Department of Pharmacology and Toxicology and the Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received September 19, 2000; accepted January 2, 2001
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ABSTRACT |
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Key Words: genistein; endometriosis; estrogen-; progesterone-receptors; uterus; rat.
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INTRODUCTION |
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Due to the dependence of endometriosis upon ovarian hormones, an increasing amount of attention has been given to the investigation of potential links between incidence or severity and exposure to chemicals that may affect the endocrine or immune systems. In humans, increased incidence of endometriosis has been associated with exposure to 2,3,7,8-TCDD (Mayani et al., 1997) or polychlorinated biphenyls (PCBs) (Koninckx et al., 1994
) via the environment. Studies in animal models simulating endometriosis have indicated that one potential contributory mechanism for such compounds in the pathogenesis of endometriosis may be antiestrogenicity (Cummings et al., 1996
). Likewise, environmental chemicals that are weakly estrogenic, such as methoxychlor, have been shown to support surgically induced endometriosis in rodents (Cummings and Metcalf, 1995
). Given the multitude of environmental compounds with the ability to modulate the endocrine or immune systems, simultaneous exposure represents the potential for synergistic action.
Another potential source of exposure to estrogenic compounds is the diet. Genistein is an isoflavonic phytoestrogen found in high concentrations in soy products, legumes, and grains. The traditional Asian diet, high in soy products, has been associated with health benefits such as prevention of hormone-dependent cancers and coronary heart disease (reviewed by Adlercreutz, 1990). Despite the numerous health advantages of a diet high in soy, a limited number of epidemiologic studies have suggested that Asian women have a higher incidence of endometriosis than other ethnic groups (Arumugam and Templeton, 1990; Miyazawa, 1976
; Sangi-Haghpeykar and Poindexter, 1995
). However, the possible role of a soy diet in the disease process of endometriosis has never been explored.
Although soy has many components, it is believed that genistein is responsible for a majority of the biological consequences. Structurally similar to estradiol-17-ß, genistein is capable of eliciting numerous biological responses that mimic those of estradiol. It has affinity for the estrogen receptors alpha (Mathieson and Kitts, 1980; Shutt and Cox, 1972
) and beta (Kuiper et al., 1998
) in receptor binding assays, induces a uterotrophic response in animals (Bickoff et al., 1962
; Cheng et al., 1953
; Folman and Pope, 1966
), and increases uterine c-fos mRNA transcripts in vivo (Santell et al., 1997
). Although cell culture studies and animal models have provided a wealth of information as to the mechanisms of action of genistein in estrogen-responsive tissues, the biological effects of a soy diet in humans are difficult to measure. Therefore, we have employed a model that simulates endometriosis in rats via implantation of uterine tissue into the peritoneal cavity (Vernon and Wilson, 1985
). Following surgery, rats were exposed to genistein, by injection or through the diet, to determine whether genistein could support the implanted tissue.
Given the hormone-dependent nature of endometriosis, alterations in ovarian steroid hormone production induced by phytoestrogen exposure have the potential to either stimulate or inhibit the growth of endometriotic lesions. Genistein and other isoflavones have been shown to induce changes in circulating estradiol in humans (Cassidy et al., 1994; Lu et al., 1996
; Nagata et al., 1998
; Petrakis et al., 1996
) and rats (Cotroneo et al., 2001
); possibly through alterations in steroid-converting enzymes such as aromatase (Adlercreutz et al., 1992
; Kao et al., 1998
; Wang et al., 1994
), 17ß-hydroxysteroid oxidoreductases (Makela et al., 1995
; 1998
) and 5
-reductase (Weber et al., 1999
). A role for steroid-metabolizing enzymes in the pathogenesis of endometriosis has been suggested, as recent studies have identified aberrations in the expression of aromatase and type 2 17ß-hydroxysteroid reductase in human endometriotic lesions (reviewed by Bulun et al., 1999). Because of the multifactorial nature of endometriosis and the numerous mechanisms by which genistein could affect lesion growth, we also investigated the potential for genistein to affect lesion growth via ovarian steroidogenesis and/or steroid receptor modulation.
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MATERIALS AND METHODS |
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Location of implants/ovariectomy.
Three weeks following implant surgery, the rats were bilaterally ovariectomized (Experiments 1 and 2) or left ovary-intact (Experiment 3). At this time, implants were located and assessed for viability by visual inspection; viable implants appeared as fluid-filled, spherical structures. In Experiment 3, where net growth of the implants was to be determined, two different colored sutures were used per rat, in order to facilitate their identification at the conclusion of the experiment. The diameter of each implant was measured using calipers. Following surgery, the rats were exposed to genistein via injections or diet.
Injections (Experiment 1).
After ovariectomy, the rats were fed a diet devoid of phytoestrogens (AIN-76A, Harlan-Teklad, Madison, WI) until the conclusion of the experiment. Subcutaneous injections of the following were given in the nape of the neck: estrone (1 µg/rat) (Sigma Chemical Co., St. Louis, MO), genistein (50 µg/g BW), genistein (16.6 µg/g BW), genistein (5.0 µg/g BW). Sesame oil (Sigma Chemical Co.) served as the vehicle for estrone; DMSO (Sigma Chemical Co.) was used for genistein. The dose response for genistein was based on preliminary experiments in our laboratory in which 50 µg/g BW produced a uterotrophic response in ovariectomized rats. Cummings (1993) reported that 1 µg estrone treatment resulted in a more reproducible biological response in the uteri of rats than 17ß-estradiol and that this treatment supported surgically induced endometriosis (Cummings and Metcalf, 1995), thus serving as a positive control. Daily injections were given for 3 weeks.
Dietary genistein (Experiments 2 and 3).
Following ovariectomy (Experiment 2) or exploratory surgery to locate and measure implants (Experiment 3), rats were fed one of the following diets for a 3-week period: 250 mg genistein/kg AIN-76A diet, 1000 mg genistein/kg AIN-76A, or AIN-76A alone (controls). Ovary-intact rats (Experiment 3) were fed control diet (AIN-76A) or 250 mg genistein/kg AIN-76A. Animals remained on the diets for 3 weeks.
Necropsy.
At the age of 15 weeks, all rats were necropsied. Ovary-intact rats were sacrificed in the estrous phase of the estrous cycle, as determined by vaginal cytology. Implants were located, measured, and assessed visually for viability. Implants presenting as fluid-filled vesicles were deemed viable. Uteri were removed and frozen in liquid nitrogen for Western blot analysis for estrogen receptor- and progesterone receptors. Serum samples were stored at 20°C until analyses for circulating estradiol, progesterone, and genistein were conducted.
Body weight analyses.
Body weights were recorded at the time of implant surgery, at ovariectomy/exploratory surgery, and at necropsy. Percent body weight gain from implant surgery to necropsy was calculated for each animal and averaged for each treatment group and compared.
Western blot analyses.
A more detailed description of the protocol may be found elsewhere (Brown et al., 1998). Uteri were homogenized in a buffer containing the following: 1% Triton-X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, and protease/phosphatase inhibitors. Equal amounts of protein were electrophoresed and transferred to nitrocellulose membranes. The membranes were blocked and incubated overnight with anti-ER-
(NHKDD) or anti-PR (Neomarkers, Fremont, CA) at 1:1000 and 1:250, respectively. Following washes with TBS-T, pH 7.5, the membranes were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase. Proteins were detected using chemiluminescence (Pierce, Rockford, IL). The relative intensity of the bands was measured using a scanner (Hewlitt Packard Scan Jet 4p, Boise, ID).
Serum estradiol-17-ß and progesterone.
RIA analyses were done by the laboratory of Dr. Larry Boots, University of Alabama at Birmingham, using commercially available kits (Pantex, Santa Monica, CA). Assays were performed according to the manufacturers' instructions.
Serum genistein.
Total (unconjugated and conjugated forms) and free (unconjugated) genistein concentrations were measured using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) (Coward et al., 1996), with a lower limit of detection of 10 pM.
Statistical analyses.
Mean values from serum steroid hormone analyses were calculated and compared using Student's t-test (Sigma Stat, Jandel Scientific, San Rafael, CA). For Western blot analyses, mean densitometric values for each treatment were compared to controls using one-way ANOVA (Sigma Stat, Jandel Scientific). Average body weight and percent body weight gain were also compared using one-way ANOVA. In Experiment 3, implant growth was compared for the two groups using Fisher's Exact test, (SAS/STAT, Cary, NC). Average growth or shrinkage of the implants was assessed using Wilcoxon Scores test (SAS/STAT).
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RESULTS |
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Serum Genistein Concentrations
Free (unconjugated) and total (conjugated and unconjugated forms) serum genistein concentrations were determined from ovariectomized rats injected (Experiment 1) or fed genistein (Experiment 2) (Table 3). For rats given injections, serum genistein concentrations exhibited a dose-responsive relationship. However, the percent free genistein was similar for the 16.6 and 50 µg/g BW doses. The lower dietary dose (250 mg genistein/kg AIN-76A) resulted in total genistein concentrations that were approximately one-half that of the higher dose (1000 mg genistein/kg AIN-76A); the percent free genistein showed a similar relationship.
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DISCUSSION |
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Human exposure to genistein occurs via the diet, not through injections. Therefore, we administered dietary genistein to rats with surgically induced endometriosis to more closely simulate a human situation. Dietary administration of genistein to ovariectomized rats did not support surgically induced endometriosis in ovariectomized rats. Based on blood genistein concentrations of Asian men (Adlercreutz et al., 1993) and women (Morton et al., 1999
) of 276 nM, the 250 mg genistein/kg AIN-76A dose resulted in total genistein concentrations that were approximately 4-fold higher. The higher dose, 1000 mg genistein/kg AIN-76A, resulted in serum genistein concentrations of 2 µM, approximately 8-fold higher than those of Asians eating a traditional diet high in soy. This dose was significantly uterotrophic and induced PR protein expression, indicative of estrogenic action. However, ER-
protein was only slightly reduced. This observation is supported by the suggestion that PR is a more sensitive uterine biomarker for estrogenic action than ER-
(Carthew et al., 1999
).
The lack of viable implants in the dietary study may be explained by bioavailability. For example, the 16.6 µg/g BW injected dose and the 250 mg dietary dose result in similar total serum genistein concentrations, 1380 nM and 1115 nM, respectively. Based on average daily food consumption of 15 g of feed per 300 g rat, rats consuming the 250 mg/kg diet are exposed to approximately 16 µg/g BW genistein per day. However, injections of 16.6 µg genistein/g BW resulted in four times as much free genistein as that of the animals in the dietary study. When administered via the diet, the fraction of free genistein circulating in the blood is reduced by the processes of absorption through the digestive tract, binding to plasma proteins, conjugation by sulfation or glucuronidation, and subsequent excretion through the urine and feces.
The advantage of using ovariectomized rats in this endometriosis model is the absence of ovarian hormones, which may confound steroid receptor analyses. However, the use of ovary-intact rats more closely simulated a physiologic situation. With an intact pituitary/hypothalamic/ovarian signaling axis, this experiment allowed for the potential to detect implant growth effects related to ovarian steroidogenesis.
In previous studies, we observed significantly increased serum estradiol and decreased progesterone following pharmacologic injections of 500 µg genistein/g BW to prepubertal female rats (Cotroneo et al., 2001). However, in the current study, which used a more physiologically relevant dose and mode of administration, genistein did not significantly alter either hormone level when compared to controls. Likewise, uterine weight, steroid receptor expression, and implant growth were not affected by dietary genistein. Because no growth effects were observed, further biochemical analyses on the implants were not performed.
We conclude that pharmacologic injections of genistein supported surgically induced endometriosis in ovariectomized rats. The action of genistein on uterine steroid hormone receptors indicated estrogen agonism. When given in the diet, evidence of estrogenic activity by genistein was observed only at a high dose, which resulted in a uterotrophic response and increased PR expression. This observation is in accordance with the work of our lab (Fritz et al., 1998) and others (Casanova et al., 1999
; Flynn et al., 2000
; Santell et al., 1997
), indicating that physiological doses of dietary genistein do not produce significant estrogen agonism or toxicity. Although the endometriosis model used in these studies provided valuable mechanistic data, its limitations in extrapolation to humans underscore the need for studies that evaluate the effects of exposure to soy or genistein on endometriosis in humans.
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ACKNOWLEDGMENTS |
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NOTES |
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