Estrogenic Endocrine Disruptors: Molecular Mechanisms Of Action

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Estrogenic endocrine disruptors: Molecular mechanisms of action - ScienceDirect



Highlights


A comprehensive summary of more than 450 estrogenic chemicals is provided.


Intracellular and intercellular signaling pathways involved in estrogen signaling are summarized.


Potential toxicity pathways that could be used for endocrine disruptor studies are discussed.



Estrogenic endocrine disruptors: Molecular mechanisms of action
Author links open overlay panelRyoitiKiyamaaYukoWada-Kiyamab
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Highlights



A comprehensive summary of more than 450 estrogenic chemicals is provided.


Intracellular and intercellular signaling pathways involved in estrogen signaling are summarized.


Potential toxicity pathways that could be used for endocrine disruptor studies are discussed.



Abstract
A comprehensive summary of more than 450 estrogenic chemicals including estrogenic endocrine disruptors is provided here to understand the complex and profound impact of estrogen action. First, estrogenic chemicals are categorized by structure as well as their applications, usage and effects. Second, estrogenic signaling is examined by the molecular mechanism based on the receptors, signaling pathways, crosstalk/bypassing and autocrine/paracrine/homeostatic networks involved in the signaling. Third, evaluation of estrogen action is discussed by focusing on the technologies and protocols of the assays for assessing estrogenicity. Understanding the molecular mechanisms of estrogen action is important to assess the action of endocrine disruptors and will be used for risk management based on pathway-based toxicity testing.

Keywords
Endocrine disruptor
Estrogen action
Molecular mechanism
Techonology
Signal transduction
Toxicity pathway
1. Introduction
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 estrogenic chemicals of natural and industrial origins, as mostly characterized 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, release, transport, metabolism, binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis 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 estrogenic. They were analyzed by a number of different assays and evaluated 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 summarized in Fig. 1 (see also Kiyama and Zhu, 2014; Kiyama et al., 2014). Estrogenic signal networks can be categorized into intracellular and extracellular 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/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.

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Fig. 1
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 receptors and stimulates signaling proteins through the non-genomic pathway, 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 characteristics of chemicals, such as biphasic and metabolic effects detected by cell proliferation assay, the activity of selective estrogen-receptor modulators (SERMs) detected by cell assay and new estrogen-responsive genes detected by DNA microarray assay (see Section 4). Thus, estrogenic 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 extracellular 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 aldehydes, acetophenones, tyrosine derivatives, phenylacetic acids, hydroxycinnamic acids, phenylpropenes, coumarins/isocoumarins/chromones, naphthoquinones, bisphenols, benzophenones, stilbenes/stilbenoids, anthraquinones, chalcones/chalconoids, flavones/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).

Table 1
Estrogenic chemical Signaling pathway Reference (assaya)
Phenolicb
Simple phenol (C6)
Alkylphenols (C6–C4–C6–C12) ER Tollefsen and Nilsen, 2008 (L)
3,4-Dichlorophenol, 4-tert-octylphenol,
pentachlorophenol (agonist) ER Li et al., 2010b (R)
4-Nitrophenol (estrogenic) ER Li et al., 2006 (A)
4-Nitrophenol, 3-methyl-4-nitrophenol,
4-Nitro-3-phenylphenol (estrogenic) ER Taneda et al., 2004(R)
p-n-Nonylphenol, p-n-octylphenol ER/NF-κB/NO/inflammation Yoshitake et al., 2008 (S)
Phenolic acid/phenolic aldehyde (C6–C1)
Octyl gallate ERα/PI3K/Akt/Alzheimer's disease Zhang et al., 2013(S)
Parabens (methyl-, ethyl-, propyl-,
isopropyl-, butyl-, isobutylparabens) ERα/PR/pituitary cancer Vo et al., 2011 (S)
3-Phenoxybenzaldehyde (estrogenic) ER McCarthy et al., 2006 (R)
Syringic acid, ellagic acid ER/NO/endothelial cells Simoncini et al., 2011 (S)
Acetophenone (C6–C2)
2,2-Dimethoxy-2-phenyl-acetophenone (estrogenic) ER Wada et al., 2004(R)
Phenylethane/phenylethanoid (C6–C2)
Acteoside, martynoside (estrogenic) ER Papoutsi et al., 2006 (R)
Tyrosine derivative (C6–C2)
Thyroid hormone TR/Integrin receptor/ERα/proliferation (crosstalk) Meng et al., 2011(S)
Phenylacetic acid (C6–C2)
Phenylacetate (anti-estrogenic) ER/CDKN1A/breast cancer Liu et al., 2007 (S)
Hydroxycinnamic acid (C6–C3)
Caffeic acid phenethyl ester ERβ Jung et al., 2010(A, C, L, P, R)
Caffeic acid phenethyl ester (SERM) ERα, ERβ/ERK/Akt/apoptosis Tolba et al., 2013(S)
Ferulic acid (estrogenic) HER2/ERα/ERK/Akt/Cyclin D1/proliferation Chang et al., 2006(S)
Rhodoeosein (p-coumaric acid) (estrogenic) ERβ Brennan et al., 2013 (R)
Phenylpropene (C6–C3)
Anethole (estrogenic), eugenol (anti-estrogenic) ER Howes et al., 2002(C)
Eugenol (antagonist) ER Anita et al., 2012(L)
Coumarin/isocoumarin/chromone (C6–C3)
4-Aryl-coumarin dimer (estrogenic) ER Nishimura et al., 2000 (C, L)
Auraptene (anti-estrogenic) ER de Medina et al., 2010 (L, R, T)
Bergapten (anti-estrogenic) TGF-β/SMAD4/ERα/proliferation Panno et al., 2012(S)
Daphnetin, esculetin (anti-estrogenic) ER/proliferation Jiménez-Orozco et al., 2011(A, C, P, T)
Icariin ER/ERK/JNK/osteoblasts Song et al., 2013(S)
Icariin TGF-β/Smad/ERK/GPER/nephropathy Li et al., 2013a (S)
Icariin, icaritin (estrogenic) GPER/EGFR/MAPK/proliferation Ma et al., 2014 (S)
Isocoumarins (estrogenic) ERβ De Angelis et al., 2005 (L, R)
SP500263 (a synthetic SERM) ER/Osteoclastogenesis Kung Sutherland et al., 2003 (S)
Umbelliferone ER Kirkiacharian et al., 2004 (L)
Naphthoquinone (C6–C4)
Alkannin (shikonin) ERα/Nrf2/NQO1/DNA damage Yao et al., 2010 (S)
Juglone ER/Pin1/ERK/LC-3/autophagy Namgoong et al., 2010 (S)
Juglone, lawsone, menadione,
1,4-naphthaquinone, plumbagin (antagonist) ER Thasni et al., 2013(C, L, T)
Menadione (vitamin K3) (anti-estrogenic) ER Jung et al., 2004(L, R, Y)
Menatetrenone (vitamin K2) ERα/vitamin K/leukemia Nishiguchi et al., 1999 (S)
Plumbagin ERα(46 kDa)/BRCA1/apoptosis Thasni et al., 2008(S)
Plumbagin NF-κB/Bcl-2/apoptosis Ahmad et al., 2008(S)
Bisphenol (C6–C1–C6)
Bisphenol A GPER/ERK/c-Fos/transcription Dong et al., 2011(S)
Bisphenols (A, B, F) (estrogenic) ER Hashimoto et al., 2001 (C, L, Y)
Bisphenol A, 4,4′-dihydroxybiphenyl, 4-hydroxybiphenyl, tetrabromobisphenol A,
tetrachlorobisphenol A (estrogenic) ERα Olsen et al., 2003(C, L, P)
Chlorinated bisphenol As (estrogenic) ER Hu et al., 2002 (L, Y)
Benzophenone (C6–C1–C6)
2,4-Dihydroxybenzophenone, 2,2′,4,4′-tetrahydroxybenzophenone
(agonist/antagonist) ER Ohta et al., 2012(A)
Stilbene/stilbenoid (C6–C2–C6)
Piceatannol (estrogenic/anti-estrogenic) ER/MAPK/ERK/Akt/PR/proliferation Vo et al., 2010 (S)
Piceid, pterostilbene, resveratrol ERβ/oxidative stress Robb and Stuart, 2014 (S)
Pinosylvin, isorhapontigenin, isorhapontin (estrogenic) ER Mellanen et al., 1996 (A, C)
Resveratrol ERα, ERβ/MAPK/atheroprotection Klinge et al., 2005(S)
Resveratrol ER/NO/cGMP/differentiation Song et al., 2006(S)
Resveratrol GPER/cAMP/PKC/K channels Dong et al., 2013b(S)
Resveratrol ERα/SIRT1/cAMP/inflammation Nwachukwu et al., 2014 (S)
Vitisin B (a resveratrol tetramer) ER/PDGF/cell adhesion Ong et al., 2011 (S)
Anthraquinone (C6–C2–C6)
Aloe-emodin, emodin (anti-estrogenic) ERα/proliferation Huang et al., 2013(S)
Anthraquinone, benzanthrone (estrogenic) ERα Machala et al., 2001 (R)
2,6-Dihydroxyanthraquinone (estrogenic) ER Matsuda et al., 2001 (C)
Emodin ERα/PI3K/Akt/JNK/Alzheimer's disease Liu et al., 2010a (S)
2-Hydroxyanthraquinone (estrogenic) ER Kurihara et al., 2005 (Y)
Mitoxantrone (estrogenic) ERα Oda et al., 2013 (T)
Chalcone/chalconoid (C6–C3–C6)
Candidachalcone ER Hegazy et al., 2011(L)
Chalcone (anti-estrogenic) ERα/MAPK/JNK/MCF-7 cells Collins-Burow et al., 2012(S)
2-Hydroxychalcone, 4-hydroxychalcone,
4′-hydroxychalcone (estrogenic) ER Kohno et al., 2005(R)
Isoliquiritigenin (estrogenic/anti-estrogenic) ERα/cell growth Maggiolini et al., 2002 (S)
Licochalcone A (estrogenic) ER Rafi et al., 2000 (C)
Nothofagin, (isovitexin; a flavone) (estrogenic) ER Shimamura et al., 2006 (P)
Phloretin (estrogenic) ER Ise et al., 2005 (T)
Phloretin Non-genomic ER/LXR/fatty liver Han et al., 2014 (S)
Phlorizin (estrogenic/anti-estrogenic) ER Wang et al., 2010(C, Y)
Flavone/flavonoid (C6–C3–C6)
Anthoxanthin (flavone and flavonol)
Apigenin (anti-estrogenic) ERβ Mak et al., 2006 (C, R, T)
Apigenin ER/TNF-α/NO/Akt/vascular diseases Palmieri et al., 2014(S)
Apigenin, luteolin ER Innocenti et al., 2007 (R)
Baicalein ER/NF-ĸB/NO/TNF-α/inflammation Fan et al., 2013 (S)
Baicalein, daidzein ER/neuroprotection Choi et al., 2013 (S)
Baicalein, quercetin (anti-estrogenic) ER/IRS-1/ROS/ERK/JNK/proliferation Lin et al., 2007 (S)
Fisetin, galangin, luteolin (estrogenic) ER Resende et al., 2013 (C, R)
Isorhamnetin, kaempferol, quercetin (estrogenic) ER Oh and Chung, 2004 (C, L, T)
Kaempferide (anti-estrogenic) ER Collins-Burow et al., 2000(L)
Kaempferol (estrogenic) ERα Guo et al., 2012 (R, T)
Luteolin (anti-estrogenic) ERα/IGF-1/PI3K/Akt/proliferation Wang et al., 2012(S)
Myricetin (estrogenic) ERα Maggiolini et al., 2005 (L, P, R, T)
β-Naphthoflavone AhR/PI3K/Akt/MAPK/ERK/ERα/carcinogenesis Wang et al., 2014(S)
Oroxylin-A ER/TNF-α/IL-1β/IL-6/NO/inflammation Wang et al., 2013c(S)
Quercetin ERβ/cyclin D1/caspase-3/apoptosis Bulzomi et al., 2012b(S)
Quercetin, naringenin (differential effects) ER Marino et al., 2012(review)
Quercetin, naringenin (anti-estrogenic) Non-genomic ERα/ERK/proliferation Virgili et al., 2004(S)
Wogonin ER/PARP/Bax/apoptosis Chung et al., 2008(S)
Flavanone
Chrysin (estrogenic) ERα Berthier et al., 2007(C, T)
Hesperetin TrkA receptor/MAPK/PKA/PKC/ERα/neuroprotection Hwang et al., 2012(S)
Liquiritigenin (agonist) ERβ Mersereau et al., 2008 (L, R, T)
Naringenin ERα,ERβ/MAPK/caspase-3/apoptosis Totta et al., 2004(S)
Naringenin (anti-estrogenic) mERα/ERK/PI3K/MAPK/proliferation Galluzzo et al., 2008 (S)
Naringin (estrogenic) ERα Pang et al., 2010(C, P, R, T)
Naringenin ERα/MAPK/Akt/caspase-3/apoptosis Bulzomi et al., 2012a(S)
Naringenin (agonist) ER Kim and Park, 2013(C, R, T)
Naringenin (anti-estrogenic) ERα/MAPK/proliferation La Rosa et al., 2014(S)
Naringenin, naringin (estrogenic/anti-estrogenic) ER Guo et al., 2011 (A, C, L, Y)
Sakuranetin ERβ Tohno et al., 2010(L)
Flavanonol
Astilbin, engeletin (estrogenic) ER Wungsintaweekul et al., 2011(C)
Taxifolin (agonist) ER Plísková et al., 2005 (R)
Flavan
Epicatechin, epicatechin 3-gallate, epigallocatechin,
epigallocatechin 3-gallate (estrogenic/anti-estrogenic) ER Kuruto-Niwa et al., 2000(R)
Epicatechin 3-gallate, epigallocatechin 3-gallate (estrogenic) ER Goodin et al., 2002(A, L, R)
Epigallocatechin 3-gallate FOXO3a/ERα/invasion Belguise et al., 2007 (S)
Epigallocatechin 3-gallate (anti-estrogenic) ER Farabegoli et al., 2007 (C, T)
Epigallocatechin 3-gallate (anti-estrogenic) ER Laschke et al., 2008(C, P)
Epigallocatechin 3-gallate (estrogenic) ERα Li et al., 2010a (C, P, T)
Epigallocatechin 3-gallate ERα/ERK/Akt/Alzheimer's disease Fernandez et al., 2010 (S)
Epigallocatechin 3-gallate ER/MAPK/Akt/caspase-3/apoptosis Park et al., 2012 (S)
Anthocyanidin
Cyanidin-3-glycoside (anti-estrogenic) ER Fernandes et al., 2010 (C, T)
Delphinidin ERα/ Src/ERK/NO/vasodilation Chalopin et al., 2010 (S)
Malvidin ER/NO/endothelial cells Simoncini et al., 2011 (S)
Isoflavone
Biochanin A (estrogenic) ERα Chan et al., 2007(C, P, R, T)
Cajanin (estrogenic) ER Umehara et al., 2008 (C, R)
Calycosin (anti-estrogenic) ERβ/IGF-1R/MAPK/PI3K/Akt/cell growth Chen et al., 2014a(S)
Calycosin, formononetin (anti-estrogenic) ERβ/IGF-1R/proliferation Chen et al., 2013(S)
Daidzein ERβ,GPER/neuroprotection Kajta et al., 2013(S)
Daidzein, genistein ERα/TNF-α/NO/inflammation Nakaya et al., 2005(S)
Daidzein, genistein, (apigenin, coumestrol) (agonist) ER Harris et al., 2005(R)
Formononetin (agonist) ER Ji et al., 2006 (C, R)
Formononetin ER/Ras/MAPK/Bax/Bcl-2/apoptosis Chen and Sun, 2012(S)
Genistein, (quercetin) (estrogenic) GPER/ERK/c-Fos/cell growth Maggiolini et al., 2004 (S)
Genistein ERα, ERβ Sotoca et al., 2011(P, T)
Genistein GPER/IL-1β/MAPK/inflammation Luo et al., 2012 (S)
Genistein (anti-estrogenic) ERα/IGF-1R/IRS-1/Akt/carcinogenesis Hwang et al., 2013(S)
Genistein (estrogenic) ERα/MAPK/NF-κB/AP-1/differentiation Liao et al., 2014 (S)
Glycitein (estrogenic) ER Song et al., 1999(A, L)
Irigenin, tectorigenin ER/CDKN1B/apoptosis Morrissey et al., 2004 (S)
Irisolidone, tectorigenin (estrogenic) ER Shin et al., 2006 (C, P, T)
Puerarin ER/PI3K/Akt/CaMKII/AMPK/NO/cardioprotection Hwang et al., 2011(S)
Puerarin (estrogenic) ER/ERK/PI3K/Akt/proliferation Wang et al., 2013a(S)
Isoflavane
Equol (estrogenic) ERα/ERK/proliferation Liu et al., 2010b (S)
Equol GPER/EGFR/ERK/Akt/NO/cardioprotection Rowlands et al., 2011 (S)
Equol, phenoxodiol ER/RhoA/Rho kinase/vasorelaxation Tilley et al., 2012(S)
Glabridin (estrogenic/anti-estrogenic) ER Tamir et al., 2000(A, C, L)
Isoflavene
Glabrene, glabridin (estrogenic) ER Somjen et al., 2004a(A, C)
Coumestan
Coumestrol (anti-estrogenic) ER Ndebele et al., 2010(C, P)
Coumestrol ERα, ERβ Chandsawangbhuwana and Baker, 2014 (L)
Psoralidin (agonist) ER Liu et al., 2014 (C, L, P, R, T)
Pterocarpan
Glycinol (estrogenic) ER Boué et al., 2009(L, R, T)
Glyceollins (antagonist) ER Burow et al., 2001(C, L)
Medicarpin (estrogenic) ERβ/differentiation Bhargavan et al., 2012 (S)
Flavonolignan
Silibinin (estrogenic) ERβ Nejati-Koshki et al., 2012(C, P, T)
Lignan/neolignan ((C6-C3)2)
Arctigenin ERα Jin et al., 2013 (L)
Enterodiol, enterolactone (estrogenic/anti-estrogenic) ER Feng et al., 2008(L)
Hydroxymatairesinol (estrogenic) ER Cosentino et al., 2007 (C, T)
Secoisolariciresinol (anti-estrogenic) ER/EGFR/BCL2/cell growth Saggar et al., 2010(S)
Sesamin, sesamolin, sesamol (estrogenic) ER Pianjing et al., 2011(C, R, T)
Diarylheptanoid (C6–C7–C6)
Curcumin (estrogenic) ER Bachmeier et al., 2010 (P, T)
Curcumin (anti-estrogenic) Erβ/proliferation Piccolella et al., 2014 (S)
Flavolan (condensed tannin) ((C6-C3–C6)n)
Propelargonidins (estrogenic) ER Chang et al., 2003(C)
Estrogenic hydrocarbon without a phenolic ring
Aniline
Alkylanilines (4-amino-benzophenone, 4-aminobiphenyl, butyl-4-aminobenzoate) (estrogenic) ER Hamblen et al., 2003 (R)
Carborane (boron estrogen)
Carboranes (agonist) ERα Endo et al., 2001(L, R)
9,10-Dimethyl-m-carborane (SERM) ER Ohta et al., 2014(C, L)
Estrone 3-carboranylmethyl ether ER Sweet, 1981 (A, L)
Indole
3,3′-Diindolylmethane AhR/ERα/endocrine disruption Liu et al., 2006 (S)
Indole-3-carbinol (agonist) ER Riby et al., 2000 (C, L, R, T)
Indole-3-carbinol (anti-estrogenic) ER/proliferation Ashok et al., 2001(S)
Indole-3-carbinol (anti-estrogenic) ERα/IGF-1R/IRS-1/proliferation (crosstalk) Marconett et al., 2012 (S)
Melatonin (anti-estrogenic) MT1(GPCR)/ERα/proliferation (crosstalk) Kiefer et al., 2005(S)
Melatonin MT2/ER/cAMP/scoliosis Letellier et al., 2008(S)
2-Phenylindoles (anti-estrogenic) ER Biberger and von Angerer, 1996 (A, L, R)
2-Phenyl-isoindole-1,3-dione ERβ Ullrich et al., 2007(L)
Serotonin Serotonin 1A receptor/GPER/depressive disorder Li et al., 2013c (S)
Metalloestrogen
Arsenic (anti-estrogenic) ERα/VEGF/Cyclin D1/CDK4/reproduction Chatterjee and Chatterji, 2010 (S)
Arsenic trioxide (anti-estrogenic) ERα Chow et al., 2004(C, L, P, R, T)
Antimony chloride, barium chloride, bis(tri-n-butyltin),
cadmium chloride, chromium chloride,
lithium hydroxide (estrogenic) ER Choe et al., 2003(C, R)
Cadmium ER/ERK/cancer risk Ali et al., 2012 (S)
Cadmium, calcium, chromium, cobalt, copper,
lead, mercury, nickel, tin ER Byrne et al., 2013(review)
Cadmium, copper, zinc (estrogenic) ER Denier et al., 2009(R)
Chromium, cobalt, copper, lead, mercury,
nickel, tin, vanadate ERα Martin et al., 2003(C, L, R)
Selenium (anti-estrogenic) ERα Lee et al., 2005 (C, L, P, R, T)
Perfluorinated compound
Fluorotelomer alcohols (estrogenic) ER Maras et al., 2006(C, T)
Fluorotelomer alcohols (6:2 FTOH, 8:2 FTOH,
NFDH) (estrogenic) ER Ishibashi et al., 2007 (Y)
Perfluorooctanesulfonic acid ER/AhR/PPAR/endocrine disruption Fang et al., 2012(S)
Perfluorobutanesulfonic acid,
perfluorooctanesulfonic acid (estrogenic) ER Lou et al., 2013 (A, T)
Perfluorooctanoate, perfluorohexane sulfonate,
perfluorooctane sulfonate (estrogenic) ER Kjeldsen and Bonefeld-Jørgensen, 2013 (R)
Perfluorooctyl iodide ER Wang et al., 2013d(A, T)
Phthalate
Bis(2-ethylhexyl) phthalate, butyl benzyl phthalate,
diisononyl phthalate, di(n-butyl) phthalate (estrogenic) ER Chen et al., 2014c(A)
n-Butyl benzyl phthalate (estrogenic) ER Hashimoto et al., 2000 (Y)
Butyl benzyl phthalate, dibutyl phthalate,
di(2-ethylhexyl) phthalate (estrogenic) ER Ghisari and Bonefeld-Jorgensen, 2009 (C, R)
Butyl benzyl phthalate (estrogenic) ER Picard et al., 2001(C, L)
Butyl benzyl phthalate, dibutyl phthalate,
diethyl phthalate, diisobutyl phthalate,
diisiononyl phthalate (estrogenic) ER Harris et al., 1997(C, R)
Butyl benzyl phthalate, dibutyl phthalate,
diethyl phthalate, diisopropyl phthalate (estrogenic) ER Parveen et al., 2008(T)
Butyl benzyl phthalate, di(n-butyl) phthalate,
di-hexyl phthalate (estrogenic) ER Zacharewski et al., 1998 (A, C, L, R)
Di(n-butyl) phthalate (crosstalk) ER/TGF-β/prostate cancer Lee et al., 2014 (S)
Polycyclic aromatic hydrocarbon (PAH)
3-Alkyl naphthalenes ER Fang et al., 2008(L)
Anthracene, benz[a]anthracene, chrysene,
benzo[b]fluoranthene, benzo[a]pyrene (estrogenic) ER Gozgit et al., 2004(L, R, T)
Benz[a]acridine, benz[a]anthracene-7,12-dione (estrogenic) ER Machala et al., 2001 (R)
Cinanthrenol A ER Machida et al., 2014 (L)
Benz[a]anthracene, benzo[a]pyrene,
fluoranthene (estrogenic) ER Kummer et al., 2008 (A)
3,9-Benz[a]anthracene diols ER Anstead and Kym, 1995 (L)
Benzo[a]pyrene (estrogenic) ER/ERK/Akt/COX-2/proliferation Tsai et al., 2004 (S)
7,12-Dimethylbenz(a)anthracene ER Garcia-Segura et al., 1992(A)
6-Hydroxy-chrysene, 2,3-benzofluorene,
benzo[a]pyrene (anti-estrogenic) ER Tran et al., 1996 (R)
2-Hydroxyfluorene, 2-hydroxyphenanthrene,
n-propyl-p-hydroxybenzoate (estrogenic) ER Kamiya et al., 2005(Y)
3-Methylcholanthrene (anti-estrogenic) AhR/ER/endocrine disruption Chaloupka et al., 1992 (S)
3-Methylcholanthrene (estrogenic/anti-estrogenic) AhR/ER/endocrine disruption Swedenborg et al., 2008 (S)
Phenanthrenes (antagonist) ER Schmidt et al., 2003(L, R)
Terpene/terpenoid
Monoterpene
Citral, geraniol, nerol (estrogenic) ER Howes et al., 2002(R)
3-(4-Methylbenzylidene) camphor ER Klann et al., 2005(A, L)
Sesquiterpene
Farnesol (estrogenic) Farnesoid X receptor/ER/proliferation Journe et al., 2008(S)
Parthenolide NF-κB/ERα/inflammation Mahmoodzadeh et al., 2009(S)
Diterpene
Retinoic acid RAR/mER/MAPK/differentiation Kauss et al., 2008(S)
Retinoic acid (anti-estrogenic) RARα/LSD1/PKA/ERα/proliferation Ombra et al., 2013(S)
Triterpene
Celastrol (anti-estrogenic) ERα/Cyclin D1/PR/c-Myb/proliferation Jang et al., 2011(S)
27-Deoxyactein ERβ Onorato and Henion, 2001 (L)
Tetraterpene
β-Carotene, lycopene, phytoene,
phytofluene (anti-estrogenic) ER Hirsch et al., 2007(C, P, R)
Sterol
Abietic acid, betulin,
β-sitosterol (estrogenic/non-estrogenic) ER Mellanen et al., 1996 (A, C)
Cholesterol LXRβ/ERα/PI3K/Akt/NO/metabolism Ishikawa et al., 2013 (S)
25-Hydroxycholesterol ERα/HIF-1α/apoptosis Lappano et al., 2011 (S)
27-Hydroxycholesterol (SERM) ER/osteoporosis DuSell et al., 2010(S)
Stigmasterol (estrogenic) ER Boldrin et al., 2013(R)
Steroid
Aldosterone (a mineralocorticoid) GPER/Ca2 +/bradycardia Brailoiu et al., 2013(S)
Progesterone (estrogenic) PR/ER/Src/ERK/PI3K/Akt/proliferation Ballaré et al., 2006(S)
Progestin (estrogenic) RP/ER/Src/Ras/ERK/proliferation (crosstalk) Migliaccio et al., 1998 (S)
Progestin (estrogenic) PR/ERβ/ERK/Akt/proliferation (crosstalk) Vallejo et al., 2005(S)
Testosterone ER-α36/ERK/Akt/carcinogenesis Lin et al., 2009 (S)
Testosterone AR/ER/Alzheimer's disease Rosario et al., 2010(S)
Testosterone AR/ER Vasconsuelo et al., 2011(review)
Saponin
Ginsenoside Rb1 ER/ERK/Akt/apoptosis Hashimoto et al., 2012 (S)
Ginsenoside Rg1 ER/IGF-1R/neuroprotection Gao et al., 2009 (S)
Ginsenoside Rg1 (estrogenic) ER Chen et al., 2012(A)
Ginsenoside Rg1 ERα/MAPK/ERK/PI3K/Akt/Alzheimer's disease Shi et al., 2012 (S)
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.

While estrogenic or anti-estrogenic activity can be detected by various 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 pathways). 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 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).


Table 2
Estrogenic chemical Signaling pathway Reference (assaya)
Food additive/dietary supplement
Active component
Aurantio-obtusin, 6-hydroxymusizin (estrogenic);
rubrofusarin, torachrysone, toralactone (anti-estrogenic) ER El-Halawany et al., 2007 (C, L, Y)
Caffeine (anti-estrogenic) ER/cAMP/PKA/osteoporosis Zhou et al., 2009(S)
Coumestrol, isoliquiritigenin, liquiritigenin (estrogenic) ER Hong et al., 2011(R)
Geniposide mER/PI3K/MAPK/apoptosis Li et al., 2014b (S)
Ginsenosides (see saponin in Table 1)
γ-Linolenic acid (antagonist) ER Menendez et al., 2004 (R)
Nectandrin B AMPK/ERα/PI3K/Akt/NO/cardioprotection Hien et al., 2011 (S)
Notoginsenoside R1 ER/PI3K/Akt/Nrf2/ARE/neuroprotection Meng et al., 2014(S)
Notoginsenoside Ft1 GR/ER/ERK/Akt/NO/vascular relaxation Shen et al., 2014(S)
Antioxidant/preservative
Butylated hydroxyanisole, propyl gallate (estrogenic) ER ter Veld et al., 2006(R)
Semicarbazide (anti-estrogenic) ER Maranghi et al., 2010 (C, R)
Color additive
β-Carotene, lycopene (anti-estrogenic) ER Prakash et al., 2001(C, T)
Lycopene ER/RAR (crosstalk) Chalabi et al., 2004(C, T)
Tartrazine, sunset yellow ER Axon et al., 2012(P, R, T)
Cosmetic
Talc ER Frazier-Jessen et al., 1996(A)
Emulsifier/thickener
Carrageenan (anti-estrogenic) ER Misiewicz et al., 1996 (A)
β-Cyclodextrin E2 (complex formed) Oishi et al., 2008(P, Y)
Flavor enhancer
Guanylate E2/ER/NO/cGMP/cell permeability Gorodeski, 2000 (S)
Δ9-Tetrahydrocannabinol (anti-estrogenic) ERα Takeda, 2014(review)
Vitamin
Niacin (vitamin B3) GPER/EGFR/ERK/inflammation Santolla et al., 2014(S)
Retinoic acid (anti-estrogenic) ER Boettger-Tong and Stancel, 1995 (A, T)
Tocopherols (anti-estrogenic) E2/ER/PPARγ/Akt/breast cancer (crosstalk) Lee et al., 2009 (S)
Vitamin C (anti-estrogenic) ER Mense et al., 2009(A)
Vitamin D ER/ERK/c-Jun/VDR/proliferation Gilad et al., 2005(S)
Vitamin D E2/ERα/PPARγ/VDR (crosstalk) Alimirah et al., 2012(S)
Vitamin D analog (JK 1624 F2-2) ER Somjen et al., 2004b(L)
Pesticide
Antifouling agent
Tributyltin ERα/MAPK/ERK/proliferation Sharan et al., 2013(S)
Antimicrobial/essential oil
Anethole, citral, geraniol, nerol (estrogenic) ER Howes et al., 2002(L, R)
Eugenol (antagonist) ER Anita et al., 2012(L)
4-Hydroxyphenyl sulfonamides (anti-estrogenic) ER/NF-κB/inflammation Sabatucci et al., 2006 (S)
Biocide
Hydroxytyrosol, oleuropein (anti-estrogenic) ER/ERK/proliferation Sirianni et al., 2010(S)
Disinfectants/sanitizer
Chloroxylenol (dettol) (estrogenic) ER Houtman et al., 2004 (R)
Hexachlorophene (anti-estrogenic) ER Jung et al., 2004(L, R, Y)
Nitrophenols (estrogenic) ER Furuta et al., 2004(A, R)
4-Phenylphenol (agonist) ER Li et al., 2010b (R)
Fungicide
Biphenyl, chlordecone, diclofop-methyl, dieldrin,
dodemorph, endosulfan, lindane, methyl parathion,
PCBs, triadimefon (estrogenic) ER Petit et al., 1997 (L, R)
Boric acid (estrogenic) ER Wang et al., 2008b(A, C, L)
Brefeldin A ER/ERK/Akt/P70S6K/cardioprotection Dong et al., 2013a(S)
Chlordecone (kepone), dicofol, fenarimol, methoxychlor,
myclobutanil, nitrofen, pyridate (estrogenic) ER Okubo et al., 2004(C)
Conazoles (anti-estrogenic) ER Kjærstad et al., 2010 (C)
Cypermethrin, malathion, propiconazole, ER Kjeldsen et al., 2013 (R)
prothioconazole, terbuthylazine (estrogenic)
Dichlorophenyls (estrogenic) ER von Angerer et al., 1980(A)
Diazinon, prothiofos, pyriproxyfen, thiabendazole,
tolclofos-methyl (estrogenic) ER Manabe et al., 2006(C, T)
Dieldrin (estrogenic) ERα, ERβ, GPER/ERK/Akt/proliferation Briz et al., 2011 (S)
Dieldrin, endosulfan, fenarimol, methiocarb,
prochloraz (agonist) ER Andersen et al., 2002 (C, R)
Diphenylamines (estrogenic) ER Ohta et al., 2008(C, L)
Fenarimol (estrogenic) ER Andersen et al., 2006 (A, T)
Fenhexamid, fludioxonil (agonist) ER Medjakovic et al., 2014 (L)
Flutolanil, isoprothiolane (estrogenic) ER Oh et al., 2007 (C, P, T)
Hexachlorobenzene (estrogenic) ERα/IGF-1/proliferation García et al., 2010(S)
Mercuric chloride (estrogenic) ER Zhang et al., 2008(A, C, L)
PBDEs ER Li et al., 2013b (L)
PCB3 (4-chlorobiphenyl) (estrogenic) ERβ Ptak et al., 2008 (P)
Procymidone (estrogenic) ER/MAPK/ROS/proliferation Radice et al., 2006(S)
Herbicide
Atrazine (antagonist) ER Eldridge et al., 2008(review)
Triclopyr (estrogenic) ER Xie et al., 2005 (P)
Glyphosate (estrogenic) ER Thongprakaisang et al., 2013(C, P, R)
Sulfonylureas mER/K channels/insulin secretion Nadal et al., 1998(S)
Triazine (antagonist) ERβ Henke et al., 2002(L, R)
Insecticide
Aldicarb, bendiocarb, carbaryl, methomyl,
oxamyl, propoxur (antagonist) ER Klotz et al., 1997(L, R)
Aldrin, quinalphos (estrogenic) ER Chatterjee et al., 1992 (A, P, T)
Benomyl, chlordane, chlordecone, o,p′-DDT, dieldrin,
endosulfan, endrin, fenarimol, fenbuconazole,
fenvalerate, methoxychlor, trans-nonachlor,
toxaphene, vinclozolin (estrogenic); aldrin,
carbaryl, chlordecone, dieldrin, endosulfan,
endrin, methoxychlor (anti-estrogenic) ER Lemaire et al., 2006(C, R)
β-BHC, δ-BHC (agonist) ERβ Kojima et al., 2004(R)
Bifenthrin, permethrin (agonist/antagonist) ER Brander et al., 2012(A, P, R)
Chlorpyrifos (estrogenic/anti-estrogenic) ERα Ventura et al., 2012(C, P)
Chlorpyrifos, dieldrin, endosulfan, fenarimol, pirimicarb,
prochloraz, tolchlofos-methyl (anti-estrogenic) ER Grünfeld and Bonefeld-Jorgensen, 2004 (C, T)
Cyhalothrin, deltamethrin, fenvalerate, permethirn (estrogenic);
cyfluthrin, etofenprox, permethrin (anti-estrogenic) ER Du et al., 2010 (C, R)
Cypermethrin, fenvalerate, permethrin (estrogenic) ER Sun et al., 2014 (R)
Diazinon, prothiofos, pyriproxyfen, thiabendazole,
tolclofos-methyl (estrogenic) ER Kojima et al., 2005(R)
Diazinon, prothiofos, pyriproxyfen, thiabendazole,
tolclofos-methyl (estrogenic) ERα Manabe et al., 2006(C, T)
Fenthion sulfoxide (estrogenic) ER Yamada et al., 2010(Y)
Fenvalerate, sumithrin (estrogenic) ER Go et al., 1999 (C, T)
Methoxychlor (estrogenic) ER (ERα/ERβ-independent) Ghosh et al., 1999(T)
Methoxychlor (estrogenic) ER/ERK/proliferation Park et al., 2009 (S)
Methoxychlor (estrogenic) ERα Lee et al., 2012 (C, T)
Methoxychlor, triclosan ER/Cyclin D1/Ras/Bax/apoptosis Kim et al., 2014 (S)
Monocrotophos (estrogenic) ER Tian et al., 2009 (A, P, T)
Ryanodine Ryanodine receptor/ER/PKCε/PKA/Ca2 +/sweat gland Muchekehu and Harvey, 2008 (S)
Tetramethrin (anti-estrogenic) ER Kim et al., 2005 (A, T)
Miticide
Dicofol (agonist) ER Hoekstra et al., 2006 (R)
Repellent
Cypermethrin, deltamethrin, fenvalerate,
permethrin (estrogenic) ER Chen et al., 2002(C, L, T)
Permethrin (estrogenic) ER Tange et al., 2014(R)
Rodenticide
Vitamin D ERβ/caveolin-1/ERK/VDR Gilad and Schwartz, 2007 (S)
Vitamin D (calcitriol) (anti-estrogenic) VDR/ER/proliferation Swami et al., 2013(S)
Pheromone
Androstenol CAR/ER/GRIP-1/differentiation Min et al., 2002 (S)
Pharmacological estrogen
Mycoestrogen
Zearalenone ER/Wnt/anti-reproduction Wagner and Lehmann, 2006 (S)
Zearalenone (estrogenic) ERα Li et al., 2012 (P, R, T)
Zeranol (α-zearalanol) (estrogenic) ERα Liu et al., 2002 (T)
Zeranol (α-zearalanol) (estrogenic) ER Takemura et al., 2007 (A, L)
Nonsteroidal
Diarylpropionitrile ERβ/cAMP/PKA/vasorelaxation Valero et al., 2011(S)
Diarylpropionitrile ERβ/PI3K/Akt/vasorepression Wu et al., 2012 (S)
Diethylstilbestrol GPER/PKA/ERK/CREB/testis Zhang et al., 2014b(S)
Pharmaceutical
Acetaminophen (estrogenic) ER (indirect) Harnagea-Theophilus et al., 1999 (A, L)
Acetaminophen (anti-estrogenic) ER Dowdy et al., 2003(C, P, R)
Acetaminophen (anti-estrogenic) ER Schwartz-Mittelman et al., 2005 (R, Y)
Acetylsalicylic acid (aspirin) ER van Aswegen et al., 1992 (L)
Amphipathic benzenes ERα Gunther et al., 2008(R)
Bezafibrate, fenofibrate, gemfibrozil (estrogenic) ERα Isidori et al., 2009(C, R)
Ephedrine (anti-estrogenic) ER Arbo et al., 2009 (A)
Fluoxetine (estrogenic) ER Müller et al., 2012(A, R)
Ibuprofen (anti-estrogenic) ER Sibonga et al., 1998(A)
Isoproterenol β-Adrenoreceptor/ER/NO/cAMP/vasorelaxation Chan et al., 2002(S)
Isoproterenol β-Adrenoreceptor/ER/cardioprotection Walters and Sharma, 2003 (S)
Oxybenzone (estrogenic) ER Coronado et al., 2008 (A, P)
Tocotrienol (vitamin E) ERβ Comitato et al., 2009 (L, P, T)
Pure ER antagonist
1,1-Dichloro-2,2,3-triarylcyclopropanes (agonist) ERα Cheng et al., 2004(L, R)
Fulvestrant (ICI 182,780) (anti-estrogenic) ERα/Wnt/β-catenin/carcinogenesis Cao et al., 2014 (S)
Fulvestrant (ICI 182,780) (anti-estrogenic) ER/HER/c-Src/proliferation Kirkegaard et al., 2014 (S)
Fulvestrant (ICI 182,780) GPER/TGF-β1/Smad/migration Li et al., 2014a (S)
Fulvestrant (ICI 182,780) GPER/ERK/calpain/cell adhesion Chen et al., 2014b(S)
Selective estrogen receptor modulator (SERM)
Arzoxifene, bazedoxifene, droloxifene, EM-800,
HMR-3339, idoxifene, lasofoxifene, levormeloxifene,
ormeloxifene, ospemifene, pipendoxifene,
raloxifene, tamoxifen, toremifene ER Shelly et al., 2008(review)
Clomifene, tamoxifen ER Han et al., 2002 (C, P)
Femarelle ER Somjen et al., 2007(A, P)
Melatonin ER Cos et al., 2008(review)
Raloxifene ROS/MAPK/CREB/HO-1/inflammation (ER-independent) Lee et al., 2011 (S)
Raloxifene ER/TNF-α/ERK/caspase-3/apoptosis Hattori et al., 2012(S)
Raloxifene GPER/Akt/Bcl-2/neuroprotection Bourque et al., 2014 (S)
Raloxifene, tamoxifen mER/ERK/NF-κB/cell protection Stice et al., 2012(S)
Tamoxifen Cell growth (ER-independent) Jiang et al., 1995(C, P, T)
Tamoxifen ER/apoptosis (ER-dependent/independent) Obrero et al., 2002(S)
Tamoxifen (anti-estrogenic) ERα/IGF-1R/EGFR/MAPK/proliferation Santen et al., 2009(S)
Tamoxifen ERα/Wnt3A/β-catenin/differentiation Gao et al., 2013 (S)
Steroidal
17β-Dihydroequilenin, 17β-dihydroequilin,
equilenin, Δ8,17β-E2 ERβ Bhavnani et al., 2008 (L, P, R)
Equilin (estrogenic) ER/NMDA receptor/cell growth Brinton et al., 1997(S)
Equilin, equilenin ER/NO/cardioprotection Novensa et al., 2010 (S)
Equilin, equilenin ERβ/antioxidant/apoptosis Bhavnani and Stanczyk, 2014 (S)
Estetrol (E4) (SERM) ER/Actin/migration Giretti et al., 2014(S)
17α-E2 ER-X/MAPK/ERK/PI3K/Akt/brain Toran-Allerand et al., 2005(S)
17α-E2 mER/Rho/ROCK/cofilin/neuroprotection Hirahara et al., 2013 (S)
17β-E2 mER/Natriuretic peptide receptor A/cGMP/Ca2 +/hepatocytes Stratton et al., 2010(S)
Estradiolquinone (estrogenic) ER/LTD4 receptor/differentiation Dietsch et al., 1996(S)
Estrone (E1), estradiol (E2), estriol (E3) mER/ERK/Ca2 +/proliferation Watson et al., 2008(S)
17α-Ethinylestradiol, 17β-E2valerate (estrogenic) ER/MAPK/cell growth Prifti et al., 2003 (S)
Mestranol, quinestrol ER/ROS/apoptosis Mann et al., 2007(S)
Moxestrol SXR/CAR/ERα/metabolism Min, 2010(S)
Plasticizer
Dicarboxylic ester (see phthalate in Table 1)
Organophosphate
Tricresyl phosphate, triphenyl phosphate (agonist) ER Kojima et al., 2013(R)
Salicylate ester
Benzyl salicylate ER Hashimoto et al., 2003 (C)
Tricarboxylic ester (trimellitate)
Tris(2-ethylhexyl) trimellitate (estrogenic) ER ter Veld et al., 2006(R)
Pollutant
Heavy metal (see metalloestrogen in Table 1)
Persistent organic pollutant (POP)
Aroclor 1221, PCB180 (estrogenic) ER Uslu et al., 2013 (A)
Aroclor 1254 ERα/cyclin D1/Bcl-2/caspase-3/apoptosis Qu et al., 2014 (S)
o,p′-DDD, p,p′-DDD, alachlor (estrogenic) ER Klotz et al., 1996(L, R)
DDE mER/ERK/reproduction Bulayeva and Watson, 2004 (S)
p,p′-DDE, o,p′-DDT, β-HCH (estrogenic) ER/Src/ERK/proliferation Silva et al., 2010(S)
o,p′-DDT (estrogenic) ER Uchida et al., 2010(A, T)
Dioxins AhR/ERα/endocrine disruption Ahmed et al., 2009(S)
β-HCH ER Steinmetz et al., 1996 (L, R)
Hexabromocyclododecane (HBCD) (estrogenic) ER Dorosh et al., 2011(C, T)
Hydroxylated PBDEs (agonist) ER Meerts et al., 2001(R)
Hydroxylated PCBs (agonist) ER Connor et al., 1997(A, C, P, R)
6-Methyl-1,3,8-trichlorodibenzofuran,
3,3′,4,4′-tetrachlorobiphenyl (SERM) AhR/ERα/endocrine disruption Liu et al., 2006 (S)
Monohydroxylated PCBs (estrogenic) ERα Arulmozhiraja et al., 2005 (Y)
PBDEs (estrogenic) ERα Mercado-Feliciano and Bigsby, 2008 (L, R)
PCB104, HO-PCB104 (estrogenic);
PCB155 (anti-estrogenic) ER Fielden et al., 1997(A, C, L, R)
PCB118 (estrogenic) ER Garritano et al., 2006 (R)
PCB126 (estrogenic) ER Mortensen and Arukwe, 2008 (P)
PCBs (agonist/antagonist) ER Zhang et al., 2014 a(C, R, Y)
3,3′,4,4′,5-Pentachlorobiphenyl (agonist) ER Abdelrahim et al., 2006 (P, R, T)
Polychlorinated dibenzofurans (anti-estrogenic) ER Krishnan and Safe, 1993 (P)
TCDD (anti-estrogenic) ER Tian et al., 1998 (T)
Tetrabromodiphenyl ether (BDE 47) (estrogenic) ER Dang et al., 2007(A, P, T)
Tetrabromodiphenyl ether (BDE 47)
(estrogenic/anti-estrogenic) ER Karpeta et al., 2014(P, T)
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 hexachloride; CAR: constitutive androstane receptor; DDD: dichlorodiphenyldichloroethane; DDE: dichlorodiphenyldichloroethylene; DDT: dichlorodiphenyltrichloroethane; E2: estradiol; ER: estrogen 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.

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 activator 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 modulation 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 modulations 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 terpenes/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 hydrogen 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 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, chromium, cobalt, copper, lead, lithium, mercury, nickel, selenium, tin, vanadate and zinc (Table 1). Perfluorinated compounds, such as fluorotelomer alcohols 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). Finally, 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 comparing 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 examining 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 unsaturated 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 effects (Table 2): They are related to various types of food additives/dietary supplements, pesticides (antimicrobials/essential oils, biocides, disinfectants/sanitizers, fungicides, herbicides, insecticides, miticides, repellents, 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 residues, 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 hydrocarbons (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 (vitamin 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, disinfectants (antimicrobials), fungicides, herbicides, insecticides, miticides, molluscicides, nematicides, predacides, piscicides, repellents, rodenticides, defoliants, desiccants and growth regulators (Randall et al., 2013). The pesticides within a particular class may share a common mode of action due to their structural similarity, and several chemical 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 membrane 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, chlorpyrifos 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 contaminated in agricultural products as a mycotoxin. Zearalenone and its derivatives interact with ERα and ERβ in a manner similar to estrogen and thus have been used as anabolic agents for animals and also proposed for hormonal replacement therapy in postmenopausal women or as oral contraceptives (Pazaiti et al., 2012). A number of pharmaceuticals, 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 number 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 esters 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 environment 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 organic compounds (e.g., those in paint and coatings), and their derivatives, 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/proliferation, 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 receptors, which transduce signals through various pathways (Table 3). Specific 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. 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 secretion of hormones or growth factors to transduce signals to different cells or tissues, which may result in completely different types of functional outcome. Feedback regulation of such signaling networks through crosstalk/bypassing and autocrine/paracrine networks would make signaling more complex, which may also contribute to pathways other than estrogen signaling and eventually result in forming a homeostatic network.

Table 3
Receptor signaling pathway Reference
Nuclear receptor
Estrogen receptor α (ERα) (see Tables 1 and 2)
4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT)/ERα Stauffer et al., 2000
Estrogen receptor β (ERβ) (see Tables 1 and 2)
2,3-Bis (4-hydroxyphenyl)-propionitrile (DPN)/ERβ Meyers et al., 2001
Estrogen-related receptor (ERR)
Apigenin, kaempferol (antagonist)/ERRγ Huang et al., 2010
Biochanin A, daidzein, genistein (agonist)/ERRα; chlordane, toxaphene (antagonist)/ERRα; diethylstilbestrol (antagonist)/ERRα, ERRβ, ERRγ Ariazi and Jordan, 2006
Chlordane, toxaphene (antagonist)/ERRα Yang and Chen, 1999
Kaempferol (antagonist)/ERRα, ERRγ Wang et al., 2013e
Resveratrol/ERα/ERRα/respiratory chain Lopes Costa et al., 2014
Membrane/cytosolic receptor
G-protein-coupled estrogen receptor (GPER) (see Tables 1 and 2)
E2/GPER/Serotonin 1A receptor/mood disorders Xu et al., 2009
E2/GPER/serotonin 1A receptor/desensitization McAllister et al., 2012
E2/GPER/serotonin 1A receptor/hippocampus Akama et al., 2013
Genistein, bisphenol A, kepone, nonylphenol, zearalenone/GPER Thomas and Dong, 2006
ER-X
17α-E2/ER-X Soltysik and Czekaj, 2013(review)
17α-E2/ER-X/MAPK/differentiation Toran-Allerand et al., 2002
17α-E2/ER-X/MAPK/ERK/PI3K/Akt/brain Toran-Allerand et al., 2005
17α-E2, 17β-E2/ER-X/Ca2 + channel/contraction Ullrich et al., 2008
ER-α36
E2/ER-α36/breast cancer Rao et al., 2011(review)
E2/ER-α36/MAPK/ERK/HER2/stem cells Kang et al., 2011
E2/ER-α36/MAPK/ERK/breast cancer Zhang et al., 2014c
E2/ER-α36/c-Src/proliferation Wang et al., 2013b
Testosterone/ER-α36/ERK/Akt/carcinogenesis Lin et al., 2009
Other
DDE/mER/ERK/reproduction Bulayeva and Watson, 2004
17α-E2/mER/Rho/ROCK/cofilin/neuroprotection Hirahara et al., 2013
17α-E2, 17β-E2/new ER/MAPK/ERK/fibroblasts Nethrapalli et al., 2005
17β-E2/mER/natriuretic peptide receptor A/cGMP/Ca2 +/hepatocytes Stratton et al., 2010
Estrone (E1), estradiol (E2), estriol (E3)/mER/ERK/Ca2 +/proliferation Watson et al., 2008
Geniposide/mER/PI3K/MAPK/apoptosis Li et al., 2014b
mER/hypothalamus Micevych and Kelly, 2012 (review)
mER/signalosome (IGFR, EGFR, Ras, c-Src, etc.)/second messengers Soltysik and Czekaj, 2013(review)
Naringenin/mERα/ERK/PI3K/MAPK/proliferation Galluzzo et al., 2008
Raloxifene, tamoxifen/mER/ERK/NF-κB/cell protection Stice et al., 2012
Retinoic acid/RAR/mER/MAPK/differentiation Kauss et al., 2008
Sulfonylurea/mER/K channels/insulin secretion Nadal et al., 1998
Other receptor/signaling (crosstalk)
AhR
Biochanin A, formononetin/AhR/ER/cancer prevention Medjakovic and Jungbauer, 2008
3-Methylcholanthrene/AhR/ER/endocrine disruption Chaloupka et al., 1992
β-Naphthoflavone/AhR/ERα/hepatocytes (bypassing) Gräns et al., 2010
β-Naphthoflavone/AhR/PI3K/Akt/MAPK/ERK/ERα/carcinogenesis Wang et al., 2014
TCDD/AhR/E2/ER/proteasome-mediated degradation Wormke et al., 2003
TCDD/AhR/E2/ER/transcription Krishnan et al., 1995
AR/PR
EGF/EGFR/AR/ER/Src/DNA synthesis Migliaccio et al., 2006
Genistein/AR/ER/transcription Takahashi et al., 2006
Piceatannol/ER/MAPK/ERK/Akt/PR/proliferation Vo et al., 2010
Progesterone/PR/ER/Src/ERK/PI3K/Akt/proliferation Ballaré et al., 2006
Progesterone, E2/PR/ER Katzenellenbogen, 2000 (review)
Progesterone, E2/PR/ER Thakkar and Mehta, 2011(review)
Progestin/PR/ERβ/ERK/Akt/proliferation Vallejo et al., 2005
IGF-1R
Calycosin, formononetin/ERβ/IGF-1R/proliferation Chen et al., 2013
E2/ERα/IRS-1/IGF-1R/PI3K/Akt/carcinogenesis Tian et al., 2012
Genistein/ERα/IGF-1R/IRS-1/Akt/proliferation Hwang et al., 2013
Ginsenoside Rg1/ER/IGF-1R/neuroprotection Gao et al., 2009
Indole-3-carbinol/ERα/IGF-1R/IRS-1/proliferation Marconett et al., 2012
Resveratrol/ERα/IGF-1R/IRS-1/Akt/cyclin D1/endocrine disruption Kang et al., 2013
Other
Androstenol/CAR/ER/GRIP-1/differentiation Min et al., 2002
Bergapten/TGF-β/SMAD4/ERα/proliferation Panno et al., 2012
Cholesterol/LXRβ/ERα/PI3K/Akt/NO/metabolism Ishikawa et al., 2013
Epigallocatechin gallate + curcumin/EGFR/VEGFR-1/ERα/cell growth Somers-Edgar et al., 2008
Ethanol/adenosine receptor/ERα/proliferation (ligand-independent) Etique et al., 2009
Hesperetin/TrkA receptor/MAPK/PKA/PKC/ERα/neuroprotection Hwang et al., 2012
Isoproterenol/β-adrenoreceptor/ER/NO/cAMP/vasorelaxation Chan et al., 2002
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: estradiol; ER: estrogen receptor; ERK: extracellular signal-regulated kinase; ERR: estrogen-related receptor; FOXO: Forkhead box O; GPER: G-protein-coupled estrogen receptor 1; GR: glucocorticoid 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.



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 addition, 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 involved in transcriptional activation of estrogen-responsive genes (AF-1); the C domain contains a two-zinc-finger structure responsible for receptor dimerization and DNA binding; and the E/F domain at the C-terminal mediates ligand binding, receptor dimerization, nuclear translocation and transactivation of target gene expression (AF-2) in association 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 ESR2are known to show splice variants, which add variation in terms of cell/tissue-specific expression, ligand-binding specificity, cell localization 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 distinguish between ERα and ERβ, some show a preference or exclusivity for one of these (Escande et al., 2006). The difference in transactivation activity 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 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 mediated by ERRs has been reported for biochanin A, daidzein, genistein and resveratrol (estrogenic), as well as apigenin, chlordane, diethylstilbestrol, 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. Canonical 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 function (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 domain, 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 testosterone 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 located at chromosome 7p22.3 with high expression in the hypothalamus, 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 aldosterone; 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, raloxifene, 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 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 proliferation, differentiation and other cell functions (Table 3). Crosstalk was implicated between mER and other receptors, such as natriuretic peptide 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 chromatin/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 proliferation, 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, mitochondrial, 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, quercetin, 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 pathways: 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).

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 (including 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 reported for the signaling pathways between estrogen and AhR (β-naphthoflavone: 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. Estrogen 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 pathways 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, methoxychlor, 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 tamoxifen 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), progesterone 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 combinations of signal inducers and receptors, such as ethanol/adenosine receptor (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 synergistically 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 estrogen action at the cell signaling level.

3.2.4. Differentiation/development
Estrogen plays critical roles in the sexual differentiation and development 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 development 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 induced 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 altering 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 (2) by the interaction between different receptors or pathways. The former case may include crosstalk through the genomic pathway, such as between ERα and AhR (Safe and Wormke, 2003). The latter case may include (2–1) direct interaction between different receptors or pathways, (2–2) one-way crosstalk, or bypassing, where signaling is uni-directional because one of the pathways involved is abortive due to the lack of appropriate effectors or signal mediators, and (2–3) paracrine and autocrine signaling, where other hormones or growth factors are involved in the signaling. Note that, in some cases, ligands are not significant or required to transduce signaling from one signaling pathway to another, where activation/inhibition of signaling is done by direct interactions between receptors, or the first signaling results in the production or the activation of the second receptor.

3.3.1. Crosstalk/bypassing
Crosstalk of ER or estrogenic signaling pathways with other receptors or receptor-mediated signaling pathways has been reported for receptors, such as AhR, EGFR, HER2 and IGF-1R, and signaling pathways, such as MAPK/ERK, c-Src, PI3K/Akt, NF-κB and NOTCH (Safe and Wormke, 2003; Ribeiro and Freiman, 2014). These pathways mostly promote the growth of cancer cells, so understanding them is essential for cancer treatment. Furthermore, signals induced by the binding of ligands (as agonists), such as biochanin A, formononetin, 3-methylcholanthrene, β-naphthoflavone and TCDD, to AhR are mediated by crosstalk between ER and AhR to contribute to various cell functions through pathways such as PI3K/AKT and MAPK/ERK signaling, or through transcription or proteasome-mediated degradation of ERα (Table 3). Biochanin A and formononetin are estrogenic chemicals, but could act as AhR agonists and thus be anti-estrogenic through the crosstalk (Medjakovic and Jungbauer, 2008). AhR signaling is bypassed by ER signaling (or crosstalk between ER and AhR is uni-directional) because inhibitory effects were observed only for β-naphthoflavone, which inhibits ER signaling, but not for 17α-ethinylestradiol, which inhibits AhR signaling (Gräns et al., 2010).

Crosstalk has been reported between ER and PR for ER ligands (genistein and piceatannol) or PR ligands (progesterone and progestin) (Table 3), or between ER and AR in a ligand-independent manner (Migliaccio et al., 2006). While ERβ and PR form a complex and crosstalk with each other (Vallejo et al., 2005), crosstalk between ER and growth factors could down-regulate PR expression (Thakkar and Mehta, 2011).

Crosstalk between ER and IGF-1R induced with estrogen or estrogenic/anti-estrogenic chemicals, such as calycosin, formononetin, genistein, ginsenoside Rg1, indole-3-carbinol and resveratrol, involves pathways, such as IRS-1 and PI3K/Akt signaling, and cell functions, such as cell proliferation and neuroprotection (Table 3).

Crosstalk between ER and other receptors, such as α1/β-adrenoceptor, CAR, GR, HER2, integrin receptor, LXRβ, natriuretic peptide receptor, PPARγ, RAR, ryanodine receptor, TGF-βR, TR and VEGFR-1, was also reported along with estrogen or estrogenic chemicals (lycopene, resveratrol, tamoxifen and tocopherols), or ligands for receptors, such as androstenol/CAR, cholesterol/LXRβ, epigallocatechin gallate/EGFR, ethanol/adenosine receptor, hesperetin/TrkA receptor, isoproterenol/β-adrenoreceptor, protopanaxadiol and protopanaxatriol/GR, ryanodine/ryanodine receptor, thyroid hormone/TR and vitamin D/VDR and Wnt/Fz (Table 3).

3.3.2. Autocrine/paracrine signaling
Since cells communicate with other cells in the neighborhood or in distant locations (by paracrine or endocrine signaling) or with themselves (by autocrine signaling) through hormones, growth factors, cytokines, ions and other mediators, it is important to examine such communications to understand the action of estrogen under physiological conditions. The signals induced by estrogen or estrogenic chemicals modulate signaling pathways for other hormones and growth factors, which are secreted and transported, and finally affect the same cell or other cells.

Autocrine/paracrine signaling was reported for receptors, such as ANF receptor, APNR, AR, CCRs, CXCR4, EGFR (ERBB4), FGFR, IGF-1R/InsR, IL6R, PDGFR and TGF-βR, where the hormones and growth factors released are ANF, adiponectin, androgen, MCP-1, CXCL12 (SDF-1), EGF (amphiregulin), FGF, IGF-1/insulin, IL-6, PDGF and TGF-β1, respectively. The signaling pathways involved are ERK, JNK, MAPK, NF-κB, PI3K/Akt/mTOR and Wnt/β-catenin signaling, which are related to cell growth and proliferation in breast and other cancer cells, and differentiation and other functions (Table 3).

In paracrine signaling, hormones and growth factors are secreted from a cell type and transported to another cell type. For example, adiponectin secreted from adipocytes is transported to epithelial cells, 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 its receptor on breast cancer cells and activates their cell division with the help of activation of ERα through PI3K/Akt/mTOR signaling (Riverso et al., 2014). Estrogen-induced activation of a procoagulant/coagulation factor further activates prothrombin to form thrombin, which is a paracrine factor that acts to develop the uterus (Jazin et al., 1990). Wnt proteins secreted from mature myometrial cells in response to estrogen and progesterone induce myometrial stem cells to develop leiomyomas in the uterus (Ono et al., 2013). Estrogen-induced secretion of growth factors and hormones from cardiomyocytes to other cells induces neovascularization and attenuates fibrosis (Mahmoodzadeh et al., 2014). The role of 17α-E2 as an autocrine/paracrine factor was implicated in brain functions and therapeutic use in association with its preferred receptor ER-X (Toran-Allerand et al., 2005). Finally, since estrogen itself is an important member of the autocrine/paracrine factors, the modulation of estrogen signaling is crucial to develop SERMs for the prevention and treatment of postmenopausal degenerative and neoplastic diseases (Härkönen and Väänänen, 2006).

3.3.3. Homeostatic networks
Extracellular networks adopt a homeostatic nature when they have a system of feedback regulation in autocrine/paracrine signaling. While such networks play important physiological and developmental roles, such as that in estrogen regulation of estrous cyclicity (Yeo and Herbison, 2014), ovulation (Wintermantel et al., 2006) and spermatogenesis (Chimento et al., 2014), too much positive or negative feedback regulation would cause the development and progression of cancer and other diseases. For example, positive feedback activation in an autocrine loop between CXCR4/CXCL12 and ERα/ERβ signaling pathways promotes the ER-dependent development of breast cancer (Sauvé et al., 2009; Boudot et al., 2011).

The feedback regulation involving estrogen can be categorized by the role of ER: (1) modulation of signaling proteins or transcription factors for growth factor and other signaling-related genes to increase/decrease the activity of signal mediators (e.g., Sauvé et al., 2009), (2) transcriptional modulation of ER genes to increase/decrease (mostly increase) the amount, and thus the sensitivity, of ERs (e.g., Wintermantel et al., 2006) and (3) modulation (mostly increasing) of estrogen synthesis by regulating aromatase activity (e.g., Wang et al., 2008a). Examples of feedback regulation have been reported for estrogen, although other estrogenic chemicals could also be involved, and such regulation of estrogen signaling could be used as targets for developing tumor-targeting drugs (Renoir et al., 2013).

4. Evaluation of estrogen action
Estrogen action is evaluated first by the structural and functional characteristics of chemicals (Tables 1 and 2), whose functional outcomes are interpreted partly by the signaling pathways that they induce (see Section 3). When these collections of information are used to predict the effect of estrogenic chemicals, we need to understand the details of the technologies, especially their advantages/disadvantages and the limitations of the methods used for the evaluation. Comparison of in vitro assays including their advantages 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, transcription assay, protein assay, cell assay, animal tests and signaling pathway analysis, which are based on the molecular and cellular mechanisms 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 pathways 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 responsive 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 binding affinity, the ratio of IC50 between the test and unlabeled E2. 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 distinguish 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 characterize 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 acetyltransferase (CAT), luciferase and green fluorescent protein (GFP); culture 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 endogenous 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 hormones effectively: TGRM-Luc cells for glucocorticoids, TM-Luc cells for progestagens, TARN-Luc cells for androgens and MMV-Luc cells for estrogens (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, progesterone and glucocorticoids (Sonneveld et al., 2005). The ER-CALUX bioassay 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 receptor or a reporter gene. Yeast cells are frequently used in this type of assay because less time is required to detect the response with significant 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 reporters, such as firefly luciferase (Leskinen et al., 2005) or GFP (Bovee et al., 2004). However, in yeast-based assays, the discrimination between 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 summarized below.

The list of estrogenic chemicals characterized by reporter-gene assay.
Acetaminophen, alkylanilines, caffeic acid phenethyl ester, chlordecone, epigallocatechin 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, 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 leucine and tryptophan. Test chemicals are added to the yeast cells to evaluate 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 estrogenic 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 synergistic 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 interaction between the B domain in AF-1 and C-terminal domain can stabilize 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 measure 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, menadione, 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 transcription 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 design could be combined by the Generally Applicable Gene-set Enrichment (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 screening of endocrine disruptors have been developed from large-scale physicochemical 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 appropriate 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 on cell functions, such as cell viability, cell proliferation, cell cycle, apoptosis 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 estrogenic 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 summarized 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 pharmaceutical 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 overestimate the risks, especially giving false-positive results, and show variability 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 inflammation 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 profound impact for environmental scientists, toxicologists and pharmacologists as well as scientists and researchers in other fields, no practical protocol has ever been established. Although a number of toxicity pathways 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 signaling (Adeleye et al., 2015) and benzo[a]pyrene and dioxin in AhR signaling (National Research Council, US, 2010), other pathways for these chemicals (such as those shown in Tables 1 and 2) are not included, indicating 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. This paradigm shift requires reliable assays, which are based on the predictability 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 signaling 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 assessment, pathway-based risk assessment would give superiority as a risk assessment 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 pathway 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 pathways to utilize the information obtained by the analysis for risk assessment 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. Meanwhile, 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 combinations of chemicals, biological outcomes and assay systems would provide quite a large number of variations to distinguish predicted outcomes of potential risks.
 
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LeeLemonoil

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Thanks, massive stuff.
I wonder why they did not scrren artificial musk and aromachemicals though
 

Amazoniac

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If you know people that wear medical gloves often (doctors, nurses, dentists, veterans, cleaners, pharmacists, cooks, barbecuelists, taboo artists, mechanics, etc.), they're likely being exposed to disruptors without realizing. In certain cases they is worm for almost the entire work journey and new ones are gotten frequently.

Flexible plastic is a red flag. But regardless of this, it doesn't matter if it's latex, vinyl, nitrile, the chances of detrimental effect are high given that it can be from the material itself, the additives, or the finishing powders, leading to a local reaction that affects the whole body or actual systemic adsorption.

- Estriol, DES, DDT, Etc

A solution is to wear grass-fed cotton gloves in between.
 
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Dave Clark

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If you know people that wear medical gloves often (doctors, nurses, dentists, veterans, cleaners, pharmacists, cooks, barbecuelists, taboo artists, mechanics, etc.), they're likely being exposed to disruptors without realizing. In certain cases they is worm for almost the entire work journey and new ones are gotten frequently.

Flexible plastic is a red flag. But regardless of this, it doesn't matter if it's latex, vinyl, nitrile, the chances of detrimental effect are high given that it can be from the material itself, the additives, or the finishing powders, leading to a local reaction that affects the whole body or actual systemic adsorption.

- Estriol, DES, DDT, Etc

A solution is to wear grass-fed cotton gloves in between.
Good Lord, and I thought the cash register receipt thing was over the top, now this. I worked in wastewater for 40 years wearing these latex, etc. gloves.....too late now. Stay tuned for the next thing to stay away from........
 

BigChad

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@High_Prob why does your post include things such as zinc, copper, olive leaf extract, oleupurein, etc. it seems like every vitamin, mineral and nutrient is listed as estrogenic there? zinc, olive leaf extract are commonly regarded as aromatase inhibitors, you are saying they have estrogenic activity? I've used zinc before and it certainly didn't seem to be estrogenic
 

Amazoniac

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It keeps getting bizarre, we have to consider motorcyclists and sportsgurus as well.
- A Case of Chromium Contact Dermatitis due to Exposure from a Golf Glove
- Understanding Glove Related Contact Dermatitis | hourglass-intl.com

It's difficult to trust anything these days, it doesn't matter if it's synthetic material or not, they find a way to make it problematic.

The person has to confirm if the cotton glove is indeed 100% without unwanted stuff, otherwise it's like the majority of them that contain some plastic in the composition or suspicious additives that are added to the final product or introduced during manufacturing.
 

BigChad

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It keeps getting bizarre, we have to consider motorcyclists and sportsgurus as well.
- A Case of Chromium Contact Dermatitis due to Exposure from a Golf Glove
- Understanding Glove Related Contact Dermatitis | hourglass-intl.com

It's difficult to trust anything these days, it doesn't matter if it's synthetic material or not, they find a way to make it problematic.

The person has to confirm if the cotton glove is indeed 100% without unwanted stuff, otherwise it's like the majority of them that contain some plastic in the composition or suspicious additives that are added to the final product or introduced during manufacturing.

Would PET bottles (bottled water) be okay? Using them since they are reverse osmosis filtered as well as ozone treated, so it seems like the water would be cleaner than simply using a reverse osmosis filter on tap water at home.

Chromium is still considered an essential nutrient? It's weird it would cause that effect from contact, I'd imagine the amount in the glove would be small
 

Amazoniac

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- The use and abuse of gloves | IIRSM
- Estrogenic and anti-androgenic endocrine disrupting chemicals and their impact on the male reproductive system
Would PET bottles (bottled water) be okay? Using them since they are reverse osmosis filtered as well as ozone treated, so it seems like the water would be cleaner than simply using a reverse osmosis filter on tap water at home.

Chromium is still considered an essential nutrient? It's weird it would cause that effect from contact, I'd imagine the amount in the glove would be small
I would avoid it, there's extensive discussions on the topic.

Regarding the second part, it was addressed in the publication.

Additives aside, how much plastic are you wearing right now? Socks almost invariably contain some and sweat doesn't help any.
 
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InChristAlone

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My beloved vitamin C antiestrogenic! Which explains why I feel like I've tanked my histamine and estrogen levels after 4 yrs of continuous use.
 

InChristAlone

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Aspirin estrogenic?
 

Amazoniac

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- Estrogenic and anti-androgenic endocrine disrupting chemicals and their impact on the male reproductive system

"Typical daily phthalate intake in humans is estimated at 1.7–52.1 μg/kg/day (Doull et al., 1999; Koch et al., 2003; McKee et al., 2004; Frederiksen et al., 2011; Martinez-Arguelles et al., 2013), whereas children exposure is 2- to 4-fold higher than adults (Koch et al., 2005; Moody et al., 2013)."​

- Phthalate Esters in Foods: Sources, Occurrence, and Analytical Methods

"Although the determination of phthalates in foods began more than 4 decades ago (Cerbulis and Ard 1967; Williams 1973b), unlike the other chemical contaminants, information on phthalates in foods is very limited, as summarized in Table 2, probably due to the challenges in the methods or high blank levels of phthalates caused by the contamination of laboratory environments."​
"In the early 1990s, samples of Canadian butter and margarine wrapped in aluminum foil-paper laminate were found to contain DBP, BBzP, and DEHP at levels up to 10.6, 47.8, and 11.9 mg/kg, respectively (Page and Lacroix 1992). These phthalates were also detected in the wrappers at levels up to 37.6, 66.0, and 25.5 mg/cm2 for DEP, BBzP, and DEHP, respectively. An analysis of unused wrappers showed 76% to 88% of the total DBP and DEHP to be present on the foil (outer) surface as a component of the protective coating (washcoat). The remainder of the DBP and DEHP was found on the food-contacting paper surface, presumably by transfer from the outer to inner surface during storage in tightly wound rolls. In the typical production of a foil-paper laminate, an adhesive is applied to the foil, the foil and paper are brought together and bonded between rollers. The washcoat can then be applied to the foil surface to increase its abrasion resistance and improve its inking properties. In order to find out the exact sources of the phthalates in butter and margarine, samples of aluminum foil, paper, adhesive, ink, a washcoat, and unprinted coated foil-paper laminate were obtained from a packaging manufacturer and analyzed, and only the washcoat was found to contain the phthalates. However, since this study was conducted in the early 1990s, and the authors did mention that suppliers of the washcoat were replacing the phthalates in their product with other plasticizers having less potential for migration, phthalates in butter and margarine wrapped in aluminum foil-paper should be re-investigated."​
"Tolerable daily intakes (TDI) have been specified by the European Food Safety Authority (EFSA) for several phthalates, and they are 0.01, 0.5, 0.05, 0.15, and 0.15 mg/kg body weight/day for DBP (EFSA 2005a), BBzP (EFSA 2005b), DEHP (EFSA 2005c), DiNP (EFSA 2005d), and DiDP (EFSA 2005e), respectively."​
"Migration from food-packaging materials plasticized with phthalates is a very important source of phthalates in foods when in contact with these packages. Although this has been investigated for various food-packaging materials, some of the information may not reflect the current situation since manufactures may have changed plasticizers in materials over the years. The regulations on the use of plasticizers in food-packaging materials vary from country to country; plasticizers banned in 1 country may be permitted for use in food-packaging materials in other countries. Thus, up-to-date studies should be conducted worldwide to investigate migration of phthalates, adipates, and other plasticizers in the current food-packaging materials."​
 

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