In Vitro Bioassays for Assessing ... - ACS Publications

Critical Review

In Vitro Bioassays for Assessing Estrogenic Substances TIM ZACHAREWSKI* Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada N6A 5C1

This past summer, United States President Bill Clinton signed into law amendments to the Safe Drinking Water Act and Food Quality Protection Act that include clauses requiring the EPA to develop testing procedures for substances that “mimic” the effect of naturally-occurring estrogens. The act requires the testing of substances found in drinking water and all pesticide materials including both active and inert ingredients as well as substances that may have a cumulative or synergistic effect when combined with pesticides. Estimates indicate that this could require the testing of as many as 70 000 chemicals currently used in the United States. EPA, industry, and public interest groups are investigating strategies to develop screening tests by August 1998 in order to implement a program by August 1999 as mandated in the legislation. This review focuses on in vitro assays such as competitive ligand binding, cell proliferation, and estrogen receptor transcription assays, which are currently being used to identify and assess chemicals alleged to possess estrogenic activity. The advantages and limitations of each assay are discussed. The review concludes that complementary in vivo and in vitro assays are required to accurately assess the potential estrogenic activity of any chemical or complex mixture.

be identified by their ability to bind to the estrogen receptor (ER) and to induce or attenuate a response. Several factors have contributed to exoestrogens becoming a contentious issue not only for regulatory agencies and industrial producers but also for the general public. Epidemiological studies have found significant increases in the incidence of breast, prostate, and testicular cancer. Other studies have reported decreasing sperm counts and semen volume and longer times to conception. These findings are complemented by field study data indicating that wildlife are also experiencing compromised reproductive fitness. Several recent scientific and popular press reports have linked these health effects to exposure to alleged exoestrogens, thus further fueling speculation of a cause and effect relationship (1-5). Although there is no consensus regarding the role of exoestrogens in these effects and no conclusive studies demonstrating that exoestrogens initiate or contribute to the development of these effects (6, 7), results from in vitro bioassays have been repeatedly used to identify chemicals that can elicit estrogenic responses, thus further implicating all exoestrogens in adversely affecting human and wildlife health. The intent of this review is to examine the in vitro assays currently being used to evaluate alleged exoestrogens and to discuss their advantages and limitations in identifying and assessing estrogenic substances.

Proposed Mechanism of Action of Estrogenic Substances Introduction Exposure to substances possessing sex steroid activities can adversely effect endocrine and reproductive systems in laboratory rodents and has been hypothesized to elicit similar responses in humans and wildlife. These effects include the development of hormone-dependent cancers, disorders of the reproductive tract, decreased levels of sperm production and semen volume, and compromised reproductive fitness. Recently, there has been heightened concern regarding the role of estrogenic substances, often referred to as xenoestrogens or environmental estrogens, in contributing to the development of these effects. In this review, estrogenic substances will be referred to as exogenous estrogens or exoestrogens in order to include environmental pollutants, industrial chemicals, natural chemicals, and estrogenic pharmaceuticals. Exoestrogens are a diverse group of substances that do not necessarily share any structural resemblance to the prototypical estrogen, 17β-estradiol (E2), but evoke agonist or antagonist responses possibly through comparable mechanisms of action. Consequently, they can * Telephone: 519-661-4026; fax: [email protected].

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The effects of estrogens are mediated by the estrogen receptor (ER), a member of the nuclear receptor superfamily (8). As illustrated in Figure 1, binding of an estrogenic compound to the ER causes the dissociation of heat shock protein 90 (hsp90), enabling occupied ERs to homodimerize. The resulting homodimer complex exhibits high affinity for specific DNA sequences referred to as estrogen response elements (EREs) located in the regulatory region of estrogen-inducible genes. Once bound to the ERE, the homodimer complex recruits transcription factors to the target gene promoter, which leads to increased gene expression. The increased levels of mRNA that are transcribed are then translated into proteins that are the ultimate effectors of the observed responses. Numerous genes have been found to be estrogeninducible including growth factors, growth factor receptors, proteases, transcription factors, and several other genes whose proteins have functions that have yet to be fully elucidated. Studies suggest these estrogen-inducible proteins may contribute to the health effects elicited by estrogenic substances. It has been proposed that exoestrogens act as ER ligand mimics that bind to the receptor, thus modulating endocrine pathways via a receptor-mediated process (1, 9). Several bioassays exploit this receptor-mediated mechanism of action

VOL. 31, NO. 3, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Proposed estrogen receptor-mediated mechanism of action of estrogenic substances. The ligand (L) passively diffuses into the responsive cell and binds to the estrogen receptor (ER) causing the dissociation of heat shock protein 90 (hsp90). The liganded receptor forms homodimer complexes and binds to specific DNA sequences referred to as estrogen response elements (EREs) located in the 5′ regulatory region of responsive genes. Binding of the homodimer complex to the ERE initiates gene transcription resulting in increased levels of gene expression. to provide a rational strategy for the identification and assessment of alleged exoestrogens that may adversely affect human health and environmental quality. However, it is also possible that exoestrogens also elicit their effects through mechanisms that are independent of the ER.

Bioassays for Estrogenic Substances Several assays have been developed to assess the activity of alleged estrogenic substances. In vivo assays use a variety of end points including effects on organ weights, cell differentiation, protein expression, and enzyme activities (1019). Although these assays are widely used, they are unsuitable for large-scale screening, and their utility is further limited due to cost, relatively poor sensitivity and modest responsiveness, and labor-intensive end point measurements. Moreover, in vivo assays that utilize highly complex responses such as induction of uterine wet weight, which is consider to be the hallmark of estrogenic activity, may be modulated through mechanisms that do not directly involve the ER and, therefore, may not be selective for substances that act through the ER (20-22). For example, administration of carbon tetrachloride (CCl4) has been reported to potentiate the action of E2 on the uterus by inhibiting E2 metabolism (23). However, CCl4 does not bind to the ER, thus, based upon the known mechanism of action, its classification as an exoestrogen is questionable, although it could be considered as an endocrine disrupter. Despite their limitations, rodent assays are an essential component of any investigation examining the estrogenic activity of a substance since they account for the various pharmacodynamic and pharmacokinetic interactions that can occur in vivo. Table 1 lists a number of in vitro assays that have been used in the assessment of alleged exoestrogens. Many of these assays are based on well-elucidated mechanisms of action and utilize more definitive end points than in vivo assays. However, in vitro assay are also plagued by drawbacks that limit their utility, especially when used to assess risk to human health and environmental quality. The following sections discuss the advantages and limitations of several in vitro assays that have been used to implicate substances as exoestrogens. Competitive Ligand Binding Assays. In vitro competitive binding assays for the estrogen receptor are well established and have been extensively used to investigate ER-ligand

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interactions. Since exoestrogens can be defined by their ability to bind and elicit an ER-mediated response, competitive binding assays should be incorporated into all assessments. Unfortunately, these assays cannot distinguish between receptor agonists and antagonists. Moreover, high concentrations of competitor ligand may result in noncompetitive displacement. In addition, binding to the ER is not sufficient to determine the estrogenicity of a substance since potency is dependent on binding affinity and the ability of the ligand to maintain nuclear receptor occupancy so as to initiate a cascade of events that culminate in an adverse response (14,24). The “sustained output” model suggests that complex responses such as the modulation of endocrine systems and/or compromised reproductive fitness may require not only early events such as ligand binding but also the continued accumulative action of ligand-occupied nuclear ER complexes (25, 26). Therefore, binding of a substance to the ER is only suggestive that it may behave as an exoestrogen but does not provide sufficient evidence to conclude that the compound will adversely affect human health or environmental quality. Subsequent in vitro and in vivo tests are required in order to fully assess the potential risks. Furthermore, binding assays are not easily amenable to automation, thereby limiting their utility as a screening tool. Cell Proliferation Assays. These assays, one of which is referred to as the E-Screen, use ER-positive, estrogenresponsive MCF-7 or T47-D human breast cancer cells. The E-Screen compares the number of cells present following a 6-day incubation period in medium supplemented with steroid-stripped dextran-coated charcoal (DCC) serum in the presence or absence of alleged exoestrogens (27). It is based on the following three premises: (i) unidentified factors in human serum inhibit the proliferation of human estrogensensitive cells, (ii) estrogens induce cell proliferation by negating the inhibitory effect of a cell proliferation repressing factor, and (iii) non-estrogenic steroids and growth factors do not neutralize the inhibitory signal present in human serum (27-29). However, in assays using fetal bovine serum (FBS), researchers have found several mitogens capable of enhancing the proliferation of human breast cancer cells (30-40). Therefore, this assay suggests but does not unequivocally demonstrate that a substance is estrogenic. Although the simplicity of this assay is attractive, many factors have been shown to affect the potency of estrogenic substances (Table 2). These include, but are not restricted to, differences in cell line clones, culture conditions, and serum lots, thus complicating standardization of the assay to ensure interlaboratory reproducibility. A comparison of data obtained from published reports indicates that these factors can significantly influence the relative potency of alleged exoestrogens when compared to E2 (Table 3). For example, the level of E2 necessary for optimal growth stimulation has been reported to range from 10 nM to 10 pM while the level of induction ranges from 0.4- to 8.0-fold (41). Moreover, cell proliferation has been measured using a number of different methods including counting cells or nuclei by Coutler Counter or hemacytometer, determining the level of tritiated thymidine incorporation, and spectrophotometrically using metabolizable dyes. It has also been shown that MCF-7 cells adapt to conditions of estrogen-depleted media by initially enhancing their sensitivity to E2 (42, 43). In addition, there have been discrepancies in the identification of alleged exoestrogens using different cell proliferation assays. For example, dibutyl phthalate ester was found to promote the proliferation of estrogen responsive T47-D cells but was negative in the MCF-7 E-Screen (29, 44). Cell proliferation assays may also have limited screening applicability for both agonist and antagonist activities due to its modest responsiveness (i.e., 5- to 10-fold maximum induction) and relatively long incubation periods (see Table 4). However, it has been successfully used in

TABLE 1. Summary of In Vitro Assays Used To Assess Alleged Exoestrogens in vitro assay competitive ER binding

cell proliferation

protein expression/enzyme activity

ERE-regulated reporter genes

chimeric receptors

yeast-based assays

substance

reference

estrogens/antiestrogens phytoestrogens zearalenone organochlorines alkylphenols phthalates polyaromatic hydrocarbons progestin androgens estrogens/antiestrogens phytoestrogens zearalenone organochlorines alkylphenols phthalates progestin androgens estrogens/antiestrogens phytoestrogens zearalenone organochlorines alkylphenols progestins androgens

129, 201-205 130-132, 204, 206-210 131, 204, 208, 211, 212 22, 146, 203, 213-220 133, 201, 221-225 44 226 67, 227 228 27, 29, 55, 64, 66, 201, 229-233 27, 29, 66, 130, 207, 208, 234,235 27, 29, 130, 208 27, 29, 55, 146 27, 29, 133, 201, 221, 223, 225, 232 44 51, 236-241 48, 228 29, 54, 55, 58-61, 64, 65, 70-72, 229, 230, 242 54, 58-61, 66, 71 60 29, 55 58, 133, 201, 223, 225, 232 51, 67, 239, 243 75, 228, 244

Recombinant Assays estrogens/antiestrogens phytoestrogens zearalenone alkylphenols phthalates oral contraceptives endogenous steroids organochlorines alkylphenols phthalates estrogens/antiestrogens phytoestrogens organochlorines alkylphenols

44, 60, 111, 130-133, 245 60, 130-132 60, 131 133 58 238-240 55, 111, 112, 138, 146, 147 55, 146, 246, 247 246 246 55, 156, 161-168 167 55, 159, 160, 167 166, 167

TABLE 2. Factors Influencing MCF-7 and T47-D Human Breast Cancer Cell Proliferation factor

reference

differences between cell line clones culture conditions receptor level differences differences in serum cell density clone heterogeneity

30, 41, 42, 230, 233, 248-251 28, 35, 41, 201, 252-256 41, 257 41, 230, 251, 258 251 250, 259-263

TABLE 3. Relative Potenciesa of Alleged Exoestrogens Derived from Cell Proliferation Assays (264) compound 17β-Estradiol zearalenone coumestrol diethylstilbestrol a

Mayr et al. (60)

Welshons et al. (204)

Soto et al. (27)

1.0 0.04 0.0030

1.0 0.0085 0.0011 0.70

1.0 0.010 0.00010 10

Potency values relative to 17β-estradiol.

structure-activity relationship studies to identify the important determinants for the antiestrogenic activities of tamoxifen (45). Although the E-Screen has been extensively used to identify and assess alleged exoestrogens, there are a lack of studies that demonstrate that the assay results are predictive of in vivo responses with previously unidentified exoestrogens. Moreover, MCF-7 cells also express androgen,

progesterone, glucocorticoid, and retinoid receptors in addition to ERs (46). This may compromise the utility of the assay if some substances are also found to bind to other receptors since it has been shown that androgens, progestins, and glucocorticoids can antagonize E2-induced cell proliferation (47-51). Despite these potential drawbacks, to date there have been few reported cases of false positive results using the MCF-7 E-Screen. Furthermore, the reported detection limit of 10 pg of E2/mL (30 pM E2) makes the MCF-7 cell proliferation assay one of the most sensitive in vitro assays for assessing the estrogenicity of exoestrogens (27). Post-Confluent Cell Accumulation and Foci Formation. The estrogen-dependent capability of confluent MCF-7 human breast cancer cells to form multicellular clusters (i.e., foci) has also been proposed as an ER-mediated response that could be used to identify and assess the potency of alleged exoestrogens. Foci are nodules of cellular outgrowth that are seen as a piling up and overlapping of cells on a confluent monolayer background. This effect has been observed with MCF-7 cells maintained in media containing FBS but not in media containing calf serum or DCC-treated FBS unless it was supplemented with estrogens (52). The assay exhibits reasonable selectivity as testosterone and dexamethasone did not induce the formation of foci at concentrations up to 10 µM, while progesterone showed limited activity at 1-10 µM. The EC50 for the E2 induction of post-confluent foci formation is reported to be 50 pM, making this assay’s sensitivity equivalent to or greater than several other assays discussed in this review (see Table 4). Furthermore, the assay exhibits a maximum induction of approximately 20-fold following


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TABLE 4. Comparison of Different In Vitro Bioassays assay

cell

E2 EC50

responsiveness

assay time (days)

reference

5 nM 9 pM 50 pM

70-fold 5-10-fold 20-fold

1 7 9

201 27, 29, 233 52

20-fold 600-fold

3 6

70 58, 61

10-fold 40-50-fold

3 3

44 138

1000-fold 2500-fold

1 2

163 168

competitive ligand binding cell proliferation foci formation

MCF-7 MCF-7 MCF-7

alkaline phosphatase vitellogenin

Protein Expression Ishikawa 11 pM rainbow trout hepatocytes 1.8 nM

ERE-regulated reporter genes chimeric receptors yeast hER and LacZ reporter gene hER and URA3 reporter gene

MCF-7 MCF-7

Recombinant Assays 50 pM 420 pM

S. cerevisiae S. cerevisiae

treatment with 1 nM E2. This is an important feature of any assay if one wishes to examine alleged exoestrogens for antiestrogenic activity since antagonistic affects will be more apparent. Although this assay has been used to examine the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin as well as two established antiestrogens, tamoxifen and LY156758 (52, 53), its use for the identification evaluation of alleged exoestrogens requires further investigation. Moreover, the assay may only be sparingly used since foci formation is only observed after 9-10 days in the presence of 1 nM E2 and after 15 days in the presence of 10 pM E2. Further studies are also required to determine if results obtained from foci formation are complementary or redundant with data obtained from cell proliferation assays and to evaluate if this assay is capable of identifying compounds that elicit in vivo responses. Induction of Protein Expression/Enzyme Activities. The induction of several proteins or enzyme activities has also been used to investigate the estrogenic potency of alleged estrogenic substances. The induction of progesterone receptor levels and the increased expression of secreted proteins such as pS2, cathepsin D, prolactin, sex hormone binding globulin, and vitellogenin have previously been used as measurable end points (13, 29, 30, 54-66). Researchers have also monitored increases in alkaline phosphatase, ornithine decarboxylase, prostaglandin F2R, and prostaglandin H synthase enzyme activities (67-72). However, expression of these proteins or enzyme activities is restricted to specific cell lines; therefore, the results may not be relevant to other tissues or species. For example, assays investigating the expression of prolactin or vitellogenin use primary cultures of immature rat pituitary cells or fish hepatocyte preparations, respectively. In addition, measurement of these end points usually involves laborious methodologies such as northern blotting, western blotting, or ELISAs. Monitoring vitellogenin expression is further complicated by variations in the immunological and structural properties of the protein, thus limiting its use within and across species. However, it may be possible to develop antibodies against conserved protein regions that could be used for a number of species while maintaining acceptable sensitivity (13). It has also been demonstrated that the expression of some proteins and enzyme activities (i.e., pS2, cathepsin D, alkaline phosphatase, prolactin, vitellogenin) may be susceptible to induction or repression via mechanisms that are removed from the ER, thus increasing the possibility of false positives (73-80). Many of these protein expression/ enzyme activity assays exhibit EC50 values in the picomolar range and are highly inducible following treatment with 1 nM E2 (see Table 4). The use of a wide variety of cell line origins also enables researchers to investigate the agonist and antagonist activities of exoestrogens in different potential target tissues. This may be an important factor in assessing the effects of an alleged

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0.8 nM 3 nM

exoestrogen, especially when extrapolating between tissues and species. Tamoxifen is an example of a target tissuespecific compound since it exhibits antiestrogenic activity in breast tissue and cells but weak agonist activity in the uterus and endometrial cells (81-84). Cross species differences are seen with the abortifacient RU486, which has been found to possess antiprogestin activities in humans while exhibiting no agonist or antagonist activity in chickens or hamsters due to a lack of receptor binding. This lack of binding is the result of the same single-point mutation within the ligand binding domain of the chicken and hamster progesterone receptor (85). Currently, there is no data indicating that exoestrogens exhibit consistent tissue- and species-specific responses. Nevertheless, the examples of the tissue-specific effect of tamoxifen and the species-specific differences observed with RU486 suggest that the judicious use of a battery of assays is warranted when assessing the potential affects of alleged exoestrogens. Recombinant Receptor/Reporter Gene Assays. Over the past decade, several pharmaceutical companies have used recombinant receptor/reporter gene assays to identify receptor-specific ligands with potential therapeutic applications. McLachlan has suggested that a comparable approach could be used to identify and assess receptor-mediated toxicants and has referred to the strategy as “functional toxicology” (9). Hence, a substance would be classified based upon its potential mechanism of action in addition to its chemistry. Recombinant receptor/reporter gene assays can be broadly categorized into (a) endogenous promoter-regulated reporter genes, (b) response element-regulated reporter genes, and (c) chimeric receptor/response element-regulated reporter genes. Their evolution is illustrated in Figure 2. Although the following discussion focuses on exoestrogens, similar strategies may be extended to any nuclear receptor-mediated response. (A) Endogenous Promoter-Regulated Reporter Gene Assays. Recombinant receptor/reporter gene assays are capable of addressing some of the drawbacks associated with monitoring protein expression or enzyme activity. In addition, due to their excellent responsiveness and sensitivity, they can be used to assess the relative potency of alleged receptor-mediated agonists and antagonists. These strategies have strived to provide a more sensitive, selective, and easily performed in vitro assay than is currently available. The first step toward these goals has been achieved by constructing recombinant reporter genes consisting of endogenous promoters from estrogen-responsive genes linked to reporter genes encoding firefly luciferase, chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), or alkaline phosphatase (see Figure 2ii). These reporter genes provide sensitive, stable, and easily measurable enzyme activities, thus eliminating the need to measure the expression of the endogenous gene product using sophisticated and sometimes laborious methodologies (86-95). Several reporter genes are

FIGURE 2. Evolution of recombinant receptor/reporter gene assays. Schematic diagrams of the stages of development of a recombinant receptor/reporter gene assay starting from an estrogen-responsive or -inducible gene (e.g., vitellogenin, progesterone receptor). (i) Estrogen responsive gene assay consists of the endogenous promoter region (also referred to as the 5′ flanking regulatory region) and the structural gene encoding the protein (ERE, estrogen response element; YFRG, your favorite responsive gene). This assay would be equivalent to measuring protein expression or enzyme activity. (ii) Endogenous promoter-regulated reporter gene assay consists of the 5′ flanking region of an estrogen-responsive gene regulating the expression of a reporter gene (cDNA, complementary DNA). The lines indicate vector sequences. Reporter gene activities are sensitive and relatively easy to measure compared to measuring protein expression or enzyme activity. (iii) ERE-regulated reporter gene assay, expression of the reporter gene is regulated by tandem EREs. Enhanced selectivity since induction can only occur through the ERE. However, the presence of serum-borne estrogens results in high levels of basal activity and other receptors such as retinoid and peroxisome proliferator-activated receptors can induce an ERE-regulated reporter gene. (iv) Chimeric receptor/reporter gene consists of two components: the chimeric receptor and the heterologous response element-regulated reporter gene (DBDy, DNA binding domain of heterologous protein transcription factor; hER, human estrogen receptor; LBD, ligand binding domain; REy, response element for heterologous protein transcription factor Y). Induction of the reporter gene can only occur through the chimeric receptor since there are no mammalian transcription factors that can bind to REy and initiate expression of the reporter gene. currently available including constructs regulated by the 5′ promoter regions of the progesterone receptor, pS2, cathepsin D, vitellogenin, complement (C3), or collagenase (96-104). Similar strategies have also been successfully developed to identify and assess other environmental pollutants including metals, oxidative stress, and dioxins and related compounds (105-110). Factors Involved in Response Variability. Studies have shown that the ability of a ligand to induce an ER-mediated response is dependent on several factors. The ER contains two activation functions, AF1 and AF2, both of which can contribute to the induction of gene expression (111-113). Activation functions (AF) are interfaces between the ER and other proteins, referred to as transcription intermediate factors (TIFs), that are believed to be responsible for transducing the induction signal to the gene expression machinery (i.e.,

polymerase complex) (114, 115). AF1 is located in the amino terminal portion of the ER (i.e., AB domains) and is a constitutive activator. Hence, AF1 is capable of inducing gene expression in the absence of a ligand when it is linked to a DNA binding domain that is bound to the response element of a reporter gene. AF2, located within the carboxy terminal ligand binding domain (i.e., domains DEF) of the ER protein, is a ligand-dependent activation function. Thus, AF2 linked to a DNA binding domain bound to a response element will induce reporter gene expression if the ligand binding domain is occupied by an agonist. However, gene induction contributions by AF1 and AF2 are dependent on the cellular environment or background (i.e., availability of TIFs within a specific cell type) and the promoter context of the responsive gene (84, 101, 116-118). Therefore, induction of a gene is dependent not only on ligand binding but also on liganddependent activation of AF2, the ability of AF1 and AF2 to interact with the available TIFs, and interactions between the ER and other factors bound to the promoter. This situation is not unique to the ER as equivalent requirements and activation functions have also been reported for the androgen, glucocorticoid, progesterone, thyroid, retinoic acid, and retinoid X receptors (119-126). Katzenellenbogen et al. (127) have recently reviewed the interactions that may occur between multiple effector sites that are the basis for cell- and promoter-specific actions of receptor ligands. The availability of the estrogen-inducible human, rat, and chicken progesterone receptor promoter-regulated reporter genes also enables us to examine the ability of an alleged exoestrogen to induce a species-specific response (97, 100, 128) using relevant cell lines derived from different tissue (e.g., breast and uterine cell lines). Therefore, these reporter gene constructs may provide important information regarding the activity of an alleged exoestrogen in different species and cellular environments. (B) Response Element-Regulated Reporter Gene Assays. Although endogenous promoter-regulated reporter genes provide more easily monitored responses, they are still afflicted by many of the same drawbacks plaguing protein expression/enzyme activity assays. The promoters that are typically used consist of several hundred base pairs of the 5′ flanking regulatory region; therefore, they are still susceptible to mechanisms of induction that are not mediated by the ER (73, 74, 77-79). Several investigators have attempted to overcome this potential pitfall by using reporter genes that are regulated by the 13 base pair (GGTCAcatTGACC) vitellogenin A2 estrogen response element (ERE) (see Figure 2iii) (44, 60, 129-133). This strategy ensures that induction of the reporter gene occurs only through the ERE. However, ERE-regulated reporter genes have been found to be extremely sensitive to the presence of serum-borne estrogens resulting in high constitutive reporter gene activity and lower overall inducibility, thus compromising the responsiveness and utility of this approach. The use of DCC-stripped serum-supplemented media was found to marginally reduce the constitutive reporter gene activity when compared to the antagonism observed with the pure antiestrogen, ICI 164,384 (134). In addition, studies have shown that receptors for retinoids, thyroxine, and peroxisome proliferators can interact with EREs and modulate reporter gene activity (135-137). Consequently, ERE-regulated reporter genes exhibit poor responsiveness due to the presence of serum-borne estrogens and are susceptible to induction, synergy, or antagonism via receptors other than the ER. (C) Chimeric Receptor/Reporter Gene Assays. Alleged exoestrogens have also been examined using chimeric receptor/reporter gene paradigms (55, 138). These approaches were initially used to investigate the structure and function of the ER as well as to determine the agonist and antagonist properties of ER ligands (111-113, 139). Chimeric receptor/reporter gene approaches have also been used to


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FIGURE 3. Schematic diagrams of the components and construction of the Gal4-human estrogen receptor chimeric receptor/reporter gene assay. (i) Domains A-F of the human estrogen receptor; (ii) the ligand binding domain (LBD) of the human estrogen receptor; (iii) the DNA binding domain of the yeast transcription factor, Gal4; (iv) the chimeric Gal4-human estrogen receptor, Gal4-HEGO consisting of the Gal4 DBD (amino acids 1-148) and the LBD of the human estrogen receptor (amino acids 250-595); and (v) the Gal4 response element (17mer)-regulated reporter gene, 17m5-G-Luc, consisting of five tandem 17mers, the rabbit β-globin basal promoter and the firefly luciferase cDNA. examine the presence of functional domains within other receptors as well as to study the activity of possible novel ligands (140-144). Some may question the value of using this approach, especially when assessments can be performed by using apparently simpler and less labor-intensive technology such as protein expression or cell proliferation. However, the chimeric receptor/reporter gene strategy provides researchers with an assay with greater selectivity and responsiveness and, therefore, a more discriminating and sensitive method to assess alleged exoestrogens. In addition, the chimeric receptor/reporter gene assay has proven to be less sensitive to the presence of serum-borne estrogens in media (138). These estrogens cause a high level of background activity, which significantly reduces the responsiveness of estrogen-inducible reporter genes. Although steroid stripping of serum by DCC treatment has been reported to remove greater than 99% of exogenously added tritiated E2 (28), functional studies indicate that there is still a significant amount of estrogen present to elicit an ER-mediated response (134). The chimeric receptor/reporter gene assay used for detecting exoestrogens, also referred to as the E2 Bioassay, involves the following two central components (Figure 3). The Gal4-HEGO chimeric receptor consists of the ligand binding domain of the ER (i.e., domains D, E and F; amino acids 250-595) linked to the DNA binding domain of the yeast transcription factor, Gal4 (i.e., amino acids 1-148). The Gal4-HEGO cDNA has been inserted into a mammalian expression vector that constitutively expresses the chimeric receptor following transfection into recipient cells (i.e., MCF7). The second component is the Gal4-regulated luciferase

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reporter gene (i.e., 17m5-G-Luc) that consists of the firefly luciferase cDNA regulated by the rabbit β-globin basal promoter and five tandem consensus Gal4 response elements (i.e., 17mer ) CGGAGGACTGTCCTCCG). The use of several tandem response elements has been previously found to improve reporter gene inducibility (145). Expression of the 17m5-G-Luc reporter gene is dependent upon the liganddependent activation of Gal4-HEGO. Both constructs (i.e., the Gal4-HEGO chimeric receptor and the 17m5-G-Luc reporter gene) have been transiently transfected into MCF-7 cells and stably transfected into HeLa cells. These models have been used to investigate the ER-mediated activities of two triazine herbicides, atrazine and simazine, and hydroxylated polychlorinated biphenyl congeners identified in human serum (55, 146). In addition, the ER-mediated activities of complex mixtures such as urban air particulate matter and samples obtained from a Kraft pulp and paper mill process have also been studied using these assays (138, 147, 148). The E2 Bioassay exploits the receptor-mediated mechanism of action of estrogenic substances. Following treatment of transfected cells, the estrogenic substance binds to the ER ligand binding domain of the chimeric receptor and transforms the Gal4-HEGO construct into an activated, high-affinity DNA binding homodimer receptor complex. The Gal4 DNA binding domain then directs the activated chimeric receptor complex to the 17m5-G-Luc reporter gene where it binds to the Gal4 response elements (17mers). Binding of the activated complex to the 17mer response elements initiates expression of the firefly luciferase cDNA which, in turn, results in the induction of luciferase activity. Consequently, determination of luciferase activity in the E2 Bioassay is a measure of the estrogenic activity of a substance. It is important to note that the ER ligand binding domain within the Gal4-HEGO chimeric receptor does not exhibit different binding affinities and/or ligand specificities when compared to the native ER based on observations that receptors are comprised of domains that can function independently from one another (8, 149). In transiently transfected MCF-7 human breast cancer cells, 1 nM E2 has been reported to induce luciferase activity 40-50-fold with an EC50 of 20 pM (138). Furthermore, since no mammalian proteins are known to bind and initiate gene expression via a 17mer response element, increases in 17m5-G-Luc reporter gene activity can occur only via Gal4-HEGO, thus ensuring that induction is mediated exclusively by the chimeric receptor. This strategy also makes it possible to reconstitute other species-specific assays. For example, the assay could be reconstituted in fish or avian cell lines using a chimeric receptor that contains the ligand binding domain of the fish or avian ER cDNAs. The development of additional speciesspecific assays may provide valuable information regarding potential differences in species sensitivities to estrogenic substances. Inherent Disadvantages. However, there are disadvantages associated with chimeric receptor/reporter gene bioassays. These assays require specialize equipment such as a luminometer and some level of technical expertise, although the latter is not beyond any competent laboratory. It has also been the experience of this laboratory that maximum induction of the bioassay can range from 20- to 80-fold, but usually falls between 40- and 50-fold. This variation in responsiveness may be due to differences in the quality of the DNA used in the transient transfection and changes in cell culture conditions and/or may be dependent on the cell passage number. Responsiveness has been found to be sensitive to the number of cell passages and the type of cells used. MCF-7 cells recently taken from frozen stocks (i.e., within the first three passages) and those beyond 10 passages after removal from frozen stocks exhibit lower overall E2induced 17m5-G-Luc activity. Therefore, the assay is most consistent when the cells used are between 3 and 10 passages.

We have found the EC50 for E2 to be 0.42 ( 0.21 nM in transiently transfected MCF-7 cells. Differences in responsiveness have also been observed when the Gal4-HEGO and 17m5-G-Luc constructs are transfected into different cell lines, further demonstrating that the cellular environment (i.e., the presence of appropriate TIFs) is an important factor in the performance of the assay. For example, maximum induction in stably transfected HeLa cells ranges from 8- to 12-fold while maximum induction in transiently transfected MCF-7 cells averages between 40- and 50-fold. Therefore, although it is possible to use this strategy to develop species- and tissuespecific systems, it is likely that there will be differences in assay performance. Differences in the level of responsiveness within experiments and between cell types may also be due to variations in ER levels. This may be the result of different transfection efficiencies and/or a reflection of the stability of the ER protein in different cells. Studies using stably transfected cells have shown that the level of responsiveness is dependent on receptor expression levels (150). There also appears to be an optimal receptor level window, below which there are insufficient receptors to attain a maximal response and above which receptors titrate or “squelch” limited protein factors that are required for maximum gene induction (117, 151). Yeast-Based Assays. The yeast strain Saccharomyces cerevisiae has been extensively used to investigate receptor structure and function as well as the activity of selected ligands (111, 139, 152-158). Yeast has several advantages over other systems including the lack of known endogenous receptors, media that is devoid of steroids, and a genetic disposition that facilitates the insertion of mammalian proteins and reporter genes. Presently, there are two variations of yeastbased estrogenic assays that have been used to identify and assess the potency of alleged exoestrogens. The more commonly employed yeast-based estrogenic assays use S. cerevisiae strains transformed with the human ER cDNA and an ERE-regulated LacZ reporter gene that encodes for the β-galactosidase enzyme (156,159-165). Although the E2 EC50 values reported in these yeast-based assays are higher than those observed in mammalian assays, they exhibit a lower limit of detection (i.e., 0.07 pM) due to the exquisite responsiveness of the system (see Table 4). This assay has also been bioengineered to allow for the secretion of the β-galactosidase enzyme into the media, thus facilitating its potential for both qualitative and quantitative assessments (166). These assays have recently been used to investigate the ER-mediated activities of a series of organochlorines and nonionic surfactants (166, 167). Another yeast-based system uses the URA3 selectable marker as the reporter gene in an ER-mediated phenotypic transactivation assay (168). The URA3 gene encodes for orotidine-5′-phosphate decarboxylase (OMPdecase), an enzyme involved in uracil synthesis. Yeast that are deficient in this enzyme activity fail to exhibit viability on minimal media unless the media has been supplemented with uracil. The recombinant S. cerevisiae strain, PL3, expresses the human ER and contains the URA3 reporter gene that is regulated by three tandem EREs. Therefore, expression of the URA3 gene and subsequent growth of PL3 on selective media lacking uracil is dependent on ER-mediated induction of OMPdecase activity. This assay can be used as a qualitative screen by monitoring growth and can be used quantitatively for assessing the potency of an alleged exoestrogen by measuring OMPdecase activity. Studies examining the efficacy of E2 indicate that this assay exhibits an EC50 of 3 nM and greater than 2500-fold induction in the presence of 10-1000 nM E2 (168). The ER-mediated activities of atrazine and simazine have also been investigated using this assay (55). Potential Problems with Yeast-Based Assays. Despite the potential of yeast-based assays, these systems possess some rather unique characteristics that must be considered

as part of the evaluation of an alleged exoestrogens. For example, tamoxifen, ICI 164,384, and ICI 182,720 exhibit significant antiestrogenic properties in both in vitro and in vivo mammalian models, yet are ineffective in blocking ERmediated reporter gene expression and have been shown to exhibit partial agonist activity in yeast (161, 165, 169, 170). Moreover, diethylstilbestrol (DES), which has been reported to have a 10-fold greater estrogenic potency in mammalian cells when compared to E2, was found to be 10-fold less potent then E2 in yeast (163). Other receptor-mediated yeast-based assays have also reported different ligand potencies when compared to mammalian counterparts (153, 171, 172). Factors such as cell wall permeability, variations in S. cerevisiae strains, differences in receptor levels and protein proteolysis mechanisms, non-receptor cell-specific factors, metabolic capabilities, multidrug resistance efflux pumps, and endogenous yeast binding proteins have been suggested as possible explanations for these differences (153, 165, 169, 171, 173175). Consequently, assessments conducted using yeast require confirmation in other in vitro and in vivo assays.

Limitations, Validation, and Utility of In Vitro Bioassays The physiological effects of estrogens are numerous. Therefore, exoestrogens could impact a variety of potential targets including the hypothalamic-pituitary-gonadal axis as well as E2 synthesis, catabolism, secretion, transport, and signal transduction. As a result of the multitude of responses that may be elicited by estrogens and the complex interplay between various mechanisms and end points, it is unrealistic to assume that one in vitro bioassays will be capable of predicting all of the potential in vivo responses elicited by an exoestrogen. For example, in vitro bioassays possess minimal metabolic capabilities relative to in vivo models that may lead to false negatives. Yet, the bioactivation and inactivation of some substances by hydroxylation, methylation, sulfonation, and aromatization have been observed in some in vitro models (29, 61, 64, 67, 70, 176-184). Moreover, some of these activities may be susceptible to induction as well as inhibition (185-192). In order to minimize potential false negatives due to a lack of metabolic activation of proestrogens, it may be prudent to also test metabolites of the alleged estrogenic substance. In vitro bioassays also do not account for bioaccumulation or interactions involving the induction of binding proteins such as sex hormone binding globulins that may modulate the uptake and metabolism of sex steroids (54, 193-196) and can not take into consideration the potential effects of exoestrogens on gene imprinting that may predispose an individual to disease at a later stage in life. Nevertheless, it will be necessary for each in vitro assay to undergo a systematic examination in order to determine its ability to identify and accurately assess substances that elicit in vivo responses. These studies should also establish specific guidelines such as what constitutes a positive or a negative response and how this information or should this information be used in the risk assessment process. For example, should any response that is statistically greater than the control be considered a positive, regardless of the magnitude of the response or the dose/concentration required to elicit the response? The bioassays described in this review specifically screen for exoestrogens that elicit ER-mediated responses. However, some estrogenic effects, such as changes in E2 metabolism and the antiestrogenic activities of TCDD and related compounds (197, 198), are removed from the ER and, therefore, would not be detected in the aforementioned in vitro bioassays. Although these assays may not detect all exoestrogens, their use is warranted given the potential of an ER-mediated pathway to elicit a broad range of responses. In vitro bioassays also provide information regarding the potential mechanism of action of an alleged exoestrogen as


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well as prioritizing candidates that require further investigation. In addition, these assays can be used to assist in the identification of an estrogenic metabolite. It is also feasible that in vitro bioassays could be used to identify an exoestrogen within a complex mixture or process stream and to assess the efficacy of remediation efforts when detailed chemical analyses are not required. Consequently, in vitro bioassays are complementary and not substitutes for in vivo tests. Many stakeholders including regulatory agencies, research scientists, trade organizations, and awareness groups have challenged the utility and validity of in vitro assays when assessing the potential hazards of a substance or complex mixture. Although the utility of in vitro assays is readily apparent, valid concerns have been expressed regarding their use in the risk assessment process. Unfortunately, few feasible alternatives have been proposed that are capable of handling the number of substances that require investigation. In most cases, there has been a lack of consensus regarding the predictive value of in vitro bioassay data and how this information should be used, if at all, in risk assessment. This has lead to an over-interpretation of the utility of these approaches that has plunged in vitro assays into disrepute among some stakeholders. However, as the list of alleged exoestrogens continues to grow and in order to comply with legislative amendments requiring the testing of estrogenicity, means of prioritizing substances through the use of in vitro assays appears to be the inevitable solution. Therefore, in order to avoid the same problems associated with in vitro equivalency factor strategies [i.e., toxic equivalency factors (TEFs)/toxic equivalents (TEQs)] or carcinogenicity tests (i.e., Ames Assay) (199, 200), it is essential that a systematic study be undertaken to evaluate the utility and validity of in vitro assays and to identify those tests that reliably contribute to the risk assessment process. Finally, it is worth repeating that in vitro assays are suggested as the initial step in an overall assessment process that must include verification of an adverse effect in vivo prior to implicating a substance as a potential exoestrogen in humans and wildlife.

Acknowledgments T.R.Z. is the recipient of a Research Career Award in Medicine from the Pharmaceutical Manufacturers of Canada-Health Research Foundation and the Medical Research Council of Canada. The author would also like to acknowledge the following individuals for their critical reading of the manuscript: J. Heinze, G. Van Der Kraak, D. Desaulniers, B. Gillesby, M. Meek, J. Clemons, and M. Fielden. Research in the author’s laboratory has been funded by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada and the Canadian Breast Cancer Foundation.

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Received for review June 18, 1996. Revised manuscript received October 3, 1996. Accepted October 3, 1996.X ES960530O X

Abstract published in Advance ACS Abstracts, December 15, 1996.


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