Bisphenol A-Induced Increase in Uterine Weight and Alterations in ...

56, 332–339 (2000) Copyright © 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Bisphenol A-Induced Increase in Uterine Weight and Alterations in Uterine Morphology in Ovariectomized B6C3F1 Mice: Role of the Estrogen Receptor Andriana D. Papaconstantinou,* Thomas H. Umbreit,‡ Benjamin R. Fisher,‡ ,1 Peter L. Goering,‡ Nicholas T. Lappas,† and Ken M. Brown* ,2 Departments of *Biological Sciences and †Forensic Sciences, George Washington University, Washington, DC 20052; and ‡Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland, 20857 Received March 15, 2000; accepted May 8, 2000

The ability of the environmental xenoestrogen bisphenol A (BPA) to increase uterine wet weight in the rodent remains controversial, and few studies have previously examined the effects of BPA on uterine morphology. Furthermore, it is not known whether BPA-induced uterotrophic effects are, similarly to ␤-estradiol (E 2), mediated through the estrogen receptor (ER). In this study, we compared the effects of BPA on uterine wet weight and morphology to those of E 2 in the B6C3F1 ovariectomized mouse. To examine whether these effects were mediated through the ER, the antiestrogen ICI 182,780 (ICI) was co-administered with BPA or E 2. We report that subcutaneous administration of BPA at doses between 0.8 and 8 mg/day over 4 days significantly increased mean uterine wet weights above those of vehicle (corn oil)-treated mice. The uterine weight data suggest that BPA acts as a partial agonist with an EC 50 of 0.72 mg/day compared to 19.4 ng/day for E 2. BPA (2 mg/day) and E 2 (40 ng/day) induced a significant increase in luminal epithelial height and in the thickness of both the stromal and myometrial layers of the uterus. The effects of 40 ng E 2/day on all endpoints studied were reversed by 20 ␮g ICI/ day. ICI at 200, but not 20 ␮g/day, was able to reverse the BPA (2 mg/day)-induced increase in both uterine wet weight and luminal epithelial height. ICI alone at 200 ␮g/day stimulated an increase in thickness of both the stroma and myometrium and did not reverse the effects of BPA (2 mg/day) on these layers. These results suggest that the BPA-induced increase in uterine wet weight and in luminal epithelial height in the ovariectomized B6C3F1 mouse are mediated by the ER. Key Words: bisphenol A (BPA); ICI 182,780 (ICI); ␤-estradiol (E 2); estrogen receptor (ER); uterotrophic effects; uterine weight; myometrium; stroma; luminal epithelium; B6C3F1 mouse.

Bisphenol A (BPA) is widely used in the manufacture of polycarbonate plastics, epoxy resins, dental sealants, and as a stabilizing agent in plastics such as polyvinyl chloride 1

Present address: Covance Laboratories, Vienna, VA. To whom correspondence should be addressed at 332 Lisner Hall, Department of Biological Sciences, George Washington University, 2023 G. St. NW, Washington, DC 20052. Fax: (202) 994-6100. E-mail: [email protected]. 2

(Staples et al., 1998). As a result, BPA is present in many household plastic products and in food- and drink-packaging materials. Possible exposure of humans to BPA due to its leaching from polycarbonate cardiotomy reservoirs (Larson et al., 1977), laboratory flasks (Krishnan et al., 1993), baby-feeding bottles (Mountfort et al., 1997), epoxy resins used for lining food cans (Brotons et al., 1995), several types of dental sealants (Olea et al., 1996), and plastic waste samples (Yamamoto and Yasuhara, 1999) have been reported. Concern about the estrogenic properties of xenoestrogens, including BPA, has escalated in recent years (Kavlock, 1999; Sonnenschein and Soto, 1998). In rodents, effects of exogenous estrogens on the uterus include hypertrophy, predominantly of the luminal but also of the glandular epithelium (Branham et al., 1993; Cooke et al., 1997; Katsuda et al., 1999), and proliferation of the myometrium (Hunter et al., 1999; Yokoyama et al., 1998) and stroma (Cook et al., 1997). In addition, estrogens cause uterine fluid imbibition (Astwood, 1938) and secretory protein production (Buchanan et al., 1999). Several studies have concluded that these effects are mediated through the estrogen receptor ␣ (ER␣) (Buchanan et al., 1999; Cooke et al., 1997; Katsuda et al., 1999; Ogawa et al., 1999; Orimo et al., 1999). The uterotrophic actions of estrogenic substances result in an increase in uterine wet weight, which is an endpoint utilized in the standard uterotrophic assay (Reel et al., 1996). The estrogenicity of BPA has been demonstrated in a number of in vitro and in vivo assays. In vitro assay endpoints include the proliferation of MCF-7 human breast cancer cells (Krishnan et al., 1993; Olea et al., 1996); an increase in levels of the progesterone receptor in human endometrial carcinoma (Bergeron et al., 1999) and MCF-7 (Olea et al., 1996) cells, which can be reversed by simultaneous treatment with the antiestrogen ICI 182,780 (Bergeron et al., 1999) or by tamoxifen (Krishnan et al., 1993), respectively; binding to the estrogen receptor (Dodge et al., 1996; Nagel et al., 1997); and the activation of

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BPA EFFECTS ON UTERINE WEIGHT AND MORPHOLOGY

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estrogen response element (ERE)-driven reporter gene constructs (Gaido et al., 1997). In vivo effects of BPA, which have been shown to mimic those of ␤-estradiol (E 2 ) in rodents, include the induction of vaginal cornification (Dodds and Lawson, 1938; Steinmetz et al., 1998); growth and differentiation of the mammary gland (Colerangle and Roy, 1997); decrease in serum cholesterol levels (Dodge et al., 1996); and increases in prolactin levels (Steinmetz et al., 1997), uterine vascular permeability (Milligan et al., 1998), and c-fos mRNA levels in the uterus and vagina (Steinmetz et al., 1998). Most in vivo studies of the estrogenicity of BPA have examined its potential uterotrophic effects in immature or ovariectomized rodents. Cook et al. (1997) observed a 67% increase in uterine weights of ovariectomized rats (strain not reported) above control values, following the ip administration of 500 mg BPA/kg/day. Gray and Ostby (1998) reported a 4-fold increase in uterine weights of ovariectomized LongEvans rats compared to controls following the sc administration of 200 mg BPA/kg/day, and Dodge et al. (1996) observed a 37% increase above controls with an oral administration of 30 mg/kg/day to ovariectomized Sprague-Dawley rats. Furthermore, Ashby and Tinwell (1998) demonstrated the ability of 400 mg BPA/kg administered po or sc to immature Alpk:AP rats to increase uterine weights. Steinmetz et al. (1998) demonstrated a significant increase in the proliferation of epithelial cells of F344 rats, following a single ip injection of 37.5 mg BPA/kg, while a much lower dose of approximately 0.3 mg/ kg/day administered by continuous release capsules induced hypertrophy of the uterine luminal epithelium and a uterine mucus secretion response in F344 but not in Sprague-Dawley rats. Gould et al. (1998) reported that up to 150 mg BPA/kg administered orally to immature Sprague-Dawley rats did not increase uterine weights, and Coldham et al. (1997) did not observe an increase in uterine weight following the sc administration of BPA to CFLP mice at the subtoxic doses of 0.05– 0.5 mg/day. The ability of BPA to induce the uterotrophic response in the above studies varied among species and strains and routes of exposure, as well as between immature and ovariectomized animals. Very few studies have examined whether BPA has an effect on uterotrophic endpoints other than wet weight gain or whether the observed uterotrophic effects of BPA were mediated, similarly to E 2, through the ER. Therefore, in this study we compared the effects of BPA and E 2 on both wet weights and morphology of the uterus of ovariectomized B6C3F1 mice. In order to assess the role of ER, the ability of the antiestrogen ICI to block uterotrophic effects of BPA and E 2 was examined.

B6C3F1 mice were supplied from Charles River Laboratories, Inc. (Wilmington, MA). Upon arrival, mice were randomly distributed into treatment groups and housed in groups of 4 to 6 in polypropylene cages with stainless steel wire lids and heat-treated chips as bedding (Cellu-Dri; Shepherd Specialty Papers, Kalamazoo, MI), with access to food (Lab Rodent Diet 5001; PMI Nutrition International, Inc., St. Louis, MO) and water ad libitum. The animal rooms were maintained on a 12 h light/dark cycle (6 A.M. to 6 P.M.) at 23 ⫾ 1°C with 30 –50% relative humidity. After an acclimation period of 1–2 weeks, animals were dosed sc with solutions of bisphenol A (Sigma Chemical, Co., St. Louis, MO), ␤-estradiol (Sigma), ICI 182,780 (Tocris, Ballwin, MO), BPA ⫹ ICI, E 2 ⫹ ICI, or with corn oil (vehicle control; Sigma) once a day between 8:30 and 11:00 A.M. for 4 consecutive days. Mice were 35– 60 days old at the start of the treatment period, and all animals in any one study were the same age. All solutions were derived from stocks in ethanol with concentrations of 0.4, 500, and 50 mg/ml for E 2, BPA, and ICI, respectively. Animals were terminated 24 h after the last treatment with carbon dioxide anesthesia followed by cervical dislocation. For the determination of an E 2 dose-response curve (study numbers 1 and 2) mice (n ⫽ 10 –16) were treated with 0.1 ml solutions of E 2 in corn oil, resulting in 0.4-, 4-, 40-, and 400-ng/day doses. These doses correspond to 0.02, 0.2, 2, and 20 ␮g/kg respectively, for the average 20-g mouse. The control animals in these experiments were treated with 0.1 ml of corn oil. In the first BPA dose-response experiment (study number 4), BPA was administered in 0.16 ml of corn oil solutions corresponding to doses of 0.02, 0.8, 2, or 8 mg/day (n ⫽ 10). Animals treated with 0.16 ml of corn oil (n ⫽ 16), or 0.16 ml of a solution of E 2 (n ⫽ 10) corresponding to a dose of 64 ng/day were also included as the negative and positive controls, respectively. When the experiment was repeated (study number 5), a dose of 0.2-mg BPA/day was added to the design. In study number 3, E 2 (40 ng/day) was co-administered with 2 or 20 ␮g ICI/day in 0.2 ml corn oil solutions. Corn oil, or 2 or 20 ␮g ICI/day alone, were included as controls. Similarly, BPA (2 mg/day) was administered alone or in combination with 20 ␮g ICI/day (study number 6) or 200 ␮g ICI/day (study number 7) in 0.2 ml solutions. Animals treated with corn oil or with 20 or 200 ␮g ICI/day in 0.2 ml solutions were included as controls for studies number 6 and 7, respectively.

MATERIALS AND METHODS

Statistical analysis. Results are expressed as means ⫾ standard deviations. One-way analysis of variance (ANOVA) was used to assess the variation of the means among the treatments. If the variation was greater than expected by chance alone, Dunnett’s test was performed for a comparison of pairs of means. Significance was established when the p value was less than 0.05.

Animals and treatment protocols. All procedures requiring the use of animals were conducted according to the National Institutes of Health⬘s Using Animals in Intramural Research: Guidelines (1998). Ovariectomized female

Uterine wet weight measurements; uterine histology and morphometry. Mice were weighed to the nearest 0.1 g on day 1 and on the day of termination. No significant differences were observed in animal weights at the beginning or at the end of the treatment period. Animals were observed daily for clinical signs of toxicity. Upon termination, uteri were removed, blotted, and weighed to the nearest 0.1 mg. A 5-mm posteriormost segment of one uterine horn was fixed overnight in 10% neutral buffered formalin, dehydrated in a graded ethanol series, cleared in xylene, embedded in paraplast, sectioned at 8 ␮m, and stained with hematoxylin and eosin (H&E). Uterine epithelial cell height was measured in uterine sections from the apical (luminal) surface to the basement membrane separating the epithelium from the stroma. The thickness of the stroma was measured from the basement membrane to the beginning of the circular layer of the myometrium, and the myometrium was measured from the beginning of the circular layer to the distal margin of the longitudinal layer, including the layer of connective tissue between the two myometrial layers. All measurements were made in areas where luminal folds were not present and care was taken to avoid measuring sections that were cut obliquely. A representative section was selected for each animal, and the average of two measurements per section was used in the calculations. Measurements were made on an Olympus BH microscope at 400⫻ magnification using an ocular micrometer. Photographs of uterine sections were taken with an Olympus C-35A camera containing Kodak Ektachrome 160T 35-mm film.

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TABLE 1 Effects of ␤-Estradiol (E 2) Alone or in Combination with ICI 182, 780 (ICI) on Uterine Wet Weight in the Ovariectomized B6C3F1 Mouse Study no. 1

2

3

Treatment

No. of animals

Mean uterus wet wt (mg) ⫾ SD

Corn oil E 2, 0.4 ng/day E 2, 4 ng/day E 2, 40 ng/day E 2, 400 ng/day Corn oil E 2, 0.4 ng/day E 2, 4 ng/day E 2, 40 ng/day E 2, 400 ng/day Corn oil E 2, 40 ng/day E 2, 40 ng/day ⫹ ICI, 2 ␮g/day E 2, 40 ng/day ⫹ ICI, 20 ␮g/day ICI, 2 ␮g/day ICI, 20 ␮g/day

16 10 10 10 9 10 9 7 8 7 8 8 8 8 7 7

7.36 ⫾ 2.99 8.85 ⫾ 3.14 16.66 ⫾ 3.83* 52.41 ⫾ 5.70* 60.49 ⫾ 10.84* 9.88 ⫾ 1.78 10.82 ⫾ 3.09 15.89 ⫾ 2.13 52.44 ⫾ 7.24* 52.01 ⫾ 8.82* 8.75 ⫾ 2.50 45.84 ⫾ 7.29* 43.01 ⫾ 3.32* 11.32 ⫾ 2.30** 8.99 ⫾ 1.71 6.42 ⫾ 1.71

*Statistically significant difference from control (corn oil) as determined by one way ANOVA and the Dunnett’s multiple comparison test (p ⬍ 0.01). **Statistically significant difference from E 2 as determined by one way ANOVA and the Dunnett’s multiple comparison test (p ⬍ 0.01).

RESULTS

Uterine Weights Mean uterine wet weights in response to E 2 treatment were increased in a dose-dependent manner (Table 1). The increase in uterine wet weight induced by 40 ng E 2/day was significantly reduced by 20 (p ⬍ 0.01) but not 2 ␮g/day of the antiestrogen ICI. The antiestrogen administered alone at these doses did not have a significant effect on the wet weight of the uterus (Table 1). BPA caused an increase in uterine weights in a dose-dependent manner (Table 2). The BPA dose of 0.2 mg/day, added in study no. 5 in order to better define the lowest observed effect level (LOEL), proved to be non-estrogenic. Therefore, the BPA LOEL for the uterotrophic assay in the B6C3F1 mouse was established at 0.8 mg/day, as it is the lowest dose used in our experimental design that produced a significant induction in studies 4 and 5 (Table 2). These studies suggest that BPA acts as a partial agonist to increase uterine weights with a maximum response that is approximately one third of that induced by E 2 and an EC 50 of 0.72 mg/day (study 4) compared to 19.4 ng/day for E 2 (study 2). The effects of 2 mg BPA/day on wet uterus weights were not significantly altered by 20 ␮g ICI/day (Table 2). However, co-administration of 200 ␮g/day of the antiestrogen lowered the BPA-induced mean uterine wet weight to a level not statistically different from the ovariectomized control (p ⬍

0.01). ICI alone at 200 ␮g/day did not affect uterine weights. Although the specific rodent diet used in this study contains detectable levels of phytoestrogens (Thigpen et al., 1999), it does not appear to have elicited a uterotrophic response in our study, since ICI alone did not reduce uterine weights. Uterine Morphology Although 0.4 ng E 2/day did not significantly increase uterine weight, an approximate 2-fold increase in uterine luminal epithelial height compared to the control was observed (data not shown). At E 2 doses greater than 0.4 ng/day, uterine height continued to increase and uterine epithelial cells changed in appearance from simple columnar (Fig. 1A) to pseudostratified columnar (as represented in Fig. 1B by the 40ng/day E 2 dose). At BPA doses between 0.8 and 8 mg/day the luminal epithelial height increased (as represented in Fig. 1D for 2 mg BPA/day) with dose, but unlike E 2, the epithelium did not become pseudostratified. Effects of 40 ng E 2/day on the epithelium were diminished by co-administration of 20 ␮g ICI/day (Fig. 1C). Co-administration of 20 ␮g ICI/day with 2 mg BPA/day

TABLE 2 Effects of Bisphenol A (BPA) Alone or in Combination with ICI 182,780 (ICI) on Uterine Wet Weight in the Ovariectomized B6C3F1 Mouse Study no. 4

5

6

7

Treatment

No. of animals

Mean uterus wet wt (mg) ⫾ SD

Corn oil BPA, 0.02 mg/day BPA, 0.8 mg/day BPA, 2 mg/day BPA, 8 mg/day E 2, 64 ng/day Corn oil BPA, 0.02 mg/day BPA, 0.2 mg/day BPA, 0.8 mg/day BPA, 2 mg/day BPA, 8 mg/day E 2, 64 ng/day Corn oil E 2, 40 ng/day BPA, 2 mg/day BPA, 2 mg/day ⫹ ICI, 20 ␮g/day Corn oil E 2, 40 ng/day BPA, 2 mg/day BPA, 2 mg/day ⫹ ICI, 200 ␮g/day ICI, 200 ␮g/day

15 14 9 10 10 10 15 9 10 10 10 9 5 14 10 10 9 10 10 10 10 10

9.72 ⫾ 1.47 10.27 ⫾ 2.43 13.66 ⫾ 2.10** 15.39 ⫾ 2.02** 15.37 ⫾ 2.30** 51.18 ⫾ 6.58** 8.07 ⫾ 1.54 7.27 ⫾ 0.81 8.29 ⫾ 1.20 10.88 ⫾ 2.09* 11.59 ⫾ 2.40** 15.76 ⫾ 2.55** 43.66 ⫾ 5.17** 4.54 ⫾ 0.83 36.07 ⫾ 2.80** 7.82 ⫾ 1.46** 7.24 ⫾ 1.36** 6.58 ⫾ 1.06 47.34 ⫾ 10.18** 12.42 ⫾ 1.84** 8.16 ⫾ 1.75*** 8.03 ⫾ 2.04

*Statistically significant difference from control (corn oil) as determined by one way ANOVA and the Dunnett’s multiple comparison test (p⬍0.05). **Statistically significant difference from control (corn oil) as determined by one way ANOVA and the Dunnett’s multiple comparison test (p⬍0.01). ***Statistically significant difference from BPA as determined by one way ANOVA and the Dunnett’s multiple comparison test (p⬍0.01).


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FIG. 1. Morphology of the uterine luminal epithelium of B6C3F1 ovariectomized mice treated with (A) corn oil; (B) 40 ng ␤-estradiol (E 2)/ day; (C) 40 ng E 2/day and 20 ␮g ICI 182,780 (ICI)/day; (D) 2 mg bisphenol A (BPA)/day; (E) 2 mg BPA/day and 20 ␮g ICI/day; (F) 2 mg BPA/ day and 200 ␮g ICI/day; (G) 20 ␮g ICI/day; and (H) 200 ␮g ICI/day. Luminal epithelium is indicated by arrows in A. The bar represents 25 ␮.

did not alter the histology of the uterine luminal epithelium compared to BPA alone (Fig. 1E); however, when 200 ␮g ICI/day were co-administered with 2 mg BPA/day, luminal epithelial height returned to control levels (Fig. 1F). Administration of 20 or 200 ␮g/day of the antiestrogen alone did not result in any significant alterations in the histology of the uterine luminal epithelium (Figs. 1G and 1H, respectively). Measurements of uterine epithelial height and stroma and myometrium thickness following treatments of 40 ng E 2/day or 2 mg BPA/day with or without ICI at 2, 20, or 200 ␮g/day are given in Figure 2. E 2 and BPA significantly increased epithelial height, stroma thickness, and myometrium thickness.

The effects of E 2 were not significantly reduced by co-administration of 2 ␮g ICI/day, but were reversed to control levels by 20 ␮g ICI/day. The effects of BPA were not significantly reduced by 20 ␮g ICI/day. The increase in epithelial height following BPA treatment was reversed to control levels by 200 ␮g ICI/day, but this concentration of the antiestrogen did not reduce BPA-induced increases in stroma or myometrium thickness. Although 20 ␮g ICI/day alone did not affect uterine morphology, 200 ␮g/day significantly increased stroma and myometrium thickness to values not significantly different from those of animals treated with BPA (2 mg/day) alone or with both BPA (2 mg/day) and ICI (200 ␮g/day).

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FIG. 2. Effects of ICI 182,780 (ICI) on ␤-estradiol (E 2)- and bisphenol A (BPA)-induced increases in luminal epithelium cell height and in the thickness of the stroma and myometrium of the uterus of ovariectomized B6C3F1 mice. Animals were treated with corn oil (CO, control), 40 ng E 2/day, 2 mg BPA/day or ICI (2, 20, 200 ␮g/day), or were co-treated with BPA or E 2 and ICI. * ,**Statistically significant difference from control (CO) as determined by one-way ANOVA and the Dunnett’s multiple comparison test (p ⬍ 0.05 and p ⬍ 0.01, respectively). a,bStatistically significant difference from E 2 or BPA, respectively, as determined by one way ANOVA and the Dunnett’s multiple comparison test (p ⬍ 0.01).

DISCUSSION

Most of the previous studies that have examined the estrogenicity of bisphenol A in vivo have utilized a standard rodent uterotrophic assay, which is based on the ability of estrogenic substances to increase uterine weight (due mainly to increased water imbibition) in immature or ovariectomized animals dosed daily over a 3 or 4 day period (Cook et al., 1997; O’Connor et al., 1996; Reel et al., 1996). These studies are inconclusive, since BPA was shown to increase uterine weights in some studies (Cooke et al., 1997; Dodge et al., 1996; Gray and Ostby, 1998) but not in others (Coldham et al., 1997;

Gould et al., 1998). Other uterotrophic effects of E 2 in the rodent include the induction of hypertrophy of uterine epithelial cells (Branham et al., 1993; Cooke et al., 1997; Katsuda et al., 1999) and stimulation of cell proliferation in the stromal (Cook et al., 1997) and myometrial (Hunter et al., 1999; Yokoyama et al., 1998) layers. Few studies have examined the effects of BPA on uterine morphology. In the present study, we compared the effects of E 2 and BPA on both uterine wet weight and morphology in the ovariectomized B6C3F1 mouse. Both BPA and E 2 were shown to significantly increase uterine wet weight in a dose-dependent


manner. Both of these compounds also significantly increased uterine luminal epithelial cell height and the thickness of both the stromal and myometrial layers of the uterus. Based on uterine weights, the estimated EC 50 for BPA was 3.7 ⫻ 10 4 times that for E 2, and BPA produced a maximal response that was approximately one third of that produced by E 2. The potency of BPA may be limited by its bioavailability. Pottenger et al. (2000) demonstrated a 20-fold reduction in BPA in the blood of F344 rats 2 h following a single subcutaneous dose of 100 mg/kg. A short half life was inferred by Steinmetz et al. (1998), who showed that the amount of uterine hypertrophy induced in F344 rats continuously exposed sc to 0.3– 0.5 mg BPA/kg over 24 h was similar to that observed following a single 50 mg/kg dose. In our study, the reduced maximum effect of BPA relative to E 2 on uterine weights suggests that BPA is a partial agonist for this particular response. Long et al. (2000) compared the effects of E 2 and BPA on the stimulation of DNA synthesis in F344 rats and observed a similar reduction in efficacy of BPA compared to E 2 as well as differences between different rat strains in the maximum response to BPA. They propose that these differences are due to differential effects on ER-mediated transcriptional activity. While other studies have demonstrated that the uterotrophic effects of E 2 are mediated by the estrogen receptor ␣ (Buchanan et al., 1999; Cooke et al., 1997; Katsuda et al., 1999; Orimo et al., 1999), the role of the ER in BPA-mediated uterotrophism has not been previously reported. To investigate whether the E 2- and BPA-induced increases in uterine wet weight and alterations of uterine morphology were mediated through the ER, we examined the potential of the antiestrogen ICI 182,780 (ICI) to attenuate these effects. This antiestrogen has been shown to bind to both the ER␣ and the ER␤ (Kuiper et al., 1997) and to antagonize estrogen activity through these receptors (Malayer et al., 1999; Wakeling and Bowler, 1992). The effects of 40 ng E 2/day on uterine wet weight and morphology were reversed by 20 ␮g ICI/day, while this dose of the antiestrogen alone did not affect these parameters. These results support previous reports that indicate a role for the ER in the mediation of these E 2-induced uterotrophic effects. They are also in agreement with studies that have demonstrated the ability of sc administration of ICI to attenuate the increase in uterine wet weight induced by E 2 (Sahlin and Eriksson, 1996), estradiol benzoate (Wade et al., 1993; Wakeling and Bowler, 1992), ethinyl estradiol (Lundeen et al., 1997) or estrone (Martel et al., 1998). While co-administration of 20 ␮g ICI/day did not alter the uterotrophic effects induced by 2 mg BPA/day, 200 ␮g ICI/day significantly reduced the increase in uterine weight and uterine luminal epithelial height to control levels and did not by itself affect these parameters. This suggests that these BPA-induced uterotrophic effects are mediated through the estrogen receptor. Furthermore, the BPA binding affinity for the ER appears to be less than that of E 2, since the amount of ICI needed to block these effects is less per mole of BPA than per mole of E 2. A reduced affinity of BPA relative to E 2

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for the estrogen receptor has also been suggested from in vitro studies (Bergeron et al., 1999; Chun and Gorski, 2000; Gould et al., 1998). Co-administration of 200 ␮g ICI/day did not significantly reduce the BPA-mediated increase in thickness of either the stroma or the myometrium. Furthermore, ICI alone at the 200-␮g/day dose significantly increased both the stroma and myometrium thickness to values comparable to those observed in animals treated with BPA alone. This apparent agonist activity of ICI on uterine stroma and myometrial tissue is somewhat surprising. However, ICI has also been shown to act as an agonist for other ER-mediated processes including bone metabolism in the rat (Sibonga et al., 1998) and the activation of reporter gene constructs transfected into HEC1A human endometrial cancer cells (Castro-Rivera and Safe, 1998) and bovine fetal uterine cells (Malayer et al., 1999). Due to the agonist properties of this high dose of ICI on uterine stroma and myometrium in the present study, it cannot be determined if BPA acts through the ER to increase the thickness of these tissues. The interaction of BPA with a region of the ER␣ that does not bind ICI (Gould et al., 1998), or to estrogen receptorrelated receptors (Das et al., 1997; Vanacker et al., 1999), or membrane estrogen receptors (Monje and Boland, 1999), which are linked to responses not antagonized by ICI, cannot be ruled out in these tissues. In summary, our findings demonstrate that the xenoestrogen bisphenol A increases uterine wet weight, luminal epithelial cell height and the thickness of both the stroma and myometrium in the ovariectomized B6C3F1 mouse. Co-administration of the antiestrogen ICI blocks the increase in uterine wet weight and epithelial cell height, and suggests that these BPAinduced events are mediated by the ER. REFERENCES Ashby, J., and Tinwell, H. (1998). Uterotrophic activity of bisphenol A in the immature rat. Environ. Health Perspect. 106, 719 –720. Astwood, E. B. (1938). A six-hour assay for the quantitative determination of estrogen. Endocrinology 23, 25–31. Bergeron, R. M., Thompson, T. B., Leonard, L. S., Pluta, L., and Gaido, K. W. (1999). Estrogenicity of bisphenol A in a human endometrial carcinoma cell line. Mol. Cell. Endocrinol. 150, 179 –187. Branham, W. S., Zehr, D. R., and Sheehan, D. M. (1993). Differential sensitivity of rat uterine growth and epithelium hypertrophy to estrogens and antiestrogens. Proc. Soc. Exp. Biol. Med. 203, 297–303. Brotons, J. A., Olea-Serrano, M. F., Villalobos, M., Pedraza, V., and Olea, N. (1995). Xenoestrogens released from lacquer coatings in food cans. Environ. Health Perspect. 103, 608 – 612. Buchanan, D. L., Setiawan, T., Lubahn, D. B., Taylor, J. A., Kurita, T., Cunha, G. R., and Cooke, P. S. (1999). Tissue compartment-specific estrogen receptor-␣ participation in the mouse uterine epithelial secretory response. Endocrinology 140, 484 – 491. Castro-Rivera, E., and Safe, S. (1998). Estrogen- and antiestrogen-responsiveness of HEC1A endometrial adenocarcinoma cells in culture. J. Steroid Biochem. Mol. Biol. 64, 287–295. Chun, T-Y., and Gorski, J. (2000). High concentrations of bisphenol A induce

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cell growth and prolactin secretion in an estrogen-responsive pituitary tumor cell line. Toxicol. Appl. Pharmacol. 162, 161–165. Coldham, N. G., Dave, M., Sivapathasundaram, S., McDonnell, D. P., Connor, C., and Sauer, M. J. (1997). Evaluation of a recombinant yeast cell estrogen screening assay. Environ. Health Perspect. 105, 734 –742. Colerangle, J. B., and Roy, D. (1997). Profound effects of the weak environmental estrogen-like chemical bisphenol A on the growth of the mammary gland of Noble rats. J. Steroid Biochem. Mol. Biol. 60, 153–160. Cook, J. C., Kaplan, M., Davis, L. G., and O’Connor, J. C. (1997). Development of a Tier I screening battery for detecting endocrine-active compounds (EACs). Reg. Toxicol. Pharmacol. 26, 60 – 68. Cooke, P. S., Buchanan, D. L., Young, P., Setiawan, T., Brody, J., Korach, K. S., Taylor, J., Lubahn, D. B., and Cunha, G. R. (1997). Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc. Natl. Acad. Sci. U.S.A. 94, 6535– 6540. Das, S. K., Taylor, J. A., Korach, K. S., Paria, B. C., Dey, S. K., and Lubahn, D. B. (1997). Estrogenic responses in estrogen receptor-␣ deficient mice reveal a distinct estrogen signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 94, 12786 –12791. Dodds, E. C., and Lawson, W. (1938). Molecular structure in relation to oestrogenic activity. Compounds without a phenanthrene nucleus. Proc. Royal Soc. London. B. Biol. Sci. 125, 222–232. Dodge, J. A., Glasebrook, A. L., Magee, D. E., Phillips, D. L., Sato, M., Short, L. L., and Bryant, H. U. (1996). Environmental estrogens: Effects on cholesterol lowering and bone in the ovariectomized rat. J. Steroid Biochem. Mol. Biol. 59, 155–161. Gaido, K. W., Leonard, L. S., Lovell, S., Gould, J. C., Babai, D., Portier, C. J., and McDonnell, D. P. (1997). Evaluation of chemicals with endocrinemodulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol. Appl. Pharmacol. 143, 205–212. Gould, J. C., Leonard, L. S., Maness, S. C., Wagner, B. L., Conner, K., Zacharewski, T., Safe, S., McDonnell, D. P., and Gaido, K. W. (1998). Bisphenol A interacts with the estrogen receptor ␣ in a distinct manner from estradiol. Mol. Cell. Endocrinol. 142, 203–214. Gray, L. E., Jr., and Ostby, J. (1998). Effects of pesticides and toxic substances on behavioral and morphological reproductive development: Endocrine versus nonendocrine mechanisms. Toxicol. Ind. Health. 14, 159 –184. Hunter, D. S., Hodges, L. C., Vonier, P. M., Fuchs-Young, R., Gottardis, M. M., and Walker, C. L. (1999). Estrogen receptor activation via activation function 2 predicts agonism of xenoestrogens in normal and neoplastic cells of the uterine myometrium. Cancer Res. 59, 3090 –3099. Katsuda, S.-I., Yoshida, M., Watanabe, T., Kuroda, H., Ando-Lu, J., Takahashi, M., Hayashi, H., and Maekawa, A. (1999). Estrogen receptor mRNA in uteri of normal estrous cycling and ovariectomized rats by in situ hybridization. Proc. Soc. Exp. Biol. Med. 221, 207–214. Kavlock, R. J. (1999). Overview of endocrine-disruptor research activity in the United States. Chemosphere 39, 1227–1236. Krishnan, A. V., Stathis, P., Permuth, S. F., Tokes, L., and Feldman, D. (1993). Bisphenol-A: An estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132, 2279 –2286.

Lundeen, S. G., Carver, J. M., McKean, M.-L., and Winneker, R. C. (1997). Characterization of the ovariectomized rat model for the evaluation of estrogen effects on plasma cholesterol levels. Endocrinology 138, 1552– 1558. Malayer, J. R., Cheng, J., and Woods, V. M. (1999). Estrogen responses in bovine fetal uterine cells involve pathways directed by both estrogen response element and activator protein-1. Biol. Reprod. 60, 1204 –1210. Martel, C., Labrie, C., Belanger, A., Gauthier, S., Merand, Y., Li, X., Provencher, L., Candas, B., and Labrie, F. (1998). Comparison of the effects of the new orally active antiestrogen EM-800 with ICI 182 780 and toremifene on estrogen-sensitive parameters in the ovariectomized mouse. Endocrinology 139, 2486 –2492. Milligan, S. R., Balasubramanian, A. V., and Kalita, J. C. (1998). Relative potency of xenobiotic estrogens in an acute in vivo mammalian assay. Environ. Health Perspect. 106, 23–26. Monje, P., and Boland, R. (1999). Characterization of membrane estrogen binding proteins from rabbit uterus. Mol. Cell. Endocrinol. 147, 75– 84. Mountfort, K. A., Kelly, J., Jickells, S. M., and Castle, L. (1997). Investigations into the potential degradation of polycarbonate baby bottles during sterilization with consequent release of bisphenol A. Food Addit. Contam. 14, 737–740. Nagel, S. C., vom Saal, F. S., Thayer, K. A., Dhar, M. G., Boechler, M., and Welshons, W. V. (1997). Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ. Health Perspect. 105, 70 –76. O’Connor, J. C., Cook, J. C., Craven, S. C., Van Pelt, C. S., and Obourn, J. D. (1996). An in vivo battery for identifying endocrine modulators that are estrogenic or dopamine regulators. Fundam. Appl. Toxicol. 33, 182–195. Ogawa, S., Chan, J., Chester, A. E., Gustafsson, J-A., Korach, K. S., and Pfaff, D. W. (1999). Survival of reproductive behaviors in estrogen receptor beta gene-deficient (betaERKO) male and female mice. Proc. Natl. Acad. Sci. U.S.A. 96, 12887–12892. Olea, N., Pulgar, R., Perez, P., Olea-Serrano, F., Rivas, A., Novillo-Fertrell, A., Pedraza, V., Soto, A. M., and Sonnenschein, C. (1996). Estrogenicity of resin-based composites and sealants used in dentistry. Environ. Health Perspect. 104, 298 –305. Orimo, A., Inoue, S., Minowa, O., Tominaga, N., Tomioka, Y., Sato, M., Kuno, J., Hiroi, H., Shimizu, Y., Suzuki, M., Noda, T., and Muramatsu, M. (1999). Underdeveloped uterus and reduced estrogen responsiveness in mice with disruption of the estrogen-responsive finger protein gene, which is a direct target of estrogen receptor ␣. Proc. Natl. Acad. Sci. U.S.A. 96, 12027–12032. Pottenger, L. H., Domoradzki, J. Y., Markham, D. A., Hansen, S. C., Cagen, S. Z., and Waechter, J. M., Jr. (2000). The relative bioavailability and metabolism of bisphenol A in rats is dependent upon the route of administration. Toxicol. Sci. 54, 3–18. Reel, J. R., Lamb, J. C., IV, and Neal, B. H. (1996). Survey and assessment of mammalian estrogen biological assays for hazard characterization. Fundam. Appl. Toxicol. 34, 288 –305.

Kuiper, G., Carlsson, B., Enmark, E., Haggblad, J., Nilsson, S., and Gustaffson, J-A. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors ␣ and ␤. Endocrinology 138, 863– 870.

Sahlin, L., and Eriksson, H. (1996). Influence of blockers for the estrogen receptor (ER) and type 1 IGF-receptor on the levels of ER, ER mRNA and IGF-I mRNA in the rat uterus. J. Steroid Biochem. Mol. Biol. 58, 359 –365.

Larson, D. F., Mayall, B., and Anderson, R. M. (1977). Hemolysis due to chemical contamination of clinical perfusion apparatus. Ann. Thorac. Surg. 23, 374 –375.

Sibonga, J. D., Dobnig, H., Harden, R. M., and Turner, R. T. (1998). Effect of the high-affinity estrogen receptor ligand ICI 182,780 on the rat tibia. Endocrinology 139, 3736 –3742.

Long, X., Steinmetz, R., Ben-Jonathan, N., Caperell-Grant, A., Young, P. C., Nephew, K. P., and Bigsby, R. M. (2000). Strain differences in vaginal responses to the xenoestrogen bisphenol A. Environ. Health Perspect. 108, 243–247.

Sonnenschein, C., and Soto, A. M. (1998). An updated review of environmental estrogen and androgen mimics and antagonists. J. Steroid Biochem. Mol. Biol. 65, 143–150. Staples, C. A., Dorn, P. B., Klecka, G. M., O’Block, S. T., and Harris, L. R.

BPA EFFECTS ON UTERINE WEIGHT AND MORPHOLOGY (1998). A review of the environmental fate, effects, and exposures of bisphenol A. Chemosphere 36, 2149 –2173. Steinmetz, R., Brown, N. G., Allen, D. L., Bigsby, R. M., and Ben-Jonathan, N. (1997). The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo. Endocrinology 138, 1780 –1786. Steinmetz, R., Mitchner, N. A., Grant, A., Allen, D. L., Bigsby, R. M., and Ben-Jonathan, N. (1998). The xenoestrogen bisphenol A induces growth, differentiation, and c-fos gene expression in the female reproductive tract. Endocrinology 139, 2741–2747. Thigpen, J. E., Setchell, K. D., Goelz, M. F., and Forsythe, D. B. (1999). The phytoestrogen content of rodent diets. Environ. Health Perspect. 107, A182–A183. Vanacker, J.-M., Pettersson, K., Gustafsson, J.-A., and Laudet, V. (1999). Transcriptional targets shared by estrogen receptor-related receptors

339

(ERRs) and estrogen receptor (ER) ␣, but not by ER␤. EMBO J. 18, 4270 – 4279. Wade, G. N., Blaustein, J. D., Gray, J. M., and Meredith, J. M. (1993). ICI 182,780: A pure antiestrogen that affects behaviors and energy balance in rats without acting in the brain. Am. J. Physiol. 265, R1392– R1398. Wakeling, A. E., and Bowler, J. (1992). ICI 182,780, a new antioestrogen with clinical potential. J. Steroid Biochem. Mol. Biol. 43, 173–177. Yamamoto, T., and Yasuhara, A. (1999). Quantities of bisphenol A leached from plastic waste samples. Chemosphere 38, 2569 –2576. Yokoyama, Y., Takahashi, Y., Hashimoto, M., Shinohara, A., Lian, Z., and Tamaya, T. (1998). Effects of sex steroids on silver stained proteins of nucleolar organizer regions (Ag-NOR) in the rabbit uterus. Biotech. Histochem. 73, 202–210.