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The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.
Volume 3, Number 2, 2001
Mary Ann Liebert, Inc.
Forum Review
Mechanisms of the Antioxidant Effects of Nitric Oxide
The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role
in tissue injury. There are a number of reactive nitrogen oxide species, such as N
and ONOO
, that can alter
critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation
chemistry mediated by reactive oxygen species such as H
and O
that occurs at physiological levels of NO.
In addition to the antioxidant chemistry, NO protects against cell death mediated by H
, alkylhydroperoxides ,
and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, ap-
pears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In ad-
dition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit
oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chem-
ical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress. An-
tioxid. Redox Signal. 3, 203–213.
NDOGENOUS FORMATION of the radical species
nitric oxide (NO) results in an array of bi-
ological effects. Over the last decade, the role
that NO plays in oxidative injury has been de-
bated (2, 8, 23, 55). In the presence of oxygen
) or reactive oxygen species (ROS) such as
superoxide (O
), NO is converted into reac-
tive nitrogen oxide species (RNOS), which can
irreversibly modify a variety of biological mol-
ecules (summarized in 54). Thus, NO is often
considered to be a toxic species. However, NO
has also been shown to abate oxidative injury
in several biological systems. This review in-
cludes a discussion of the diverse chemistry of
NO, which can result in processes as disparate
as abatement of oxidative stress and mediation
of cell death and tissue injury. Also discussed
are the mechanisms by which NO modifies cel-
lular processes that protect cells and tissue
from oxidative damage. Finally, the effects of
NO treatment in various disease models are re-
One of the fundamental determinants of the
role of NO
in vivo
is its chemistry. As NO is a
radical, it has a number of potential chemical
pathways in biological systems. As discussed
in previous articles, the complex chemistry of
NO can be divided primarily into the two cat-
Tumor Biology Section, Radiation Biology Branch, National Cancer Institute and
Surgery Branch, NINDS,
Bethesda, MD.
Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030-6125.
Division of Neurology, Duke University Medical Center, Durham, NC.
Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA.
egories of direct and indirect effects (54). Direct
effects are comprised of those reactions in
which NO interacts directly with the biological
target. Conversely, indirect effects are medi-
ated by RNOS formed from the reaction of NO
with O
or O
One advantage to categorizing the reactions
of NO in this manner is that the concentration
of NO can be used to predict the contribution
of each effect. Direct effects require low con-
centrations of NO, whereas indirect effects oc-
cur at much higher NO concentrations (54).
Toxicity largely occurs under conditions where
NO-derived species are formed via the indirect
route. Thus, deleterious effects derived from
the chemistry of NO are often confined to con-
ditions of high local NO concentrations. The
antioxidant effects of NO are primarily a result
of direct effects at lower NO concentrations.
An antioxidant is a substance that prevents
oxidant formation or scavenges oxidants pro-
duced under conditions of oxidative stress. The
primary source of oxidative stress is ROS, such
as O
and peroxide. These species often pro-
duce oxidants through Fenton-type reactions.
RNOS, such as peroxynitrite (ONOO
), nitro-
gen dioxide (NO
), and nitroxyl (HNO), are an-
other potential source of oxidative stress, al-
though they are less potent oxidants than those
formed as a result of Fenton-type reactions.
Further, lipid peroxidation perpetuates oxida-
tive stress through formation of a variety of
lipid-oxy and -peroxy adducts. The versatile
chemistry of NO provides antioxidant mecha-
nisms against all three of these types of oxi-
dants predominantly through radical–radical
and ligand–metal interactions.
Fenton chemistry
In general, Fenton-type reactions occur be-
tween peroxide and transition metals and re-
sult in formation of hydroxyl radicals or hy-
pervalent peroxo- or oxo-metal complexes.
1 H
OH, Fe
O, FeO
These reactive intermediates can then alter bi-
ological substances such as proteins, nucleic
acids, and lipids, resulting in tissue injury (54).
NO can abate Fenton-mediated oxidative stress
by direct scavenging of oxidants, prevention of
peroxide reaction, and scavenging of reducing
equivalents supplied by O
(Fig. 1).
Hydroxyl radicals and high valent metal
complexes are scavenged by NO at near diffu-
sion control (
, .10
for NO 1
4). However, reactions between these highly re-
active compounds and substances other than
NO can also proceed with similarly high sec-
ond-order rate constants (21, 22). Therefore, the
determining factor for the antioxidant proper-
ties of NO is the relative concentration of NO
with respect to other potential reaction sites
, the pseudo first-order rate constants). As
discussed below, lipid peroxidation is abated
through a radical–radical reaction mechanism
at biologically relevant concentrations of NO
(48). Conversely, the reaction products of the
Fenton reaction generally must be produced in
close proximity to a macromolecule to induce
DNA strand breaks or protein oxidation.
Therefore, diffusion of NO to these regions
would likely not yield high enough effective
NO concentrations to out-compete the delete-
rious reactions between oxidants and biomole-
cules. Under these conditions, NO would not
FIG. 1. Antioxidant mechanism of NO in the Fenton-
type reactions.
be a good scavenger of oxidants and probably
would not have an antioxidant role.
The antioxidant properties of NO also in-
clude prevention of oxidant formation. For in-
stance, formation of a metal nitrosyl complex
can prevent addition of peroxide to the metal
coordination site. Metals such as copper and
nickel have been invoked as mediators of ox-
idative stress in some cases, however, iron com-
plexes are the predominant catalyst for this re-
in vivo
, generally through Fenton-type
reactions. To understand better the relationship
between iron nitrosyl complex formation and
prevention of oxidant production, it is useful
to consider the environment of the interacting
metal and ligands.
Hardsoft acid–base rules state that hard li-
gands, which donate their electrons less read-
ily than soft ligands, tend to associate more
strongly with hard, low polarizable metals. For
instance, ferric iron, which is a hard metal, will
bind more tightly to the hard ligand hydroxide
than will the softer ferrous ion. However, bind-
ing of soft ligands can result in softening of
hard metals. The ability of ligands to alter the
metal environment affects the reactivity of
metal-catalyzed reactions, which depend on
the oxidation state of the metal as well as the
ligand field.
For instance, hydroxyl radical is the primary
product of the reaction between peroxide and
soft ferrous iron surrounded by a hard ligand
field. In softer ligand fields, such as provided
by heme complexes, both the ferric and ferrous
states principally produce oxidizing species
such as Fe
and Fe
oxo complexes rather
than hydroxyl radicals. As NO is a soft ligand,
it will readily bind to ferrous complexes to form
a metal-nitrosyl species, but as a general rule,
if peroxide can react with the metal site, NO
can also bind. The resulting nitrosyl complex
inhibits the reaction between peroxide and the
metal, thereby preventing ROS production.
As described above, formation of hydroxyl
radicals or metal-oxo species often requires
. However, iron is more stable in the ferric
valence state
in vivo
. Reduction of Fe
to Fe
(Haber–Weiss chemistry) can involve O
which can provide the electrons to facilitate cat-
alytic oxidation of biological compounds (20).
NO and O
react at diffusion control to form
, which rapidly rearranges to nitrate in
the absence of other reactive species (45). Shunt-
ing of O
to nitrate thus inhibits reduction of
ferric iron and prevents catalytic formation of
ROS. This may be an important mechanism for
abatement of Fenton-type reaction-mediated
oxidative stress by NO.
Although scavenging of O
by NO can pre-
vent production of ROS via the Fenton reac-
tion, ONOO
is itself an oxidant. Exposure to
synthetic ONOO
has been shown to induce
tissue injury through reactions such as DNA
damage and lipid peroxidation (46). Therefore,
formation of ONOO
from the reaction be-
tween O
and NO has been speculated to in-
duce oxidative stress
in vivo
(2, 46). However,
the chemistry of the NO/O
reaction can of-
ten give different results from exposure to syn-
thetically generated ONOO
The oxidative chemistry of the NO/O
action was found to depend on the relative rate
of production of the two radicals (41, 59). For
instance, in the presence of a constant O
from xanthine oxide/hypoxanthine (XO), oxi-
dation of dihydrorhodamine or glutathione
(GSH) increased with increasing NO until the
rates of formation of these radicals were equiv-
alent. Further increases in NO concentration,
produced from a class of compounds known as
NONOates, which release NO in a controlled
manner over specific time periods (31), resulted
in marked decreases in dihydrorhodamine or
GSH oxidation. These data demonstrate that, in
the presence of excess NO, the reactive inter-
mediates formed during decomposition of
are scavenged by NO. This reaction
ultimately results in production of the ni-
trosating species N
(Fig. 2; 59). These find-
ings indicate that the oxidative chemistry of the
reaction is confined to a small range
of fluxes because excess NO can convert ox-
idative RNOS to nitrosating species.
More recently, carbon dioxide (CO
) was
shown to react with ONOO
to form a potent
adduct, CO
, which has oxidative prop-
erties similar to those of ONOO
(17, 36, 38).
However, CO
activation of ONOO
is pH-in-
dependent, unlike formation of the reactive
species HOONO in the absence of CO
. Excess
NO also quenches the oxidation chemistry of this
adduct (28). The CO
adduct has been proposed
to decompose to the carbonate radical (CO
and NO
(38). Reaction of excess NO with NO
formed in this manner would again result in pro-
duction of N
(Fig. 2; 18, 28, 54). These stud-
ies suggest that the spatial positioning as well as
timing of formation of these two radicals will de-
termine the extent of RNOS-mediated oxidation
and that oxidative stress and nitrosative stress
are balanced within the NO/O
Therefore, nitrosative stress may provide an op-
timal antioxidant environment.
Lipid peroxidation
The process of lipid peroxidation results in
formation of a variety of lipid-oxy and -peroxy
adducts (21). Perpetuation of lipid oxidation by
these species can result in cell membrane com-
promise (Fig. 3). Reaction of NO with these per-
oxy and oxy radicals terminates lipid peroxi-
dation via Eq. 2 and results in protection
against ROS (45):
1 NO
where LOO
is the lipid peroxy radical. Chain
termination also prevents oxidation of low
density lipoproteins in both endothelial cells
(52) and macrophages (26). Reduction in oxi-
dized cholesterol is thought to reduce initiation
of atheroscleroses mediated by foaming mac-
rophages. Other inflammatory processes such
as production of leukotrienes are also effected
by NO (54, and references therein). Lipoxyge-
nase, which mediates a variety of lipid oxida-
tion, is inhibited by NO (54, and references
therein). Thus, termination of lipid peroxida-
tion may be one of the most important antiox-
idant properties of NO.
Although NO often acts as an antioxidant,
the chemistry of NO can also affect cellular pro-
cesses such that cells or tissue becomes more
susceptible to oxidative stress. This complexity
of potential NO reactions within the cell has led
to differing opinions on the role of NO in ox-
idative stress (23). The effect of NO on the tox-
icity of hydrogen peroxide (H
), alkylhy-
droperoxide, and O
at the cellular level was
therefore examined by clonogenic assay, which
is the primary means for determining the cy-
totoxicity of chemical substances as it accounts
for both necrotic and apoptotic death.
Cytotoxic effects of NO/H
mediates oxidation of biological mole-
cules, which can result in tissue damage. Al-
though NO does
react directly with H
(55), it can protect cells against H
toxicity (19, 55, 56, 57). Exposure of lung fibro-
FIG. 2. Chemistry of reaction between NO and O
FIG. 3. Mechanism for termination of lipid peroxida-
tion by NO.
blasts to increasing concentrations of H
duced marked increases in cytotoxicity (55).
Addition of NONOate compounds surpris-
ingly resulted in protection against the cyto-
toxicity of H
(55). Pre- or posttreatment
with these NO donor complexes did not result
in protection; in fact, the by-product of the de-
composition of NO, nitrite, increased the cy-
totoxicity of H
. Similar observations were
made in neuronal (55), hepatoma (56), and en-
dothelial cells (6, 19).
These protective effects of NO were not re-
stricted to NONOates, as endogenous forma-
tion of NO in endothelial cells was also sug-
gested to be involved in protection against
damage to vascular smooth muscle mediated
by H
(37). Further, the
-nitrosothiolglutathione (GSNO)
-acetylpenicillamine (SNAP)
also protected against H
-mediated toxicity
(58). However, nitrovasodilators commonly
used in the clinic, such as 3-morpholinosyd-
nonimine (SIN-1) and sodium nitroprusside
(SNP), increased the toxicity of H
(14, 58).
Angeli’s salt (AS; Na
), which is struc-
turally similar to the NONOate compounds but
donates nitroxyl (NO
) instead of NO, signifi-
cantly potentiated the toxicity of H
These results demonstrate that the common
putative NO donors modulate the toxicity of
differently, and so caution is required in
the interpretation of experimental results.
The diverse effects exhibited by the NO
donors examined may be explained by vari-
ance in donor effect on cellular antioxidant de-
fenses and in amount and flux of NO produced.
One of the major cellular defenses against H
is consumption of this oxidant by the enzymes
glutathione peroxidase and catalase (21, 22).
When the kinetics for the disappearance of
were examined in the presence of the dif-
ferent NO donors, cellular consumption of
was noted to be inhibited to varying de-
grees by several of the donors. For instance, the
amount of time required to decompose 0.75
in the presence of SNP, the
NONOate Et
NN(O)NO (DEA/NO), AS, and
SNAP increased by 30–200% (58). Conversely,
SIN-1 and GSNO retarded H
by as much as 400%. Thus, enhancement of
-mediated toxicity by AS and SIN-1 might
be partially explained by inhibition of H
consumption. However, this cannot be the sole
mechanism by which NO enhances or protects
against H
, because GSNO, SNAP, and
DEA/NO also decreased the rate of decompo-
sition of H
, but proved to be cytoprotective.
Furthermore, cellular exposure to the NO
donors resulted in varied reduction of intra-
cellular levels of GSH. Exposure of V79 cells to
1 m
nitrite, SNAP, SIN-1, GSNO, DEA/NO,
or AS for 1 h resulted in varying degrees of de-
pletion of intracellular GSH (60). For instance,
SNAP, GSNO, and DEA/NO resulted in only
modest GSH decreases (,30%), which were re-
covered rapidly. Conversely, SIN-1 and AS de-
creased intracellular GSH levels by as much as
85%, whereas nitrite decreased GSH levels by
50% in these cells. This reduction in cellular
GSH may be the mechanism by which SIN-1,
AS, and nitrite enhance H
The observed differences in protective effects
among the various chemical NO donors may
also be a reflection of the actual flux of NO pro-
duced by each compound. The temporal pro-
files of NO release by the different compounds,
as assessed with an NO-selective electrode,
demonstrate that the amount of NO released
over time is quite variant (58). Both the
NONOates and
-nitroso complexes, which
protected against H
toxicity, released NO
over the entire time course of exposure to H
(1 h). However, SIN-1, SNP, and AS did not
produce measurable NO (,1 m
) under these
experimental conditions, coincident with a lack
of protection against H
SNP appears to increase the toxicity of ROS
by yet another mechanism. Chemical interaction
with SNP can result in formation of not only NO,
but also cyanide (CN
) and free iron. The iron
chelator desferrioxamine (DF) completely pro-
tected cells from H
, yet only partially pro-
tected against the toxicity of H
with SNP (58). This discrepancy may be ac-
counted for by enhanced release of CN
SNP. Monocytes and polymorphonuclear leuko-
cytes have also been shown to facilitate release
of CN
from SNP, which is a phenomenon be-
lieved to be mediated by H
(5). A transition
metal complex with a labile ligand was sug-
gested to oxidize substrates further via Fenton-
type catalysis (58). Further evidence supporting
this hypothesis comes from Imlay and col-
leagues, who showed that bacteria became more
sensitive to H
in the presence of CN
The fact that DF completely protected against
the toxicity of CN
suggests that metal–perox-
ide reactions are required to initiate cytotoxic-
ity. Thus, the DF-insensitive enhancement of
-mediated toxicity by SNP could be attrib-
uted to an iron complex, which cannot be bound
by DF, and so could catalyze the Fenton oxida-
tion chemistry of cellular molecules.
Cytotoxic effects of NO/alkylperoxides
NO was also found to act as an antioxidant
against XO-mediated lipid peroxidation (49). We
examined the effect of NO on organic hy-
droperoxide-mediated toxicity, which is thought
to be mediated by oxidation of lipophilic mem-
branes (57). Our studies further illustrate the im-
portance NO production for the duration of ex-
posure to oxidants. DEA/NO, which is a
NONOate with a half-life of
2 min at 37°C, pro-
vided minimal protection against either
butyl hydroperoxide or cumene hydroperoxide,
whereas the longer lived PAPA/NO (half-life of
15 min at 37°C) exhibited marked protection. The
different effects of these two NONOates on cy-
totoxicity can be attributed to the timing of de-
livery of NO as exposure to organic peroxides
usually requires up to 2 h to induce observable
toxicity (57). As alkylhydroperoxides require
longer times to penetrate cells and thereby exert
their damage, longer sustained fluxes of NO are
more protective.
Several potential mechanisms may be in-
volved in the protection by NO against organic
hydroperoxide-mediated toxicity. Intracellular
metalloproteins, such as those containing heme
moieties, react quickly with organic peroxides
to form hypervalent complexes (22). Upon de-
composition, these complexes release intracel-
lular iron, which in turn can catalyze damage
to macromolecules such as DNA. NO can react
with these hypervalent metalloproteins at near
diffusion control, which may restore these ox-
idized species to the ferric form (16, 50). Re-
duction of these metal-oxo proteins prevents
both their oxidative chemistry and their release
of free iron (16, 29, 56), thus limiting intracel-
lular damage mediated by oxidative stress.
Interestingly, although NO can protect
against the toxicity of H
in mammalian cells,
the opposite effect is observed in
E. coli
. Deliv-
ery of H
either as a bolus or through the en-
zymatic activity of XO, exhibited only modest
bactericidal activity (44). However, simultane-
ous exposure to both H
and NO, delivered
either as gas or by a NONOate complex, in-
creased bactericidal activity by four orders of
magnitude. Addition of catalase abated toxicity,
whereas exposure to superoxide dismutase
(SOD) had no effect. These results demonstrate
that the synergistic action of NO and H
rather than O
was responsible for this bacte-
ricidal activity. Thus, the combination of NO
and H
may be ideally suited to combat
E. coli
infections due to the additional protective effect
of NO for the host. This mechanism may also
apply to other species of bacteria, albeit with dif-
ferent kinetics. For instance, the cytotoxicity of
was abrogated by NO at
early time points, yet NO helped sustain toxicity
at longer time intervals. The maximal effect was
dependent upon the timing of exposure to NO,
and O
(30). These findings may explain
why NO and ROS are produced by immune ef-
fector cells at different times following exposure
to different pathogens.
The diametric responses of mammalian cells
and prokaryotes to the combination of
may reflect their different cellular
structures and complement of metalloproteins.
Bacteria utilize iron-sulfur clusters to a greater
extent than do mammalian cells, and these
types of proteins are especially susceptible to
degradation mediated by NO or RNOS (12, 24).
E. coli
, decomposition of iron complexes oc-
curs in the periplasmic space, which is in close
proximity to the cytoplasm. This relative lack of
compartmentalization may allow iron to bind
to and oxidize DNA. However, due to the or-
ganelle structure of mammalian cells, metal la-
bilization may be limited to the cytoplasm and
mitochondria. In such a cellular arrangement,
metals would be required to travel a large dis-
tance to reach the nucleus and bind to DNA.
Cytotoxic effects of NO/O
Treatment of cells with ONOO
results in
cell death in both bacterial (62) and mammalian
systems (reviewed in 46). However, treatment
of lung fibroblasts and neurons with O
NO did not exhibit appreciable toxicity (55).
Other studies showed that ovarian carcinoma
cells exposed to 5 m
SIN-1, which is a si-
multaneous generator of NO/O
, did not re-
sult in appreciable toxicity (14). In fact, cells
treated concomitantly with O
and NO re-
leasing compounds resulted in protection
against O
-mediated toxicity and
did not
play appreciable toxicity due to ONOO
mation (58). These results suggest that there
is a distinct difference between treating cells
with bolus concentrations of synthetic ONOO
(millimolar) and generating ONOO
Part of the discrepancy between bolus ad-
ministration and
de novo
synthesis of ONOO
can be explained in terms of reactant concen-
trations. Beckman and co-workers described
that penetration into cells by bolus treatment
required high concentrations of ONOO
cause the cell membrane forms a formidable
barrier for ONOO
penetration to intracellular
targets (62). Although extracellular generation
of NO and O
results in ONOO
the short lifetime of this species in solution does
not allow accumulation of high enough con-
centrations to penetrate the cell. Therefore, the
amount of ONOO
that could cross the cellu-
lar membrane under more biologically relevant
conditions than bolus administration, despite
production of stoichiometrically high amounts
over a prolonged time period, is dramatically
reduced. The cell membrane thus limits the
contribution of extracellular ONOO
to toxi-
cological mechanisms.
Another factor to consider with respect to tox-
icity mediated directly by ONOO
chemistry is
that the reaction between NO and ONOO
forms NO
, as was discussed above. Competi-
tion for O
by cellular components such as SOD
and redox proteins increases the amount of NO
required to form ONOO
. As the NO flux then
exceeds the O
flux, ONOO
is converted to
potent nitrosating agents. Hence, the chemistry
of extracellular formation of ONOO
by excess
NO converts ONOO
to nitrite. Direct necrotic
cell death mediated by oxidative chemistry of
from exposure of simultaneous
derived from NADPH is thus unlikely.
In addition to abating oxidative stress chem-
ically, NO can protect against oxidative stress
at the cellular level. For example, pretreatment
with NO several hours prior to exposure can
confer protection against H
(43). In bacte-
ria, treatment with NO resulted in up-regula-
tion of the SOXR and subsequent expression of
protective proteins against ROS (43). Hepato-
cytes also became resistant to peroxide injury
following treatment with NO (33). Expression
of hemoxygenase as well as other enzymes may
participate in NO-induced protection against
peroxide (33). In addition to stimulating ex-
pression of protective proteins, NO has a num-
ber of effects on cellular functions that may
influence oxidative stress. For instance, the
presence of NO may limit ROS production by
preventing assembly of NADPH oxidase,
which is one of the major contributors to ox-
idative stress in various immune responses (9).
In addition to limiting interfering with ox-
idative stress, NO has been shown to prevent
the induction of some genes that are induced
from ROS. For instance, oxidative stress can in-
duce early growth response-1, which through
the extracellular signal–regulated kinase path-
way can activate a number of adhesion mole-
cules. It has been shown that NO derived from
a donor or endothelial NO synthase (eNOS)
abates the up-regulation of this system (7).
NO can also inhibit oxidative stress pro-
cesses at physiological levels, which can result
in protection against tissue injury. NO has been
shown to attenuate ischemia–reperfusion in-
jury in numerous organs such as heart, intes-
tine, liver, kidney, and lung. To illustrate, NO,
either from donors or produced endogenously,
was shown to protect against myocardial isch-
emiareperfusion injury (39, 51). Endogenous
NO can also attenuate lung damage after in-
testinal ischemia. These reports suggest that
under different conditions where ischemia
reperfusion is involved NO can abate tissue
Neutrophil and leukocyte adhesion is also
inhibited by NO (34, 39). Leukocyte adhesion
and extravasation to the endothelium is one of
the primary mechanisms by which tissue dam-
age is mediated in ischemia–reperfusion. Pre-
venting an increase in activated leukocytes in
these regions is thought to protect tissue from
ischemia–reperfusion injury (34, 39). Increased
leukocyte adhesion and infiltration involve a
number of proinflammatory molecules, in-
cluding vascular cell adhesion molecule, in-
tercellular adhesion molecule (ICAM), and
CD11/CD18 expression (1, 11, 32, 53), and are
induced by ROS formation such as superfused
XO in the rat mesentery (15). This response
could be inhibited by treatment with either
SOD or NO donors, which scavenged O
, thus
preventing activation of the cell adhesion mol-
ecule, P-selectin. It also has been shown that
monocyte chemotactic protein-1 induced by
oxidative stress is inhibited by NO derived ei-
ther from an NO donor or from NOS-3 (53). In
summary, oxidative stress-induced leukocyte
adhesion mechanisms are inhibited by the
presence of NO.
NO also abates oxidative chemistry from
ROS, other than O
, which can lead to leuko-
cyte adhesion. Oxidants from the Fenton reac-
tion enhance production of the proinflamma-
tory molecules platelet-activating factor and
leukotriene B4, as well as increase the adhesion
molecules P-selectin, E-selectin, and ICAM-1
(35). Oxidant activation of platelet activating
factor and leukotriene B4 can activate nuclear
factor-kB, which can result in synthesis of in-
terleukin-8 and other adhesion molecules. In-
hibition by NO of the oxidative chemistry in an
analogous manner as described above for pro-
tection against peroxide-mediated toxicity also
prevents the cascade of events that leads to in-
creased leukocyte activation. Thus, the ability of
NO to limit ROS chemistry prevents expression
of different proinflammatory molecules and
subsequently limits leukocyte activation and ox-
idative stress in ischemia–reperfusion-mediated
tissue injury.
The role of NO in ischemia–reperfusion in-
jury in the brain has been controversial (13).
toxicity has been invoked as the
causative factor responsible for reperfusion-
mediated tissue damage (2, 10). However, as
discussed above, NO can be a powerful anti-
oxidant. A recent study assessed the value of
administering NO donor complexes at the
time of reperfusion in both a focal and global
brain ischemiareperfusion injury in rat mod-
els (40). The major problem with systemic ad-
ministration of most NO donors is that the re-
sulting dramatic reduction of blood pressure
places the patient at risk. This problem can be
overcome by delivering NONOates with very
short half-lives directly to the brain through
intracarotid injection.
Infusion of DEA/NO in this manner, at the
time of reperfusion subsequent to 5–20 min of
ischemia, resulted in a dramatic decrease of the
infarct area (40). A concurrent dramatic reduc-
tion in salicylate oxidation was also observed.
Electrochemical monitoring of the brain NO
levels showed that an initial NO burst (from
eNOS) declined with time in the absence of NO
donors. This reduction of NO production cor-
responded to an increase in salicylate oxida-
tion. In the presence of the NO donor, NO
levels were maintained throughout the experi-
ment, and salicylate oxidation was reduced.
From these data it was concluded that the pri-
mary function of NO in ischemia–reperfusion
injury is as an antioxidant, which in part was
responsible for reduction in infarct size. There-
fore, NO donors have two benefits: restoration
of blood flow and limiting oxidative stress in
the injured tissue. Endogenous production of
NO by eNOS limits infarct volume as do other
antioxidants, such as Tempol (40, 47). These
studies suggest that NO has a powerful anti-
oxidant role in ischemia–reperfusion injury in
the brain. Thus, for stroke, NO administration
following treatment with antithrombotic
agents may be beneficial.
Although the role of NO in ischemia–reper-
fusion injury represents acute tissue damage,
atherosclerosis depicts a chronic disease that is
mediated by oxidative stress. Although ather-
osclerosis has been proposed to be improved
by the presence of NO, the positive role of NO
in such diseases may be multifaceted, exerting
protective effects chemically, biochemically,
and physiologically (25, 61). Chemically, NO
can prevent oxidative damage and limit lipid
peroxidative chain propagation (25, 48, 49).
This is similar to the factors that limit alkylhy-
droperoxide toxicity as discussed above. Ter-
mination of lipid peroxidation by NO may de-
crease low-density lipid oxidation whose prod-
ucts promote foaming macrophage and in-
crease plaques. Furthermore, low-density lipid
oxidation can lead to mast cell degranulation,
which can induce further inflammation. Bio-
logically, NO can inhibit platelet aggregation,
prevent mast cell degranulation and leukocyte
adhesion, and thereby is beneficial with respect
to diseases that involve lipid oxidation (42).
Thus, NO may have multiple effects in main-
taining the health of the vascular wall, which
may play a critical role in a number of acute
and chronic injuries to tissue.
The ability of NO to abate oxidative stress
plays an important role in both physiological
and pathophysiological mechanisms involving
properties at the chemical, cellular, and physi-
ological levels. The relatively low concentra-
tion of NO required to be an antioxidant sug-
gests that in addition to its involvement with
cyclic GMP, this radical molecule serves to
counterbalance oxidative stress. This balance
between NO and oxidative stress provides an
important regulatory mechanism in numerous
physiological effects. Imbalance in this redox
symbiotic relationship can lead to different
pathophysiological conditions.
AS, Angeli’s salt (Na
); CN
, cyanide;
, carbon dioxide; DEA/NO, Et
DF, desferrioxamine; eNOS, endothelial nitric
oxide synthase; GSH, glutathione; GSNO,
nitrosothiolglutathione; H
, hydrogen per-
oxide; ICAM, intercellular adhesion molecule;
, lipid peroxy radical; NO, nitric oxide;
, nitrogen dioxide; O
, superoxide;
, peroxynitrite; RNOS, reactive nitro-
gen oxide species; ROS, reactive oxygen species;
SIN-1, 3-morpholinosydnonimine; SNAP,
-acetylpenicillamine; SNP, sodium ni-
troprusside; SOD, superoxide dismutase; XO,
xanthine oxide/hypoxanthine.
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Address reprint requests to:
David A. Wink, Ph.D.
Radiation Biology Branch
National Institutes of Health/National Cancer
Building 10, Room B3-B69
Bethesda, MD 20892
Received for publication October 2, 2000; ac-
cepted November 20, 2000.
... However, exceeding certain concentrations can cause antioxidant fatigue, and hence lead to oxidative damage, as evidenced by the MDA content at high concentrations. NO is a free radical that also serves as an antioxidant, able to react with metal sites and the resulting nitroso complex to inhibit the reaction between the peroxide and the metal, thus preventing the production of ROS [40]. We observed a dose-dependent decrease in NO enzyme activity both in the hepatopancreas and in the plasma. ...
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There is growing evidence that long-term exposure to prometryn (a widely used herbicide) can induce toxicity in bony fish and shrimp. Our previous study demonstrated its 96 h acute toxicity on the crab Eriocheir sinensis. However, studies on whether longer exposure to prometryn with a lower dose induces toxicity in E. sinensis are scarce. Therefore, we conducted a 20 d exposure experiment to investigate its effects on the hepatopancreas and intestine of E. sinensi. Prometryn reduce the activities of antioxidant enzymes, increase the level of lipid peroxidation and cause oxidative stress. Moreover, long-term exposure resulted in immune and detoxification fatigue, while short-term exposure to prometryn could upregulate the expression of genes related to immunity, inflammation and detoxification. Prometryn altered the morphological structure of the hepatopancreas (swollen lumen) and intestine (shorter intestinal villi, thinner muscle layer and thicker peritrophic membrane). In addition, prometryn changed the species composition of the intestinal flora. In particular, Bacteroidota and Proteobacteria showed a dose-dependent decrease accompanied by a dose-dependent increase in Firmicutes at the phylum level. At the genus level, all exposure groups significantly increased the abundance of Zoogloea and a Firmicutes bacterium ZOR0006, but decreased Shewanella abundance. Interestingly, Pearson correlation analysis indicated a potential association between differential flora and hepatopancreatic disorder. Phenotypic abundance analysis indicated that changes in the gut flora decreased the intestinal organ’s resistance to stress and increased the potential for opportunistic infection. In summary, our research provides new insights into the prevention and defense strategies in response to external adverse environments and contributes to the sustainable development of E. sinensis culture.
... NOS2 generates NO at a high rate and for prolonged periods, which favours a shift in the cellular redox potential to a more oxidised state. Evidence reveals that low concentrations of NO, such as would result from CCT downregulating NOS2, protect against ROS-associated injury [67]. Reports show that NO restrains lipolysis via the oxidative modification of adenylyl cyclase (AC), mimicking antioxidant actions, and it has been suggested that NO suppresses lipolysis by reducing cyclic adenosine monophosphate (cAMP) and PKA activation [30,68], thus avoiding HSL and perilipin-1 phosphorylation. ...
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Crocetin (CCT) is a natural saffron-derived apocarotenoid that possesses healthy properties such as anti-adipogenic, anti-inflammatory, and antioxidant activities. Lipolysis is enhanced in obesity and correlates with a pro-inflammatory, pro-oxidant state. In this context, we aimed to investigate whether CCT affects lipolysis. To evaluate CCT’s possible lipolytic effect, 3T3-L1 adipocytes were treated with CCT10μM at day 5 post-differentiation. Glycerol content and antioxidant activity were assessed using colorimetric assays. Gene expression was measured using qRT-PCR to evaluate the effect of CCT on key lipolytic enzymes and on nitric oxide synthase (NOS) expression. Total lipid accumulation was assessed using Oil Red O staining. CCT10μM decreased glycerol release from 3T3-L1 adipocytes and downregulated adipose tissue triglyceride lipase (ATGL) and perilipin-1, but not hormone-sensitive lipase (HSL), suggesting an anti-lipolytic effect. CCT increased catalase (CAT) and superoxide dismutase (SOD) activity, thus showing an antioxidant effect. In addition, CCT exhibited an anti-inflammatory profile, i.e., diminished inducible NOS (NOS2) and resistin expression, while enhancing the expression of adiponectin. CCT10μM also decreased intracellular fat and C/EBPα expression (a transcription factor involved in adipogenesis), thus revealing an anti-adipogenic effect. These findings point to CCT as a promising biocompound for improving lipid mobilisation in obesity.
... Higher TCA cycle activity causes an increase in TCA cycle acids, which are antioxidants [144]. Glutathione, nitric oxide produced from arginine, and conjugated linoleic acid isomers, though important antioxidants [145][146][147], may be less compatible with sustained hypotonic conditions where protein and lipid breakdown are reduced, nitric oxide-mediated vasodilation is less needed, and NF-κB-dependent cytokine expression [148,149] is downregulated by hypotonic conditions [150,151]. Reduced breakdown of the dipeptides anserine and carnosine to beta-alanine favors anserine and carnosine antioxidant capacity. ...
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Background/aims: Cells adapt to chronic extracellular hypotonicity by altering metabolism. Corresponding effects of sustained hypotonic exposure at the whole-person level remain to be confirmed and characterized in clinical and population-based studies. This analysis aimed to 1) describe changes in urine and serum metabolomic profiles associated with four weeks of sustained > +1 L/d drinking water in healthy, normal weight, young men, 2) identify metabolic pathways potentially impacted by chronic hypotonicity, and 3) explore if effects of chronic hypotonicity differ by type of specimen and/or acute hydration condition. Materials: Untargeted metabolomic assays were completed for specimen stored from Week 1 and Week 6 of the Adapt Study for four men (20-25 years) who changed hydration classification during that period. Each week, first-morning urine was collected after overnight food and water restriction, and urine (t+60 min) and serum (t+90 min) were collected after a 750 mL bolus of drinking water. Metaboanalyst 5.0 was used to compare metabolomic profiles. Results: In association with four weeks of > + 1 L/d drinking water, urine osmolality decreased below 800 mOsm/kg H2O and saliva osmolality decreased below 100 mOsm/kg H2O. Between Week 1 and Week 6, 325 of 562 metabolic features in serum changed by 2-fold or more relative to creatinine. Based on hypergeometric test p-value <0.05 or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway impact factor >0.2, the sustained > + 1 L/d of drinking water was associated with concurrent changes in carbohydrate, protein, lipid, and micronutrient metabolism, a metabolomic pattern of carbohydrate oxidation via the tricarboxylic acid (TCA) cycle, instead of glycolysis to lactate, and a reduction of chronic disease risk factors in Week 6. Similar metabolic pathways appeared potentially impacted in urine, but the directions of impact differed by specimen type. Conclusion: In healthy, normal weight, young men with initial total water intake below 2 L/d, sustained > + 1 L/d drinking water was associated with profound changes in serum and urine metabolomic profile, which suggested normalization of an aestivation-like metabolic pattern and a switch away from a Warburg-like pattern. Further research is warranted to pursue whole-body effects of chronic hypotonicity that reflect cell-level effects and potential beneficial effects of drinking water on chronic disease risk.
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The Manila clam ( Ruditapes philippinarum ), as one of the shellfish living in the intertidal zone, is known for its strong ability to withstand air exposure. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been shown to be useful for antioxidant and immune regulation in aquatic animals. In this study, an untargeted metabolomics (LC–MS/MS) technique was employed for the first time in Manila clam to analyze the metabolic and histological impacts after air exposure and the positive effects of SNP pretreatment. During air exposure, a significant increase in taurine, L-glutamate, and several polyunsaturated fatty acids in clams was detected, which indicates that clams may experience inflammatory reactions, oxidative stress, and an increase in blood ammonia content. When clams were exposed to SNP for 6 h, arginine, spermine, L-glutamic acid, and glutathione content were all upregulated, indicating that the SNP exposure induced NO production and improved antioxidant capacity in clams. When the clams were exposed to air after SNP pretreatment, there were no significant differences in the levels of taurine, L-glutamate, or aliphatic acids between the experimental and control groups. Gill tissue was more severely damaged in clams directly exposed to air than in those that experienced air exposure after SNP pretreatment, especially in clams exposed to air for a long time (72 h). Both metabolomics and tissue section structure indicated that SNP pretreatment decreased the stress responses caused by air exposure in R. philippinarum . These findings provided fresh insights and a theoretical foundation for understanding the tolerance to air exposure and physiological functions of SNP (or NO) in R. philippinarum .
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Over 1 million Americans are currently living with T1D and improvements in diabetes management have increased the number of adults with T1D living into later decades of life. This growing population of older adults with diabetes is more susceptible to aging comorbidities, including both vascular disease and osteoporosis. Indeed, adults with T1D have a 2- to 3- fold higher risk of any fracture and up to 7-fold higher risk of hip fracture compared to those without diabetes. Recently, diabetes-related vascular deficits have emerged as potential risks factors for impaired bone blood flow and poor bone health and it has been hypothesized that there is a direct pathophysiologic link between vascular disease and skeletal outcomes in T1D. Indeed, microvascular disease (MVD), one of the most serious consequences of diabetes, has been linked to worse bone microarchitecture in older adults with T1D compared to their counterparts without MVD. The association between the presence of microvascular complications and compromised bone microarchitecture indicates the potential direct deleterious effect of vascular compromise, leading to abnormal skeletal blood flow, altered bone remodeling, and deficits in bone structure. In addition, vascular diabetic complications are characterized by increased vascular calcification, decreased arterial distensibility, and vascular remodeling with increased arterial stiffness and thickness of the vessel walls. These extensive alterations in vascular structure lead to impaired myogenic control and reduced nitric-oxide mediated vasodilation, compromising regulation of blood flow across almost all vascular beds and significantly restricting skeletal muscle blood flow seen in those with T1D. Vascular deficits in T1D may very well extend to bone, compromising skeletal blood flow control, and resulting in reduced blood flow to bone, thus negatively impacting bone health. Indeed, several animal and ex vivo human studies report that diabetes induces microvascular damage within bone are strongly correlated with diabetes disease severity and duration. In this review article, we will discuss the contribution of diabetes-induced vascular deficits to bone density, bone microarchitecture, and bone blood flow regulation, and review the potential contribution of vascular disease to skeletal fragility in T1D.
The presence of hydrogen peroxide along with ferrous iron produces hydroxyl radicals that preferably oxidize polyunsaturated fatty acids (PUFA) to alkyl radicals (L•). The reaction of L• with an oxygen molecule produces lipid peroxyl radical (LOO•) that collectively trigger chain reactions, which results in the accumulation of lipid peroxidation products (LOOH). Oxygenase enzymes, such as lipoxygenase, also stimulate the peroxidation of PUFA. The production of phospholipid hydroperoxides (P-LOOH) can result in the destruction of the architecture of cell membranes and ultimate cell death. This iron-dependent regulated cell death is generally referred to as ferroptosis. Radical scavengers, which include tocopherol and nitric oxide (•NO), react with lipid radicals and terminate the chain reaction. When tocopherol reductively detoxifies lipid radicals, the resultant tocopherol radicals are recycled via reduction by coenzyme Q or ascorbate. CoQ radicals are reduced back by the anti-ferroptotic enzyme FSP1. •NO reacts with lipid radicals and produces less reactive nitroso compounds. The resulting P-LOOH is reductively detoxified by the action of glutathione peroxidase 4 (GPX4) or peroxiredoxin 6 (PRDX6). The hydrolytic removal of LOOH from P-LOOH by calcium-independent phospholipase A2 leads the preservation of membrane structure. While the expression of such protective genes or the presence of these anti-oxidant compounds serve to maintain a healthy condition, tumor cells employ them to make themselves resistant to anti-tumor treatments. Thus, these defense mechanisms against ferroptosis are protective in ordinary cells but are also potential targets for cancer treatment.
Background: Nickel is a heavy metal that is regarded as a possible hazard to living organisms due to its toxicity and carcinogenicity. Nickel chloride (NiCl2), an inorganic divalent Ni compound, has been shown to cause oxidative stress in cells by altering the redox equilibrium. We have investigated the effect of NiCl2 on isolated human erythrocytes under in vitro condition. Methods: Isolated erythrocytes were treated with different concentrations of NiCl2 (25-500 µM) for 24 h at 37 ºC. Hemolysates were prepared and several biochemical parameters were analyzed in them. Results: Treatment of erythrocytes with NiCl2 enhanced the intracellular generation of reactive oxygen species (ROS). A significant increase in hydrogen peroxide levels and oxidation of proteins and lipids was also seen. This was accompanied by a reduction in levels of nitric oxide, glutathione, free amino groups and total sulfhydryl groups. NiCl2 treatment impaired both enzymatic and non-enzymatic defense systems, resulting in lowered antioxidant capacity and diminished ability of cells to quench free radicals and reduce metal ions. NiCl2 exposure also had an inhibitory effect on the activity of enzymes involved in pathways of glucose metabolism (glycolytic and pentose phosphate shunt pathways). Increased level of methemoglobin, which is inactive in oxygen transport, was also seen. The rate of heme breakdown increased resulting in the release of free iron. Exposure to NiCl2 led to considerable cell lysis, indicating damage to the erythrocyte membrane. This was supported by the inhibition of membrane bound enzymes and increase in the osmotic fragility of NiCl2 treated cells. NiCl2 treatment caused severe morphological alterations with the conversion of normal discocytes to echinocytes. All changes were seen in a NiCl2 concentration-dependent manner. Conclusion: NiCl2 generates cytotoxic ROS in human erythrocytes which cause oxidative damage that can decrease the oxygen carrying capacity of blood and also lead to anemia.
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Nitric oxide (NO) is a gaseous molecule that has a central role in signaling pathways involved in numerous physiological processes (e.g., vasodilation, neurotransmission, inflammation, apoptosis, and tumor growth). Due to its gaseous form, NO has a short half-life, and its physiology role is concentration dependent, often restricting its function to a target site. Providing NO from an external source is beneficial in promoting cellular functions and treatment of different pathological conditions. Hence, the multifaceted role of NO in physiology and pathology has garnered massive interest in developing strategies to deliver exogenous NO for the treatment of various regenerative and biomedical complexities. NO-releasing platforms or donors capable of delivering NO in a controlled and sustained manner to target tissues or organs have advanced in the past few decades. This review article discusses in detail the generation of NO via the enzymatic functions of NO synthase as well as from NO donors and the multiple biological and pathological processes that are modulated by NO. The methods for incorporation of NO donors into diverse biomaterials including physical, chemical, or supramolecular techniques are summarized. Then, these NO-releasing platforms are highlighted in terms of advancing treatment strategies for various medical problems.
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The mechanism of cytotoxicity of the NO donor 3-morpholino-sydnonimine toward a human ovarian cancer cell line (OVCAR) was examined. It was found that the NO-mediated loss of cell viability was dependent on both NO and hydrogen peroxide (HO). Somewhat surprisingly, superoxide (O) and its reaction product with NO, peroxynitrite (OONO), did not appear to be directly involved in the observed NO-mediated cytotoxicity against this cancer cell line. The toxicity of NO/HO may be due to the production of a potent oxidant formed via a trace metal-, HO-, and NO-dependent process. Because the combination of NO and HO was found to be particularly cytotoxic, the effect of NO on cellular defense mechanisms involving HO degradation was investigated. It was found that NO was able to inhibit catalase activity but had no effect on the activity of the glutathione peroxidase (GSHPx)-glutathione reductase system. It might therefore be expected that cells that utilize primarily the GSHPx-glutathione reductase system for degrading HO would be somewhat resistant to the cytotoxic effects of NO. Consistent with this idea, it was found that ebselen, a compound with GSHPx-like activity, was able to protect cells against NO toxicity. Also, lowering endogenous GSHPx activity via selenium depletion resulted in an increased susceptibility of the target cells to NO-mediated toxicity. Thus, a possible NO/HO/metal-mediated mechanism for cellular toxicity is presented as well as a possible explanation for cell resistance/susceptibility to this NO-initiated process.
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Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O2 were nearly equivalent. The presence of superoxide dismutase reversed O2-mediated inhibition, while catalase had no effect. Increasing the flux of O2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and O2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.
Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O-2(radical anion), while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O-2(radical anion). A solution of glutathione (GSH), a model thiol containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O-2(radical anion) were nearly equivalent. The presence of superoxide dismutase reversed O-2(radical anion)-mediated inhibition, while catalase had no effect. Increasing the flux of O-2(radical anion) yielded oxidized glutathione (GSSG), peaking when the flux of NO and O-2(radical anion) were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.
The role of nitric oxide (NO) in inflammation represents one of the most studied yet controversial subjects in physiology. A number of reports have demonstrated that NO possesses potent anti-inflammatory properties, whereas an equally impressive number of studies suggest that NO may promote inflammation-induced cell and tissue dysfunction. The reasons for these apparent paradoxical observations are not entirely clear; however, we propose that understanding the physiological chemistry of NO and its metabolites will provide a blueprint by which one may distinguish the regulatory/anti-inflammatory properties of NO from its deleterious/proinflammatory effects. The physiological chemistry of NO is complex and encompasses numerous potential reactions. In an attempt to simplify the understanding of this chemistry, the physiological aspects of NO chemistry may be categorized into direct and indirect effects. This type of classification allows for consideration of timing, location, and rate of production of NO and the relevant targets likely to be affected. Direct effects are those reactions in which NO interacts directly with a biological molecule or target and are thought to occur under normal physiological conditions when the rates of NO production are low. Generally, these types of reactions may serve regulatory and/or anti-inflammatory functions. Indirect effects, on the other hand, are those reactions mediated by NO-derived intermediates such as reactive nitrogen oxide species derived from the reaction of NO with oxygen or superoxide and are produced when fluxes of NO are enhanced. We postulate that these types of reactions may predominate during times of active inflammation. Consideration of the physiological chemistry of NO and its metabolites will hopefully allow one to identify which of the many NO-dependent reactions are important in modulating the inflammatory response and may help in the design of new therapeutic strategies for the treatment of inflammatory tissue injury.