686 Current Drug Metabolism, 2008, 9, 686-696

1389-2002/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.

Classical Inhibitors of NOX NAD(P)H Oxidases Are Not Specific

Elisabetta Aldieri, Chiara Riganti, Manuela Polimeni, Elena Gazzano, Cristina Lussiana, Ivana Campia and

Dario Ghigo*

Department of Genetics, Biology and Biochemistry, University of Torino, Italy

Abstract: NAD(P)H oxidases (NOXs) are a family of enzymes catalyzing the univalent reduction of oxygen to produce the superoxide anion radical, which in turn can be converted in other reactive oxygen species (ROS) and may participate to the formation of reactive ni-

trogen derivatives, such as peroxynitrite. By virtue of their activity, NOXs may represent a double-edged sword for the organism’s ho-meostasis. On one hand ROS participate in host defence by killing invading microbes and may regulate several important physiological

functions, such as cell signalling, regulation of cell growth and differentiation, oxygen sensing, angiogenesis, fertilization and control of vascular tone. On the other hand ROS may play an important role in pathological processes such as hypertension, atherosclerosis, diabe-

tes, cancer, ischemia/reperfusion injury, neurodegenerative diseases. Many roles suggested for NOXs in various tissues and physiopa-thological situations have been inferred by the in vitro and in vivo effects of several NOX inhibitors. In particular, most studies are based

on the use of two compounds, diphenyleneiodonium and apocynin. Aim of this review is to describe the main features of these two com-pounds, to show that they cannot be used as specific NOX inhibitors and to solicit researchers to find other tools for investigating the role

of NOXs.

Keywords: NAD(P)H oxidases, reactive oxygen species, diphenyleneiodonium, apocynin.

THE NAD(P)H OXIDASE (NOX) FAMILY

NAD(P)H oxidase (NOX) enzymes are a family of transmem-brane hemoproteins, which carry electrons across a cell membrane from a cytosolic electron donor (generally, NADPH or NADH) to an electron acceptor (oxygen) in the extracellular or lumenal space. The result of their activity is the univalent reduction of dioxygen to produce the superoxide anion radical (O2• ) [1].

O2• is a short-lived (t1/2: 1 ms) and cell-impermeant molecule that rapidly dismutates to hydrogen peroxide (H2O2) either sponta-neously or via the catalytic intervention of superoxide dismutase (SOD) [2, 3]. O2• may also react with the free radical nitric oxide (NO) to form peroxynitrite (ONOO ), a much more potent oxidiz-ing agent, whereas H2O2 may react with chloride in the myeloper-oxidase (MPO)-catalyzed formation of hypochlorous acid (HClO) or may be splitted into hydroxyl anion (OH ) and hydroxyl radical (OH•) by effect of two non-enzymatic processes, the iron-catalyzed Fenton reaction and the superoxide-dependent Haber-Weiss reac-tion [2, 3]. O2• , H2O2, OH• and HClO are the most important reac-tive oxygen species (ROS) primarily generated in the cells [4].

ROS production may be beneficial for the organism, playing a key role in the innate immunity [2]. When professional phagocytes such as neutrophils and macrophages are exposed to microbes, they consume large amounts of oxygen: this “respiratory burst” has been found to be mainly dependent on the activation of the phagocyte NOX, leading to enhanced production of ROS, significant changes of phagosome’s pH and ionic environment and subsequent micro-bial killing [5]. On the other hand, an exceeding NOX-mediated production of ROS could trigger a state of chronic inflammation and favour the onset of degenerative and neoplastic diseases. Many well-known and widespread pathologies such as diabetes, tumors, atherosclerosis, hypertension, ischemia-reperfusion injury, inflam-matory and neurodegenerative diseases, as well as the aging proc-esses, are governed by ROS generation [6]. This makes NOX, one of the main producers of ROS, a double-edged sword whose physiopathological role may be greatly different, depending on the extent and duration of its activation.

More recently, other important functions of ROS have emerged, rendering the role of NOX much more complex. In fact, ROS are

*Address correspondence to this author at the Dipartimento di Genetica,

Biologia e Biochimica (Sezione di Biochimica), Via Santena, 5/bis, 10126

Torino, Italy; E-mail: dario.ghigo@unito.it

involved not only in cellular damage and killing of pathogens, but also as signalling molecules in a large number of reversible regula-tory processes in virtually all cells and tissues [2], including gene expression, regulation of cell death and growth, oxygen sensing, synthesis of biomolecules, regulation of redox potential, regulation of membrane potential, reduction of metal ions, regulation of metal-loproteinases, angiogenesis and modulation of the NO signalling [1]. It has been postulated, for instance, that the O2• generated by blood vessels functions as a blood pressure regulator by consuming NO, a well-known hypotensive agent (O2• and NO react at a diffu-sion-limited rate): NOX activity has been suggested to serve as a component of oxygen sensors in various tissues [2]. Thus, a dis-regulation of NOX activity has been proposed to be responsible for a wide array of diseases [5, 7]. Besides the original phagocytic NOX (now named NOX2), other six NOX isoforms, expressed in most non-phagocytic cells, have been found (identified as NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2) [2]. Furthermore, several regulatory proteins should be added to the NOX system, such as the organizer subunits p47

phox and NOXO1, the activator

subunits p67phox

and NOXA1, etc. [for an exhaustive review, see 2].

The mechanisms of activation of NOX2 have been intensively studied, while as to the other NOX/DUOX isoforms they are still poorly clarified. Briefly, NOX2, also known as gp91

phox, constitu-tively associates in the membranes with the p22

phox protein. The

activation of NOX2/p22phox

occurs through a complex series of protein/protein interactions and may be elicited by several different kinases, such as protein kinase C, MAP kinases and tyrosine kinases. At least four cytosolic factors have to translocate to the NOX2/p22

phox complex: the small GTPase Rac, the NOX organizer

p47phox

, the NOX activator p67phox

and the modulatory p40phox

[2, 8, 9] (Fig. (1)).

NOX INHIBITORS

The use of specific inhibitors of NOXs is deemed to be an im-portant tool to check whether these enzymes are involved in any physiopathological event investigated either in vivo or in vitro.Moreover, the modulation of NOX/DUOX activity has been sug-gested as a new intriguing perspective in the treatment of several pathologies (in particular, cardiovascular diseases). On the other hand, a really specific inhibitor of NOXs is still missing, unfortu-nately. The acknowledgement of this statement is important, since most studies concerning the NOXs’ role in physiology and pathol-

Classical Inhibitors of NOX NAD(P)H Oxidases Are Not Specific Current Drug Metabolism, 2008, Vol. 9, No. 8 687

ogy rely on the use of inhibitors only, which too many authors still claim to be specific.

This review is aimed at providing an updated revision of the scientific literature concerning the non specific effects of the most used NOX inhibitors, i.e. diphenyleneiodonium and apocynin. We will not take into consideration several other compounds sometimes used to inhibit NOXs, such as 4-(2-aminoethyl)benzenesulfonyl-fluoride, neopterin, protein kinase C inhibitors, angiotensin convert-ing enzyme inhibitors, angiotensin receptor blockers, MAP kinase inhibitors, eicosanoids, phosphodiesterase inhibitors, corticosteroids [for a review see: 2, 10]. These compounds cannot be considered abinitio a specific tool to inhibit NOX: they are indirect inhibitors, interfering with signalling pathways that may affect the operation of other target molecules besides NOX.

DIPHENYLENEIODONIUM (DPI)

Diphenyleneiodonium (DPI) (Fig. (2)) and the structurally re-lated compound diphenyliodonium (DIP) are uncompetitive inhibi-tors of flavoenzymes. Firstly identified as an hypoglycaemic agent able to block gluconeogenesis and respiration in rat liver [11], DPI was subsequently shown to inhibit the activity of the NADH dehy-drogenase (mitochondrial complex I) in rat liver mitochondria (IC50

= 18 M, 2 min) [12] and in bovine heart (IC50 = 23-30 nmol/mg of isolated complex I, 1 h) [13]: the consequent inhibition of ATP synthesis was thought to be responsible for the ability of the drug to inhibit hepatic gluconeogenesis, causing in vivo hypoglycaemia. About a decade later, Cross and Jones [14] observed that DPI could inhibit the NADPH-dependent production of O2• by the solubilized oxidase of pig neutrophils: this "respiratory burst oxidase" was already known to be a flavoenzyme, and Cross and Jones found that

it formed with DPI a covalent adduct stable to electrophoresis. At concentrations above 10 M, inhibition was complete within few seconds [14]. Both DPI and DIP inhibited also the phorbol 12-myristate 13-acetate-elicited generation of O2• in rat peritoneal macrophages after a few min preincubation [15]. In spite of a very similar structure, DIP showed a weak inhibitory activity (IC50 = 80

M) on superoxide production by neutrophils stimulated with phor-bol myristate acetate, when compared to DPI (IC50 = 0.9 M) [15, 16]. Whereas several authors were using DPI to establish an animal model (for instance, in rat 1.5 mg/kg/day for 4-5 weeks) of mito-chondrial myopathy (subsequent to the impairment of NADH de-hydrogenase activity) [17, 18], other researchers employed the same compound to gain the first insights into the mechanism of electron transport in the human NAD(P)H oxidase [19] and to pro-vide the first evidence that NOX might play a role as an O2 sensor in the rat carotid body [20].

I+

Fig. (2). Structure of diphenyleneiodonium (DPI).

DPI INHIBITS THE SYNTHESIS OF BOTH OXYGEN- AND NITROGEN-DERIVED REACTING SPECIES

At the end of the 1980s, DPI and DIP were assessed as inhibi-tors of both mitochondrial respiration and ROS synthesis. In the subsequent decade, the number of their potential targets increased exponentially. In 1991, Stuehr et al. found that DPI and its ana-logues inhibit two isoforms of NO synthases (NOS), a family of

Fig. (1). Schematic representation of the NAD(P)H oxidase activation in a professional phagocyte (neutrophil, macrophage). In the resting cell, three compo-

nents — p40phox, p47phox and p67phox — exist in the cytosol, whereas the other two components — p22phox and gp91phox — are located in the membranes of in-

tracellular vesicles. Upon activation in response to appropriate stimuli, the cytosolic proteins fuse rapidly with the plasma membrane or phagosome membrane.

During the activation process, also a Rac GTP-binding protein translocates to the membrane, and several components of the NAD(P)H oxidase complex, such

as p47phox, are phosphorylated. Activation requires also the participation of at least two other proteins, Rho-GDI (Guanine nucleotide Dissociation Inhibitor),

and Rap1A, not shown in the figure. After the assembly, the NAD(P)H oxidase complex is able to generate a respiratory burst, discharging superoxide anions

to the exterior (or to the lumen of a phagosome). In the novel NOX terminology, gp91phox is called NOX2.

688 Current Drug Metabolism, 2008, Vol. 9, No. 8 Aldieri et al.

flavoenzymes generating the free radical NO: iNOS (inducible, macrophage) and eNOS (endothelial, constitutive) [21]. NO is an important mediator in a wide range of physiological and pathologi-cal processes: for instance, it is a vasodilator, a neurotransmitter and a modulator of cell apoptosis, proliferation and differentiation [22, 23]. The NO synthesis in either semi-purified enzyme prepara-tions or activated macrophage cytosol was irreversibly inhibited after a 30-min incubation with 10 M DPI at 37°C; the IC50 of DPI was 50 nM after a 3-h incubation with semi-purified macrophage iNOS, whereas DIP was 3 times less potent [21]. DPI inhibited also eNOS, as assessed from its ability to prevent the acetylcholine-induced relaxation of norepinephrine-constricted rabbit aortic rings (IC50 = 0.3 M, 30-min incubation) [21].

NO plays a bidirectional cross-talk with the NOX-derived O2•in many biological systems [7], for instance in the cell sur-vival/apoptosis balance [24] and in the modulation of vascular tone [25]. NO and O2• may scavenge each other, but, on the other hand, when reacting under equimolar amounts, they may generate the potent oxidizing molecule peroxynitrite (ONOO ) [22]. Thus, de-pending on the relative concentrations, NO and O2• may either induce a reciprocal neutralization or synergize to generate oxidative stress. A large body of data suggests that excessive levels of ROS may elicit vasoconstriction and hypertension: the prevailing hy-pothesis is that vascular ROS reduce the bioavailability of NO, thus impairing its relaxing effect on vascular tone [7]. On the other hand, in porcine pulmonary arteries the NOX expression is blocked by NO produced by eNOS and iNOS [26], and in cultured rat aortic smooth muscle cells NO has been proposed to inhibit NOX via a cGMP-mediated mechanism [27]: this suggests that NO may act as a protective factor, preventing the increase of NOX expression and activity in response to inflammatory stimuli. Finally, ROS are known to activate the redox-sensitive transcription factors NF-kB and AP-1, which may elicit increased expression of the iNOS gene in mouse and man [28]. Indeed, DPI was observed to inhibit the IL-1-induced NO synthesis in bovine articular chondrocytes by two distinct mechanisms: by inhibiting the NOS activity (IC50 = 0.03

M, 18-h incubation) and by preventing the iNOS expression through the blockade of NF-kB activation (almost complete after a 6-h incubation with 10 M DPI) [29]. The ability of DPI to inhibit NF-kB activation has been correlated to the blockade of ROS syn-thesis [29]. Thus, depending on the cell type and the metabolic conditions, ROS may induce NOS via NF-kB or AP-1 activation, whereas NO may inhibit NOX via a not yet well defined mecha-nism. Finally, in some cells, such as human intestinal Caco-2 cells, NO can be metabolized via a microsomal dioxygen- and NADPH-dependent DPI-sensitive cytochrome P450 oxidoreductase, that yields nitrate: by inhibiting this enzyme (see below), DPI could increase the bioavailability of NO [30].

In such a complex cross-talk, the use of DPI may produce op-posite effects on the ROS/NO balance, depending on the experi-mental system investigated, and cannot give meaningful results in order to clarify the role of NOX-derived ROS and NOS-derived NO in cell signalling, both in vivo and in vitro. Yet, in many experimen-tal works DPI continues to be proposed and used as a potent inhibi-tor of either NOS or NOX only, despite its ambivalent modulatory role.

DPI INHIBITS MANY OTHER FLAVOPROTEINS, THUS CAUSING COMPLEX CHANGES IN THE CELL FUNC-

TION

In 1992, Doussière and Vignais [31] reported that DPI inhibits also xanthine oxidase (IC50 = 0.3-0.5 mol/40 g enzyme, 15 min), a non-haem iron flavoprotein obtained from bovine neutrophils, and one year later Tew [32] showed that DIP inhibits cytochrome P450 reductase (Ki = 2.8 mM) from bovine liver. O'Donnell and coll. [33, 34] and Tew [32] provided evidence, using electron paramagnetic resonance techniques, that phenyl radicals are formed during reac-tion of iodonium compounds with reduced free flavin and protein-

bound (cytochrome P450 reductase or xanthine oxidase) flavin. Kinetic analysis indicated these compounds to be uncompetitive inhibitors of reduced flavoproteins functioning in one-electron transfer [32]. DPI was reported to inhibit the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) on the substrate side of the Fe-S clusters by reacting irreversibly with FMN [35]. In 1997, pro-toporphyrinogen IX oxidase, which catalyzes the oxidation of pro-toporphyrinogen IX to protoporphyrin IX in the penultimate step of haem biosynthesis, was also observed to be inhibited by DPI (Ki = 67.5 nM) with an essentially irreversible slow-binding kinetics, probably associated with formation of a covalent adduct to reduced FAD [36]. In lung epithelial cells DPI (10 M) has been observed to inhibit the FAD-containing enzyme thioredoxin reductase [37]. Subsequently, also haem groups, such as the haem b of NOX, have been found to react with DPI and DIP [38]. Taken together, these reports suggest that the electron transport through the flavin moie-ties of these enzymes causes reduction of DPI to its radical form, followed by irreversible phenylation of either the flavin or the adja-cent amino acids and haem groups.

Such ability to inhibit a wide range of flavoproteins makes it very difficult to interpret the effects of DPI on ROS generation in mammalian cells. Indeed, various DPI-sensitive enzymes may be responsible for the production of O2• and H2O2. It has been found that the three NOS isoforms iNOS [39], eNOS [40] and neuronal NOS (nNOS) [41] may generate O2• : in eNOS and nNOS this oxi-dase activity is dependent on the level of the cofactor tetrahydro-biopterin [40-41]. The pre-incubation of purified iNOS with 20 MDPI totally blocked the O2• generation [39]. Also xanthine oxidase and cytochrome P450 may generate ROS: via this mechanism, for instance, they cooperate with NOX and NOS to the myocardial ischemia/reperfusion injury [42]. In human unstimulated mono-cytes/macrophages DPI inhibited the production of O2• and H2O2

by mitochondrial respiration at the concentrations (0.5-2.5 M, 30 min) which inhibited also NOX in the same cells stimulated with phorbol ester: DPI was as potent as rotenone in reducing the pro-duction of ROS by mitochondria, probably through the inhibition of NADH-ubiquinone oxidoreductase [43], although a recent paper has reported that flavin is not likely to be the target of DPI in com-plex I (some evidence suggests that DPI can act at the Q-binding site of the complex) [44]. This is a relevant information, since mito-chondria are known to be a major source of cellular ROS. Recently it has been observed that in mitochondria isolated from rat skeletal muscle 5 M DPI strongly inhibits in a few seconds the O2• pro-duction by complex I during reverse electron transport from succi-nate, without affecting O2• generation during forward electron transport from NAD-linked substrates [44]. Taking all these data into account, it seems quite hazardous to affirm that the inhibitory effect of DPI on ROS generation in mammalian cells is an evidence of the involvement of NOX in the oxidative stress. Yet, this af-firmation keeps on appearing in the scientific literature.

Interestingly, 30 M DPI almost completely inhibits diapho-rases, a family of NAD(P)-dependent oxidoreductases not yet well characterized: these enzymes are responsible for the metabolism of some NO-donor vasodilators, such as glyceryl trinitrate, favouring the release of NO from these drugs, and they are also able to reduce 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a well known NO-scavenger, thus eliminating its NO-scavenging effect [45]. This observation provides a further caveat to the inter-pretation of results obtained incubating cells with DPI together with either NO donors or PTIO.

DPI MAY EXERT EFFECTS APPARENTLY NOT DEPEN-DENT ON FLAVOPROTEINS INHIBITION

Since NO is an important vasodilator, DPI could modulate car-diovascular functions either by inhibiting the NOX-dependent syn-thesis of ROS (thus decreasing the scavenging of NO) or by inhibit-ing the NOS activity. Actually, DPI may elicit in vivo cardiovascu-lar effects, but they are not always relatable to its NOX/NOS-

Classical Inhibitors of NOX NAD(P)H Oxidases Are Not Specific Current Drug Metabolism, 2008, Vol. 9, No. 8 689

inhibiting activity. For instance, bolus injections of DPI (0.07 mg/kg) caused a pressor response in conscious rats by activating the sympathetic nervous system, but such an effect was not related to the potential inhibitory action of DPI on neuronal NO synthesis [46]: the pressor and tachycardic responses to DPI were due instead to the release of norepinephrine and epinephrine from nerve termi-nals and adrenal medullae [47], by an unknown mechanism. Fur-thermore, in isolated rat aortic rings, 5 M DPI, like the NOS in-hibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mM), in-hibited the endothelium-dependent vasodilation elicited by acetyl-choline. Both L-NAME and DPI initiated a small constriction of rings with endothelium when added at resting tension. However, when applied to preconstricted aortic rings, these compounds ex-erted distinct effects. While L-NAME produced a further sustained contraction of partially preconstricted rings, the contraction in re-sponse to DPI was transient and was followed by a prolonged re-laxation. The contraction initiated by DPI was likely due to removal of NO, since it was abolished by L-NAME pretreatment. In con-trast, the endothelium-independent relaxation caused by DPI was unrelated to NO, since it was not affected by L-NAME pretreat-ment. This relaxant effect was attributed by the authors, at least partly, to the activation of soluble guanylate cyclase, since its time course was delayed by the pretreatment with the guanylate cyclase inhibitor methylene blue [48]. The authors concluded that a compo-nent of the relaxant effect of DPI is the NO-independent activation of guanylate cyclase in vascular smooth muscle. Methylene blue is not a specific inhibitor of guanylate cyclase, it can also generate O2• [49] and inhibit NOS [50]: however, O2• generation and NOS inhibition by methylene blue were not involved in the DPI-induced relaxation of preconstricted rings [48]. Furthermore, these results were consistent with previous findings of Pettibone et al. [51], showing that the analogue DIP (10-300 M) stimulated guanylate cyclase activity in rat lung, decreased blood pressure and total pe-ripheral resistance in anaesthetized dogs and relaxed isolated rabbit aorta with or without endothelium.

DPI (10 M, 1 h) has been observed to block the hypoxic exci-tation of the rat carotid body and its blockade of hypoxic vasocon-striction in the pulmonary vasculature, leading to the suggestion that NOX might act as an oxygen tension sensor in rat type I carotid body cells [20, 52] and rat pulmonary smooth muscle cells [53]. But in 1994 DPI (3-10 M) was demonstrated to act as a non-selective blocker of ionic (potassium and calcium) channels in pulmonary artery smooth muscle cells: such mechanism of action did not in-volve inhibition of ROS formation. The ability of 3 M DPI to block calcium currents was considered sufficient to explain its inhi-bition of hypoxic excitation of the carotid body and the hypoxic pulmonary vasoconstriction [52, 53]. Furthermore subsequent works showed that O2 sensing is preserved in pulmonary arteries and carotid body of knockout mice lacking the gp91

phox subunit of

NOX [54, 55], suggesting that NOX2 is not a primary O2 sensor in arterial chemoreception in mice.

DPI may interact with other channels. The binding of the ago-nist MK-801 to the rat brain N-methyl-D-aspartate (NMDA) recep-tor was significantly inhibited by the addition of 100 M DPI (IC50

= 45.2 M) or DIP (IC50 = 5.8 M). Both compounds were sug-gested to modulate synaptic responses through inhibition of open-ing processes of the calcium channel domain of NMDA, in a man-ner independent from the synthesis of NO and binding to flavopro-teins [56]. DIP has been proposed as a new antagonist to the NMDA receptors: it would protect neurons (EC50 = 3 M) against glutamate toxicity due to a direct blocking of the Ca

2+ influx [57].

This conclusion is supported by the stereochemical similarity be-tween DIP and the NMDA receptor antagonist dizocilpine maleate [57]. In T24 bladder carcinoma cells 10 M DPI stimulated a rapid efflux of intracellular reduced glutathione (GSH). The loss of GSH (about 50% after a 2-h incubation and 70% after 4 h) was blocked with bromosulfophthalein, an inhibitor of the canalicular GSH

transporters, suggesting that DPI induces such an effect by modu-lating a specific transport channel. This phenomenon was related to the proapoptotic effect exerted by DPI on T24 cells, and its mecha-nism is still matter of investigation [58].

The plasma membrane oxidoreductase (PMOR) is an ubiqui-tous redox system that transports electrons from intracellular NADH to physiological extracellular electron acceptors thought to include oxygen, disulfides and ascorbate free radical. The PMOR modulates the cellular redox status and redox-sensitive cellular processes such as cell growth, intracellular signalling and apoptosis [59], and it maintains plasma membrane antioxidant systems [60]. PMOR activity in intact cells is readily quantifiable using artificial membrane impermeant electron acceptors such as ferricyanide (FeCN) and 2,6-dichloroindophenol (DCIP). The pretreatment of chick forebrain neurons with 2.5 M DPI for 10 min inhibited FeCN reduction but had no effect on DCIP reduction, suggesting that these substrates are reduced by distinct PMOR redox pathways [61].

The O-quinone cofactor pyrroloquinoline quinone (PQQ), also called methoxatin, is synthesized by numerous bacteria and as-sumed by animals with foods [62]. Although the function of PQQ in animals remains unclear, its ability to carry out continuous redox cycling suggests a role for PQQ as a cofactor or antioxidant. Mice fed with diets devoid of PQQ but otherwise nutritionally adequate have impaired neonatal growth and abnormal features [62]. PQQ has been isolated from guinea pig neutrophils, wherein it could play a possible role, direct or indirect, in the respiratory burst [63]. DPI and DIP have been observed to sequester synthetic PQQ and inhibit its redox-cycling activity in an in vitro model system (IC50 = 1.5

M and 10 M, respectively), in guinea pig neutrophils and in HL-60 human monocytes [63-65]. This has been suggested as a further mechanism by which DPI and DIP inhibit the respiratory burst in neutrophils.

The observation that DPI, in the range of 1-100 M (IC50 = 12-15 M), inhibits the elicitor-induced production of ROS by several types of plant cells has been used as evidence that an enzyme ho-mologous to the leukocyte NOX is present in the plasma membrane of plant cells and might be responsible for the oxidative burst evoked by pathogens or chemical elicitors [66]. Instead, Frahry and Schopfer have shown that DPI inhibits the NADH-dependent H2O2

production by horseradish peroxidase in the same concentration range (5-50 M) as previously used for the inhibition of putative NOX activity in plants [66]. Thus, the conclusion of the authors was that this inhibitor cannot be used to discriminate between a mammalian-type NOX and peroxidases in mediating the pathogen-induced oxidative burst in plants.

The DPI-mediated inhibition of ROS and NO production does not account for all in vivo and in vitro effects of the drug. For in-stance, 0.3 M (15 min) DPI inhibited the pharmacological actions of glyceryl trinitrate and D-isoidide dinitrate (IIDN) in isolated rat aorta via inhibition of the bioactivation of these prodrugs: the abil-ity of DPI to modulate the tolerance to the vasodilatory effect of these organic nitrates was not related to the inhibition of O2• gen-eration [67]. DPI (12 M, 60 min) caused rapid, pronounced, and reversible disassembly of the microtubule cytoskeleton in Rat1 fibroblasts. The failure of DIP and L-NAME to cause disassembly of the microtubule cytoskeleton indicated that the activity of DPI on the microtubules was not due to effects on ROS or NO levels. This was further substantiated by the failure of overexpression of the ROS scavenging enzyme catalase to cause microtubule disassem-bly. Similar to other microtubule drugs, DPI (6 M, 120 min) showed to block mitotic spindle assembly and mitotic cell division [68].

DPI MAY INDUCE AN OXIDATIVE STRESS

DPI is known to inhibit the oxidative burst in activated neutro-phils and macrophages [14, 15] and to decrease the production of

690 Current Drug Metabolism, 2008, Vol. 9, No. 8 Aldieri et al.

ROS by mitochondria [43, 44], but in particular conditions it may also increase the generation of ROS. This different effect could depend either on the DPI concentration and incubation time or on the cell type investigated. For instance, DPI (100 M, 10-60 min) in whole human promyelocytic leukemia HL-60 cells and isolated rat-heart submitochondrial particles induced increased generation of O2• and subsequent apoptosis [69]: the precise mitochondrial site of action of DPI in these experiments has not yet been identified. In rat pulmonary artery endothelial cells, DPI elicited increased ex-pression of the stress protein haem oxygenase-1, a frequent event in cells exposed to oxidative stress [70]. Furthermore, in porcine aortic endothelial cells DPI (10 g/ml, 1 h) potentiated the generation of ROS, both basally and after stimulation with vascular endothelial growth factor [71]. A 24-h incubation with 10 M DPI caused in-creased generation of ROS in human retinal pigment epithelial cells [72].

Riganti et al have observed that DPI (1-100 M; IC50 = 1 M) inhibits the pentose phosphate pathway (PPP) in N11 glial cells after a 3-h incubation [73]. PPP is a metabolic route operating in all tissues: its first, oxidative phase converts glucose 6-phosphate into ribulose 5-phosphate and CO2, leading to the synthesis of NADPH, a redox cofactor for many antioxidant enzymes. The metabolic flux through PPP is a sensitive index of the cell exposure to oxidant molecules, since glucose 6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pathway, is activated by any oxidative stress, via a decrease of the NADPH/NADP

+ ratio. The

inhibition of PPP by 0.1 mM DPI or DIP, respectively, had been already observed in hepatic endothelial cells [74] and Kupffer cells [75] from rats treated with endotoxin in vivo, and a similar effect was induced by 1 M DPI in rat peritoneal macrophages stimulated with phorbol myristate acetate [76]: this phenomenon was previ-ously thought to be the consequence of the inhibition of NOX and the subsequent reduced consumption of NADPH. This mechanism was not likely in N11 cells, since the PPP activity in cells stimu-lated with the oxidizing agents menadione and H2O2 was signifi-cantly inhibited by DPI as well, thus ruling out that DPI inhibits the PPP activity by blocking NOX [73]. Instead, DPI (1-100 M) was shown to inhibit directly the activity of the regulatory enzyme G6PD. In parallel, DPI induced in N11 cells a concentration-dependent increase of ROS generation and lipoperoxidation, an increased leakage of lactate dehydrogenase in the extracellular me-dium, a decrease of the GSH/glutathione disulfide (GSSG) ratio, and an increased efflux of glutathione out of the cells [73]. This suggests that DPI causes oxidative stress and is cytotoxic: these effects were prevented when cells were loaded with glutathione. Similar results were observed using DIP instead of DPI, and also in other cell types, such as murine alveolar macrophages MH-S, hu-man monocyte-like U937 cells and human erythrocytes [73]. The oxidative cytotoxic damage caused by DPI was subsequent to the G6PD and PPP inhibition. Furthermore, also NAD-dependent en-zymes, such as lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase, were inhibited by the incubation with

DPI: the subsequent blockade of glycolysis could account, at least partly, for the inhibition of glucose flux through the tricarboxylic acid (TCA) cycle observed in N11 cells [73]. Taken as a whole, these data suggest that DPI inhibits the cell metabolic activity not only by blocking flavoenzymes such as NADH dehydrogenase and NOX, but also by impairing the activity of non-flavinic NAD(P)-dependent enzymes. The concentration of DPI (10 M) necessary to achieve the half-maximal inhibition of these enzymes was within the concentration range usually utilized to inhibit NOX. A conse-quence of this effect is the inhibition of one of the main antioxidant pathways of the cells, i.e. PPP, causing the onset of an oxidative stress. It is likely that, when NOX is not maximally activated, the prevailing effect of DPI is to stimulate, rather than to inhibit, ROS generation. Furthermore, these results provide a new mechanism by which DPI could inhibit the respiratory burst, i.e. by decreasing the availability of NADPH via PPP [73]. Also the ability of DPI to inhibit the cell proliferation, observed in many experimental works, could be, at least partly, related to the inhibition of PPP, which provides the cell with ribose 5-phosphate and NADPH, both neces-sary to the synthesis of deoxyribonucleotides.

APOCYNIN

The aromatic ketone 4-hydroxy-3-methoxyacetophenone (triv-ial names: apocynin, acetovanillone) is an ortho-methoxy-substituted catechol, constituent of root extracts of the medicinal herb Picrorhiza kurroa which grows in the Himalayan mountains. In 1990 it was shown to be a strong inhibitor of neutrophil O2•release in vitro and to exert antiinflammatory activity in vivo [77]: when rats were treated orally with low doses of apocynin (0.3

g/ml of drinking water) the severity of collagen-induced arthritis was significantly reduced [78]. Soon it was clear that this drug in-hibits NOX activity in neutrophils and eosinophils (complete inhi-bition after a 7-min incubation with 300 M apocynin), by interfer-ing with the intracellular translocation to the membrane of two cytosolic components of NOX2, p47

phox and p67

phox [79]. Apocynin

showed to be a potent inhibitor of NOX: in stimulated human neu-trophils its IC50 was 10 M [80]. The reaction of apocynin with ROS and peroxidases is critical for the drug to exert the inhibitory effect: in a proposed chemical reaction involving ROS and peroxi-dase, ortho-methoxy-substituted catechols are demethoxylated and replaced with a hydroxyl group, by which the corresponding catechols are formed [80]. These products have been suggested to react with essential thiol groups in the NOX subunits, preventing the oxidase complex from assembling and activating [81]. Alterna-tively, it has been proposed that peroxidases (MPO is a very effi-cient catalyst for the oxidation of apocynin) would convert apo-cynin to a symmetrical dimer, diapocynin, through the formation of a 5,5' carbon-carbon bond, as shown in Fig. (3), and, via further oxidation and hydroxylation, to a trimer [82]: both GSH and L-cysteine inhibit the dimerization [83]. Diapocynin would be the active compound that prevents NOX complex assembly and activa-tion, by reacting with thiol groups [83]. On the other hand, when

OH

OCH3

CH3C O

apocynin

MPO/H2O2

O

OCH3

CH3C O

apocynin

radical

radical

coupling

OH

CO CH3

H3OCOCH3

COH3C

OH

apocynin

dimer

Fig. (3). MPO-mediated dimerization of apocynin (modified from Ximenes et al. [82]).

Classical Inhibitors of NOX NAD(P)H Oxidases Are Not Specific Current Drug Metabolism, 2008, Vol. 9, No. 8 691

injected in rats apocynin was not significantly converted in vivo to diapocynin [84].

Ximenes et al. [82] have recently proposed the putative path-ways by which apocynin and/or its dimer may inhibit the activation of the cytosolic p47

phox, a process wherein MPO plays an important

role. They started from the observation that neither apocynin nor the dimer and trimer derivatives are able to conjugate with GSH or other thiols, but thiol groups may react with apocynin radical and/or its dimer radical, which are formed during MPO-catalyzed oxida-tion [82]. Thus, essential thiols of p47

phox could be oxidized with

the following mechanism:

1) apo OH + H2O2

MPO

apo O• + 2 H2O

2) 2 apo O• dimer OH

3) dimer OH + H2O2

MPO

dimer O• + 2 H2O

4a) p47 SH + apo O• p47 S SR

4b) p47 SH + dimer O• p47 S SR

In this hypothetic scheme, R could be another protein thiol or glutathione (modified from Ximenes et al. [82]).

IS APOCYNIN A SPECIFIC INHIBITOR OF NOX ASSEM-BLY?

At the beginning of 1990s, when DPI was definitely abandoned as a specific inhibitor of NOX, apocynin became very soon the new selective inhibitor of NOX. Since apocynin showed a very low toxicity in vivo, it could be used for long time periods and at high doses: this made apocynin the election drug for chronic treatments aimed at maintaining NOX inhibited in animal models. In the meanwhile, experimental evidence was showing that cardiovascular cells possess NOX activity critically involved in ROS generation, and that the subsequent oxidative stress in the vascular wall is in-volved in the pathogenesis of hypertension and atherosclerosis. Many in vitro studies showed that, in endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts, apocynin blocks O2• generation [85]. Thus, notwithstanding very few reports inves-tigating its mechanism of action are available (to our knowledge, only five) [79-83], apocynin has been used in the last 15 years (with exponentially increasing popularity) as a specific inhibitor of NOX in several hundreds experimental works. This incredible unbalance between the knowledge of a compound and its use can be explained with the high need of researchers to inhibit the activity of an en-zyme family involved in so many critical physiopathological proc-esses.

On the basis of the above mentioned mechanism, apocynin was soon proposed as a selective inhibitor of NOX, and particularly of the leukocyte-type NOX, subsequently named NOX2. Thus, the observation that apocynin could inhibit the translocation of p47

phox

to the membrane of human endothelial cells exposed to atherogenic levels of low-density lipoproteins was provided in support of the hypothesis that a functionally active leukocyte-type NOX (i.e. NOX2) is present in endothelial cells [86]. Yet, apocynin has been also observed to inhibit NOX activity in cells expressing isoforms different from NOX2. For instance, apocynin inhibited the ROS production in human aortic smooth muscle cells stimulated with tumour necrosis factor- (apocynin: 1 mM, 12 h) [87] and in human fibroblasts stimulated with polyunsaturated fatty acids (apocynin: 2.5 M, 4 h) [88]: in both cell models NOX activity was dependent on NOX4 expression. On the other hand, in human embryonic kid-ney HEK-293 cells transfected with NOX4 apocynin (contrary to DPI) was not effective in inhibiting NOX activity (IC50 > 100 M, 60 min) [89]. Furthermore, in the human oesophageal adenocarci-noma cell line SEG1 apocynin (100 M, 30 min) has been reported to inhibit the generation of ROS, which was mainly dependent on

NOX5 activation and expression [90]. NOX5 does not need cytoso-lic organizer or activator subunits and has been shown to function in a cell-free system without the requirement of any cytosolic pro-teins [2]. Instead, in HEK-293 cells stably expressing NOX5 63 Mapocynin (60 min) was not effective in inhibiting NOX activity, while DPI inhibited the ROS generation [91]. The weak effect of apocynin on NOXs expressed in HEK-293 cells [89, 91] has been explained with the absence of MPO, which should be necessary to activate apocynin in these cells. However, taken together, these data suggest that the inhibition of the NOX complex assembly is not likely to be the only mechanism of action of apocynin.

Indeed, recent scientific evidences provide suggestions for other mechanisms of apocynin-mediated NOX inhibition. In human monocytes, the incubation with apocynin (100 g/ml, 4 h) de-creased the expression of p47

phox [92], and a similar effect was

observed on p67phox

expression in THP-1 monocytes [93], thus suggesting that the inhibitory effect of apocynin on NOX2 could be exerted also via regulation at transcription level. In isolated rat hearts apocynin (given 2 mg/kg i.p. 4 h before the experiment) de-creased the generation of ROS induced by angiotensin II and abol-ished the angiotensin II-mediated increase in p22

phox and gp91

phox

expression [94], and in H9C2 embryonal rat heart-derived cells apocynin (100 M, 24 h) inhibited the angiotensin II-induced NOX activation and increase in p47

phox expression [95]. Apocynin inhib-

ited also in vivo the p47phox

expression in rabbit heart (apocynin: 15 mg/day for 4 weeks) [96] and in angiotensin II-infused hypertensive rat penes (apocynin: 10 mM in the drinking water for 4 weeks) [97]. These experimental works did not explain how apocynin may in-hibit the expression of NOX subunits. A possible explanation has been provided recently by Muzaffar et al [98] who demonstrated that O2• itself upregulates gp91

phox expression in pulmonary artery

endothelial cells: this means that, at least in the vascular tissues, O2• may create, through the upregulation of NOX, a self-amplifying positive feedback loop leading to a progressively in-creasing generation of ROS [98].

APOCYNIN DOES NOT INTERFERE WITH NOX ACTIV-ITY ONLY

Since the beginning of its use, apocynin was also observed to exert effects not related to the inhibition of NOX. Indeed, apocynin affected the arachidonic acid metabolism in guinea pig pulmonary macrophages (the formation of thromboxane A2 was inhibited, whereas the release of prostaglandins E2 and F2 was stimulated) and in bovine platelets (the arachidonic acid-induced aggregation was potently inhibited) [99]. Furthermore, apocynin was shown to increase the blood flow in rabbit eyes [100], inhibited the cyto-chrome P450 activity in endothelial cells [101], interfered with actin polymerization and cytoskeletal rearrangement in polymor-phonuclear granulocytes [102]. Apocynin inhibited also the expres-sion of the inducible cyclooxygenase (Cox2) in human macro-phages via a NOX-independent (at least partly) mechanism [92].

No mechanism was provided for these effects of apocynin. It has been suggested that the inhibitory effect of apocynin on cy-toskeletal rearrangements and cell migration is subsequent to the drug oxidation by peroxidases, yielding oligophenolic and quinone-type compounds. These apocynin derivatives may inhibit not only NOX but also the small G protein Rac1, that, besides participating to the NOX2 assembly, controls also cell migration: in breast can-cer cells these derivatives (but not apocynin itself) caused a signifi-cant rearrangement of the actin cytoskeleton, cell rounding, and decreased the levels of active Rac1 and its related G protein Cdc42 [103]. No evidence was given that NOX inhibition was actually implicated in the effect of apocynin derivatives on cell migration.

Very recently, apocynin has been also observed to decrease the ROS level via a NOX-independent mechanism. In HEK-293 cells overexpressing NOX1, NOX2 or NOX4, apocynin (10-600 M, 20-40 min) failed to inhibit O2• generation, while it acted as a radical

692 Current Drug Metabolism, 2008, Vol. 9, No. 8 Aldieri et al.

scavenger (at 0.1-1 mM) in different assay systems generating ROS, such as pyrogallol and xanthine/xanthine oxidase [104]. In rat smooth muscle cells, directly stimulated with either H2O2 or the ROS generator menadione, apocynin impaired the ROS-induced activation of p38 mitogen-activated protein kinase, as well as of the Akt kinase and the extracellular signal-regulated kinase 1/2 [104]. Furthermore, apocynin is a prodrug that in leukocytes is activated by MPO, resulting in the formation of apocynin dimers (see above): endothelial cells and smooth muscle cells failed to form these di-mers. Diapocynin formation was, however, observed in NOX-overexpressing HEK-293 cells when MPO was supplemented. As a consequence of these results, it has been concluded that apocynin should only inhibit NOX in leukocytes, whereas in vascular cells the compound could act as an antioxidant in a NOX-independent way and should not be used as a specific NOX inhibitor [104]. This statement needs further confirmation: indeed it assumes that diapo-cynin is the only active compound able to prevent the NOX com-plex assembly [83], but it cannot be excluded that other apocynin derivatives (such as the apocynin radical) [80, 81] may be responsi-ble for NOX inhibition in vascular cells.

APOCYNIN PER SE INDUCES OXIDATIVE STRESS

In human monocytes (activated by phorbol ester and zymosan) apocynin (100 g/ml, 1 h) caused a decrease of the GSH/ GSSG ratio, which was significantly related with the inhibitory effect of apocynin on Cox2 transcription; indeed, such inhibition was fully abrogated when the intracellular levels of GSH were increased [92]. After this first observation suggesting that apocynin could elicit an oxidative stress, Vejra ka et al. [105] showed that apocynin (100

M, 10-60 min) stimulates the generation of ROS in rat vascular fibroblasts and inhibits the respiratory burst in rat monocytes stimu-lated with zymosan. Vejra ka and coll. supposed that apocynin could act as both an inhibitor of phagocyte NOX and a stimulator of ROS production in non-phagocyte cells [105]. Furthermore, in N11 glial cells apocynin was reported to induce, in a concentration-dependent way (30-1200 M, 6 h), a significant increase of both malonyldialdehyde level (index of lipid peroxidation) and lactate dehydrogenase release (index of a cytotoxic effect) [106]. Apocynin (300 M, 6 h) significantly evoked also an increase of H2O2 con-centration and a decrease of the intracellular GSH/GSSG ratio, accompanied by augmented efflux of glutathione. Apocynin in-duced the activation of both PPP and TCA cycle, which was pre-vented when cells were incubated with GSH together with apo-cynin. The co-incubation with GSH prevented also the apocynin-induced increase of malonyldialdehyde generation and lactate de-hydrogenase leakage. Apocynin elicited an oxidative stress also in a cell-free system: indeed, in aqueous solution it evoked a faster oxi-dation of the thiols GSH and dithiothreitol, and elicited the genera-tion of ROS, mainly O2• [106]. These results suggested that apo-cynin per se can induce an oxidative stress and exert a cytotoxic effect in N11 cells and other cell types, such as human erythrocytes and epithelial cells. Thus, both DPI and apocynin may affect the activity of central metabolic pathways such as PPP and TCA cycle, and elicit an oxidative stress: while DPI and DIP induced oxidative stress via inhibition of G6PD and PPP [73], on the contrary the apocynin-induced activation of both PPP and TCA cycle was sub-sequent to the oxidative stress, since the presence of GSH in the medium together with apocynin actually prevented the activation of both metabolic pathways [106]. The mechanism by which apocynin induces oxidative stress is still under investigation. Apocynin con-tains a phenolic structure and several phenolic molecules have shown to be cytotoxic. It has been suggested that the one-electron oxidation of phenolic compounds by cell oxidoreductases, resulting in the generation of phenoxyl radicals, may be an important con-tributor to the cytotoxic effects. These radicals would be readily reduced to phenols by intracellular reductants such as ascorbate and thiols, triggering a redox cycling [107, 108]. As described above, apocynin has been suggested to require enzymatic conversion to

inhibit NOX [80, 81, 83]: on the contrary, cell-free experiments showed that apocynin per se may oxidize GSH and dithiothreitol and generate ROS [106]. The strong decrease of ROS concentration in the presence of SOD and in air-evacuated tubes suggested that apocynin favours the one-electron reduction of molecular oxygen to O2• ; furthermore, nitrilotriacetic acid, which chelates di- and triva-lent metal ions, inhibited the apocynin-evoked increase of ROS level, suggesting that a metal triggers the apocynin-elicited produc-tion of ROS. Iron was excluded, whereas copper showed to be a likely candidate [106]. Subsequently, it has been shown that, after oxidation in the presence of MPO/H2O2, apocynin generates radi-cals able to oxidize GSH and sulfhydryl groups of albumin [109].

APOCYNIN INDUCES NO SYNTHESIS

More recently, apocynin, within a range of concentrations (30-1200 M, 6-24 h) commonly used in in vivo and in vitro experi-ments, has been reported to induce also the accumulation of nitrite, the stable derivative of NO, in the extracellular medium of N11 mouse glial cells cultures, and to increase the intracellular NOS activity and iNOS mRNA: the apocynin-induced NO synthesis was dependent on the generation of an oxidative stress, since it was completely inhibited by catalase, and was mediated by the activa-tion of the redox-sensitive transcription factor NF-kB [110]. Indeed, binding sites for NF-kB have been found in the promoter region of both mouse and human iNOS gene [111]. The NF-kB inhibitor SN50 prevented the apocynin-induced nuclear translocation of NF-kB and the increase of nitrite accumulation and NOS activity. The apocynin-elicited NO synthesis was responsible for the cytotoxic effect exerted by apocynin on N11 cells. Apocynin increased the nuclear translocation of NF-kB and the NO synthesis also in MH-S murine alveolar macrophages and in A549 human lung epithelial cells [110]. These data suggest that apocynin induces increased NO synthesis by eliciting a generation of ROS, which in turn activate NF-kB and evoke increased expression of iNOS. It has been re-ported that apocynin reduces the NF-kB activation in H9c2 rat car-diac cells and in C2C12 mouse myoblasts (apocynin: 250 nM, 6 days) [112], and in rat aortic smooth muscle cells (apocynin: 3.5 mM, 1 h) [113]: the different experimental conditions (respectively: longer time periods of incubation, higher concentrations of apo-cynin) may account for the discrepancy of these results with those of Riganti et al. [110].

Previously, apocynin has been shown to increase NO levels in different biological models, particularly in vascular systems: this observation has been interpreted as a consequence of the inhibition of NOX, causing a decreased generation of O2• , which is a poten-tial NO scavenger. Apocynin has been found to cause relaxation of arteries in many in vivo experiments, suggesting that the NO/O2•balance is a key regulator of endothelial function. This observation has been used as an evidence of the role of NOX-derived O2• in lowering NO effects in diabetes, aging, hypertension, etc., and has raised interest about the possibility to use apocynin or other NOX inhibitors in the treatment of cardiovascular pathologies. Instead, the results of Riganti et al. [110] suggest that apocynin may favour NO synthesis without involving the NOX system, and that any con-clusion concerning apocynin-induced improvement of NO signal-ling should take into account the possibility that the higher avail-ability of NO might depend on the drug-elicited induction of iNOS, rather than on the reduced scavenging of NO by ROS.

SPECIFIC NOX INHIBITORS ARE STILL MISSING

Table 1 summarizes the principal target molecules of DPI and apocynin and reports several examples of effective inhibiting con-centrations. At present, no specific NOX inhibitors exist [2]. In sharp contrast with this statement, many studies aimed at investigat-ing the role of NOXs in a great deal of physiopathological proc-esses are based on the use of NOX inhibitors, mainly DPI and apo-cynin. Recently, new potential NOX inhibitors have been proposed,

Classical Inhibitors of NOX NAD(P)H Oxidases Are Not Specific Current Drug Metabolism, 2008, Vol. 9, No. 8 693

such as the synthetic compounds VAS2870 and S17834, the natu-rally occurring antibiotic peptide PR-39 and the chimeric peptide gp91ds-tat [for a review, see 10]. The last one has been shown to interfere with the assembly of gp91

phox and p47

phox, and it is pres-

ently the most promising candidate for the nomination to specific NOX inhibitor: the docking sequence (ds) of 9 amino acids mimics a region of gp91

phox that interacts with p47

phox, whereas the other 9

amino acids correspond to a specific tat sequence in the HIV viral coat, which allows the peptide to be internalized by cells [115]. Unfortunately, gp91ds-tat has some limitations. The peptide is a low-efficacy inhibitor in whole cells [10], and may lack specificity, since the region targeted by gp91ds-tat is homologous in other NOX isoforms [2, 10, 116]. Finally, the tat sequence of the peptide has been shown to cause side effects affecting cellular activity and signalling [116]. The knowledge of the pharmacokinetics of gp91ds-tat is limited, and further pharmacological studies need to be completed in vivo [10].

An approach alternative to inhibitors is the use of transgenic animals knock-out for a NOX protein [116] or the knocking-down of NOX transcripts in cultured cells using the small interference RNA (siRNA) technique. Both techniques cause the lack of expres-sion of a specific NOX protein, but in many tissues two or more NOX isoforms coexist: the abolition of only one of them could be devoid of effect, since the other isoform(s) could replace the func-tion of the missing NOX. Thus, the quest for a specific inhibitor of the NOX family continues to be greatly important. In the mean-while, any evidence obtained with the presently available putative NOX inhibitors should be supported by experiments excluding that their effects are NOX-independent.

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Table 1. Principal Target Molecules of DPI and Apocynin. Owing to the Huge Amount of Experimental Works Concerning the Activity of these

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1. DPI

Target Inhibiting Concentrations Reference

NADH dehydrogenase (mitochondrial complex I) IC50 = 18 M (rat liver)

IC50 = 23-30 nmol/mg of protein (bovine heart)

[12]

[13]

NOX IC100 > 10 M (pig neutrophils)

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[15, 16]

[33]

NOS IC50 = 50 nM (semi-purified macrophage iNOS)

IC50 = 0.3 M (eNOS, aortic rings)

IC50 = 0.03 M (iNOS, condrocyte)

[21]

[21]

[29]

xanthine oxidase IC50 = 0.3-0.5 mol/40 g enzyme (bovine neutrophils) [31]

cytochrome P450 reductase Ki = 2.8 mM (bovine liver) [32]

protoporphyrinogen IX oxidase Ki = 67.5 nM (yeast mitochondria) [36]

thioredoxin reductase IC100 = 10 M (lung epithelial cells) [37]

diaphorase activity IC100 > 30 M (from Clostridium kluyveri) [45]

cationic channels IC100 = 3-10 M (rat carotid body cells and pulmonary smooth muscle cells) [52, 53]

NMDA receptor IC50 = 45.2 M (rat brain synaptic membranes) [56]

PMOR IC100 = 2.5 M (chick forebrain neurons) [61]

PQQ redox cycling IC50 = about 50 nM (guinea pig neutrophils)

IC50 = 10 M (HL-60 human monocytes)

[63]

[65]

G6PD IC50 = 10 M (purified enzyme) [73]

2. Apocynin

Target Inhibiting Concentrations Reference

NOX IC50 = 10 M (human neutrophils) [80, 114]

ROS radical scavenging at 0.1-1 mM (HEK-293 cells) [104]

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Received: July 07, 2008 Revised: August 08, 2008 Accepted: August 13, 2008