Determination of the estrogenic potency of
phyto- and synthetic estrogens
using in vitro bioassays
in Natural and Synthetic Estrogens Aspects of the Cellular and Molecular Activity 2002
[eds.: E. Dopp, H. Stopper, G. Alink; Transworld Research Network, Trivandrum, India]
Juliette Legler Institute for Environmental Studies, Vrije Universiteit, De Boelelaan 1087 1081 HV Amsterdam, The Netherlands
Correspondence/Reprint request: Dr. Juliette Legler, Institute for Environmental Studies, Vrije Universiteit, De Boelelaan 1115 1081 HV Amsterdam, The Netherlands`. Phone: (31) 20 4449516, Fax: (31) 20 4449553, E-mail: email@example.com
Many compounds found in the environment, including plant estrogens, pharmaceuticals and industrial chemicals, have been found to mimic estrogens by binding to estrogen receptors and influencing estrogen-signaling pathways. Bioassays based on the common mechanisms of action of estrogens can provide a very useful means of identifying and measuring the biological potency of a suspected estrogen or mixture of estrogens. The objective of this article is to provide a brief review of the most widely used in vitro bioassays for the determination of estrogenic potency of substances. These assays include competitive binding assays, cell proliferation assays and recombinant receptor/reporter gene assays. A novel in vitro reporter gene assay for measuring the estrogenic potency of chemicals, the estrogen receptor-mediated chemical activated luciferase gene expression (ER-CALUX) assay, is described in more detail. The in vitro estrogenic potency of phyto-, synthetic and xenobiotic estrogens measured with the ER-CALUX assay is provided.
In 1992, Carlsen, Skakkebaek and colleagues published a controversial study reporting a significant, worldwide decrease in sperm quality over the past 50 years (Carlsen et al., 1992). In addition to decreasing sperm counts, increased incidence of testicular cancer and cryptorchidism were reported (Giwercman et al., 1993). Evidence also indicated that deleterious effects on male reproductive health in wildlife species were occurring. In Florida panthers, males were found with low sperm counts and cryptorchidism (Facemire et al., 1995). Male alligators in Lake Apopka in Florida demonstrated reproductive abnormalities including reduced penis size, abnormal testis morphology and abnormal testicular steroidogenesis (Guillette et al., 1994). In seagulls, altered sex ratio and feminization of sexual behaviour in males were observed (Fry and Toone, 1981). In wild fish populations, intersex (defined as the simultaneous presence of both male and female gonadal characteristics) and testis abnormalities have been reported in a high proportion of male fish sampled in rivers, estuaries and coastal waters in the U.K. (Jobling et al., 1998; Lye et al., 1997; Allen et al., 1999) as well as in localized freshwater sites in The Netherlands (Vethaak et al., 2002). These alarming reports led to the hypothesis that declines in sperm counts and related disorders of the male reproductive system in both humans and wildlife could have arisen because of exposure of the developing fetus or during neonatal life to estrogens (Sharpe and Skakkebaek, 1993; Colborn et al., 1993). One potential source of increased estrogen exposure is via exposure to compounds in the environment that can mimic natural estrogens, thereby disrupting endocrine systems. Endocrine disruption has been officially defined as process by which an exogenous substance "causes adverse health effects in an intact organism or its progeny consequent to changes in endocrine function" (European Commission, 1996). This wide definition includes not only the estrogenic substances, but also all substances that can affect endocrine function via interference with hormone (e.g. androgen, thyroid, retinoid or progesterone) pathways.
The reports of endocrine disruption in humans and wildlife (recently reviewed by Golden et al., 1998; Tyler et al., 1998; Vos et al., 1999) did not go unnoticed by the media and resulted in a shock wave of concern in the general public. Numerous newspaper articles as well as television reports (e.g. the BBC documentary "Assault on the Male" in 1993) raised public awareness, culminating in 1996 with the publication of the book "Our Stolen Future" by Colborn and colleagues. In response to the heightened public concern, research efforts were launched internationally to test the hypothesis that environmental chemicals can disrupt male reproductive function. In fact, very little evidence actually existed to unequivocally demonstrate that environmental substances could interfere with endocrine processes, with the exception of the effects of DES on male offspring (reviewed by Cheek and McLachlan, 1998). Regulatory agencies worldwide, including the European Chemical Industry Council (CEFIC), the Organization of Economic Cooperation and Development (OECD) and the U.S.
Environmental Protection Agency, launched ambitious programs to test existing and new chemicals for their potential to disrupt endocrine systems. In The Netherlands, The Health Council published advisory reports on the effects of endocrine-disrupting chemicals in humans (Health Council, 1997) and ecosystems (Health Council, 1999), and the Netherlands Research Platform on Endocrine-Disrupting Compounds (Vethaak et al., 2000) was initiated. One general theme has arisen from these regulatory initiatives; there is a definite need for the development of rapid, biologically relevant methods to test chemicals for endocrine-disrupting potential.
Objectives of this study
The objective of this article is to provide a brief review of the most widely used in vitro bioassays for determination of estrogenic potency of substances such as phytoestrogens, synthetic estrogens and xenoestrogens. Because these bioassays are based on
the molecular and cellular mode of action of estrogens, a brief description of the role of estrogens in vertebrate physiology and the cellular mechanism of action of estrogens is given. The in vitro estrogenic potency of phyto- and synthetic estrogens measured with a sensitive reporter gene assay (ER-CALUX) is provided.
Role of estrogens in vertebrate physiology
|Figure 1. Structures
of natural, synthetic, and xenobiotic estrogenic chemicals
Estrogens are steroid hormones made primarily in the female ovaries. Estrogens are found in greater amounts in females than males and are thus referred to as the female steroid hormones. In all vertebrates, estrogens play an important role in many reproductive and developmental processes as they influence growth, development and behavior (puberty), regulate reproductive cycles (menstruation, pregnancy) and affect many other body parts (bones, skin, arteries, the brain). Recently, estrogens have been shown to be essential for male fertility as well (Hess et al., 1997). 17ß-estradiol (E2; structure pictured in Figure 1) is the major endogenous estrogen.
Cellular mechanism of action of estrogens
Estrogens are transported through the blood mainly bound to sex hormone binding globulins. Free, non-bound estrogens can exert their action through diffusing through cell membranes and binding estrogen receptors (ER). In addition to the two known ER subtypes (ERa and ERb), recently a third form was identified in the marine fish Atlantic croaker (ERg, Hawkins et al., 2000). These three ER subtypes differ in amino acid sequence, as well as tissue distribution, ligand binding affinities and transactivation properties. ERs are found in many tissues, including reproductive organs and accessory sex organs, brain, bone and liver. Inactive ERs exist in large complexes associated with heat shock proteins (Figure 2). Upon binding of estrogens to the ER, the heat shock proteins disassociate, inducing a conformational change that activates the receptor. Following dimerization of two ER-ligand complexes, binding to estrogen response elements (ERE) of genes in the nucleus takes place. This binding stabilizes the binding of transcription factors involved in gene activation and transcription (Figure 2). Gene transcription can be modified by cellular coactivators, repressors and modulators (Stoney,1996). Following transcription, mRNA is translated into protein by ribosomes. By inducing the synthesis of new proteins that alter cellular functions, estrogens can have profound effects on cell function and physiology (Ing and O'Malley, 1995).
Figure 2: The mechanism of steroid hormone receptor action in the cell. Inactive receptors are associated with heat shock proteins (hsp). Hormone freely enters the cell and binds to inactive receptor, inducing lisp dissociation and conformational change. Activated dimers bind to hormone response elements (HRE) of genes in the nucleus. Transcriptional factors (B,D,E,F) and RNA polymerase 11 (Pol II) are recruited for transcription. Messenger RNA is subsequently translated into proteins by ribosomes. These new protein may alter cell function and physiology (from Ing and O'Malley 1995).
Phyto-, synthetic and xeno-estrogens
In addition to the endogenous estrogens such as estradiol, estrone and estriol, other compounds found in the environment can mimic estrogens by binding to estrogen receptors and influencing estrogen-signaling pathways. These compounds include natural plant chemicals such as the mycoestrogens and phytoestrogens. Synthetic estrogens are pharmaceuticals specifically synthesized to mimic estrogens, and include diethystilbestrol and the anti-conception pill component ethynylestradiol (pictured in Figure 1). A number of xenobiotic chemicals ("xeno-estrogens") have been shown to mimic estrogens, and include the organochlorine pesticide o,p'DDT, the industrial surfactant 4-nonylphenol, plasticizers such as di-(2-ethylhexyl)phthalate (DEHP) and Bisphenol A, a compound used in the manufacture of polycarbonate. Structures of some of these chemicals are shown in Figure 1. Some compounds have also been identified with an anti-estrogenic mode of action; that is, they can block, prevent and/or alter binding to estrogen receptors. More detailed lists of phyto- and mycoestrogens, as well as suspected xeno-estrogenic chemicals are provided in Colborn et al., 1993 and Tyler et al., 1998. Recently, the Health Council of The Netherlands reported 34 groups of estrogenic chemicals as potential hormone disrupters that may form a risk for ecosystem health in the Netherlands (Health Council, 1999).
Bioassays for the detection of biologically active (xeno-)estrogens
The large difference in chemical structures of estrogenic compounds makes prediction of estrogenic activity based on structure difficult. However, estrogenic compounds may share a common mechanism of action with endogenous estrogens, such as binding to the estrogen receptor and induction of the ER signal transduction pathway. They may also share a common estrogen-mediated biological effect in an intact organism, such as inducing the proliferation of estrogen-sensitive cells and tissues, feminization of male gonads or effects on reproduction. Biological test systems or "bioassays" which make use of these common mechanisms of action can provide a very useful means of measuring the biological potency of a suspected estrogen or mixture of estrogens.
A number of in vitro bioassays have been developed to screen substances for estrogenic potency. The most widely used bioassays include competitive ligand binding assays, cell proliferation assays, and recombinant receptor/reporter gene assays. The principles of these assays, as well potential drawbacks that limit may limit their usefulness as a screen for (anti-)estrogenic compounds are outlined below. For an exhaustive review of these and other types bioassays, the reader is referred to Zacharewski, 1997.
Competitive ligand binding assay
This extensively used assay investigates the ability of compounds to bind in vitro to the ER, thereby displacing (radioactive) labeled E2 from the ER (Korach et al., 1978; Berthois et al., 1986; Migliaccio et al., 1992, Schwartz and Skafar, 1993). Though binding of a substance to the ER is an important step in the biological mode of action of estrogenic chemicals, this type of assay has the disadvantage that it cannot distinguish between receptor agonists and antagonists. Furthermore, the ability of a substance to initiate the molecular cascade of events involved in gene transcription and protein production related to adverse effects is not measured with this assay.
Cell proliferation assays
This type of assay, also known as the "E-screen", measures the growth of estrogen- sensitive MCF-7 or T47D human breast cancer cells in medium containing serum stripped of estrogens (Soto et al., 1995). Prolonged (6 day) incubation of these cells with estrogenic compounds induces cell proliferation. Though this assay is very sensitive, its use may be limited due to lack of estrogen specificity, as mitogens other than estrogens are able to influence the proliferation of human breast cancer cells (van der Burg et al., 1988; Dickson and Lippman, 1995). In addition, factors such as differences between cell line clones, culture condiction, serum and cell density may greatly affect cell proliferation and thus reproducibility and repeatability of the assay (Zacharewski, 1997).
Recombinant receptor/reporter gene assays
This type of assay involves the use of mammalian or yeast cells which have been transfected or transformed with recombinant DNA contructs, so that they contain constitutively expressed ER sequences and/or ERE-driven reporter genes. Reporter genes, such as firefly luciferase or ß-galactosidase, are sensitively and relatively easy to measure compared to measuring endogenous protein expression. These assays have the advantage that they are based on the receptor-mediated mechanism of action of estrogens and reporter gene expression is a culmination of the molecular cascade of events involved in receptor transactivation.
A number of recombinant receptor and reporter gene assays in yeast cells have been developed (Connor et al., 1995; Arnold et al., 1996; Routledge and Sumpter, 1996). The recombinant yeast estrogen screen developed by Glaxo, U.K. and first published by Routledge and Sumpter, 1996, has been widely used to rapidly screen various estrogenic compounds. In this assay, yeast cells have been stably transformed with human ER-a cDNA and an ERE-regulated expression plasmid (lac-Z). Interaction of an estrogenic compound with the ER results in expression of the reporter gene lac-Z and secretion of the enzyme ß-galactosidase in the yeast medium. This assay is easy to perform, rapid (3 day exposure) and relatively sensitive, however disadvantages include the inability to distinguish estrogens and anti-estrogens (Legier et al., 2002b) as well as differences in permeability of compounds through the yeast cell wall (Lyttle et al., 1992).
The use of recombinant reporter gene assays based on stably transfected mammalian cell lines may circumvent many of the disadvantages listed above for the various in vitro assays. The recently developed estrogen receptor (ER)-mediated Chemical Activated Luciferase gene expression (ER-CALUX) assay uses T47-D human breast adenocarcinoma cells expressing endogenous ER a and ß, which are stably transfected with an estrogen-responsive luciferase reporter gene (Legier et al., 1999). Exposure of stably transfected T47-D cells to (xeno-)estrogens results in transactivation of the ER and consequent induction of the luciferase gene, which is easily assayed by lysing cells and adding the substrate luciferin, and measuring light output. In comparison to other recombinant receptor and/or reporter gene assays using stably or transiently transfected mammalian cells, the ER-CALUX assay is one of the most sensitive and responsive stably transfected estrogen-responsive cell line (Legier et al., 1999). The assay detects as low as 0.5 pM E2 following 24 hour exposure, and demonstrates up to 100-fold induction relative to controls. The ER-CALUX also shows higher sensitivity and responsiveness when compared with a competitive ER ligand binding assay and the recombinant yeast screen (Murk et al., 2002).
Estrogenic potency of phyto- and synthetic estrogens in the ER-CALUX assay
A number of suspected estrogenic compounds have been measured in the ER-CALUX assay. An overview of their potency relative to estradiol is provided in Table 1. With the exception of the synthetic estrogen ethynyl-estradiol, which is slightly more potent than estradiol itself, the industrial chemicals such as the alkylphenols, pesticides and phthalates are generally more than 10,000 times less potent than E2 (Table 1). The phyto-estrogens tested, including biochanin A, genistein, diadzein, genistin, coumestrol and formononetin, are more potent luciferase inducers in the ER-CALUX assay than the industrial chemicals, with a relative potency of 10-5 to 10-4 in comparison to estradiol (Table 1).
When comparing the widely used bioassays to detect substances with an estrogenic mode of action, the ER-CALUX assay is a very promising new method. The ER-CALUX assay is rapid and simple, and detection in microtiter volumes is possible, allowing for qualitative and quantitative assessment of luciferase induction in a high throughput setup. The assay can be used as a biomarker of internal exposure to (xeno-) estrogens (Legier et al., 2002a) as well as for screening complex environmental samples for unexpected estrogenic activity that could be further identified by chemical analysis. This in vitro assay has proven to be very effective in detecting estrogenic activity in complex environmental mixtures, such as extracts of sediments, water, biota, and effluents (Legier, 2001). For these reasons, the ER-CALUX assay has been implemented in various field surveys in The Netherlands to determine estrogenic activity in a wide variety of environmental matrices from various freshwater and marine locations (Murk et al., 2002, Vethaak et al., 2002).
Despite the many advantages of using in vitro assays to determine biological potency of (mixtures of) substances, one of the main disadvantages is their simplification of the in vivo situation. In vitro assays cannot completely reflect complex in vivo events, such as bioavailability and toxicokinetics, of a compound. In addition, cross-talk with other mechanisms not directly related to the interaction of substances with the ER signal transduction pathway are not included in the ER-CALUX assay. The breast cancer cell line used in the ER-CALUX assay expresses both ERA and ERP subtypes, which may be problematic for the prediction of tissue-specific effects. Tissues such as prostate and ovary have been found to contain prominent ERP expression (Kuiper et al., 1997). Differences in the binding affinities of compounds, such as phytoestrogens, as well as in the transcriptional activity for both ER subtypes have been demonstrated (Kuiper et al., 1998). In addition, the possibility of extrapolating the estrogenic potency predicted in the ER-CALUX assay using human cells to other species, such as fish, birds and reptiles, requires further study. For example, differential binding affinities of polychlorinated biphenyls using human, rainbow trout and reptilian ERs have been recently demonstrated (Matthews and Zacharewski, 2000). For these reasons, in vivo bioassays are essential for determining species-specific effects of (xeno-)estrogenic compounds (Kavlock et al., 1996; Ankley et al, 1998). However, the use of in vitro assays to screen compounds for their potency estrogenic mode of action and further direct and refine in vivo studies is an essential initial step in the overall risk assessment process.
Table 1. Potency of natural, phyto- and synthetic estrogens relative to estradiol in the ER-CALUX assay
Relative Relative Compound potency Ref Compound potency Ref Natural Estrogens: Synthetic estrogens: 17ß-Estradiol 1 Ethynyl-estradiol 1.2 a 17a-Estradiol 5.6x10-2 a Diethylstilbestrol 0.1 d Estrone 1.6x10-2 a Estriol 1.0 a Alkylphenols: Estradiol 3B-D- n.c.* a 4-nonylphenol (NP) 2.3x10-5 a glucuronide 4-octylphenol (OP) 1.4xl0-6 c Phyto-estrogens: 4-tert-pentylphenol 2.3x10-5 d Genistin 2.6x10-4 e NP1EO# 3.8x10-6 c Diadzein 1.3x10-4 e NP2EO# 1.1x10-6` c Formononetin 1.1x10-4 e NP4EO# 1.1X10-7 c Biochanin A 5.3x10-4 e NP10EO# n.c.* c Genistein 6.0x10-5 b NP1EC# n.c.* c NP2EC# n.c.* c Pesticides: OP8/9EO# n.c.* c o,p'-DDT 9.1x10-6 b o,p'-DDE 2.3x10-6 c Phthalates: Methoxychlor 1.0x10-6 b dimethylphthalate n.c.* c Dieldrin 2.4x10-7 b diethylphthalate 3.2x10-8 c Endosulfan 1.0x10-6 b dibutylphthalate 1.8x10-8 c Chlordane 9.6x10-7 b butylbenzylphthalate 1.4x10-6 c Simazine n.c.* c di(2- n.c.* c ethylhexyl)phthalate Atrazine n.c.* c dioctylphthalate n.c.* c Desethylatrazine n.c.* c Deisopropylatrazine n.c.* c bisphenol A 7.8x10-6 b Kepone 5.2x10-6 d Lindane n.c.* d Benzo(a)pyrene 1.9x10-6 a References: a) Legler et al., 2002a b) Legler et al., 1999 c) Legler et al., 2002b d) Legler et al., in preparation e) Belfroid et al., unpublished results *n.c.: not calculated; no luciferase induction observed, relative potency is <1.0x10-8 #nEO or nEC refers to number of ethoxylate or carboxylate sidechains
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Natural and Synthetic Estrogens Aspects of the Cellular and Molecular Activity, 2002:1-11 ISBN: 81-7736-138-4 Editors: Elke Depp, Melon Stepper and Gerrit IL Alink
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