12 R. Kiyama, Y.

Wada-Kiyama / Environment International 83 (2015) 11–40

4. Evaluation of estrogen action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

4.1. Assays for detecting estrogen action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.1.1. Ligand-binding assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.1.2. Reporter-gene assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.1.3. Yeast two-hybrid assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.1.4. Transcription assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.1.5. Protein assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.1.6. Cell assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.1.7. Animal test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.1.8. Signaling pathway analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.2. Pathway-based risk assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5. Conclusion and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1. Introduction hormones in the body responsible for the maintenance of homeostasis

Estrogen is a female hormone that plays a role in menstrual and
estrous reproductive cycles. The role of estrogen is thus complex and
includes involvement in the physiology of reproductive organs and
tissues (e.g., breast, ovary and endometrium), lipid metabolism, protein
synthesis, behavior (e.g., lordosis behavior) and diseases (e.g., cancer
and neurodegenerative/cardiovascular diseases) (Deroo and Korach,
2006; Gillies and McArthur, 2010). While three major estrogens,
estrone, estradiol and estriol, are known, there are a number of estro-
genic chemicals of natural and industrial origins, as mostly character-
ized by comparing them with the major estrogens. Furthermore,
estrogenic chemicals are those that are not only directly responsible
for activating or inhibiting estrogen action, but also those indirectly
modulating its action; thus, a variety of chemicals should be considered
to understand its action. Estrogenic chemicals are thus considered as a
type of important chemicals acting as an endocrine disruptor, which is
defined as “an exogenous agent that interferes with the production, re-
lease, transport, metabolism, binding, action or elimination of natural
and the regulation of developmental processes” (Kavlock et al., 1996).
During our research for characterization of estrogenic chemicals, we
found that quite a number of chemicals have been reported to be estro-
genic. They were analyzed by a number of different assays and evaluat-
ed by different criteria to judge that they are estrogenic. Furthermore,
their mechanisms of action seem to be quite complicated. As we failed
to find a comprehensive list of estrogenic chemicals in literatures, we
examined potentially estrogenic chemicals one by one to find why
they were reported to be estrogenic.
2. Estrogenic chemicals
The mechanism of estrogenic signaling at the cellular level is sum-
marized in Fig. 1 (see also Kiyama and Zhu, 2014; Kiyama et al., 2014).
Estrogenic signal networks can be categorized into intracellular and ex-
tracellular types. The genomic pathway, which involves transcription of
target genes, and the non-genomic pathway, which rapidly transduces
signals mediated by membrane-bound estrogen receptors (ERs) and/

Intracellular Network Extracellular Network

Genomic Pathway Non-genomic Pathway Autocrine/Paracrine
Signaling
Hormone/
Estrogen Estrogen
GF/Cytokine

Membrane Other
ER Receptor
Crosstalk
Bypassing
Signaling
Signaling Signaling Signaling-pathway
Protein
Proteins Protein Analysis (S)
Yeast Two-
hybrid Assay (Y)

Ligand-binding Pol II
Estrogen
Assay (L)
Co-regulator
Nuclear Transcription Protein Function
ER

ERE Target Gene

Protein Cell Assay (C)
Assay (P)
Animal Test (A)
Reporter-gene Transcription
Assay (R) Assay (T)

Fig. 1. Signaling pathways, crosstalk and autocrine/paracrine/homeostatic signaling networks of estrogenic chemicals.
 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40
or other receptors through crosstalk and/or bypassing, belong to the
former, while the pathways of autocrine and/or paracrine signaling,
which involve other hormones, growth factors and cytokines, belong
to the latter.
The genomic pathway, or the classical pathway, is initiated by
the binding of chemicals (or ligands) with the nuclear ERs, ERα
and ERβ, and the ligand-bound ERs act as transcription factors to
up-regulate or down-regulate the transcription of target genes or
estrogen-responsive genes, in transcription machinery containing RNA
polymerase II and co-regulators of transcription, such as RIP-140,
p300/CBP and SRC-1 (Parker, 1998; Moggs and Orphanides, 2001). On
the other hand, estrogen binds to the membrane or cytoplasmic recep-
tors and stimulates signaling proteins through the non-genomic path-
way, which occurs rapidly, sometimes just minutes after stimulation,
and involves a number of signaling pathways. This type of signaling
starts by the binding of ligands to membrane/cytoplasmic ERs, and
several ERs, including G-protein-coupled estrogen receptor 1 (GPER),
have been identified. However, several new receptors and variants
of known receptors were identified, such as ER-X and ER-α36 (see
Section 3), increasing the complexity of estrogenic signaling. Estrogen-
related receptors also mediate estrogenic signaling. Meanwhile, recent
studies have revealed that intracellular networks cooperate with various
types of autocrine and/or paracrine signaling, and thus cells in different
tissues or locations are also involved in the extracellular networks
(see Section 3).
One of the reasons why a number of new signaling pathways were
identified recently is the availability of a variety of new technologies
to detect estrogenic chemicals (Fig. 1); they revealed the novel charac-
teristics of chemicals, such as biphasic and metabolic effects detected by
cell proliferation assay, the activity of selective estrogen-receptor mod-
ulators (SERMs) detected by cell assay and new estrogen-responsive
genes detected by DNA microarray assay (see Section 4). Thus, estro-
genic chemicals include chemicals or their metabolites that have not
only estrogenic effects but also anti-estrogenic ones, and they transduce
signals to estrogen signaling through crosstalk, bypassing and extracel-
lular networks.
2.1. Structure-based categorization of estrogenic chemicals
To understand the action of estrogenic chemicals at the molecular
level, especially at the cell signaling level, we categorized estrogenic
chemicals based on several characteristics. First, the estrogenic
chemicals categorized by structure are summarized in Table 1. A
number of estrogenic chemicals are phenolics, and phenolic
estrogens include simple phenols, phenolic acids/phenolic alde-
hydes, acetophenones, tyrosine derivatives, phenylacetic acids,
hydroxycinnamic acids, phenylpropenes, coumarins/isocoumarins/
chromones, naphthoquinones, bisphenols, benzophenones, stil-
benes/stilbenoids, anthraquinones, chalcones/chalconoids, fla-
vones/flavonoids, lignans/neolignans, diarylheptanoids, flavolans
and hydroxylated polycyclic aromatic hydrocarbons (PAHs). On the
other hand, chemicals without a phenolic ring can also show estrogenic
activity, which include anilines, carboranes, indoles, metalloestrogens,
perfluorinated compounds, phthalates, PAHs and terpenes/terpenoids
(monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes,
sterols, steroids, saponins and meroterpenes).
While estrogenic or anti-estrogenic activity can be detected by vari-
ous methods (see Section 4), they have limitations in their sensitivity
(e.g., concentration-dependent activity), distinguishing agonists/
antagonists and specificity (e.g., cell/tissue types and signaling path-
ways). Thus, the same estrogenic chemicals can show contradictory
results (listed in Tables 1 and 2), such as differences in estrogenic and
anti-estrogenic, agonistic and antagonistic, and ligand/pathway-
dependent and -independent responses, differences in increased and
decreased gene expression or enzymatic activity, as well as differences
13
in assay systems, which were reported for the chemicals summarized
below.
The list of chemicals showing contradictory results in estrogenicity.
Phlorizin (Wang et al., 2010), quercetin/naringenin (Marino et al., 2012), naringin
(Guo et al., 2011), epigallocatechin 3-gallate (Kuruto-Niwa et al., 2000), glabridin
(Tamir et al., 2000), enterodiol/enterolactone (Feng et al., 2008),
3-methylcholanthrene (Swedenborg et al., 2008), abietic acid/botulin/β-sitosterol
(Mellanen et al., 1996), methoxychlor (Lemaire et al., 2006), bifenthrin/permethrin
(Brander et al., 2012), raloxifene (Lee et al., 2011), tamoxifen (Jiang et al., 1995;
Obrero et al., 2002), PCBs (Zhang et al., 2014a) and BDE 47 (Karpeta et al., 2014).
Biphasic activity, where estrogenic activity was observed at low
concentrations and anti-estrogenic activity at high concentrations, has
been reported for phytoestrogens (Wang, 2002). Enterolactone, a lignin,
stimulates DNA synthesis in MCF-7 cells at 10–50 μM, but inhibits it at
higher concentrations. Enterolactone was shown to be a selective ER ac-
tivator through its interaction with ER (Penttinen et al., 2007). SERMs
are the chemicals that show differential (agonist or antagonist) effects
on different tissues or cells, where the effects are based on the modula-
tion of receptor functions by differential interactions between the
receptor and the ligand, which would be beneficial for the treatment of
hormone-responsive cancer and osteoporosis (Maximov et al., 2013).
Such effects are likely to occur at downstream pathways, such as in the
case of raloxifene (Lee et al., 2011) and tamoxifen (Jiang et al., 1995;
Obrero et al., 2002), where ER-dependent and -independent modula-
tions of signaling were observed.
2.1.1. Phenolic estrogens
Phenolic estrogens are categorized by the number of skeletal
carbons (Table 1; Vermerris and Nicholson, 2008). While phenol
itself is not estrogenic, simple modifications, such as chlorination
(pentachlorophenol), nitration (nitrophenol) and alkylation
(nonylphenol), confer estrogenicity on the derivatives. The derivatives
of phenolic acid and phenolic aldehyde, such as gallic acid, parabens,
benzaldehydes, syringic acid and ellagic acid, show estrogenicity. More
complex phenolics and their derivatives, including phenylethanoids
(acteoside and martynoside), tyrosine derivatives (thyroid hormone),
phenylacetic acid, naphthoquinones, bisphenols, benzophenones and
phenylpropanoids, also show estrogenicity. Phenylpropanoids are a
group of chemicals synthesized by plants from phenylalanine, and
include hydroxycinnamic acids (caffeic acid and ferulic acid),
phenylpropenes (anethole and eugenol), coumarins and related
chemicals (auraptene, bergapten, daphnetin, esculetin, icariin and
icaritin), stilbenes/stilbenoids, anthraquinones, chalcones/chalconoids
and flavones/flavonoids (anthoxanthins, flavanones, flavanonols, flavans,
anthocyanidines, isoflavones, isoflavanes, isoflavenes, coumestans,
pterocarpans and flavonolignans).
PAHs are known to show estrogenicity, which is partly explained by
the presence of hydroxylated phenolic rings (3,9-benz[a]anthracene
diols and cinanthrenol A; Table 2) or by the introduction of a hydroxyl
group after hydroxylation or metabolization within a cell.
2.1.2. Non-phenolic estrogens
A number of non-phenolics have been identified as estrogenic
chemicals, which include anilines, carboranes (boron estrogens), indoles,
metalloestrogens, perfluorinated compounds, phthalates, PAHs and ter-
penes/terpenoids (Table 1). Anilines are aromatic amines and used as
precursors to polyurethane, synthetic dyes and other industrial
chemicals. Because of similarities in their structure and biological activity,
anilines were expected to show estrogenicity (Hamblen et al., 2003).
Carboranes are a group of chemicals composed of boron, carbon and hy-
drogen atoms in polyhedral forms, and have advantages for designing
new ER agonists/antagonists and SERMs due to their unique three-
dimensional structure and superior synthetic flexibility (Armstrong and
Valliant, 2007). Indoles consist of a benzene ring fused to a pyrrole ring.
Some indoles show estrogenicity without having a phenolic hydroxyl
18 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40

Table 1 (continued)

Estrogenic chemical Signaling pathway Reference (assaya)

Saponin
Ginsenoside Rg1 (estrogenic) ER Gao et al., 2014 (P, R, T)
Ginsenoside Rg3 ER/PI3K/Akt/AMPK/NO/cardioprotection Hien et al., 2010 (S)
Ginsenoside Rh2 (estrogenic) ER Kovalchuk et al., 2006 (Y)
Ginsenoside Re ER/NO/K channel/vasodilation Nakaya et al., 2007 (S)
Gypenoside XVII ER/PI3K/Akt/Nrf2/ARE/autophagy Meng et al., 2014 (S)
Protopanaxadiol, protopanaxatriol ERβ/GR/NO/cardioprotection Leung et al., 2009 (S)
Soysaponin I (estrogenic) ER Ali et al., 2009 (R)
Withaferin A ERα/MAPK/p53/apoptosis Zhang et al., 2011 (S)
Meroterpene
Bakuchiol (estrogenic) ER Lim et al., 2011 (C, L, R)

Estrogenic chemicals categorized by structure are shown along with their effects, such as estrogenic and anti-estrogenic.
a
Abbreviations for assays are: animal test (A), cell-proliferation assay (C), ligand-binding assay (L), proten assay (e.g., Western blotting and immunoassay) (P), reporter-gene assay (R),
signaling pathway analysis (S), transcription assay (e.g., RT-PCR and DNA microarray assay) (T) and yeast two-hybrid assay (Y).
b
Phenolics are categorized by the number of skeletal carbons according to Vermerris and Nicholson (2008). AhR: arylhydrocarbon receptor; AMPK: AMP-activated protein kinase;
CaMKII: Ca2+/calmodulin-dependent protein kinase; EGFR: epidermal growth factor receptor; ER: estrogen receptor; ERK: extracellular signal-regulated kinase; FOXO: Forkhead box O;
GPCR: G-protein-coupled receptor; GPER: G-protein-coupled estrogen receptor 1; GR: glucocorticoid receptor; HIF-1α: hypoxia-inducible factor 1; IGF-1R: insulin-like growth factor 1
receptor; IL: interleukin; IRS-1: insulin receptor substrate-1; JNK: c-Jun N-terminal kinase; LXR: liver X receptor; MAPK: mitogen-activated protein kinase; mER: membrane estrogen
receptor; MT: melatonin receptor; NO: nitrogen oxide; PARP: poly(ADP-ribose) polymerase; PDGF: platelet-derived growth factor; PI3K: phosphoinositide 3-kinase; PKA: protein kinase
A; PPAR: peroxisome proliferator-activated receptor; PR: progesterone receptor; RARα: retinoic acid receptor α; SERM: selective estrogen receptor modulator; TGF-β: transforming
growth factor β; TNF-α: tumor necrosis factor α; TrkA receptor: neurotrophic tyrosine kinase receptor type 1.

group (Table 1). Metalloestrogens are inorganic metal ions that bind to
and activate ERs, and are classified into groups of metal/metalloid anions
and bivalent cationic metals (Byrne et al., 2013). Estrogenic activity has
been reported for arsenic, antimony, barium, cadmium, calcium, chromi-
um, cobalt, copper, lead, lithium, mercury, nickel, selenium, tin, vanadate
and zinc (Table 1). Perfluorinated compounds, such as fluorotelomer al-
cohols and perfluorohexane/perfluorooctane sulfonates, are
fluorocarbon-based oligomers, synthesized by telomerization. Volatile
fluorotelomer alcohols form perfluorinated carboxylic acids, such as
perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA), by
biodegradation, which persist in the environment and are found in the
blood of humans and wildlife. They have been shown to be estrogenic.
Phthalate esters are esters of phthalic acid and alcohols, and widely
used as plasticizers. Various phthalate esters, such as bis(2-ethylhexyl)
phthalate, butyl benzyl phthalate, dibutyl phthalate, diethyl phthalate,
diisobutyl phthalate, diisononyl phthalate and di(n-butyl) phthalate,
were found to show estrogenicity by various technologies (Table 1). Fi-
nally, terpenes/terpenoids are a group of chemicals having isoprene
units, (C5H8)n, and their derivatives, such as mono-, sesqui-, di-, tri-
and tetra-terpenes, as well as a diverse group of meroterpenes/sterols/
steroids/saponins. Some of them are estrogenic (Table 1).
2.1.3. Chemical basis for estrogen action
The structural basis of estrogenicity has been examined by compar-
ing the activity of chemicals before and after substituting chemical
groups. Anstead & Kym examined benz[a]anthracene and its analogs
by molecular modeling, and found that a polycyclic planar ring system
mimicking the steroid AB-ring is an important feature for the binding
affinity for ERs and carcinogenicity (Anstead and Kym, 1995). Routledge
& Sumpter examined the estrogenicity of alkylphenols with different
structural features (differences in size, branching and position) by a
yeast reporter gene assay, and found that alkylphenols substituted at
the 4-position with 6- to 8-carbon-containing, tertiary alkyl groups
exhibited higher activities (Routledge and Sumpter, 1997). By examin-
ing 120 aromatic chemicals, Schultz et al. found the importance of the
hydroxyl groups at positions 3 (C3–OH; A-ring) and 17 (C17–OH;
D-ring) of 17β-estradiol and hydrophobicity of its B- and C-rings
(Schultz et al., 2002). Hamblen et al. further examined the importance
of C3–OH by substituting it (4-substituted phenols) with the amino
group (4-substituted anilines) and found that 4-substituted anilines
showed activities at least 105 times lower than that of 17β-estradiol
(Hamblen et al., 2003). C3–OH, which is present in 17β-estradiol,
17α-estradiol and 17α-ethinylestradiol but absent in mestranol and
quinestrol, is suggested to confer anti-apoptotic behavior (Mann et al.,
2007) and to be important for the development of SERMs (Baker,
2013), while B-ring, which is saturated in the above compounds but un-
saturated in equilin and equilenin, is associated with the affinity for ERs
and the differential actions between ERα and ERβ (Bhavnani et al.,
2008; Bhavnani and Stanczyk, 2014).
2.2. Function-based categorization of estrogenic chemicals
Estrogenic chemicals can be categorized by applications, usage and ef-
fects (Table 2): They are related to various types of food additives/
dietary supplements, pesticides (antimicrobials/essential oils, biocides,
disinfectants/sanitizers, fungicides, herbicides, insecticides, miticides, re-
pellents, rodenticides and pheromones), pharmacological estrogens
(mycoestrogens, nonsteroidals, pharmaceuticals, pure ER antagonists,
SERMs and steroidals), plasticizers (phthalate/trimellitate/salicylate
esters and organophosphates) and pollutants (heavy metals or
metalloestrogens, persistent organic pollutants or POPs, pesticide resi-
dues, pharmaceutical pollutants and PAHs). Note that these categories
are arbitrary, so many estrogenic chemicals may belong to more than
one category.
2.2.1. Food additives/dietary supplements
The categories of food additives and dietary supplements contain a
number of chemicals with structural diversity (Table 2). The categories
of active components, antioxidants/preservatives, color additives,
cosmetics, emulsifiers/thickeners, flavor enhancers and vitamins consist
of phenolics and chemicals having a phenolic ring (e.g., coumestrol,
gallate, hydroxyanisole and tetrahydrocannabinol), unsaturated hydro-
carbons (e.g., carotenes, linoleic acid and lycopene), azo compounds
(e.g., tartrazine and sunset yellow), polysaccharides/sugar derivatives
(e.g., carrageenan, cyclodextrins and guanylate), saponins (ginsenosides
and notoginsenosides), and vitamins and their metabolites (e.g., niacin
and tocopherols). The chemicals in these categories have roles in various
biological effects: osteoporosis (caffeine; Zhou et al., 2009), apoptosis
(geniposide; Li et al., 2014b), cardioprotection (nectandrin B; Hien et al.,
2011), neuroprotection (notoginsenoside R1; Meng et al., 2014), vascular
relaxation (notoginsenoside Ft1; Shen et al., 2014), cell proliferation (vita-
min D; Gilad et al., 2005) and inflammation (niacin; Santolla et al., 2014).
2.2.2. Pesticides
Pesticides are substances intended for preventing, destroying,
repelling or mitigating any pest, and include avicides, bactericides, disin-
fectants (antimicrobials), fungicides, herbicides, insecticides, miticides,
molluscicides, nematicides, predacides, piscicides, repellents,
22 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40

Table 2 (continued)

Estrogenic chemical Signaling pathway Reference (assaya)

Pesticide residue (see pesticide shown above)

Pharmaceutical pollutant (see pharmacological estrogen
shown above)
Polycyclic aromatic hydrocarbon (see Table 1)

Estrogenic chemicals categorized by applications, usages and effects are shown along with their effects, such as estrogenic and anti-estrogenic. Characterization of estrogenicity, such as
estrogenic/anti-estrogenic and agonist/antagonist, is based on the description in each reference. Only a representative category for each chemical is shown.
a
Abbreviations for assays are: animal test (A), cell-proliferation assay (C), ligand-binding assay (L), proten assay (e.g., Western blotting and immunoassay) (P), reporter-gene assay (R),
signaling pathway analysis (S), transcription assay (e.g., RT-PCR and DNA microarray assay) (T) and yeast two-hybrid assay (Y). AhR: arylhydrocarbon receptor; BHC: benzene hexachlo-
ride; CAR: constitutive androstane receptor; DDD: dichlorodiphenyldichloroethane; DDE: dichlorodiphenyldichloroethylene; DDT: dichlorodiphenyltrichloroethane; E2: estradiol; ER: es-
trogen receptor; ERK: extracellular signal-regulated kinase; GPER: G-protein-coupled estrogen receptor 1; HCH: hexachlorocyclohexane; IGF-1R: insulin-like growth factor 1 receptor;
IRS-1: insulin receptor substrate-1; MAPK: mitogen-activated protein kinase; mER: membrane estrogen receptor; NO: nitrogen oxide; PBDE: polybrominated diphenyl ether; PCB:
polychlorinated biphenyl; PI3K: phosphoinositide 3-kinase; PKA: protein kinase A; PKC: protein kinase C; PPAR: peroxisome proliferator-activated receptor; RAR: retinoic acid receptor;
ROS: reactive oxygen species; SERM: selective estrogen receptor modulator; SXR: steroid and xenobiotic receptor; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TGF-β: transforming
growth factor β; TNF-α: tumor necrosis factor α; VDR: vitamin D receptor; VEGF: vascular endothelial growth factor.

rodenticides, defoliants, desiccants and growth regulators (Randall
et al., 2013). The pesticides within a particular class may share a com-
mon mode of action due to their structural similarity, and several chem-
ical groups, such as carbamates, organochlorides, organometallic
compounds, organophosphates, pyrethroids and sulfonylureas, have
been used. Many of them show estrogenic activity (Table 2), such as
those summarized below.
The list of pesticides showing estrogenic activity.
Aldicarb, bendiocarb, carbaryl, methiocarb, methomyl, oxamyl, pirimicarb and
propoxur (carbamates); aldorin, atrazine, chlordane, chlordecon, chloroxylenol,
DDT, dichlorophenyls, dicofol, dieldrin, endosulfan, endrin, fenarimol, fenvalerate,
hexachlorobenzene, hexachlorocyclohexanes, hexachlorophene, methoxychlor,
nitrofen, procymidone, propiconazole, terbuthylazine, toxaphene, triadimefon,
triclopyr, triclosan and vinclozolin (organochlorides); tributyltin (an organometallic
compound); chlorpyrifos, diazinon, fenthion, malathion, parathion and quinalphos
(organophosphates); eugenol, nitrophenol and 4-phenylphenol (phenolics);
bifenthrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, etofenprox,
permethrin, sumithrin and tetramethrin (pyrethroids); and sulfonylureas.
Estrogenic pesticides bind to ERs and affect the downstream signaling
pathways, such as the ER/ERK pathway for cell proliferation (tributyltin,
hydroxytyrosol, oleuropein, diedrin, procymidone and methoxychlor),
the ER/ERK/Akt/P70S6K pathway for cardioprotection (brefeldin A), the
ER/IGF-1 pathway for cell proliferation (hexachlorobenzene), the mem-
brane ER/K channel pathway for insulin secretion (sulfonylurea), the
ER/cyclin D1/Ras/Bax pathway for apoptosis (methoxychlor) and the
ryanodine receptor/ER/PKCε/PKA/Ca2+ pathway in the sweat gland
(ryanodine). Note that the same chemical may act as an estrogenic or
an anti-estrogenic depending on the assay used (e.g., bifenthrin, chlorpyr-
ifos and permethrin) or the receptors involved (such as methoxychlor)
(see Table 2).
2.2.3. Pharmacological estrogens
Pharmacological estrogens can be categorized into the groups of
mycoestrogens, nonsteroidals, pharmaceuticals, pure ER antagonists,
SERMs and steroidals (Pazaiti et al., 2012). Mycoestrogens are estrogens
produced by fungi, and include zearalenone, which is widely contami-
nated in agricultural products as a mycotoxin. Zearalenone and its de-
rivatives interact with ERα and ERβ in a manner similar to estrogen
and thus have been used as anabolic agents for animals and also pro-
posed for hormonal replacement therapy in postmenopausal women
or as oral contraceptives (Pazaiti et al., 2012). A number of pharmaceu-
ticals, such as those used for the replacement or addition of estrogen
(diethylstilbestrol, pure ER agonists, SERMs and steroids) or for other
purposes (acetaminophen, aspirin, bezafibrate, ephedrine, fenofibrate,
gemfibrozil and tocotrienol), are estrogenic (Table 2). Note that a num-
ber of chemicals other than those listed in Table 2 have been reported to
be SERMs, which include those categorized as synthetic estrogens and
phytoestrogens, such as bisphenol A, methoxychlor, naringenin,
nonylphenol, octylphenol and resveratrol, based on the differences in
gene activation in different cells (Ruh et al., 1995; Gould et al., 1998;
Gaido et al., 1999; Yoon et al., 2001; Safe et al., 2001).
2.2.4. Plasticizers
Plasticizers are used to increase the plasticity or fluidity of mainly
plastics, and some dicarboxylic and tricarboxylic esters, such as phthalate
and trimellitate esters, salicylate esters, such as benzyl salicylate (a tissue
conditioner), and organophosphates, such as tricresyl phosphate and
triphenyl phosphate, used as plasticizers, show estrogenicity (Table 2).
Phthalates and trimellitates are di- or tri-carboxylic acid derivatives of
benzene, respectively, and they do not have a phenolic hydroxyl group.
Estrogenicity is increased when the mass of participating alcohols in es-
ters becomes large (Parveen et al., 2008). Meanwhile, organophosphates
are phosphoric acid esters of alcohols and used widely for solvents,
pesticides and plasticizers, but they may cause adverse neuropsychiatric
and neurological effects, such as on the neurobehavioral development of
fetuses and children, by inhibiting acetylcholinesterase in nerve cells
(Chen, 2012). Tricresyl phosphate and triphenyl phosphate are esters of
cresol or phenol, respectively, and while there is no phenolic ring, they
interact with the ER and show anti-proliferative effects through the
NF-κB pathway (Sabatucci et al., 2006).
2.2.5. Pollutants
Pollutants can be tentatively categorized into heavy metals and
organic compounds, although any compounds found in the environ-
ment can be considered as pollutants. The organic pollutants include
POPs (Wang and Needham, 2007), pesticide residues, pharmaceutical
pollutants and PAHs, such as the chemicals derived from industrial
products, including pesticides, pharmaceuticals, plastics and volatile or-
ganic compounds (e.g., those in paint and coatings), and their deriva-
tives, and most of the categories include estrogenic chemicals
(Table 2). Estrogenic chemicals of industrial origins are those listed in
the categories of pesticides, pharmacological estrogens and plasticizers
(see above), while those of other industrial origins or their byproducts
are DDT, dioxins/dioxin-like compounds (Aroclors, PCBs and TCDD),
hexachlorocyclohexanes (HCHs) and PBDEs/PCDEs.
3. Molecular mechanisms of estrogenic signaling
The mechanism of estrogenic signaling can be divided into pathways
and networks (Fig. 1). The pathways that direct the signal to specific
functional outcomes, such as apoptosis, carcinogenesis, cell growth/pro-
liferation, differentiation/development and inflammation, are mostly
initiated by the binding of estrogen or estrogenic chemicals to ERs.
ERs can be classified into the groups of nuclear and membrane recep-
tors, which transduce signals through various pathways (Table 3). Spe-
cific signaling pathways, such as PI3K, MAPK/ERK and NF-κB signaling
(Ribeiro and Freiman, 2014), can be found in the pathways for different
outcomes as functional modules, where specific signaling pathways and
functional outcomes can be selected by selecting specific effectors.
24 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40

Table 3 (continued)

Receptor signaling pathway Reference

Other (continued)
Melatonin/α1/β-adrenoceptor/ER/cAMP/pinealocytes Hernández-Díaz et al., 2001
Methyl-amoorain + DMBA/ER/Wnt/β-catenin/carcinogenesis Mandal et al., 2013
Protopanaxadiol, protopanaxatriol/ERβ/GR/NO/cardioprotection Leung et al., 2009
Resveratrol/ER/TGF-β2/Smad/lung epithelial cells Suenaga et al., 2008
Ryanodine/ryanodine receptor/ER/PKCε/PKA/Ca2+/sweat gland Muchekehu and Harvey, 2008
Tamoxifen/EGFR/HER2/MAPK/Akt/ER/proliferation Osborne et al., 2005 (review)
Thyroid hormone/TR/ERα/proteolysis Alarid et al., 2003
Thyroid hormone/TR/integrin receptor/ERα/proliferation Meng et al., 2011
Autocrine/paracrine signaling
Autocrine signaling
E2/ER/Akt/IGF-1R/InsR/breast cancer Fox et al., 2013
E2/ER/ERBB4/PR/cell growth Zhu et al., 2006
E2/ER/IGF/IGF-1R/breast cancer Hamelers et al., 2003
E2/ER/IGF-1R/MAPK/cyclin D1, cyclin E/cell growth Kashima et al., 2009
E2/ER/MAPK/PDGF/PDGFR/proliferation Barbarisi et al., 2001
E2/ER/PDGF/PDGFR/ERK/proliferation Finlay et al., 2003
Exemestane/ER/amphiregulin/EGFR/MAPK/proliferation Wang et al., 2008a
Wnt/EGFR/ERK/ER/proliferation (crosstalk) Schlange et al., 2007
Paracrine signaling
Adiponectin/APNR/ERβ/differentiation Rahal and Simmen, 2011
E2/ER/Factor X/coagulation factor VII/Thrombin/development Jazin et al., 1990
E2/ER/FGF/FGFR/Tbx3/carcinogenesis Fillmore et al., 2010
E2/ER/TGF-β1/TGF-βR/Smad2/thyroid Gantus et al., 2011
E2/ER/Wnt/β-catenin/AXIN2/proliferation Ono et al., 2013
E2/ERα/JNK/cardioprotection Mahmoodzadeh et al., 2014
MCP-1/CCRs/PI3K/Akt/mTOR/ERα/proliferation (ligand-independent) Riverso et al., 2014
Autocrine/paracrine signaling
Androgen/AR/E2/ERα, ERβ/spermatogenesis Pearl et al., 2011
E2/ER/ANF/ANF receptor/cGMP/anti-hypertrophy Babiker et al., 2004
E2/ER/IGF-1/IGF-1R/osteoblasts Kassem et al., 1998
E2/ER/NF-κB/cytokines/cytokine receptors/inflammation, etc. Härkönen and Väänänen, 2006 (review)
E2/ER/STAT3/IL-6/IL6R/apoptosis Wang et al., 2001
E2/ERα/CXCL12/CXCR4/MAPK/proliferation Hall and Korach, 2003
E2/ERβ/CXCL12/CXCR4/MAPK/proliferation Sauvé et al., 2009
17α-E2/ER-X/MAPK/ERK/PI3K/Akt/brain Toran-Allerand et al., 2005

Estrogen-signaling pathways are categorized by the types of receptors and the ways of signaling. AhR: arylhydrocarbon receptor; ANF: atrial natriuretic factor; APNR: adiponectin receptor;
AR: androgen receptor; CAR: constitutive androstane receptor; CCRs: CC chemokine receptors; DDE: dichlorodiphenyldichloroethylene; DMBA: 7,12-dimethylbenz(a)anthracene; E2: es-
tradiol; ER: estrogen receptor; ERK: extracellular signal-regulated kinase; ERR: estrogen-related receptor; FOXO: Forkhead box O; GPER: G-protein-coupled estrogen receptor 1; GR: glu-
cocorticoid receptor; IGF: insulin-like growth factor; IGF-1R: IGF-1 receptor; IL-1: interleukin 1; IL6R: interleukin 6 receptor; InsR: insulin receptor; IRS-1: insulin receptor substrate-1;
JNK: c-Jun N-terminal kinase; LXR: liver X receptor; MAPK: mitogen-activated protein kinase; mER: membrane estrogen receptor; mTOR: mammalian target of rapamycin; NO: nitrogen
oxide; PDGF: platelet-derived growth factor; PDGFR: PDGF receptor; TrkA receptor: neurotrophic tyrosine kinase receptor type 1; PI3K: phosphoinositide 3-kinase; PKA: protein kinase A;
PKC: protein kinase C; PR: progesterone receptor; RAR: retinoic acid receptor; SERM: selective estrogen receptor modulator; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TGF-β:
transforming growth factor β; TGFBRs: TGF-β receptors; TR: thyroid hormone receptor; VDR; vitamin D receptor; VEGF: vascular endothelial growth factor; VEGFR-1:VEGF receptor 1.

Signaling pathways can influence each other not only through signaling
pathways by crosstalk (exchanging signals between different receptors
and/or pathways) and/or bypassing (signaling uni-directionally from
one pathway to another) at the intracellular level, but also through the se-
cretion of hormones or growth factors to transduce signals to different
cells or tissues, which may result in completely different types of func-
tional outcome. Feedback regulation of such signaling networks through
crosstalk/bypassing and autocrine/paracrine networks would make sig-
naling more complex, which may also contribute to pathways other
than estrogen signaling and eventually result in forming a homeostatic
network.
3.1. Receptors
Estrogenic chemicals first bind to ERs to initiate cell signaling. There
are three major ERs, in which ERα and ERβ are nuclear ERs and the G-
protein-coupled estrogen receptor 1 (GPER) is a membrane ER. In addi-
tion, recent studies have revealed the presence of other ERs, such as the
estrogen-related receptors (ERRs), variants of ERα or ERβ (ER-X and
ER-α36) and uncharacterized ERs. These receptors share some common
features, although there are differences in structure, signaling pathways,
cell specificity and functional outcomes.
3.1.1. Nuclear receptors
There are two major nuclear ERs, ERα and ERβ, which are encoded
by different genes, ESR1 located at 6q25.1 and ESR2 located at
14q23.2–q23.3 on human chromosomes, respectively. These receptors
have similar molecular sizes (595 amino acids for ERα and 530 amino
acids for ERβ) and share a common structural architecture composed
of three functional domains: the A/B domain at the N-terminal is in-
volved in transcriptional activation of estrogen-responsive genes (AF-
1); the C domain contains a two-zinc-finger structure responsible for re-
ceptor dimerization and DNA binding; and the E/F domain at the C-
terminal mediates ligand binding, receptor dimerization, nuclear trans-
location and transactivation of target gene expression (AF-2) in associ-
ation with co-regulators (Nilsson et al., 2001). While no ligand binds to
the A/B domain and thus AF-1 is ligand-independent, ligands bind to the
E/F domain and thus AF-2 is affected by ligands. Furthermore, ESR1 and
ESR2 are known to show splice variants, which add variation in terms of
cell/tissue-specific expression, ligand-binding specificity, cell localiza-
tion and cell functions (Taylor et al., 2010). Transactivation of target
genes is mediated by the binding of ERs with estrogen-responsive
genes with specific sequence motifs, such as GGTCACAGTGACC (Klein-
Hitpass et al., 1986), often with the help of other transcription factors,
such as Sp1 and AP1 (Safe, 2001).
The estrogenic chemicals that transduce signals through ERα and/or
ERβ are summarized in Tables 1 and 2. While they usually cannot distin-
guish between ERα and ERβ, some show a preference or exclusivity for
one of these (Escande et al., 2006). The difference in transactivation ac-
tivity of various compounds between ERα and ERβ was investigated,
where some phytoestrogens showed significant preferences in the
binding affinity (Kuiper et al., 1998). Such differences in estrogenic
 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40
action between ERα and ERβ have been investigated by using selective
antagonists against each receptor, such as 4,4ʹ,4″-(4-propyl-[1H]-
pyrazole-1,3,5-triyl)trisphenol (PPT) for ERα (Stauffer et al., 2000)
and 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN) for ERβ (Meyers
et al., 2001) (Table 3).
Recently, another nuclear receptor family, estrogen-related receptor
(ERRs), was found to be partly involved in estrogen signaling. ERRs
(ERRα, ERRβ and ERRγ) act as ligand-dependent transcription factors,
although no natural ligand has been identified. ERRs regulate the
genes that are distinct from ER-regulated genes and are associated
with physiological functions, such as negative regulation of osteoblast
differentiation (Gallet and Vanacker, 2010). Estrogenic signaling medi-
ated by ERRs has been reported for biochanin A, daidzein, genistein
and resveratrol (estrogenic), as well as apigenin, chlordane, diethylstil-
bestrol, kaempferol and toxaphene (Table 3). Crosstalk between ER and
ERRs was reported for resveratrol-induced functional modulation of the
respiratory chain (Lopes Costa et al., 2014).
3.1.2. Membrane receptors
The presence of membrane ERs (mERs) was evidenced by rapid, thus
non-genomic, mechanisms of estrogen signaling, which were observed
as rapid alterations in the levels of cAMP, cGMP and Ca2+, and in the
activity of enzymes (Soltysik and Czekaj, 2013). There are several
mERs, which are classified by their structure: canonical ERα and ERβ
translocated to the membrane after modification, variants of ERα and
ERβ (e.g., ER-X and ER-α36), GPER and less characterized mERs. Canon-
ical ERα and ERβ bind to caveolin-1 after being palmitoylated, which are
translocated to the membrane and anchored there as a dimer (Soltysik
and Czekaj, 2013). They mediate rapid signaling for hypothalamic func-
tion (Micevych and Kelly, 2012).
ER-X is a 62–63 kDa membrane protein likely to be derived from
ERα as a splicing variant (Soltysik and Czekaj, 2013). ER-X is activated
with 17α-E2, and associated with the function of the brain and the
uterus through MAPK and ERK signaling (Table 3). ER-X could be
involved in the E2-induced inhibition of Ca2+ influx and contraction in
murine cardiomyocytes derived from ERα/ERβ-knockout mice (Ullrich
et al., 2008).
ER-α36 is a splice variant of ERα lacking both AF-1 and AF-2 but
retaining the DNA-binding domain and the partial ligand-binding do-
main, and is predominantly localized at the membrane. ER-α36 inhibits
wild-type ERα (ER-α66) and ERβ in a dominant-negative manner and
thus contributes to the resistance of breast cancer to endocrine therapy
(Rao et al., 2011). ER-α36 mediates estrogenic signaling through the
MAPK/ERK pathway in breast cancer cells and c-Src-mediated signaling
in gastric cancer cells (Table 3). ER-α36 is involved in testosterone-
initiated carcinogenesis by transducing estrogenic signals after testos-
terone is converted to estrogen (Lin et al., 2009).
GPER, previously known as G-protein-coupled receptor 30 (GPR30),
is a G-protein-coupled receptor (GPCR) encoded by the GPER gene lo-
cated at chromosome 7p22.3 with high expression in the hypothala-
mus, pituitary gland, adrenal medulla, renal pelvis and ovary (Hazell
et al., 2009; Soltysik and Czekaj, 2013). GPER is a 7-membrane-spanning
protein with high affinity for E2 and other chemicals such as aldoste-
rone; thus, the binding of estrogen to GPER induces rapid non-
genomic signaling. Other than the cell membrane, GPER has been
reported to be located at the endoplasmic reticulum, Golgi apparatus
and even in the nucleus (Soltysik and Czekaj, 2013). GPER can be
activated directly by the binding of ligands other than estrogen, such
as bisphenol A, daidzein, dieldrin, diethylstilbestrol, equol, fulvestrant,
genistein, icariin, icaritin, kepone, niacin, nonylphenol, quercetin, ralox-
ifene, resveratrol and zearalenone as agonists to the receptor (Table 3).
Kajta et al. used two tetrahydro-3H-cyclopenta[c]quinoline analogs,
G15, a selective GPER antagonist, and G1, a selective GPER agonist, to
study the neuroprotective effect of daidzein (Kajta et al., 2013). GPER
is involved in the signaling pathways mediated by other receptors,
such as serotonin/serotonin 1A receptor (Li et al., 2013c), and crosstalk
25
with other signaling pathways, such as GPER/TGF-β1 crosstalk in
fulvestrant-induced cell migration (Li et al., 2014a) or icariin-induced
expression of type IV collagen (Li et al., 2013a), GPER/IL-1β crosstalk
in genistein-induced inflammatory response (Luo et al., 2012), and
GPER/EGFR crosstalk in equol-induced NO synthesis (Rowlands et al.,
2011), icariin- or icaritin-induced cell proliferation (Ma et al., 2014) or
niacin-induced inflammatory response (Santolla et al., 2014). GPER is
known to inhibit serotonin 1A receptor selectively (Xu et al., 2009;
McAllister et al., 2012; Akama et al., 2013) and to accelerate the efficacy
of its inhibitor fluoxetine (Li et al., 2013c) in neuronal cells, so it is a
target to treat mood disorders.
3.1.3. Other membrane receptors
Several groups of researchers found that cell signaling is initiated by
unidentified or unspecified membrane receptors (Soltysik and Czekaj,
2013). Some of them are likely to be variants of ERα or ERβ, while
the others are likely to be factors other than ERα, ERβ, ER-X or GPER
(Nethrapalli et al., 2005). Endogenous estrogens (E1, E2 and E3) and
estrogenic chemicals, such as DDE, 17α-E2, geniposide, naringenin
(anti-estrogenic), raloxifene, retinoic acid, sulfonylurea and tamoxifen,
bind to mER and transduce signals through pathways, such as ERK,
NF-κB and PI3K/MAPK signaling, to contribute to apoptosis, cell prolifer-
ation, differentiation and other cell functions (Table 3). Crosstalk was
implicated between mER and other receptors, such as natriuretic pep-
tide receptor (Stratton et al., 2010), RAR (Kauss et al., 2008), and IGFR
and EGFR in signalosomes (Soltysik and Czekaj, 2013) or K channels
(Nadal et al., 1998).
3.2. Cell functions and signaling pathways
There are a number of signaling pathways linked to estrogen and
estrogenic chemicals (a comprehensive list is shown in Kiyama et al.,
2014), which are associated with specific cell functions, such as chroma-
tin/epigenesis, apoptosis, autophagy, cellular metabolism, translational
control, cell cycle/DNA damage/cytoskeletal formation, immunology/
inflammation response, neurological diseases and development/
differentiation. Here, we discuss further the role of estrogenic chemicals
in some cell functions: apoptosis, carcinogenesis, cell growth and prolif-
eration, differentiation/development and inflammation.
3.2.1. Apoptosis
Estrogen is known to stimulate growth and inhibit apoptosis
(see Section 3.2.3). However, recent progress has revealed that estrogen
can induce apoptosis through several pathways, such as Fas/FasL, mito-
chondrial, NF-κB and PI3K/Akt pathways (Lewis-Wambi and Jordan,
2009). Fas/FasL plays a key role in the extrinsic pathway, a type of
signaling of external origin, while the mitochondrial pathway is induced
by various stresses and involves p53, Bax and other pro-apoptotic
proteins. Estrogenic chemicals are also related to either the inhibition
or the induction of apoptosis (Tables 1 and 2): Aroclor 1254, caffeic
acid phenethyl ester, epigallocatechin 3-gallate, formononetin, 25-
hydroxycholesterol, irigenin, mestranol, naringenin, plumbagin, quer-
cetin, quinestrol, tamoxifen, tectorigenin, withaferin A and wogonin
(pro-apoptotic); and equilenin, equilin, geniposide, ginsenoside Rb1,
methoxychlor, raloxifene and triclosan (anti-apoptotic). Furthermore,
estrogenic chemicals transduce signals through specific signaling path-
ways: Aroclor 1254 (ERα/cyclin D1/Bcl-2/caspase-3), epigallocatechin
3-gallate (ER/MAPK/Akt/caspase-3), formononetin (ER/Ras/MAPK/
Bax/Bcl-2), naringenin (ERα, ERβ/MAPK/caspase-3), plumbagin (ER/
NF-κB), quercetin (ERβ/caspase-3), withaferin A (ERα/MAPK/p53)
and wogonin (ER/PARP/Bax) (pro-apoptotic); and E2 (ER/STAT3/IL-6/
IL6R), geniposide (mER/PI3K/MAPK), ginsenoside Rb1 (ER/ERK/Akt),
methoxychlor and triclosan (ER/cyclin D1/Ras/Bax) and raloxifene
(ER/TNF-α/ERK/caspase-3) (anti-apoptotic).
26 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40
3.2.2. Carcinogenesis
Estrogen plays significant roles in the development of breast and
other cancers, and blockade of estrogen action with antagonists reduces
the risk (Villa, 2008; Yue et al., 2013). The estrogenic chemicals (includ-
ing those that modulate estrogenic signaling) that are associated with
carcinogenesis or cancer functions are as follows: cadmium, di(n-butyl)
phthalate, parabens and testosterone (positive effects); and genistein,
β-naphthoflavone, phenylacetate and tocopherols (negative effects)
(Tables 1 and 2). The signaling pathways involved in estrogen-related
carcinogenesis or cancer functions are: cadmium (ER/ERK), di(n-butyl)
phthalate (ER/TGF-β), E2 (ER/Akt/IGF-1R/InsR, ER/IGF/IGF-1R, ERα/IRS-
1/IGF-1R/PI3K/Akt and ER-α36/MAPK/ERK), methyl-amoorain + DMBA
(ER/Wnt/β-catenin), testosterone (ER-α36/ERK/Akt) and tocopherols
(E2/ER/PPARγ/Akt). Crosstalk of signaling in breast cancer has been re-
ported for the signaling pathways between estrogen and AhR (β-naph-
thoflavone: Wang et al., 2014), IGF-1 (genistein; Hwang et al., 2013),
TGF-β (Band and Laiho, 2011), PR (parabens; Vo et al., 2011) or PPARγ
(di(n-butyl) phthalate; Lee et al., 2009) (Table 3). The cancer-related
signaling induced by estrogen or estrogenic chemicals can be modulated
by other chemicals, such as testosterone (Lin et al., 2009), tocopherols
(Lee et al., 2009) and TGF-β (Lee et al., 2014).
3.2.3. Cell growth and proliferation
Cell growth and differentiation are key steps for the differentiation
and development of cells, tissues and organs and thus include a number
of cellular events, such as cell division, cell adhesion, cell movement, cell
survival and cellular response to a variety of stimuli and stresses. Estro-
gen promotes the growth and proliferation of various types of cell for
various cellular events, and thus, cell growth and proliferation have
been examined for various estrogenic chemicals and the signaling path-
ways related to them (Tables 2 and 3). Estrogenic chemicals associated
with cell growth and differentiation are summarized below.
The list of estrogenic chemicals associated with cell growth and differentiation.
Benzo[a]pyrene, bisphenol A, p,p'-DDE, o,p'-DDT, dieldrin, equilin, equol, estrone
(E1), estradiol (E2), estriol (E3), 17α-ethinylestradiol, 17β-E2 valerate, ferulic acid,
genistein, β-HCH, hexachlorobenzene, icariin, icaritin, methoxychlor, puerarin,
procymidone, quercetin and tributyltin (estrogenic); and aloe-emodin, baicalein,
bergapten, calycosin, emodin, formononetin, celastrol, curcumin, daphnetin,
esculetin, epigallocatechin gallate + curcumin, fulvestrant, hydroxytyrosol,
indole-3-carbinol, luteolin, naringenin, oleuropein, quercetin, secoisolariciresinol
and tamoxifen (anti-estrogenic).
Isoliquiritigenin and piceatannol show dose-dependent estrogenic
and anti-estrogenic effects (Maggiolini et al., 2002; Vo et al., 2010).
The major signaling pathways identified for estrogenic chemicals are
ER/Akt (benzo[a]pyrene, bisphenol A, dieldrin, ferulic acid, luteolin
and puerarin), ER/ERK (baicalein, benzo[a]pyrene, p,p'-DDE, o,p'-DDT,
dieldrin, E1, E2, E3, equol, ferulic acid, β-HCH, hydroxytyrosol, methoxy-
chlor, naringenin, oleuropein, puerarin, quercetin and tributyltin), ER/
MAPK (17α-ethinylestradiol, 17β-E2 valerate, naringenin, piceatannol,
procymidone and tributyltin) and GPER/ERK (dieldrin, genistein and
quercetin). There are cases where signals induced by estrogenic
chemicals affect other signaling pathways: bisphenol A, calycosin,
formononetin, hexachlorobenzene, indole-3-carbinol, luteolin and ta-
moxifen for IGF-1 signaling; equilin for NMDA signaling; icariin, icaritin
and secoisolariciresinol for EGF signaling; celastrol and piceatannol for
progesterone signaling; epigallocatechin gallate + curcumin for VEGF
signaling; and fulvestrant for Wnt/β-catenin signaling. Furthermore,
estrogenic signaling can be modulated by chemicals in other signaling
pathways by crosstalk, such as bergapten in TGF-βR/ERα (Panno et al.,
2012), farnesol in farnesoid × receptor/ER signaling (Journe et al.,
2008), melatonin in MT1/ERα signaling (Kiefer et al., 2005), progester-
one and progestin in PR/ER signaling (Migliaccio et al., 1998; Vallejo
et al., 2005; Ballaré et al., 2006), retinoic acid in RARα/ERα signaling
(Ombra et al., 2013), 3,5,3′-triiodothyronine in integrin receptor/ERα
signaling (Meng et al., 2011) and vitamin D in VDR/ER signaling (Gilad
et al., 2005; Swami et al., 2013), while the signals induced by estrogen
affect other signaling pathways through autocrine/paracrine networks
involving the secretion of hormones and growth factors, such as
ER/CXCL12/CXCR4 (Hall and Korach, 2003; Sauvé et al., 2009), ER/
EGFR (Osborne et al., 2005), ER/ERBB4 (Zhu et al., 2006), ER/IGF-1R
(Kashima et al., 2009), ER/PDGFR (Barbarisi et al., 2001; Finlay
et al., 2003) and Wnt/β-catenin signaling (Ono et al., 2013). There
are cases of ligand-independent activation of ER, where combina-
tions of signal inducers and receptors, such as ethanol/adenosine re-
ceptor (Etique et al., 2009) and MCP-1/CCRs (Riverso et al., 2014),
affect ERα signaling in the absence of its ligands for the induction
of cell proliferation. On the other hand, exemestane, an aromatase
inhibitor, modulates estrogen synthesis and affects ER and EGFR syn-
ergistically by autocrine signaling for cell proliferation (Wang et al.,
2008a). Note that silent estrogens, a type of estrogenic chemicals
without explicit cell proliferation activity, were observed for some
chemicals including brefeldin A, revealed by DNA microarray assay
(Dong et al., 2013a; Kiyama and Zhu, 2014). The presence of such a
type of estrogenic chemicals suggests that signaling pathways related
to estrogen action, such as cell growth, can be separated or differentially
manifested not only by SERMs but also by different types of chemicals,
and such a new type of chemicals could be explored to modulate estro-
gen action at the cell signaling level.
3.2.4. Differentiation/development
Estrogen plays critical roles in the sexual differentiation and devel-
opment of the brain, breast, prostate and other tissues and organs
(Ohtani-Kaneko, 2006; McPherson et al., 2008; Simões and Vivanco,
2011). Estrogenic chemicals associated with cell differentiation and de-
velopment are adiponectin, estradiolquinone, genistein, medicarpin,
resveratrol and tamoxifen (estrogenic or synergistic), and androstenol
and retinoic acid (anti-estrogenic or inhibitory), and the associated
signaling pathways are ER/NO/cGMP (resveratrol), ERα/MAPK/NF-κB/
AP-1 (genistein), ERα/Wnt3A/β-catenin (tamoxifen) and mER/MAPK
(retinoic acid) (Tables 2 and 3). Crosstalk was observed for the ER
signaling with CAR (androstenol; Min et al., 2002), LTD4 receptor
(estradiolquinone; Dietsch et al., 1996) and APNR (adiponectin; Rahal
and Simmen, 2011).
3.2.5. Inflammation
Estrogen has anti-inflammatory and vasoprotective effects when
it is administered, and several signaling pathways, such as the TNF-α/
NF-κB/JNK pathway, have been suggested to mediate its effects (Xing
et al., 2009). Estrogenic chemicals affect the inflammatory response
positively (daidzein and genistein) or negatively (baicalein, genistein,
4-hydroxyphenyl sulfonamides, niacin, p-n-nonylphenol, p-n-
octylphenol, oroxylin-A and resveratrol), or modulate the signaling in-
duced by estrogen (parthenolide, an NF-κB inhibitor) (Tables 1 and
2). The signaling pathways involved are ER/NF-κB (4-hydroxyphenyl
sulfonamides), ER/NF-κB/NO (p-n-nonylphenol and p-n-octylphenol),
ER/NF-ĸB/NO/TNF-α (baicalein), ER/TNF-α/IL-1β/IL-6/NO (oroxylin-
A), ERα/TNF-α/NO (daidzein and genistein), GPER/EGFR/ERK (niacin)
and GPER/IL-1β/MAPK (genistein). The anti-inflammatory effects of
an SERM, raloxifene, are ER-independent (Lee et al., 2011), suggesting
the presence of a new anti-inflammatory target. Resveratrol shows
pathway-selective ER signaling, where it activates the inflammatory
pathway but not the cell proliferation pathway, presumably by alter-
ing the way of recruiting the coregulators associated with ERα
(Nwachukwu et al., 2014).
3.3. Signaling networks
Estrogen receptors or estrogenic signaling can crosstalk with other
receptors or signaling, either (1) by modulating estrogenic signaling
(mostly induced with E2) by signals induced with other chemicals or
 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40 27

(2) by the interaction between different receptors or pathways. The for- Autocrine/paracrine signaling was reported for receptors, such as
mer case may include crosstalk through the genomic pathway, such as ANF receptor, APNR, AR, CCRs, CXCR4, EGFR (ERBB4), FGFR, IGF-1R/
between ERα and AhR (Safe and Wormke, 2003). The latter case may InsR, IL6R, PDGFR and TGF-βR, where the hormones and growth factors
include (2–1) direct interaction between different receptors or path- released are ANF, adiponectin, androgen, MCP-1, CXCL12 (SDF-1), EGF
ways, (2–2) one-way crosstalk, or bypassing, where signaling is uni- (amphiregulin), FGF, IGF-1/insulin, IL-6, PDGF and TGF-β1, respectively.
directional because one of the pathways involved is abortive due to The signaling pathways involved are ERK, JNK, MAPK, NF-κB, PI3K/Akt/
the lack of appropriate effectors or signal mediators, and (2–3) para- mTOR and Wnt/β-catenin signaling, which are related to cell growth
crine and autocrine signaling, where other hormones or growth factors and proliferation in breast and other cancer cells, and differentiation
are involved in the signaling. Note that, in some cases, ligands are not and other functions (Table 3).
significant or required to transduce signaling from one signaling path- In paracrine signaling, hormones and growth factors are secreted
way to another, where activation/inhibition of signaling is done by from a cell type and transported to another cell type. For example,
direct interactions between receptors, or the first signaling results in adiponectin secreted from adipocytes is transported to epithelial cells,
the production or the activation of the second receptor. and binding of adiponectin to its receptor and synergistic activation of
ERβ with genistein facilitate epithelial cell differentiation (Rahal and
Simmen, 2011). MCP-1 secreted from normal epithelial cells binds to
3.3.1. Crosstalk/bypassing
its receptor on breast cancer cells and activates their cell division with
Crosstalk of ER or estrogenic signaling pathways with other recep-
the help of activation of ERα through PI3K/Akt/mTOR signaling
tors or receptor-mediated signaling pathways has been reported for
(Riverso et al., 2014). Estrogen-induced activation of a procoagulant/co-
receptors, such as AhR, EGFR, HER2 and IGF-1R, and signaling pathways,
agulation factor further activates prothrombin to form thrombin, which
such as MAPK/ERK, c-Src, PI3K/Akt, NF-κB and NOTCH (Safe and
is a paracrine factor that acts to develop the uterus (Jazin et al., 1990).
Wormke, 2003; Ribeiro and Freiman, 2014). These pathways mostly
Wnt proteins secreted from mature myometrial cells in response to es-
promote the growth of cancer cells, so understanding them is essential
trogen and progesterone induce myometrial stem cells to develop
for cancer treatment. Furthermore, signals induced by the binding
leiomyomas in the uterus (Ono et al., 2013). Estrogen-induced secretion
of ligands (as agonists), such as biochanin A, formononetin, 3-
of growth factors and hormones from cardiomyocytes to other cells in-
methylcholanthrene, β-naphthoflavone and TCDD, to AhR are mediated
duces neovascularization and attenuates fibrosis (Mahmoodzadeh et al.,
by crosstalk between ER and AhR to contribute to various cell functions
2014). The role of 17α-E2 as an autocrine/paracrine factor was implicat-
through pathways such as PI3K/AKT and MAPK/ERK signaling, or
ed in brain functions and therapeutic use in association with its pre-
through transcription or proteasome-mediated degradation of ERα
ferred receptor ER-X (Toran-Allerand et al., 2005). Finally, since
(Table 3). Biochanin A and formononetin are estrogenic chemicals, but
estrogen itself is an important member of the autocrine/paracrine fac-
could act as AhR agonists and thus be anti-estrogenic through the
tors, the modulation of estrogen signaling is crucial to develop SERMs
crosstalk (Medjakovic and Jungbauer, 2008). AhR signaling is bypassed
for the prevention and treatment of postmenopausal degenerative and
by ER signaling (or crosstalk between ER and AhR is uni-directional) be-
neoplastic diseases (Härkönen and Väänänen, 2006).
cause inhibitory effects were observed only for β-naphthoflavone,
which inhibits ER signaling, but not for 17α-ethinylestradiol, which
3.3.3. Homeostatic networks
inhibits AhR signaling (Gräns et al., 2010).
Extracellular networks adopt a homeostatic nature when they have
Crosstalk has been reported between ER and PR for ER ligands
a system of feedback regulation in autocrine/paracrine signaling.
(genistein and piceatannol) or PR ligands (progesterone and progestin)
While such networks play important physiological and developmental
(Table 3), or between ER and AR in a ligand-independent manner
roles, such as that in estrogen regulation of estrous cyclicity (Yeo and
(Migliaccio et al., 2006). While ERβ and PR form a complex and crosstalk
Herbison, 2014), ovulation (Wintermantel et al., 2006) and spermato-
with each other (Vallejo et al., 2005), crosstalk between ER and growth
genesis (Chimento et al., 2014), too much positive or negative feedback
factors could down-regulate PR expression (Thakkar and Mehta, 2011).
regulation would cause the development and progression of cancer and
Crosstalk between ER and IGF-1R induced with estrogen or
other diseases. For example, positive feedback activation in an autocrine
estrogenic/anti-estrogenic chemicals, such as calycosin, formononetin,
loop between CXCR4/CXCL12 and ERα/ERβ signaling pathways pro-
genistein, ginsenoside Rg1, indole-3-carbinol and resveratrol, involves
motes the ER-dependent development of breast cancer (Sauvé et al.,
pathways, such as IRS-1 and PI3K/Akt signaling, and cell functions,
2009; Boudot et al., 2011).
such as cell proliferation and neuroprotection (Table 3).
The feedback regulation involving estrogen can be categorized by the
Crosstalk between ER and other receptors, such as α1/β-
role of ER: (1) modulation of signaling proteins or transcription factors for
adrenoceptor, CAR, GR, HER2, integrin receptor, LXRβ, natriuretic
growth factor and other signaling-related genes to increase/decrease the
peptide receptor, PPARγ, RAR, ryanodine receptor, TGF-βR, TR and
activity of signal mediators (e.g., Sauvé et al., 2009), (2) transcriptional
VEGFR-1, was also reported along with estrogen or estrogenic chemicals
modulation of ER genes to increase/decrease (mostly increase) the
(lycopene, resveratrol, tamoxifen and tocopherols), or ligands for recep-
amount, and thus the sensitivity, of ERs (e.g., Wintermantel et al., 2006)
tors, such as androstenol/CAR, cholesterol/LXRβ, epigallocatechin
and (3) modulation (mostly increasing) of estrogen synthesis by regulat-
gallate/EGFR, ethanol/adenosine receptor, hesperetin/TrkA receptor,
ing aromatase activity (e.g., Wang et al., 2008a). Examples of feedback
isoproterenol/β-adrenoreceptor, protopanaxadiol and protopanaxatriol/
regulation have been reported for estrogen, although other estrogenic
GR, ryanodine/ryanodine receptor, thyroid hormone/TR and vitamin
chemicals could also be involved, and such regulation of estrogen signal-
D/VDR and Wnt/Fz (Table 3).
ing could be used as targets for developing tumor-targeting drugs (Renoir
et al., 2013).
3.3.2. Autocrine/paracrine signaling
Since cells communicate with other cells in the neighborhood or 4. Evaluation of estrogen action
in distant locations (by paracrine or endocrine signaling) or with them-
selves (by autocrine signaling) through hormones, growth factors, Estrogen action is evaluated first by the structural and functional
cytokines, ions and other mediators, it is important to examine such com- characteristics of chemicals (Tables 1 and 2), whose functional out-
munications to understand the action of estrogen under physiological comes are interpreted partly by the signaling pathways that they
conditions. The signals induced by estrogen or estrogenic chemicals mod- induce (see Section 3). When these collections of information
ulate signaling pathways for other hormones and growth factors, which are used to predict the effect of estrogenic chemicals, we need
are secreted and transported, and finally affect the same cell or other cells. to understand the details of the technologies, especially their
28 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40
advantages/disadvantages and the limitations of the methods used
for the evaluation. Comparison of in vitro assays including their ad-
vantages and limitations was already discussed (Mueller, 2004;
Kiyama and Zhu, 2014). Here, we summarize the methods used
for the evaluation of estrogen action.
4.1. Assays for detecting estrogen action
Estrogenic activity can be detected by various methods, such as
ligand-binding assay, reporter-gene assay, yeast two-hybrid assay, tran-
scription assay, protein assay, cell assay, animal tests and signaling
pathway analysis, which are based on the molecular and cellular mech-
anisms of estrogen action (Fig. 1). The assays used and the pathways
identified for various estrogenic chemicals are summarized in Tables 1
and 2. Note that signaling pathway analysis is categorized separately
from other assays because it usually consists of the assays categorized
in other methods, and focuses on the identification of signaling path-
ways and functional outcomes rather than the evaluation of
estrogenicity alone. Here, we briefly discuss the assays used to detect
and characterize the estrogenic activity of chemicals.
4.1.1. Ligand-binding assay
Ligand-binding assay is a well-established method to detect
receptor–ligand interactions. An extract from hormonally respon-
sive tissues, such as the uterus and liver (Song et al., 1999;
Tollefsen and Nilsen, 2008), an extract from cultured cells expressing
ERs (Olsen et al., 2003) or a recombinant receptor protein (Goodin
et al., 2002) is used as a source of ERs. The assay quantifies the ability
of a test chemical to compete with 3H-labeled 17β-E2 in binding to
ERs, and the result is often expressed as the concentration showing
50% inhibition, or IC50. The result is also expressed as a relative bind-
ing affinity, the ratio of IC50 between the test and unlabeled E 2. In
place of radioactive ligands, assays using non-radioactive ligands
have been developed, such as the fluorescence polarization method
(Jung et al., 2004; Hashimoto et al., 2001; Mersereau et al., 2008)
and the competitive enzyme immunoassay (Nishizuka et al., 2004).
While such assays are easy and fast to perform, they cannot distin-
guish between agonists and antagonists. Recently, McLachlan et al.
developed a biosensor that can distinguish agonists and antagonists,
where two halves of a split Venus fluorescent protein fused to either
end of the ERα ligand-binding domain produce a fluorescent signal
after conformational changes induced by binding estrogenic
chemicals to the ligand-binding domain (McLachlan et al., 2011).
The quantitative structure–activity relationship (QSAR) method has
been developed on the basis of the molecular dynamic structure of the
compound and used to predict estrogenic activity and to explore the
interaction between ERs and ligands (Li and Gramatica, 2010). To obtain
more detailed information about ER–ligand interactions at the atomic
level, computational molecular modeling is a powerful tool. Li et al.
carried out a molecular modeling study combining molecular docking,
molecular dynamics stimulation and binding free energy calculation to
examine the interaction between hydroxylated polychlorinated
biphenyls (HO-PCBs) and ERs, and found that several amino acid
residues formed hydrophobic and hydrogen–bond interactions with
the ligand (Li et al., 2013d). Ligand-binding assay was used to character-
ize the estrogenicity of the chemicals (Tables 1 and 2), which are
summarized below.
The list of estrogenic chemicals characterized by ligand-binding assay.
Acetaminophen, acetylsalicylic acid, aldicarb, 3-alkyl naphthalenes, alkylphenols,
anethole, benzo[a]pyrene, bisphenol A, boric acid, chlordecone, citral,
dichlorophenyls, 9,10-dimethyl-m-carborane, diphenylamines, enterodiol,
enterolactone, epigallocatechin 3-gallate, equilenin, geraniol, glabridin, glycitein,
β-HCH, indole-3-carbinol, isocoumarins, isorhamnetin, kaempferide, kaempferol,
metalloestrogens, 3-(4-methylbenzylidene) camphor, myricetin, nerol, PBDEs,
phenanthrenes, 2-phenylindoles, 2-phenyl-isoindole-1,3-dione, phthalates,
quercetin, triazine, umbelliferone and zeranol (by means of competitive binding of
ERs against 3H–E2); caffeic acid phenethyl ester (by means of recombinant hERα);
auraptene, 3,9-benz[a]anthracene diols, carboranes, coumestrol, DDT, eugenol, glycinol,
juglone, naringenin, naringin, PBDEs, psoralidin, phenanthrenes and tocotrienol
(by means of molecular modeling); and arctigenin, bisphenol A, hexachlorophene,
liquiritigenin and menadione (by means of fluorescence polarization).
4.1.2. Reporter-gene assay
In a reporter-gene assay, the receptor, upon binding to ligands, binds
to a specific response element on DNA and induces transcription of the
downstream reporter gene. There are variations of this assay, such as in
terms of the reporters: β-galactosidase (lacZ), chloramphenicol acetyl-
transferase (CAT), luciferase and green fluorescent protein (GFP); cul-
ture systems; receptor/reporter gene constructs; and the protocols for
the transfection of reporter constructs into host cells. There are two
types of culture system, dependent on the status of the expression of en-
dogenous ERs. MVLN cells were developed from human breast cancer
MCF-7 cells constitutively expressing ERs by introducing a reporter
gene expressing firefly luciferase under the control of the estrogen-
responsive element derived from the 5′-flanking region of the Xenopus
vitellogenin A2 gene (Pons et al., 1990). New cell lines for luciferase-
based reporter-gene assay have been developed to detect steroid hor-
mones effectively: TGRM-Luc cells for glucocorticoids, TM-Luc cells for
progestagens, TARN-Luc cells for androgens and MMV-Luc cells for es-
trogens (Willemsen et al., 2004). They were used to assess estrogenic
and androgenic endocrine disruptors in sport supplements (Plotan
et al., 2012). More recently, a panel of mammalian reporter cell-line-
based CALUX (Chemically Activated LUciferase eXpression) bioassays
was developed to detect chemicals with androgen, estrogen, progester-
one and glucocorticoids (Sonneveld et al., 2005). The ER-CALUX bioas-
say utilizes human osteosarcoma (U2-OS) cells, which have been
stably transformed with specific estrogen-response elements linked to
a luciferase reporter gene, and evaluates estrogenicity by quantifying
luciferase protein.
When cells do not express endogenous receptors, it is necessary to
transfect two different plasmids constitutively expressing either a re-
ceptor or a reporter gene. Yeast cells are frequently used in this type
of assay because less time is required to detect the response with signif-
icant reproducibility. In the yeast estrogen screen (YES) assay, yeast
cells (Saccharomyces cerevisiae) were stably transfected with DNA
of two plasmid constructs containing the human ER gene and the
estrogen-response element-linked lacZ, and β-galactosidase activity
was detected colorimetrically using chromogenic substrates, such as
o-nitrophenyl-β-D-galactoside (ONPG) (Arnold et al., 1996). However,
the colorimetric assays take several days to perform and require cell
lysis. These problems were solved by using fluorescence-based re-
porters, such as firefly luciferase (Leskinen et al., 2005) or GFP (Bovee
et al., 2004). However, in yeast-based assays, the discrimination be-
tween agonists and antagonists is not effective, and the differences in
membrane permeability, transport proteins and signal transduction
pathways may cause differences in the response to chemicals between
yeast cells and humans. Reporter-gene assay was used to characterize
the estrogenicity of the chemicals (Tables 1 and 2), which are summa-
rized below.
The list of estrogenic chemicals characterized by reporter-gene assay.
Acetaminophen, alkylanilines, caffeic acid phenethyl ester, chlordecone, epigallo-
catechin 3-gallate, fisetin, galangin, isocoumarins, luteolin, menadione, naringenin,
naringin, parabens, pentachlorophenol and 3-phenoxybenzaldehyde (by means of
β-galactosidase-based reporters); acteoside, anthraquinone, apigenin, auraptene,
benz[a]acridine, benzanthrone, benzo[a]pyrene, biochanin A, butylated
hydroxyanisole, cajanin, carboranes, β-carotene, daidzein, dieldrin, ephedrine,
epigallocatechin 3-gallate, formononetin, genistein, ginsenoside Rg1, glycinol,
glyceollins, glyphosate, β-HCH, hexachlorophene, hydroxychalcones, insecticides,
γ-linolenic acid, liquiritigenin, luteolin, kaempferol, malathion, martynoside,
metalloestrogens, naringenin, naringin, PBDEs, PCBs, perfluorinated compounds,
phenanthrenes, 2-phenylindoles, phthalates, propyl gallate, psoralidin,
 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40
rhodoeosein, sesamin/sesamolin/sesamol, tartrazine, tricresyl phosphate and
zearalenone (by means of luciferase-based reporters); indole-3-carbinol and
metalloestrogens (by means of CAT-based reporter genes); and bifenthrin,
chloroxylenol, insecticides and taxifolin (by means of ER-CALUX).
4.1.3. Yeast two-hybrid assay
Yeast two-hybrid assay was originally designed to detect protein–
protein interaction in vivo and to identify the genes encoding the
interacting proteins using transcriptional activators such as yeast
GAL4 protein. To evaluate estrogenic activity, two plasmids are used: a
plasmid carrying a gene encoding a fusion protein containing the
GAL4 DNA-binding domain (GAL4DBD) and the human ERα/β ligand-
binding domain (hERα/β LBD) and another plasmid containing a
cDNA fragment encoding the ER-binding domain of a co-regulator
with a region encoding GAL4 transactivation domain (GAL4AD) fused
to the lacZ gene. Yeast cells are transformed with DNA of both plasmids,
and the transformants are selected by growth on a medium lacking leu-
cine and tryptophan. Test chemicals are added to the yeast cells to eval-
uate β-galactosidase activity. Since hERα/β LBD and the co-regulator
interact with each other only in the presence of a ligand, the expression
of the lacZ gene is dependent on the ligand binding. The assay revealed
that the ER also binds to nuclear receptor co-regulators, such as RIP140,
SRC-1, TIF1 and TIF2, in response to chemicals according to their estro-
genic activity, and demonstrated that the interaction between steroid
hormone receptors and co-regulators can be a useful tool for identifying
chemicals that interact with the receptors (Nishikawa et al., 1999). The
entire hERα in combination with the nuclear receptor-binding domain
of co-regulators, SRC-1 or TIF2, resulted in a higher response to estrogen
than GAL4DBD-hERαLBD (Lee et al., 2006). This suggests that the syner-
gistic action of AF-1 and AF-2 of hERα mediates the recruitment of
co-regulators, such as SRC-1, and also that a ligand-dependent direct in-
teraction between the B domain in AF-1 and C-terminal domain can sta-
bilize the cooperative interaction between ERα and co-regulators
(Métivier et al., 2001). The yeast two-hybrid assay was compared with
a commercial enzyme-linked immunosorbent assay (ELISA) kit to mea-
sure estrogenic activity or total estrogen concentrations, respectively, in
natural and waste waters (Allinson et al., 2011). While the two assays
showed a very good correlation and the ELISA kit is a reasonably rapid
tool for preliminary screening of water samples, the yeast two-hybrid
assay may not be accurate enough to represent mammalian or human
systems, and ELISA may not be able to detect unknown compounds.
Yeast two-hybrid assay was used to characterize the estrogenicity of
acetaminophen, bisphenols, n-butyl benzyl phthalate, β-cyclodextrin,
fenthion sulfoxide, hexachlorophene, 2-hydroxyanthraquinone, mena-
dione, naringenin, naringin, PCBs and phlorizin (Tables 1 and 2).
4.1.4. Transcription assay
Analysis of the transcripts of genes, or transcriptomic analysis, is one
of the most effective approaches to predict the toxicity of chemicals, and
various transcription assays, such as DNA microarray assay, Northern
blotting, RNA-seq, RT-PCR and the serial analysis of gene expression
(SAGE), have been developed (Kiyama and Zhu, 2014). These assays
quantify the expression of ER genes or specific marker genes, such as
APOA1 (ApoA-1), BRCA2, CCDN1 (cyclin D1), PGR and TFF1 (pS2) (by
Northern blotting and RT-PCR), or sets of genes responding to estrogen
or estrogen-responsive genes (by DNA microarray assay, RNA-seq and
SAGE). DNA microarray-based evaluation of endocrine disruptors has
been discussed previously (Tanji and Kiyama, 2004; Kiyama and Zhu,
2014). One of the advantages for DNA microarray assay over other tran-
scription assays is that it can give gene-expression profiles of the
chemicals examined, which could be used to compare the effects of
chemicals by comparing the genes related to various cell functions.
Comparative evaluation of DNA microarray, RT-PCR and real-time
RT-PCR showed DNA microarray assay has the advantage over the
others in effectiveness and quickness in diagnosis (Sultankulova et al.,
2014). Gene expression datasets of different size and experimental
29
design could be combined by the Generally Applicable Gene-set Enrich-
ment (GAGE) method and used to predict estrogen signaling pathways
(Luo et al., 2009). Recently, DNA microarrays have been developed for
non-mammalian genes and used for evaluation of estrogenic chemicals
(Baker et al., 2013; Hao et al., 2013). Transcription assay was used to
characterize the estrogenicity of the chemicals (Tables 1 and 2), which
are summarized below.
The list of estrogenic chemicals characterized by transcription assay.
Apigenin, arsenic, benzo[a]pyrene, biochanin A, curcumin, cyanidin-3-glycoside,
daphnetin, DDT, dieldrin, epigallocatechin 3-gallate, esculetin, fenarimol,
fluorotelomer alcohols, flutolanil, ginsenoside Rg1, glycinol, hydroxymatairesinol,
insecticides, isorhamnetin, kaempferol, liquiritigenin, lycopene, myricetin,
naringenin, naringin, irisolidone, parabens, perfluorobutanesulfonic acid,
perfluorooctyl iodide, psoralidin, quercetin, sesamin/sesamolin/sesamol, silibinin,
tartrazine, TCDD, tectorigenin, tocotrienol, zearalenone and zeranol (by RT-PCR);
curcumin, genistein, juglone, phloretin and phthalates (by means of DNA microarray
assay); and β-carotene, indole-3-carbinol, insecticides, lycopene and SERMs
(by Northern blotting).
4.1.5. Protein assay
Various analytical matrices for qualitative and quantitative screen-
ing of endocrine disruptors have been developed from large-scale phys-
icochemical methods, such as mass spectrometry (MS), to bench-scale
approaches, such as Western blotting and immunohistochemistry. In
order to detect protein quantitatively, radio-, enzyme- and fluoro-
based immunoassays (IAs), ELISA, liquid chromatography (LC), gas
chromatography (GC), MS and combined MS with immunoassay
(MSIA) are available. It should be noted that a single or several protein
markers are often used in these assays, whereas sets of protein markers,
such as those used for expressional profiling in transcription assays, are
rarely used, mainly due to the difficulty in preparing tens or hundreds of
antibodies or peptides at low cost and in predicting estrogen action by
proteomic profiling. Thus, ER proteins and markers, such as APOA1
(ApoA-1), CCDN1 (cyclin D1), PGR (PR), TFF1 (pS2) and vitellogenin,
are often detected alone by protein assay. Protein assay was used to
characterize the estrogenicity of the chemicals (Tables 1 and 2), which
are summarized below.
The list of estrogenic chemicals characterized by protein assay.
Acetaminophen, biochanin A, caffeic acid phenethyl ester, β-carotene, coumestrol,
curcumin, daphnetin, epigallocatechin 3-gallate, esculetin, flutolanil, genistein,
glyphosate, insecticides, irisolidone, metalloestrogens, myricetin, naringin, parabens,
PCB3, tartrazine, tectorigenin, tocotrienol and zearalenone (by Western blotting);
bisphenol A, β-cyclodextrin, genistein, insecticides, nothofagin, oxybenzone, SERMs,
silibinin and triclopyr (by immunoassay); myricetin (by ELISA); and epigallocatechin
3-gallate, ginsenoside Rg1 and psoralidin (by ChIP assay).
4.1.6. Cell assay
Cell growth and proliferation can be detected by a variety of
methods: visualization of cell sizes and shapes by microscopy after ap-
propriate staining, counting live cell numbers by the dye exclusion
method using trypan blue or by cytometry, such as fluorescence-
activated cell sorting (FACS), distinguishing dead/viable cells, cell
types, cell differentiation and biomarkers, and detecting metabolic
or redox activity by colorimetric (MTT) or fluorimetric (resazurin)
methods. The formazan-based MTT assay is a colorimetric assay for
assessing cell viability. MTT, a yellow tetrazole derivative, is reduced to
purple formazan by NAD(P)H-dependent oxidoreductase. In contrast,
resazurin-based assays, such as alamarBlue assay (Desaulniers et al.,
1998), exhibit greater sensitivity than cell counting, microfluorometric
DNA determination assay and even MTT assay. Other colorimetric
chemicals, such as MTS (a tetrazolium) and sulforhodamine B (SRB),
have been used in cell assays.
Cell lines derived from breast cancer have been used in cell assays.
Estrogenic breast cancer cell lines, such as MCF-7, T-47D and MDA-
MB-231 cells, are commonly used to examine the effects of chemicals
30 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40
on cell functions, such as cell viability, cell proliferation, cell cycle, apo-
ptosis and cell migration. For the determination of estrogenic activity,
cell proliferation assays are widely used. The E-screen assay developed
by Soto et al. (1995) is based on the growth promotion of MCF-7 cells
in the presence of estrogen, and is widely used as a screening tool for es-
trogenic chemicals (Grünfeld and Bonefeld-Jorgensen, 2004; Maras
et al., 2006; Resende et al., 2013). Cell assay was used to characterize
the estrogenicity of the chemicals (Tables 1 and 2), which are summa-
rized below.
The list of estrogenic chemicals characterized by cell assay.
Alachlor, anethole, arsenic, boric acid, citral, eugenol, geraniol, glyphosate,
licochalcone A, metalloestrogens, nerol, nitrophenols, PCB118, 4-phenylphenol,
phthalates and soysaponin I (by YES assay); 4-aryl-coumarin dimer, biochanin A,
2,6-dihydroxyanthraquinone, epigallocatechin 3-gallate, formononetin,
glyceollins, 6-hydroxymusizin, insecticides, juglone, naringenin, naringin,
pharmaceuticals, phlorizin, pinosylvin, propelargonidins, silibinin and β-sitosterol
(by MTT assay); apigenin, epigallocatechin 3-gallate and naringin (by MTS assay);
benzyl salicylate, conazoles, cyanidin-3-glycoside, dieldrin and SERMs (by SRB
assay); cajanin (by alamarBlue assay); arsenic (by trypan blue assay); coumestrol
and dieldrin (by using CellTiter); β-carotene, glyphosate, indole-3-carbinol,
lycopene, metalloestrogens and phthalates (by using cell counters); bezafibrate,
bisphenol A, daphnetin, esculetin, fenofibrate, fisetin, fluorotelomer alcohols,
flutolanil, galangin, gemfibrozil, insecticides, irisolidone, isorhamnetin luteolin,
kaempferol, metalloestrogens, PCBs, quercetin, semicarbazide and tectorigenin
(by E-screen assay); and β-carotene, epigallocatechin 3-gallate,
hydroxymatairesinol, juglone and lycopene (by flow cytometry).
4.1.7. Animal test
Animal tests have been used for basic research, such as genetics,
developmental biology and behavioral studies, as well as applied
research, such as biomedical research, xenotransplantation, drug testing
and toxicology tests. Some of the abnormality in animals used for
aminal tests, such as ovotestis, was found originally by the observation
of wild animals, such as alligators and fish. In vivo biomarkers, such as
vitellogenin and aromatase, have been used as physiological indicators
to estimate efficiently the impact of estrogenic chemicals, although
careful interpretateion is needed (Nadzialek et al., 2011). Meanwhile,
animal tests for toxicology testing have been conducted by pharmaceu-
tical companies and animal testing facilities, and one million animals are
used every year in Europe in toxicology tests (Commission of the
European Communities, 2007), to examine industrial products, such
as those listed in Table 2. Test results indicate acute, subchronic or
chronic effects or the damage to specific organs, such as eye and skin
irritancy, mutagenicity, carcinogenicity, teratogenicity and reproductive
problems. The results are expressed using variables such as LD50, the
concentration of chemicals at which 50% of the test animals are killed.
Animal tests have several problems: they cost several million dollars
per substance, take three or four years to complete, can potentially over-
estimate the risks, especially giving false-positive results, and show var-
iability in the results between the effect of high doses of chemicals in
animals and the effects of low doses in large numbers of humans, as
well as in the rationale of how to use data on one species to predict
the risk in another.
Animal tests are useful to screen endocrine disruptors to evaluate
adverse endocrine-mediated reproductive and/or developmental
effects in multi-generation reproduction studies. In general, the use of
living organisms allows the variation of assays based on a wide range
of species with different end-points. The rodent uterotrophic bioassay
is the most widely used in vivo screening assay, based on the response
of the estrogen-sensitive uterus. Animals are injected with the test
substances and uterotrophic activities are estimated by the uterine
weight (Zacharewski et al., 1998; Tamir et al., 2000; Dang et al., 2007;
Jiménez-Orozco et al., 2011). Similarly, Hershberger assay has been
used to identify potential (anti-)androgenic compounds (Yamada
et al., 2000) and frog embryo teratogenesis assay (FETAX) to detect
developmental toxicants in the environment (Fort et al., 2001). These
animal tests are time-consuming and most require the use of live
animals. Animal tests were used to characterize the estrogenicity of
the chemicals (Tables 1 and 2), which are summarized below.
The list of estrogenic chemicals characterized by animal tests.
Acetaminophen, benzo[a]pyrene, boric acid, caffeic acid phenethyl ester,
daphnetin, 2,4-dihydroxybenzophenone, esculetin, ephedrine, epigallocatechin
3-gallate, estrone 3-carboranylmethyl ether, fenarimol, fluoxetine, ginsenoside Rg1,
glabridin, glycitein, insecticides, naringenin, naringin, 4-nitrophenol, PCBs,
2-phenylindoles, phthalates and zeranol (by rodent uterotrophic assay); pinosylvin
and β-sitosterol (by rainbow trout feeding assay); 3-(4-methylbenzylidene) camphor
and perfluorobutanesulfonic acid (by using Xenopus); and DDT, oxybenzone,
perfluorooctyl iodide and phthalates (by using medaka).
4.1.8. Signaling pathway analysis
Signaling pathway analysis consists of a variety of methods and
protocols, which have variations depending on the signaling pathways,
signaling networks and functional outcomes. Various types of cell
signaling induced by estrogenic chemicals are summarized in Tables 1
to 3, and the signaling pathways related to apoptosis, carcinogenesis,
cell growth/differentiation, differentiation/development and inflamma-
tion are discussed in Section 3. Signaling pathway analysis was used to
characterize the estrogenicity of the chemicals (Tables 1 and 2), which
are summarized below.
The list of estrogenic chemicals characterized by signaling pathway analysis.
Aroclor 1254, epigallocatechin 3-gallate, formononetin, geniposide, ginsenoside
Rb1, methoxychlor/triclosan, naringenin, plumbagin, quercetin, raloxifene,
withaferin A and wogonin (for apoptosis); cadmium, di(n-butyl) phthalate,
methyl-amoorain + DMBA, testosterone and tocopherols (for carcinogenesis);
baicalein, benzo[a]pyrene, bisphenol A, p,p′-DDE, o,p′-DDT, equol,
17α-ethinylestradiol, 17β-E2 valerate, dieldrin, ferulic acid, genistein, β-HCH,
hydroxytyrosol, luteolin, methoxychlor, naringenin, oleuropein, piceatannol,
procymidone, puerarin, quercetin and tributyltin (for cell growth and proliferation);
adiponectin, androstenol, estradiolquinone, genistein, resveratrol, retinoic acid and
tamoxifen (for differentiation/development); and baicalein, daidzein genistein,
4-hydroxyphenyl sulfonamides, niacin, p-n-nonylphenol, p-n-octylphenol and
oroxylin-A (for inflammation).
4.2. Pathway-based risk assessment
Replacing animal tests with in vitro assays has been accelerated in
basic science as well as in the industrial technology field. In 2007, the
US National Research Council (NRC) released a report “Toxicity Testing
in the 21st Century: A Vision and a Strategy” (National Research Council,
2007), in which they called for a transformation of toxicity testing from
animal tests to a system based on in vitro assays where perturbations in
key toxicity pathways in response to chemicals can be evaluated.
Current and future toxicity testing can be categorized into four options:
status quo animal testing as Option I, animal testing partly assisted by in
silico and in vitro screens as Option II, minimum in vivo testing largely
assisted by in vitro screens as Option III and complete in vitro (in silico)
testing without animal tests as Option IV. While this report had a pro-
found impact for environmental scientists, toxicologists and pharmacol-
ogists as well as scientists and researchers in other fields, no practical
protocol has ever been established. Although a number of toxicity path-
ways have already been identified, most are still partial and do not have
sufficient annotations for their use for risk assessment, such as that in
human exposure to chemicals. Although prototype protocols for risk
assessment were examined for quercetin in DNA-damage-related sig-
naling (Adeleye et al., 2015) and benzo[a]pyrene and dioxin in AhR sig-
naling (National Research Council, US, 2010), other pathways for these
chemicals (such as those shown in Tables 1 and 2) are not included, indi-
cating the difficulty of such an approach.
What is important to use pathway-based risk assessment is not how
well such a strategy can mimic animal testing, but how to shift from
outcome-based assessment, where only the result of assays or the fate
of animals is evaluated, to mechanism-based assessment, where the
process occurring in cells is evaluated before outcomes have emerged.
 R. Kiyama, Y. Wada-Kiyama / Environment International 83 (2015) 11–40
This paradigm shift requires reliable assays, which are based on the pre-
dictability of the risk and are as valuable as the fate of animals. However,
as mentioned above, the variability of signaling pathways is so limited at
this moment that the variability of chemicals is restricted to limited
numbers of categories, so the characteristics of each chemical cannot be
properly evaluated. Interesting and suggestive approaches, however, can
be found in cancer genomics, where pathway-based pharmacogenomics
has been a key strategy for developing personalized cancer medicine
(Chin et al., 2011), and in the US National Cancer Institute (NCI) 60
human tumor cell line anticancer drug screen (NCI60), where more
than 100,000 chemicals were examined for screening candidate drugs
using 60 human tumor cell lines (Shoemaker, 2006). The characteristics
of each chemical could be evaluated to give sufficient predictability,
which is also crucial for personalized medicine.
5. Conclusion and future prospects
Here, we summarized estrogenic chemicals, which are categorized by
structure (Table 1) or by characteristics other than structure (Table 2)
(Section 2). Estrogenic activity is understood by the mechanism-based
characterization of estrogenic chemicals as summarized in Fig. 1, so we
characterized the signaling pathways in which each estrogenic chemical
is involved and the methods by which each estrogenic chemical is
assayed (Section 3). Signaling pathways are further characterized by
the receptors and the networks involving other receptors and signaling
pathways as well as the pathways involving autocrine/paracrine signal-
ing and homeostatic networks (Table 3). We found here that a number
of chemicals with quite diversified structures, phenolics and non-
phenolics of natural or industrial origin, show estrogenicity, and a variety
of signaling pathways and networks, owing not only to the differential
roles of ERs but also to the complexity of pathways, effectors and cells/
tissues, are associated with estrogenicity. While this kind of diversity
and complexity would be an obstacle to understand clearly the effect of
estrogenic chemicals and to establish the methodology for risk assess-
ment, pathway-based risk assessment would give superiority as a risk as-
sessment technology, once established, because sensitivity would be
higher due to multiple monitoring points in signaling pathways,
which are then integrated into a single or a few outcomes, such as
those detected by animal assay, cell-proliferation assay, ligand-
binding assay, protein assay, reporter-gene assay and signaling path-
way analysis (see Section 4). Furthermore, complex systems such as
in animal tests are not required, providing less expensive, quick, high-
throughput and highly sensitive assays.
However, we still need to understand more about signaling path-
ways to utilize the information obtained by the analysis for risk assess-
ment and to refine the methodology based on the pathways, which
need to be reasonably developed in order to replace and reduce animal
tests. First, the current information about genes is quite large, although
it is mostly descriptive and explanatory, and lacks predictability. Mean-
while, pathway-based risk assessment could be used as a partial
replacement of or as a preliminary screening step for animal tests,
such as uterotrophic assay and life-cycle assay, to detect alterations at
early stages before using the uterus and other tissues/organs. The com-
binations of chemicals, biological outcomes and assay systems would
provide quite a large number of variations to distinguish predicted
outcomes of potential risks.
Acknowledgments
This research was supported partly by a Knowledge Cluster Initiative
program and a Grant-in-aid for Basic Areas from the Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan, and by the Guide-
line Program for Medical Device Development from the Ministry of
Economy, Trade and Industry of Japan.
31
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