The Spectrum of Phytoestrogens in Nature

7/31/2019 The Spectrum of Phytoestrogens in Nature...

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Phyergen 173

Address for correspondence:P. Moutsatsou, Department of Bioogica Chemistry, MedicaSchoo, University of Athens, 75 Mikras Asias Str., 115 27Goudi, Athens, Greece, Te.-Fax: +30 210 7462682,e-mai: [email protected]

Received 03-03-07, Revised 05-05-07, Accepted 06-06-07

The spectrum of phytoestrogens in nature: our knowledge

is expanding

Paraskevi Moutsatsou

Department of Biological Chemistry, Athens University Medical School, Athens, Greece

Review

HORMONES 2007, 6(3):173-193

ABSTRACT

The classical phytoestrogens, so far known, constitute a group of plant-derived compounds

which include mainly isoflavones, lignans, coumestanes, stilbenes and the flavonoids quer-

cetin and kaempherol. The discovery of many more novel estrogen-like compounds in the

plant kingdom demonstrates that the spectrum of phytoestrogens in nature is expanding. The

classical as well as the novel phytoestrogens show a complex mode of action via interaction

with the nuclear estrogen receptor isoforms ER and ER, exhibiting either estrogen-agonist

or estrogen-antagonist effects. Their final biological activity, assessed by cell culture assay

systems, animal studies and clinical trials, depends on multiple factors such as the chemical

structure of the phytoestrogen, the kind of tissue and cell type, the intrinsic estrogenic status,

the route of administration, the metabolism as well as the time and the level of exposure. Theyare characterized by high tissue specificity and dose-dependent activity. However, although

phytoestrogen intake as food or dietary supplements, in particular soya products and the iso-

flavones genistein and daidzein, has been associated with health promoting effects, some data

indicate increased disease risk. Evidently, phytoestrogen supplementation should be viewed

with caution until further studies satisfactorily delineate the effects of individual phytoestro-

gens on human health and disease.

Key words: Breast cancer, Estrogen receptor apha, Estrogen receptor beta, Coon cancer, Coro-

nary heart disease, Favonoids, Osteoporosis, Phytoestrogens, Prostate cancer, SERMs

INTRODUCTION

Estrogens are steroid hormones with a compex

mode of action, characterized by high tissue specificityand dose-dependent activity. They exert peiotropic

effects on a diverse range of tissues, such as ovary,

testis, prostate, breast, uterus, bone, iver, immune

system, cardiovascuar and centra nervous system.1

Estrogens promote breast and endometria cancer inwomen and exacerbate autoimmune diseases, whereasthe oss of estrogens during menopause has been

correated with osteoporosis, coronary heart disease,depression and neurodegeneration. Compounds whichantagonize the estrogenic effects (antagonists) in sometissues, such as breast and uterus, whie mimicking


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174 P.Moutsatsou

the estrogens effects (agonists) in other tissues, suchas bone, brain and cardiovascuar ces, are known asseective estrogen receptor moduators (SERMs).2

The substitution during menopause of endogenousestrogens with exogenous estrogens, widey known ashormone repacement therapy (HRT), is of enormousimportance. However, the Womens Heath Initiativestudy raised serious concerns about the safety of HRT,resuting in ow compiance of women due to the fearof breast and uterus cancer deveopment.3 SERMs

are considered as a new aternative postmenopausa

therapy, since they improve bone function with mini-ma risk for breast and uterine cancer. A arge body ofevidence has demonstrated that endogenous estrogens

and SERMs mediate their effects by moduating theintraceuar receptor subtypes estrogen receptor a-

pha (ER) and estrogen receptor beta (ER), both

members of the nucear receptor superfamiy. The

binding of estrogens or SERMs to estrogen receptors(ER and ER) initiates a series of events resuting

in transcriptiona reguation of specific genes, RNA

and protein synthesis.

Phytoestrogens are a arge famiy of pant-de-

rived estrogens possessing significant estrogen ago-

nist/antagonist activity. These, naturay occurring

moecues, incude the isofavonoids, the ignans,the coumestanes, the stibens and the favonoids

quercetin and kaemphero. Their effects, mediated

via the estrogen receptor subtypes ER and ER,

have been shown to be ce type/tissue specific and

dose-dependent. The so far known phytoestrogens

may act as natura SERMs and may possiby be

considered for the prevention of postmenopausa

osteoporosis and cardiovascuar disease without an

adverse effect on breast and uterus. However, since

data are sti controversia, further cinica studies are

needed to deineate satisfactoriy the heath effects ofphytoestrogens in specific popuation groups.4

Current data demonstrate that many more nove

compounds are continuousy being identified in na-

ture exhibiting estrogenic/ antiestrogenic properties,indicating that our knowedge about phytoestrogensin nature is expanding.

This review outines the major categories of the

so far known phytoestrogens, provides the current

knowedge regarding their overa bioogica effects

and briefy describes their estrogen-ike mediated

mechanism of action as we as their potentia in trig-gering other signaing pathways. The review presentsthe aboratory evidence for the nove pant-derived

compounds and their moduatory activity on the ERsubtypes. In order to have a better understanding of

the moecuar mechanisms by which phytoestrogens

moduate estrogen receptor-mediated activity, it wasconsidered essentia to briefy expicate the structureof estrogen receptors and their encoding respective

genes as we as some key stages in the estrogen

signaing cascade.

In this review the in vitro and in vivo assays, as-

sessing the estrogenic/antiestrogenic potentia of phy-

tochemicas, are aso presented and their advantagesand disadvantages are discussed.

ESTROGEN RECEPTORS

Structure

Estrogens exert their effects via their respective

receptors estrogen receptor apha (ER) and beta

(ER), which are members of the nucear receptor

superfamiy.5,6 Estrogen receptors have been identi-

fied in a types of human ces and have distinct

tissue expression patterns.1

Target organs with ahigh estrogen receptor content are those reated to

reproductive functions such as the mammary gand,

the ovaries and the uterus, known to be cassica

targets for estrogens. Bone, the cardiovascuar sys-

tem, the brain, the immune system and the iver are

characterized by ower ER content.

The primary structure of the estrogen receptors hasbeen fuy characterized and their respective genes,

ESR1 and ESR2 have been coned and sequenced.7,8The encoded protein moecues contain six functiona

domains (Figure 1): the transcriptiona reguation(A/B) domain (AF-1) which is highy immunoreactive,the DNA binding domain (C) which is responsibe

for binding to specific DNA sequences known as

estrogen response eements (ERE), the domain D

(between C and E domains, hinge region) and the

domains E and F responsibe for igand binding,

dimerization and transcriptiona activation (AF-2).

ER and ER differ in size (595 and 530 amino

acids, respectivey). ER and ER exhibit high ho-

moogy in their DNA binding domain (96%), partia


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Phyergen 175

homoogy in the igand binding domain (53%), but

very ow in their A/B domain (30%) ocated in the

amino-termina part of the moecues. Due to high

homoogy in the C domain, ER and ER are ikey

to bind to various igands with simiar specificity

and affinity. In ER and ER the conformation of

the -heix 12 (H12) strongy depends on the natureof the igand and evidenty different igands induce

different receptor conformations, thus affecting the

agonist/antagonist potency of igands.9 Differentia

spicing of the ER and ER genes resuts in variousER and ER spicing variants acking certain exons.Moore et a (1998) described five ER isoforms (ER1-5), R1 corresponding to the previousy describedER-530.10 These isoforms are co-expressed with thewid type receptors in various tissues and pertinent

data indicate that they are bioogicay active. Their

roe in estrogen-dependent diseases is thus underinvestigation.1

Mechanisms of estrogen action

Estrogens exert their effects on estrogen respon-

sive ces via activation of estrogen receptor (ER)

and, directy or indirecty, reguation of transcriptionof target genes invoved in the ce function. There

foows a brief description of the mechanisms of es-

trogen action, as reviewed by Nisson et a (2001).9

The term ER hereinafter refers to both ER and

ER subtypes.

Direct action of estrogen receptors

on the genome

Endogenous estrogens, synthetic estrogen anaogsor phytoestrogens enter the ces, where they bind tothe estrogen receptor (Figure 2). The conformation ofthe bound receptor depends strongy on the nature ofthe igand. Binding resuts in the reease of the recep-tor associated proteins, such as hsp90, dimerization

of the receptor (ER and ER may be present as

homodimers or heterodimers), rapid nucear oca-

ization of the activated ER-estrogen compex and

binding of the dimer to its specific DNA sequences

(EREs). The DNA bound compex interacts with other

coactivator proteins such as p160 and the proteins ofthe CBP/P300 famiy, as we as other transcription

factors, resuting in the remodeing of chromatin andinitiation of transcription by the basa transcriptionamachinery and RNA poymerase.11,12 The resut is

either induction or repression of genes and increase

or decrease in protein synthesis, respectivey. The

chromatin decompaction is a key step required for

the initiation of transcriptiona activation and requireshistone acetytransferases (HATs).13 Many coactiva-tor proteins required for ER activity are HATs (p160

famiy, p300, CBD).14 Another group of enzymes,known as histone deacetyases (HDACs), deacetyatehistones to promote ER-mediated transcriptiona

repression.14,15

Figure 1. Schematic representation of human estrogen recep-tor and and their respective isoforms (Matthews J, Gustafs-son JA, 2003 Moecuar Interventions 3:281-292), used by kindpermission.

Figure 2. The genomic cassica (via EREs) and non-cassi-ca (via AP-1 and viaNF-kB response eements) actions of theestrogen receptor (Nisson et a, 2001. Physio Rev 81: 1535-1565), used with kind permission.


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176 P.Moutsatsou

sponsibe for tissue-specific responses to estrogenic

compounds.9,23

Cross talk between estrogen and other signaling

pathways; Estrogen-independent activationof ER

Phosphoryation is one of the most frequent post-transationa modifications of proteins on serine andthreonine residues and ess on tyrosines. Phosphory-ation is reguated by ceuar enzymes (kinases and

phosphatases), which transfer a phosphate group

from ATP onto target proteins or remove it cor-

respondingy. Phosphoryation of the estrogen re-

ceptors is infuenced by the cognate igand and it

affects receptor functions positivey or negativey,

such as transcriptiona activity, stabiity and nuceo-cytopasmic shutting.24

Avaiabe data indicate that in severa ce types

extraceuar signas such as insuin, IGF-I, EGF andTGF-, via the mitogen activated protein kinases

(MAPK), phosphoryate the AF-1 region of ER andER resuting in transactivation of the receptors in

the absence of the igand.25 Studies have shown that

ER is phosphoryated by growth factors resuting in

the initiation of ERE-mediated gene expression.26,27

For fu activity of ER AF-1, the phosphoryation fSer-118 is very important, whereas for ER this effectis mediated via Ser-124. Phosphoryation may aso

take pace at tyrosine residues of ER, especiay at

Tyr-537 which reguates the igand binding step.9

ER and ER are aso activated by neurotrans-

mitters (dopamine) as we as by reguators of the

genera ceuar phosphoryation state such as proteinkinase A (PKA) and protein kinase C (PKC), which

aso phosphoryate ERs.28,29 The cycins, cycin A andD1, which are reguatory subunits of cycin-dependent

kinases (CDKs), have aso been shown to be ERactivators.30-32 The presence of factors other than thecassica hormone, activating ERs, accompanied by anincreased receptor phosphoryation, may moduate

the activity of ERs in response to diverse physioogi-ca signas.33,34 The estrogen-independent activation

of estrogen receptors may be important when the

concentration of growth factors is ocay increased

or when estrogens are too ow.

An aternative pathway of ER and ER action

has been reported by which the receptors infuence

gene transcription, not through the ERE-directed

cassica pathway, but independenty of ER binding to

its consensus response eements. This is accompishedby interaction of the receptors with the transcriptionfactor AP-1 (c-fos/c-jun dimers) by way of protein-

protein interactions, thus moduating the binding of

AP-1 to AP-1 sites on the DNA9,16 (Figure 2). Currentresearch indicates that binding of SERMS to ER

transactivates the transcription through binding to

EREs, whereas binding of SERMs to ER stimuatesthe AP-1 pathway, demonstrating that ER and ERrespond differenty to certain igands at an AP-1 ee-ment.17 Of note, some antiestrogens, such as tamoxifen,

are strong agonists of AP-1 dependent transcriptionin certain tissues, whereas in other tissues they act asantiestrogen via the cassica EREs. The estrogenic

effects of tamoxifen resuting in tissue growth and

differentiation, as for exampe in uterus and endo-

metrium, may be attributed to the AP-1 mediated

pathway, a major route via which estrogens promotegrowth and differentiation effects.18

Estrogen receptor interacts with another importanttranscription factor, nucear factor-kB (NF-kB), resut-

ing in suppression of genes, such as the intereukin-6gene, or activation of genes, such as the serotonin

receptor 1A gene16,19,20 (Figure 2). This suggests that

estrogen receptor reguates the NF-kB mediated geneexpression in a ce type-specific manner. Intereukin-6,a cytokine transcriptionay up-reguated by NF-kB,

reguates bone metaboism and endotheia ce func-tion, therefore its estrogen-mediated repression mayhave important impications in osteoporosis, infam-matory processes and atheroscerosis.20-22 Evidenty,

the interaction of ER with the transcription factor

NF-kB as we as with the AP-1 is subtype and tis-sue specific and is considered to be invoved in the

inhibitory effects of estrogens on proinfammatory

cytokine activity.21

In concusion, the two ER subtypes exhibit distinctinteractions with common or specific cofactors, bindin a variabe manner to a variety of igands and may

act via different response eements exhibiting ateredspecificity and affinity. These characteristics and thedifferentia expression of ER and ER may be re-


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Phyergen 177

Membrane and mitochondrial estrogen

receptors

Accumuated evidence strongy supports the pres-ence and importance of pasma membrane estrogen

receptors in a variety of ces. The membrane ER is

possiby simiar to the nucear receptor, which has

transocated to a particuar ocation in the membrane,known as caveoae, aowing its cross tak with other

signaing moecues.

The binding of 17-estradio (E2) to ce surface

proteins triggers downstream signaing cascades re-

suting in cacium fux, adenyate cycase activation

and cAMP generation, phosphoipase C activation

and IP3 generation. Given that these signas are

emanating from the activation of G protein reatedpathways and because G proteins and ER exist in

caveoae, it is ikey that an interaction may take

pace within this membrane domain. Aternativey,

E2 via ER may activate another G-protein couped

receptor (GPCR) in the membrane, thus affecting

G protein activation.35,36

Additiona signa transduction pathways, being

rapidy responsive to E2 and originating from the

membrane, invove the stimuation/inhibition (beingdependent on the ER or ER subtype) of phos-

phoinosito-3 hydroxy kinase (PI3K) and the famiy ofMAP kinases, such as extraceuar-reguated kinase(ERK), p38 isoform, as we as the c-Jun N-termi-

na kinase (JNK).37 Of note, the signaing from the

membrane can aso extend to other intraceuar

transcription factors such as AP-1 or NF-kB, resut-

ing in gene repression or activation. In concusion,

signas emanating from the membrane are transducedto the nucear compartment and reguate important

functions, such as ce growth and surviva, migration,angiogenesis and apoptosis.1,35,36

Finay, current data documenting the presence

of ER subtypes in the mitochondria of anima and

human ces impicate a roe of ER signaing in energyproduction, thus further emphasizing the compexityof ER-signaing.38,39

ER and ER expression

ERa and ER are co-expressed in many tissues.ER is primariy expressed in the uterus, iver, heart,kidney, whereas ER is primariy expressed in the

ovary, prostate, ung, gastrointestina tract, bad-

der, centra nervous system and hematopoietic sys-tem.1,9

Characterization of mice acking either ER r

ER r both (ERKO, ERKO and ERKO) hasyieded vauabe information about the unique roe

of each subtype in estrogen action in vivo.40 Briefy,

resuts showed that ER is required for norma

mammary gand deveopment and mediates estro-

gen-stimuated increase in bone formation, whie

ERs roe in bone is ess cear. ER is required for

protection of vascuar and endotheia ces against

injury as we as for cardioprotection in a myocar-

dia ischemia mode. ER appears to pay a roe in

adipocyte growth and proiferation, the ER aso

possiby invoved in adipogenesis.1

ER and ER areexpressed in various brain areas and ER-mediatedeffects are considered to provide neuroprotection. Ina mouse mode of brain injury, estradio treatment

protects wid-type and ER-nu mice from brain

injury, whie this protection is aboished in ER-numice, thus suggesting an important roe for ER.41

wever, recent data indicate that ER-mediates

decreased microgia activation, suggesting a roe of

ER in neuroprotection.42 On the other hand, ER

or ER seective agonists have been shown to protect

hippocampa neurons against gutamate-induced cedeath and enhanced bc-2 expression in hippocampaneurons, suggesting that activation of either ER or

ER can promote neuroprotection.43 Taken together,as ER is highy expressed in the brain compared to

the reproductive organs in femaes, ER-seective

agonists may be of benefit and protect, at east in part,from some neurodegeneration eading pathways.44

Accumuating evidence supports the key roe of ERsubtypes in some cancers, the ER expression being

significanty reduced in breast, prostate and coon

cancer.1 In ovarian cancer, ER is expressed in tumorsof both epitheia and stroma origin, whereas ER

is abundanty expressed predominanty in granuosa

ce-derived tumors and to a esser extent in epitheiaorigin tumors.45 Maignant ovarian tumors originat-

ing from epitheia surface constitute about 90% of

ovarian cancers and express ow eves of ER com-

pared to norma tissues. Recent data point out that

restoration of ER in ovarian cancer ces eads to

inhibition of proiferation and enhancement of apop-tosis, thus suggesting that ER may pay a possibe


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178 P.Moutsatsou

tumor-suppressor roe in ovarian carcinogenesis.46

Taken together, severa ines of evidence indicate

that ER exerts an inhibitory action on ER-medi-

ated gene expression and in many instances opposes

the actions of ER.23

Since many phytoestrogens bind to ER with

higher affinity than ER and because their potentia totransactivate ER is higher than ER, phytoestrogensmay be of benefit in tissues in which ER-mediating

signaing pays a significant roe.44

PHYTOESTROGENS

Structure/Classification

The main casses of phytoestrogens are the iso-favones (genistein, daidzein, gycitein, equo and

biochanin A), the ignans (enteroactone, enterodio),the coumestanes (coumestro), the favonoids (quer-cetin, kaempfero), the stibenes (resveratro) and themycotoxins (zearaeno)47 (Figure 3). They are eitherof dietary pant origin or ingested as precursors and

then metaboized in vivo by the microfora of the

human gut. Indeed, equo is produced from daidzeinby the intestina microfora, whie the mammaian

ignans, enterodio and enteroactone, are formed

from pant-ignan gycoside precursors by the activityof the gut microfora in the proxima coon.48,49 Of

note, in some studies myco-estrogens are not incudedin the group of phytoestrogens because they are not

funga-derived but pant devired estrogens.49 The

aforementioned compounds are a poyphenos andstructuray resembe the natura estrogens.

Poyphenos are present in a pants, whie there

are >8000 phenoic structures identified in nature

which may be categorized in more than 10 casses.

These may be simpe moecues (such as phenoic

acids) or highy poymerized (i.e. tannins). Favonoids,the most common poyphenos in the pant kingdom,contain more than 5000 compounds subdivided into

six major casses such as favones (e.g. apigenin,

uteoin), favonos (e.g. quercetin, kaemphero),

favanones (e.g. naringenin, hesperidin), favanos

(e.g. catechins, epicatechin, gaocatechin), anthocya-nidins (e.g. cyanidin) and isofavones (e.g. genistein,daidzein).50

Favonoids do not exhibit an estrogenic effect;

however, the isofavones (genistein, daidzein) and

some favones, favanones and favonos (apigenin,

kaempfero and naringenin) activate estrogen-recep-tor-mediated signaing.

Absorption and Bioavailability

Exporation of the bioavaiabiity of isofavones

(the most common form of phytoestrogens), particu-ary genistein and daidzein, reveaed that it dependson the gut microfora activity. Data show that, de-

spite the considerabe degradation of isofavones in

the gut, their pasma concentrations are significant.

Isofavones are mainy present as inactive gycosidesand, after remova of the sugar residue in the gut

by bacteria (beta-gycosidases), they become active

compounds, taken up by enterocytes and entering theperiphera circuation.51 Foowing absorption, they

are reconjugated in the iver, mainy to gucuronic

acid and to a esser degree to suphuric acid.52,53

There is a arge variabiity in genistein and daidzein

metaboism in humans, which has been attributed tovariabe individua intestina fora and transit time. Inthe Japanese, genistein has been found in micromoarconcentrations, but in western popuations, eves aremuch ower.49 Of note, Setche and Cassidy (1999)

have shown that in an adut, consumption of 50 mg/day

of isofavones may give rise to pasma concentrationsranging from 200-3000 nM.54 Severa other studies

assessing pasma concentrations of phytoestrogens

have demonstrated that the pasma eves in humansrange from 10nm-10M.55 With regard to ignans, theyare metaboized to enteroactone and enterodio in

the gut. In the Finnish popuation, consumption of

whoegrain bread (200g/day) has resuted in variabepasma concentrations of enteroactone (15-41.4

nmo/).49

A recent study assessing the effect of an 8-week

dietary soy intervention on urinary isofavone excre-tion among young girs (8-14 years od) reveaed a

very high urinary isofavone excretion as compared

to adut women. Such data suggest ess intestina deg-radation and/or greater absorption of isofavones in

young peope, emphasizing that further investigationsinto the pharmacokinetics and age at consumption

of isofavones are needed.56

Soy infant formuas are widey used and may resutin a daiy exposure of infants to isofavones (genistein,


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Phyergen 179

daidzein and equo) that is 6- to 11-fod higher on a

body weight basis from the dose that has hormona

effects in aduts consuming soy foods. The mean

pasma concentration of tota genistein in infants

fed soy-based formua has been reported to be 2,500pmo/m (684 ng/m) as compared to 276 pmo tota

genistein/m bood concentration found in Asians ona traditiona diet that is high in soy content.57,58 It hasbeen reported that the circuating concentration of

isofavones in infants fed soy-based formuas were

13,000-22,000 times higher than pasma estradio

concentrations in eary ife and may exert bioogica

Figure 3. The chemica structure of the so far known phytoestrogens.


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180 P.Moutsatsou

effects, whereas the contribution of isofavones frombreast mik and cow mik was negigibe.57 In addition,it has been shown that infants fed soy-based formuamaintain a high steady state pasma concentration

of isofavones, which may possiby be expained byreduced intestina biotransformation, as evidenced

by ow or undetectabe concentrations of equo and

other metaboites.59

In a recent report, isofavones in breastfed infants,whose mothers consumed soy for 2 to 4 days, were

significanty increased in the infants urine (from

29.8 to 111.6nmo/mg creatinine) and in breast mik

increased from 5.1 to 70.7nmo/l.60 Of note, in somecountries such as Israe, the soy-based formua is

widespread, with rates far beyond cinica indications(in case of suspicion of cows mik aergy).61 Since

there is a broad spectrum of infant foods becoming

avaiabe, there may be a high exposure to dietary

isofavones during the first year of ife. Due to the

ack of definitive evidence of hormona activities of

isofavones at doses reevant to the soy-fed infants,

further studies in experimenta animas and human

popuations exposed to isofavone-containing prod-

ucts and in particuar soy-based infant formuas are

necessary.62

Favonoids are mainy found conjugated withsugars (gycosides) in pants and occasionay as

agycones. Evidence supports that the agycone formof favonoids as we as their gycosides show a con-

siderabe degree of intestina absorption, ranging

from 10-60%, depending on the type and the dietarysource of the favonoid. For exampe, the absorptionof querquetin from tea has been shown to be haf thatof onions. Given that querquetin in onions is found

mainy as a gycoside moiety, it has been suggested

that the gycosidic form of favonoids may enhance

absorption.63

Current evidence indicates that manyof the metaboites of phytoestrogens and favonoids

formed, either in the gut or in iver, may be aso

bioogicay active and may mediate estrogen-signa-ing.64,65 Taken together, the bioavaiabiity of dietary

substances determines their activity in vivo. However,the reevance of in vitro studies to the in vivo situ-

ation shoud be considered with caution. Athough

metaboism in periphera tissues aso takes pace,

most of the conversion seems to occur at the eve

of the gut and iver, therefore the target tissue eves

may be anaogous to those in pasma.

Intake

Severa studies have investigated the dietary in-

take of poyphenos and phytoestrogens; resuts,however, are contradictory. Numerous factors, such

as ight, environmenta conditions, pant genetics,

ripeness and species variety, affect the formation

and the content of favonoids and phytoestrogens in

pants. Moreover, different methods assessing the

content of poyphenos in pants have given variabe

resuts.63 The most widey studied phytoestrogens arethe isofavones genistein and daidzein from soy food(mainy consumed in the Orient), whereas ignans aremainy consumed by Europeans, since foods such as

cerea brans, egumes and vegetabes (known to berich in ignans) pay important roe in the European

diet. Soya beans contain 560-3810mg/kg isofavones,depending on variety and growing conditions. Soya

mik, bean sprouts etc., contain 13-2030mg isofavones/kg, depending on the starting raw materia and fina

water content. Soy foods, however, are now widey

avaiabe to European consumers and soya-based

European foods contain 38-3000mg isofavones/kg,

depending on the source of soya and diution with

other ingredients.66 In the iterature, various reports

point out that the estimated daiy intake of favonoidsdiffers among countries, ranging from 20-100mg/day.63Dietary assessment of phytoestrogens gives estimatesvarying between an intake ranging from 50mg to 200mg of phytoestrogen/day in a traditiona Japanese

diet.67,68 In Asian countries, an average daiy dietary

intake of soy and isofavones is estimated to be 50g

and 20-150mg, respectivey, whie in western coun-

tries the intake is ower, i.e. 1g daiy and 2mg daiy,

respectivey.69 It must be underined that women

consuming the commerciay avaiabe phytoestrogen

preparations are taking doses ranging from 50-150mgagycones/day.70

In view of the above, it becomes evident that whenscreening phytochemicas to assess their possibe es-trogenic activity, the eves of the compounds tested

must be at a range that is thought to be achievabe inhumans in vivo. In this regard, many factors shoud

be taken into account, such as the daiy intake of the

phytochemica, the rates of metaboism and cearanceas we as the pasma eves, factors which are known


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Phyergen 181

to vary consideraby between individuas.

Phytoestrogens are modulators of ER and

ER-mediated activity

A arge body of evidence demonstrates variousfeatures of phytoestrogens: estrogen-ike action, es-

trogen receptor binding, ER-transactivation, estrogen-dependent target genet expression or ceuar growtheffects. Different studies, however, provide variabe

resuts regarding the estrogen-ike potencies of the

phytoestrogens. This may be due to the fact that the

potencies of severa phytoestrogens have been testedin various ce systems using different techniques (i.e.radioigand binding assays, transactivation assays,

target gene expression) under different conditions

(such as different dosages, in presence or in absenceof estradio), so that resuts are not comparabe.

Given the conficting resuts in the iterature,

Mueer et a (2004) empoyed a human endometriaIshikawa ce ine that staby expresses human ER

or ER to determine the potencies of the best knownphytoestrogens in one comparabe system for both

ER and ER activity.64 They found that the soy-

derived genistein, coumestro and equo dispayed

a preference for transactivation of ER-ERE re-

sponses compared to ER-ERE responses and were10- to 100-fod ess potent than diethystibestro.

Resveratro, enteroactone and its human metabo-

ite 6-OH enteroactone and human metaboites of

daidzein were weak agonists to both ER and ER.

Interestingy, they showed that phytoestrogens affectthe transcriptiona activity of ER and ER in an

ERE sequence-dependent manner,64,71 thus impy-

ing a degree of promoter dependency. Their igand

binding measurements reveaed that genistein, cou-

mestro and equo had high binding affinity to ER

and ER and with a distinct preference for ER.64

Quercetin, a common phytoestrogen, has been shownto induce ERE-dependent transactivation in MCF-7ces through both ER and ER but with a higher

capacity, ike genistein, to stimuate ER responses ascompared to the stimuation ER responses.72 It mustbe stressed that Kosteac et a (2003) reveaed that

the isofavones genistein and daidzein, its metaboiteequo, and coumestro were abe to moduate the

binding of both ER and ER to estrogen-response

eements (EREs).73 Of note, genistein and daidzein

preferentiay activated the binding of ER to ERE,

whereas coumestro and equo showed ony a sight

preference in the binding of ER to ERE compared

to the binding of ER to ERE. Such data ead to the

concusion that phytoestrogens differ not ony in theirERE-transactivation potencies and their ER-bindingaffinities, but aso in their abiity to increase the ER

binding onto DNA-response eements (EREs).

On the other hand, Harris et a (2005), in MCF-

7 ces transfected with ER or ER, evauated the

dose-dependent responses of various phytoestrogensin the absence and presence of 17-estradio.55 Theirresuts reveaed that genistein, daidzein, apigenin andcoumestro had a differentia and robust transactiva-

tion of ER- and ER-induced transcription, withup to 100-fod stronger activation of ER. Equo,

naringenin and kaempfero were weaker agonists. Inthe presence of 0.5 nM 17-estradio, the addition ofgenistein, daidzein and resveratro superstimuated

the system, whie kaempfero and quercetin acted

as antagonists at the highest doses. In the same ine,

Muer et a (2004) have shown that in the presence

of diethystibestro, resveratro dispayed a biphasic

activity: at ow doses it increased the DES-induced

activity of ER and ER, but at high doses it inhibited

the activity of ER and ER.64

It is of interest that the abiity of phytoestrogens

to moduate ceuar proiferation is aso dose-de-

pendent and estrogenic status-dependent. Resvera-

tro, genisteina and quercetin have shown biphasic

moduation with regard to proiferation of ER positivebreast cancer ce ines. They stimuate proiferation

at ow concentration (physioogicay reevant), the

ER possiby invoved in adverse ce proiferative

effects.74-76 However, they inhibit proiferation at

concentrations higher than 50-60, the inhibitory

ceuar growth effects considered to be exerted byway of ER-independent pathways. Of note, Shao et

a (2000) showed that genisteins anti-proiferative

effects are more pronounced in the presence of es-

tradio, impying that the good estrogen action of

genistein is reevant to chemoprotection.77

Anima studies have aso shown that phytoestro-

gens exert variabe effects depending on the absenceor presence of endogenous estradio. For exampe, agroup of immature, ovariectomized Sprague-Dawey


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182 P.Moutsatsou

rats ingesting a commerciay avaiabe, cosed-formuadiet containing high amounts of genistein and daid-zein (21mg genistein and 14mg daidzein per 100g of

rat feed) faied to exhibit a uterotrophic response to

administered estradio. This finding suggests that theresponse was aready stimuated to neary maxima

eves by phytoestrogens before the animas were

treated with estradio.78 However, in another study

invoving immature, intact Sprague-Dawey femaes

exposed to higher doses of genistein (37.5 or 75mg

per 100g body weight of a modified AIN-76 diet), nodetectabe increase in uterine weight was observed

when compared to contros fed the same diet.79 This

resut impies that, at east with respect to this as-

say, intact femaes are much ess responsive than

ovariectomized femaes to dietary phytoestrogens.Such findings emphasize the importance of variabe

dosages and the impact of estrogenic status in the

overa estrogenic effect of phytoestrogens.

Phytoestrogens, simiar to estrogens, have been

shown to act via severa membrane-initiated signaingmechanisms in ce ines expressing the membrane

version of ER (mER), resuting in the activation

of ERK kinases.80-82 Phytoestrogens have been shownnot ony to exert direct effects on ER activity but aso

to affect the formation of endogenous 17-estradio,thus reguating indirecty estrogen-signaing. Indeed,some inhibit aromatase, an enzyme that catayzes

the conversion of testosterone to estradio, thus

protecting against breast cancer via ER-independentmechanisms.83,84 Metaboites of genistein and daid-

zein isoated from human urine (i.e. gucuronides,

sufates) have aso demonstrated interactions with

estrogen receptor and , indicating that metaboismof isofavones not ony by enteric bacteria but aso byhepatic enzymes may affect estrogenic activity.65

In concusion, the so far known phytoestrogens andsome of their human metaboites, produced either

by enteric bacteria or hepatic enzymes, are seective

estrogen receptor moduators, exhibiting variabe po-tencies towards ER- or ER-mediated responses, theER-mediated activity preferentiay induced. Finay,most phytoestrogens moduate many other signaingcascades in various ce types invoving MAP kinase

and NF-kapa B signaing pathways, AP-1-mediated

signaing, ce cyce reguation, apoptosis and other

nucear receptor-mediated signaing.85-89

In view of the above data, care shoud be taken

when defining the estrogenic/antiestrogenic of a

compound. The intrinsic estrogenic status and the

dose shoud be considered, especiay in the context

of using a compound to prevent symptoms associ-

ated with estrogen deficiency during menopause

or to prevent hyperestrogenic effects in breast and

endometria cancer. Evidenty, it is essentia to assessthe phytoestrogens effects at mutipe eves, in vitroand in vivo, in order to obtain a fu picture which maybe reevant to different physioogica or pathoogicastates in humans (i.e. menopause, premenopause,

breast cancer).

NOVEL ESTROGEN-LIKE COMPOUNDS

OF PLANT ORIGIN

Apart from the known phytoestrogens, accumuat-ing evidence attests to the fact that there are many

more phytoestrogens in nature. A detaied presenta-tion of the recenty identified nove phytoestrogens

in nature is given beow. Their chemica structures

are shown in Figure 4.

Haabaaki et a (2000) isoated three nove aryo-benzofurans, namey ebenfuran I, ebenfuran II and

ebenfuran III, from the pant Onobrychis ebenoides.A three compounds showed high binding affinity forthe native ER.90 Further investigation by Papoutsi eta (2004) reveaed that ebenfuran II, at ow concentra-tions (10-6-10-9M), demonstrated a seective estrogenreceptor moduator profie, being antiestrogenic in

MCF-7 breast cancer ces and estrogenic in osteobasts(KS483 ce ine), without any stimuatory activity in

uterine ces, impying that it may be considered for

the prevention and treatment of breast cancer and

osteoporosis.91 Acteoside and martynoside, which arepant phenypropanoid gycosides, have been shown

to antagonize both ER and ER-mediated signa-

ing.92 Papoutsi et a (2006), by using the appropriate

end-point assays, demonstrated that martynoside wasanti-estrogenic in MCF-7 ces, induced mineraiza-

tion in osteobasts and inhibited endometria ce

proiferation, thus impying its potentia as natura

SERM.92

Eagic acid, a dietary poypeno, present in abun-dance in strawberries, has shown significant anties-

trogenic activity in MCF-7 breast cancer ces at ow


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Phyergen 183

concentration (10-9M-10-7M) and estrogenic activity

in osteobasts (induced mineraization), without anyeffects on endometrium-derived epitheia ces. In

this regard, eagic acid may be considered as naturaSERM.93

Deoxybenzoins, intermediates in the biosynthesisof isofavones, are found in severa pants and marinesources, such as Gycyrrhiza sp., Trifoium subter-

raneum and Ononis spinosa and have structura

simiarities to isofavones. Fokiaakis et a (2004)

showed that deoxybenzoins constitute a new cass

of phytoestrogens exhibiting affinities for ER and

ER, grossy comparabe to those of daidzein, whiesome demonstrated considerabe seectivity and

transcriptiona bias towards ER. Their activity, in

stimuating the proiferation of ER-positive breast

cancer ces and reguating the expression of endo-

genous and staby transfected reporter genes, differedconsideraby, with some inhibiting ce proiferation,whie effectivey inducing gene expression at the

same time.94

4-Ethoxymethypheno (4) is another nove

Figure 4. The chemica structure of some nove phytoestrogens. a. arybenzofurans, b. acteoside, c. martynoside, d. eagic acid, e.deoxybenzoines, f. ethoxymethypheno, g. catechin, h. epicatechin, i. gyecoins,j. indeyin, k. gRhi, l. gabrene, m. gabridin, n.isoiquitireginin chaone, o. soyasapogeno A, p. soyasapogeno B.


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184 P.Moutsatsou

phytoestrogen, found in Macura pomifera that acts asan agonist of ER and ER in MCF-7 breast cancer

ces and in Hela ces. The presence of coactivatorsSRC-1, CBP and E6-AP, enhanced, ike estrogen, the

4EM-mediated transcription of ER. The activity

of 4EM was specific for ER and did not activate the

transcription of the progesterone receptor.95

Tea catechins, which are favonoids, incude cat-

echin, epicatechin, epicatechin gaate, epigaocat-

echin and the epigaocatechin gaate. Experimentaevidence indicates that in Hela ces, transienty

transfected with an ERE-reguated uciferase reporterand an ER or ER expression vector, tea catechinsaone did not induce uc activity in either of ERs.

However, high concentrations of ECG or EGCG(5X10-6M) reduced the E2-induced estrogen effects,whereas ower concentrations increased significantythe E2-induced uc activity via ER and ER. In viewof the ower doses found in human pasma after tea

drinking aong with their estrogenic effects, the roe ofusua tea drinking might be hepfu for the preventionof estrogen deficient diseases, such as menopausa

disorders and osteoporosis.96

Phytoaexins constitute a chemicay heteroge-

neous group of antimicrobia substances. They are

synthesized de novo, accumuate in pants as a stressresponse and are products of a pants secondary

metaboism. The gyceoins represent a group of

phytoaexins whose biosynthesis is increased in re-

sponse to stress signas. Gyceoin accumuates in

high concentrations in soybeans under stress condi-

tions. The gyceoin isomers I-III are derived from

the precursor daidzein in the gyceoin pathway. Thegyceoin can represent as much as 56% of the tota

isofavone composition in soybean. In vitro data showthat gyceoins dispay ony sight estrogenic activ-

ity, but they cause a dose-dependent suppression ofE2-induced transactivation and MCF-7 proiferation.Gyceoins suppressed estrogen activity through bothER and ER in transienty transfected HEK 293

ces. Such data impy that gyceoins, being unique

antagonists on ER in both HEK 293 and MCF-7

ces, may represent an important component of the

heath effect of soybased foods.97

The phytochemica indeyin isoated from Rhei

rhizoma (rhubarb), a herba medicine extract tradi-

tionay used in Chinese medicine, acts as an antago-nist (10-5M) equay to both ER and ER, being a

nove phytoestrogen. However, it had no effect on

TR or PPAR, suggesting that the ER interactions

are specific. lindeyin has a phenoic backbone andis a gycoside obtained from Aeonium indreyi, a

crassuacea endemic to the Canary Isands.98

Ginseng is a popuar herba medicine that has

been used for over 2000 years in orienta countries. Acomponent of ginseng, saponin, known as gingenoside(gingenoside-Rh1), has been shown to activate the

transcription of the ERE-responsive uc-reporter genein MCF-7 breast cancer ces and CV-1 ces at high

concentration (50 ), impying that this compound

is a weak phytoestrogen, presumaby by binding andactivating the estrogen receptor.99

Consumption of Gycyrrhiza gabra, the icorice

pant, can be traced back 6000 years. There is evidencethat compounds isoated from the icorice root exhibitvarying degrees of ER action in different estrogen

responsive tissues. Briefy, resuts indicated that

gabrene, isoiquiritigenin (IlC) and gabridin bind

ER. The stimuatory effects of gabrene in vivo were

tissue specific and simiar to those of estradio. The

effects of concomitanty increasing concentrations of

gabrene and IlC on the growth of breast tumor ceswere variabe, promoting ER-dependent growth at

ow concentrations (10nM-10) and exerting ER

independent antiproiferative activity at concentra-

tions >15 .100,101

Saponins are, inter aia, important components ofsoy. Recent evidence has shown that a soy sapogenohas the potentia to induce estrogen receptor medi-

ated signaing.102

BIOLOGICAL EFFECTS

Epidemioogica, cinica and anima studies as

we as studies in vitro have provided data on the ef-

fect of phytoestrogens on human heath and disease.Accumuating evidence, based on epidemioogica

and anima studies, has indicated that phytoestrogensintake may have protective effects against various

cancers. However, controversia resuts have aso

been reported.49,103 Phytoestrogens, originating fromvarious pant sources, have shown variabe effects on


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Phyergen 185

different types of cancer. It is noteworthy that many

of the epidemioogica and experimenta studies havefocused on isofavones and soy products. It is ony

recenty that interest is increasing in the protective

effects of other casses of phytoestrogens, such as

ignans.49

It has been shown that in adut women the con-

sumption of phytoestrogen-rich foods such as soy, a

source of isofavones, is not or ony sighty protec-

tive against breast cancer. No negative effects of soy

on breast cancer have been reported, whie it may

be beneficia if consumed before puberty or dur-

ing adoescence or at very high doses.49,104,105 Such

findings are in agreement with concusions derived

from studies of immigrants and other epidemioogi-ca studies.49,106 On the other hand, a diet based on

whoegrain products, ow in ignans, resuting in ow

pasma enteroactone eves, has been associated withincreased breast cancer risk. A high pasma entero-

actone (30-70 nmo/) is probaby protective againstbreast cancer, at east in Nordic countries with high

whoegrain intake.49,107 In a recent prospective study,high genistein circuating eves were associated withreduced breast cancer risk in the Dutch popuation,

whereas no effects of ignans on breast cancer risk were

observed.108

It is important to mention that anima andce cuture studies have demonstrated that certainphytoestrogens ike genistein and resveratro not onyreduce but aso stimuate estrogen-dependent growthsuch as uterine growth and breast cancer growth,

depending on dose and timing of exposure.49,76,109-113

Severa other potentia adverse heath effects have

aso been reported.114,115 However, severa other inesof evidence indicate that genistein and resveratro

can protect against breast cancer in rat by reguat-

ing important mammary growth and differentiation

pathways, the timing of exposure being critica forthe mammary protective effects of genistein.116 Takentogether, the roe of phytoestrogens in breast canceris controversia and their overa cinica efficacy is

questionabe, whie negative effects on the breast

cannot be competey excuded.

Prostate cancer, one of the main causes of death

among men in western societies, has been studied

intensivey in reation to phytoestrogen intake. Epi-

demioogica, anima and ce cuture studies have

demonstrated that dietary phytoestrogens, mainy

soy and soy-derived isofavones, may pay a protectiveroe against prostate cancer.49 In a recent prospectivestudy in 43,509 Japanese men (age 45 to 74 years), whogeneray have a high intake of isofavones, intakes

of genistein, daidzein and soy food decreased risk ofocaized prostate cancer, whereas positive associa-

tions were seen between isofavones and advanced

prostate cancer.117 We-designed trias in human

subjects, however, assessing the roe of ignans in

prostate cancer are not avaiabe, thus any definite

concusions for this cass of phytoestrogens can not

be drawn.49,118

Data on the effect of soy and isofavones on cooncancer, based on experimenta rat modes, are confict-

ing, showing either no effect or even a stimuatoryone. Ryebran or isoated ignans seem to protect

against coon cancer and the formation of poyps.49

The positive findings from animas, however, can-

not be extrapoated to humans, thus, we-designed

experimenta studies are needed to define the impactof phytoestrogens on human coon cancer.

Severa anima and in vitro studies indicate that

phytoestrogens, mainy daidzein and genistein, preventbone oss. Human studies, however, which are of shortduration (6 months) and incude a sma number of

subjects, have demonstrated that phytoestrogens exertony moderatey beneficia effects on bone.67,68 Basedon data from the EU-funded project phytoheath,

there is a suggestion, but no concusive evidence,

that phytoestrogens, primariy genistein and daidzein,given as soyabean-protein isoates, whoe-soyabean

foods or extracts, suppements or pure compounds,

have a beneficia effect on bone heath.119 However, itis considered that ong-asting, we-designed cinicatrias are needed to prove a specific roe of phytoes-

trogens in the prevention of osteoporosis.

The phytoestrogen intake and the incidence of

hot fushes and vagina dryness in postmenopausa

women has been studied extensivey, yieding either

favorabe effects or no effect at a.112,119,120

Anima studies have ed to the concusion that iso-favones (in particuar their metaboic product equo)ead to infertiity (mainy in femaes) and endome-

tria hyperpasia in catte, sheep and the guinea-pig.

Despite the fact that phytoestrogens are impicated

in adverse effects upon fertiity in various animas,


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186 P.Moutsatsou

there are no simiar reports on humans consuming

arge amounts of these compounds (such as Asian

women). In humans, intake of phytoestrogens has

been reported to interfere with the reguation of the

menstrua cyce in premenopausa women (increasedength of the menstrua cyce and reduced lH, FSH

and progesterone eves).47

There is growing evidence inking the intake of

soy-based foods and genistein with a reduction of

coronary heart disease. Many of the studies have

shown that whoe-soyabean foods exert favorabe

effects on cardiovascuar disease, especiay on serumipoprotein profie, whie isoated isofavones seem

to improve ony the endotheia function.67,70,119 The

cardioprotective effect of the individua phytoestro-gens, however, has yet to be substantiated by ong-

term controed trias.121 Finay, in ce cutures it hasbeen shown that genistein may exert neuroprotectivefunctions or induce neurotoxicity.122-124 Recent in vivofindings strengthen the idea that soy induces neuro-

degeneration.125 Epidemioogica data demonstrate

a positive correation between tofu consumption andbrain atrophy.126 It is concuded that further studies

are needed to deineate the effects of phytoestrogen

on cognitive functions.

The variation between studies and the confictingresuts obtained may be due to mutipe factors, such asthe soy product used or the type of the individua phy-toestrogen used, their pant origin, the dose deivered,the time and duration of exposure, the metaboism,

the route of administration, the bioavaiabiity, as weas the intrinsic estrogenic status. Given that differ-

ences in the above parameters may resut in variabeand in many instances divergent effects, it becomes

evident that, at present, definite concusions on the

heath effects of individua phytoestrogens cannot be

drawn. The studies conducted so far are imited tosoya isofavones and soya products. Further systematicresearch is needed to evauate the cinica effects of

each individua phytoestrogen at the physioogica

and pharmacoogica eves, taking aso into accountwhether these substances woud be deivered in the

context of food or as dietary suppement or even as

pharmaceutica agents.127,128

At present, athough there are no approved heathcaims for phytoestrogens, many preparations in the

form of pant extracts or mixtures, containing varyingamounts of isoated phytoestrogens, are commerciayavaiabe on the market in numerous forms as dietarysuppements and as heath food products.70,129 Given

that these products are not under any reguatorycontros, their composition is not consistent, their

components vary greaty and, since there have not

been systematic dose-ranging studies, no dietary

recommendations for each of these products can be

made; therefore, their safety and cinica effectivenessare questionabe. Evidenty, the use of the so-caed

heath-promoting phytoestrogen-enriched products

shoud be viewed with caution.

TEST SYSTEMS ASSESSING

PHYTOESTROGENS ESTROGENIC ACTIVITY

In vitro test systems

The estrogenic/antiestrogenic potentia of phy-

toestrogens is usuay assessed by an array of in vitro

test systems as described beow:

1) Radiometric competitive receptor binding

assay.

Given that the first step in the ER transduction

cascade is binding of a igand to ER, the measurement

of igand binding is an important assay characterizingthe potentia of a test compound to interact with the

ER. The cassic igand binding assay uses radioac-

tivey abeed estradio, which competes with the testcompound for ER binding sites. This competitive

binding assay provides reative binding affinities

of test compounds to ER or ER compared to

estrogens, which is usuay unabeed estradio or

diethystibestro (DES). The use of a fuorescein

abeed estradio instead of radioactive abe made itpossibe to use fuorescence poarization to measure

binding affinities. Data indicate that reative bindingaffinity rankings of various compounds are identica

in both assays. The advantage of the radioactive bind-ing assay is that both quantification of ER binding

sites of any tissue or ce sampe expressing ER can

be obtained, and binding affinities of compounds

may aso be determined. In contrast, fuorescence

poarization requires the use of purified ER due to

the high background fuorescence in crude extracts.

The advantage of fuorescence poarization is that

it is a high-throughput assay which can screen many


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Phyergen 187

compounds for ER binding affinity within a day or

ess.

2) The uciferase reporter gene assay using ces

acking endogenous ER.The binding of the iganded ER dimer to the pro-

moter region of its target gene on DNA specific sites,i.e. the estrogen response eement (ERE) is a cruciastep in the ER signaing pathway. Transient transacti-

vation assays or recombinant ce assays, in which cesare co-transfected with the cDNA for ER, or R

and a reporter gene containing an ERE, are widey

used to measure igand-induced ER-mediated gene

activation. In this assay, a mammaian ce ine ack-

ing endogenous ER is transfected with an expression

pasmid carrying the cDNA of ER or ER togetherwith ERE inked to a uciferase reporter DNA. Addi-tion of test compounds, candidates for estrogen-ike

activity, induces dose-dependent transcription of thereporter uciferase that can easiy be monitored and

quantified. It is a very sensitive assay whereby very

weak to highy potent estrogens can be anayzed for

their estrogenicity and antiestrogenicity.

3) The uciferase reporter gene assay using ces

containing endogenous ER.

In the transient transactivation assays, exogenousreceptor (ER or ER) is forced into a ce accus-

tomed to the ack of ER; therefore, some effects

measured may not refect the physioogica responseof the anayzed ce type. These assays measure the

potentia of a candidate estrogen to trigger ER

signaing at the eve of the ER/ERE or ER/ERE

pathway. To account for these imitations, ce ines

with endogenous ER expression (ike MCF-7) can beused to measure the potentia of a candidate estro-gen to transactivate the native ER/ERE pathway.

4) Ce ines with endogenous ER expression

measuring the expression of endogenous ER-target

genes (end point assays).

Ce ines with endogenous ER expression can asobe used to measure the expression of endogenous ERtarget genes, i.e. the anaysis of end-points markers,

such as mRNA or protein expression. The measure-

ment of endogenous ER target gene (or protein)

expression represents a vauabe physioogica assay

for tissue specific estrogenicity or antiestrogenicity.

In this type of assay, a steps of the ER signaing

transduction pathway are considered, i.e. ER igand

binding, ER expression, ER dimerization, nucear

transocation, nucear binding, avaiabe co-activators,

etc. It is important to mention that the right protein(or gene) marker has to be monitored dependent onthe chosen ce ine.

5) Proiferation assay using an estabished ce inethat is known to respond to estrogens.

The proiferation assay assesses the growth promot-ing effects of a candidate estrogen, since estrogensare known stimuants of ceuar growth, whereas

antiestrogens arrest the growth of ces (depending onthe ce ine). This assay system measures the potentia

of a candidate estrogen to trigger generaized ef-fects on growth, which are the refection of compex

transcriptiona factors and may differ from the effectsof the candidate estrogen on igand-mediated regu-ation of a singe target gene or protein.130,131

Avaiabe data regarding the estrogenic/anties-

trogenic potency of compounds are predominanty

obtained with igand-binding, transactivation assays

and proiferation assays. Evidence shows that 1) it

is difficut to compare data about the estrogenic po-

tentia of compounds evauated by different methodsusing different end points; 2) the reative estrogenic

potency of compounds determined with different as-says might vary greaty; and 3) it cannot be expected

that the correation of the resuts obtained is aways

good. It is important to mention that the estrogenic

potencies of compounds determined by different as-says are not comparabe, whereas the sensitivity of

the different assays represented by the EC50 vaues

for the standard estrogen 17-estradio differ greaty(by two or three orders of magnitude), the ce free

binding assays being the ess sensitive.132

The aforementioned broad spectrum of in vitro

test systems may be usefu to eucidate the mecha-

nism of action of the test phytoestrogens and to set

priorities for further assessment. It is important

that the combination of severa in vitro test systems

be required in order to predict vaidy the effects

in vivo. However, the in vitro assays do not incude

either metaboites of compounds or aspects of the

absorption of compounds, thus they may generate

fase-negative or fase-positive effects. In vivo stud-


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188 P.Moutsatsou

ies are ony abe to predict the action of a substance

in the organism, since under in vivo conditions the

substance is exposed to absorption processes and

mutipe metaboic transformation.

In vivo test systems

Various in vivo assays (anima modes) are used tocharacterize the estrogenic potency of phytoestrogensand their mechanism of action. The animas used areusuay immature, hypophysectomized or ovariect-

omized rats, mice or rabbits. The test phytoestrogen

is given oray or subcutaneousy. Among the most

common anima modes are severa tumor modes, theuterotrophic assay and the anaysis of estrogen sensi-tive endpoints (such as morphoogica, histoogica,

biochemica and moecuar endpoints) in the uterusand other estrogen sensitive target tissues, such as

the vagina, the mammary gand, the iver, the bone,

the cardiovascuar system and the brain.

The uterotrophic assay assesses the abiity of phy-toestrogens to stimuate uterine growth. However, thisassay may not be exacty suitabe for assessing estro-

genicity, since there are compounds, ike raoxifene,

that exert tissue-specific estrogen-ike activity withouteffects on the uterus.133 Tumor modes are mainy used

to assess the potentia chemopreventive propertiesof phytoestrogens. There are severa approaches,

such as spontaneous carcinogenesis, chemica car-

cinogen-induced tumor modes and tumor modes

by xenotranspantation of tumor ces. Spontaneous

carcinogenesis may be appied for prostate and the

endometrium. Chemica carcinogen-induced tumor

modes invove the exposure of rats to DMBA or

NMU for the deveopment of mammary carcinomas.Finay, some tumor ce ines, if xenotranspanted toimmunodeficient nude mice or rats, grow tumors at

the ectopic site which eventuay metastasize throughbood or ymphatics. This mode is usuay used forbreast, prostate and endometria tumors.134

Additionay, there are more specific anima

modes to evauate the possibe beneficia effects of

phytoestrogens in osteoporosis, atheroscerosis and

neurodegeneration. These incude the ovariectomizedadut rat mode for osteoporosis, the rabbit mode forhigh-choestero induced atheromatosis and severa

anima modes for brain injury.135-145

In concusion, the use of a suitabe pane of dif-

ferent in vitro test systems combined with a fina

assessment in anima modes wi predict the rea

estrogenic potentia of phytoestrogens.

CONCLUSION

A high-phytoestrogen diet may resut in various

heath effects; however, some of the resuts obtainedare sti controversia. It must be stressed that pres-

ent data are imited in characterizing the bioogica

effects of soy products and the isofavones genistein,daidzein, whie ony a few data exist on ignans.

laboratory evidence indicates that apart from the

so far known phytoestrogens, e.g. the isofavones,

ignans, coumestanes, the stibenes and the favonoidsquercetin and kaempfero, there are many more phy-toestrogens identified in nature; thus the spectrum

of phytoestrogens is expanding and their bioogica

roe awaits determination. The experimenta meth-

odoogy foowed is of high importance in defining

the estrogenic effect of phytochemicas, the in vitro

studies providing the first cues about the estrogenic

potency, whereas the use of anima modes indicatesthe rea estrogenic potentia of phytoestrogens. Manydata have shown a wide range of hormona activities of

phytoestrogens (estrogenic/antiestrogenic via the ERsubtypes). Overa, however, phytoestrogen bioogicaactivity depends on mutipe factors, e.g. the chemicaform of the phytoestrogen, the route of administration,the metaboism, the intrinsic estrogenic status and thetime and the eve of exposure. On the market there

are severa preparations containing varying concentra-tions of individua phytoestrogens of different originand their use as dietary suppements may resut in

high eves of phytoestrogens, reative to those pro-

vided in whoe foods. In addition, the idea that what

is natura is aso safe may not prove vaid.

In this respect, the consequences of phytoestrogenintake may not necessariy be beneficia and in somecases may actuay increase disease risk.

Because of the ack of we-designed, prospectivestudies in humans for individua phytoestrogens anddue to the fact that the commercia preparations of

phytoestrogens are not subjected to any reguatory

contros, dietary recommendations for individua


17/21

Phyergen 189

phytoestrogens cannot be given. Caution is rather

suggested for their consumption and each preparationshoud be thoroughy evauated individuay before

it is used as a dietary suppement or as a therapeutic

agent.

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The Spectrum of Phytoestrogens in Nature