Current Medicinal Chemistry, 2006, 13, 87-98

87

Comparative Studies of the Antioxidant Effects of Cis- and TransResveratrol

F. Orallo*
Departamento de Farmacologa, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela (La Corua), Spain
Abstract: Resveratrol (3,4',5-trihydroxystilbene, RESV) is a natural phenolic compound that exists as cis and trans isomers [c-RESV or (Z)-RESV and t-RESV or (E)-RESV, respectively]. t-RESV is a natural component of Vitis vinifera L. (Vitaceae), abundant in the skin of grapes (but not in the flesh) and in the leaf epidermis, and present in wines, especially red wines. In in vitro, ex vivo and in vivo experiments t-RESV exhibits a number of biological activities, including anti-inflammatory and anticarcinogenic properties. RESV also exists in wines as a cis isomer, which (unlike t-RESV) is not currently available commercially; as a result, little is known about this isomers pharmacological activity. In this review, I will focus on the few comparative studies of the antioxidant effects of the two RESV isomers in different experimental models.

Keywords: Resveratrol isomers, NAD(P)H oxidase, xanthine oxidase, inducible nitric oxide synthase, murine macrophages, antioxidant activity, free radicals, wine. 1. INTRODUCTION Resveratrol [3,4',5-trihydroxystilbene, RESV, (Fig. 1)] is a natural phenolic compound that exists as cis and trans isomers [the c-RESV or (Z)-RESV diasteromer, and t-RESV or (E)-RESV diasteromer, respectively], facilitated by the double bond in its chemical structure [1-3]. t-RESV is a natural component of Vitis vinifera L. (Vitaceae) that is abundant in the skin of grapes (but not in the flesh) and in the leaf epidermis, and that is present in wines, especially red wines. t-RESV is not unique to Vitis but is also present in at least 72 other plant species (distributed in 12 families and 31 genera, e.g. Veratrum, Arachis, Morus, Vaccinium and Trifolium), some of which are components of the human diet, such as mulberries and peanuts [1,4-6]. t-RESV was first identified in 1940 by Michio Takaoka [7] as a constituent of the roots of white hellebore [Veratrum grandiflorum (Maxim. ex Baker) Loes. (Liliaceae)], and later (in 1963) found by Nonomura et al. [8] (together with its 3O--D-glucoside -glucoside, so-called trans-piceid or transpolydatin) in the dried roots of Japanese knotweed Polygonum cuspidatum Sieb. et Zucc. (Polygonaceae), called Ko-jo-kon (or Itadori) in Japanese and Chinese folk medicine, in which it is used for the treatment of suppurative dermatitis, gonorrhea favus, hyperlipemia, favus, athlete's foot (tinea pedis), arteriosclerosis, as well as in allergic and inflammatory diseases and other pathologies [see, e.g., refs. 1-4]. Later, in 1976, t-RESV was detected in grapevines by Langcake and Pryce [9], who found that it is synthesized by leaf tissues in response to fungal infection (mainly by Botrytis cinerea) or exposure to ultraviolet light. In 1992, Siemann and Creasy [10] reported the presence of this polyphenol in wines (basically red wine). At the same time, a number of large-scale epidemiological studies were clearly suggesting that prolonged moderate consumption of wine (especially red wine) by the Southern French and other Mediterranean populations was associated with a very low incidence of cardiovascular diseases, notably coronary heart disease, despite a high-fat diet, little exercise and widespread smoking: this was the so-called French paradox [see, e.g., ref. 11]. When it became known that the protective effects of wine consumption were independent of alcohol content, a number of studies were initiated with the aim of identifying the component(s) responsible. Since then, the pharmacological activity of t-RESV has been extensively investigated. In in vitro, ex vivo and in vivo experiments t-RESV has shown a number of biological activities including anti-inflammatory and anticarcinogenic properties [for reviews, see, e.g., refs. 1,3,4,6,12-14]. Several studies within the last few years have demonstrated that t-RESV may protect against coronary heart disease as a result of different effects, including significant antioxidant activity, modulation of lipoprotein metabolism, and vasodilatory and platelet antiaggregatory properties [see, e.g., refs. 12,15-18]. Besides t-RESV, other polyphenolic compounds (the socalled flavonoids) have been implicated in the cardioprotective effects of moderate wine consumption [19]. Flavonoids are a large group of polyphenols present in plants, regularly consumed foods (e.g. vegetables and fruits), and beverages like tea and wine. These low molecular weight substances are phenylbenzo--pyrones (phenyl--chromones) with an assortment of structures based on a common threering nucleus. They are usually subdivided according to their substituents into several subclasses including anthocyanidins, flavanones, flavones, flavonols, flavanonols (or dihydroflavonols), chalcones, isoflavones and flavanols (also called catechins) (Table 1). The biological activities of flavonoids (including their antioxidant properties) have been
2006 Bentham Science Publishers Ltd.

*Address correspondence to this author at the Departamento de Farmacologa, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur, E-15782 Santiago de Compostela (La Corua), Spain; Tel: +34-981-563100; Ext: 14895. Fax: +34-981594595; E-mail: fforallo@usc.es 0929-8673/06 $50.00+.00

88 Current Medicinal Chemistry, 2006, Vol. 13, No. 1

F. Orallo

Fig. (1). Chemical structures (panel A) and 3D molecular configurations (panel B) of the cis and trans isomers of RESV.

reviewed comprehensively elsewhere [see, e.g., ref. 19] and, therefore, will not be covered here. As noted, RESV also exists as a cis isomer, which (unlike t-RESV) is not currently available commercially [see, e.g., refs. 1,2]; as a result, little is known about this isomers pharmacological activity. In most studies [see, e.g., refs. 20,21], the cis isomer has not been detected in grapes, unlike t-RESV [though see ref. 22], but is present in wines at variable concentrations, suggesting that it may be produced from t-RESV by yeast isomerases during fermentation, or released from RESV polymers called viniferins, or from RESV glucosides (mainly the 3-O--Dglucoside) [for reviews, see, e.g., refs. 1,4]. c-RESV can also be obtained from t-RESV by exposure to ultraviolet radiation [for more details, see, e.g., refs. 23-25]. The few comparative studies reported in the scientific literature on the biological effects of cis- versus t-RESV have generally demonstrated only quantitative, not

qualitative differences, in the activities of the two forms [for more details, see ref. 26]. For example, Varache-Lembge et al. [27] reported that t-RESV is more efficient than c-RESV for inhibiting human platelet aggregation induced by a number of aggregatory agents (arachidonic acid, ADP and collagen), whereas Pettit et al. [28], in a preliminary structure-activity relationship study of the potential antineoplastic activity of various stilbene derivatives, found that the cis isomer exhibits slightly weaker inhibitory effects than the trans isomer in a number of cancer cell lines. Bearing in mind the above considerations, in this review I will first focus on the remarkable antioxidant activity exhibited by c- R E S V and t-RESV in thioglycollateprestimulated rat peritoneal macrophages, which has been a central concern of my groups research at the University of Santiago de Compostela. I will then briefly discuss what has been discovered about other antioxidant effects exhibited by the RESV isomers in different comparative studies and in different experimental models.

Comparative Studies of the Antioxidant Effects of Cis- and Trans-Resveratrol

Current Medicinal Chemistry, 2006, Vol. 13, No. 1

89

Table 1.

Chemical Structures of the Most Common Flavonoids. GLU = Glucose

ANTHOCYANIDINS Examples Apigenidin Cyanidin
3' 2' 8 4' O 2 3 5 4 6'

3 OH GLU OH OH OH OH OH 3 OH 5 OH OH OH OH OH OH OMe 5 OH OH OH OH OH 3

5 OH OH GLU OH OH OH OH OH 5 OH OH OH OH OH OH 6 OH OMe 7 OH OH OH OH OH OH 5

7 OH OH OH OH OH OH OH OH 7 Rutinose Rutinose OH OH Rutinose OH

3 OH OH OH OMe OMe OH 3 OH OH OH OH 3 OH OH OH 4 OH OH OH OH OH 3

4 OH OH OH OH OH OH OH OH

5 OH OMe OMe 4 OH OMe OMe OH OH OH 4 OH OMe OMe OH OMe 5 OH 4

Cyanin Delphinidin
5'

7 6

Malvidin Pelargonidin Peonidin Petunidin

FLAVANONES 3' 2' 8 7 6 5 O O 2 3 6' 4' 5'

Examples Eriocitrin Hesperidin Hesperetin Naringenin Naringin Taxifolin

FLAVONES
3' 2' 8 7 6 5 O O 2 3 6' 4' 5'

Examples Apigenin Baicalein Chrysin Diosmetin Diosmin Luteolin Tangeretin

7 OH OH OH OH Rutinose OH OMe 2 OH 7

8 OMe 3 OH OH OH

FLAVONOLS
3' 2' 8 7 6 5 O O 2 6' OH 4' 5'

Examples Fisetin Galangin Kaempferol Myricetin Morin Quercetin Examples

FLAVANONOLS

3' 2' 8 7 6 5 O 1 O 2 6' OH 4' 5'

Dihydrofisetin

OH

OH

OH

OH

Dihydroquercetin

OH

OH

OH

OH

OH

90 Current Medicinal Chemistry, 2006, Vol. 13, No. 1

(Table 1). contd.....

F. Orallo

CHALCONES 3 5' 4' 3' 2' O 6' 7 8 2 1 6 4

Examples

Butein 5 Floretin Floridzin

OH

OH

OH

OH

OH GLU 5

OH OH 7

OH OH 4

OH OH 5

ISOFLAVONES

Examples

8 7 6 5

Genistein
2 1' 4 2' 3' 4' 5'

OH

OH

OH

Daidzein

OH

OH

6'

Orobol FLAVANOLS
3' 2' 8 7 6 5 4 OH 1 O 6' 4' 5'

OH 3 5

OH 7 3

OH 4

OH 5

Examples

(+)-Catechin (-)-Epicatechin (-)-Epigallocatechin

OH(S) OH(R) OH(R)

OH OH OH

OH OH OH

OH OH OH

OH OH OH

OH

2. ANTIOXIDANT EFFECTS OF c-RESV VERSUS t-RESV IN MURINE PERITONEAL MACROPHAGES Prior to 2002 there had been no previous studies comparing the potential inhibitory effects of c-RESV and t-RESV on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), e.g. via inhibition of the enzymatic activity and/or expression of oxidant enzymes such as nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase [NAD(P)H oxidase], xanthine oxidase (XO) and nitric oxide synthase (NOS). In 2002 we therefore initiated a detailed comparative study of the possible in vitro effects of the RESV isomers on ROS and RNS generation during the respiratory burst of thioglycollate-elicited rat peritoneal macrophages. The principal results obtained in this project, which have been previously published [25,29], will be briefly outlined in what follows. 2.1. Effects of the RESV Isomers on Extracellular ROS Production by Inflammatory rat Peritoneal Macrophages Stimulated with Phorbol 12-Myristate 13-Acetate (PMA) The first series of experiments was designed to study the effects of c-RESV and t-RESV on extracellular ROS

production in thioglycollate-prestimulated rat peritoneal macrophages. It has been reported that, in phagocytic cells, PMA basically activates protein kinase C (PKC), which in turn induces ROS production [basically generation of superoxide radicals (O2 )] by two main pathways (Fig. 2).

First, O2 generation may occur as a result of activation of an NAD(P)H oxidase (see below) that is assembled from different membrane and cytosolic subunits at the plasma membrane, and that catalyzes the vectorial synthesis of O2 from molecular oxygen (O2 ) and cellular NADPH (the preferred substrate in phagocytic cells) supplied by the hexose monophosphate shunt (2O2 + NADPH 2O2 + NADP + + H+ ) [see, e.g., refs. 30-32]. The structure and function of NAD(P)H oxidase was characterized initially in neutrophils, in which it includes two protein subunits located in the cell membrane, a glycoprotein of 91 kDa (gp91phox), and a non-glycosylated protein of 22 KDa (p22phox), together forming the so-called cytochrome b558. Other important components include the cytoplasmic subunits p47phox, p40phox, p67phox and the small G proteins Rac and Rap1A. When phagocytic cells are activated, the cytosolic subunits translocate to cytochrome b 558 at the membrane, which results in activation of the oxidase and the well-characterized oxidative burst (Fig. 2). Two events are required for activating NAD(P)H oxidase: exchange of GTP for GDP on the small G protein Rac, and phosphorylation of the p47phox subunit and possibly other

Comparative Studies of the Antioxidant Effects of Cis- and Trans-Resveratrol

Current Medicinal Chemistry, 2006, Vol. 13, No. 1

91

Fig. (2). Extracellular production of ROS (basically O2 ) induced by PMA in thioglycollate-elicited rat peritoneal macrophages. Steps which may be interfered with by c-RESV and t-RESV are indicated by arrows. H2HFF/BSA = OxyBURST Green H2HFF/BSA reagent.

subunits (e.g. p67phox) by PKC, which triggers a change in the conformation of the cytosolic complex. This activated cytoplasmic complex then associates with cytochrome b558 in the membrane to form a functional enzyme that is thought to include one copy of each phox subunit, as well as Rac and Rap1A [see, e.g., refs. 30-36]. Second, O2 generation may occur through stimulation by PKC of xanthine oxidoreductase, an enzyme mainly located in the cytosol which catalyzes the oxidative hydroxylation of purine substrates [e.g. xanthine or hypoxanthine (HX)] at the molybdenum center (the reductive half-reaction) with production of uric acid, and the subsequent reduction of O 2 at the flavin center with generation of either O2 or hydrogen peroxide (H2O2) (the oxidative half-reaction) (Figs. 2) and (3)] [for more details, see, e.g., refs. 37-39]. This enzyme has two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase, which transfer electrons from xanthine to two different preferred acceptors, O2 and NAD+ respectively [38]. The contribution of XO to phagocyte O2 generation during the respiratory burst is more questionable than the contribution of NAD(P)H oxidase [40].

OxyBURST Green H2 HFF/BSA [i.e. bovine serum albumin (BSA) coupled to dihydro-2,4,5,6,7,7hexafluorofluorescein (H2HFF), a cell-impermeant protein conjugate], which reacts with O2 under physiological conditions, to produce fluorescence (Fig. 2) [see, e.g., ref. 29].

Using this reagent, we have demonstrated that both RESV isomers (1-100 M) markedly reduced extracellular levels of O2 following PMA stimulation of thioglycollateprestimulated rat peritoneal macrophages, i.e. they exhibit a typical antioxidant activity [for more details and numerical data, see refs. 25,29].

The above results i.e. reduced extracellular levels of O2 may be attributable either to effects on enzyme activities [XO and/or NAD(P)H oxidase] or to direct scavenging of O2 by c-RESV and t-RESV. To distinguish between these possibilities we used assays based on the HX-XO system.

Once O2 have been synthesized, large quantities are immediately released to the extracellular space. This extracellular release of O2 can be detected using

Under standard assay conditions for this system, using the standard commercial form of XO from buttermilk, XO converts HX to xanthine, H2O 2 and O2 , and xanthine to uric acid, H2O2 and O2 [see, e.g., ref. 41]; O2 generated in this way then chemically reduces nitroblue tetrazolium (NBT) to produce the colored compound formazan; the amount of O2 generated by the assay system is estimated

92 Current Medicinal Chemistry, 2006, Vol. 13, No. 1

F. Orallo

Fig. (3). Summarized reaction steps in the HX/XO system. As noted in the text, our experiments indicate that neither c-RESV nor t-RESV are scavengers of O2 or inhibitors of commercial (buttermilk) XO.

on the basis of spectrophotometric determination of formazan at 560 nm (Fig. 3) [see, e.g., ref. 42]. In parallel assays, the production of uric acid by XO may be estimated on the basis of spectrophotometric determination at 265 nm. When a test substance lowers the amount of O2 (i.e. decreases the rate of NBT reduction) and at the same time does not affect the formation of uric acid, it can be considered a selective scavenger of O2 ; on the other hand, reduced levels of both O2 and uric acid indicate that the test substance is inhibiting XO activity. In our experiments, and unlike the reference O2 scavenger superoxide dismutase (SOD, 0.1-10 U/ml), c-RESV and t-RESV (1-100 M) did not decrease the rate of NBT reduction. In addition, and unlike allopurinol (1-10 M), a well-known reference inhibitor of XO, neither c-RESV nor t-RESV (1-100 M) had significant effects on uric acid production in this assay system [for more details and numerical data, see refs. 25,29]. These results clearly indicate that c-RESV and t-RESV do not selectively scavenge O2 or directly inhibit commercial XO, suggesting that the reduction in extracellular O2 levels induced in inflammatory macrophages by the RESV isomers is due to inhibition of NAD(P) oxidase activity. These results agree with those obtained by Hung et al. [43], who found that low concentrations of t-RESV (10 M) do not scavenge O2 generated by the HX/XO system. In contrast, our results do not agree with those of Zhou et al. [44], who found that t-RESV inhibits the enzymatic activity of XO with an IC50 of about 11 M.

allopurinol (1-10 M), a reference inhibitor of XO] had no effects on O2 generation from xanthine (Fig. 2). These data confirm that c-RESV and t-RESV do not affect XO activity [in this case native XO, as opposed to commercial (buttermilk) XO]. However, both c-RESV and t-RESV (10100 M) [like diphenyleneiodonium (DPI, 10-100 M), a reference inhibitor of NAD(P)H oxidase] inhibited NAD(P)H oxidase activity, i.e. the specific chemiluminescence signal emitted by the reaction between lucigenin and O2 generated from NADPH (Fig. 2). These results strongly indicate that a decline in NAD(P)H oxidase activity may be responsible, at least in part, for the observed inhibitory effects of the RESV isomers on extracellular ROS (basically O2 ) production [for more details and numerical data, see ref. 25].

We have observed similar effects of c-RESV and t-RESV on NAD(P)H oxidase activity in rat aortic homogenates [45,46]. In addition, we have recently obtained similar results using rat aortic myocytes and human umbilical-vein endothelial cells as sources of enzymes (unpublished data). These data agree with those obtained by Liu et al. [47], who found that t-RESV inhibits mechanical-strain-induced NAD(P)H oxidase activity in endothelial cells isolated from human umbilical cords. However, our results disagree with those obtained by Poolman et al. [48], who found that t-RESV does not affect NAD(P)H oxidase activity in human monocytes (differentiated U937 cells). 2.2. Effects of c-RESV on Intracellular ROS Production by Inflammatory Rat Macrophages Stimulated with Kluyveromyces Lactis Cells The second series of experiments was designed to study the effects of the RESV isomers on intracellular ROS production in thioglycollate-prestimulated rat peritoneal

To confirm these findings, we performed experiments to assess the possible effects of the RESV isomers on XO and NAD(P)H oxidase activities (as measured by lucigenin-enhanced chemiluminescence) in extracts from thioglycollate-prestimulated rat peritoneal macrophages. In these experiments, c-RESV and t-RESV (1-100 M) [unlike

Comparative Studies of the Antioxidant Effects of Cis- and Trans-Resveratrol

Current Medicinal Chemistry, 2006, Vol. 13, No. 1

93

Fig. (4). Summarized effects of the RESV isomers on the intracellular production of ROS (basically O2 ) induced by Kluyveromyces lactis cells in thioglycollate-prestimulated rat peritoneal macrophages. Steps which may be interfered with by c-RESV and t-RESV are indicated by arrows. KL/H2DCF = Kluyveromyces lactis cells/OxyBURST Green H2DCFDA complex.

macrophages. When phagocytosable particles [in this case Kluyveromyces lactis cells conjugated to OxyBURST Green H2DCFDA (2,7-dichlorodihydrofluorescein (H2DCF) diacetate)] bind to membrane receptors on phagocytic cells, they are internalized (by endocytosis) into the phagovacuole. In addition, a number of host defense mechanisms are activated [see, e.g., refs. 36,49,50]. The first and the limiting step in a series of reactions designed to produce a number of ROS (toxic oxidants that damage and destroy the phagocytosed particles) is the stimulation of the NAD(P)H oxidase-mediated oxidative burst (see above), which leads to the massive generation of O2 and subsequent release into the phagosome (Fig. 4). In the present experiments, using OxyBURST Green H2DCFDA, these O2 radicals will oxidize the nonfluorescent H2DCF molecules to fluorescent dichlorofluorescein (DCF), thus generating fluorescence within the phagosome [see, e.g., refs. 51,52].

These effects may be basically due to the inhibitory action of the RESV isomers on NAD(P)H oxidase activity described in the previous section. 2.3. Effects of c-RESV and t-RESV on RNS Production by Inflammatory rat Macrophages Stimulated with Lipopolysaccharide (LPS)/Interferon Gamma (IFN- ) The next series of experiments was designed to study the effects of c-RESV and t-RESV on RNS production in rat thioglycollate-prestimulated macrophages. Stimulation of murine macrophages with bacterial wall components such as LPS and cytokines like IFN- induces them to express large amounts of inducible NOS (iNOS or NOS-2) in the cytosol, which leads to strong endogenous nitric oxide (NO ) production (nano- to micromolar range) from the terminal guanidino nitrogen atom of L-arginine (Fig. 5) [for reviews, see, e.g., refs. 53-59]. Thereafter, the process involves the diffusion of NO into the phagosome, where NO acts as a toxic agent. NO can rapidly react with O2 synthesized by phagocytic cells [via XO and/or NAD(P)H oxidase, see above, which may be activated by LPS and IFN-] to form the powerful oxidant and antimicrobial compound

In these experiments, c-RESV (like t-RESV) (1-100 M) showed typical antioxidant activity, i.e. it concentrationdependently inhibited (though less effectively than t-RESV) the intracellular production of ROS induced by Kluyveromyces lactis cells/OxyBURST Green H2DCFDA [for more details and numerical data, see refs. 25,29].

94 Current Medicinal Chemistry, 2006, Vol. 13, No. 1

F. Orallo

Fig. (5). The L-arginine-NO pathway in phagocytic cells (murine macrophages). Steps which may be interfered with by cRESV and t-RESV are indicated by arrows.

peroxynitrite (ONOO ) [60,61]. This is the principal pathway for inactivation of NO in the human body. In addition, NO may be rapidly oxidized to RNS which also induces cell toxicity.

O N O O reacts with and damages many important biological molecules including thiols, lipids, proteins and nucleic acids, by a number of mechanisms [62,63]. Three different SOD isoforms may dismute O2 into H2O2 and O2 under physiological conditions in vascular and non-vascular cells (Fig. 5) [for more details, see, e.g., refs. 64-66].

receptors on phagocytic cells, PKC and other protein kinases (e.g. IB kinase) are activated [see, e.g., refs. 70,71,75,76]. These kinases may in turn induce the phosphorylation of IB, which results in its degradation and dissociation from the complex. The released NF-B (p50-p65 heterodimer) then translocates to the nucleus, where it binds with high affinity to specific sequences (B sites) in the promoter or enhancer regions of target genes (including iNOS gene), thus triggering their transcription (Fig. 6). The RESV isomers (10-100 M) once again exhibited a typical antioxidant activity, i.e. they markedly inhibited the generation of RNS by inflammatory rat macrophages stimulated with LPS and IFN- [for more details and numerical data, see refs. 25,29] (Fig. 5). This effect could be antagonized in part by the inhibitory effects of the RESV isomers on NAD(P)H oxidase activity, a subsequent decrease in basal cellular O2 biosynthesis, and thus a decrease in NO biotransformation.

The induction of iNOS is usually mediated by activation of the nuclear trancription factor NF-B, which is a common regulatory element in the promoter region of many enzymes. The role of NF-B transcription factor activation in the induction of iNOS and other enzymes has been reviewed comprehensively elsewhere [see, e.g., refs. 67-69]. Briefly, under resting conditions, mammalian NF-B consists of two subunits [p50 (also called NF-B1) and p65 (also called Rel A)] that are complexed with an anchorin-domaincontaining protein (IB) in the cytoplasm. LPS is recognized by a complex of cell surface receptors composed of CD14, MD-2 and Toll-like receptor 4 (TLR4) [for more details, see, e.g., refs. 70-72], whereas the IFN- receptor is composed of two subunits, IFN-R1 and IFNR2 [73,74]. When LPS and/or IFN- bind to membrane

The above-described inhibitory effects of the RESV isomers on RNS production may be related at least in part to effects on iNOS gene or protein expression, since c-RESV (100 M) inhibited iNOS mRNA production (determined by semiquantitative RT-PCR) and iNOS protein synthesis (determined by slot blot assay) in murine macrophages treated with LPS and IFN- [for more details and numerical

Comparative Studies of the Antioxidant Effects of Cis- and Trans-Resveratrol

Current Medicinal Chemistry, 2006, Vol. 13, No. 1

95

Fig. (6). Hypothesized interference of c-RESV and t-RESV with the LPS and IFN-/NF-B/NO signal transduction pathway in murine macrophages (for more details see text).

data, see ref. 25]. Similar results have been obtained for tRESV by other authors [see, e.g., refs. 77-78] and by us (unpublished results). These effects of the RESV isomers on iNOS gene and protein expression may be mediated by inhibition of NF-B activation, mainly as a result of inhibition of the degradation and phosphorylation of IB, as previously reported for tRESV by Murakami et al. [79] (Fig. 6). The precise effects of c-RESV and t-RESV on the NF-B signaling pathway are currently being evaluated in our laboratory. The preliminary results obtained (basically using a DNA hybridization array containing 96 NF-B-related genes) indicate that c-RESV provokes an increase in the IB gene expression and a down-regulation of two genes of the NFB family, NF-B1 (p50) and NF-B2 (p52) [for more details, see ref. 80]. Similar results have been obtained with t-RESV (unpublished results). 3. DPPH-SCAVENGING ACTIVITY OF c-RESV AND t-RESV The potential capacity of c-RESV and t-RESV to scavenge free radicals other than O2 has been studied using the stable free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH). In these assays, the free-radical-scavenging activity of an antioxidant is evaluated by measurement of the decrease in absorbance at 515 nm. Because of its odd electron, the DPPH radical shows a strong absorbance band at this visible-light wavelength (a deep purple color). When

this electron is paired off (by acceptance of an electron or hydrogen radical from an antioxidant compound), a stable DPPH-H molecule is generated, leading to a reduction in absorbance (color change from purple to yellow) proportional to the number of electrons taken up [see, e.g., refs. 81,82]. Several authors have studied the free-radical-scavenging properties of t-RESV and c-RESV using DPPH assay. Basly et al. [83] found that c-RESV is more efficient than t-RESV as a scavenger of DPPH. However, this finding contradicts the results of Fauconneau et al. [84] and Waffo-Teguo et al. [85], who found that c-RESV is less efficient than t-RESV for scavenging DPPH, as well as the results of Stivala et al. [24], who found that the two isomers scavenge DPPH with similar efficiency. 4. EFFECTS OF c-RESV AND t-RESV ON LIPID PEROXIDATION Lipid peroxidation is initiated by the attack on a fatty acid or fatty acyl side chain by any chemical species (usually a free radical) that has sufficient reactivity to abstract a hydrogen atom from a methylene carbon in the side chain. Most biological studies of lipid peroxidation involve transition metal ions, added to the reaction mixtures. When ferrous ions, cuprous ions, or certain chelates of these ions (e.g. Fe2+ -ADP) are added to liposomes, lipoproteins, cultured cells or isolated biological membranes (such as microsomes, mitochondrias, plasma-membrane fractions,

96 Current Medicinal Chemistry, 2006, Vol. 13, No. 1

F. Orallo

etc.), peroxidation occurs [see, e.g., refs. 86,87]. The oxidized forms of these transition metal ions (e.g. Fe3+ , Cu 2+) can also accelerate peroxidation if a reducing agent (e.g. ascorbate) is present. Free radical generators such as 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) [88] and tert-butylhydroperoxide (TBHP) [89] are also widely used in assays of this type. A number of free radicals (mainly peroxyl radicals which then initiate peroxidation) can usually be detected in these various reaction mixtures [for more details, see, e.g., ref. 87]. Several authors have studied the effects of the isomers of RESV on lipid peroxidation under different experimental conditions. Fauconneau et al. [84] found that t-RESV is more effective than c-RESV for reducing Fe2+-induced lipid peroxidation in rat liver microsomes and Cu2+-induced lipid peroxidation in human low-density lipoproteins (LDL). Similar results were obtained by Waffo-Teguo et al. [85] in human LDL, by Belguendouz et al. [23] in porcine LDL, and by Stivala et al. [24] in rat liver microsomes (using Fe 2+ -ascorbate in place of Fe2+ alone) and in cultured normal human fibroblasts (using TBHP). By contrast, Belguendouz et al. [23] found that c-RESV and t-RESV inhibit AAPH-mediated porcine LDL oxidation with similar potency. These antioxidant effects of the RESV isomers may be due to one or more of the following mechanisms: 1. 2. Direct quelation by RESV of metal ions (e.g. Cu2+) [see refs. 1,23]; Inhibition by RESV of free radical generation by metal ions (e.g. Fe2+ ) or other generators (e.g. TBHP); Scavenging of the free radicals by RESV.

significantly interfere with macrophage function. Specifically, both c-RESV and t-RESV exhibit typical antioxidant activity, i.e. they block extra- and intracellular production of ROS (basically O2 ) by inflammatory rat peritoneal macrophages, through inhibition of NAD(P)H oxidase activity, and inhibit the production of NO (at least in part by inhibition of iNOS gene and protein expression). In adition, in in vitro studies, c-RESV and t-R E S V scavenge the stable free radical DPPH and inhibit both lipid peroxidation (measured under different experimental conditions) and citroneal thermo-oxidation.

These inhibitory effects of c-RESV and t-RESV are qualitatively similar. Therefore, the different spatial conformation of c-RESV [versus that of the trans isomer, (see Fig. 1 )] does not seem to markedly modify its interaction with the potential cellular targets. c-RESV and t-RESV seem to have different hepatic metabolisms (specifically, regio- and stereoselective glucuronidation, catalyzed by different UDPglucuronosyltransferase isoforms) [90]; but notwithstanding this, c-RESV (like t-RESV) has been reported to be effectively absorbed after oral administration in rats, and to accumulate in rat tissues such as the heart, liver and kidney [91,92]. In addition to free RESV isomers (c-RESV and t-RESV) present at variable concentrations in red wines, a number of RESV derivatives (mainly the 3-O--D-glucosides) are also present [see, e.g., refs. 1,4]. These may be absorbed directly, as reported for the rat small intestine [93,94], and/or hydrolysed before absorption by glucosidases present in the human intestinal tract, with subsequent release of free RESV [95,96; for reviews, see, e.g., refs. 4,97]. These RESV derivatives may contribute to the biologically available RESV dose. For these reasons, Bertelli et al. [98] have concluded that an average drinker of wine can absorb a sufficient amount of RESV, at least in the long term, to explain the beneficial effects of red wine on health. In addition, it is interesting to note that increased macrophage activation [e.g. as a result of overproduction of ROS and RNS, or abnormally high NAD(P)H oxidase or iNOS activity] and abnormally high oxidative stress (e.g. LDL oxidation) have been observed in different stages of coronary heart disease, and in a number of inflammatory processes (including atherosclerosis) [see, e.g., ref. 99]. Bearing in mind the above considerations and assuming that the RESV isomers show similar behaviour in humans and in vivo, it can be concluded that: 1. The apparent beneficial (cardioprotective) effects of moderate red wine consumption may be due to the combined effects of t-RESV and c-RESV; The RESV isomers may be of value as structural templates for the design and development of new drugs useful for reducing pathogenesis of cardiovascular diseases and other oxidative-stressrelated pathologies.

3.

Further studies are required to clarify the precise mechanisms by which the RESV isomers inhibit lipid peroxidation. 5. OTHER ANTIOXIDANT EFFECTS OF c-RESV VERSUS t-RESV Finally, Stivala et al. [24] have studied the possible antioxidant effects of the RESV isomers using citroneal thermo-oxidation. In this assay, the aldehyde (-)-citronellal is used as the oxidation substrate: it is subjected to heating and intensive oxygenation in chlorobenzene (which acts as an oxidant agent), and its disappearance with the consequent formation of degradation products is monitored by gas chromatography. The antioxidant efficacy of the RESV isomers was measured by determining the efficient quantity (EQ), i.e. the concentration required for each isomer to double the half-life of citronellal with respect to the control reaction (citronellal without antioxidant). The results indicated that t-RESV inhibited citroneal thermo-oxidation more effectively than c-RESV (EQ = 135 8.81 and 241 38.0 M, respectively). 6. CONCLUDING REMARKS In summary, the studies described in this review indicate that c-RESV and t-RESV at micromolar concentrations

2.

However, further studies are required to confirm that the antioxidant effects of the RESV isomers observed in vitro likewise occur in vivo (after long-term treatment), with the

Comparative Studies of the Antioxidant Effects of Cis- and Trans-Resveratrol

Current Medicinal Chemistry, 2006, Vol. 13, No. 1

97

aim of confirming the possible therapeutic applications mentioned above. Until conclusive long-term clinical, epidemiological and toxicological studies with RESV have been yet carried out in healthy and unhealthy human volunteers, the RESV isomers should be considered as fashionable dietary components with interesting antioxidant and other biological properties (e.g. antineoplastic activity) which may be therapeutically beneficial. 7. ACKNOWLEDGMENTS I apologize for failing to cite many relevant primary papers because of space constraints. The work in my laboratory was supported in part by grants from the Spanish Ministerio de Ciencia y Tecnologa (SAF2002-0245), Almirall-Prodesfarma Laboratories (Pharmacology Award 2003) and the Xunta de Galicia (PGIDIT02BTF20301PR and PGIDIT05BTF20302PR), Spain. I am especially grateful to Almirall-Prodesfarma Laboratories and the Spanish Pharmacological Society for granting me the 2003 Pharmacology Award. NON-STANDARD ABBREVIATIONS AAPH BSA c-RESV DCF DPI DPPH EQ H2DCFDA H2HFF HX IFN- LDL LPS = 2,2'-azobis(2-amidinopropane) dihydrochloride = Bovine serum albumin = Resveratrol, cis isomer = Dichlorofluorescein = Diphenyleneiodonium chloride = 1,1-diphenyl-2-picryl-hydrazyl = Efficient quantity = 2,7-dichlorodihydrofluorescein (H2DCF) diacetate = Dihydro-2,4,5,6,7,7-hexafluorofluorescein = Hypoxanthine = Interferon gamma = Low-density lipoproteins = Lipopolysaccharide

RESV RNS ROS RT-PCR SOD TBHP t-RESV XO REFERENCES

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]

= Resveratrol = Reactive nitrogen species = Reactive oxygen species = Reverse transcription-polymerase chain reaction = Superoxide dismutase = Tert-butylhydroperoxide = Resveratrol, trans isomer = Xanthine oxidase

NADH/NADPH = Nicotinamide adenine dinucleotide/ nicotinamide adenine dinucleotide phosphate oxidase NBT NF-B NO iNOS O2

= Nitroblue tetrazolium = Nuclear factor-B = Nitric oxide = Inducible nitric oxide synthase = Superoxide radical = Peroxynitrite = Protein kinase C = Phorbol 12-myristate 13-acetate

ONOO PKC PMA

Frmont, L. Life. Sci., 2000, 66, 663. Pervaiz, S. FASEB J., 2003, 17, 1975. Aggarwal, B.B.; Bhardwaj, A.; Aggarwal, R.S.; Seeram, N.P.; Shishodia, S.; Takada, Y. Anticancer Res., 2004, 24, 2783. Soleas, G.J.; Diamandis, E.P.; Goldberg, D.M. Clin. Biochem., 1997, 30, 91. Soleas, G.J.; Diamandis, E.P.; Goldberg, D.M. Adv. Exp. Med. Biol., 2001, 492, 159. Alarcn de la Lastra, C.; Villegas, I. Mol. Nutr. Food Res., 2005, 49, 405. Takaoka, M.J. J. Fac. Sci. Hokkaido Imp. Univ., 1940, 3, 1. Nonomura, S.; Kanagawa, H.; Makimoto, A. Yakugaku Zasshi., 1963, 83, 988. Langcake, P.; Pryce, R. J. Physiol. Plant Pathol., 1976, 9, 77. Siemann, E.H.; Creasy, L.L. Am. J. Enol. Vitic., 1992, 43, 49. Renaud, S.; De Lorgeril, M. Lancet, 1992, 339, 1523. Wu, J.M.; Wang, Z.R.; Hsieh, T.C.; Bruder, J.L.; Zou, J.G.; Huang, Y.Z. Int. J. Mol. Med., 2001, 8, 3. Granados-Soto, V. Drug News Perspect., 2003, 16, 299. Ulrich, S.; Wolter, F.; Stein, J.M. Mol. Nutr. Food Res., 2005, 49, 452. Hao, H.D.; He, L.R. J. Med. Food, 2004, 7, 290. Bradamante, S.; Barenghi, L.; Villa, A. Cardiovasc. Drug. Rev., 2004, 22, 169. Delmas, D.; Jannin, B.; Latruffe, N. Mol. Nutr. Food Res., 2005, 49, 377. Olas, B.; Wachowicz, B. Platelets, 2005, 16, 251. Middleton, E.Jr.; Kandaswami, C.; Theoharides, T.C. Pharmacol. Rev., 2000, 52, 673. Palomino, O.; Gmez-Serranillos, M.P.; Slowing, K.; Carretero, E.; Villar, A. J. Chromatogr. A, 2000, 870, 449. Burns, J.; Yokota, T.; Ashihara, H.; Lean, M.E.; Crozier, A. J. Agric. Food Chem., 2002, 50, 3337. Wang, Y.; Catana, F.; Yang, Y.; Roderick, R.; van Breemen, R.B. J. Agric. Food Chem., 2002, 50, 431. Belguendouz, L.; Frmont, L.; Linard, A. Biochem. Pharmacol., 1997, 53, 1347. Stivala, L.A.; Savio, M.; Carafoli, F.; Perucca, P.; Bianchi, L.; Maga, G.; Forti, L.; Pagnoni, U.M.; Albini, A.; Prosperi, E.; Vannini, V. J. Biol. Chem., 2001, 276, 22586. Leiro, J.; lvarez, E.; Arranz, J.A.; Laguna, R.; Uriarte, E.; Orallo, F. J. Leukoc. Biol., 2004, 75, 1156. Orallo, F. In Resveratrol in Health and Disease, Aggarwal, B.B.; Shishodia, S., Eds.; CRC Press: Boca Raton, USA, 2005, pp. 577600. Varache-Lembge, M.; Waffo-Teguo, P.; Richard, T.; Monti, J.P.; Deffieux, G.; Vercauteren, J.; Mrillon, J.M.; Nuhrich, A. Med. Chem. Res., 2000, 10, 253. Pettit, G.R.; Grealish, M.P.; Jung, M. K.; Hamel, E.; Pettit, R.K.; Chapuis, J.C.; Schmidt, J.M. J. Med. Chem., 2002, 45, 2534. Leiro, J.; lvarez, E.; Garca, D.; Orallo, F. I n t . Immunopharmacol., 2002, 2, 767. Cai, H.; Griendling, K.K.; Harrison, D.G. Trends Pharmacol. Sci., 2003, 24, 471. Griendling, K.K. Heart, 2004, 90, 491. El-Benna, J.; Dang, P.M.; Gougerot-Pocidalo, M.A.; Elbim, C. Arch. Immunol. Ther. Exp. (Warsz), 2005, 53, 199.

98 Current Medicinal Chemistry, 2006, Vol. 13, No. 1 [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] Lassgue, B.; Clempus, R.E. Am. J. Physiol. Regul. Integr. Comp. Physiol., 2003, 285, R277. Geiszt, M.; Leto, T.L. J. Biol. Chem., 2004, 279, 51715. Brandes, R.P.; Kreuzer, J. Cardiovasc. Res., 2005, 65, 16. Segal, A.W. Annu. Rev. Immunol., 2005, 23, 197. Harrison, R. Free Radic. Biol. Med., 2002, 33, 774. Berry, C.E.; Hare, J.M. J. Physiol., 2004, 555, 589. Hille, R. Arch. Biochem. Biophys., 2005, 433, 107. Martin, H.M.; Hancock, J.T.; Salisbury, V.; Harrison, R. Infect. Immun., 2004, 72, 4933. Cos, P.; Ying, L.; Calomme, M.; Hu, J.P.; Cimanga, K.; Van Poel, B.; Pieters, L.; Vlietinck, A.J.; Berghe, D.V. J. Nat. Prod., 1998, 61, 71. Robak, J.; Gryglewski, R.J. Biochem. Pharmacol., 1988, 37, 837. Hung, L.M.; Su, M.J.; Chu, W.K.; Chiao, C.W.; Chan, W.F.; Chen, J.K. Br. J. Pharmacol., 2002, 135, 1627. Zhou, C.X.; Kong, L.D.; Ye, W.C.; Cheng, C.H.; Tan, R.X. Planta Med., 2001, 67, 158. Orallo, F.; lvarez, E.; Camia, M.; Leiro, J.M.; Gmez, E.; Fernndez, P. Mol. Pharmacol., 2002, 61, 294. Rivadulla, E.; lvarez, E.; Leiro, J.M.; Quezada, E.; Uriarte, E.; Orallo, F. The Pharmacologist, 2002, 44, suppl. 2, A144, 92.5 (abstract). Liu, J.C.; Chen, J.J.; Chan, P.; Cheng, C.F.; Cheng, T.H. Hypertension, 2003, 42, 1198. Poolman, T.M.; Ng, L.L.; Farmer, P.B.; Manson, M.M. Free Radic. Biol. Med., 2005, 39, 118. Allen, L.A. Microbes Infect., 2003, 5, 1329. Voyich, J.M.; Musser, J.M.; DeLeo, F.R. Microbes Infect., 2004, 6, 1117. Ischiropoulos, H.; Gow, A.; Thom, S.R.; Kooy, N.W.; Royall, J.A.; Crow, J.P. In Nitric Oxide. Part C. Biological and Antioxidant activities. Methods in Enzymology, Packer, L., Ed.; Academic Press: New York, 1999; Vol. 301, pp. 367-373. O'Malley, Y.Q.; Reszka, K.J.; Britigan, B.E. Free Radic. Biol. Med., 2004, 36, 90. Frstermann, U.; Gath, I.; Schwarz, P.; Closs, E.I.; Kleinert, H. Biochem. Pharmacol., 1995, 50, 1321. Marn, J.; Rodrguez-Martnez, M.A. Pharmacol. Ther., 1997, 75, 111. Hobbs, A.J.; Higgs, A.; Moncada, S. Annu. Rev. Pharmacol. Toxicol., 1999, 39, 191. Domenico, R. Curr. Pharm. Des., 2004, 10, 1667. Mariotto, S.; Menegazzi, M.; Suzuki, H. Curr. Pharm. Des., 2004, 10, 1627. Ghafourifar, P.; Cadenas, E. Trends Pharmacol. Sci., 2005, 26, 190. Korhonen, R.; Lahti, A.; Kankaanranta, H.; Moilanen, E. Curr. Drug Targets Inflamm. Allergy, 2005, 4, 471. Tiefenbacher, C.P.; Kreuzer, J. Curr. Vasc. Pharmacol., 2003, 1, 1231. Lindgren, H.; Stenman, L.; Tarnvik, A.; Sjstedt, A. Microbes Infect., 2005, 7, 467. Murphy, M.P.; Packer, M.A.; Scarlett, J.L.; Martin, S.W. Gen. Pharmacol., 1998, 31, 179. Drge, W. Physiol. Rev., 2002, 82, 47. Muscoli, C.; Cuzzocrea, S.; Riley, D.P.; Zweier, J.L.; Thiemermann, C.; Wang, Z.Q.; Salvemini, D. Br. J. Pharmacol., 2003, 140, 445. Faraci, F.M.; Didion, S.P. Arterioscler. Thromb. Vasc. Biol., 2004, 24, 1367. [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99]

F. Orallo Petersen, S.V.; Enghild, J.J. Biomed. Pharmacother., 2005, 59, 175. Hayden, M.S.; Ghosh, S. Genes Dev., 2004, 18, 2195. Shishodia, S.; Aggarwal, B.B. Biochem. Pharmacol., 2004, 68, 1071. Xiao, C.; Ghosh, S. Adv. Exp. Med. Biol., 2005, 560, 41. Beutler, B. Nature, 2004, 430, 257. Takeda, K.; Akira, S. Int. Immunol., 2005, 17, 1. Theofilopoulos, A.N.; Baccala, R.; Beutler, B.; Kono, D.H. Annu. Rev. Immunol., 2005, 23, 307. Schroder, K.; Hertzog, P.J.; Ravasi, T.; Hume, D.A. J. Leukoc. Biol., 2004, 75, 163. Rosenzweig, S.D.; Holland, S.M. Immunol. Rev., 2005, 203, 38. Blanchette, J.; Jaramillo, M.; Olivier, M. Immunology, 2003, 108, 513. Viatour, P.; Merville, M.P.; Bours, V.; Chariot, A. Trends Biochem. Sci., 2005, 30, 43. Tsai, S.H.; Lin-Shiau, S.Y.; Lin, J.K. Br. J. Pharmacol., 1999, 126, 673. Chan, M.M.; Mattiacci, J.A.; Hwang, H.S.; Shah, A.; Fong, D. Biochem. Pharmacol., 2000, 60, 1539. Murakami, A.; Matsumoto, K.; Koshimizu, K; Ohigashi, H. Cancer Lett., 2003, 195, 17. Leiro, J.; Arranz, J.A.; Fraiz, N.; Sanmartin, M.L.; Quezada, E.; Orallo, F. Int. Immunopharmacol., 2005, 5, 393. Matthus, B. J. Agric. Food Chem., 2002, 50, 3444. Aruoma, O.I. Mutat. Res., 2003, 523-524, 9. Basly, J.P.; Marre-Fournier, F.; Le Bail J.C.; Habrioux, G.; Chulia, A.J. Life Sci., 2000, 66, 769. Fauconneau, B.; Waffo-Teguo, P.; Huguet, F.; Barrier, L.; Decendit, A.; Mrillon, J.M. Life Sci., 1997, 61, 2103. Waffo-Teguo, P.; Fauconneau, B.; Deffieux, G.; Huguet, F.; Vercauteren, J.; Mrillon, J.M. J. Nat. Prod., 1998, 61, 655. Halliwell, B.; Chirico, S. Am. J. Clin. Nutr., 1993, 57, 715S. Rice-Evans, C.; Leake, D.; Bruckdorfer, K.R.; Diplock, A.T. Free Radic. Res., 1996, 25, 285. Yoshida, Y.; Itoh, N.; Saito, Y.; Hayakawa, M.; Niki, E. Free Radic. Res., 2004, 38, 375. Azorn, I.; Bella, M.C.; Iborra, F.J.; Fornas, E.; Renau-Piqueras, J. Free Radic. Biol. Med., 1995, 19, 795. Aumont, V.; Krisa, S.; Battaglia, E.; Netter, P.; Richard, T.; Mrillon, J.M.; Magdalou, J.; Sabolovic, N. Arch. Biochem. Biophys., 2001, 393, 281. Bertelli, A.A.; Giovannini, L.; Stradi, R.; Bertelli, A.; Tillement, J.P. Int. J. Tissue React., 1996, 18, 67. Bertelli, A.A.; Giovannini, L.; Stradi, R.; Urien, S.; Tillement, J.P.; Bertelli, A. Int. J. Clin. Pharmacol. Res., 1996, 16, 77. Andlauer, W.; Kolb, J.; Siebert, K.; Furst, P. Drugs Exp. Clin. Res., 2000, 26, 47. Kuhnle, G.; Spencer, J.P.; Chowrimootoo, G.; Schroeter, H.; Debnam, E.S.; Srai, S.K.; Rice-Evans, C.; Hahn, U. Biochem. Biophys. Res. Commun., 2000, 272, 212. Goldberg, D.M.; Ng, E.; Karumanchiri, A.; Diamandis, E.P.; Soleas, G.J. Am. J. Enol. Vitic., 1996, 47, 415. Soleas, G.J.; Angelini, M.; Grass, L.; Diamandis, E.P.; Goldberg, D.M. Methods Enzymol., 2001, 335, 145. Wenzel, E.; Somoza, V. Mol. Nutr. Food Res., 2005, 49, 472. Bertelli, A.; Bertelli, A.A.E.; Gozzini, A.; Giovannini, L. Drugs Exp. Clin. Res., 1998, 24, 133. Stocker, R.; Keaney, J.F.Jr. Physiol. Rev., 2004, 84, 1381.

[52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65]

Received: May 17, 2005

Revised: September 5, 2005

Accepted: September 10, 2005

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.