Drugs modulating the biological effects of peroxynitrite and related nitrogen species

Drugs Modulating the BiologicalE¡ects of Peroxynitrite and Related

Nitrogen Species

Ana Olmos, Rosa M. Giner, Salvador Manez

Departament de Farmacologia, Universitat de Valencia, Valencia, Spain

Published online 2 June 2006 in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/med.20065

!

Abstract: The term ‘‘reactive nitrogen species’’ includes nitrogen monoxide, commonly called

nitric oxide, and some other remarkable chemical entities (peroxynitrite, nitrosoperoxycarbonate,

etc.) formed mostly from nitrogen monoxide itself in biological environments. Regardless of the

specific mechanisms implicated in their effects, however, it is clear that an integrated

pharmacological approach to peroxynitrite and related species is only just beginning to take

shape. The array of affected chemical and pathological processes is extremely broad. One of the

most conspicuous mechanisms observed thus far has been the scavenging of the peroxynitrite anion

by molecules endowed with antioxidant activity. This discovery has in turn lent great significance

to several naturally occurring and synthetic antioxidants, which usually protect not only against

oxidative reactions, but also from nitrating ones, both in vitro and in vivo. This has proven to be

beneficial in different tissues, especially within the central nervous system. Taking these results and

those of other biochemical investigations into account, many research lines are currently in

progress to establish the true potential of reactive nitrogen species deactivators in the therapy of

neurological diseases, ischemia-reperfusion damage, renal failure, and lung injury, among others.

� 2006 Wiley Periodicals, Inc. Med Res Rev, 27, No. 1, 1–64, 2007

Key words: peroxynitrite; reactive nitrogen species; tyrosine nitration; antioxidants; NOS; PARP

inhibitors

1 . I N T R O D U C T I O N

In 1985, Blough and Zafiriou were the first to describe the manner in which two of the most

important free radicals in mammal physiology, namely nitrogen monoxide and superoxide, react

to form the aggressive peroxynitrite anion (Reaction of superoxide with nitric oxide to form

Contract grant sponsor: SpanishMinistryof Scienceand Technology;Contract grant number: SAF 2002-00723;Contract

grant sponsor:Generalitat Valeciana;Contract grant number: CTBPRA/2002/56 (to A.O.)

Correspondence to: Salvador Ma¤ n‹ ez, Departament de Farmacologia, Facultat de Farma' cia,Universitat deVale' ncia, Av.Vicent

Andre¤ s Estelle¤ s s/n, 46100 Burjassot,Valencia Spain.E-mail: [email protected].

Medicinal Research Reviews, Vol. 27, No. 1, 1^64, 2007

� 2006 Wiley Periodicals, Inc.

peroxonitrite in alkaline aqueous solution. Inorg Chem 1985;24:3502–3504). Later, Beckman et al.1

postulated that this reaction could have a pivotal role in cardiovascular physiology. Since then, a great

deal of effort has been put into determining the key events that regulate the genesis and reactivity of

peroxynitrite. In addition to an obligatory reference to its intrinsic oxidant properties, one of the

generally admitted suppositions concerning the role of the anion is that its influence in degenerative

and inflammatory diseases is tightly correlated to its ability to nitrate aromatic amino acids—mostly

tyrosine residues—in proteins. Nevertheless, it is obvious that other, less reactive nitrogen species

such as nitrite can also be endowed with nitrating activity upon activation by metals and peroxides.

Another remarkably important point in the natural history of peroxynitrite is its ability to induce

DNA degradation, which in turn activates poly (ADP-ribose) polymerase, an enzyme that depletes

NADþ and consequently exhausts cell ATP sources, thus leading to cell death. In addition, the

activation of poly (ADP-ribose) polymerase results in a greater release of inflammatory mediators

through the activation of AP-1 and nuclear factor-kB.

Furthermore, some investigations concentrate on the control of nitrogen monoxide synthesis

from arginine by nitric oxide synthase as a very early step that may even trigger the entire process.

Although many substances are known inhibitors of inducible nitric oxide synthase, several are of

particular interest because they also exhibit peroxynitrite-scavenging activity, as is the case of

mercaptoguanidines and allied derivatives.

For the purpose of outlining the current knowledge on the chemical diversity, pharmacody-

namics, and therapeutic projection of the drugs that counteract the harmful effects of reactive

nitrogen species, we will review and discuss the pertinent results that have appeared since 1994 up to

the present.

2 . A C H E M I C A L I N T R O D U C T I O N T O T H E B I O S Y N T H E S I S A N D T U R N O V E RO F R E A C T I V E N I T R O G E N S P E C I E S

This section constitutes a preface that tries to summarize some of the essential issues necessary for

understanding one of the most exciting interfaces between inorganic chemistry and pharmacology.

With the aim of reaching this goal, we wish to give a cursory, perhaps even abrupt, overview on the

chief reactive nitrogen species (RNS).

A list of biologically relevant RNS would regularly include three oxides, namely NO (nitrogen

monoxide, nitric oxide), N2O3 (dinitrogen trioxide, nitrous anhydride), and NO2 (nitrogen dioxide),

as well as three anions, to wit, NO� (oxidonitrate, nitroxyl anion), ONOO� (oxidoperoxidonitrate,

peroxonitrite, peroxynitrite), and ONOOCO�2 (1-carboxylato-2-nitrosodioxidane, nitrosoperoxy-

carbonate). However evident it may seem, the fact that these are all oxygenated nitrogen species

bears repeating since they can thus be qualified as a special class of reactive oxygen species (ROS).

To date, a high number of biological interactions have been described for the whole family of

RNS; however, because of their elusive and short-lived character, determining the specific cellular

sources for each relevant RNS is an extremely difficult task. Since there is evidence that NO is an

antecedent for each of them, a widely held view qualifies their effects as indirect effects of NO.

A. NO

Reactive nitrogen species are interconnected through a series of reactions, which could

conventionally be admitted to start with the generation of NO from the guanidino portion of

arginine by means of the intervention of nitric oxide synthase (NOS). This enzyme exists in three

distinct isoforms: inducible NOS (iNOS or NOS2), which produces large amounts of NO,

constitutive neuronal NOS (nNOS or NOS1), and endothelial NOS (eNOS or NOS3), both of which

are responsible for the low basal levels of NO. A more recently discovered isoform is the constitutive

2 * OLMOS, GINER, AND MAN‹ EZ

mitochondrial NOS (mtNOS), which is associated to the inner membrane and is considered to be a

post-transcriptionally modified nNOS.2 The enzyme transforms arginine into the ureido-aminoacid

citrulline after consuming 2 moles of O2 and 1.5 moles of NADPH per mole of substrate. The

molecule of NO, a gas discovered by Priestley in 17843, is a free radical that has served as a model to

explain the nature of the covalent bonding orbitals as well as the existence of unpaired electrons in a

simple inorganic molecule. For this reason alone it has been a mandatory topic in general chemistry

books for years. Moreover, nearly two centuries after being discovered, it came to be known as a key

mediator for a huge number of physiological functions.

In the NO molecule, the distance between the N and O nuclei is 1.154 A, which corresponds to a

conventional bond order of 2.5, with 60% of the electron spin density on the nitrogen atom. At strong

alkaline pH, NO can exhibit reducing properties, according to the following reaction:4

NO�2 þ H2O þ e� ! NO þ 2OH�ðEo ¼ �0:46VÞ

Although NO is a radical, it is not as reactive as generally supposed. Instead, it diffuses away the

cellules in which it has been synthesized without being altered, unless it meet other molecules with

unpaired electrons, for example, molecular oxygen. It should be taken into account that the half-life

of NO at physiological concentrations (<4 mM) is considerably higher than that measured in

saturated water dissolution (� 2 mM).5 Regarding the biochemical role of NO, its most prominent

characteristic is its reactivity with transition metals. In living systems, NO normally bonds to only one

metal atom (M), in the schematic form M–N–O, a formula that accounts for several linear or bent

resonant structures with a different formal distribution of charges.4 The formation of iron complexes,

which are among the most salient metal nitrosyl derivatives, is pivotal for the activity of enzymes

containing a heme group, such as the ubiquitous guanylate cyclase (GC), but detrimental for others,

such as catalase. Several other effects based on direct reactivity are definitely antioxidant; for

example, NO reacts with oxygen free radicals implicated in lipid peroxidation, thereby deactivating

them. It is also able to reduce metalloxo heme-hypervalent states of hemoproteins (hemoglobin,

myoglobin, cytochromes, etc) to give nitrite (NO�2 ) or nitrate (NO�

3 )

Fe4þ ¼ O þ NO ! Fe3þ þ NO�2

B. NO�

As mentioned before, full functioning of NOS generates NO. However, in certain circumstances NOS

can also lead to the synthesis of NO�, particularly in the absence of tetrahydrobiopterin. This cofactor

allows for maximum efficiency in the stoichiometrically-adjusted production of NO because it

lessens the extent of the NADPH-mediated reduction of O2 to superoxide anion radical (O�2 ).

Experimental evidence from Schmidt et al.6 has suggested that NOS activity may first generateNOpre,

a kind of pre-NO metabolite, which, in the presence of superoxide dismutase (SOD), would give NO.

Proof of the existence of NOpre may be the concomitant production of certain metabolites such as

NH2OH or N2O, which are unlikely to arise from NO, but compatible with the NO� lineage (Fig. 1).

On the basis of the formation of a ferrous rather than a ferric heme nitrosyl complex of

tetrahydrobiopterin-deprived NOS, NO� has been fully recognized as NOpre.7,8 This nitroxyl anion

oxidizes different molecules, such as thiols or amines, participates in reductive nitrosylation of

metals, and seems responsible for several cytotoxic effects by means of other oxygen-dependent

reactions.9

The effective importance of NO�, or rather, its protonated actual form (HNO), in cardiovascular

physiopathology is a subject of current research.10,11 A preliminary consideration is that, unlike what

one could intuitively suppose, the conversion between the so called orthogonal siblings12 NO and

NO� is not easy, and they initiate independent signaling pathways in regulating vascular tone.

Experiments carried out with dogs have shown that, while infusion of NO donors such as

DRUGS MODULATING THE BIOLOGICAL EFFECTS OF PEROXYNITRITE * 3

diethylamine/NO adduct or nitroglycerin increase cGMP levels, analogous treatment with Angeli’s

salt (Na2N2O3)—the most widely used NO� donor—does not alter the production of cGMP at all, but

results in an increase in calcitonin-gene related peptide (CGRP) levels. As CGRP is a potent

vasodilator neuropeptide that acts through cAMP-based signaling, the orthogonality between NO

and NO� is no more than a sign of the orthogonality between cGMP and cAMP as cardiovascular

second messengers.13

C. N2O3

As a preliminary step in the synthesis of N2O3, NO is transformed into NO2 upon reacting with

molecular oxygen. The complete process leading to N2O3 occurs in hydrophobic environments when

the activity of NOS, mainly iNOS14 supplies sufficient amounts of NO to allow coupling with NO2

2NO þ O2 ! 2NO2; NO þ NO2 ! N2O3

In the presence of water, N2O3 easily decomposes to nitrous acid (HNO2). Given that the pKa of

HNO2 is 3.36, this acid only exists in the non-dissociated form at very low pH values.

The fact that the nitrosation of amines (e.g., morpholine) by NO in cell-free systems15 required

the additional presence of oxygen was one of the clues indicating that N2O3 is responsible for the

nitrosation of the amine or thiol groups present in many biomolecules. Furthermore, the evidence that

chloride and phosphate anions (X�) decreased the rate of the reaction suggests an active role for

N2O3, which would be scavenged according to the following reaction:

N2O3 þ X� ! NO�2 þ XNO

Since HNO2 decomposes to nitrite at physiological pH, hydrolysis of N2O3 increases to equilibrate

HNO2 concentration, which, according to Lewis et al.16, means that nitrite is a sink for the N2O3

arising from the oxidation of NO. Consequently, nitrosation and hydrolysis are two mutually

competing chemical events here, so that if a given molecule of N2O3 does not bond quickly to an

amine or a thiol, it will lead to NO�2 . It should be remembered that the main products of nitrosation—

nitrosamines and nitrosothiols, respectively—are medically relevant: nitrosamines are dietary

carcinogens, while nitrosothiols, which are NO-carriers, act as important mediators in cardiovascular

pharmacology.

When the production and effects of N2O3 are studied in macrophage cultures,16 the chemical

complexity increases. This is due to the increased intervention of other released oxygen species such

as O�2 , which, as discussed below, participates in the genesis of ONOO�.

Figure 1. Chemicalandphysiologicalorthogonalitybetweennitogenmonoxideandntroxylanion.BasedonWinketal. (2003)and

Feelisch (2003).


D. NO2

The NO2 molecule contains two N–O bonds that form an angle of 134� and its unpaired electron spin

density is mainly (53%) located on the nitrogen atom. In aqueous solution, NO2 tends to aggregate

and form dinitrogen tetroxide (N2O4), which is far more soluble in water than NO2 itself. This

dimerization process habitually precedes the hydrolysis to tri- and pentavalent nitrogen anions17

N2O4 þ H2O ! NO�2 þ NO�

3 þ 2Hþ

Nitrogen dioxide is present in, and partially responsible for, the toxicity of polluted air and cigarette

smoke. Further, it can be endogenously generated from NO�2 by the activity of phagocyte

myeloperoxidase (MPO) under oxidative stress. The high reactivity of NO2, which like NO is a free

radical, has two significant characteristics. First, it is a strong oxidant, capable of initiating oxidation

of unsaturated fatty acids and exhausting the plasmatic reserves of ascorbate, glutathione, some other

thiols, and a-tocopherol. Second, it potently nitrates aromatic rings present in aminoacids, typically

tyrosine, through the formation of an intermediate hydroxyphenyl (tyrosyl) radical.18

The deleterious effect of inhaled NO2 on airway epithelia is certainly the best-known trait of the

compound. The signs of toxicity observed to date involve fibrosis, ciliar damage, hypertrophy of

bronchiolar and alveolar endothelium, and emphysema. Experimental studies confirm that death

occurs after pleural effusion and hemorrhage.19

E. ONOO�

After coupling with O�2 , NO is converted into ONOO�. Such a simple affirmation essentially

condenses the biological origin of ONOO�, at least from a physiological point of view.1 However, the

anion was born to the science in the field of inorganic photochemistry. The oldest records says that in

1901, Baeyer and Villiger discovered an oxidant product obtained by mixing HNO2 and H2O2, but it

was not until 1935 that the resulting species was identified as the conjugated acid HOONO

(hydrogenoxidoperoxidonitrate, peroxynitrous acid) by Gleu and Hubold. Some years later, one of

the multiple branches of the Manhattan Project, which was designed to fabricate nuclear weapons, led

to the finding that ionizing radiation transformed solid nitrates into nitrites, with a concomitant

liberation of dioxygen. Subsequent studies demonstrated that ONOO� was also formed in the process

and it is now clear that ONOO� can be readily obtained by UVirradiation of nitrates. At physiological

temperature, HOONO ionizes at pH 6.7–6.8 to give ONOO�, which presents two conformers,

namely the cis conformer, which is more stable, and the less stable trans conformer. The order of the

central N–O bond is 1.3.20

The combination of NO and O�2 is thermodynamically irreversible and implies the liberation of

22 kcal/mole.5 The reaction rate is considerably high: 1.9 � 0.2� 1010/M/sec 21, a fact that renders

the formation of ONOO� a serious competitor of SOD, the ubiquitous enzymatic remover of the O�2

regularly formed from cellular respiration. Provided that the expression of the inducible isoform of

iNOS is essentially variable, and therefore that the production of NO is markedly irregular, a high

level of ONOO� synthesis depends directly on that of NO. It should be stressed that the presence of

NO in the proximity of an O�2 source is a requisite for the effective production of ONOO�, and,

perhaps more important, the ONOO�-dependent oxidative stress is mainly confined to this area. This

occurs because as ONOO� diffuses, protonates and decomposes to NO�3 , far less reactive.22

As seen, the life of ONOO� has an eventful origin and, unless maintained in strongly alkaline

solutions, also a sudden end. Nevertheless, ONOO� leaves chemical signs of extreme reactivity

wherever it has been. Because it is a peroxide, peroxynitrite acts as an oxidant, and, since it is a source

of NO2 after protonation to HOONO and subsequent homolytic rupture, it also possesses energetic

nitrating properties.

One of the most characteristic oxidative manifestations of ONOO� is the reaction with thiol

groups from very small peptides such as glutathione or proteins. Maintaining an appropriate


intracellular concentration of glutathione constitutes a guarantee against ONOO�-induced damage,

which is particularly visible in tissues suffering high oxidative stress, such as brain or liver. When

thiol groups are fundamental for the proper functioning of a protein, as is the case in enzymes

containing zinc-thiolate active sites or transcription factors with zinc fingers, oxidation by ONOO�

often impairs the function of such proteins. One example of the enzymatic activity associated with

zinc-thiolate complexes is eNOS, a form of constitutive NOS. It has been demonstrated that bovine

aortic eNOS loses its NO-synthesizing activity in the presence of ONOO�, a phenomenon directly

related to the release of zinc, measured in terms of the complexation of Zn2þ with 4-(2-

pyridylazo)resorcinol. This disruption of eNOS results in an increase in the ratio O�2 /NO produced by

this enzyme.23

Additional oxidative processes induced by ONOO� include lipid peroxidation to give hydroxy

and peroxy fatty acid derivatives, conjugation of dienes and malondialdehyde, and severe

transformation of DNA nucleosides due to deoxyribose oxidation and formation 8-oxoguanine (8-

OG) derivatives.24–26 For a comprehensive review on the mechanisms underlying oxidation

processes brought about by HOONO/ONOO�, see Pryor and Squadrito.27

The key position of ONOO� is strengthened by the fact that it can decompose to the very reactive

species NO� and a singlet oxygen, or, by reacting with CO2, undergo transformation into

ONOOCO�2 . Although the anions NO�

2 and NO�3 are the major final stable metabolites of ONOO�

and NO2, it must be taken into account that NO�2 can be readily transformed into NO2 in the presence

of peroxides, hemoproteins, or even free heme, and thereby return to the RNS pool (Fig. 2).

F. ONOOCO�2

The generation of ONOOCO�2 is thought to be the quickest ONOO�-removing mechanism in vivo.28

As early as the first years of research on the biological role of RNS, Radi et al.29 envisaged the

possibility that ONOO� could react with bicarbonate. Shortly thereafter it was demonstrated that

the reaction in fact took place with CO2, which is in equilibrium with bicarbonate in plasma

([CO2]/[HCO�3 ] ¼ 0.052). The anion ONOOCO�

2 can thus decompose to the stable species CO2 and

NO�3 , but it can also suffer a minority homolytic rupture to give the radicals NO2 and carbonate

(CO�3 ), both with strong reactivity towards many substrates (e.g., thiols, amines, purines, ascorbate,

ferrocyanide, etc.).17,30 It has been pointed out that the combination of ONOO� with CO2 is

responsible for most of the oxidative reactions of ONOO� at neutral pH because spontaneous

decomposition of this anion is too slow to compete with its capture by CO2. In contrast, at acidic pH,

Figure 2. Schemeof the generationand turnoverof biological reactivenitrogenspecies.


such as that present in ischemic tissues or phagocytes, oxidation by ONOO� should run

independently of CO2.17

3 . I N T E R A C T I O N O F R E A C T I V E N I T R O G E N S P E C I E S W I T HB I O M O L E C U L E S : P H Y S I O P A T H O L O G I C A L C O N S E Q U E N C E S

A. Binding to Metal Atoms

Nitrogen monoxide is a necessary mediator for the vasorelaxant effect of a number of agonists,

among them bradykinin, histamine, substance P, or acetilcholine. Not so many years ago, this idea

was increasingly popular, not only in the field of basic pharmacological research, but also in all of the

biological sciences. Now it constitutes a dogma of sorts, and the biochemical steps involved in this

process are reasonably well-known. The essential target is GC, or more strictly the soluble isoform,

which is also referred to as NO-sensitive GC due to the fact that its rate of cGMP formation increases

200-fold upon interaction with NO. This interaction consists of the formation of a nitroso–ferrous

heme complex and the disruption of an axial iron-histidine bond, thereby causing the required

conformational change.31 This process was supposed to underlie the therapeutical effect of the so-

called nitrovasodilators, for example, nitroglycerin, which, after sublingual, oral, or epicutaneous

administration, are metabolized to NO and S-nitrosothiols, which are also able to activate the enzyme.

However, there is not yet a definitive link between vascular relaxation and NO/nitrosothiol

production, since the NO production seems too modest to account for the relaxing activity.32 Some

authors are now proposing that the effect of nitroglycerin arises not from NO-heme complexation, but

from heme oxidation. This latest hypothesis comes after Artz et al.33 observed the different shifts in

the electronic absorption spectra of NO- and nitroglycerin-activated GC, and the analogous electron

paramagnetic resonance spectra of nitroglycerin-activated GC and potassium ferricyanide. The

synthesis of cGMP is also important in other physiological areas, such as the adhesion of pro-

inflammatory blood cells, long-term potentiation, non-adrenergic-non-cholinergic neurotransmis-

sion, and retinal phototransduction.26,34

NO also regulates the activity of several other enzymatic heme proteins, with cyclooxygenase

(COX) being just one example. Thus, though COX activity is increased by low NO levels, possibly

due to O�2 scavenging, nitrosylation of Fe3þ leads to the suppression of the synthesis of eicosanoids.

The formation of iron-nitroso compounds also serves to explain the inhibition of catalase, which in

turn increases the effectiveness of antimicrobial and antitumoral defenses, albeit at the expense of

enhanced oxidative stress. Furthermore, NO feedback regulates its own production through

nitrosylation of NOS.26

Probably the most salient case of the interaction of NO with non-heme iron is the regulation of

the activity of iron regulatory proteins (IRPs). When intracellular iron levels fall, IRPs recognize and

bind untranslated regions of mRNA for the transferrin receptor and ferritin. In contrast, iron overload

causes IRP-1 to lose its mRNA-binding ability and transforms it into a protein with aconitase activity.

Iron regulatory protein-1 is fairly homologous to mitochondrial aconitase, which is the enzyme

responsible for catalyzing the transit of citric acid to isocitric acid in the Krebs’ cycle.35 The essential

structural change that makes the diversion to aconitase—more specifically, cytosolic aconitase—

activity possible is the formation of a 4Fe–4S cluster, which suffers disassembling in the presence of

NO. In a study with J774A.1 macrophages and recombinant IRP-1 that used SIN-1 as a donor of NO

or ONOO�, it was demonstrated that SIN-1 at 0.25 mM switches IRP-1 to an RNA-binding form that

can recover aconitase activity after addition of cysteine and ferrous ammonium sulfate. However,

SIN-1 at 0.5 mM did not allow for a recuperation of aconitase activity, probably because excessive

ONOO� not only disassembles Fe–S clusters, but also impedes their subsequent reconstruction

(Fig. 3). When macrophages were supplemented with hemin, aconitase activity increased, to then be

inhibited by SIN-1 treatment. As SOD did not modify the deactivation of aconitase, it is thought that


both NO and ONOO� deactivate this enzyme. In the presence of the iron chelator desferrioxamine,

the aconitase activity decreased while the IRP-1 activity increased. In this case, no effect was

observed for SIN-1 plus SOD, whereas SIN-1 alone decreased IRP-1 activity, a result that indicates

that at low iron ONOO� (but not NO) generates a null protein, that is, a protein with no function.36

It has recently been determined that the suppression of aconitase of IRP-1 by ONOO� is a process

that involves nitration of tyrosine residues, not solely after treatment of the isolated protein with

ONOO�, but also in cultured macrophages stimulated with interferon-g (IFN-g)/lipopolysaccharide

(LPS)/12-O-tetradecanoyl-phorbol 13-acetate (TPA). Furthermore, nitration of IRP-1 depends not

only on NOS activity, as would be expected, but also on MPO activity. Interestingly, when the

substrate cis-aconitate is added to the cytosol fraction prior to the addition of ONOO�, the consequent

stabilization of 4Fe–4S clusters makes the protein resistant to nitration.37

B. S-Nitrosylation

The chemical interaction of NO with proteins, however, does not merely encompass the mechanisms

based on nitrosyl-heme complexation; indeed, research is uncovering an increasing number of

biological processes that are known to be regulated by nitrosylation of thiol groups. It has been

postulated that S-nitrosylation is a general redox system of post-transcriptional modification of

proteins occurring in many different cellular processes including kinase activity, growth regulation,

glutamatergic neurotransmission, antimicrobial defense, etc. It should be emphasized that

nitrosylation of thiols is not an indiscriminate way of blocking possibly reactive sites, but rather

that it occurs selectively on cysteine residues surrounded by acidic and alkaline aminoacids.38

The influence of S-nitrosylation has recently been well characterized within the regulation of

caspases, the C-aspartases that culminate the apoptosis cascade. By using a technique of NO trapping

by thiocarbamate along with subsequent electron spin resonance analysis, Rossig et al.39

demonstrated that when transfected COS-7 cells were treated with the NO donor S-nitroso-L-

cysteine, caspase-3 suffers nitrosylation of Cys-163. Different NO-donors caused a reduction of

apoptotic death, which in this case was being examined in human umbilical vein endothelial cells

(HUVEC), but that this reduction was higher than the percent of inhibition of caspase-like enzymatic

activity. Another pro-apoptotic enzyme that was found to be nitrosylated and thereby deactivated was

the apoptosis signal-regulating kinase 1 (ASK-1). The activity of ASK-1, which was stimulated by

IFN-g, was reduced by the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), an effect that was

absent when Cys-869 was replaced by alanine.40

The role of NO in inflammation is bipartite (dual) in nature. Thus, while it mediates many

inflammatory conditions by its transformation into ONOO�, the direct effect is anti-inflammatory

due to its repression of nuclear factor-kB (NF-kB), a transcription factor responsible for the synthesis

Figure 3. Transformationsof iron regulatoryprotein, according toCairoand Pietrangelo (2000) andCairo etal. (2002).


of pro-inflammatory enzymes, adhesion molecules, cytokines, and their receptors. Nuclear factor-kB

actually consists of a small family of five factors with a homologous Rel domain, which, once

deprived of its cytosolic association with IkB proteins, binds DNA and activates transcription.41 As is

the case in several other factors, the binding to DNA depends on the presence of a reduced cysteine

residue, which, in the case of NF-kB, is Cys-62 in the p50 monomer. Detailed studies performed by

Marshall and Stamler42,43 have shown that S-nitrosylation of this critical cysteine at the nucleus

inhibits NF-kB-dependent transcription and signaling, as does the apoptosis induced by tumor

necrosis factora (TNF-a). The authors noted that different cellular types show different susceptibility

to NO-induced apoptosis, perhaps due to their nitrosothiol degrading capacity. They also raised the

possibility that nitrosylation could affect NF-kB signaling at a cytosolic level, a point that has

recently been confirmed by Reynaert et al.44 in their investigations on Ikb kinase (IKK). In fact, the

combined treatment of Jurkat cells with NO donors and TNF-a resulted in the S-nitrosylation of Cys-

179 within IKKb, one of the subunits of IKK that is to a large degree responsible for IkB degradation.

Other cellular mechanisms regulated by S-nitrosylation include endothelial granule exocy-

tosis,45 osteopontin expression,46 and activation of ATP-sensitive potassium channels.47

C. Tyrosine Nitration

Although we have touched upon it briefly during our explanation of the essential features of NO2 in

Section 2, it bears repeating that the most conspicuous chemical target of RNS is without a doubt the

phenolic ring of tyrosine. We will not examine the multiple biological implications—demonstrated

or putative—of tyrosine nitration in depth here, partly due to space constraints, but mostly because of

the pharmacological emphasis of the present review. For the purpose of reference, however, mention

should be given to Ischiropoulos’ excellent review (1998),48 in which the author analyzes eight

different ways in which tyrosine can be transformed into 3-nitrotyrosine (3-NT) in the biological

context. In most cases the direct nitrating agent is NO2, although it may arise from NO, NO�2 , or

ONOO� to mediate O2, hemoproteins, or Hþ, respectively. Furthermore, 3-NT is also derived from

nitrosotyrosine, through in situ oxidation (Fig. 4). Provided that tyrosine residues are structurally

important for the function of a number of proteins, and that nitration is not an indiscriminate process,

a physiopathological significance for such a notable transformation was soon hypothesized. Indeed,

when Ischiropoulos published his research, 3-NTwas reported to be detectable in more than 20 varied

human diseases, including atherosclerosis, pulmonary fibrosis, and some neurodegenerative and

autoimmune diseases.

Figure 4. Different ways leading to tyrosinenitration.Basedon Ischiropoulos (1998).


Nitration of tyrosine residues often causes inhibition of enzyme activity, as it occurs with

prostacyclin synthase, mitochondrial Mn-SOD, glutamate-ammonia ligase (glutamine synthetase,

GS),49 and tyrosine hydroxylase (TH).50 This last enzyme, which limits the rate of the biosynthesis of

dopamine and other catecholamines, has been investigated by Kuhn et al.51 who, although they

admitted that nitration occurred, suggested that the inhibition of activity depends mainly on the

oxidation of thiol groups. Using PC12 cells, they actually demonstrated that subtotal glutathionyla-

tion of cysteine residues of the enzyme led to strong reduction of catalytic activity.52

Apart from its being implicated in a growing number of unrelated enzymatic or structural

proteins, there is evidence that tyrosine nitration performs a regulatory role in the signaling pathways

initiated or mediated by tyrosine phosphorylation, including signaling by receptor and non-receptor

tyrosine kinases. Although this hypothesis has a solid chemical rationale, the construction of a

coherent generalizable theory faces several obstacles, most notably the fact that common nitrating

agents such as ONOO� also exert a powerful oxidative activity, making it almost impossible to

evaluate the true influence of aromatic nitration alone. Further, the modifications of the activity of

protein phosphatases, which comprise the physiological counterbalance of kinases, add another

degree of complication to the schema. The effect of ONOO� on members of the mixed threonine-

tyrosine mitogen-activated protein kinase (MAPKs) family is generally positive, either through

activation of growth factor receptors or activation of MAPK kinases, which helps explain the role of

that anion in apoptosis.53 However, there are other cases, such as that of the human T-lymphocytes, in

which nitration of tyrosine residues is clearly detrimental for tyrosine phosphorylation signaling, a

fact that is reflected in the reduction of cellular viability and proliferation by ONOO�.54

The assembly of microtubules, which are the cytoskeletal polymers of tubulin, represents a

special version of how the formation of 3-NT modifies protein function. The key here is that tubulin

can undergo a series of simple, though numerous, post-translational derivations, among them

tyrosination by the enzyme tubulin tyrosine ligase (TTL). The process of cycling tyrosination/

detyrosination is of pivotal importance for cell differentiation and proliferation, as seen by the fact

that cells lacking TTL are prone to suffer tumoral growth. As TTL does not discriminate clearly

between phenylalanine, tyrosine, halotyrosines, or nitrotyrosine, the incorporation of this last

aminoacid, which results from the catabolism of nitrated proteins or from nitration of free tyrosine,

results in serious alterations of morphogenesis.55

As it currently stands, there are solid arguments favoring the hypothesis that tyrosine nitration is

not merely an imprint of oxidative damage, but also a process of intracellular signaling. Still, a vast

amount of work remains to be done to demonstrate just how finely controlled the production of

nitrating species is and whether a regulated system of denitration actually exists. Selectivity in

targeting proteins has mainly been attributed to an apparently unselective factor, namely the

proximity to the source of nitrating species, as described in a model of transcytosis (i.e., the transport

of the enzyme across an epithelium). In this model, nitration of fibronectin was found to colocalize

with MPO, the hemoprotein that catalyzes the process. As for the role of more selective factors, the

presence of negative charges in the steric vicinity has been found to lend a degree of specificity to the

affected tyrosine residues. While elimination of nitrated proteins occurs either by ordinary

proteolytic mechanisms or by specialized lymphocytes56 pure denitration without further protein

transformation is still a matter of controversy.57

D. Modification of Structural Elements of Nucleic Acids

Peroxynitrite causes chemical modifications to purine and pyrimidine bases, as typically exemplified

by the transformation of guanine into 8-nitroguanine (8-NG) and 8-hydroxyguanine (8-OG in the

lactam form), which can in turn lead to genetic mutations. The formation of 8-OG is a prelude to

further degradation as this compound is more sensitive to ONOO� than guanine itself and is a

substrate for a number of imidazolones, imidazolidinones, and oxazolinones. This kind of reaction is


favored by the presence of CO2 as opposed to the attack to deoxyribose, which occurs mainly in the

absence of CO2.24 Strands of DNA containing nitrated purines undergo spontaneous depurination

under physiological conditions with various kinetic profiles. Thus, the half-life of 8-NG generated by

reaction with ONOO� is 4 hr, whereas that of 8-nitroxanthine (8-NX) generated in the presence of

nitryl chloride (NO2Cl) is 2 hr. 8-NX should therefore be more deleterious than 8-NG because it gives

way to mutagenic abasic sites faster.58

Although it is much less aggressive than ONOO�, NO can also cause important changes in DNA

sequences by performing deamination of both purine and pyrimidine bases. However, this reaction,

which produces uracil from cytosine and xanthine from guanine, is weak and requires the

intermediate formation of N2O3. It has been demonstrated that double-stranded DNA reacts ten times

more slowly than single-stranded DNA, a fact which suggests that base pairing in the Watson–Crick

helix confers genomic stability against nitrosative stress.59

Oxidative damage of both supercoiled plasmid and mammalian cellular DNA by ONOO� results

in strand rupture, thus causing cytotoxic effects.60 The mechanism of ONOO�-induced cellular

injury involves the activation of poly(adenosine 5 0-diphosphate[ADP]-ribose) polymerase (PARP),

also known as poly (ADP-ribose) synthetase (PARS) or poly (ADP-ribose) transferase (PADPRT).

This enzyme has been implicated in multiple cellular functions, among them cell differentiation,

inflammatory signaling pathways, and cytoskeletal organization. The general term PARP designates

a family of nuclear and cytosolic enzymes whose principal member, PARP-1, functions as a DNA

damage sensor. It is a 116 kDa protein consisting of three main domains: the N-terminal DNA-

binding domain, the automodification domain, and the C-terminal catalytic domain. This enzyme,

which is constitutively expressed in most cell types, is responsible for ADP-ribosylation of acceptors

such as histones, transcription factors, or the enzyme itself, and catalyses the reaction between NADþ

and an (ADP-D-ribosyl)n-acceptor to form nicotinamide and (ADP-D-ribosyl)nþ 1-acceptor.

The activation of PARP in response to DNA single strand breakage initiates an energy-consuming

metabolic cycle by transferring ADP units to nuclear proteins, which results in a massive

poly(ADP) ribosylation and an immediate depletion of intracellular NADþ and ATP pools. This, in

turn, impairs glycolysis and mitochondrial respiration and ultimately leads to cellular dysfunction

and death. Furthermore, the nicotinamide formed by PARP activation is recycled back to NAD,

thus consuming even more ATP. This whole process has been termed ‘‘the PARP Suicide

Hypothesis.’’61

Nitroxyl (NO�/HNO) has been also reported to cause DNA strand breakage and be cytotoxic in

cultured cells, probably because it can be converted, under physiological conditions, to NO and other

ROS and RNS, for example, H2O2 and ONOO�, respectively.

4 . M O D U L A T I O N O F F R E E A N D P R O T E I N T Y R O S I N E N I T R A T I O N

A. Phenolic Compounds

Flavonoids, hydroxycinnamic, and hydroxybenzoic acids are phenolic compounds that are

universally distributed in the plant kingdom. Many studies have been carried out to demonstrate

their antioxidant and free radical scavenger activities, which are due to their hydrogen-donating and

metal-chelating properties. The extention of the conjugation and the number of hydroxyl groups are

the main characteristics that define their efficacy.62–64

1. Flavonoids and Catechins

It has been reported that flavonoids and related C-15 compounds (Tables I–III) are able not only to

capture the precursors of ONOO�, NO,65 and O�2 ,66 but also to block the nitration of a wide variety of

targets.


Table I. Structures of Catechins

ECG, epicatechin gallate; EGC, epigallocatechin; EGCG,

epigallocatechingallate.

Table II. Structures of Flavones and Flavonols

GlcA,glucuronyl; HEQ,hydroxyethylquercetin; HER,hydroxyethyl rutoside; Rut, rutinosyl.


Some catechin polyphenols, present in high amounts in dietary sources such as green tea, certain

chocolates, and red wine, have been studied as possible ONOO� scavengers by measuring the

decrease of tyrosine nitration. Catechin, epicatechin, epigallocatechin (EGC), their gallate

derivatives, and gallic acid have been demonstrated to be potent ONOO� scavengers, most of

them more effective than the standards (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

(trolox, Fig. 5) or the endogenous antioxidants, ascorbic acid, and glutathione). Essentially, there are

two possible ways in which compounds can inhibit ONOO�-mediated nitration: either by their own

nitration or through an oxidation reaction with the consequent formation of a quinone. In the

ultraviolet spectra of the mix of catechin with ONOO�, an increase in the absorbance at 430 nm has

been observed, indicative of the formation of a nitrophenol. This finding seems to support the idea that

the protection of this kind of compound is produced by its direct competition with tyrosine for

nitration.67,68 It is likely that the nitration occurred mainly at the C-2 0, C-5 0, and, to a lesser extent, the

C-6 0 of the B ring. Due to the presence of two meta-hydroxyl groups, the nitration in the chromanol

ring is generally diminished. Of all the catechins tested, EGC was the least effective in protecting

against nitration, probably because of the presence of an additional hydroxyl group at the 5 0 position

of the B ring. The gallate esters exhibit more activity as ONOO� scavengers possibly due to the fact

that they can also suffer nitration reactions on the gallic acid moiety. This leads to the conclusion that

the actual number of sites favorable for nitration is an important factor in the ability to inhibit ONOO�

action.

Furthermore, some epicatechin oligomers of various sizes have been studied as possible nitration

inhibitors, with the tetrameric compound exhibiting the most activity. The changes observed in the

UV spectrum suggest the nitration of the compounds, but it seems that they do not react directly with

ONOO�, but most likely with reactive intermediates such as the tyrosyl radical.69,70 Tibi et al.71

proposed that the flavonoids are not ONOO� scavengers, based on the fact that the reactions of this

anion with the phenolic compounds are first order with respect to ONOO�, but zero-order with

Table III. Structures of Isoflavones and Dihydroflavonols

Figure 5. Structures of simple tocopherol analogs.


respect to the compounds themselves. The changes produced in the flavonoid spectra might not have

been caused by ONOO�, since by that time it has already disappeared. Moreover, in vivo, ONOO�

reacts with CO2 to form an unstable adduct whose rate of disappearance was not influenced by the

phenolic compounds.

As mentioned above, different researchers have demonstrated that epicatechin and some related

oligomers are active in preventing ONOO�-tyrosine nitration in a hydrophilic system, but no one had

demonstrated their activity in a hydrophobic ambient until Schroeder et al.72 investigated the effect of

these compounds on the reaction of ONOO� with a hydrophobic tyrosine analog (N-t-BOC L-

tyrosine tert-butyl ester) that had been incorporated into liposomes. The protection obtained with

epicatechin was similar to that found in the hydrophilic system, but none of the oligomers were as

efficient as the monomer in the hydrophobic system. This may be due to the fact that oligomers do not

have easy access to the lipid bilayer. The same authors73 demonstrated that, due to the amphiphilic

properties of epicatechin (octanol/aqueous buffer partition coefficient of 1.5), murine aortic

endothelial cells (MAECs) are able to remove epicatechin from the culture medium, thereby

producing a significant protection against tyrosine nitration by ONOO�, even after several cell

washings. The calculated IC50 is similar to that obtained when epicatechin is present in the medium to

which ONOO� is added. This corroborates previous findings that this flavonoid is an effective agent

in protecting against ONOO�-induced nitration in both hydrophilic and hydrophobic environments.

Physiological concentrations of bicarbonate can modify the ability of compounds to avoid the

reactions mediated by ONOO�, possibly due to the reaction between ONOO� and CO2—in

equilibrium with bicarbonate—and the formation of more complex anions (see Section 2.F) which

are able to nitrate tyrosine with the consequent increase in 3-NT formation. Thus, as distinguished

from earlier studies, Ketsawatsakul et al.74 chose to perform several assays in the presence of 25 mM

bicarbonate. For catechin and epicatechin there was no significant difference in their ability to inhibit

ONOO�-mediated tyrosine nitration.75 However, at concentrations higher than 50mM, the efficacy of

gallic acid to avoid this process decreased. It is thus important to take the presence of added

bicarbonate into account in every invitro study that tests the ability of a compound to inhibit ONOO�-

mediated reactions.

These groups of compounds have been also assayed in other systems in which ONOO� is

involved, such as collagen nitration. Using Western blotting and ELISA techniques, Kato et al.76 have

demonstrated that both epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) exhibit

strong inhibitory effects on collagen nitration. Moreover, it has been demonstrated that epicatechin

efficiently protects against ONOO�-induced nitration in cell-free and cellular systems such as

isolated proteins (glyceraldehyde 3-phosphate dehydrogenase and soybean lipoxygenase-1), MAEC

lysate, and MAEC cultured cells, possibly by means of interaction with a tyrosil radical rather than by

direct interference with ONOO�.77

Neuronal protein nitration in some neurodegenerative diseases may in part be related to

hemin or heme-containing proteins, such as peroxidase, hemoglobin, and myoglobin. This is most

likely due to the fact that the hemin/nitrite/H2O2 system induces nitration of brain homogenate

proteins. Quercetin, catechin, and baicalein have all shown a dose-dependent inhibition of brain

homogenate nitration, with quercetin being the most effective. Since the extent of this activity

is more or less the same as that of their radical scavenging, nitration inhibition may actually be

related with their antioxidant activity.78 To study the effect of epicatechin on peroxidase-catalyzed

nitration of cellular proteins, Wippel et al.75 used TPA-stimulated HL-60 cells, which express

large amounts of MPO. The potency of the flavonoid is very similar in both the inhibition of free

tyrosine nitration by ONOO� and that of protein-bound tyrosine in the presence of nitrite in TPA-

stimulated HL-60 cells. This suggests that the same reactive intermediate may be involved in both

systems.

Recently, Ferroni et al.79 have demonstrated that several phenolic antioxidants with a catechol

structure, namely quercetin, catechin, and epicatechin, were very active in the inhibition of ONOO�/


CO2-mediated nitration of low density lipoproteins (LDL); moreover, their efficacy in the presence of

bicarbonate was similar to that observed in its absence. The authors also tested some polyphenol-rich

beverages, red wine and alcohol-free red wine (the phenolic content was calculated as quercetin

equivalent). Both proved to be only slightly less active than phenols with a catechin structure. As

electron paramagnetic resonance (EPR) studies revealed, the formation of an o-semiquinone after red

wine was treated with ONOO� increased in the presence of bicarbonate. Based on these results, the

authors suggested that polyphenols are better scavengers of radicals derived from ONOO�-CO2

interactions than from HOONO.

Another flavonoid that has been under study is genistein, the principal isoflavone in soybeans

(Glycine max, Fabaceae). Immunohistochemical localization of 3-NT in terminal epithelial cells

from guinea pigs with trinitrobenzene sulfonic acid-induced ileitis was reduced after the

administration of genistein at a dose of 0.1 mg/kg. One of the possible explanations for the anti-

inflammatory activity of this compound may have to do with the attenuation of ONOO� formation.80

Boersma et al.81 have investigated the mechanism by which the isoflavones genistein and daidzein

interact with ONOO� and hypochlorous acid (HOCl)/nitrite. To know precisely where the nitration is

produced, they also included in the study a methylated analog of genistein, biochanin-A. Using

different analytical techniques, they demonstrated that while a nitrated product was obtained from

genistein and daidzein, no nitrated product was detected with biochanin-A. Because tyrosine is

nitrated at the C-3 position, the authors speculated that the nitration of genistein and daidzein occur at

the analog position, namely C-3 0 of the B ring. Moreover, as the only structural difference between

genistein and biochanin-A is the methylation of the OH group at the C-4 0 position of biochanin, they

also surmised that the nitration of biochanin-A by ONOO� was hindered by this group.

2. Hydroxycinnamates

Another group of phenolic compounds that have been under study are the hydroxycinnamates or

simple phenylpropanoids (Table IV). Both the ability of p-, m-, o-coumaric, chlorogenic (3-

caffeoylquinic), ferulic, and caffeic acids to decrease ONOO�-mediated nitration along with their

mechanism of scavenging ONOO� has been determined.82 The most active group of substances

Table IV. Structures of Hydroxycinnamic Acids


proved to be the 3,4-disubstituted hydroxycinnamates, caffeic and chlorogenic acids. These catechol

derivatives do not exhibit any spectrophotometric change in the visible region after being exposed to

ONOO�. Subsequent high performance liquid chromatography (HPLC) analysis of caffeic acid

exposed to ONOO� indicated that the catechol compounds act by donating electrons to form a

quinone.82,83 On the other hand, the monohydroxycinnamates (ferulic and the coumaric acids) are

preferentially nitrated, as has been determined by means of UV, HPLC,82 and liquid chromatography-

mass spectrometry (LC-MS) analysis.76

As with the flavonoids, Ketsawatsakul et al.74 studied the effect of the addition of 25 mM of

bicarbonate on the ability of hydroxycinnamates to protect free tyrosine from ONOO�-mediated

nitration. In this case, the presence of bicarbonate decreased both the efficacy and the potency of the

compounds to prevent ONOO�-dependent nitration, a result that leads to the conclusion that they

react more slowly with the nitrating species generated from bicarbonate-ONOO� interaction than

with those obtained from ONOO� alone. Thus, once again the importance of the local bicarbonate

concentration is evident.

p-Coumaric, sinapic, and caffeic acid all show more inhibitory activity against collagen nitration

by ONOO� than do the standards tocopherol and ascorbic acid.76,84 The inhibitory action of sinapic

acid, but not of caffeic acid, seems to be due to its one-electron oxidation; this hypothesis has been

corroborated by evidence from HPLC, proton-nuclear magnetic resonance (1H-NMR), and LC-

MS.84 The same authors85 also examined 12 phenolic hydroxycinammate compounds, 11 of which

were p-coumaric acid derivatives, isolated from corn steep liquor as possible inhibitors against

ONOO�-mediated nitration of LDL. In general, these phenolic compounds also showed a stronger

inhibitory activity than the typical antioxidants such as tocopherols or ascorbic acid.

It has been demonstrated that sinapic acid isolated from Brassica juncea (Brassicaceae)

attenuated in a dose-dependent manner the nitration of tyrosine as well as that of bovine serum

albumin (BSA) and LDL, both physiologically relevant proteins, by an electron donation between the

acid and ONOO� as Niwa et al.84 proposed.86

3. Other Phenolics

In 1996, Whiteman and Halliwell evaluated the activity of several phenolic compounds that would be

later used as standards in the inhibition of ONOO�-mediated nitration of free tyrosine. Uric acid was

found to be more effective than ascorbate, gluthatione, or trolox. At physiological concentrations,

ascorbate is able to scavenge ONOO� and its derived species, thus protecting against tyrosine

nitration.87 Avitamin E metabolite, 2,5,7,8-tetramethyl-2-(2 0-carboxyethyl)-6-hydroxychroman (a-

CEHC, Fig. 5), has been shown to have reactivities against ONOO� similar to those of trolox; it has

thus been proposed that in biphasic systems that include cell membranes it would be better to use the

former compound instead of trolox because its higher liposolubility.88

Three prenylhydroquinones (Table V) and four di-O-caffeoylquinic acids (Table VI) isolated

from Phagnalon rupestre (Asteraceae) have also been studied as possible inhibitors of tyrosine

nitration. This was the first time that carbon-NMR (13C-NMR) spectroscopy was used to determine

the manner in which a compound actually inhibits ONOO� action. 1H-NMR, 13C-NMR, and UV

spectral data all indicated that isoprenylhydroquinone glucoside, the most active compound, suffers

its own nitration by ONOO�. In contrast, ONOO� leads to oxidation of caffeoylquinic derivatives.89

Two phenolic compounds (Fig. 6), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (TDB)

isolated from the algae Symphyocladia latiuscula (Rhodomelaceae)90 and lithospermate B, the most

potent ONOO� scavenger of all the components isolated from Salvia miltiorrhiza (Lamiaceae),91

were tested as possible inhibitors of tyrosine, BSA, and LDL nitration by ONOO�. Pre-incubation of

TDB and lithospermate B inhibited aminoacid and protein nitration, probably due to electron

donation. The hydroxyl groups along with the double bounds for the lithospermate B seem to be

responsible for the ONOO� scavenging activity of the phenolic compound TDB.


The hydroxyindoles 5-hydroxytryptamine, 5-hydroxy-L-tryptophan, and N-acetyl-5-hydroxy-

tryptamine were more effective in protecting BSA nitration by ONOO� than the methoxyindole

derivatives 5-methoxytryptamine and 5-methoxyindole-3-acetate. UVanalysis and the yellow color

characteristic of 3-NT indicated that the compounds protect against BSA nitration by means of

autonitration.92

Alaternin and nor-rubrofusarin glucoside (Fig. 7), the phenolic active components from

Cassia tora (Caesalpiniaceae), are effective in inhibiting tyrosine nitration through an electron

donation. Alaternin, but not nor-rubrofusarin glucoside, was shown to attenuate BSA nitration in

a dose-dependent manner. An explanation for this may lie in the number of aromatic hydroxyl

Table V. Structures of Prenylhydroquinones

Abbreviations: IPH, 2-isoprenylhydroquinone-1-O-glucoside; IPHH,

2-(3 0 -hydroxy)isoprenylhydroquinone-1-O-glucoside; IPHC, 2-iso-

prenylhydroquinone-1-O-(4 00-O-caffeoyl)-glucoside.

Table VI. Structures of Di-O-Caffeoylquinic Acids

3,5-CQM,3,5-di-O-caffeoylquinicacidmethylester;4,5-CQM,4,5-di-O-caffeoyl-

quinicacidmethyl ester; 3,5-CQ,3,5-di-O-caffeoylquinicacid; 4,5-CQ,4,5-di-O-

caffeoylquinicacid.


groups found in the compounds; alaternin has four whereas nor-rubrofusarin glucoside only has

two.93

B. Non-Phenolic Compounds

In addition, several non-phenolic compounds have been tested as possible inhibitors of ONOO�-

mediated nitration reactions. Once again, tyrosine is the target of choice for many authors to test the

capacity of a number of compounds to reduce nitration caused by ONOO�.

Thiourea, dimethilthiourea,94 and 3-mercapto-2-methylpentan-1-ol, a constituent of Allium

cepa (Liliaceae),95 significantly inhibited tyrosine nitration at each of the concentrations tested to a

greater extent than did either of the positive controls, gluthatione or trolox. Aminoethylcysteine

ketimine decarboxylated dimer (AECK-dimer, Fig. 8), but not its oxidation product, AECK-dimer

sulfoxide, was found to completely inhibit tyrosine nitration at 100 mM while at lower concentrations

it produced a significant reduction of aminoacid nitration, similar to that exerted by gluthatione and

N-acetylcysteine, but higher than that of methionine. This difference in the effect of oxidized and

non-oxidized compounds suggests that the thiol ether group is necessary for the scavenger effect.96

The natural histidine-containing dipeptides, carnosine and anserine, along with three synthetic

sulfonamide pseudopeptides, tauryl-histidine, tauryl-3-methyl-histidine, and tauryl-1-methyl-

histidine (Table VII), are all able to decrease ONOO�-induced tyrosine nitration significantly, with

no significant changes in the presence of 25 mM bicarbonate. The part of the molecule responsible for

the activity is the imidazole moiety, which is in turn an effective inhibitor of ONOO�-induced

tyrosine nitration. However, the most important finding of these studies is that the synthetic

pseudopeptides may be important inhibitors of ONOO� action, not only because they maintain the

properties of the natural dipeptides, but also because they are resistant to the rupture caused by serum-

peptidases. Furthermore, they have been shown to moderately prevent antiproteinase inactivation in

the range 0–20 mM.97 a-Lipoic and dihydrolipoic acids inhibit tyrosine nitration in a similar way,

whereas other thiol compounds such as glutathione and penicillamine are much more effective than

their corresponding disulfides.98 Nakagawa et al.99,100 studied more than 40 natural and synthetic

Figure 6. Structures of TDB (A) and lithospermate B (B).

Figure 7. Structures ofalaternin (A) andnor-rubrofusaringlucoside (B).


compounds as possible inhibitors of ONOO�-induced tyrosine nitration or oxidation, measuring the

formation of 3-NTand dityrosine, respectively. Lipoic acid and five indole derivatives: L-tryptophan,

melatonin, 5-methoxytryptamine, tryptamine, and tetrahydro-b-carboline all had a selective

inhibitory effect on tyrosine nitration without affecting oxidation, which suggests not only that

these reactions occur in a different way, but also that the compounds react only with the nitrating

species derived from ONOO�. In contrast, the rest of the compounds were found to be efficient

inhibitors of both 3-NT and dityrosine formation, which indicates that they have the same inhibition

potency for both nitration and oxidation and that they scavenge the common species in both

processes.

Several assays designed to test the inhibition of ONOO�-induced nitration of enzymes such as 5-

lipoxygenase (5-LOX) and TH have also been carried out. 5-Lipoxygenase, for example, possesses

many tyrosine residues near the active site that can be nitrated, which could explain the loss of the

enzyme activity by ONOO�. Thus, zileuton, a reversible 5-LOX inhibitor, was found to block direct

nitration of BSA, 5-LOX recombinant enzyme, and 5-LOX in intact cells. The exact mechanism by

which zileuton reduces 3-NT formation is as yet unknown. Since other reducing 5-LOX inhibitors,

including A63162 (anN-hydroxyurea) and nordihydroguaiaretic acid, exhibit effects similar to those

of zileuton in the inhibition of 5-LOX nitration, it is possible that this effect has to do with their

quenching the oxidative effect of ONOO�. However, in the presence of an excess of ONOO�, the

reducing effects of zileuton are not overcome. Another possible explanation could be that zileuton is

able to bind the active site of the enzyme by blocking the nitration of tyrosine residues. However,

given that it also completely prevents BSA from ONOO�-induced nitration, this hypothesis is

unsatisfactory; thus, the mechanism remains undetermined.101

Tyrosine hydroxylase can be nitrated and inactivated not only by ONOO�, but also by NO2.

Tetrahydrobiopterin (BH4, Fig. 9) and several of its analogs prevented both ONOO� and NO2-

Figure 8. Structure of AECKdimer.

Table VII. Structures of Natural Histidine-Containing Dipep-

tides and Synthetic Sulfonamide Pseudopeptides


induced nitration of TH. In fact, only the fully oxidized form of BH4 and 5,6,7,8-tetrahydropterin

(biopterin and pterin, respectively) showed no effect on TH nitration. Moreover, the products

generated by the oxidation of BH4 by ONOO� were effective in preventing ONOO�-induced

nitration of TH. It is possible that instead of interacting with TH, the pterins react directly with

ONOO� or NO2, an idea which is bolstered by the fact that while the tetrahydropterins tested were TH

cofactors, the dihydropterins tested were not.102

In a different study, mice treated with gadolinium chloride and dextran sulfate 24 hr before the

administration of acetaminophen showed a decrease both in the hepatotoxicity and in the formation

of NT-protein adducts in the centrilobular cells of the liver. In contrast, the latter phenomenon did

occur in the mice treated with acetaminophen only.103

Another pathological model in which ONOO� has been implicated is the zymosan-induced rat

model of arthritis. Both, zymosan and authentic ONOO� produce a significant increase in nitrated

proteins in the joint exudates. Uric acid, a ONOO� scavenger, causes an important reduction of 3-NT

levels, which have been associated with the prevention of articular cartilage damage. In contrast,

while the iNOS inhibitor L-NAME is able to reduce nitrated protein in the joint, it does not prevent

articular cartilage damage. It has thus been proposed that while NO can act in a protective manner in

experimental arthritis, it can also, as a consequence of ONOO� generation, promote joint damage in

experimental, zymosan-induced arthritis in rats. This observation implicates a role for the RNS in the

arthritic joint.104

5 . C O N T R O L O F P R O - O X I D A N T A C T I V I T Y O F O N O O �

This section, which treats the oxidative reactions mediated by ONOO� and, much less commonly, by

other RNS, is divided into two parts according to the different ways of examining the interactions

observed. The first part (Section 5.1) analyzes the use of the universally described method to measure

the ability of compounds to inhibit ONOO�-mediated oxidation: the attack on dihydrorhodamine

(DHR) 123 and related compounds such as dichlorodihydrofluorescein (DCDHF). The second part

(Section 5.2) includes various different techniques used for evaluating antioxidant activities over

multiple substrates, most of which are of biological interest.

A. Interventions on Oxidation of Fluorescein Analogs

1. Flavonoids and Catechins

Many studies have been carried out to determine the ONOO� scavenger activity of flavonoids by

measuring the inhibition of DHR 123 oxidation. The oxidation of this non-fluorescent compound to

fluorescent rhodamine is a useful probe both in vitro and in vivo to detect the formation of ONOO�.

The formation of the oxidized product is linear in the range from 1 to 1000 nM of ONOO� and does

not depend on pH in the range between 4.2 and 8.3, although it decreases at higher pH values.105,106

As is the case with the inhibition of tyrosine nitration, the importance of the number of free

aromatic hydroxyl groups, which are responsible for the activity, is also a key point in DHR 123

oxidation (Tables I–III). Quercetin, in which the aromatic hydroxyl groups are all free, is thus the

Figure 9. Structure of tetrahydrobiopterin.


most potent compound. In contrast, tetrahydroxyethyl rutoside, in which all the aromatic hydroxyl

groups are substituted, exhibits the least activity. Haenen et al.107 were the first authors to describe the

two pharmacophoric groups responsible for the activity of these kinds of compounds as ONOO�

scavengers. Having observed that quercetin, rutin, and monohydroxyethyl rutoside share a very

similar ONOO� scavenging activity, they surmised that the catechol group (B ring), which is found in

all the three compounds, is the feature mainly responsible for this activity and proposed a much lesser

role for the hydroxyls at positions 3 and 7.107,108 In fact, the substitution of one (dihydroxyethyl

rutoside) or both (trihydroxyethyl rutoside) ortho-hydroxyl groups in the B ring leads to an important

reduction in the activity. Several years later, the same team demonstrated that catechol (1,2-

dihydroxybenzene), but not phenol (hydroxybenzene), is a potent scavenger of ONOO�. The

inhibitory activity of hydroquinone indicates that the OH group does not need to be in an ortho

position for efficient ONOO� scavenging to take place.109 Using 31 flavonoids, Choi et al.110

extended this study of the structure-activity relationship and concluded that the o-dihydroxyl group in

either the A or B ring is the most important feature for the scavenging activity of flavonoids. This is the

case with the 5,6-dihydroxyl flavone baicalin, for example, which exhibits a scavenging activity

similar to that of 3 0-4 0-dihydroxylated flavonoids.

The second pharmacophore is the A–C ring system, with the hydroxyl group at position 3

playing the most important role, as can be seen from the different effects caused by trihydroxyethyl

quercetin (IC50 ¼ 0.58 mM) and trihydroxyethyl rutoside (IC50 ¼ 162 mM). In a subsequent study

with a synthetic and a selected group of several natural flavonols, Heijnen et al.109 concluded that a

substituent at positions 5 and 7 plays a pivotal role in the reactivity of the 3-OH group. The high

activity of kaempferol and galangin, flavonoids that lack the catechol group, but which have a

hydroxyl at position 3, along with the poor ONOO� scavenger activity of the synthetic flavonoid

(TUM 9761) and 3-hydroxyflavone, which only posses a hydroxyl group at C-3, could be explained

by invoking an intramolecular rearrangement that takes place when the 5-OH group is present to give

a catechol-like structure in ring C, which would be the active form (Fig. 10). Moreover, flavonoids

that only have a hydroxyl at C-5 or C-7 (apigenin, chrysin, or trihydroxyethyl rutoside) are poor

ONOO� scavengers, indicating that lone hydroxyl groups at these positions have only a minor

contribution.107,109

Choi et al.110 added that the C-2-C-3 double bond does not contribute to the scavenging activity

of flavonoids as can be seen with aromadendrin and taxifolin, both of which are active flavonoids

without a C-2-C-3 double bond.

Green tea is a better scavenger of ONOO� than its fermented form, black tea. The components

responsible for the action are principally the catechins, with the most active ones being ECG, EGCG,

and the mixed theaflavin fraction (MTF) on a molar basis.111 Thus, in terms of molarity, procyanidin

oligomers of various sizes (monomer through nonamer) isolated from the seeds of Theobroma cacao

(Sterculiaceae) are more effective than the oligomers, with the tetramer being by far the most potent

(IC50 ¼ 83 nM). In contrast, by weight, (�)-epicatechin is more effective than all the oligomers.69,70

Some of these compounds have also been evaluated as inhibitors of DCDHF oxidation by SIN-1

or of ONOO� itself. For example, (�)-epicatechin (IC50 ¼ 1.1 mM) protects against oxidative

reactions in cellular systems, although it is more effective against tyrosine nitration; the IC50 is more

Figure 10. Tautomersofgalangin.


than two orders of magnitude (IC50 ¼ 0.02 mM), lower than those for oxidation.73,77 Moreover, some

dimers of EGC, (�)-epicatechin, or (� )-gallocatechin isolated from Apocynum venetum

(Apocynaceae) have been shown to be more active than monomers in the inhibition of DCDHF

oxidation by ONOO�. Epigallocatechin-(4b-8)-epicatechin has the highest activity, followed by

epicatechin-(4b-8)-gallocatechin and procyanidin B-2. These compounds were more effective

scavengers than penicillamine, an effective scavenger of ONOO� in vitro.112 Moreover, it was found

that catechins with a galloyl group isolated from the leaves of green tea not only inhibit ONOO�

formation by SIN-1, but also scavenge ONOO� itself. The green tea components (�)-

epigallocatechin 3-O-gallate and (�)-gallocatechin 3-O-gallate, which have two galloyl groups,

show the highest ONOO� scavenging activity (IC50 � 20 mM, � 3 times more potent than

penicillamine). This indicates that the galloyl group plays an important role in the ability of tannins to

scavenge ONOO� since gallate-free molecules exhibit much lower activity.113

2. Hydroxycinnamates

In contrast with the work carried out on the nitration of tyrosine, few studies on the inhibition of DHR

123 oxidation have dealt with hydroxycinnamic compounds (Table IV). The importance of the

presence of aromatic ortho-dihydroxylated groups has again been patent in two compounds obtained

from the leaves ofEriobotrya japonica (Rosaceae), chlorogenic acid and its methyl ester. The latter is

both a more potent inhibitor of ONOO� formation by SIN-1 and a better scavenger of authentic

ONOO� than the free acid. A possible explanation could be that the chlorogenic acid forms inter-

molecular hydrogen bonds between carboxylic acid and a hydroxyl group in its aromatic ring. In

contrast, methylation of the carboxyl group in the methyl chlorogenate hinders the formation of such

intermolecular bonds.108

3. Other Phenolics

The importance of the caffeoyl moiety found in some phenolic compounds for the inhibition of DHR

123 oxidation has been demonstrated by comparing the activity of three prenylhydroquinones

(Table V) and four di-O-caffeoylquinic acids (Table VI) isolated from Phagnalon rupestre. Thus,

while the caffeoylquinic derivatives were found to have IC50 values ranging from 2.1 to 9.3 mM, the

IC50 values of the two prenylhydroquinones without caffeoyl groups were more than 40 mM.89

Of 28 herbs screened for their ONOO� scavenging activities, witch hazel bark (Hamamelis

virginiana, Hamamelidaceae) produced the strongest effect. Indeed, the active component of this

herb, hamamelitannin, significantly inhibits rhodamine formation either with SIN-1 or authentic

ONOO�.114 Other compounds which showed ONOO� scavenging activities strong enough to inhibit

DHR 123 oxidation included alaternin (IC50 ¼ 2.70 mM) and nor-rubrofusarin glucoside

(IC50 ¼ 1.78 mM) (Fig. 7);93 the hydroxyindoles 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxy-

tryptamine, and 5-hydroxytryptamine (IC50 ¼ 0.73, 0.98 and 1.03 mM, respectively), but not the

methoxyindole derivative 5-methoxyindole-3-acetate (IC50 ¼ 174mM);92 and a novel diaminouracil

derivative, CX-659S (IC50 ¼ 8 mM) (Fig. 11), which has a hydroxyl group in the chroman moiety.

However, when the hydroxyl is methylated, as in CX-659-052 (IC50> 100mM), the activity is low.115

The NADPH-oxidase inhibitor apocynin (4-hydroxy-3-methoxy-acetophenone), the O�2

scavenger TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy, Fig. 12), and the NOS inhibitor

aminoguanidine are all potent inhibitors of DHR 123 oxidation by murine macrophages,

which when stimulated with LPS and IFN-g release high amounts of NO and O�2 , most probably

leading to ONOO� formation. However, neither apocynin nor aminoguanidine are scavengers of

ONOO�, O�2 , or NO as evidenced by the fact that neither of the compounds was found to inhibit SIN-

1-mediated DHR 123 oxidation. Therefore, the production of ONOO� in immunostimulated

macrophages is associated with both NO production and NADPH-oxidase-dependent O�2

formation.116


4. Non-Phenolic Compounds

Several hydroxyguanidines, for example, hydroxyguanidine and NG-hydroxy-L-arginine, reduce the

oxidation not only of DHR 123 (IC50 ¼ 0.7–16 mM), but also of cells exposed to ONOO�. On the

contrary, except for aminoguanidines, which exhibit modest effects (IC50 ¼ 20–67 mM), the rest of

the guanidines tested showed no effect whatsoever (IC50> 3,000 mM). The presence of the

hydroxyimino/hydroxyamino (¼N–OH/–NH–OH) group is important, although by itself it is not

enough to confer activity, as it happens with hydroxylamine and acetone oxime.117

Sulindac is an anti-inflammatory sulfoxide prodrug that in vivo is converted to the metabolites

sulindac sulfide and sulindac sulfone. All three inhibit ONOO�-induced DHR 123 oxidation slightly

and in a concentration-dependent manner, with sulindac sulfide being the most active scavenger

(IC50 ¼ 483 mM). This suggests that metabolism of sulindac increases its scavenging activity.118

B. Oxidation of Various Substrates of Biological Significance

Applying two experimental models based on the oxidation of NADH and the chemiluminescence of

liver homogenates, both induced by ONOO�, Boveris et al.119 were able to gain insight into the

activity of phenolics of dietary importance, known as such due to their presence in fruits and red

wines. (�)-Epicatechin, (þ)-catechin, and myricetin were all shown to inhibit chemiluminescence

generated by OH*

, with crescent IC50 values ranging from 2 to 20 mM. In the NADH oxidation test,

however, the flavonol myricetin (IC50 < 50 mM) proved to be much more potent that the two

catechins (IC50 � 250–320 mM). Caffeic acid and its quinic conjugate, chlorogenic acid, showed

intermediate potencies (IC50 � 150–200 mM). The authors proposed that wine flavonoids may,

provided that they reach sufficient plasmatic levels, protect LDL from the oxidative and inflammatory

damage leading to atherosclerosis.

Chemiluminescence was also used as a tool to measure the antioxidant properties of green tea

catechins by Van Dyke et al.120 To this end, luminol was subjected to oxidation by ONOO� or its

donor SIN-1. At a concentration of 4.5 mM, (�)-epicatechin, EGCG, and racemic catechin almost

Figure 11. Structure of CX-659S.

Figure 12. Structures of piperidinyloxyderivatives.


abolished the reaction initiated by ONOO�, whereas ECG produced a reduction of only 40%. When

SIN-1 was used instead ONOO�, homogenous inhibition values (84%–97%) were obtained for all

the catechins, with the apparent paradoxical exception of EGCG, which in fact stimulated

luminescence by 35%. For that compound, which is the only one containing two trihydroxylated,

gallic, aromatic rings, the ability to generate light per se would predominate over its relatively poor

antioxidant activity. In this vein, it should be pointed out that SIN-1 generates ONOO� constantly;

therefore, its behavior differs from that of ONOO� itself.

Ferroni et al.79 carried out a broad study on the effects of various natural food antioxidants

present in the so-called Mediterranean diet, particularly in vegetables, wines, and olive oil. Oxidative

transformations of LDL were measured as the decrease of bound-tryptophan fluorescence. In general,

the phenolic compounds assayed were not particularly effective, with only quercetin reaching a 41%

inhibition at 100 mM. These results discouraged the authors from proposing an actual protective role

for this class of antioxidants against cardiovascular disease.

When ONOO� reacts with dopamine it is transformed into either 6-nitrodopamine by nitration,

or 6-hydroxyindole-5-one by oxidation, which are both of interest as chemical markers in brain

diseases related to altered dopaminergic neuronal systems. Kerry and Rice-Evans121 studied the

effect of catechin and hydroxycinnamic acids on several schemes of dopamine transformation by

ONOO� in vitro and concluded that both catechin and caffeic acid competitively inhibited the

oxidation of dopamine, as seen from the inverse correlation found between the exhaustion of either of

these phenolics and that of dopamine. Ferulic and caffeic acids reduced the amounts of reacted

dopamine by 60%–40%, with very slight differences between 100 and 1000 mM, but their

effectiveness was always far below that of ascorbic acid. Also related with the field of dopamine

research is the work by Kuhn and Geddes102 (see also Section 6.A) on the effect of pterins on

oxidative transformations of the thiol groups of TH, the enzyme that catalyzes the synthesis of

dihydroxyphenylalanine (DOPA) from tyrosine. These authors demonstrated that tetrahydrobiop-

terin, but not biopterin, inhibits oxidation of cysteine residues caused by ONOO� and NO2 (the latter

originating from nitrite and H2O2) in TH. This is relevant because tetrahydrobiopterin localizes

specifically in dopaminergic neurons in appreciable amounts.

As explained in Section 2, glutathione is possibly our main physiological instrument for

eliminating different toxic oxidants. Within the glutathione molecule, the cysteine residue is crucial,

not only because it bears the active thiol group, but also because it is the rate-limiting aminoacid for

glutathione synthesis. It is precisely for this reason that cysteine donors such as N-acetylcysteine, 2-

oxothiazolidine-4-carboxylate, and g-glutamil-cysteine ethyl ester (GCEE) have received

pharmacological attention. A thorough study of this class of compounds was performed with gerbil

brain synaptosomes, which were treated with ONOO� to measure the affect on GS as well as any

conformational changes in the membrane proteins. The findings demonstrated that GCEE confers a

certain degree of protection against oxidative injury. However, other direct chemical parameters such

as protein carbonyl levels and dichlorofluorescein oxidation were only slightly modified.122

One unusual way to evaluate antioxidant activity related to ONOO� is based on the analysis of

the interaction of a given compound with NO2 and CO3

*�, which, as explained in Section 2, come from

decomposition of ONOOCO�2 . Alternatively, both species can be obtained in the laboratory by pulse

radiolysis of sodium carbonate and sodium nitrite solutions saturated with dinitrogen oxide (N2O).

This last substance accepts one hydrated electron (eaq�) and gives rise to the necessary intermediate,

free radical OH*

:

N2O þ H2O þ e�aq ! OH� þ OH� þ N2

CO2�3 þ OH

� ! CO�3� þ OH�

NO�2 þ OH

� ! NO2 þ OH�


Using this approach, Zhao et al.123 described the scavenger effect of two flavonols, quercetin and

rutin; two phenyletanoid glycosides, p-hydroxyphenetyl glucoside (saliodroside) and verbascoside

(Fig. 13), both from Pedicularis species (Scrophulariaceae); and glucosyl ferulate, from

Aristolochia manshuriensis (Aristolochiaceae). Several new, unidentified molecules were found

by visible spectroscopy analysis. The two flavonols and verbascoside, which also has 3 0,4 0-dihydroxyphenyl substitution, showed the highest rate of formation of the adducts with NO2 and

CO�3� (0.6–1.9� 108/M/sec and 2.8–3.4� 108/M/sec, respectively).

6 . E F F E C T S R E L A T E D T O G E N E R A T I O N O F N O

The NO pathway is involved in many fundamental regulatory processes such as endothelial

regulation of smooth muscle tone, skeletal muscle relaxation, and calcium current regulation in

cardiomyocytes through modulation of cGMP production. When derived from either nitrosothiols or

the constitutive isoforms of NOS, NO has been shown to modulate bronchial tone while in contrast,

the NO derived from iNOS seems to be a pro-inflammatory mediator with immunomodulatory

effects. The production of NO under oxidative stress conditions generates RNS, which in turn, may

enhance the inflammatory response or modulate the development of chronic inflammatory

diseases.124

Since the bioactivity of NO is altered under pathological conditions, understanding its regulation

mechanisms can provide a novel target in the prevention and treatment of diverse diseases. Also of

great interest is the fact that, under certain conditions, NO can react with O�2 to form ONOO�, which

can then initiate cytotoxic processes. It is therefore quite likely that selective inhibition of NOS by

preventing excessive NO and ONOO� formation can also provide therapeutic benefits. Among the

various approaches for dealing with the NO pathway, the inhibition of NO synthesis by arginine

derivatives, competing with the natural substrate L-arginine, represents one of the most interesting

success in this field. Furthermore, agents that directly interfere with ONOO� may have therapeutical

potential in diseases associated with increased ONOO� formation such as neurodegenerative

diseases, acute lung injury, atherosclerosis, ischemia/reperfusion, bacterial infections, and chronic

inflammation.

A. Amidines, Isothioureas, and Derivatives

Southan et al.125 have reported that a series of S-substituted isothioureas are not only potent inhibitors

of NOS enzymes, but also that this effect is selective with regard to the various isoforms. Thus, in

competition with L-arginine, S-methylisothiourea (MIT), S-(2-aminoethyl)isothiourea (AIT), S-

ethylisothiourea (EIT), and S-isopropylisothiourea (IIT) (Table VIII) all inhibited iNOS activity in

J774.2 macrophages that had been activated with a bacterial endotoxin possessing a potency lower

than that of Ng-methyl-L-arginine (L-NMA) and Ng-nitro-L-arginine (L-NO2Arg). However, as

compared to the inhibition of eNOS activity caused by L-NMA in homogenates of bovine aortic

endothelial cells, EIT and IIT were more potent, MIT was similarly potent, and AIT was less potent

than L-NMA. The structure-activity relationship of the potencies of ethyl-, isopropyl-, n-propyl-,

Figure 13. Structure of verbascoside.


t-butyl-, and n-butyl-isothioureas showed that when the length of the side chain was greater than 2

carbon atoms, the inhibitory activity against iNOS was not as great.

Several guanidines, amidines, S-alkylisothioureas, and mercaptoalkylguanidines (Table VIII)

have been described as inhibitors of NO generation from L-arginine by NOS. Of these, some

guanidines and S-alkylisothioureas (e.g., aminoethylisothiourea) actually exhibit iNOS selectivity,

which is unusual for L-arginine based inhibitors. Aminopropylisothiourea (APIT), for instance,

significantly inhibited NO�2 formation by immunostimulated J774 macrophages, with EC50 values

ranging from 6 to 30 mM, as compared to those obtained for L-NMA (159 mM) and Ng-nitro-L-

arginine (>1000 mM).126 At physiological pH, certain aminoalkylisothioureas undergo spontaneous

rearrangement to form mercaptoalkylguanidines such as mercaptoethylguanidine (MEG). These, in

turn, exhibit degrees of iNOS inhibition similar to those of their isomer compounds. Moreover, the

oxidation of MEG yields guanidinoethyldisulfide (GED), which is a competitive inhibitor of iNOS

activity. This compound has proven to be a potent inhibitor of the NOS activity of purified iNOS,

eNOS, and nNOS enzymes, with Ki values of 4.3, 18, and 25 mM, respectively. As for selectivity, at

the enzyme level GED shows a four-fold preference for iNOS over eNOS.127

Mercaptoethylguanidine shows potency in the same range as the ONOO� scavengers

glutathione, cysteine, cysteine methyl ester, and penicillamine, and also inhibits both ONOO�-

induced hydroxylation of benzoate and nitration of 4-hydroxyphenylacetic acid. Aminoguanidine

inhibited both reactions but failed to stop cytochrome c2þ oxidation, which suggests that it reacts with

either HOONO or NO2.128 Further studies have demonstrated that MEG protects against the

Table VIII. Structures of Guanidines, Isothioureas, and Derivatives

AIT,S-(2-aminoethyl)isothiourea; APIT,aminopropylisothiourea;EIT,S-ethylisothiourea;GED,guanidinoethyldisulfide; ITT,S-isopropylisothiourea;

MIT,S-methylisothiourea.


suppression of mitochondrial respiration and DNA single strand breakage induced by ONOO�. In

addition, it acts as an anti-inflammatory agent with diverse mechanisms in many experimental

models. For example, in studies with rats, MEG inhibited the inflammatory response in carrageenan-

induced paw edema and pleurisy, reduced iNOS activity in the lungs, decreased NO�2 /NO�

3

production in the exudate, and completely inhibited 3-NT immunostaining in the inflamed lung

tissue.129 It also reduced the development of arthritis in a collagen-induced model,130 protected

against trinitrobenzene sulfonic acid-induced colonic damage131 and caused a dose-dependent

inhibition of COX activity.132 At a concentration of 1 mM, MEG also had a protective effect on a1-

antiproteinase activity, although at concentrations lower than 100 mM it had the opposite effect, as the

deactivation by ONOO� was actually enhanced.133 This phenomenon had previously been observed

for other thiols such as cysteine and penicillamine, and was attributed to the formation of ONOO�-

derived sulfur free radicals.134 It has been reported that daily treatment with aminoguanidine (20 mg/

kg i.p.) relieved the inflammatory lesion in the lungs produced by intratracheal administration of the

anticancer drug bleomycin to rats. Apart from determining the 3-NT immunoreactivity of the lung

tissue, histological damage was evaluated through microscopic examination of sections stained with

hematoxylin-eosin and with Sirius red for type I and type III collagen. Aminoguanidine was found to

inhibit 3-NT generation in epithelial alveolar cells and interstitial macrophages, and also to reduce

fibrosis and afflux of inflammatory cells.135

Other mercaptoalkylguanidines such as N-methyl-mercaptoethylguanidine, N,N 0-dimethyl-

mercaptoethylguanidine, S-methyl-mercaptoethylguanidine, and GED also inhibited LPS-stimu-

lated 6-keto-PGF1a production, with IC50 values ranging between 34 and 55 mM. In contrast,

aminoguanidine, L-NAME, and L-NMA had no effect on the production of prostaglandins. In a

porcine model of severe hemorrhagic shock, MEG was found to exert several other beneficial effects,

including improvement of survival rate, reduction of lipid peroxidation and neutrophil accumulation,

and a decrease in blood pressure.136 In a porcine model of long-term hyperdynamic endotoxemia, it

did not affect the endotoxin-related impairment of the hepato-splanchnic metabolism, although it

prevented the progressive fall in blood pressure and reduced the development of both systemic and

regional acidosis.137 In addition, GED also prevented the development of diabetes, probably by

inhibiting 3-NT formation in the islet b-cells.138

NO may also play a key role in excitotoxic neuronal injury in the central nervous system. The

activation of excitatory amino acid receptors leads to increased cellular calcium levels. This may be

followed by both activation of NOS and generation of free radicals, which subsequently leads to

ONOO� production. Malonate, a reversible inhibitor of succinate dehydrogenase, induces

excitotoxic lesions in the striatum, similar to those caused by Huntington’s disease. These lesions

are attenuated by the NOS inhibitor N-nitro-L-arginine. 139 For its part, the neurotoxicant MPTP is

oxidized by monoamine oxidase B to form MPPþ, an active metabolite that inhibits mitochondrial

complexes I, III, and IV, which in turn produces pathological neurochemical effects mimicking those

of Parkinson’s disease. The administration of MPTP produces TH nitration in mouse striatum,

resulting in the loss of enzymatic activity and consequent dopamine synthesis failure. However, TH

was not nitrated in mice that over-expressed copper/zinc SOD, a finding that corroborates the role for

O�2 in TH nitration.50 Having observed that reduced, but not oxidized forms of the nicotinamide

adenine dinucleotide cofactors NADH and NADPH prevented ONOO�-induced nitration of TH,

Kuhn and Geddes have emphasized the influence of the redox status of nicotinamide nucleotides in

protein modification brought about by ONOO�.140 Administration of the relatively selective nNOS

inhibitor S-methylthiocitrulline (Fig. 14) not only protected against malonate lesions—an effect that

was blocked by L-arginine—but also attenuated the increase in 3-NT produced by malonate. It also

slowed the depletion of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and

homovanillic acid in MPTP-induced dopaminergic neurotoxicity. All of these findings point to a

potential role for relatively selective nNOS inhibitors in the treatment of neurodegenerative

diseases.141


It has been reported that ONOO� is an initiator of the expression of IL-8, a potent pro-

inflammatory neutrophil-activating chemokine. More specifically, ONOO� functions as an

intracellular messenger that mediates, via activation of the transcription factors NF-kB and AP-1,

IL-8 gene expression in human leukocytes stimulated with LPS, TNF-a, or IL-1b. In contrast, NO

released by SNAP and SNP did not induce cytokine release, unless O�2 was simultaneously generated

by a xanthine/xanthine oxidase system. In LPS-stimulated human blood cells, NOS inhibitors such as

aminoguanidine and L-NAME inhibited IL-8 production, but had no effect on that of IL-1b or TNF-

a.142 Still, both compounds blocked NF-kB and AP-1 activation and inhibited both IL-8 gene

expression and IL-8 release in leukocytes challenged with IL-1b or TNF-a.143

B. Phenolic Compounds

Phenolcarboxylic acids such as caffeic, p-coumaric, and ferulic acids not only inhibited NO

production by LPS-activated macrophages, but also actively scavenged NO itself. Interestingly,

while caffeic acid was the most efficient inhibitor of NO production, it was the least efficient NO

scavenger. It is also worth noting that the dehydrogenation polymers of caffeic and p-coumaric acids

inhibited NO production more efficiently than did the corresponding monomers.144 Caffeic acid

phenethyl ester, an active component of honeybee propolis, inhibited the iNOS expression induced

by the combination of LPS and IFN-g, which in turn inhibited NO production. In fact, the ester was

found to inhibit iNOS gene expression at the transcriptional level through suppression of NF-kB

activation, directly inhibiting the catalytic activity of the enzyme.145

[6]-Gingerol (1-[4 0-hydroxy-3 0-methoxyphenyl]-5-hydroxy-3-decanone, Fig. 15), the major

pungent constituent in the ginger rhizome (Zingiber officinale, Zingiberaceae), was shown to protect

against ONOO�-induced damage and markedly decrease iNOS protein induction in LPS-activated

J774.1 macrophages, probably by inhibiting AP-1. It also prevented SIN-1 and ONOO�-induced

oxidation of DCDHF, nitration of protein tyrosyl residues in BSA and J774.1 macrophages,

and oxidative single strand breaks in supercoiled pTZ 18U plasmid DNA.146 Previously, [6]-gingerol

had already been proven to be an inhibitor of epidermal growth factor (EGF)-induced AP-1 DNA

binding activity in mouse epidermal JB6 cells.147 Similarly, the yellow-colored spice curcumin from

the turmeric rhizome (Curcuma longa, Zingiberaceae) inhibited both NO production and iNOS

induction in LPS- and IFN-g-activated RAW 264.7 macrophages and suppressed TPA-induced c-jun/

AP-1 activation.148

Rotenone (Fig. 16), an inhibitor of mitochondrial complex I activity that is widely used as a

pesticide, has for some time now been thought to induce Parkinson’s disease. Thus, chronic

intravenous administration of rotenone infusion in rats induced selective pathological and

biochemical changes in the nigro-striatal dopaminergic system, as indicated by a reduction of

striatal dopamine levels and a loss of substantia nigra TH-positive nigral cells. Rotenone also induced

Figure 14. Structure of S-methylthiocitrulline.

Figure 15. Structure of [6]-gingerol.


selectively increased nNOS expression and 3-NT production in the striatum and increased NOS

activity in both the striatum and substantia nigra.139

Certain crude drugs including Sanguisorbae Radix (Sanguisorba officinalis, Rosaceae),

Caryophylli Flos (Syzigium aromaticum, Myrtaceae), Gambir (Uncaria gambir, Rubiaceae), Granati

Cortex (Punica granatum, Punicaceae), Gallae Rhois (Rhus javanica, Anacardiaceae), Rhei

Rhizoma (Rheum officinale, Polygonaceae) and Cinnamomi Cortex (Cinnamomum zeylanicum,

Lauraceae), which contain tannins as major constituents, in addition to Coptidis Rhizoma (Coptis

chinensis, Ranunculaceae), which contains mainly alkaloids, exhibited a direct NO scavenging

activity using SNP as an NO donor in vitro.149 Sanguisorbae Radix was reported to attenuate renal

dysfunction in vivo by suppressing iNOS activity as well as by decreasing excessive NO and ONOO�

levels. In subsequent studies, sanguiin H-6 (Fig. 33), a tannin constituent of this crude extract, was

found to exhibit a direct NO scavenger effect. It was also demonstrated to be the most active

component in inhibiting NO production in LPS-activated macrophages due to its concomitant

inhibition of iNOS mRNA induction and enzyme activity.150,151

C. Other Compounds

Peroxynitrite is considered a key factor in the pathogenesis of renal ischemia-reperfusion injury. In

addition to the alterations in both the structure and the function of proteins caused by the nitration,

ONOO� spontaneously decomposes to generate OH*

, which causes additional oxidative injury to the

tissues. Studies with the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL, Fig. 17)

demonstrated that ONOO� formation actually mediates the development of the renal injury, since

administration of L-NIL (3 mg/kg) to treat rats that had been subjected to ischemia-reperfusion

improved their renal function in terms of plasma creatinine levels. This treatment also decreased

ONOO� generation, measured as 3-NT. However, administration of L-NIL at higher doses (10 mg/

kg) had no effect.152

The traditional Chinese medication Wen-Pi-Tang, which contains Rhei rhizoma (Rheum

officinale, Polygonaceae), Ginseng radix (Panax ginseng, Araliaceae), Aconiti tuber (Aconitum

japonicum, Ranunculaceae), Zingiberis rhizome (Zingiber officinale, Zingiberaceae), and Glycyr-

rhizae radix (Glycyrrhiza glabra, Fabaceae), is considered to be a therapeutic agent against

pathological renal disorders associated with ONOO�.153 When administered orally to rats subjected

to LPS stimulation plus renal ischemia-reperfusion, this drug protected against ONOO� formation by

Figure 16. Structure of rotenone.

Figure 17. Structure of L-N-(1-iminoethyl)lisine.


reducing plasma levels of 3-NT in vivo; however, it did not affect the activity of iNOS or xanthine

oxidase, which are major sources of NO and O�2 , respectively. Still, Wen-Pi-Tang extract reduced

the levels of tyrosine isomers produced by OH*

as secondary reactive end products and attenuated the

induced damage not only by scavenging the radicals themselves, but also by increasing the activity

and efficiency of the antioxidative enzymes SOD, catalase, and glutathione peroxidase (GPX) in

renal tissues. It also ameliorated renal dysfunction by decreasing the urea nitrogen and chromium

levels. Previous studies in vitro had already demonstrated its protective effect against renal

impairment induced by oxidative stress and ONOO�, NO, and O�2 scavenging activity.

The generation of ONOO� has also been implicated in the dopaminergic neurotoxicity produced

by methamphetamine. Treatment with this drug produced significant mitochondrial damage and

increased the formation of 3-NT, which is correlated with dopamine depletion in the striatum. It has

previously been demonstrated that pre-treatment with either the relatively selective nNOS inhibitor

7-nitroindazole (7-NI, Fig. 18), antioxidants such as selenium and melatonin, or ONOO�

decomposition catalysts (see Section 8) protects against the methamphetamine-induced neurotoxi-

city.154–156 It has subsequently been observed that pre- and post-treatment of mice with L-carnitine,

which carries long-chain fatty acyl groups into the mitochondria for b-oxidation, produces a

significant reduction in the production of 3-NT in the atria of mouse hearts that have been treated

with methamphetamine. These protective effects may be due to the enhancement of mitochondrial

metabolism, which can prevent the generation of ONOO� and other free radicals generated

by methamphetamine and/or by the scavenging of ONOO� itself.157 Furthermore, in mice treated

with multiple doses of methamphetamine, no significant production of 3-NT in the striatum was

observed for those subjects which either lacked the nNOS gene or overexpressed copper–zinc

SOD.158

Likewise, treatment with 7-NI was found to attenuate excitotoxic lesions induced by malonate as

well as reduce the increase of NOS activity, block the increase of 3-NT, and protect against rotenone-

induced neurotoxicity of the nigro-striatal pathway. These effects are probably all mediated by the

increased generation of NO.139 MPTP-induced neurotoxicity is also attenuated by 7-NI treatment, or

alternatively by using nNOS deficient mice.141

Pyrrolidine dithiocarbamate (Fig. 19), an inhibitor of NF-kB activation, diminished the

induction of the IL-8 gene expression and IL-8 release induced by LPS, IL-1b, TNF-a, and

ONOO�. 142,143 Pro-inflammatory cytokine release and the nuclear translocation of NF-kB induced

by ONOO� in human monocytes was blocked by the cell-permeable ONOO� scavenger, 5,10,15,20-

tetrakis(4-sulfonatophenyl)prophyrinato iron III chloride, but was unaffected by the protein nitration

inhibitor EGC.159 Iho et al.160 demonstrated that nicotine induces the generation of free radicals and

stimulates neutrophils to produce IL-8 in dose- and time-dependent manners in vitro. The nicotine-

induced IL-8 production was abrogated by antioxidants such as N-acetyl-L-cysteine and pyrrolidine

dithiocarbamate, as well as by the specific NOS inhibitor L-NAME and the NF-kB inhibitor

Figure 18. Structure of 7-nitroindazole.

Figure 19. Structure of pyrrolidine dithiocarbamate.


dexamethasone, but not by the AP-1 inhibitor curcumin (Fig. 20). These researchers found that IL-8

production was mediated by the interaction of nicotine with nicotinic acetylcholine receptors and

subsequent activation of NF-kB. Furthermore, pyrrolidine dithiocarbamate, reported to be a potent

NF-kB inhibitor, attenuated IL-8 production, reduced iNOS activity, as well as 3-NT formation

and PARP activation in carrageenan-induced lung pleurisy and in collagen-induced arthritis.161

Selenium-containing compounds such as selenomethionine, selenocystine, and the synthetic

organoselenium compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) were more effective

in inhibiting 4-hydroxyphenylacetate nitration and rhodamine formation by ONOO� than their sulfur

analogs methionine, cystine, and ebsulfur.162

These same selenium-containing compounds attenuated IL-8 gene expression and IL-8

production in both polymorphonuclear and mononuclear human leukocytes by preventing ONOO�-

mediated activation of the transcription factors NF-kB and AP-1.163

1H-(1,2,4)Oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, Fig. 21) is a potent GC inhibitor that

slightly enhanced NO�2 /NO�

3 production in immunostimulated cells. This finding shows that GC does

not suppress iNOS induction and that the role of cGMP in this process depends on the stimulus and the

cell type assayed. In studies on rats, ODQ had no effect on the suppression of mitochondrial

respiration in response to either LPS/IFN-g stimulation in the aortic smooth muscle cells or NO or

ONOO� challenge. ODQ protected against LPS-induced relaxation caused by iNOS expression in

endothelium-denuded thoracic aortic rings pre-contracted with norepinephrine. It also restored

in vitro the suppression of the changes in vascular contractility of rat aortic rings to norepinephrine in

response to LPS. ODQ also improved the survival rate in mice that had been subjected to endotoxic

shock.164

Nicaraven (2(R,S)-1,2-bis(nicotinamido)propane, Fig. 22) is a known OH*

scavenger that

weakly interferes with the NO pathway. It slightly inhibited, in a dose dependent manner, NO

production in LPS-stimulated macrophages, but had no effect against DHR 123 oxidation induced by

ONOO�, thus failing as a scavenger of this anion.165

7 . P R E V E N T I O N O F R N S - I N D U C E D D N A D A M A G E

A. Inhibition of PARP and DNA Breakage

Many of the physiological functions attributed to PARP (see Section 3.D) were first deduced from the

results of investigations on its deactivation. Nicotinamide (Fig. 23), for example, which exerts

a feedback inhibition to PARP, is a weak and non-specific PARP inhibitor that was initially used

to prove PARP function. Subsequent studies with structurally related compounds such as

Figure 20. Structure ofcurcumin.

Figure 21. Structure of ODQ.


3-aminobenzamide (3-AB, Fig. 23) and its derivatives demonstrated the involvement of PARP in

gene expression, as well as DNA replication, rearrangement, differentiation, and mutagenesis. Both

nicotinamide and 3-AB showed IC50 values of approximately 30 mM on the isolated PARP-1

enzyme.166–169

A large number of studies on the effects of 3-AB, the main member of the aminobenzamide

family, on PARP activity have been reported. This drug is effective against ischemia-reperfusion

injury of the brain and heart. It also provides protection against reperfusion injury in the intestine,

kidneys, skeletal muscle, liver, retina, and in experimental models of shock. Nicotinamide was also

found to be effective against ischemia-reperfusion injury, shock, and stroke. It has been reported that

both compounds protect various cell types against oxidative stress in response to ROS, NO donors,

and ONOO�.170 They are also effective in inhibiting pro-inflammatory mediators and preventing the

development of diabetes. Szabo et al.171 investigated the role of PARP in the process of neutrophil

recruitment and in the development of local and systemic inflammation. They found that 3-AB

reduced the development of acute172 and chronic inflammation.171 Most results from pharmaco-

logical studies have been confirmed by experiments using genetically engineered mice lacking

functional PARP enzyme. Thus, treatment of normal rats with 3-AB reduced the non-septic shock and

multiple organ failure induced by i.p. injection of a high dose of zymosan. The same results were

obtained in untreated PARP-knock out mice. The effects of PARP inhibition were mainly due to

interference with polymorphonuclear leukocytes (PMNs) post-adhesion phenomena. The reduction

of neutrophil recruitment has been related to the prevention of endothelial oxidant injury and

inhibition of the expression of adhesion receptors. The anti-inflammatory effects exerted by PARP

inhibition were also corroborated by the efficacy of 3-AB in inhibiting the inflammatory response

against carrageenan-induced pleurisy. Thus, treatment of rats with 3-AB dose-dependently reduced

edema formation, mononuclear cell infiltration and histological injury, NO�2 /NO�

3 concentrations in

the pleural exudate, and 3-NT staining in the lungs.172

In general, it has been assumed that benzamides inhibit PARP by interfering with the binding of

NAD to the PARP active site, but they also prevent PARP activation by binding to DNA and avoiding

detection of DNA breakage by PARP. However, they have other pharmacological effects

independently from PARP inhibition, including antioxidant effects. Nicotinamide also acts as a

substrate for other NAD-metabolizing enzymes, and, as a vitamin with an established safety profile, it

can be administered to humans. However, at the high doses required to provide PARP inhibition, it

would probably exert toxic effects.

Southan and Szabo173 and, more recently, Jagtap and Szabo174 exhaustively overviewed the

structures and pharmacological actions of different classes of compounds that inhibit the catalytic

Figure 22. Structure ofnicaraven.

Figure 23. Structuresofnicotinamide (A) and 3-aminobenzamide (B).


activity of PARP. Apart from nicotinamide and 3-AB, Southan and Szabo divided PARP inhibitors

into six groups according to their related chemical skeletons: isoquinolinones and dihydroisoqui-

nolinones; benzimidazoles, indoles, and related compounds; phthalazin-1-(2H)-ones and quinazo-

linones; isoindolinones; and phenanthridinones and miscellaneous compounds.

Among isoquinolinones and dihydroisoquinolinones, those containing a lactam structure in the

fused cyclic system are more effective PARP inhibitors than 3-AB. The aromatic ring system bearing

the carboxamide group with a hydrogen on the amide nitrogen are important chemical features for the

competitive inhibition of PARP at the catalytic site. The potency was enhanced by substitution in

position 5, with the hydroxyl group having the highest effect (IC50 ¼ 100 nM). After evaluating a

synthesized series of dihydroisoquinolinones, rigid analogs of 3-substituted benzamides, and a series

of 2,3-disubstituted benzamides, Suto et al.175 suggested that the orientation of the amide with respect

to the substituent on the aromatic ring, which must be cis, was critical for optimum inhibitory activity.

Thus, 3,4-dihydro-5-methyl-isoquinolin-1(2H)-one has an IC50 value of 140 nM against the purified

enzyme, approximately 50 times lower than that for 3-AB.176 Both 1,5-dihydroxyisoquinoline

(Fig. 24) and 3,4-dihydro-5-[4-(piperidin-1-yl)butoxy]isoquinolin-1(2H)-one (DPQ, Fig. 24) are

more potent PARP inhibitors, but they must first be dissolved in dimethylsulfoxide, which is itself a

hydroxyl radical scavenger and PARP inhibitor. The 5-amino derivative 5-aminoisoquinolin-1-(2H)-

one (5-AIQ) has been shown to be a water-soluble inhibitor of PARP activity in a cell-free

preparation.

In fact, 5-AIQ was used to demonstrate the contribution of PARP activation to the organ injury

and dysfunction associated with severe hemorrhage and resuscitation.177 However, while 5-AIQ

reduced the multiple organ injury and dysfunction in a dose dependent manner, it had no effect on the

circulatory failure associated with hemorrhagic shock.

GPI 6150 (1,11b-dihydro-[2H]benzopyrano [4,3,2-de]isoquinolein-3-one, Fig. 25) is an

extensively studied PARP inhibitor belonging to the related tetraheterocyclic lactam family. In

enzyme kinetic analysis, GPI 6150 exhibited a typical competitive inhibition mode, competing with

NADþ for the PARP catalytic site (Ki ¼ 60 nM).166 This compound dose-dependently reduced H2O2

cytotoxicity, inhibited H2O2-induced PARP activation, and maintained the cellular NADþ

concentration in P388D1 cells. Moreover, it has shown a remarkable efficacy in reducing tissue

damage in rodent models of ischemic or traumatic models of brain injury. GPI 6150 also protected

dopaminergic neurons from damage caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Furthermore, that compound was beneficial in regional myocardial ischemia, septic shock, and

streptozotocin-induced diabetes.166,178 It also inhibited carrageenan-induced paw edema and

adjuvant-induced paw arthritis, and reduced the mortality, morphological injury, and neutrophil

infiltration in zymosan-induced multiple organ failure.170 In addition, in studies on the efficacy of

GPI 6150 against colitis caused by intra-colonic administration of dinitrobenzensulfonic acid, the

compound attenuated in a dose dependent manner the extent and severity of colon injury, the degree

Figure 24. Structuresof1,5-dihydroxyisoquinoline (A) andDPQ (B).


of hemorrhagic diarrhea and immunostaining for PARP, neutrophil recruitment and accumulation, as

well as cytokine production.61 Administration of GPI 6150 also exhibited multiple protective effects

in rats subjected to splanchnic artery occlusion (SAO) shock. It significantly improved mean arterial

blood pressure, prevented tissue infiltration of neutrophils, and reduced the degree of immunostain-

ing for PARP, P-selectin, and ICAM-1 in the reperfused intestine. Most importantly, it actually

improved the survival rate.179

As far as phenantridinones are concerned, the potent PARP inhibitor PJ34 (N-(oxo-5,6-dihydro-

phenanthridin-2-yl)-N,N-dimethylacetamide, Fig. 26) is one of the most intensively investigated.

This compound improved endothelium-dependent vascular relaxant function in vascular injury

associated with chronic ischemic heart failure,180 diabetes,181 hypertension and aging, but not

atherosclerosis.182 It also improved cardiac function and increased the survival rate for LPS-induced

cardiac shock.183 PJ34 demonstrated a marked protection in an experimental porcine model of sepsis

induced by Escherichia coli, abolishing PARP activation, reducing TNF-a in plasma, attenuating

bacteremia, increasing systemic and pulmonary vascular resistance, and improving survival and

cardiovascular conditions.184 Pharmacological inhibition of PARP-1, either with PJ34 or by genetic

deletion, reduced LPS-induced increases of the following: cytokines TNF-a, IL-1b, and IL-6;

chemokines macrophage inflammatory proteins (MIP)-1a and MIP-2; alveolar neutrophil

accumulation; lung hyperpermeability; NO production; and lipid peroxidation. Lung damage was

also attenuated.185 PJ34 also showed protective effects in a murine asthma model, suppressing both

inflammatory cell migration and the production of TNF-a and IL-12 (but not of IL-5 or IL-13). In

addition, it downregulated MIP-1a, but not MIP-2.186

Recently, PARP activation has also been identified as an important mechanism in the oxidative

stress-related development of diabetic retinopathy and neuropathy. Two structurally unrelated PARP

inhibitors, 3-AB and 1,5-isoquinolinediol, counteract the overexpression of diabetes- and hypoxia-

induced retinal vascular endothelial growth factor in both streptozotocin-diabetic rats and in human

retinal pigment epithelial cells exposed to hypoxia. PARP-deficient (PARP(�/�)) mice were

Figure 25. Structure of GPI 6150.

Figure 26. Structure of PJ34.


protected from functional and metabolic changes in the diabetic neuropathy as well as streptozotocin-

induced diabetic rats treated with both PARP inhibitors.187

Naturally occurring phenolic compounds are considered to be active dietary constituents that

protect against diseases induced by oxidative damage. Ohshima et al.188 studied the effects of

flavonoids on DNA damage induced by NO, ONOO�, and NO�. They demonstrated that most of the

flavonoids tested acted as antioxidants to inhibit ONOO�-mediated DNA strand breakage and

nitration of guanine; however, in the presence of NO, certain flavonoids exerted pro-oxidant effects.

Concurrent incubation of plasmid DNAwith an NO-releasing compound such as sodium (z)-1-(N,N-

diethylamino)diazen-1-ium-1,2-diolate (DEA-NO) and a polyhydroxy aromatic compound with a

catechol or pyrogallol group leads to synergistic induction of DNA strand breakage whereas none of

them alone induces such breakage. Phenolic compounds such as flavonoids and anthocyanidins with

an o-trihydroxyl group in either the A ring (baicalein and quercetagetin) or the B ring (delphinidin,

EGCG, and myricetin) exhibited strong activity in the presence of NO. This pro-oxidant effect of

certain phenolics might be due to RNS generation by the reaction between NO and semiquinone/

quinone derivatives. However, this hypothesis seems paradoxical, considering the usual antioxidant

effects of the phenolics evaluated. Nevertheless, catechin, cyanidin, epicatechin, ECG, EGCG,

myricetin, and rutin all inhibited ONOO�-mediated DNA strand breakage by 80%. Moreover, most

of the compounds tested almost completely inhibited ONOO�-mediated formation of 8-NG in calf-

thymus DNA, as measured with the HPLC-electrochemical detection method. However, they were

less effective in inhibiting the single-strand breakage induced by Angeli’s salt. Previously, Fiala

et al.67 demonstrated that EGCG inhibited ONOO�-induced 3-NT and 8-oxodeoxyguanosine

formation in calf-thymus DNA much more efficiently than the endogenous antioxidants ascorbic acid

and glutathione.

Chlorogenic acid is a potent o-dihydroxyphenolic free radical scavenger that efficiently inhibits

ONOO�-induced DNA damage. This effect is notably enhanced in the presence of horseradish

peroxidase, a heme-containing enzyme that catalyses ONOO�-decomposition, probably due to the

fact that chlorogenic acid acts as an electron donor to regenerate the active form of the enzyme.189

Cyanidin-3-O-glucoside (kuromanin), is a glycosilated polyhydroxy flavilium salt that

represents approximately 80% of the total anthocyanidin content in blackberry (Rubus sp.,

Rosaceae) juice. Cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, cyanidin-3-O-xyloside,

malvidin-3-O-glucoside, and pelargonidin-3-O-glucoside have also been characterized as minor

constituents of the extract. Serraino et al.190 demonstrated that blackberry juice and cyanidin-3-O-

glucoside as chloride are both ONOO� scavengers and exert a protective effect against ONOO�-

induced endothelial dysfunction and vascular failure. Pre-treatment of HUVEC with different

dilutions of blackberry juice (containing 80, 40, and 14.5 ppm of cyanidin-3-O-glucoside) and

cyanidin-3-O-glucoside as chloride (0.085, 0.028, and 0.0085 mM) reduced the ONOO�-induced

DNA damage and PARP activation as well as the suppression of mitochondrial respiration.

Blackberry juice and cyanidin-3-O-glucoside also improved ONOO�-induced vascular dysfunction.

Both protected against the ONOO�-mediated suppression of vascular contractility and endothelial

dysfunction in the thoracic aortic rings of rats. Immunohistochemical analysis in pre-treated aortic

rings revealed a complete inhibition of ONOO�-induced 3-NT formation and of PARP activation.

The main isoflavones in soybeans, namely genistin, daidzin and their aglycones genistein

and daidzein, all of which were previously reported to protect against LDL oxidation induced by

ONOO�,191 were evaluated as inhibitors of plasmid and cellular DNA damage induced by SNP and

ONOO�. The isoflavones significantly inhibited the induction of SNP- and ONOO�-mediated RAW

264.7 cell genotoxicity, with genistein showing the highest potency. Both genistein and daidzein

inhibited ONOO�-mediated fX174 DNA degradation in a dose-dependent manner. In addition,

treating macrophages with SNP elevated cellular glutathione levels; however, no significant

differences in glutathione content or a reduced/oxidized glutathione ratio were observed when these

cells were exposed to genistein and daidzein in the presence of SNP. Taking into account these results


as well as the important role that the glutathion redox cycle plays in scavenging ROS and RNS, it was

suggested that the inhibitory effects of isoflavones on SNP- and ONOO�-induced DNA damage

might be associated with their prevention of antioxidant enzyme deactivation and their NO and

ONOO� scavenger abilities.192 More recently, it has been reported that soy isoflavones inhibit iNOS

activity and exhibit ONOO� scavenging activity.193 The isoflavones genistein and daidzein along

with extracts from soy-based products such as miso, soybeans, black soybeans, soy milk, tofu, and

yuba, exhibited potent antioxidant activity in vitro and inhibited RNS-mediated damage. They

protected against cellular DNA damage induced by SNP and ONOO�, inhibited NO production from

LPS-induced macrophages, and showed NO scavenging activity. The efficacy of the extracts was

correlated with their isoflavone content. Oral administration of the isoflavones genistein and

daidzein, and also of soybean and yuba extracts to rats for 1 week prior to LPS treatment attenuated

serum NO�2 , NO�

3 , and 3-NT concentrations, suggesting that supplementing the diet with isoflavones

may regulate the metabolism of RNS in LPS-treated rats.

Acidified NO�2 is known to cause damage to DNA bases. Previous work has demonstrated that

exposure of DNA bases to NaNO2 under acidic conditions (pH ¼ 1), such as those found in the

stomach, resulted in deamination of adenine and guanine to form hypoxanthine and xanthine,

respectively. Among the phenolic compounds tested against this phenomenon, EGCG was the most

potent inhibitor of hypoxanthine and xanthine formation. Quercetin, caffeic acid, and other catechin

compounds such as catechin, epicatechin, and EGC exhibited a similar efficacy, whereas catechol,

gallic acid, and 3,4-dihydroxyphenylacetic acid showed a weaker effect.194 Ascorbic acid failed in

inhibiting acidic NO�2 -induced base deamination.

Antioxidants maintain hemoproteins and lipoyl dehydrogenase, the enzyme involved in the

reductive modification of the nitrated DNA pathway, in their reduced states, which are the last

reducing agents for nitrated DNA bases. Chen et al.195 investigated the abilities of biological and

dietary antioxidants in scavenging NO2Cl and decreasing the subsequent DNA nitration by

incubating calf thymus DNA with antioxidants in the presence of a mixture of NO�2 /HOCl. The

inhibitory effect was determined by measuring the decrease in 8NX formation by means of HPLC-

DAD. The antioxidants evaluated were those from dietary sources such as ascorbate, folic acid,

diallyldisulfide, and a-tocopherol, as well as phenolic compounds such as ferulic acid, quercetin,

rutin, and caffeic acid; in addition, the effects of NADH and uric acid, along with sulfur-containing

compounds at various oxidation states including sulfhydryls (dihydrolipoic acid,N-acetil-L-cysteine,

and reduced glutathione), sulfides (diallylsulfide, L-methionine, and N-acetil-L-methionine),

disulfides (a-lipoic acid and oxidized glutathione), sulfoxide (dimethyl sulfoxide), and sulfonic

acid (taurine) were examined. Among the sulfur-containing compounds, those that were more highly

reduced proved to be the stronger inhibitors of DNA nitration. Dihydrolipoic acid was the most

effective in preventing DNA nitration, followed by N-acetil-L-cysteine (IC50 ¼ 0.1 and 0.46 mM,

respectively). Among the rest of the antioxidants evaluated, folic acid and ferulic acid showed IC50

values of 0.5 and 1.0 mM, respectively.

In glaucoma, ONOO� plays a role in the glutamate-induced retinal excitotoxicity, which is

mediated by over-stimulation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. This

activation increases intracellular calcium concentrations, which subsequently increases iNOS

expression, NO and O�2 production, and ONOO� generation, which in turn cause lipid peroxidation,

mitochondrial dysfunction, DNA damage, and cell death. El-Remessy et al. 196 have demonstrated

the neuroprotective effects of the psychotropic principle of marijuana D9-tetrahydrocannabinol (D9-

THC, Fig. 27) and a non-psychotropic cannabinoid, cannabidiol (CBD, Fig. 27), against NMDA-

induced retinal injury in rats. The protective effects are caused by the compounds’ ability to reduce

lipid peroxidation and slow the production of NO and ONOO�.

Tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy, Fig. 12), an SOD mimetic and O�2

and OH*

scavenger, and the iNOS inhibitor L-NAME also showed retinal neuroprotective effects.

Even earlier, it was known that both D9-THC and the synthetic cannabinoid WIN55,212-2 (Fig. 27)


protected against neurotoxicity mediated by glutamate and NMDA in neurons and the brain by

activating the cannabinoid receptor CB1. Furthermore, D9-THC, CBD, and the synthetic derivative

HU-211 (Fig. 27) all possess antioxidant and/or NMDA receptor antagonist effects that prevent

glutamate-induced death and oxidative stress in neuron cultures.

Cuzzocrea et al.197 demonstrated the protective effects of endogenous gluthatione against

ONOO�- and LPS-induced vascular failure by evaluating whether L-buthionine-(S,R)-sulfoximine

(BSO), a specific inhibitor of g-glutamylcysteine synthetase, affects ONOO�-induced endothelial

and vascular smooth muscle injury in vitro and endotoxic shock in vivo. They found that pre-

treatment of HUVEC and rat aortic smooth muscle cells with BSO significantly enhanced the

ONOO�-induced suppression of mitochondrial respiration, DNA single-strand breaks and PARP

activation, tyrosine nitration, and protein oxidation. However, pre-treatment of cells with glutathione

or gluthathione ethyl ester attenuated ONOO�-induced cellular injury. BSO treatment of the isolated

thoracic aortic rings of rats enhanced the ONOO�-induced reduction of the contractions in response

to noradrenaline and the ONOO�-induced impairment of the endothelium-dependent relaxations in

response to acetylcholine, while these effects were attenuated by gluthathione treatment. In BSO-

pretreated rats, LPS treatment caused an enhancement of vascular hyporeactivity and endothelial

dysfunction. The administration of BSO also increased the degree of 3-NT staining in the aorta after

LPS treatment.

Ergothioneine (N,N,N-trimethyl-2-mercaptohistidine, Fig. 28), a natural antioxidant found in

humans at concentrations of up to 1–2 mM and probably absorbed from the diet, was found to protect

against oxidative base modifications in isolated calf thymus DNA as well as in DNA from a human

Figure 27. Structuresofnatural andsynthetic cannabinoidderivatives.

Figure 28. Structure of ergothioneine.


neuronal hybridoma cell line exposed to ONOO�. Ergothioneine inhibited the formation of DNA

adducts along with that of xanthine and hypoxanthine, the deaminated products of guanine and

adenine, respectively.198 Previously, this thiol had proven to be an effective ONOO� scavenger by

preventing against nitration of tyrosine and deactivation of a1-antiproteinase by this species.199 An

ubiquitous inducible protein with antioxidant properties, metallothionein prevented plasmid DNA

damage and LDL oxidation induced by SIN-1 and ONOO�.200

Tyrphostin AG 126, a tyrosine kinase inhibitor, attenuated the degree of the multiple organ

dysfunction syndrome associated with zymosan-induced peritonitis in rats by reducing ONOO�

formation, PARP activation, iNOS expression, production of pro-inflammatory cytokines TNF-a and

IL-1b, recruitment of neutrophils, and tissue injury.201

The Chinese medicine Wen-Pi-Tang, whose constituents were described in Section 6, scavenged

ONOO� in a dose-dependent manner, thus protecting renal tubular LLC-PK1 cells from apoptotic

cell death. It increased cell viability, decreased DNA fragmentation, attenuated cellular

morphological changes, and restored the cell-cycle, when it was arrested by SIN-1, all of which

indicates a potential role for this extract in the prevention and treatment of renal injury.202

Nicaraven (Fig. 22) exerted a weak dose-dependent inhibition of PARP, probably by interfering

with the catalytic active site of the enzyme, and protected against the suppression of mitochondrial

respiration in ONOO�-stimulated RAW macrophages.165

It has been demonstrated that PARP-1 facilitates DNA repair and reduces the resistance of cancer

cells to certain DNA-damaging agents. The compound AG14361 (Fig. 29) is considered to be the first

high-potency PARP-1 inhibitor (Ki < 5 nM), with both the specificity and in vivo activity to enhance

the effectiveness of human cancer therapy. While it did not affect cancer cell gene expression or

growth, it did increase the antiproliferative activity of temozolomide and topotecan.203 PARP-1,

together with DNA-dependent protein kinase (DNA-PK), play an important role in radio- and chemo-

resistance and are therapeutic targets for anticancer drug development. New findings have shown that

AG14361 actually restores sensitivity to temozolomide in mismatched repair-deficient cells.204

Specific inhibitors of the enzymes that repair DNA, such as NU7026, for DNA-PK, and AG14361, for

PARP-1, also act as potent radiosensitizers.205,206

Miknyoczki et al.207 demonstrated the chemopotentiating ability of the PARP-1 inhibitor CEP-

6800 (Fig. 30) when used in combination with temozolomide, irinotecan, and cisplatin against

carcinoma xenografts and cell lines. A series of potent non-toxic PARP-1 inhibitors, including

benzimidazole-4-carboxamides and tricyclic lactam indoles with structural modifications,208,209

were also found to be powerful chemopotentiators of temozolomide and topotecan in cell lines. Other

PARP inhibitors that proved effective in resistance-modifying agents in human tumor cell lines were

2-aryl-1H-benzimidazole-4-carboxamides,210 3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-ones,

and 3,4-dihydropyrrolo[4,3,2-de]isoquinolin-5-(1H)-ones.211 In addition, 8-hydroxy-2-methyl-

quinazolin-4-[3H]one (NU1025) and 2-methylbenzimidazole-4-carboxamide (NU1064) enhanced

Figure 29. Structure of AG14361.


the cytotoxicity of the DNA-methylating agent MTIC.212 It has been demonstrated that the

cytotoxicity induced by temozolomide and PARP inhibitors such as 3-AB or NU1025 can be

improved by a fractionated modality of drug treatment.213 6(5H)-Phenanthridinone modulates the

cytotoxicity of anticancer agents such as bleomycin, carmustin, and doxorubicin differently

depending on the cell type and the drug.214

Despite the extensive work on PARP, there are no drugs on the market as yet, although several are

currently in clinical testing. Inotek has developed an ultrapotent parenteral inhibitor of PARP, INO-

1001, with an IC50 value of 1 nM. INO-1001 has shown remarkable protection effects in experimental

acute ischemic stroke, septic shock, and acute lung injury. Treatment with INO-1001 improves the

cardiac and vascular dysfunction associated with advanced aging.215 It also improves the recovery of

myocardial and endothelial function after hypothermic cardiac arrest and reduces the pulmonary

injury associated with extracorporal circulation.216 In addition, INO-1001 attenuates various aspects

of the pathophysiological response in ovine models of sepsis,217 and of injuries related to burns and

smoke inhalation.218 It has also been found that INO-1001 inhibited myocardial ischemia-

reperfusion induced by PARP activation in circulating leukocytes.219 This drug is currently being

evaluated for a variety of critical care diseases associated with reperfusion injury and inflammation,

including ischemic stroke, acute respiratory distress syndrome, thoracoabdominal aortic aneurysm

repair surgery, and for the prevention of complications associated with cardiopulmonary bypass

surgery, among others.

Inotek has also developed an orally bioavailable ultrapotent PARP inhibitor, WW-46, which has

just entered in clinical trials for prevention of diabetic vascular dysfunction. Pre-clinical studies have

revealed that Inotek’s PARP inhibitor blocks the development of diabetes-induced retinopathy,

peripheral neuropathy, and endothelial vascular dysfunction.

B. Apoptosis and the Role of PARP

Apoptosis, a mode of programmed cell death that occurs under certain physiological and pathological

conditions, is characterized by cell shrinkage, membrane blebbing, and if a nucleus is present, nuclear

pyknosis, chromatin condensation, and degradation of DNA into oligonucleosomal fragments.

Apoptotic endothelial cell death has been observed in a variety of pathophysiological conditions such

as acute inflammation, atherosclerosis, transplant rejection, and allograft arteriopathy.220

The role of PARP in the development of apoptosis is controversial. PARP serves as a substrate for

diverse enzymes such as the caspases, which are implicated in apoptotic processes. Presumably, the

effect of PARP inhibitors depends on the apoptotic trigger, as well as the metabolic status and type of

the cells studied. It has been shown that exposure of human intestinal epithelial (T84) and RAW

murine macrophage cells to ONOO� caused cell death either via apoptosis or necrosis, depending on

the dose and the incubation time. Thus, while short-term incubation of cells with ONOO�

concentrations lower than 300 mM induced apoptosis, doses higher than 300 mM caused necrosis.

However, overnight exposure of cells to lower ONOO� concentrations (<75 mM) resulted in

apoptosis whereas higher concentrations (>75 mM) induced necrosis. In these cell types, then,

apoptosis occurs at lower doses of ONOO� and over a longer period of time than necrosis. Sandoval

et al.221 hypothesized that ascorbic acid may provide a detoxification pathway for ONOO� in these

Figure 30. Structure of CEP-6800.


protocols, noting that while physiological concentrations of ascorbic acid may be insufficient to

prompt a scavenging action, at doses higher than 1 mM this acid induces apoptosis, probably by

acting as a pro-oxidant in the presence of transition metals. Thus, administration of ascorbic acid at

approximately 5–10 times its physiological concentration (500 mM)—either directly to the media or

via pre-incubation for 2 hr and subsequent washing—attenuated ONOO�-induced apoptosis in T84

and RAW cells. These results indicate a protective role for this antioxidant and a probable benefit

from dietary supplementation with ascorbate.

Szabo et al.220 observed that the delayed DNA fragmentation in HUVEC after 24 hr in response

to low concentrations of ONOO� was not affected by inhibition of PARP with 3-AB, indicating that

inhibition of PARP failed to affect the course of the delayed apoptotic process.

Virag et al.222 reported that the degree of PARP activation is key in diverting the apoptotic cell

towards necrosis. Thus, exposure of thymocytes to low concentrations of ONOO� resulted in

apoptosis, as evidenced by DNA fragmentation and caspase activation, and did not affect cellular

ATP levels. In contrast, higher concentrations led to loss of membrane integrity, indicative of necrosis

and reduced cellular ATP levels. Considering the fact that apoptosis is dependent on ATP, higher

levels of oxidant stress may inhibit the process, not least of all by reducing the activity of the enzymes

involved. In fact, suppression of cellular ATP levels and cellular necrosis was observed when using

either the PARP inhibitor 3-AB or thymocytes from PARP-deficient animals, indicating that in the

absence of PARP, cells divert to apoptosis. The crucial role of caspase-3 as a regulator of apoptosis

has also been demonstrated. In one study, treatment of HL-60 cells with the specific tetrapeptide

caspase inhibitor DEVD-fmk completely blocked ONOO�-induced apoptotic DNA fragmentation.

The effect of PARP inhibitors such as 3-AB and 5-iodo-6-amino-1,2-benzopyrone was also

dependent on the dose of ONOO�. These drugs increased DNA fragmentation caused by low dose of

ONOO�, whereas the effect was the opposite at high dose of ONOO�.223

Different studies have demonstrated that exposure of cells to SIN-1 or directly to ONOO� caused

apoptotic cell death, mediated in part by activation of p38 MAPK. In fact, it was noted that inhibiting

this compound partially reduced the activation of caspase-3 and the entire apoptotic process. On the

other hand, ONOO� causes irreversible inhibition of respiration by oxidizing and nitrating

polyunsaturated fatty acid on mitochondrial membranes, mitochondrial DNA, enzyme complexes in

the respiratory chain, and thiol in protein.224 It has also been reported that these structural and

functional changes to the mitochondria lead to cytochrome c release, which activates caspase-9 and,

consequently, caspase-3.225 The initiation of ONOO�-induced apoptosis may actually occur in the

mitochondria, where a chain of events including mitochondrial depolarization, O�2 production, and

release of apoptotic mediators takes place.

Free 3-NT, released from ONOO�-nitrated proteins, is considered to promote DNA damage and/

or apoptosis. Apoptogenic factors derived from mitochondria, such as apoptosis-inducing factor

(AIF), along with cytochrome c triggered by ONOO� mediate both caspase dependent and caspase

independent apoptosis.2

Morphine inhibited nitration of tyrosine induced by ONOO� in a dose-dependent manner. It also

prevented the cell death and DNA fragmentation induced by ONOO� and SIN-1 in human

neuroblastoma SH-SY5Y cell cultures, but not those induced by the NO donor N-ethyl-2-(1-ethyl-2-

hydroxy-2-nitroso-hydrazino)-ethanamine (NOC12).226 Morphine protected cells from damage

caused by ONOO� through a direct scavenging action and not via opioid receptors since neither

naloxone nor other selective ligands for opioid receptor subtypes were found to alter the effect. In

subsequent studies morphine was shown to protect primary rat neuronal astrocytes against death

induced by NO and ONOO�.227 Morphine significantly protected these cells from apoptosis

mediated by SIN-1 and SNP in a dose-dependent manner, but did not protect others such as C6

glioma, RAW 264.7, or HL-60 cells. The protective effect of morphine on SIN-1-induced cell death,

but not on that induced by SNP, was antagonized by naloxone. Morphine also protected astrocytes

from glutathione depletion by BSO, an inhibitor of g-glutamylcysteine synthetase. The protective


effect of morphine on SIN-1-induced cell death was inhibited by pertussis toxin, which deactivates Gi

protein by blocking its coupling with receptors. On the other hand, pre-treatment of astrocytes with

PI3 kinase inhibitors abrogated the effects of morphine on SIN-1-induced cytotoxicity, indicating

that PI3 kinase partially mediated these effects. These findings thus suggest that morphine protects

primary rat astrocytes against oxidative stress via intracellular signaling cascades involving G protein

and PI3 kinase.

Reactive oxygen species play an important role in the cascade of events leading to neuronal

apoptosis, which is implicated in the pathogenesis of neurodegenerative processes such as

Alzheimer’s and Parkinson’s diseases, amyotrophic lateral sclerosis, and epilepsy. Wei et al.228

demonstrated that ONOO� is a relevant mediator of NO-induced neurotoxicity. They evaluated the

protective effects of the antioxidants L-ascorbic acid, 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-

trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1, Fig. 31),

SOD, and the NO scavenger hemoglobin on immature cerebellar granule cells after incubation with

the NO donors, S-nitrosoglutathione (GSNO) and SNP. EPC-K1 is a phosphate ester derivative of

vitamin C and vitamin E that has been reported to act as an OH*

scavenger and lipid peroxidation

inhibitor. The researchers found that hemoglobin prevented the decrease of mitochondrial

transmembrane potential and intracellular ATP content induced by exposure of cells to NO donors;

they thus surmised that the release of NO caused the mitochondrial dysfunction. EPC-K1 and SOD

prevented NO-induced mitochondrial dysfunction and protected cells from NO-induced neurotoxi-

city by scavenging O�2 /ONOO� and its breakdown products.

Pistafolia A (Fig. 32), a gallotannin isolated from the leaf extract of Pistacia weinmannifolia

(Anacardiaceae), effectively attenuated ONOO�-induced oxidative neuronal damage and apoptosis.

Pre-treatment of primary cultures of rat cerebellar granule cells with pistafolia A prevented lysis and

nuclear morphological alterations caused after exposure to the ONOO� donor SIN-1.229 This

gallotannin scavenges both OH*

and O�2 in a dose-dependent manner with IC50 values of 50.4 and

14.4 mM, respectively, versus IC50 values of 202.6 and 50.2 mM obtained for the tocopherol analog,

trolox.

It has been shown that ONOO� initiates lipid peroxidation and consequently induces the

formation of thiobarbituric acid-reactive substances (TBARS), malonaldehyde (MDA), and other

aldehydes that can react with aminoacids and DNA to establish cross-linkages between proteins and

nucleic acids. Treatment of rat thymocytes with trolox and three other phenolic antioxidants (3-tert-

butyl-4-hydroxyanisole, butylated hydroxytoluene, and 2,6-diisopropylphenol) after exposure to

ONOO� reduced the formation of TBARS and DNA-protein crosslinks and prevented apoptosis.

These results indicate that intracellular oxidation plays a central role in ONOO�-mediated apoptotic

cell death.230

Phycocyanin, one of the major constituents of the blue–green algae Spirulina platensis

(Cyanobacteria, Oscillatoriales) is a biliprotein that contains an open chain tetrapyrrole chromophore

known as phycocyanobilin, which is covalently attached to the apoprotein. Earlier studies established

that phycocyanin not only scavenges ROO*

, OH*

, and O�2 radicals, but also acts as a potent

antioxidant and inhibits lipid peroxidation mediated by ROS. Bhat and Madyastha60 demonstrated

that both phycocyanin and its chromophore are ONOO� scavengers, which phycocianin being the

more efficient compound. Still, they both inhibited bleaching of pyrogallol red in a concentration-

Figure 31. Structure of EPC-K1.


dependent manner (IC50 ¼ 22 and 31 mM, respectively, vs. 5 mM for glutathione). Phycocyanobilin

also inhibited ONOO�-mediated DNA damage (IC50 ¼ 3 mM) in a dose-dependent fashion.

Structurally, phycocyanobilin is very similar to bilirubin, which is considered to be an important

physiological antioxidant against ROS that inhibits oxidative modification of plasma proteins and

aromatic amino acid residues. As occurs when bilirubin is treated with ONOO�, resulting in a rapid

destruction of the pigment, phycocyanobilin likewise interacts with ONOO� and undergoes

oxidative degradation.

The protective effects of sanguiin H-6 (Fig. 33), a tannin component of Sanguisorbae Radix

crude extract, against ONOO�-induced oxidative stress in renal mitochondria and apoptosis have

also been evaluated. Sanguiin H-6 inhibited the apoptosis caused by caspase-3 activity in LPS-treated

rats subjected to ischemia-reperfusion. This compound also scavenged ONOO� and attenuated the

oxidative mitochondrial damage by increasing glutathione levels and decreasing the huge increase of

TBARS.224

Figure 32. Structure of pistafolia A.

Figure 33. Structure of sanguiin H-6.


Tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), a heavy metal chelator, showed protec-

tive effects against cell death induced by both ONOO� and SIN-1. These effects were abolished in

presence of equimolar Zn2þ. This compound also inhibited PARP activation in a dose-dependent

manner and reduced ONOO�-induced mitochondrial alterations without actually scavenging this

anion. TPEN inhibited ONOO�-induced necrosis by zinc chelation and, in part, by inhibition of

PARP.231

Purines such as adenosine, inosine, and hypoxanthine have been described as potential

endogenous PARP inhibitors. They dose-dependently inhibited ONOO�-induced PARP activation as

well as the activity of the purified enzyme and also inhibited mitochondrial respiration and NO�3

production in IFN-g/LPS-stimulated macrophages. Hypoxanthine, the most potent purine, prevented

ONOO�-induced necrosis in thymocytes and inhibited mitochondrial alterations; however, it also

induced caspase activation and DNA fragmentation.232

8 . M I S C E L L A N E O U S I N T E R A C T I O N S

A. Peroxynitrite Decomposition Catalysts

As noted above, unless stabilized as an alkaline salt, ONOO� quickly isomerizes to NO�3 , which at

the cellular level amounts to a detoxification mechanism. Since iron porphyrin complexes can

accelerate this reaction, they are often called ONOO� decomposition catalysts (PDCs). Their

mechanism of action corresponds to a true catalysis mechanism: ONOO� forms an adduct with the

Fe(III) at the porphyrin core (Fe(III)–ONOO), after which this adduct breaks into NO2 and

Fe(IV)¼O, which can in turn reorganize to a Fe(III)–ONO2 structure. It is from this last structure that

NO3 is liberated. In the presence of antioxidants, the recuperation of the initial ferriporphyrin is

favored. As can be logically supposed, natural hemoproteins such as MPOs can perform an analogous

catalytic process.233

By monitoring the characteristic ONOO� absorbance at 302 nm as a test to evaluate its

decomposition, the coordination compounds 5,10,15,20-tetrakis(N-methyl-4 0-pyridyl)-porphyri-

nato iron (III) (FeTMPyP, Fig. 34), protoporphyrin IX iron(III) chloride (FePPIX), and 5,10,15,20-

tetrakis(4-sulfonatophenyl)-porphyrinato manganese(III) (MnTSPP) were found to be the most

effective catalysts among a series of porphyrins with different transition metallic atoms. Their

respective rate constants were 7.9, 3.7, and 3.3� 105/M/sec. It should be noted that 5,10,15,20-

tetrakis(N-methyl-4 0-pyridyl)-porphyrinato manganese(III) (MnTMPyP) is very easily oxidized by

ONOO� (pseudofirst order rate ¼ 3.6� 106/M/sec) and therefore loses part of its potential catalytic

properties. These can, in turn, be increased in the presence of antioxidants like ascorbate or trolox.

The same four compounds were also the best activators of tyrosine nitration in a tripeptide (GYA).

This effect is directly related to the formation of NO2 and either Mn(IV)¼O or Fe(IV)¼O complexes,

which mediate the formation of tyrosyl radicals.

Extensive research on PDCs has demonstrated their effectiveness not only in destroying

ONOO�, but also in improving certain pathological conditions that have been experimentally

induced in animals. This is the case of 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-

porphyrinato iron (III) (FeTMPS, Fig. 34), which was active in the ischemia-reperfusion process

derived from occlusion of the mesenteric artery and the celiac trunk in rats. Administration of

FeTMPS (10 mg/kg, i.v.) 30 min before reperfusion diminished to less than one tenth the extent of

tyrosine nitration in the ileum, and almost abolished the up-regulation of P-selectin and ICAM-1,

all of which were measured with immunohistochemical methods. Plasmatic concentration of

MDA, IL-1b, and TNF-alfa were all reduced, thus indicating effective inhibition of both oxidative

stress and the inflammatory process. Moreover, survival time, which was 90 min in the non-treated

group, lasted longer than the experiment schedule (240 min) in the group of animals treated with

FeTMPS.234


In a model of intestinal damage induced by bacterial endotoxin, FeTMPS, FeTMPyP, and the

SOD-mimetic SC-55858 (Fig. 35) all reduced the main parameters indicative of toxicity in rat

duodenum and jejunum. These parameters include plasma extravasation, lipid peroxidation, and

death of epithelial cells. Roughly equivalent effects were measurable at SC-55858 doses that were 30

times lower than those of PDCs. It is also worth noting that, unlike SC-55858, PDCs did not affect

leukocyte infiltration, as measured in terms of MPO activity.235

The activity of FeCl tetrakis-2-(triethyleneglycol monomethylether) pyridyl porphyrin

(Fe2T(PEG3)PyP or FP-15, Fig. 36) on severe inflammatory diseases in mice was studied by

Mabley et al.236 They found that at a dose of 3 mg/kg, this compound reduced the lesional

manifestations of colitis induced by orally-administered dextran sodium sulfate; thus, not only was

rectal bleeding reduced, but histological scores of inflammation extent, severity, and crypt damage

were also lowered significantly. Still, the inhibition of biochemical parameters of neutrophil

infiltration (MPO) and lipid peroxidation (MDA) was not as great. FP-15 was also active on a model

of arthritis induced by collagen plus Freund’s adjuvant, remarkably reducing arthritic scores

(swelling and joint inflammation) and concentrations of IL-12 and MIP-1 in paw tissue. Indeed, the

reduction in MPO and MDA levels was higher than that observed in the colitis model.

Simultaneously, authors of the same team headed by Szabo237 described in detail both the

preparation and purification of FP-15, along with the multiple effects of this porphyrin on various

chemical, cellular, and physiopathological models related to the role of ONOO� in diabetes. They

placed particular emphasis on the cardiovascular consequences of the loss of endothelial function in

streptozotocin-induced experimental diabetes in mice.

One of the complications associated with heart and lung transplants is obliterative bronchiolitis,

an inflammatory condition that can seriously compromise respiratory function. By using a model

based in the implant of trachea and first-order bronchi from Brown–Norway rats (allograft) or Lewis

Figure 34. Structuresof FeTMPyP (A) and FeTMPS (B).

Figure 35. Structure of SC-55858.


rats (isograft) into Lewis animals, Naidu et al.238 demonstrated that daily treatment with FP-15

(i.p., 1 mg/kg) led to better conservation of the epithelia and reduced 3-NT levels, peribronchial

inflammation, and the loss of airway cross-sectional area.

In the cardiovascular system, FP-15 produced beneficial effects in a porcine model of myocardial

ischemia induced by ligation of the coronary artery. This agent, administered as an i.v. bolus (1 mg/

kg) and as an infusion of 1 mg/kg/hr, reduced the infarct area by 35%.239 Furthermore, the same agent

was significantly active against the deleterious effects of the antitumoral drug, doxorubicin, on the

heart. It reduced both 3-NT staining in cardiomyocytes and the matrix metalloproteinase levels in

whole-heart homogenates of mice. A general improvement in hemodynamic cardiac parameters

(ejection fraction, stroke volume, left ventricle systolic pressure, etc) was also observed.240

Peroxynitrite decomposition catalysts were also active in inhibiting the chemical interactions of

ONOO� with neuronal constituents, as was the case in the formation of 3-NT in mice brain striatum

upon treatment with methamphetamine. After administration of FeTMPyP or FeTMPS, the extent of

tyrosine nitration was strongly reduced, as was, albeit to a lesser extent, the simultaneous depletion of

dopamine. It should be remembered that methamphetamine toxicity is correlated to imbalanced

oxidative stress, a basic condition for ONOO� generation, and stimulation of N-methyl-D-aspartate

(NMDA) receptors, which are known to be implied in the stimulation of nNOS.155,156

Another catalytic process of ONOO� transformation, but one different from that observed for

porphyrin complexes, is the reaction with OH*

, as follows:

ONOO� þ OH� ! NO þ O2 þ OH�

At acidic pH, this reaction competes with ONOO� protonation, which in turn is a route for NO2

production. The N-oxypiperidine radical, tempol (Fig. 12), previously described as a radical

scavenger of some pharmacological significance, performs the oxygen generation from ONOO�

independent of pH, by giving one electron to OH*

(Fig. 37). As NO and NO2 react to produce N2O3

(see Section 2.D), tempol acts as an inductor of aromatic nitrosation, in detriment to aromatic

nitration.241

B. Antiproteinase Inactivation

Much of the tissue damage provoked by exacerbation of the immune—mostly unspecific—cellular

response comes from the release of proteinases such as elastase, collagenase, gelatinase, and

Figure 36. Structure of FP-15.


stromelysin. These enzymes do not act wholly unopposed, but rather are subjected to control by

plasmatic proteins, for example, a1-antiproteinase, or tissue proteins such as the tissue inhibitor of

metalloproteinase 1 (TIMP-1). Several years ago, researchers were able to demonstrate that certain

tyrosine residues of a1-antiproteinase are sensitive to nitration; in fact, it is through this mechanism

that this protein loses its activity as an elastase inhibitor,242 although attack to methionine residues

has also been implicated.243 The physiological mediator of the antiproteinase deactivation is

ONOO�, which, as explained above, is formed in the oxidative environment of tissues affected by

chronic inflammatory diseases.

In a study designed to establish the ability of common biological antioxidants to prevent elastase

inhibition, ascorbate and glutathione were found to be almost equally effective at various

concentrations ranging from 0 to 1 mM.87 It was later discovered that several non-steroidal anti-

inflammatory drugs (NSAIDs) used in the treatment of rheumatoid arthritis may act, not only as

inhibitors of prostanoid synthesis, but also as inhibitors of a1-antiproteinase inactivation by ONOO�.

The most efficient compounds, also in the range of 0–1 mM, were indomethacin, naproxen, and

paracetamol, three drugs with extremely varied potency as inhibitors of COX, and penicillamine, a

classical antirheumatic drug. Of these compounds, indomethacin and paracetamol inhibited nitration

of free tyrosine by ONOO�. No relationship was found between the effectiveness of this class of

drugs in protecting elastase activity and in inhibiting tyrosine nitration.244 In a similar study carried

out with antibiotics belonging to various groups, tetracycline and its analogs doxycycline and

minocycline, followed by rifampicin (all tested at 1 mM), were found to be the most effective

inhibitors of ONOO�-induced deactivation of a1-antiproteinase. As in the case of anti-inflammatory

drugs, no relationship existed between this activity and the inhibition of tyrosine nitration.

One natural agent which has proven to be a potent preserver of enzyme activity is the thiazine

AECK-dimer (see Section 4.B, Fig. 8). Provided that it interacts with ONOO� prior to its contact

with a1-antiproteinase, AECK-dimer exerts a dose-dependent effect at concentrations lower than

0.1 mM.96

Ambroxol is a widely used expectorant drug, which, apart from its mucolytic activity, has proven

its effectiveness against several oxidative and degradative phenomena that characterize respiratory

inflammation. For this reason, any potential interaction with the elastase/a1-antiproteinase system is

of special interest. In a comparative study with thiol antioxidants and NSAIDs, ambroxol showed a

moderate effect, with its best pharmacological value being attained in the field of ROS production by

leukocytes.245

Gallic acid, along with several widespread plant phenylpropanoids, including five hydro-

xycinnamic acids, two catechins, and the flavonol quercetin (Tables I–III), were described by

Ketsawatsakul et al.74 as a1-antiproteinase protectors. The IC50 values estimated for these agents

ranged homogeneously between 30 and 70 mM, except for ferulic acid, which had an IC50 of 112 mM,

and o-coumaric acids, which were inactive. All the compounds tested lost their activity when the

aggressiveness of ONOO� was refined by adding 25 mM of bicarbonate. Under these conditions, the

Figure 37. Schemeof tempol cycling in thepresence ofperoxynitrite.


most potent phenolics were caffeic acid (IC50 ¼ 55 mM) and its quinic conjugate chlorogenic acid

(IC50 ¼ 58 mM). The most dramatic decrease in activity corresponded to the readily oxidizable

molecules catechin and epicatechin.

C. Aconitase Inhibition

In a study with endogenous and synthetic thiols, Cheung et al.246 demonstrated that N-(2-

mercaptopropionyl)-glycine reversed the inhibition of myocardial aconitase (see Section 3.A)

induced by 100 mM of ONOO�. This effect, which was determined by measuring the enzymatic

activity present in rat ventricle homogenates, was concentration-dependent between 0.1 and 1 mM.

Unlike the results for cysteine, the presence of ferrous sulfate did not increase the protective effect of

N-(2-mercaptopropionyl)-glycine, possibly because the formation of dinitrosyl-iron complexes limit

the free thiol availability.

D. Nitrosation

The idea that the intake of nitrites could be a cause of cancer acquired general acceptance roughly

three decades ago. In fact, conclusions about the spontaneous nitrosation of amines at acidic pH and

the role of antioxidant vitamins in preventing the formation of mutagenic nitrosamines had been

drawn well before researchers started looking into the mechanisms of the biological role of RNS,

which has been summarized in previous sections.247 Today, however, the search for exogenous

substances that interact in this process seems to have lost its former appeal. Nevertheless, in a paper

published at the beginning of the period covered in this review, Kono et al.248 revealed that at high

concentrations (0.2–0.5 mM), chlorogenic acid and its phenolic counterpart, caffeic acid, inhibited

the N-nitrosation of 2,3-diaminonaphthalene induced by NaNO2 in acetate buffer, pH ¼ 4.4. This

process was monitored by measuring the formation of the highly fluorescent product, 2,3-

naphthotriazole. The fact that chlorogenic acid was nitrated in the presence of NO�2 /Hþ and also by

the mixture NO/NO2, but not by either gas alone, led the authors to conclude that nitration was

mediated by N2O3.

E. Effects on Lipid Peroxidation

Unsaturated fatty acids are fairly sensible to degradation by oxygen free radicals—including oxygen

itself—which ultimately leads to the breaking of olefinic bonds and the subsequent formation of

carbonyl compounds. This multi-step process, called lipid peroxidation because of the participation

of alkyl radical peroxides (R � OO*

) and alkyl hydroperoxides (R-OOH), is basically an oxidative

process with markedly typical features. Because lipid peroxidation is extremely important in

environmental toxicity, inflammation, and cellular senescence, it seems appropriate to include a

section devoted to RNS-induced lipid peroxidation in this manuscript.

Incubation of rat brain synaptosomes with SIN-1 allows for the evaluation of the formation of

phospholipid hydroperoxides by ONOO� generated in situ. Thus, within the range 0–2 mM, SIN-1

exhibited a quick and powerful increase in the concentration of phosphatidylethanolamine

hydroperoxide and, to a much lesser extent, of phosphatidylcholine hydroperoxide. For both

hydroperoxides, time-dependent clearance by phospholipase A2 was observed. As for regulation by

natural antioxidants, accurate HPLC measurements demonstrated that ONOO�-mediated oxidation

of a-tocopherol to a-tocopherol-quinone could be one way to control neuronal damage in this model

of lipid peroxidation.249

By using the combined reactions of N-methyl-2-phenylindole with the end products of lipid

peroxidation, namely malonaldehyde and 4-hydroxynonenal, Zou et al.86 studied the effect of sinapic

acid on the degradation of LDL by SIN-1. This phenolic acid, isolated from Brassica juncea

(Brassicaceae), produced a slight, concentration-dependent inhibition of the process in the range of

5–100 mM (76% at 100 mM). Another related compound, salicylic acid, inhibited peroxidation in a


similar fashion, but with lower potency (IC50 � 1 mM). In contrast, 2,3- and 2,5-dihydroxybenzoic

acids, which are metabolites resulting from the hydroxylation of salicylic acid by SIN-1, showed IC50

values below 0.1 mM.250 The three acids also prevented SIN-1-induced alteration in LDL

electrophoretic mobility. Results relating to salycilic acid and its metabolites are of interest because

this drug is used in long-term curative or preventive treatment of diseases derived from

atherosclerosis, including myocardial ischemia.

After having purified the LDL fraction from human blood serum, Kostyuk et al.251 studied the

influence of flavonoids on the lipid peroxidation induced by NO�2 and MPO combined with an H2O2-

generating system. As part of the study itself, the authors had previously demonstrated that after only

a few seconds, 50 mM NO�2 caused nearly a four-fold increase in conjugated dienes, which are one of

the final products of lipid peroxidation, in comparison with nitrite-free samples. Each of the seven

flavonoids tested (the flavone luteolin; the flavonols kaempferol, quercetin, rutin, and morin; and the

flavans epicatechin and taxifolin) inhibited lipid peroxidation in the micromolar range. The most

potent compounds were those possessing an ortho-dihydroxyl substitution in the B ring (see Tables II

and III), namely quercetin (IC50 ¼ 2.2 mM), rutin (IC50 ¼ 3.0 mM), and taxifolin (IC50 ¼ 3.8 mM).

Although other species may participate, it is generally admitted that the pro-oxidant effect of NO�2 /

MPO/H2O2 is associated with NO2 formation, (see Section 2.D). The authors therefore proposed that

flavonoids are oxidized by NO2 to different extents, a hypothesis that they were able to corroborate by

means of UV-vis spectral analysis.

F. Peroxisome Proliferator-Activated Receptor gg Ligands and Inflammation

In any discussion about the newest potential therapeutical alternatives in the field of inflammatory

and autoimmune diseases, a place should be reserved for the specific ligands of peroxisome

proliferator-activated receptor g (PPARg). This class of compounds is chemically heterogeneous, but

comprises, for example, certain thiazolidinediones, the glitazones (Fig. 38), that have been marketed

for the treatment of type II diabetes.252,253 The beneficial effects of PPARg ligands are linked in part

to the inhibition of iNOS expression, although it appears that this activity does not solely depend on

their intrinsic effect on PPARg. In fact, rosiglitazone, pioglitazone, and GW347845X all inhibited

iNOS expression in homozygous PPARg-defective murine macrophages.254 As for the influence of

these drugs on pathological models in vivo, rosiglitazone was effective in pulmonary injury induced

by bleomycin in mice, decreasing 3-NT, PARP, and iNOS staining in lung tissues.255 Moreover, both

rosiglitazone and pioglitazone decreased the formation of 3-NT in ankle and temporomandibular

joints of mice suffering adjuvant-induced arthritis. Complementary studies on cultured RAW 264

macrophages also showed inhibition of iNOS, COX-2, and ICAM-1 expression, as mediated through

modulation of NF-kB.256

G. Mitochondrial Calcium Efflux

Provided that mitochondria ordinarily produce a discrete excess of O�2 , stressful conditions that lead

to high NO production also produce a rise in the concentration of ONOO�, which poses a real danger

for the integrity and function of this organelle.257 In a study concerning the mechanisms involved in

Figure 38. Structures of rosiglitazone (A) andpioglitazone (B).


the effects of the widely used immunosuppressant cyclosporin A on membrane calcium turnover,

Packer and Murphy258 described the inhibition of the mitochondrial calcium loosening. They found

that at 0.5 mM, cyclosporin A markedly inhibited both calcium efflux and swelling induced by

250 mM ONOO� through a pore in the inner membrane.

H. Gap Junctional Communication

Transcytoplasmatic communication between adjunct cells occurs through the so-called gap junctions

(GJs), which comprise a sort of membrane protein channel constituted by the coupling of six units of

connexins. These gap junctions have been found to be very important for cell-cycle regulation and

proliferation. In fact, a number of studies have demonstrated that mediators of oxidative stress

execute post-translational modifications in connexins, causing inhibition of GJ communication.259

Furthermore, it has been suggested that the carcinogenic potential of several pesticides, organic

pollutants, strong oxidants, and some toxic heavy metals may come from the fact that these

substances disturb the integrity of GJs.

Some years ago, Schieke et al.260 demonstrated that ONOO� causes fast activation of P38-

MAPK, leading to the formation of protein-bound 3-NT in WB-F334 rat liver epithelial cells. These

effects were alleviated by treating the cultured cells with sodium selenite, which improved the

synthesis of selenoproteins, most notably GPX. Shortly thereafter, Sharov et al.261 working with

the same cells, discovered how selenite restores GJ damaged by ONOO� itself or by SIN-1, and also

the manner in which it favors GPX activity. Intercellular communication in both cases was evaluated

by fluorescence dying, either by scrape-loading or microinjection with Lucifer Yellow CH. Because

selenite is intrinsically cytotoxic at concentrations above 1 mM, it is worth noting that in this system it

was effective at 0.1 mM.

I. Neuronal Cytoxicity

The term lazaroid describes a class of lipophilic antioxidant and iron-chelating substances that have

been pharmacologically characterized by their protective activity against oxidative stress in the brain

and other organs. Although, some papers refer to these compounds as being synonymous with 21-

aminosteroid, this is only partially true. Thus, while many lazaroids have this particular structure

(e.g., tirilazad or PNU-74006F, a pyrimidine steroid, Fig. 39), other non-steroidal, structurally related

agents have also been synthesized. As for the effects of these substances, Fici et al.262 reported on

their role in a model of ONOO�-induced cytotoxicity in cultured rat cerebellar granular cells.

Depletion of glutathione with L-buthionine-(S,R)-sulfoximine led to the exhaustion of natural

Figure 39. Structure of tirilazad.


antioxidant defense, with the uptake of a-methyl-[3H]-aminoisobutyric acid serving as an indicator

of cellular viability. The pyrrolopyrimidines U-101033E and U-91736B (Fig. 40), which at 10 mM

produced total cell viability, were markedly more active than tirilazad and its 21-aminosteroid

congener, U-74500A. Addition of the thiol penicillamine resulted in additive synergy with the

lazaroids assayed. Additionally, the compound U-101033E greatly reduced the extent of protein

tyrosine nitration induced by ONOO� in red blood cell membranes, without affecting protein

oxidation as manifested by cross-linking and a decrease in spectrin staining.263

9 . F U T U R E P R O S P E C T S

This short section attempts to give a prospective conclusion and therefore lacks any ambition of being

comprehensive. The future is as long and far away as one wants to imagine; as such, its limits are

imprecise. For the purpose of a scientific review it can be limited at the whim of the authors, but their

predictions should always rest on the exclusive basis of what is currently known. As has surely

become obvious, the present paper inextricably combines chemical and physiological aspects of RNS

with their possible pharmacological control. For this reason, it now seems appropriate to layout, on

the one hand, the main subjects for future research on the role of RNS and, on the other, the potential

therapeutical interventions derived therefrom.

One of the major points of growing interest is whether the imbalance in the levels of ONOO�

with respect to those of NO is important for the development of coronary and peripheral vascular

disease. It is known that ONOO� disrupts the zinc-thiolate centers of eNOS, causing uncoupling of

the enzyme and increasing O�2 production, which in turn leads to an increase in additional ONOO�.

As this anion nitrates and inactivates prostacyclin synthase, an enzyme that, like eNOS, is pivotal for

maintaining adequate blood flow, both elements are intimately linked in the genesis of vascular

insufficiency typically associated with pathological conditions such as diabetes mellitus.264 Another

result of the imbalance of ONOO�/NO in cardiovascular function that merits further study is the

regulation of lipid peroxidation and atherogenesis, especially in relation to the activity of

lipoxygenases. Such a class of non-heme iron enzymes is present in different cellular types, and is of

interest as a possible connection between inflammation and atherosclerosis.265

We have already addressed at length the role of NO and its filial RNS in inflammation and

apoptosis. However, it is appropriate to dedicate here some words concerning the modulation of the

fate of macrophages, a subject on which Bosca et al.266 have published a sound and very recent

review. The authors point out that sustained production of NO inhibits apoptosis, whereas explosive

donation has the opposite effect. This explains in part why macrophages, a rich source of NO, can

resist proapoptotic signals from their environment and remain admirably alive. The subsequent

Figure 40. Structuresof U-101033EandU-91736B.


resolution of inflammation involves macrophage apoptosis, a process which may be regulated by

RNS and prostaglandins by mechanisms not yet well described.

As outlined in Section 3, the nitration of peptide tyrosine residues by RNS is an ongoing issue,

one that most certainly will provide results in the near future. Nevertheless, what we have yet to

ascertain is whether the more or less brilliant discoveries in the field of protein structural biochemistry

will parallel significant results in the field of cellular signaling and, perhaps more importantly, in that

of pharmacology. In this vein, it should be emphasized that the studies on the participation of heme

groups are of the utmost importance in describing the mechanisms involved in the nitration of

tyrosine and tryptophan, not only because of the ubiquity of heme-enzymes and other heme-proteins,

but also due to their implication in human diseases.267

According to the strictest, albeit inexact, meaning of the term pharmacology, the nature of drugs,

or better yet, the chemical nature of drugs, is nuclear for every applied approach. For this reason, it is

necessary to establish criteria to help to select which kind of compounds would have the highest

values. We consider that safety, specificity, potency, and availability are four rarely coinciding key

characters that define the potential therapeutical interest of a new drug. Additionally, the mere

novelty is often a reason for increased attention.

Given that naturally occurring phenols usually combine antioxidant and nitration-preventing

activities, they form a group endowed with a number of effects related to RNS turnover. Their

potential lies in the fact that they are present in many dietary vegetables and may therefore play an

essential role in protecting against cardiovascular and neoplastic diseases in humans. By way of

example, the body of research on [6]-gingerol (Fig. 15) has demonstrated its cancer chemopreventive

effect, which is probably linked to both ONOO� scavenging as well as the inhibition of COX-2

expression, the latter being an enzyme induced by tumor promoters through the p38 MAP kinase-

NF-kB pathway.268 The interest in other phenolics, for example, the flavonoids, as cancer

chemopreventive agents is enhanced by their properties as inhibitors of certain protein tyrosine

kinases implicated in cellular proliferation. 269 We also strongly feel that phenolic acids and their

derivatives merit further investigations in the field of vascular flow homeostasis to determine whether

their demonstrated activity in inhibiting chemical modification of the atherogenic particles (LDL) is

therapeutically relevant.

During the last decade many natural and synthetic drugs have been declared to be anti-

inflammatory due to their inhibition of NO synthesis. Although such compounds, per se, are not

covered in this review, some of them have shown additional RNS scavenger activities. Thus the

importance of these compounds has been discussed in Section 6, which highlighted MEG as a

remarkable example. Many attempts have been made in the last decade to determine the applicability

of MEG as well as that of some of its analogs on different experimental models of inflammatory and

vascular diseases. Nevertheless, accurate predictions as to further discoveries related to MEG and its

allies are difficult to make at present.

Although the influence of ONOO� on the fate of a cell is considerable, it is impossible to explain

in a simple fashion since, on the one hand, it induces apoptosis, but on the other, it causes necrosis by

activating PARP. As was shown in Section 7, much valuable research has been carried out with PARP

inhibitors. It seems evident that counteracting ONOO� destruction of DNA is one way to oppose

cellular and histological damage manifested in ischemia-reperfusion and in a number of inflammatory

diseases influenced by PARP activation. It is worth noting here that among the drugs being tested in

this field by the Inotek Pharmaceuticals Corporation, several (e.g., INO-1001) are extremely potent,

and are therefore promising as therapeutical agents for certain respiratory and cardiac diseases. Thus,

with the implication of this enzyme in many pathological processes, great advances in the field of

PARP inhibitors have been made. Furthermore, given that they enhance the cytotoxic effects of

topoisomerase inhibitors, these drugs may also be useful in the treatment of neoplastic diseases.

In our opinion, one of the most interesting points of this review, at least from a pharmacodynamic

perspective, is the existence of ONOO� decomposition catalysts, a class of heme derivatives—


among them the thoroughly analyzed FP-15—that accelerate the isomerization of ONOO� to the

pentavalent form NO�3 . Such a peculiar mode of action should exclude potential interactions with

earlier biological processes in the life of RNS. Still, possible toxicity associated with the deposition of

heme compounds on serum or tissue proteins must be carefully assessed. In any event, a broad

panorama of possible applications in the fields of inflammation and heart–lung pharmacology

appears to be open. Recent papers, like that of Lancel et al.270 continue in this vein by evaluating

interactions with leukocyte adhesion, I-kB degradation, or MPO activity, among others.

As a final comment, it must be pointed out that the control of either signaling or destructive

pathways derived from the release of nitrogen species has a profound pharmacological significance.

Although formerly considered only as air and water pollutants, nitrogen oxides and their derivatives

are currently also known as endogenous redox mediators in inflammation, vascular homeostasis and

apoptosis. For this reason, we feel that a systematic study of the molecular mechanisms involved in

the activity of the drugs concerned is required to establish both their safety profile and their

therapeutical projection.

A C K N O W L E D G M E N T S

This work has been supported by the Spanish Ministry of Science and Technology (Project SAF

2002-00723). Ana Olmos is a recipient of a predoctoral research fellowship from the Generalitat

Valenciana (CTBPRA/2002/56).

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AnaOlmos studied pharmacy and obtained her degree in 2001 at the University of Valencia. Nowadays she is a

Ph.D. student at the Department of Pharmacology at the same University and she is working on the nitration of

proteins and chemistry of peroxynitrite. She obtained the Advanced Studies Degree in 2003 and she has done

research stays at the Department of Biochemistry and Molecular Biology, Royal Free Hospital, University

College of London; and at the Department of Pharmacology and Pathophysiology, Faculty of Pharmaceutical

Sciences, University of Utrecht.

Rosa M. Giner studied pharmacy and obtained her Ph.D. in 1988 at the Department of Pharmacology,

University of Valencia (Spain) for the characterization of active natural products. She did a 1-year post-doctoral

stint in the Phytochemistry Research Laboratories at the University of Strathclyde (Glasgow, UK). Since 1993,

sheworks as aTitular professor at theDepartment of Pharmacology,University of Valencia. She joined for 1 year

theWilliamHarvey Research Institute, Division of Pharmacology of theMedical School,QueenMaryUniversity

of London (UK) doing research on the field of annexins. Her present research interest includes mechanism of

action of anti-inflammatory natural products.

Salvador Manez got his degree in pharmacy at the University of Granada (1977). Shortly after, he became

specialized in structural analysis of flavonoids and read his Ph.D. thesis at the University of Valencia (1981). In

this institution, hewas appointed Titular professorof theDepartment of Pharmacology in 1987. Since then he has

developedmany research projects on the anti-inflammatory activity of plant principles, particularly in the field of

protein kinase C modulators and eicosanoid synthesis inhibitors. His current research interest is focused on the

interactions of natural products with reactive nitrogen species.


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Drugs modulating the biological effects of peroxynitrite and related nitrogen species