Regulation of cellular signals from nutritional molecules: a specific role for phytochemicals, beyond antioxidant activity

Free Radical Biology & Medicine 45 (2008) 1205–1216

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Review Article

Regulation of cellular signals from nutritional molecules: a specific role for phytochemicals, beyond antioxidant activity Fabio Virgili a,⁎, Maria Marino b a b

National Institute for Food and Nutrition Research, Via Ardeatina, 546, I-00178 Roma, Italy Department of Biology, University Roma Tre, Viale G. Marconi, 446, I-00146 Roma, Italy

a r t i c l e

i n f o

Article history: Received 16 May 2008 Revised 21 July 2008 Accepted 1 August 2008 Available online 8 August 2008 Keywords: Antioxidants Polyphenols Signal transduction pathways Nuclear receptors

a b s t r a c t Phytochemicals (PhC) are a ubiquitous class of plant secondary metabolites. A “recommended” human diet should warrant a high proportion of energy from fruits and vegetables, therefore providing, among other factors, a huge intake of PhC, in general considered “health promoting” by virtue of their antioxidant activity and positive modulation, either directly or indirectly, of the cellular and tissue redox balance. Diet acts through multiple pathways and the association between the consumption of specific food items and the risk of degenerative diseases is extremely complex. Recent literature suggests that molecules having a chemical structure compatible with a putative antioxidant capacity can actually “perform” activities and roles independent of such capacity, interacting with cellular functions at different levels, such as affecting enzyme activities, binding to membrane or nuclear receptors as either an elective ligand or a ligand mimic. Inductive or signaling effects may occur at concentrations much lower than that required for effective antioxidant activity. Therefore, the “antioxidant hypothesis” is to be considered in some cases an intellectual “shortcut” possibly biasing the real understanding of the molecular mechanisms underlying the beneficial effects of various classes of food items. In the past few years, many exciting new indications elucidating the mechanisms of polyphenols have been published. Here, we summarize the current knowledge of the mechanisms by which specific molecules of nutritional interest, and in particular polyphenols, play a role in cellular response and in preventing pathologies. In particular, their direct interaction with nuclear receptors and their ability to modulate the activity of key enzymes involved in cell signaling and antioxidant responses are presented and discussed. © 2008 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of dietary components on human health . . . . . . . . . . . . . . . . . . . . . . . . . . Bioavailability of dietary polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential mechanisms for nutritional molecules . . . . . . . . . . . . . . . . . . . . . . . . . . Nutritional molecules: “not necessarily antioxidants” . . . . . . . . . . . . . . . . . . . . . . Improving endothelial function by diet: the case of wine . . . . . . . . . . . . . . . . . . . . Nutritional molecules as estrogen receptor ligands . . . . . . . . . . . . . . . . . . . . . . . Nutritional molecules as ligands of other nuclear receptors . . . . . . . . . . . . . . . . . . . The peroxisome proliferator-activated receptors and the modulation of energy homeostasis by The pregnane X receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The estrogen-related receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Fax: +390651494550. E-mail address: [email protected] (F. Virgili). 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.08.001

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Introduction In the past few decades, this journal has witnessed the increasing interest attracted by reactive oxygen–nitrogen species (RONS) because of their importance in normal cellular and tissue physiology [1] and also their putative role in the development of a wide number of degenerative pathologies, including cardiovascular disease (CVD), neurodegenerative diseases, chronic inflammation, and several cancers [2–4]. RONS have been also considered major determinants in tissue degeneration and dysfunction associated with aging [5]. The role of RONS in the pathogenesis of different degenerative diseases was originally attributed to a “nonspecific” oxidative damage of biological targets leading to tissue degeneration and loss of function [6]. Later on, a second more specific mechanism for the role of RONS in cellular activities was clearly identified and described, owing to the direct participation of reactive oxygen and nitrogen species to intracellular signaling [7,8]. In fact, several RONS, including nitric oxide (NO) and hydrogen peroxide (H2O2), participate in cell signaling, through narrow concentration ranges that eventually signal for a repair response and also for opposite responses, i.e., proliferation or apoptosis. Moreover, alterations in oxidative metabolism have long been known to occur during differentiation and development [9–12]. The discovery of specific genes and pathways affected by oxidants led to the hypothesis that RONS serve as intracellular messengers in gene regulatory and signal transduction pathways [13]. Several enzymes and ion channels (e.g., calcium channels) that are involved in cell signaling mechanisms are potential targets of RONS.

Enzymes include guanylyl cyclase, phospholipase C, phospholipase A2, and phospholipase D. Activating protein-1 (AP-1), nuclear factor κB (NF-κB), human insulin receptor kinase activity, c-Src family kinases, Jun N-terminal kinase, and p38 mitogen-activated protein kinase (MAPK) are components of some of the most well-known signaling mechanisms that respond to changes in the thiol/disulfide redox state [14]. In addition, several growth factors, cytokines, or other ligands act as triggers of RONS production in nonphagocytic cells through their corresponding membrane receptors. RONS generation induced by extracellular stimuli has been reported to mediate a positive feedback effect on signal transduction from these receptors, because intracellular signaling is often enhanced by RONS (see [15] for review) or by a pro-oxidative shift of the intracellular thiol/disulfide redox balance in the breast cancer cell line MCF-7 [16]. H2O2 is one of the most important RONS in cellular systems. It is endogenously synthesized in specific cell types as a response to activation by specific cytokines or growth factors, acting as a second messenger to stimulate protein kinase cascades coupled to inflammatory gene expression or in control of the cell cycle [17]. Exposure to high concentrations of H2O2 as well as a decreased ratio between thiol and thiol disulfide levels generally leads to sustained tyrosine phosphorylation in numerous proteins, in part owing to the oxidative inhibition of protein tyrosine phosphatases in various cultured cell types [18,19]. Significant inhibition of phosphatase activity, associated with an increased net phosphorylation level of receptor tyrosine kinases, has also been reported to be induced by various types of oxidative stressors, including high doses of RONS, UV irradiation, or

Fig. 1. (a and b) The classical view of oxidant–antioxidant effects on cell signaling and response. RONS can act on cell signaling, either directly or indirectly, through changes in the redox cellular equilibrium (e.g., a decrease in the GSH to GSSG ratio). Antioxidants modulate the RONS-mediated cell response by shielding their reactivity or reducing their availability, at both the extracellular and the intracellular level. (c and d) Various nutritional molecules (phytochemicals) interact with cell signaling thanks to mechanisms independent of their antioxidant properties, by directly affecting the activities of a wide spectrum of cellular targets, including key enzymes and membrane and nuclear receptors. In the presence of perturbations in the cellular redox status, a combination of the two paths occurs, resulting in a prevalence of RONS-driven cellular signaling and response, with respect to phytochemical-mediated signals.

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alkylating agents [18,19]. P38 MAPK and related gene expression has been reported to be activated by H2O2 in fibroblasts and in MCF-7 breast cancer cells, modulating cellular senescence [20]. Similarly, Lu and co-workers [21] reported that the p38 pathway modulates the induction of the activating transcription factor 3 (ATF3) by oxidant signals, playing an important role in the proapoptotic cellular response in HeLa cells and in mouse embryonic fibroblasts. In the same paper, H2O2 was also shown to inhibit phosphatases, probably through the direct oxidation of cysteine in the active site of these enzymes [21]. Various molecules of nutritional interest having a chemical structure compatible with putative antioxidant properties in vivo, in particular flavonoids, isoprenoids, and methyl-tocols, among other dietary phytochemicals (PhC), have been considered able to modulate cellular responses to various stimuli interacting with RONS-mediated intracellular signaling by (i) scavenging reactive oxygen species or suppressing their generation, either by inhibiting enzymes or by chelating trace elements involved in free radical production [22,23], or (ii) protecting antioxidant defenses or up-regulating intracellular signaling resulting in the antioxidant cellular response [24–27]. This twofold “antioxidant” activity has usually been considered beneficial for human health and, according to this hypothesis, a huge number of preparations are commercially available on the market in the form of plant extracts or mixtures, containing varying amounts of isolated PhC as dietary supplements and as health food products. The commercial success of these supplements claimed as antioxidants is evident, even though several activities and mechanisms, in part or totally independent of PhC participation in the intracellular redox balance, have been also described [28,29]. In fact, a spectrum of cellular effects not directly related to antioxidant capacity has been recently reported for both tocopherols and tocotrienols, a family of nutritional compounds sharing vitamin E activity [30,31], and for flavonoids [32–34], widening the perspective of research on the relationship between nutrition and health. Therefore, distinct complex mechanisms of actions, possibly interacting with one another, for nutritional PhC on cell signaling and response can be hypothesized not necessarily depending on antioxidant mechanisms (Fig. 1). In the rest of this paper, some of the current knowledge of the mechanisms by which specific food items or molecules of nutritional interest play a role in preventing degenerative pathologies will be summarized. In particular, we will focus on the activity of red wine on endothelial cell functions and the activity of isolated flavonoids on signaling that is dependent on various nuclear receptors as illustrative paradigmatic examples of the complex interactions between nutritional molecules and cell signaling and response. Effects of dietary components on human health The defense against the detrimental effects of RONS achieved by nutritional antioxidant molecules has been considered pivotal in determining the risk of disease [35,36]. Even though the real contribution of dietary antioxidants to the total antioxidant capacity at the tissue level is not yet clear, a “typical” conclusion drawn from epidemiological and supplementation studies is that “high intake of antioxidant-rich foods is inversely related to cancer risk” (see, e.g., [36]). The close relationship between diet and cancer is suggested by the large variation in rates of specific cancers in different countries and by the spectacular changes observed in the incidence of cancer in migrating populations [37,38]. These observations are strengthened by many experimental data obtained from studies using cellular and animal models [38–40]. The chemopreventive activity of plant-based food has been suggested to be associated with the ability to block the progression of latent microtumors [38]. This effect could be elicited via the modulation of the enzymatic systems responsible for neutralizing free radicals [41,42] and/or by directly inducing cancer cell death by

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apoptosis. For example, phenethyl isothiocyanate from cruciferous vegetables, curcumin from turmeric, resveratrol from grapes, and naringenin from oranges have all been shown to possess strong proapoptotic activity against cells isolated from a variety of tumors [33,43,44]. However, PhC, originating from various plant sources, have shown variable effects on different types of cancer [45,46]. Epidemiological, animal, and cell culture studies have demonstrated that soy-derived isoflavones, rye bran, or isolated lignans may play a protective role against several types of cancer [47–49]. Moreover, soy-derived isoflavones have been reported to be more beneficial against cancer if consumed before puberty or during adolescence or at very high doses [47,50,51]. Similarly, high vitamin E intake has been proposed to reduce the risk of some forms of cancer, including prostate and colon cancer [52,53]. It is important to mention that studies based on animal and cell cultures have demonstrated that specific PhC, like genistein and resveratrol, may have opposite effects, not only inhibiting cell proliferation but also stimulating tissue growth such as uterine and breast cancer growth, depending on dose and timing of exposure [54,55]. Several animal and in vitro studies indicate that isoflavones, and in particular daidzein and genistein, prevent bone loss [56,57]. However, long-lasting, well-designed clinical trials are needed to unambiguously prove a specific beneficial role for PhC in the prevention of osteoporosis. Several studies have also addressed the role of antioxidant intake in aging-associated diseases [58]. Surveys indicate that aged people are particularly at risk for marginal deficiency of vitamins and trace elements. Recently, it has been demonstrated that dietary habits compatible with high antioxidant intake may have a preventive role in age-associated cancer, due to the modulation of the expression of a number of different cytokines and molecules driving cell-to-cell junction [59]. These findings concur with epidemiologic, clinical, and animal studies in building up the hypothesis that high intake of specific food items is associated with a reduced risk of cardiovascular disease and cancer, the leading causes of morbidity and mortality among the elderly. Food phenolics have attracted great interest in the past few decades, owing to their apparent beneficial effects on human health and, in particular, for their favorable effects on the risk of cardiovascular disease [60], but significant differences have been reported between the effects of the intake of complex food matrices and that of purified molecules. For example, many studies have shown that whole-soybean foods exert favorable effects on cardiovascular disease, especially on the serum lipoprotein profile, whereas isolated purified isoflavones seem to improve only cultured endothelial cell functions [61]. The cardioprotective effect of isolated PhC, however, has yet to be substantiated by long-term controlled trials. Similarly, data on the protective role of nutritional antioxidant molecules in models of atherosclerosis have not been fully confirmed in humans. Observational and epidemiological data, as well as randomized trials, provide no clear-cut indications for cardiovascular protection. Despite the lack of a general consensus, there is a general, though not fully justified, agreement that the intake of antioxidant-rich foods can contribute to vascular functions [62,63]. Bioavailability of dietary polyphenols The two main classes of polyphenols are flavonoids and phenolic acids. Flavonoids are further classified as flavones, flavonols, flavanols, flavanones, isoflavones, proanthocyanidins, and anthocyanins [64]. Polyphenols are widespread constituents of fruits, vegetables, cereals, dry legumes, chocolate, and plant-derived beverages, such as tea, coffee, and wine, representing, therefore, the most abundant “minor components” in the diet [64]. On the basis of the 24-h dietary recall in the NHANES 1999–2002 study and

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utilizing the database of the U.S. Department of Agriculture, it has been calculated that the average total flavonoid intake of the subjects older than 19 years was about 190 mg [65], but higher intakes of total polyphenols, up to 1000 mg/day, have been calculated by other authors in the presence of a dietary profile rich in fruit and vegetables [66]. Data on tissue distribution are very scarce even in experimental animals. At 1–6 h after a single load of radiolabeled polyphenols (quercetin, epigallocatechin gallate, quercetin 4′-glucoside, resveratrol) in rats and mice, radioactivity was mainly recovered in blood and tissues of the digestive system, such as the stomach, intestine, and liver [67]. However, polyphenols have also been detected by HPLC analysis in a wide range of tissues in mice and rats, including brain [68], endothelial cells [69], heart, kidney, spleen, pancreas, prostate, uterus, ovary, mammary gland, testes, bladder, bone, and skin [70], in concentrations ranging from 30 to 3000 ng aglycone eq/g tissue depending on the dose administered and the tissue considered. Quercetin aglycone and quercetin metabolites are widely distributed in various tissues, in a concentration on the order of nanomoles per gram of tissue, with the highest concentration in lungs and the lowest in brain and spleen of rats and pigs, after the administration of high doses (up to the equivalent of several grams in an adult man) of quercetin aglycone [71]. Very few studies have addressed the distribution of polyphenol in humans [72,73]. Available data indicate that plasma concentrations are not directly correlated with concentrations in target tissues and therefore that plasma concentrations should not be considered biomarkers of exposure (see Manach et al. for review [74]). The majority of available in vitro studies addressing the understanding of polyphenol mechanisms of action have considered concentrations between 10 and 100 μM [75,76], which are largely too high to be achieved in the circulation under physiological conditions. Indeed, at least in theory, these concentrations could be observed in specific tissues such as skin, in which high amounts of polyphenols can be topically applied, or in the gastrointestinal tract after a meal [77]. Moreover, chemical and structural modifications due to gastrointestinal absorption and metabolization have usually not been taken into account. The comprehensive understanding of the protective mechanisms exerted by polyphenols is hindered by the lack of complete knowledge about their bioavailability. Information about absorption, distribution, metabolism, and excretion of individual flavonoids is in fact scarce. According to these considerations, a really major issue still open for the understanding of the molecular mechanism(s) underlying the effects of food on human health is the impact of PhC metabolism on their biological activities. However, it is important to remark that no study is available at present that addresses the effects of food metabolites in the form circulating in the body, once ingested, absorbed, and distributed to target tissues and organs. Even though a few studies have addressed the effects of biotransformed polyphenols [44,78], the majority of available in vitro studies have been designed and performed by adding to experimental cultured cells food items “as they are in the food”. Alternatively, single, purified phenolic compounds, either as glycone or in their aglycone form, have been added to the cultured cells. These approaches are obviously somehow “naïve” and unable to take into account either the extensive metabolism of polyphenols during gastrointestinal absorption or the possible interactions of different molecules, therefore excluding the assessment of possible synergic/cooperative activities within the same food item. These experimental weaknesses can provide only a very nebulous picture. As an obvious consequence of their complex metabolism and poor bioavailability, the direct transfer of in vitro observations to in vivo conclusions must be cautious. The molecular effects of polyphenols in the form found in food detected in vitro could be not necessarily relevant in vivo, as suggested by our laboratories [44,79] and others [78,80].

Potential mechanisms for nutritional molecules Nutritional molecules: “not necessarily antioxidants” PhC might protect against cancer and/or heart disease through inhibition of oxidative damage [36,45]. The theoretical basis of this protection is very well known. Flavonoids can be taken as an illustrative example of such activity. Their chemical structure is compatible with a one-electron donor activity. They have been demonstrated to function as antioxidants in vitro in both cell cultures and cell-free systems by scavenging superoxide anion, singlet oxygen, and lipid peroxy-radicals and/or by stabilizing free radicals involved in oxidative processes through hydrogenation or complexing with oxidizing species [81]. As a product of this reaction, flavonoids themselves become free radicals, but their conjugated structure allows the remaining orbital electron to be relatively inactive. A theoretical underpinning for the efficacy of flavonoids as antioxidants in vivo comes from the inhibition of low-density lipoprotein (LDL) oxidation. However, the antioxidant efficiency of dietary polyphenols seems to be associated not only with their reductive capacity, but also with their protein-binding properties. It has been suggested that the availability of flavonoids at specific oxidizable sites on LDL may block the occurrence of oxidative attacks and prevent LDL damage in vivo. Potential protein binding and interference with oxidative processes by flavonoids have been proposed to explain their ultimate antioxidant roles in vivo [82]. In some cases, the evidence of antioxidant properties due to a specific chemical structure has opened the avenue to a biased or simplified approach to the understanding of the real role and functions of molecules of nutritional interest in human health and disease. For instance, flavonoids have been considered responsible for one of the most celebrated available examples of the protective role of wine drinking in determining the low incidence of CVD in the presence of the unfavorable high-saturated-fat diet in France, often referred as the “French paradox.” This beneficial effect has been widely attributed to the putative antioxidant capacity of red wine polyphenols [83]. In general, other than wine, any of the beneficial effects observed in epidemiological or intervention studies in association with fruit and vegetable intake have been interpreted on the basis of the putative antioxidant activity of these food items [84], simply ignoring the fact that the antioxidant capacity as a chemical property of a given molecule or of a whole food item is not necessarily associated with an equivalent biological function [85]. Accordingly, more recently, the real impact of flavonoids and in general of polyphenols as antioxidants has been reconsidered and questioned, opening up to the evidence that the molecular basis of their activity is much larger than originally considered [86]. Diverse mechanisms have been proposed to explain the biological activity of polyphenols, including the capacity to bind protein and eventually affect enzyme activity by either competitive or allosteric interactions [87,88]; regulation of signal transduction; modulation of redox-sensitive transcription factors, including Nrf2, NF-κB, and AP-1 [89,90]; glutathione biosynthesis [91]; and gene expression in general [92–94]. Improving endothelial function by diet: the case of wine Red wine is considered one of the major sources of polyphenols in the Mediterranean diet and therefore suggested as a “healthy food” by a large number of medical associations at least in specific groups of populations at risk for cardiovascular disease [95]. Several reports indicated that light-to-moderate wine consumption reduces the risk of mortality by CVD [96]. The protective effects associated with polyphenols have been attributed to various mechanisms leading to the inhibition of the progression of early atherosclerotic lesions to advanced plaque. Several studies have demonstrated that the consumption of polyphenols reduces the development of

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atheromatous lesions. Supplementation of dealcoholized wine, catechins, or quercetin reduced the size of these lesions in apoE-deficient mice. These effects are associated with reduced LDL uptake by macrophages, lower oxidation of isolated LDL, and decreased susceptibility of LDL to aggregation [97]. The administration of purified polyphenols to rats produced a progressive decrease in blood pressure in normal and hypertensive rat [98,99]. Moreover, red wine polyphenols have been shown to inhibit platelet aggregation in vivo and in vitro [100], preventing thrombosis. Short-term administration of red wine polyphenolic compounds has been reported to induce a decrease in blood pressure through an NO-dependent pathway [101]. The antioxidant capacity of red wine polyphenols constitutes the most popular hypothesis on their beneficial effects. However, as mentioned above, experiments in vitro have shown multiple biological activities independent of antioxidant activity, such as inhibition of platelet aggregation and vasorelaxation [102,103] and the ability to modulate the adhesion process [75,76] and promote fibrinolysis [104]. Overall, these considerations strongly suggest the need for models that are able to mimic the complex metabolism as it occurs in humans, to be applied to cellular models for the study of the molecular mechanisms of complex food matrices undergoing significant modification during metabolism. We have recently proposed a novel model to study the effect of complex food matrices, as in this case red wine, on the molecular aspects of cell functions according to a more physiological approach, utilizing healthy human subjects as “bioreactors.” According to this experimental model, subjects are fed red wine and, at appropriate time points after drinking (i.e., 40 min), blood is withdrawn and serum (RWS) utilized to enrich the culture medium of human primary endothelial cells (HUVEC) [79]. According to this experimental approach, we have reported that a group of genes involved in cell adhesion and fibrinolysis (VCAM, ICAM, MCP-1, t-PA, PAI-1, and PAI-2), chosen on the basis of previous in vitro experiments dealing with the beneficial effects of red wine and the regulation of

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the expression of genes involved in the early and progressive stages of atherosclerosis, is differentially expressed after 12 h incubation with RWS in HUVEC. In addition, we reported that the administration of a volume of wine added to cultured cell medium to reach an amount of alcohol (0.015% w/v) in the same range as that utilized in experiments conducted with RWS was associated with a completely different effect on gene expression [79]. The administration of RWS was in fact associated with a significant down-regulation of the expression of the genes considered, with the exception of MCP-1, with respect to controls. The administration of wine “as we drink it” was associated with a very different gene expression profile in comparison to the administration of serum collected after wine drinking. In this case, the level of mRNAs encoding the cell adhesion factors VCAM, ICAM, and MCP-1 was significantly and dramatically up-regulated (several hundredfold in comparison to control, see Fig. 2a). On the other hand, the levels of mRNA encoding the fibrinolytic factors t-PA and PAI-1 were significantly decreased, similar to the administration of RWS, but at a much higher and evidently nonphysiological extent [79]. Upstream of the effect on gene expression, the activation profiles of the transcription factors NF-κB and AP-1 after a proinflammatory stimulus (TNF-α) are also significantly modified by pretreatment with RWS. The incubation of HUVEC with serum containing red wine metabolites induced NF-κB and AP-1 nuclear translocation after 12 h of incubation in the absence of any classical stimulus (Fig. 2b). The treatment with TNF-α in the presence of RWS is associated with a delay in the activation pattern of these transcription factors in comparison with a control serum. Moreover, red wine metabolites also modulate the activation of other transcription factors downstream of cAMP generation, therefore affecting the expression of a number of genes related to this cellular signal (R. Canali, personal communication). Data obtained from this model suggest that the effects of red wine metabolites present in plasma after drinking on the profile of gene expression and transcription factor activation are the results of the combined effect of all wine components, significantly affecting the

Fig. 2. (a) Effects of red wine and serum containing red wine metabolites (RWS) on gene expression in human umbilical vein endothelial cells (HUVEC). RWS was isolated from healthy subjects after 40 min of red wine supplementation. The amounts of red wine and RWS added to cell culture media contained the same range of alcohol, but had different polyphenol contents. 12 h after treatment, adhesion molecule gene expression was assessed using real-time PCR. The addition of red wine to the medium induced a very high upregulation of mRNA levels, whereas treatment with RWS was associated with a down-regulation of the expression of the same genes. (b) RWS-dependent activation of nuclear translocation of transcription factors. 12 h treatment with RWS induced the nuclear translocation of AP-1 and NF-κB in HUVEC (assessed by electromobility shift assay). The effects of the treatment with RWS compared with the treatment with serum from the same subject collected before wine administration (CS) are shown.

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consequences of a proinflammatory stimulus in a direction different from that previously considered. On the other hand, the addition of red wine “as we drink it” to the cell culture induced a very strong, and evidently not physiological, inflammatory and procoagulant pattern of expression in primary endothelial cells [79]. This evidence emphasizes the significant difference in biological activity of complex food matrix components “as they are in food” compared to the same matrix metabolized during gastrointestinal absorption. Experiments in vitro should consider the food–organism interaction in its entire complexity to understand the mechanisms underlying the effects of diet in human health. Biotransformation, and possibly synergism and cross activity between different components, must be taken into account. However, it seems more and more evident that it is not a single dietary factor that acts as a “magic bullet,” but the overall dietary pattern that has a pivotal role in cardiovascular outcome by interplaying with other risk factors, including lifestyle and individual genetic profile. Nutritional molecules as estrogen receptor ligands Estrogens, including 17β-estradiol (E2), are steroid hormones with a peculiar and complex mode of action. They exert pleiotropic effects on a diverse range of tissues, such as ovary, testis, prostate, breast, uterus, bone, liver, and the immune, cardiovascular, and central nervous systems [105]. Estrogens promote breast and endometrial cancer in women and exacerbate autoimmune diseases, whereas the loss of estrogens during menopause has been correlated with osteoporosis, coronary heart disease, depression, and neurodegeneration [105]. Compounds that antagonize the estrogenic effects (antagonists) in some tissues, such as breast and uterus, while mimicking the estrogen effects (agonists) in other tissues, such as bone, brain, and cardiovascular cells, are known as selective estrogen receptor modulators (SERMs) [106]. Two estrogen receptors (ERs)—ERα and ERβ—coded on different genes have been identified, although numerous mRNA splice variants exist for these receptors in both diseased and normal tissue [107,108]. Several molecules of plant origin are able to interact with and affect the cellular responses mediated by ERα and ERβ and thus are now defined as “dietary phytoestrogens” [109]. In vitro studies indicate that ERα and ERβ display marked differences in binding affinity and activation by SERMs [110,111]. Although the affinity of flavonoids for both ERα and ERβ (in their aglycone forms) is lower than that of E2, competition binding studies performed by several authors confirmed that nutritional molecules (e.g., genistein, coumestrol, daidzein, and equol) showed a distinct preference for ERβ [110–112]. On the other hand, in comparison with genistein, 8-prenylnaringenin, a prenylated chalcone occurring only in hops, was found to be 100 times more potent an ERα agonist but a much weaker agonist of ERβ in estradiolcompetition assays for receptor binding [113]. E2 displacement by flavonoids and in particular isoflavones could be equally well explained by their binding to a secondary site on the ER, thereby altering the Kd value of E2, rather than there being competitive binding between these compounds [114]. A confirmation of the genistein mechanism has been provided by X-ray diffraction crystal structure indicating that genistein binds to the ligand-binding domain of ERα [115]. Later, the crystal structure for the genistein–ERβ complex was also resolved [116]. As far we know these are the only crystal structures reported for PhC and no information is available yet on the other components or on their binding to ERs. Similar to other steroid receptors with which ERs share analogous domain structure, ERα and ERβ are transcription factors that regulate gene transcription through binding to the consensus estrogen response element (ERE) on DNA. E2 and estrogenic substances can also regulate transcription, promoting the ER binding to other promoter elements on the DNA such as AP-1 binding sites, CRE, and Sp1 response element [117] to which other transcription factors bind

[118]. Interestingly there is also some evidence that the potency of the two different ERs on non-ERE binding sites versus ERE binding sites differs [119,120]. SERMs such as tamoxifen enhanced AP-1-mediated transactivation through both ERα and ERβ [121]. It is interesting to note that all these transcription factors have been proposed to be somehow modulated by RONS [20,122]. In both the direct and the indirect action modes, the agonist-activated ER is not the transcription controller. In fact, ERs need to interact with a coregulatory protein complex, as either coactivators or corepressors [123]. Thus, the collaborative efforts of E2, receptor isoforms, ERE, and coactivators or corepressors can all contribute to the activation of genes in target tissues and organs [120,123]. Several investigators, including our group, reported that daidzein, genistein, naringenin, and quercetin increase the activity of an ERE– luciferase reporter gene construct in cells that express, coexpress, or even overexpress ERα or ERβ [32,33,44], whereas these flavonoids completely impair ERα interaction with Sp1 and AP-1 transcription factors [32,124]. By utilizing a DNA array approach in MCF-7 cells, a significant number of genes responding to estrogen were identified and characterized [125]. Cluster analysis performed on the same cell line indicated that very similar profiles were obtained with 10 μM genistein [125]. In contrast, the expression of only five genes was affected by daidzein with respect to E2 in TM4 Sertoli cells. These five genes were related to cell signaling, cell proliferation, and apoptosis, suggesting a possible correlation with the inhibition of cell viability reported after treatment with daidzein [126]. A variety of cell types respond to estrogens in a very short time, making the classical ER-mediated mechanism of action unlikely. These E2-induced rapid signals require an estrogen binding site localized at the plasma membrane. Debate continues over whether structural changes target ERα and ERβ to separate pools localizing them to the membrane [127,128] or whether an atypical receptor(s) could account for rapid E2-induced signals. Among others, the ability of E2 to activate G proteins through an orphan G-protein-coupled receptor-30 (GPR30) has been reported [129,130]. GPR30 shows a low binding capacity for E2, supporting a modest generation of cAMP [131], whereas genistein is a highly effective competitor for E2 binding to GPR30, with an IC50 of 133 nM [132]. More recent data indicate that cells from the ERα and ERβ knockout mice (i.e., DERKO mice) fail to show endogenous membrane or nuclear ERα or ERβ, and rapid signaling [133], thus favoring the idea that the membrane-localized ER is the same protein present in the nucleus. We recently demonstrated that ERα and ERβ undergo S-palmitoylation [134,135], which allows ER anchoring to plasma membrane and association with caveolin-1 and which accounts for the ability of E2 to rapidly activate different signaling pathways (e.g., c-Src, Shc, MNAR) and thus initiate nongenomic effects that lead to ERK/MAPK, PI3K/AKT, and p38/ MAPK pathway activation [128,135]. The ability of PhC to influence rapid actions of E2 in both reproductive and nonreproductive E2-target tissues and how such effects may impact the normal development and physiological properties of cells largely have not been tackled until very recently [136,137]. We have demonstrated that both quercetin and naringenin hamper ERα-mediated rapid activation of signaling kinases (i.e. ERK/ MAPK and PI3K/AKT) and cyclin D1 transcription [32] only when HeLa cells, devoid of any ER isoforms, were transiently transfected with a human ERα expression vector. In the same cell system, naringenin and quercetin activated the rapid phosphorylation of p38/MAPK and, in turn, the induction of a proapoptotic cascade (i.e., caspase-3 activation and PARP cleavage). Thus, naringenin decouples ERα action mechanisms, preventing the activation ERK/MAPK and PI3K/AKT signal transduction pathways and driving cells to apoptosis [33]. It is possible that flavonoids, having a strong affinity to protein binding, could induce conformational changes in ER, precluding the activation of rapid signaling cascades. In agreement with this hypothesis, we have demonstrated that naringenin prevents ERα palmitoylation and

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Fig. 3. Model representing the naringenin (Nar) effects on cell signaling through estrogen receptor α (ERα). Left: Under steady-state conditions, ERα is palmitoylated (black triangle) and localized at the plasma membrane associated with caveolin-1 (cav1). Right: Upon Nar stimulation, ERα undergoes rapid depalmitoylation, which impairs ERα reallocation at the plasma membrane and its association with other signaling proteins important for proliferation. However, Nar promotes ERα association with p38 and p38 activation triggering apoptosis. For details, see text.

reduces its association with membrane caveolin-1, therefore impairing the activation of rapid signals [134] (Fig. 3). On the other hand, naringenin does not inhibit the ERα-mediated transcriptional activity of an ERE-containing promoter [32,33,138]. All together these data indicate that a single flavonoid, naringenin, has a very complex spectrum of activities. It should not be considered a “natural” SERM, which function as ER agonists in some tissues and antagonists in other organs. Rather, naringenin could function as an ERα agonist on certain pathways in all organs, eliciting effects downstream of these pathways. Although available data are currently limited, this may be important in providing a conceptual background for the putative protective effect of PhC against cancer. Other mechanisms elicited by nutritional molecules have been proposed. After the early report that genistein inhibited the epidermal growth factor receptor (EGFR) tyrosine kinase activity [139], many investigators attributed various biological effects of genistein to the inhibition of other tyrosine kinases. However, in most reports, a direct demonstration of such inhibition was not shown. Indeed, although EGF stimulation of EGFR tyrosine autophosphorylation in prostate and breast cancer cells was blocked by tyrphostin (a synthetic tyrosine kinase inhibitor), genistein had no effect [140,141]. Rats treated with genistein displayed a reduced reactivity of the EGFR with antiphosphotyrosine antibodies but no reduction in the amount of EGFR protein [142], suggesting that genistein affects transcriptional processes rather than directly inhibiting tyrosine kinase activity. As an alternative mechanism, it has been proposed that phytoestrogens have a protective effect on the initiation and/or progression of breast cancer by inhibiting the local production of estrogens from circulating precursors in breast tissue. In fact, it has been shown that flavonoids, and in particular flavones and flavonones, inhibit in vitro the activity of key steroidogenic enzymes (i.e., aromatase and 17βhydroxysteroid dehydrogenase) involved in the synthesis of estradiol from circulating androgens and estrogen sulfate [143]. Evidently, it is essential to assess the phytoestrogens' effects at multiple levels, in vitro and in vivo, to obtain a full picture, which may be relevant to various physiological or pathological states in humans (i.e., menopause, premenopause, cancer). Nutritional molecules as ligands of other nuclear receptors Although it had been understood for many years that PhC acted as phytoestrogens, little was done to evaluate the possibility that flavonoids, and in particular isoflavones, activated other nuclear receptors (NRs).

NRs are ligand-activated transcription factors sharing a common evolutionary history [144], having similar sequence features at the protein level. A specific corresponding endogenous ligand for some of the NRs is not known, and therefore these receptors have been named “orphan receptors.” Whenever ligands have been recognized for any of the orphan receptors, they have then been categorized and grouped as “adopted” orphan receptors. This group includes the lipid-regulating peroxisome proliferator-activated receptors (PPARs), the liver X receptor, the farnesoid X receptor, and the pregnane nuclear receptor (PXR). Fibrates or glitazones, oxysterols, bile acids, and xenobiotics could activate the orphan receptors of the NR1 and NR3 subfamilies and produce effects that resemble some of the actions caused by PhC intake, suggesting that nutritional molecules could activate orphan NRs [145]. The peroxisome proliferator-activated receptors and the modulation of energy homeostasis by PhC Similar to other nuclear hormone receptors, PPARs act as ligandactivated transcription factors. PPARs (NR1C, α, γ, and δ) have distinctly different tissue distributions and have been shown to impact lipid metabolism in the liver, muscle, adipocytes, and macrophages, among other tissues, and energy homeostasis in general [145,146]. PPARs have been shown to be activated by dietary fatty acids (FAs), particularly polyunsaturated FAs, and regulate various aspects of metabolic processes. Coregulators of PPAR signaling have been proposed to act as metabolic switches, which regulate metabolic pathways through their pleiotropic interactions with nuclear receptors and other transcription factors [147]. Accordingly, the peroxisome proliferator activator receptor-γ coactivator 1 (PGC-1) is one of the major candidates for diabetes-related metabolic phenotypes, contributing to decreased expression of nuclear-encoded mitochondrial genes in muscle and adipose tissue. Recent reports indicate that obesity and dietary factors, such as saturated fatty acids, decrease PGC-1 and mitochondrial gene expression and function via p38 MAPK-dependent transcriptional pathways [148]. In fact, PGC-1α and-β have been reported to be reduced in obesity in both genetically determined obesity (Lep(ob)/Lep(ob)) and obesity induced by a highfat diet. Resveratrol, a polyphenolic phytoalexin, has been found to selectively activate PPARα and PPARγ transcriptional activity by 15to 30-fold above control levels. This activation was dose dependent at quite high concentrations (10, 50, and 100 μM) in endothelial cells [146]. In a mouse model of stroke, treatment with resveratrol and PPARα agonists has been reported to reduce brain infarct volume. This

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attenuation was not observed when the same treatments were given to PPARα-knockout mice, suggesting that the protective effect of resveratrol may specifically involve PPARα [149]. Resveratrol administration to various animal models (Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster) produces molecular changes associated with longer life span, including increased insulin sensitivity, reduced insulin-like growth factor-1 levels, increased AMP-activated protein kinase and PGC-1α activity, and increased mitochondrial number and physical activity scores [150]. These effects, at least in part dependent on the activity of sirtuins, a family of protein deacetylases [151], are able to revert to normal the majority of metabolic pathways altered by a high-calorie diet. Howitz and coworkers [152] also reported that resveratrol administration to S. cerevisiae lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD+and increases survival by stimulating SIRT1-dependent deacetylation of p53. Similar results have been reported by Terzibasi and collaborators in the short-lived fish Nothobranchius furzeri [153]. Lagouge and collaborators reported that mice treated with resveratrol have a significantly increased aerobic capacity, as indicated by increased physical activity rate and oxygen consumption in muscle [154]. These effects were associated with the induction of the expression of genes encoding proteins involved in oxidative phosphorylation and mitochondrial biogenesis and were consistent with a decrease in PGC-1α acetylation and an increase in PGC-1α activity. This mechanism matches the hypothesis that resveratrol activates the protein deacetylase SIRT1 and is corroborated by the observed lack of effect of resveratrol treatment in SIRT1−/− mouse embryonic fibroblasts. Importantly, resveratrol treatment protected mice against diet-induced obesity and insulin resistance. Fat cells seem to interfere with aging processes through a number of mechanisms and white adipose tissue now emerges as being pivotal in controlling the life span of many organisms [151]. In this perspective, sirtuins could be considered one of the major factors linking the reduction in white adipose tissue mass to life-span extension. In fact, it has been shown that human SIRT1 represses (PPAR-γ) transactivation and thereby inhibits lipid accumulation in adipocytes [155]. In white adipose tissue, PPAR-γ inhibition by SIRT1 acts as a repressor not only of genes involved in fat storage, but also of genes controlling adipocyte differentiation [155]. The presence of allelic variants of the SIRT1 gene associated with different energy homeostases has also been detected in Finnish subjects. This observation strengthens the importance of the SIRT1 gene and its modulation by nutritional factors such as PhC in regulating energy metabolism and homeostasis [154]. Similarly, the potential benefits of green tea and its most abundant phenolic components catechin and epigallocatechin gallate (EGCG) in obesity and type 2 diabetes mellitus, as major and interlinked healthcare problems, are increasingly being investigated [156]. An extract from green tea containing catechin, EGCG in a pure form has been observed to prevent and counter diet-induced obesity and to increase plasma levels of glucose, triglycerides, and leptin in C57BL/6J mice and Sprague–Dawley rats. In mice fed EGCG, the investigators found a significant decrease in fatty acid synthase and acetyl-CoA carboxylase-1 mRNA levels in adipose tissue and a dose-dependent inhibition of adipocyte differentiation in vitro [157]. The same extract has been reported to beneficially affect cellular dysfunctions in type 2 diabetic db/db mice and ZDF rats. EGCG treatment was found to be associated with improved oral glucose tolerance and blood glucose in a dose-dependent manner and to reduce plasma concentrations of triacylglycerol and enhance glucose-stimulated insulin secretion. In the same study, a gene array approach on H4IIE rat hepatoma cultured cells indicated that EGCG treatment is associated with the down-regulation of the expression of genes involved in gluconeogenesis and in fatty acid, triacylglycerol, and cholesterol synthesis. In both H4IIE cells and the liver and adipose tissue of db/db mice, EGCG treatment led to a significant decrease in mRNA expression of the

gluconeogenic enzyme phosphoenol pyruvate carboxykinase and to the up-regulation of glucokinase mRNA expression [158]. Using whole-genome microarrays, de Boer and co-workers [159] demonstrated that different pathways of fatty acid catabolism, including β-oxidation and ketogenesis, are up-regulated in rat lungs by long-term (41 weeks) high quercetin supplementation. The expression of several genes involved in lipid synthesis and metabolism (mitochondrial HMG-CoA synthase, enoyl-CoA hydratase, acylCoA oxidase, the α subunit of propionyl-CoA carboxylase, lipoprotein lipase, and mitochondrial acetyl-CoA acyltransferase) was significantly up-regulated. In the same study, the authors observed a significant decrease in plasma free fatty acid levels in rats fed the quercetin diet. Even though the amount of quercetin utilized in these experiments is far from a level achievable through a normal diet (1% diet weight), these data demonstrate that dietary quercetin significantly modulates (at least in rats) fatty acid catabolism in vivo. It is interesting to note that the chemical structure of isoflavones appears quite similar to that of GW501516 and, in particular, to that of fibrate agonists of PPARα, used to treat hyperlipidemia and type 2 diabetes [160]. In agreement with observations in humans treated with antidiabetic PPARα agonists, female obese Zucker rats consuming a high isoflavone diet improved their glucose tolerance and displayed liver triglyceride and cholesterol and plasma cholesterol levels significantly lower than those of controls [161]. These findings are consistent with the hypothesis that soy isoflavones improve lipid metabolism and have an antidiabetic effect by activating PPAR receptors. Experimental results confirmed that genistein could act as a ligand for PPARγ with a Ki comparable to that of some known PPARγ ligands [162,163]. The pregnane X receptor The nuclear receptor PXR (NR1I2) has been shown to be activated by a chemically and structurally diverse set of xenobiotic and endogenous compounds. PXR regulates gene expression pathways involved in metabolism and transport of these same classes of compounds [164,165]. Notably, PXR has been shown to directly regulate the cytochrome P450 3A gene, a phase I drug metabolism gene whose product is responsible for the oxidative metabolism of N 50% of all drugs [164,165]. Consistent with the function of PXR as a xenobiotic sensor, virtually all PXR ligands isolated to date demonstrated agonist activity. PXR mediates the effects of the botanical hyperforin (an active component of the herb St. John's wort) on induction of cytochrome P450 3A activity, which is associated with clinically significant drug interactions [146]. In some instances, these interactions were life-threatening or lethal [166]. Mixtures of isoflavones and isolated isoflavones have been reported to induce PXR transcriptional activity [145]. Other authors reported that genistein, formononetin, kaempferol, and apigenin did not exhibit PXR ligand activity [167]. Thus, at present the contribution of PXR signaling to the PhC effects on human health remains elusive. The estrogen-related receptors Although the three members of the estrogen-related receptor (ERR) family, ERRα (i.e., NR3B1), ERRβ (i.e., NR3B2), and ERRγ (i.e., NR3B3), do not bind estrogens, they share a significant homology with ERs, in particular at the DBD and LBD sites [168,169]. ERRα is found almost ubiquitously in adult tissues and was initially described as a regulator of fatty acid oxidation, mitochondrial biogenesis, and oxidative phosphorylation [170]. More recent reports suggest a role for ERRα as a modulator of ER signaling [169]. ERRβ is essential for reproduction, being present in a subset of cells in extraembryonic ectoderm designed to make up the chorion of the early developing placenta. In mice lacking ERRβ, trophoblast stem cell differentiation is impaired and the placenta fails to develop correctly [169]. ERRβ synthesis is highly restricted in postnatal life, and low levels have been detected only in liver, stomach, skeletal muscle, heart, and kidney of adult mice [169]. Human ERRγ transcript has been detected at very

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high levels in fetal brain and at lower levels in kidney, lung, and liver. In adult human tissues, the gene encoding ERRγ is widely expressed and has been detected in brain, lung, and bone marrow [169]. Despite their significant homology with ERs in the LBD, ERRs do not (or only very weakly) respond to E2 [169]. F-urthermore, whereas ERs are ligand-activated receptors, ERRs are constitutively active [171]. However, several amino acid residues crucial for E2 recognition are conserved among members of the ER and ERR families, suggesting that ER and ERR ligands could be structurally related. This hypothesis has been validated through the discovery that the ER agonist diethylstilbestrol can act as an inverse agonist on all three ERR isoforms [172], whereas the SERM 4-hydroxytamoxifen acts as an isoform-specific ERR inverse agonist [173,174]. 4-Hydroxytamoxifen binds to and deactivates ERRβ and ERRγ, but does not recognize ERRα. Computer docking analyses and mammalian two-hybrid experiments indicate that isoflavones (i.e., genistein, daidzein, and biochanin A) and one flavone (trihydroxyflavone) are relatively poor ligands of ERRγ but they can act as agonists of ERRα and ERRβ activity [175]. The effects of phytoestrogens on ERRα may not be easily observed when functionally active ERα or ERβ are present owing to the putative preference of these PhC for ERs. Therefore, the effects of phytoestrogens mediated by the interaction with ERRα in women's breast and bone could be detected when the function of ERs was suppressed, such as through the use of antiestrogens [175]. Conclusion and perspectives The diet–health relationship is obviously very complex and food items most probably act through multiple pathways, and the isolation of the specific role of a single component is very difficult or essentially impossible. The interaction between diet and cell signaling is even more complex. Remarkably, the association between the consumption of specific food items (in general fruits and vegetables) and the risk of degenerative diseases has been almost unanimously attributed to their capacity to provide a high intake of components having a putative antioxidant capacity. In the light of the recent literature, this conclusion can be considered in some cases an intellectual shortcut, biasing the real understanding of the molecular mechanisms underlying the beneficial effects of different classes of food items. A wide spectrum of molecules having antioxidant properties is provided by these foods, but it is necessary to consider that: (1) The antioxidant capacity is frequently lost (or dramatically reduced) just after absorption owing to the metabolic transformation often associated with (multiple) conjugation of the original molecules. (2) As described above, molecules having a chemical structure compatible with a strong putative antioxidant capacity can actually “perform” activities and roles totally independent of such capacity and interact with cellular functions at different levels, such as affecting enzyme activities and binding to membranes or nuclear receptors as either elective ligand or ligand mimic. (3) There is another important point to consider in the different effects exerted by purified molecules vs the effects of complex matrices containing a wide spectrum of candidate potential effectors. In this context, it is possible that a sort of natural predisposition of researchers to attempt to simplify the problem to build up and pursue hypotheses that are easily tested, leads to the underestimation of the importance of exposure to complex food matrices in favor of single dietary components. (4) “Omic” approaches and high-throughput methodologies obviously play a pivotal role in this context, providing a tool to carry over a “problem-driven” research, taking into account the combined complexity of human nutrition and the human genome.

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Further investigations into the potential role of nutritional molecules in impacting cellular signals to improve human health are warranted. However, once a robust data base to be utilized as a starting point for understanding the effects of complex matrices is achieved, there will often be the need to move back to simplified models to isolate and explore in depth the mechanism of action of purified single components. The final goal would be to come back to the more complex experimental molecular and clinical approaches for consideration as the bases of specific nutritional recommendations. Focusing on specific molecules also opens up the avenue to the transfer of knowledge from the relationship between nutrition and health to pharmacology. Acknowledgments Some experimental concepts described in this paper are based on work conducted in the laboratories of the authors. These experimental studies were supported by grants from the Ministry of Education, University, and Research of Italy (PRIN-COFIN 2006 to M.M. and FISR “Safe-Eat” to F.V.). The authors thank past and present members of their laboratories who contributed with data and discussions to the ideas presented here. References [1] Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T.; Mazur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39:44–84; 2007. [2] Koutsilieri, E.; Scheller, C.; Grunblatt, E.; Nara, K.; Li, J.; Riederer, P. Free radicals in Parkinson's disease. J. Neurol. 249 (Suppl. 2):II1–II5; 2002. [3] Closa, D.; Folch-Puy, E. Oxygen free radicals and the systemic inflammatory response. IUBMB Life 56:185–191; 2004. [4] Valko, M.; Rhodes, C. J.; Moncol, J.; Izakovic, M.; Mazur, M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160:1–40; 2006. [5] Wickens, A. P. Ageing and the free radical theory. Respir. Physiol. 128:379–391; 2001. [6] Slater, T. F. Free-radical mechanisms in tissue injury. Biochem. J. 222:1–15; 1984. [7] Cadenas, E. Mitochondrial free radical production and cell signaling. Mol. Aspects Med. 25:17–26; 2004. [8] Linnane, A. W.; Eastwood, H. Cellular redox regulation and prooxidant signaling systems: a new perspective on the free radical theory of aging. Ann. N. Y. Acad. Sci. 1067:47–55; 2006. [9] Dennery, P. A. Effects of oxidative stress on embryonic development. Birth Defects Res. C Embryo Today 81:155–162; 2007. [10] Villalobo, A. Nitric oxide and cell proliferation. FEBS J. 273:2329–2344; 2006. [11] Daftary, G. S.; Taylor, H. S. Endocrine regulation of HOX genes. Endocr. Rev. 27:331–355; 2006. [12] Lechner, M.; Lirk, P.; Rieder, J. Inducible nitric oxide synthase (iNOS) in tumor biology: the two sides of the same coin. Semin. Cancer Biol. 15:277–289; 2005. [13] Droge, W. Free radicals in the physiological control of cell function. Physiol. Rev. 82:47–95; 2002. [14] Hehner, S. P.; Hofmann, T. G.; Dienz, O.; Droge, W.; Schmitz, M. L. Tyrosinephosphorylated Vav1 as a point of integration for T-cell receptor-and CD28mediated activation of JNK, p38, and interleukin-2 transcription. J. Biol. Chem. 275:18160–18171; 2000. [15] Allen, R. G.; Tresini, M. Oxidative stress and gene regulation. Free Radic. Biol. Med. 28:463–499; 2000. [16] Filomeni, G.; Graziani, I.; G.; R.; Ciriolo, M. R. trans-Resveratrol induces apoptosis in human breast cancer cells MCF-7 by the activation of MAP kinases pathways. Genes Nutr. 2:245–310; 2007, doi:10.1007/s12263-007-0059-9. [17] Stone, J. R.; Yang, S. Hydrogen peroxide: a signaling messenger. Antioxid. Redox Signaling 8:243–270; 2006. [18] O'Loghlen, A.; Perez-Morgado, M. I.; Salinas, M.; Martin, M. E. Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide: potential regulation of eIF2α phosphorylation in differentiated PC12 cells. Arch. Biochem. Biophys. 417:194–202; 2003. [19] Hao, Q.; Rutherford, S. A.; Low, B.; Tang, H. Selective regulation of hydrogen peroxide signaling by receptor tyrosine phosphatase-α. Free Radic. Biol. Med. 41:302–310; 2006. [20] Dasari, A.; Bartholomew, J. N.; Volonte, D.; Galbiati, F. Oxidative stress induces premature senescence by stimulating caveolin-1 gene transcription through p38 mitogen-activated protein kinase/Sp1-mediated activation of two GC-rich promoter elements. Cancer Res. 66:10805–10814; 2006. [21] Lu, D.; Chen, J.; Hai, T. The regulation of ATF3 gene expression by mitogenactivated protein kinases. Biochem. J. 401:559–567; 2007. [22] Hanasaki, Y.; Ogawa, S.; Fukui, S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radic. Biol. Med. 16:845–850; 1994.

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