Bioactive substances of plant origin in food â€“ impact on genomics

Review

Bioactive substances of plant originin food – impact on genomics

Arkadiusz ORZECHOWSKI, Piotr OSTASZEWSKI*, Michal JANK,Sybilla Jacqueline BERWID

Department of Physiological Sciences, Faculty of Veterinary Medicine,Warsaw Agricultural University, Poland

Abstract — In the past decade, substantial progress has been made concerning our knowledge of bioac-tive components in plant foods and their links to health. Human diets of plant origin contain many hun-dreds of compounds which cannot be considered as nutrients, but appear to play a role in the main-tenance of health. These substances are called nutraceuticals. In some cases where the disease processis at least partially understood, elements of protection can be related to a single compound or struc-turally related group of compounds in the diet. Bioactive components of food which are of special inter-est include the following groups: polyphenols, phytoestrogens, phytosterols, phytates and polyun-saturated fatty acids. Most of them are featured by antioxidant properties. In the first part of thisreview, we indicate the main groups of bioactive compounds giving a description of their localisation,chemical properties and biological actions. Recently, it was shown, however, that the bioavailabil-ity of potential antioxidants from plant foods is generally too low to have any substantial directeffect on reactive oxygen species. As a result of that it is postulated that dietary compounds, even invery low concentrations, may have a far greater impact than previously appreciated on the regulationof gene expression. The second part of this paper concerns the action of the literally most importantbioactive substances on the molecular mechanisms of the control of genes which in turn affect cel-lular metabolism. A few current studies on the action of selected nutraceuticals on the activity of tran-scription factors such as AP-1, NF-kB, SREBPs, PPARs as final targets in the signal transduction cas-cade and gene regulation are included. A detailed analysis of numerous factors of dietary originwith their targets is far beyond the scope of this paper. However, continuing research on the effectsof nutraceuticals on gene expression should provide insight into the mechanisms of prevention of dis-eases such as obesity, diabetes, atherosclerosis, hypertension and cancer by dietary manipulations.

bioactive compounds / antioxidants / transcription factors / AP-1 / NF-kB / PPARs / SREBPs /gene expression

Reprod. Nutr. Dev. 42 (2002) 461–477 461© INRA, EDP Sciences, 2002DOI: 10.1051/rnd:2002034

* Correspondence and reprintsE-mail: [email protected]

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1. INTRODUCTION

Wild primates, close relatives to humans,consume as a rule diets high in fiber, vita-mins, minerals, and with variable levels ofproteins and fatty acids [66]. Even in cap-tivity but more in the wild, they sponta-neously have plenty of exercise. In turn, thecurrent lifestyles of humans almost every-where in the world are in sharp contrast, andas a consequence, humans suffer from alarge number of chronic diseases. In thepast, infectious diseases killed our ances-tors early on, often younger than age 40, sothey did not display the current epidemic ofchronic diseases that arise in older ages.Now medical status has been improved. Theproblem of infectious diseases has beensolved due to the amelioration of medicalcare (vaccination programs, antibiotics).Nowadays, people live longer and thereforeexpress symptoms of chronic diseases asso-ciated with senescence and lifestyle (alsocalled civilisation diseases) such as obesity,diabetes, hypertension, coronary heart dis-ease, and cancer.

2. NATURAL BIOACTIVECOMPOUNDS OF PLANTS

Bioactive components of food which areof special interest include the followinggroups: polyphenols, phytoestrogens, phy-tosterols, phytates, lectins, oligosaccharidesand polyunsaturated fatty acids (PUFA)[30]. These groups consist of many relatedcompounds, each with slightly differentproperties. It is important to stress that theprotection against cancer and cardiovascu-lar disease is undoubtedly the result of thecumulative action of many natural sub-stances present in the diet. Since each plantcontains different bioactive components, theeating of various foods seems to be impor-tant but needs further evidence. Taking thisinto account, we may enjoy a lower risk ofoccurrence of modern diseases.

2.1. Polyphenols

Polyphenolic compounds are mainlyfound in fruits and vegetables and are one ofthe most important sources of bioactivecomponents of the human diet [76] Over8000 polyphenols have been identified andamong them more than 2000 are found innature. Plants need them for pigmentation,growth, reproduction, resistance to pathogensand for many other functions. One of themost important groups of polyphenols isflavonoids. They can be divided into thefollowing subgroups: flavones/flavonones,anthocyanins and catechins/flavonols. Inplants, flavonoids usually form complexeswith various sugars which are called glyco-sides. Flavones/flavonones have been iso-lated from almost all fruits and vegetableswith their highest concentrations beingfound in the outer layers. Therefore flavonoidconsumption can be dramatically reducedif the peel of an apple is removed. How-ever, there are fruits like oranges for exam-ple that have high amounts of flavonoidsalso present in the pulp. In most Europeancountries, the average daily consumption offlavones/flavonones does not exceed 25 mgper day. Anthocyanins are the largest groupof water-soluble pigments in plants. Theyare widely distributed in the human dietthrough crops, beans, fruits, vegetables andred wine [31]. Tsuda et al. [98] showed thatanthocyanins can inhibit the formation ofthe nitrated tyrosine and scavenge perox-ynitrites. Moreover anthocyanins express apotent antioxidant activity and protectiveeffect against hepatic ischemia-reperfusioninjury in vivo.

Catechins are unique flavonoids foundin large quantities in green tea. In black teathe level of catechins is about 30% that ofgreen tea. Green tea extracts are described asprotective against experimentally inducedcancer in animals. They act as stronginhibitors of the in vitro nitrosation of sec-ondary amines and therefore lower tumorinitiation [94]. High amounts of catechins

Bioactive substances of plant origin

and other non-hormonal cancers, cardio-vascular diseases and osteoporosis [5].Recently, Karamsetty et al. [46] found thatsoybean phytoestrogens genistein anddaidzein act like estrogens in restoring nitricoxide-mediated relaxation in hypoxic ratpulmonary arteries and moreover, this effectis not mediated by the inhibition of tyrosinekinases.

It is recommended to consume moder-ate amounts of phytoestrogens in their nat-ural form as plant foods. If their intake istoo high they could be potentially harmful tohuman health although this is unlikely tohappen.

2.3. Phytosterols

Phytosterols are bioactive non-nutrientsubstances structurally similar to choles-terol. They exist in two forms: (1) unsatu-rated, common, present in many plants and(2) saturated, called stanols, which are foundonly in small amounts in cereals, fruits andvegetables [71]. Northern European dailyconsumption of phytosterols is in the range100–400 mg and comes mainly from veg-etable oils, bread, fruits and vegetables. Insouthern Europe the intake may be evenhigher as a result of high consumption ofvegetable oils and nuts. The health effects ofphytosterols are the result of their structuralsimilarity to cholesterol; therefore plantsterols compete with cholesterol absorptionfrom the intestinal tract. When typicalamounts of sterols (240–320 mg) are con-sumed, only about 5% are absorbed fromthe small intestine [53]. Thus, the dietaryintake of phytosterols causes an increasedexcretion of both dietary and biliary choles-terol in humans [35]. In addition to reducingthe absorption of cholesterol, plant stanolsinhibit the absorption of other plant sterols[32]. In humans, this inhibition of intesti-nal cholesterol absorption is accompaniedby a compensatory increase in cholesterolsynthesis, as reflected in the increase ofserum cholesterol precursors, lathosterol

present in tea are also found in red wine andchocolate, which may contribute signifi-cantly to the daily intake of polyphenols[100]. It is estimated that the daily intakeof polyphenols does not exceed 200 mg perday, which is relatively high compared tothe intake of other antioxidant nutrients,such as vitamins E, C or A. Polyphenoliccompounds have beneficial health effectsbecause of their antioxidant properties andtheir inhibitory role in the various stages oftumor development [38]. There are cohortstudies indicating a possible protectiveaction against coronary heart disease [49]and strokes [45]. Polyphenols act throughthe scavenging of free radicals (reactive oxy-gen species, ROS) and therefore are con-sidered to be powerful antioxidants.

2.2. Phytoestrogens

Phytoestrogens have become one of themore topical areas of interest in clinicalnutrition. They mimic human estrogens andtherefore are considered as natural selectiveestrogen receptor modulators (SERMs) [90].There are two subclasses of polyphenols(isoflavonoids and lignans) isolated fromvarious plants [65]. The main consumableplant sources of phytoestrogens includeisoflavonoids and lignans found mainly insoybeans and flaxseed, respectively. Othersources of phytoestrogens include sunflowerand sesame seeds, various nuts, berries, gar-lic and carrots [86]. Plant lignans are alsofound in many cereals, grains, fruits andvegetables [6]. Since Asians consume a lotof soybean products, their daily isoflavonoidintake is 25–100 mg. In western Europeisoflavonoid consumption is usually a fewmg per day due to a much lower intake ofsoybean products. Phytoestrogens may pre-vent cancer in humans. In countries withhigh consumption of soybean products therisk for hormone-related prostate and breastcancer is much lower than in populationswith a low consumption of soybeans [68].Phytoestrogens also protect against bowel

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and desmosterol. However, the net effect isstill a reduction in serum cholesterol. Thebeneficial effects of vegetable oils are due tothe high amount of phytosterols which lowerserum cholesterol. Moreover, the interac-tion between dietary fibre and phytosterolscould explain why diets rich in fibre mayreduce the risk of coronary heart disease.The use of a special margarine increases thedietary intake of phytosterols to 1–3 g perday which is a therapeutic amount. In 1995,the Finns introduced plant stanol esters(PSE) in margarine, as dietary adjuncts tolower cholesterol by more than 10% [15].

2.4. Phytates

This group of bioactive substances is alsocalled substances with antinutritional prop-erties although this term is also appropriatefor flavonoids.

Phytates are present in seeds which are animportant source of plant phosphorus storedthere in the form of phytic acid (myo-inos-itol hexaphosphate acid, InsP6) [61]. Theantinutritional effects of phytic acid are pri-marily related to the strong chelating asso-ciated with its six reactive phosphate groups.Its ability to complex with proteins and par-ticularly with minerals has been a subjectof investigation from chemical and nutri-tional viewpoints [99]. High contents ofphytates are observed in cereal grains,legumes and nuts whereas in vegetables theirconcentrations are low. The hydrolysis ofphytates into inositol and phosphates orphosphoric acid occurs as a result of phy-tase action [79] or nonenzymatic cleavage,i.e. food processing [2]. Enzymes capableof hydrolysing phytates are widely dis-tributed in micro-organisms, plants and ani-mals. Phytases, naturally present not onlyin plant foods, but also in yeasts or othermicroorganisms implemented in food pro-cessing, act in a stepwise manner to catalysethe hydrolysis of phytic acid. To reduce oreliminate the chelating ability of phytate,dephosphorylation of hexa- and penta-phos-

phate forms is essential since a high degreeof phosphorylation is necessary to bind min-erals. In developing countries where the dietis almost wholly based on cereals andlegumes, iron and zinc deficiency are fre-quently observed. Phytate-related mineraldeficiencies are also reported in people fromdeveloped countries, such as pregnantwomen, infants and adolescents. There areseveral methods of decreasing the inhibitoryeffect of phytic acid on mineral absorption(germination, fermentation, soaking, autol-ysis and malting). Unfortunately heat pro-cessing at home during cooking or duringfood manufacturing does not affect phy-tates. Therefore, the selection of starter cul-tures to improve phytate degradation is veryimportant. In some cases, commercial phy-tase can be added to remove phytate, espe-cially from infant foods.

In recent years, dietary phytate hasreceived increased attention due to its role incancer prevention and/or therapy and itshypocholesterolemic effect [54]. In turn, bybinding an excess of free iron in the smallintestine, phytates may prevent the formationof free radicals by the Fenton reaction inthe colon and consequently decrease ironabsorption for all who need less iron.

2.5. Lectins

In the past, the main scientific interestwas focused on the toxicity of lectins, forexample ricin. Nowadays, these bioactivecompounds are recognized as natural com-ponents of the human diet. It has been shownthat dietary lectins, which bind avidly andare endocytosed by cells of the brush borderepithelium, are powerful growth factors forthe gut [83], induce changes in its digestive/absorptive functions, modify the state ofglycosylation of luminal receptors, alter theexpression of genes coding for digestiveenzymes, transport and structural proteinsand interfere with both the bacterial ecol-ogy and the immune response of the gut tofood antigens. Furthermore, they stimulate

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2.7. Polyunsaturated Fatty Acids(PUFA)

Polyunsaturated fatty acids consist of twoparent compounds: linoleic acid, a fatty acidof the w-6 family with 18 carbon atoms andtwo double bonds (18:2n6) and a-linolenicacid, a fatty acid of the w-3 family with18 carbon atoms and 3 double bonds(18:3n3). These fatty acids have differentmetabolic effects. Linoleic acid (LA) canbe elongated to arachidonic acid (AA), afatty acid with 20 carbon atoms and 4 dou-ble bonds (20:4n6) with two intermediarymetabolites termed g-linolenic acid (18:3n6,GLA) and dihomo-g-linolenic acid (20:3n6,DHLA) while a-linolenic acid can be elon-gated to either eicosapentaenoic acid (EPA),a fatty acid with 20 carbon atoms and 5 dou-ble bonds (20:5n3) or docosahexaenoic acid(DHA), a fatty acid with 22 carbon atomsand 6 double bonds (22:6n3). The fatty acidswith 20 carbon atoms, AA and EPA play animportant role in prostaglandin metabolismand may influence the thrombotic process.Cohort studies [39] indicate that the intakeof a-linolenic acid is inversely related tocoronary heart disease. Whether this effectis independent of other unsaturated fattyacids e.g. linoleic acid, is difficult to estab-lish because different unsaturated fatty acidsare present in the same foods e.g. soybeanoil. However, the hypothesis of a protectiveeffect of a-linolenic acid in relation tocoronary heart disease is supported by theresults of the Lyon trial. In this interventionstudy, a Mediterranean diet enriched witha-linolenic acid was strongly protective inrelation to coronary heart disease [23, 84].However, more data is needed before defi-nite statements can be made about the pos-sible protective effect of a-linolenic acid.

2.8. Other mechanisms of action

A number of human intervention studiesconcerning antioxidant and/or anti-genotoxiceffects of various polyphenols have shown

pancreatic growth and have profoundeffects on the immune system [81]. Plasmacells involved in a multiplicity of immunefunctions express high and variable levelsof endogenous membrane lectins, mostof which are used in cell-to-cell communi-cation.

2.6. Oligosaccharides

Oligosaccharides represent a structurallydiverse class of macromolecules of a rela-tively widespread occurrence in nature. Theyare mainly present as glucans with differ-ent types of glycosidic linkages, while oth-ers mostly bind to protein residues asoligosaccharide-protein complexes [25].The most promising biopharmacologicalactivities of these biopolymers are theirimmunomodulation and anti-cancer effects.Oligosaccharides and oligosaccharide-pro-tein complexes are considered as multicy-tokine inducers that are able to induce thegene expression of various immunomodu-latory cytokines and cytokine receptors.Numerous anti-tumor polysaccharides havebeen discovered from mushrooms, fungi,yeasts, algae, lichens and plants and at pre-sent are intensively studied [75]. Fructo-oligosaccharides (FOS) are short-chain poly-mers of fructose which are producedcommercially by hydrolysis of inulin or byenzymatic synthesis from sucrose or lac-tose. They are not hydrolyzed in the humansmall intestine but degraded in the colon bythe resident microflora. They are mainlyknown for their ability to increase theendogenous growth of intestinal lactobacilliand bifidobacteria in humans and animalswhich is recognized as beneficial to health.[8]. In vivo studies in rats have shown thatFOS increase the proportion of butyratewhich in turn stimulates water and sodiumabsorption and modulates intestinal motility.FOS also increase Ca, Mg and Fe absorptionand enhance bone calcium stores in rats [74].

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no convincing results [37, 50, 101]. Alsoextensive studies with orally administeredhigh rates of plant extracts rich in phenolicshave failed to demonstrate antioxidanteffects, except for a transient improvement inthe amount of trapped free radicals [49, 107].According to estimated daily intake offlavonoids, the range of 100–200 mg.day–1

is a very low level when compared with thelevels used in the aforementioned studies.Recent work has shown that the bioavail-ability of potential antioxidants is too low tohave any substantial direct effect on reac-tive oxygen species (Fig. 1). Moreover, thevast number of phenolics present in food-stuffs are glycosides and the free radicalscavenging activity decreases with the pres-ence of a sugar moiety, so glycosides arenot antioxidants, although their corre-sponding aglycons are. However, it is wellrecognized that many polyphenols that donot show antioxidant effects show anti-inflammatory, anti- or pro-estrogenic, anti-mutagenic and anti-carcinogenic effects.Therefore, it is postulated that dietary com-pounds, even in very low concentrations,may have a far greater impact than previ-

ously anticipated, most likely by the regu-lation of gene expression. This in turn canaffect cellular metabolism with profoundeffects on detoxification mechanisms andcell proliferation, differentiation, survivaland death.

On the contrary, deleterious effects ofpolyphenolic compounds have also beenobserved, and are associated with the abilityto bind and precipitate macromoleculesincluding protein and carbohydrates andreduce the digestibility of food. The colour-ing pigments in plants called flavonoids arethe best known and best characterized ofthese groups. Flavonoids have been con-sidered antinutrients because they have beenshown to inhibit the activity of a wide rangeof enzymes including digestive enzymessuch as hydrolases, but also isomerases,oxygenases, oxidoreductases, polymerases,phosphatases, protein kinases and aminoacid oxidases. Failure to scavenge free rad-icals in vitro does not necessarily mean thatsome flavonoids will not trigger biologicaleffects in vivo. Flavonoids might interferewith various transduction signal cascades

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Figure 1.Relationship between bioavailibility of potential antioxidants and cell functions.


(XME) of phase I (CYP1A1, CYP1A2), andphase II (NADP(P)H-menadione oxidore-ductase, aldehyde dehydrogenase, UDP-glucuronosyl-transferase, glutathione S-trans-ferase) xenobiotic elimination. There arespecific transcription factors, which in turnspecifically bind to XRE. Once activatedby the assembly with aromatic hydrocar-bons or halogenated derivatives such asdioxin (TCDD), the cytosolic protein calledthe aryl hydrocarbon receptor (AhR) translo-cates to the nucleus where it heterodimerizeswith the aryl hydrocarbon nuclear translo-cator forming a transcription factor thatbinds to the XREs present in the 5’-pro-moter [33]. Dietary flavonoids are ligands ofthe AhR and affect cyp1a1expression, withquercetin being a very potent activatorwhereas kaempferol and green tea polyphe-nols (GTP) – catechins; especially (-)-epi-gallocatechin gallate (EGCG) inhibit cyp1a1transcription induced by TCDD [19, 104].Alternatively, flavonoids are reported to actthrough the transcriptional regulation ofgenes by directly affecting the antioxi-dant/oxidant response element (ARE/ORE)in the promoter regions of some of the genes(gsta1, cyp1a1, cyp1a2) of the XME [108].It should be noted that ARE/ORE is the elec-trophile response element, so flavonoidsmay act directly on ARE/ORE as phenolicradicals or indirectly by the effects on oxida-tive stress. Flavonoids have been observedto repress intrinsic antioxidant systems asa feedback mechanism exerted on antioxi-dant enzymes eventually pointing to theimportance of intracellular prooxidant-antioxidant homeostasis.

There are promoter regions of severalgenes (including XME) that posses anotherresponse element that is activated by gluco-corticoid and glucocorticoid-like structures.This, termed the glucocorticoid response ele-ment (GRE) is induced either by the gluco-corticoid receptor-ligand transcription factoror by the glucocorticoid receptor-indepen-dent mechanism [60]. There is also the pos-sibility that transcription factors formed bythe glucocorticoid receptor-glucocorticoid

by affecting the eicosainoid synthesis viacyclooxygenase/lipoxygenase pathways[56], or protein tyrosine kinases [40, 85].Some of them may also form complexeswith metal cations, thereby interfering withthe absorption of minerals such as iron orcopper [14]. The ability to bind mineralsmay be beneficial in some cases, since cop-per and iron are the initiators of hydroxylradical formation by the Fenton reaction[96]. Except in extreme cases, undernour-ishment in western societies may actuallylead to beneficial effects such as the pre-vention of obesity and genomic stability.

3. IMPACT ON GENOMICS

Polyphenols including flavonoids are tobe considered as xenobiotics and as suchmay profoundly affect the activation andexcretion of exogenous carcinogens. Cer-tain polyphenols may directly or indirectlyinduce phase II enzymes such as glutathionetransferases (GSTs), NAD(P)H:quinonereductases, epoxide hydrolases, and UDP-glucuronosyltransferases that will enhancethe excretion of oxidising species [29, 108].Concomitantly, flavonoids significantlydecrease the activity of antioxidant enzymesglutathione reductase (GR), catalase (CAT)and glutathione peroxidase (GPx) in the redblood cells of rats [11]. They are also reportedto influence the expression and the activityof cytochrome P450 (CYP) [19, 104].Antioxidant activities have shown little orno relationship to the above-mentionedantimutagenic/anticarcinogenic activities offlavonoids [34].

How do flavonoids induce such numer-ous and multidirectional modifications inthe intracellular biochemical apparatus? Itseems likely that the effects of some of themmay indirectly occur through the action onresponse elements in the regulatory regionsof the genes. The xenobiotic response ele-ment (XRE) is localized in the promoterregions of several genes encoding proteinssuch as xenobiotic metabolizing enzymes

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interaction influence XRE the regulation ofgene expression. Flavonol quercetin hasbeen reported to selectively inhibit GRE-dependent gene regulation [58].

The activation of xenobiotic responseelements by dietary agents has been knownfor some time, but the effects driven by theantioxidant response elements and throughnuclear transcription factors such as AP-1and nuclear factor kappa B (NF-kB) familiesare only now being recognized.

3.1. Modulation of signal transductioncascades

One important mechanism of regulationappears to be the inhibition by dietary agentsof one or more of the kinase families ofenzymes involved in the respective signal

transduction. This may occur either by directinhibition of the kinases itself or via theredox sensitivity of the kinase protein. Atpresent, little is known about the molecu-lar mechanisms of specific genes codingfor proteins responsible for the observedbeneficial health effects of flavonoids. Thecandidates to play the key role in the regu-lation of cell life and death at the transcrip-tional level includes NF-kB and AP-1 tran-scription factors. Conflicting data describethe effects of catechins and teaflavins onthe activation of extracellular signal-regu-lated kinase (ERK2) and c-Jun N-terminalkinase (JNK1) and the expression of c-junand c-fos mRNA as well as the activityof the activator protein 1 (AP-1) (Fig. 2).There is also contradicting evidence forthe induction of AP-1 and NF-kB by the

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Figure 2.Suggested metabolic pathway of nutritive and non-nutritive dietary agents. NF-kB – nuclearfactor kappa B; AP-1 – activating protein-1, PPARs – peroxisome proliferator-activated receptors;SREBPs – sterol regulatory element – binding proteins; ARE/ORE – antioxidant response ele-ment/oxidant response element.


mechanisms leading to the enhanced expres-sion of genes responsible for cell resistanceto stress and apoptosis. The chemical struc-ture of flavonols is characterized by the pres-ence of the 2-phenylbenzen-g-pyron ring.The ultimate difference between quercetinand kaempferol is confined to the presenceof an additional hydroxyl (OH) residue inthe 3’ position of the B ring [1, 95]. Thus,one who compares the effect of quercetinor kaempferol on colorectal cancer cellsmight easily distinguish the role of thehydroxyl group present or absent in the par-ticular flavonol. Anticarcinogenic proper-ties of flavonols resulted in part from theinhibition of NF-kB activity [78]. NF-kBis a ubiquitous regulator of transcription inalmost every cell and it modulates the activ-ity of genes that are characterized by thepresence of the NF-kB consensus sequencein the regulatory (enhancer/promotor) regionsof DNA [9]. Activation of NF-kB has beenreported to suppress cell death, while theblockade leads to the amplification of thecytotoxic effects of TNF-a and promotesapoptosis [113]. When stimulated, NF-kBpromotes transcription, whereas the inactiveform resides within the cytoplasm, blockedby the IkB subunit (with the exception oflymphocytes – B cells, where NF-kB is con-stitutively expressed in the nucleus) [26,64]. According to the differences in thestructure, at least five isoforms of NF-kBhave been identified but the most abundantare subunits p50 and p65 that form homo- orheterodimers which can bind to DNA [64].On the contrary, TNF-a is a proinflamma-tory cytokine, which is known to induce celldeath. TNF-a acts on the cell by the acti-vation of the membrane receptors TNF-R1or TNF-R2. Association with the receptorsleads to conformational changes (oligomer-ization into trimers) and the receptors areable to recruit a signaling complex calledthe DISC (death-initiated signaling com-plex) composed of the TRADD (TNF-R1-associated death domain) and FADD (Fas-associated death domain). Simultaneouslyor alternatively, conformational changes in

commercially used phenolic antioxidantsbutylated hydroxyanisol (BHA) and t-butyl-hydroquinone (tBHQ). They either activateNF-kB (measured by the electrophoreticmobility shift assay) with the formation ofH2O2 [82], or phenoxyl radicals and/or theirderivatives [51] or inhibit NF-kB DNAbinding [10]. Phenolics trigger c-Jun N-ter-minal kinase (JNK1) and/or extracellularsignal regulated protein kinase (ERK2) ina dose-dependent fashion [108]. In contrastto Yu et al. [108] who observed activation,Chung et al. [18] found the inhibition ofERK2, JNK1, and AP-1 activity. SinceJNKs are strongly and preferentially acti-vated by stress stimuli, this signaling path-way as one of the stress responses and isfunctionally involved in cellular survivaland/or apoptosis [47]. It is probable that theeffect of quercetin is also dose-dependenton the regulation of MAPKs and leads tothe induction/repression of gene expressionand cell survival or cell death. At certain lev-els quercetin might be an indirect NF-kBinducer by targeting several kinases (i.e.MAP kinases), which activate NF-kB.Upstream activators include NIK, MEKK1,MEKK2, MEKK3, TAK1, proteinkinase Cz, and S6 kinase [55, 69, 72, 88,112]. Similarly, contradicting results wereobtained from studies with quercetin andother phenolics on the activity of NF-kB,the key regulator of cellular antioxidantdefence systems. Sato et al. [87] or Ishikawaet al. [43] reported the suppression of NF-kBactivation by quercetin in human synovialcells, or glomerular cells, respectively, butthe cells were studied in serum free medium,with additional one day fasting as the pre-treatment period to induce cell quiescence.We reported transient activation of this tran-scription factor by quercetin in conditionsfavoring cell proliferation [77]. It thereforeappears, that quercetin-induced NF-kB acti-vation is characterized by cell specificity.Actually, phenolic antioxidants at high dosesalso activate ICE/Ced-3 caspases [51, 52].An NF-kB RelA/p65 subunit acts as a signalfrom cytosol, which initiates transcription

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the receptors can recruit a signaling com-plex composed of TRADD and TRAF2(TNF-associated factor 2) and/or RIP (recep-tor interacting protein) for survival [91].After the association, TRAF2 and RIP couldactivate kinase NIK (NF-kB inducingkinase) that stimulates NF-kB indirectly bythe activation of IkBa or -b kinases (IKKa,IKK b). IKKb kinase i.e. phosphorylates theIkBa inhibitory subunit in positions 32 and36 of serine residues. After phosphoryla-tion IkBa can be ubiquitinated and prote-olytically degraded by the proteasome. Thus,NF-kB is not sequestered any more andtranslocates into the nucleus [10, 26, 47, 59,91]. NF-kB activation improves cell sur-vival whereas inhibition enhances cytopathicand apoptotic effects of TNF-a indicating aconsiderable role of functional NF-kB incell viability. The protective effect of NF-kBis in turn dependent on mRNA and proteinsthat regulate the activity of antiapoptoticgenes. Obviously, NF-kB directly activatesBcl-2/A1 (a homologue of Bcl-2) the proteinthat plays an important role in the blockadeof apoptosis associated with the activity ofthe mitochondria [113]. Additionally, theactivation of TNF-R1 by TNF-a is associ-ated with the increased activity of PI-3Kand PKB, the most powerful antiapoptotickinases. On the contrary, the inhibition ofNF-kB was shown to occur as a conse-quence of proteasome inhibitors, corticos-teroids, and factors that are known to blockNIK and IKK [36], as well as under theinfluence of STAT-1, a tyrosine kinase thatappears to be a component of the signalingcomplex of TNF-R1 and TRADD. Appar-ently, STAT-1 is recruited by TNF-R1 andenables the formation of the DISC complex;furthermore it reveals apoptotic domains ofdeath-mediating proteins with the concomi-tant inhibition of the assembly of the sur-vival complex, which releases NF-kB [103].Substantial interest to study the physiologi-cal role of the STAT-1 resulted from its dualrole as the non-receptor tyrosine kinase andtranscription factor (the STAT acronymstands for that meaning). One should bear

in mind, however, that flavonoids are knownas the most powerful inhibitors of tyrosinekinases. Whether flavonols are potentinhibitors/activators of STAT-1 and effectsignal transduction form TNF-a to NF-kBwith simultaneous activation of genes sup-porting cell viability, is a matter of debateand needs experimental verification which iscurrently in progress in our laboratory.

The developing resistance of tumor cellsto chemotherapy is a challenge to contem-porary medicine. A number of drug resis-tance mechanisms are not known as well asthe origin of this phenomenon. Apparently,apoptosis is a hallmark of an efficient cyto-toxic effect of chemotherapy or radiationtherapy. Apoptosis is also widely acceptedas a mechanism leading to cell eliminationinduced by TNF-a. Therefore, TNF-a either/or chemotherapy as well as radiation therapymight be considered as important activatorsof NF-kB. It has been demonstrated thatinhibition of NF-kB supports the therapiesbased on the action of TNF-a [7, 102]. Amarked role of NF-kB has been observedin developing resistance to chemotherapyin the following cases: Hodgkin lymphoma,juvenile myelomonocytic leukemia, prostatecancer, virus-mediated leukemic T cells andtumor cells transformed with Ras oncogene.Tumor cells are also characterized by ahigher nuclear representation of NF-kB,moreover the genes regulated by NF-kB areoften constitutively upregulated in neoplas-mas [59, 70, 93]. NF-kB is thus linked totumor growth, because it inhibits apopto-sis. Several experimental data supportthe evidence of a profound role played byNF-kB in TNF-a-mediated apoptosis. Over-expression of IkB renders tumor cells sig-nificantly less susceptible to TNF-a-inducedcell death [28]. The reaction was observed intumor cell lines such as Jurkat T cells,human urine bladder line T24 and breastcancer MCF7. On the contrary, flavonoidsare cytopathic to tumor cells. The puzzlingissue of the developing resistance of tumorcells to cytopathic actions of flavonols in thepresence of TNF-a-stimulated NF-kB activ-ity remains unexplained and ambiguous [77].

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balance between the protein products ofgenes controlling cell life and death. Thekey roles whether an individual cell dies orremains alive may be the modulation ofantioxidant defences [12]. Quercetin hasbeen suggested to be beneficial for health,however, studies have shown that manyantioxidants can also exhibit, a prooxidantbehavior [16, 24]. Plant polyphenols includ-ing quercetin aglycon may interfere withthe cellular redox state by the inhibition ofglutathione reductase [110] and the effluxof glutathione S-conjugates [111]. The finaleffect of plant phenolics on the viability ofcells is therefore variable. At low concen-trations quercetin and derivatives seem toexert a stimulatory action on cell viabilityand survival, whereas at high doses (100 mMand higher) they are apoptogenic and cyto-toxic [3, 4, 51, 52, 85]. From a dietary pointof view, it remains to be established whatdoses could be considered as beneficial forhealth.

3.2. PUFA as ligandsof transcription factors

The development of obesity and associ-ated insulin resistance involves a multitudeof gene products, including proteinsinvolved in lipid synthesis and oxidation,thermogenesis and cell differentiation [20].The dietary w-6 and w-3 polyunsaturatedfatty acids (PUFA) suppress lipogenesis inthe liver while they simultaneously inducethe transcription of genes encoding proteinsof lipid oxidation and thermogenesis [21].Furthermore, the lipoprotein metabolic path-way is altered by peroxisome proliferator-activated receptors (PPARs). The PPARsare a member of the steroid hormone recep-tor superfamily. Three types of PPARs havebeen described: PPARa, PPARb/d (Nuc1),and PPARg. PPARa and PPARb are ubiq-uitously expressed in body tissues that pre-dominantly catabolize fatty acids (i.e. heart,liver, muscle, brown adipose tissue) whereasPPARg is selectively expressed in adipose

Another possible mechanism of cell tox-icity of flavonoids seems to be quite similarto that reported by Serrano et al. [89] forother phenolic compounds such as gallicacid and its esters, which inhibit proteintyrosine kinases (PTKs). Similar findingswere obtained by Kawada et al. [48] on thebasis of studies with quercetin and resvera-trol in cultured rat stellate and Kupfer cells.In their studies, the action of quercetin agly-con was associated with suppressed inositolphosphate metabolism, tyrosine phospho-rylation, reduced level of cell cycle proteincyclin D1 and mitogen-activated (MAP)kinase activation in PDGF/BB stimulatedstellate cells.

In certain cases quercetin aglycon couldpromote tumorigenesis and tumor growth[62] possibly by oxidative DNA damage inthe presence of Cu2+ [106]. We suggest thatthe anticarcinogenic activity of quercetinaglycon is dose dependent and is influencedby the presence of cytotoxic agents as wellas serum survival factors (cytokines, oxy-gen free radicals – OFR) [63, 86]. Pheno-lics stay in the first line of antioxidantdefence, donating electrons to OFR with aresultant formation of phenoxyl radicals[16]. Prooxidant phenoxyl radicals co-oxi-dise NADH and GSH, which in turn are nolonger able to inhibit NF-kB activation [17].It appears that apoptosis may be initiatedby phenolics. According to recent advancesin the interpretation of events that occur dur-ing programmed cell death, the generationand spreading of ROS within the cell areconsequences of the increased permeabil-ity of the mitochondrial membrane [80,109]. Lepley and Pelling [57] during a novelcell culture study with apigenin (a quercetinderivative), obtained evidence that flavonoidantioxidants may enhance apoptosis in cer-tain tumor cell lines. Hydrogen peroxide(H2O2), similarly to quercetin has beenreported to stimulate the activity of the mito-gen-activated protein kinases (MAPKs)ERK and JNK, and the expression of theproto-oncogenes c-fos and c-jun [97]. Cellelimination or survival is then a matter of

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tissues (recently found in other tissuesincluding skeletal muscle) and seems to beassociated with the differentiation ofadipocytes. PPARa is activated by PUFAsuch as eicosapentaenoic acid (w-3; EPA)or linoleic acid (w-6, LA) and heterodimer-izes with the 9-cis-retinoic acid receptor(RXRa). After ligand binding (EPA), it func-tions as a transcription factor in the regula-tion of adipogenesis and insulin-mediatedglucose transport. There is a positive cor-relation between the expression of Cu, Zn-dependent superoxide dismutase (SOD-1)and PPARa [42]. Moreover, the same groupobserved that an increased liver PPARamRNA level confers a reduction of theplasma TBARS levels indicating thecausative role of reactive oxygen species inthe pathology of insulin resistance [41].PUFA are not only strong ligands to PPARa,but also strong activators of PPARg andPPARb [13]. Ligand binding enhanced theinteraction of PPAR with its DNA-bindingdomains [44] called the PPAR response ele-ment (PRE). Functional PRE that reside inthe 5’-flanking region have been found toexist in several genes including thoseinvolved in the oxidation of fatty acids andthermogenesis (uncoupling proteins UCP-1and UCP-3), thus additionally supportingthe evidence for its anti-diabetic and anti-obesity function [27, 92]. Repartitioning ofmetabolic fuels away from storage andtowards oxidation reflects the fact thatPUFA co-ordinately suppress the transcrip-tion of lipogenic genes, while simultane-ously inducing the transcription of genesencoding proteins of lipid oxidation (b-oxi-dation of fatty acids) and thermogenesis [20,22]. This effect of PUFA is in turn medi-ated by the transcriptional and translationalsuppression of another group of transcriptionfactors termed sterol regulatory element-binding proteins (SREBPs) [73, 105]. Thus,PUFA play a beneficial role in health by ahypolipidemic action by lowering plasmacholesterol and preventing atherosclerosis,hypertension, cardiovascular diseases,obesity and insulin resistance. This is further

corroborated in studies performed by Mohanet al. [67], who observed that oral supple-mentation with oils rich in w-3 and w-6PUFA could protect animals against alloxan-induced diabetes mellitus. It is thought thatPUFA exert the aforementioned effect byenhancing the antioxidant status and sup-pressing the production of cytokines (TNF-a in particular). Apparently, PUFA play arole as intrinsic ligands in activating thePPARg – transcription factor, which up todate has been known to be activated merelyby thiazolidinediones (antidiabetic drugs).

4. PERSPECTIVES

In recent years research has been revo-lutionized by the implementation of rapidlydeveloping technologies. Examples are theconstruction of DNA/RNA arrays, the devel-opment of proteomics, the widespread avail-ability of probes for important signalmolecules, the insertion of reporter genesdownstream of regulatory sequences andthe use of gene knock-out models. All ofthese are having a major impact on the studyof disease and disease development at thegenome level. The same technologies areproviding a unique opportunity for estab-lishing the role of diet and dietary agents inprotecting humans against diseases and dis-orders. The effects of food-derived com-pounds on the regulation of a broad spec-trum of metabolic activities can thus beinvestigated, often simultaneously.

ACKNOWLEDGEMENTS

This work was supported by a grant No 3P06T 054 22 from the State Committee for Sci-entific Research in Poland.

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List of abbreviations

AA – arachidonic acid; AhR – aryl hydrocarbon receptor; AP-1 – activator protein 1; ARE/ORE –antioxidant/oxidant response element; Bcl-2/A1 – Bcl-2 antiapoptotic protein A1; BHA –butylated hydroxyanisol; CAT – catalase; CYP – cytochrome p450; CYP1A1, CYP1A2 –isoforms of cytochrome p450; DHA – docosahexaenoic acid; DHLA – dihomo-gamma-linolenicacid; DISC – death-initiated signaling complex; EGCG – epigallocatechin gallate; EPA –eicosapentaenoic acid; ERK – extracellular signal-regulated kinase; FADD – Fas-associated deathdomain; FOS – fructo-oligosaccharides; GLA – gamma-linolenic acid; GPx – glutathioneperoxidase; GR – glutathione reductase; GRE – glucocorticoid response element; GSH – reducedform of glutathione; GST – glutathione transferase; gsta1, cyp1a1, cyp1a2– genes for GST,CYP1A1, and CYP1A2, respectively; GTP – green tea polyphenols; ICE – interleukin 1 betaconverting enzyme/caspase-1; IKKa, IKKb – IkBa, IkBb kinase; IkBa – inhibitor of NF-kBalpha; JNK – c-Jun N-terminal kinase; LA – linoleic acid; MAPKs – mitogen-activated proteinkinases; MEKK1, MEKK2, MEKK3 – mitogen-activated protein kinase/ERK kinase 1, 2, 3;NAD(P)H – nicotinamide-adenine dinucleotide phosphate; NF-kB – nuclear factor kappa B; NIK –nuclear factor kappa B inducing kinase; OFR – oxygen free radicals; PDGF/BB – platelet derivedgrowth factor type BB; PI-3K – phosphatidylinositol 3-kinase; PKB – protein kinase B; PPAR –peroxisome proliferator-activated receptor; PRE – PPAR response element; PSE – plant stanolesters; PUFA – polyunsaturated fatty acids; RIP – receptor interacting protein; ROS – reactiveoxygen species; RXRa – 9-cis-retinoic acid receptor; SOD1- Cu, Zn-dependent superoxidedismutase; SREBP – sterol regulatory element-binding protein; STAT1 – signal transducer andactivator of transcription 1; TBARS – thiobarbituric acid reactive substances; tBHQ – t-butyl-hydroquinone; TCDD – 2,3,7,8-tetrachlorodibenzo-p-dioxin; TNF-R1, 2 –tumor necrosis factoralpha receptor 1, 2; TNF-a – tumor necrosis factor alpha; TRADD – TNF-R1-associated deathdomain; TRAF2- TNF-associated factor 2; UCP1, UCP3 – uncoupling protein 1, 3; UDP- uridinedinocleotide phosphate; XME – xenobiotic metabolising enzyme; XRE – xenobiotic responseelement.

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