechanism in estrogen neuroprotection PNAS, September 30, 2003; 100(20): 11741 - 11746. [Abstract] [Full Text] [PDF]
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 P J Barnes Chronic obstructive pulmonary disease * 12: New treatments for COPD Thorax, September 1, 2003; 58(9): 803 - 808. [Full Text] [PDF]
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L. Ding-Zhou, C. Marchand-Verrecchia, B. Palmier, N. Croci, P.-E. Chabrier, M. Plotkine, and I. Margaill Neuroprotective Effects of (S)-N-[4-[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2-thiophenecarboximid-amide (BN 80933), an Inhibitor of Neuronal Nitric-Oxide Synthase and an Antioxidant, in Model of Transient Focal Cerebral Ischemia in Mice J. Pharmacol. Exp. Ther., August 1, 2003; 306(2): 588 - 594. [Abstract] [Full Text] [PDF]
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 J. Pfeilschifter, W. Eberhardt, and A. Huwiler Nitric Oxide and Mechanisms of Redox Signaling J. Am. Soc. Nephrol., August 1, 2003; 14(90003): S237 - 240. [Abstract] [Full Text] [PDF]
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V. Koshkin, X. Wang, P. E. Scherer, C. B. Chan, and M. B. Wheeler Mitochondrial Functional State in Clonal Pancreatic {beta}-Cells Exposed to Free Fatty Acids J. Biol. Chem., May 23, 2003; 278(22): 19709 - 19715. [Abstract] [Full Text] [PDF]
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 A. Ceriello New Insights on Oxidative Stress and Diabetic Complications May Lead to a "Causal" Antioxidant Therapy Diabetes Care, May 1, 2003; 26(5): 1589 - 1596. [Abstract] [Full Text] [PDF]
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 S. T. L. Ting, B. Earley, and M. A. Crowe Effect of repeated ketoprofen administration during surgical castration of bulls on cortisol, immunological function, feed intake, growth, and behavior J Anim Sci, May 1, 2003; 81(5): 1253 - 1264. [Abstract] [Full Text] [PDF]
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I. Medved, M. J. Brown, A. R. Bjorksten, J. A. Leppik, S. Sostaric, and M. J. McKenna N-acetylcysteine infusion alters blood redox status but not time to fatigue during intense exercise in humans J Appl Physiol, April 1, 2003; 94(4): 1572 - 1582. [Abstract] [Full Text] [PDF]
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I. V. Turko and F. Murad Protein Nitration in Cardiovascular Diseases Pharmacol. Rev., December 1, 2002; 54(4): 619 - 634. [Abstract] [Full Text] [PDF]
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 C. Goodyear-Bruch and J. D. Pierce Oxidative Stress in Critically Ill Patients Am. J. Crit. Care., November 1, 2002; 11(6): 543 - 551. [Abstract] [Full Text] [PDF]
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 D. Kobayashi, K. Kondo, N. Uehara, S. Otokozawa, N. Tsuji, A. Yagihashi, and N. Watanabe Endogenous Reactive Oxygen Species Is an Important Mediator of Miconazole Antifungal Effect Antimicrob. Agents Chemother., October 1, 2002; 46(10): 3113 - 3117. [Abstract] [Full Text] [PDF]
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 R. Kohen and A. Nyska Invited Review: Oxidation of Biological Systems: Oxidative Stress Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantification Toxicol Pathol, October 1, 2002; 30(6): 620 - 650. [Abstract] [PDF]
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 U. Raj and L. Shimoda Oxygen-dependent signaling in pulmonary vascular smooth muscle Am J Physiol Lung Cell Mol Physiol, October 1, 2002; 283(4): L671 - L677. [Abstract] [Full Text] [PDF]
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 J. Pleiner, F. Mittermayer, G. Schaller, R. J. MacAllister, and M. Wolzt High Doses of Vitamin C Reverse Escherichia coli Endotoxin-Induced Hyporeactivity to Acetylcholine in the Human Forearm Circulation, September 17, 2002; 106(12): 1460 - 1464. [Abstract] [Full Text] [PDF]
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 J. J. Lysiak, Q. A. T. Nguyen, and T. T. Turner Peptide and Nonpeptide Reactive Oxygen Scavengers Provide Partial Rescue of the Testis After Torsion J Androl, May 1, 2002; 23(3): 400 - 409. [Abstract] [Full Text] [PDF]
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M. Nishida, K. L. Schey, S. Takagahara, K. Kontani, T. Katada, Y. Urano, T. Nagano, T. Nagao, and H. Kurose Activation Mechanism of Gi and Go by Reactive Oxygen Species J. Biol. Chem., March 8, 2002; 277(11): 9036 - 9042. [Abstract] [Full Text] [PDF]
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 F. Visioli Effects of vitamin E on the endothelium: equivocal? {alpha}-tocopherol and endothelial dysfunction Cardiovasc Res, August 1, 2001; 51(2): 198 - 201. [Full Text] [PDF]
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J.L. Mehta, H.J. Chen, and D.Y. Li Protection of Myocytes From Hypoxia-Reoxygenation Injury by Nitric Oxide Is Mediated by Modulation of Transforming Growth Factor-{beta}1 Circulation, May 7, 2002; 105(18): 2206 - 2211. [Abstract] [Full Text] [PDF]
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