Introduction

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Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] is a chlorinated hydrocarbon pesticide structurally similar to dichlorodiphenyltrichloroethane [DDT; 1,1,1-trichloro-2,2-bis(chlorophenyl)ethane]. Like o,p'-DDT, methoxychlor is estrogenic in vivo (Bulger et al., 1978; Gray et al., 1989; Alm et al., 1996; Chapin et al., 1997; Cummings, 1997; Hall et al., 1997). However, methoxychlor has low affinity for the estrogen receptor (ER) and the in vivo estrogenic activity is caused by metabolism to phenolic estrogenic metabolites. The primary estrogenic metabolite of methoxychlor is 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). HPTE competes with estradiol for binding to ER, induces ornithine decarboxylase and uterotrophic activity in ovariectomized rats, and is approximately 100-fold more active than methoxychlor (Bulger et al., 1978; Ousterhout et al., 1981; Shelby et al., 1996).

Estrogenic responses are mediated through two separate estrogen receptors, ER and ER. These two receptors have homologous DNA and ligand binding regions (Kuiper and Gustafsson, 1997; Tremblay et al., 1997; Ogawa et al., 1998), and most compounds have similar binding affinities and transcriptional activities with ER and ER (Kuiper et al., 1996, 1998; Mosselman et al., 1996; Tremblay et al., 1997).

We previously demonstrated that HPTE is an ER agonist and an ER antagonist in HepG2 human hepatoma cells transfected with estrogen-responsive reporter constructs (Gaido et al., 1999). This unique activity of HPTE makes it an ideal compound with which to evaluate the in vitro and in vivo differences in ER-subtype dependent responses. We have also shown that HPTE is an androgen receptor (AR) antagonist in vitro (Maness et al., 1998). The differential activity of HPTE with ER, ER, and AR may explain why some of the responses induced by methoxychlor in vivo differ from those induced by estradiol. For example, the ability of methoxychlor to act as an ER antagonist in the ovary (Hall et al., 1997) may be caused by the high level of ER expression relative to ER in this tissue (Saunders et al., 1997).

The physiological consequences of a chemical that is an ER agonist, an ER antagonist, and an AR antagonist are unknown, and HPTE can serve as a model for investigating the effects of an agent that modulates multiple endocrine pathways. Additional studies with HPTE and structural analogs may lead to further insights on ligand specificity for ER, ER, and AR. Therefore we compared the ER, ER, and AR activity of HPTE and structural analogs and show that some chemicals similar in structure to HPTE also demonstrate unique ER, ER, and AR activity.

	Materials and Methods

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Chemicals. HPTE was synthesized by dissolving 1 g of methoxychlor (Aldrich Chemical Co., Milwaukee, WI) in 100 ml of methylene chloride and then treating with excess boron tribromide in methylene chloride (Aldrich) for 24 h. Water (5 ml) was carefully added, and crude HPTE was isolated in methylene chloride. The residue (0.8 g) was purified by preparative thin-layer chromatography (TLC). The resulting HPTE was >97% pure as determined by gas-liquid chromatography.

Plating and Transfection. Transfection experiments were performed as described previously (Maness et al., 1998; Gaido et al., 1999). HepG2 human hepatoma cells (ATCC, Rockville, MD) were plated in triplicate in 24-well plates (Falcon Plastics, Oxnard, CA) at a density of 10⁵ cells/well in complete medium consisting of phenol red-free Eagle's minimal essential medium (GIBCO/BRL, Grand Island, NY) supplemented with 10% resin-stripped fetal bovine serum (Hyclone, Logan, UT), 2% L-glutamine, and 0.1% sodium pyruvate. Cells were incubated overnight at 37°C in a humidified atmosphere of 5% CO₂/air and then transfected after the Superfect procedure (Qiagen, Valencia, CA) with three plasmids. For detection of ER activity, cells were transfected with human ER expression plasmid, plus an estrogen-responsive complement 3-luciferase (C3-Luc) reporter plasmid, and a constitutively active cytomegalovirus (CMV)--galactosidase reporter plasmid (transfection and toxicity control) (Tzukerman et al., 1994; Gaido et al., 1999). For detection of ER activity, cells were transfected with a human ER expression plasmid, a C3-Luc reporter plasmid, and CMV--galactosidase reporter plasmid (Gaido et al., 1999; Hall and McDonnell, 1999). For detection of AR activity, cells were transfected with a human AR expression plasmid, plus an androgen-responsive MMTV-Luc reporter plasmid, and CMV--galactosidase reporter plasmid (Maness et al., 1998). Transfected cells were rinsed with PBS and dosed with various concentrations of test chemical and dimethyl sulfoxide (vehicle control; Sigma) in complete medium. After a 24 h incubation, cells were rinsed with PBS and lysed with 65 µl of lysing buffer (25 mM Tris-phosphate, pH 7.8, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 0.5% Triton X-100, 2 mM dithiothreitol). Lysate was divided into two 96-well plates for luciferase and -galactosidase determination.

Statistical Analysis. Unless otherwise noted, values presented in this study represent the means ± S.E. resulting from at least three separate experiments with triplicate wells for each treatment dose. Dose-response data were analyzed using the sigmoidal dose-response function of the graphical and statistical program Prism (GraphPad, San Diego, CA).

	Results

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We compared the activity of HPTE and structural analogs in HepG2 cells transfected with expression vectors for human ER, ER, and AR along with the appropriate reporter plasmid (Fig. 1). HepG2 cells were dosed with set concentrations of chemical alone and in combination with an inducing dose of either 17-estradiol (E2) or dihydrotestosterone (DHT) for determining antagonistic activity with ER/ and AR, respectively. HPTE (Fig. 1C) exhibited ER agonist, and ER and AR antagonist activity as described previously (Maness et al., 1998; Gaido et al., 1999). HPTE does display some partial ER agonist activity of approximately 13% of that obtained with a maximally inducing dose of estradiol (Maness et al., 1998). The monohydroxy metabolite of methoxychlor, as well as the mono- and dihydroxy analogs of p,p'-DDE (Fig. 1, B, H, and I), also had ER agonist and ER and AR antagonist activity. Bisphenol A exhibited ER and ER agonist activity but did not have antiandrogenic activity (Fig. 1K). Replacement of the trichloromethyl of HPTE or dichloromethylene group of dihydroxy-DDE results in a conversion from ER antagonist activity to full ER agonist activity but retains ER agonist and AR antagonist activity (Fig. 1, C and I versus L). Trimethoxy and trihydroxy ring substituted compounds (Fig. 1, D and E) exhibited minimal ER agonist activity and did not affect ER or AR-dependent responses.

View larger version (69K): [in this window] [in a new window]   Fig. 1.   ER, ER, and AR activity of methoxychlor analogs. HepG2 cells were transiently transfected with expression plasmids for human ER, ER, and AR plus C3-Luc and a constitutively active -galactosidase expression plasmid (transfection and toxicity control). Cells were treated with increasing concentrations of methoxychlor analogs alone for detecting agonist activity (hatched bars) and with an inducing dose of either estradiol or DHT for detecting antagonist activity (shaded bars). Luciferase activity was normalized to -galactosidase activity. Values represent the means ± S.E. of three separate experiments and are presented as percentage response, with 100% activity defined as the activity achieved with 107 M estradiol for ER and ER, and 107 M DHT for AR. The abscissa represents log molar concentration. Agonists cause an increase in percentage response (rising hatched bars) with increasing concentration, whereas antagonists cause a decrease in percentage response (declining shaded bars) with increasing concentration.

Concentration-response curves for selected ER and ER agonists are presented in Fig. 2, A and B. EC₅₀ values for ER and ER agonist activity are presented in Table 1. HPTE and dihydroxy-DDE were most potent as ER agonists and were approximately 17- and 25-fold less potent, respectively, than estradiol. HPTE and dihydroxy-DDE were followed in ER agonist potency by monohydroxy methoxychlor, bisphenol A, monohydroxy-DDE, bishydroxyphenylmethane, and bishydroxyphenylethane. Bishydroxyphenylmethane and bishydroxyphenylethane were equally potent as ER agonists and were approximately 285-fold less potent than estradiol.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Dose response of selected methoxychlor analogs with ER (A) and ER (B). Experiments were performed as described in Fig. 1. Luciferase activity was normalized to -galactosidase activity. Values represent the means ± S.E. of three separate experiments. , E2; , monohydroxy-methoxychlor; , monohydroxy-DDE; , dihydroxy-DDE; , bishydroxyphenyl methane; , bishydroxyphenyl ethane.

View this table: [in this window] [in a new window]   TABLE 1 A comparison of the agonist and antagonist potencies of methoxychlor analogs with ER, ER, and AR

We characterized the ER antagonist activity of selected compounds in HepG2 cells by determining the effect of various concentrations across a complete estradiol dose-response range (Fig. 3, A-C). Each of the tested compounds caused parallel shifts in the estradiol dose-response curve, indicating competitive antagonism. Schild regression analyses were performed and equilibrium dissociation (K_B) values determined (Table 1). HPTE, monohydroxy methoxychlor, monohydroxy-DDE, and dihydroxy-DDE demonstrated relatively similar antagonist potencies.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of various concentrations of methoxychlor analogs on an estradiol dose-response curve with ER. Experiments were performed as described in Fig. 1 with 1010 to 105 M E2 either alone or in combination with the indicated concentrations of monohydroxy-methoxychlor (A), monohydroxy-DDE (B), and dihydroxy-DDE (C). Luciferase activity was normalized to -galactosidase activity. Values represent the means of three separate experiments.

Similar experiments were performed to characterize AR antagonist activity (Fig. 4, Table 1). HPTE, dihydroxy-DDE, and p,p'-DDE demonstrated similar AR antagonist potencies. Monohydroxy-DDE, bishydroxyphenylmethane, and bishydroxyphenylethane were approximately 3- to 5-fold less potent than HPTE, dihydroxy-DDE, and p,p'-DDE.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of various concentrations of methoxychlor analogs on a DHT dose-response curve with AR. Experiments were performed as described in Fig. 1 with 109 to 105 M DHT either alone or in combination with the indicated concentrations of HPTE (A), bishydroxyphenylmethane (B), bishydroxyphenylethane (C), monohydroxy-DDE (D), and dihydroxy-DDE (E). Luciferase activity was normalized to -galactosidase activity. Values represent the means of three separate experiments.

	Discussion

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ER and ER bind structurally diverse classes of chemicals, and it is difficult to compare structure-dependent receptor binding or transactivation between chemical classes. In contrast, structure-activity correlations within a single structural class such as the triphenylethanes can be used to design ER agonists and antagonists (e.g., tamoxifen) for clinical applications as selective estrogen receptor modulators. For a series of mono-, di-, and trihydroxyphenyl ethane/ethylene analogs structurally related to methoxychlor, the order of potency as ER agonists was dihydroxyphenyl > monohydroxyphenyl > trihydroxyphenyl, suggesting that the optimal structure for ER agonist activity contained two p-hydroxyphenyl groups substituted at a single ethane or ethylene carbon atom (i.e., bis-substitution). In contrast, the fully methylated metabolites (e.g., Fig. 1, A versus C; Fig. 1, G versus D) were significantly less active as ER agonists; this is consistent with previous studies with methoxychlor and HPTE (Bulger et al., 1978; Ousterhout et al., 1981; Shelby et al., 1996). A similar pattern was observed for the substituted diphenylmethane analogs even though only a limited number of these compounds were tested (Fig. 1, J and N versus M).

With few exceptions (Fig. 1, K and L), the bishydroxy/methoxyphenyl ethane or ethylene analogs were not significant ER agonists, and only two bishydroxyphenylmethanes (Fig. 1, J and N) induced measurable ER-dependent reporter gene activity. Thus, our results demonstrate that this series of bishydroxyphenyl-substituted ethanes, ethylenes, and methanes are preferential ER agonists and exhibit weak to nondetectable ER agonist activity. These results are unique because previous studies for ER subtype-dependent ligand binding and transactivation report similar ER and ER activity for various structural classes of estrogenic compounds (Kuiper et al., 1996, 1998; Mosselman et al., 1996; Tremblay et al., 1997).

The results of our studies also demonstrate that methoxychlor and structurally related analogs exhibit minimal ER antagonist activity but that three compounds (Fig. 1, B, H, and I) in addition to HPTE (Fig. 1C) are highly effective ER antagonists. Structural features required for this response include bis(4-hydroxyphenyl) or bis(4-hydroxyphenyl),(4-methoxyphenyl) groups attached to chlorine-substituted ethane/ethylene moieties. Additional compounds are required to more accurately define structural requirements for ER antagonist activities; however, our results clearly demonstrate remarkable structure-dependent differences among these compounds for activity as ER antagonists.

Several compounds investigated in this study exhibited antiandrogenic activity. Four analogs that were ER antagonists (Fig. 1, B, C, H, and I) were among the most active antiandrogens and exhibited activity similar to that observed for p,p'-DDE. Interestingly, both p,p'-DDE (Fig. 1F) and dihydroxy-DDE (Fig. 1I) exhibited similar antiandrogenic activities, and the interchange of two p-chloro and two p-hydroxyl substituents had minimal effects on this AR response. In contrast, the two p-hydroxyl groups (but not p-chloro substituents) conferred both ER agonist and ER antagonist activity on dihydroxy-DDE, demonstrating that subtle substituent changes can affect some but not all ligand-activated hormone receptor action.

Methoxy-DDE and monohydroxy-DDE are impurities in technical grade methoxychlor (Bulger et al., 1985), and dihydroxy-DDE is formed during the metabolism of methoxychlor in mice (Kapoor et al., 1970). HPTE, monohydroxy-methoxychlor, monohydroxy-DDE, and dihydroxy-DDE have previously been shown to compete with estradiol for ER binding in vitro and demonstrate uterotropic activity in vivo (Bulger et al., 1978, 1985; Ousterhout et al., 1981). Thus, exposure to methoxychlor results in a complex interaction of multiple metabolites with different activities at ER, ER, and AR.

The molecular mechanism by which a ligand can act as an ER agonist and an ER antagonist is of both toxicological and pharmacological interest. The overall structure of the ER ligand-binding domain is very similar to that of ER (Pike et al., 1999), and most compounds demonstrate similar binding affinities and transcriptional activities with ER and ER (Kuiper et al., 1996, 1997; Mosselman et al., 1996; Tremblay et al., 1997). The helix 12 region present on both receptors plays an important role in the mechanism of ER action (Darimont et al., 1998). This region folds over the ligand binding pocket and exposes a region on both receptors involved in coactivator binding. ER and ER antagonists such as raloxifene and hydroxytamoxifen contain bulky constituents that reposition helix 12 and block receptor interaction with coactivators (Brzozowski et al., 1997; Pike et al., 1999). HPTE analogs used in this study do not have substituents of the size and character of raloxifene and consequently less likely to physically reposition helix 12. However, X-ray crystallography and sequence analysis comparison of the ligand-binding domains of ER and ER suggest that the agonist orientation of helix 12 in ER may be unstable and thus easier to antagonize than ER (Pike et al., 1999). The ER agonist/ER antagonists identified in this study may be able to stabilize helix 12 in the agonist orientation for ER but not for ER. X-ray crystallographic studies are needed to confirm this hypothesis.

The R,R-enantiomer of tetrahydrochrysene (R,R-THC) has also recently been shown to have differential ER and ER activity (Meyers et al., 1999; Sun et al., 1999). Like HPTE, R,R-THC behaves as an ER agonist and an ER antagonist. In contrast, the S,S-enantiomer (S,S-THC) is an agonist with both ER and ER. The equilibrium dissociation value (K_B) for R,R-THC has not been determined, and whether R,R-THC is an ER competitive antagonist remains to be demonstrated. THC compounds differ considerably in structure from the methoxychlor analogs presented in this study, and this class of compounds will provide additional information regarding the ligand specificity of ER and ER binding and transactivation.

Less is known about the mechanism of AR antagonism by AR ligands. AR antagonists are generally thought to prevent or reduce binding of AR to DNA (Kelce et al., 1995, 1998). However, the specific mechanisms responsible for this inhibition of AR-DNA binding remain unknown.

Much still remains to be determined about the precise roles of ER, ER, and AR in reproductive development and endocrine function, especially in humans; the physiological consequences of exposure to chemicals that are ER agonists, ER antagonists, and AR antagonists are unknown. HPTE and its structural analogs give us further insights into the ligand specificity of ER, ER, and AR and serve as model chemicals for investigating ER, ER, and AR steroid hormone receptor interactions.