* Division of Neurotoxicology and
Division of Biochemical Toxicology, National Center for Toxicological Research, 3900 NCTR Drive, Jefferson, Arkansas 72079
Received September 3, 2002; accepted January 2, 2003
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ABSTRACT |
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Key Words: endocrine disruptors; isoflavones; pituitary; phytoestrogens; estrogens; blood pressure.
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INTRODUCTION |
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Although their total fluid intake was increased under these conditions, there was no general polydipsia. In a separate test, the same groups of rats, regardless of the type or dose of endocrine disruptor to which they were exposed, showed a preference for saccharin in a two-bottle test, but no overall increase in fluid consumption. We hypothesized that alterations in vasopressin (also known as antidiuretic hormone, which promotes the retention of water by the kidney) might underlie the effects of the endocrine disruptors.
Previous research has indicated that acute exposure to 17ß-estradiol stimulates the synthesis of the mRNA encoding arginine-8-vasopressin (vasopressin) in hypothalamic paraventricular and supraoptic magnocellular neurons (Roy et al., 1999). The brain-derived vasopressin is then transported to the posterior lobe of the pituitary gland, which secretes the peptide into the circulation (Hashimoto et al., 1981;
Roy et al., 1999
). The circulating vasopressin can then cause increases in a number of important physiological parameters such as blood pressure and fluid retention (Silverman et al., 1990
). These effects of estradiol exposure were only temporary (during the period immediately after exposure), and they were reversible with suspension of dosing.
There is also some evidence for more permanent alterations in hypothalamic peptides produced by estrogenic compounds (Brawer et al., 1993). For example, exposure to a single 2-mg/kg (im) injection of estradiol valerate resulted in a loss of 60% of ß-endorphin immunopositive neurons in the arcuate nucleus of the hypothalamus when measured 8 weeks later. (Desjardins et al., 1993
). The estradiol valerate procedure also resulted in polycystic ovaries, with reduced binding of gonadotrophins to cystic thecal cells (Convery and Brawer, 1991
).
Because vasopressin and ß-endorphin have been useful biomarkers of certain reversible, as well as more permanent effects, of estrogenic compounds, we hypothesized that they might be responsive to the effects of exposure to the phytoestrogen genistein, as well. We reasoned that altered functioning of the vasopressin system may have been responsible for the increased salt intake observed to occur after exposure to estrogenic endocrine disrupters (Flynn et al., 2000). Therefore, we sought to determine if dietary exposure to genistein would indeed increase hypothalamic vasopressin content, as estradiol had been shown to do. In addition we wished to determine if the dose-response relationship by which genistein increased hypothalamic vasopressin content was similar to the dose-response relationship by which genistein increased sodium intake.
Since ß-endorphin content had also been a useful biomarker for revealing neuronal loss in the hypothalamus following exposure to estradiol valerate (Desjardins et al., 1993), we measured it as well.
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MATERIALS AND METHODS |
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Drug and dose exposure.
Genistein was obtained from Toronto Research Chemicals (Ontario, Canada) with purity greater than 99%, as determined from 1H- and 13C-nuclear magnetic resonance, electron ionization/mass spectrometry, melting point, and thin layer chromatography analyses.
The basal (control) diet was irradiated, 5K96 meal that contains 3.13 kcal/g, 1822% protein, 3.8% fiber, and 4.6% fat (Purina Mills, Inc., St. Louis, MO). This diet is similar to the standard NIH 31 except that the soymeal and alfalfa components are replaced by casein, soy oil is replaced by corn oil, and the vitamin content is adjusted to compensate for irradiation effects. The genistein (0.54 µg/g) and daidzein (0.48 µg/g) content in this feed was determined using liquid chromatography/electrospray/mass spectroscopy analysis after complete hydrolysis of glucoside conjugates (not shown). The amounts of genistein added to the experimental diets to obtain 25, 250, and 1250 ppm were confirmed by gas chromatography/mass spectrometry. Genistein concentrations in fortified diets were measured using liquid chromatography-/UV (260 nm detection). Complete details regarding feed consumption and body weights from a related dose range-finding study have been published elsewhere (Delclos et al., 2001). The doses of genistein used in the present study were chosen to be the same as in the studies of Flynn et al., 2000
and Delclos et al., 2001
, so that the neuropeptide results could be compared to other previously described effects of genistein.
Dissection and preparation of brain tissue.
Two control litters, as well as four 25-ppm litters, four 250-ppm litters, and four 1250-ppm litters were evaluated. One male and one female from each litter, for a total n of 28 rats, were sacrificed by asphyxiation with carbon dioxide between 0800 and 1200 h (lights on), and their brains were rapidly removed. The brains were immediately frozen on dry ice and stored at 70°C. The whole hypothalamus was later dissected out (Glowinski and Iversen, 1966) and placed in a microfuge tube containing 1.0 ml of cold 0.1 N HCl. Following sonication for 5 s each, the samples were centrifuged at 12,000 x g for 15 min at 5°C in a refrigerated microcentrifuge (Eppendorf Model 5810R, Hamburg, Germany). The supernatants were divided into 100-µl aliquots and stored at -70°C until assayed in duplicate by ELISA for ß-endorphin and arginine-8-vasopressin, using kits from Peninsula Laboratories (San Carlos, CA) designed for the direct assay of acid extracts of tissue samples.
Competitive ELISA procedure.
Either a specific antivasopressin or anti-ß-endorphin antiserum (25 µl) was added to an immunocoated 96-well plate designed to bind and immobilize rabbit IgG. Then 25 µl of a known amount of biotin-labeled peptide was added to the well, together with 50 µl of either an unknown sample extract or a known concentration of a standard peptide solution (prepared from serial dilutions across a range of 01000 ng/ml). A 2-h incubation allowed the unlabeled peptide (either a standard or an unknown) to compete with the biotin-labeled peptide for binding to the immobilized antipeptide IgG. The addition of 100 µl streptavidin horseradish peroxidase for 1 h, then bound HRP to the immobilized IgG-biotin complex. Following a thorough rinse, 100 µl of the HRP substrate tetramethylbenzidine dihydrochloride (TMB) was added to each well, forming a blue color. An absorbance plate-reader (HTS-7000, Perkin-Elmer Corp, Wellesley, MA) was used to read the results at 450 nm. A standard curve (10 points between 0 and 1000 ng/ml) was fit to the data using nonlinear regression (Graphpad Prism, Graphpad Software, San Diego CA) and the peptide concentrations in the unknown samples were computed by reference to the standard curve. The minimum detectable amount of ß-endorphin was about 5 pg/well, while the ED50 for ß-endorphin was around 100 pg/well. The vasopressin assay was a little more sensitive, with a minimum detectable amount of 1 pg/well of vasopressin, and an ED50 of about 16 pg/well. Intra-assay coefficients of variation (CV) were 68%; no interassay CVs were computed.
Statistical analysis.
The ß-endorphin and vasopressin data were evaluated by analysis of variance using Sigmastat Software (SPSS Science, Chicago, IL). Two-way analyses of variance (with sex and dose as the two independent "between" variables) revealed no differences between the sexes in hypothalamic levels of either peptide. Therefore, the ß-endorphin and vasopressin data were also analyzed using one-way ANOVAs with 4 levels of the variable, dose, and omitting the sex variable. Post hoc comparisons were performed according to Fishers least significant difference (LSD) approach, using a level of significance of p = 0.05.
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RESULTS |
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DISCUSSION |
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Previous research has reported that exogenous estrogenic compounds may either increase or decrease hypothalamic ß-endorphin levels, depending on the circumstances. Thus, a single dose of 10 mg of estradiol benzoate, injected (im) into ovariectomized and adrenalectomized rats, elevated ß-endorphin in the hypothalamus. This estradiol benzoate regimen also induced sexual receptivity in a subgroup of the treated animals; the increases in ß-endorphin were largest in the arcuate, ventromedial, and preoptic hypothalamic regions of the sexually receptive females (Medina et al., 1998).
In contrast, a single injection (im) of 2 mg of estradiol valerate (Brawer et al., 1993;Schipper et al., 1994
) resulted in neuronal loss (primarily of ß-endorphin immunopositive cells) in the arcuate nucleus of the hypothalamus of intact females or males. The neuronal loss was detectable by radioimmunoassay as a large decrease in hypothalamic content of ß-endorphin immunoreactivity (Desjardins et al., 1993
).
The studies reporting neuronal loss and decreased ß-endorphin immunoreactivity used a 200-fold higher dose of estradiol than the study that showed an estradiol-induced increase of ß-endorphin immunoreactivity. The decreased ß-endorphin immunoreactivity was a result of the pathological loss of peptide neurons following large doses of estradiol, while the increase in ß-endorphin immunoreactivity occurred at a much smaller dose of estrogen given to an adrenalectomized and ovariectomized animal. It was uncertain what effects, if any, to expect on hypothalamic ß-endorphin immunoreactivity, since we were using a phytoestrogen compound added chronically to the diet. The normal ß-endorphin levels we observed in the whole hypothalamus in the present study does suggest the absence of any pathological loss of ß-endorphin neurons. As in the Desjardin paper (1993) and in our own previous study of mice with hypothalamic lesions (Caputo et al., 1996), we should have measured decreased hypothalamic ß-endorphin if such a lesion was present. However, our data provided no evidence of any genistein-induced increases of ß-endorphin immunoreactivity in the hypothalamus, either. In the present study, the animals were both gonadally and adrenally intact and may have reacted differently to estrogenic exposure compared to the study of Medina et al., 1998
, which measured ß-endorphin in microdissected regions of the hypothalamus and examined the effects of a replacement dose of estradiol injected into ovariectomized rats. Our study investigated the effects of a phytoestrogen added to the diet of an intact animal on ß-endorphin in the whole hypothalamus. Thus these data are not in conflict, since they stem from studies involving different estrogenic compounds and dose ranges of exposure and are methodologically distinct. Our ß-endorphin data may have been hampered somewhat by the large individual variation between animals, perhaps due to variation in the hypothalamic dissection. If this error-term variance were to be reduced, perhaps a genistein-induced effect on ß-endorphin could yet be demonstrated.
Vasopressin
The levels of hypothalamic vasopressin we observed by ELISA were somewhat lower than in previous reports using radioimmunoassay methods (Epstein et al., 1983;van der Sluis et al., 1986
). We are uncertain whether variation between rat strains, sample storage conditions, etc. may have accounted for the differences; however, all our samples were handled identically and as a single batch throughout the analyses.
In several previous studies (Crowley and Amico, 1993Hashimoto et al., 1981;
Kucharczyk, 1984;
Levin and Sawchenko, 1993;
Roy et al., 1999;
Shapiro et al., 2000
), exogenous estrogens given to ovariectomized, or ovariectomized/adrenalectomized female rats have altered the levels of hypothalamic vasopressin. Likewise, reports have indicated differences in hypothalamic vasopressin between female rats sacrificed during different stages of the estrous cycle (Kucharczyk, 1984
). In the present study, we did not evaluate the stage of the estrous cycle in our female rats at the time of sacrifice. However, variability due to estrous stage might reduce the statistical power of our tests, but it could not be expected to produce a systematic increase limited only to the high-dose genistein group. Also, the high-dose genistein male group showed a similar response to that of the female group, allowing this data to be combined for presentation in Figure 2
. Our present data is in agreement with previous reports indicating no prominent differences in vasopressin levels between untreated, gonadally intact male and female rats (Krajnak et al., 1998;
Magnusson and Meyerson, 1996
).
Vasopressin is not the only neurosecretory peptide that might be affected by exposure to estrogenic endocrine disruptor compounds. For example, some hypothalamic magnocellular neurons express and secrete primarily oxytocin (OT) instead of vasopressin. Physiologically, oxytocin mediates such things as milk elaboration and letdown reflexes in the breast, as well as uterine contractions. Using single-cell RT-PCR, Glasgow et al.(1999) showed that neurons primarily expressing OT mRNA also expressed some vasopressin mRNA, and vice versa. Analyses of these neurons for coexisting peptide mRNAs also revealed corticotropin releasing hormone (CRH), cholecystokinin, galanin, dynorphin, and the calcium-binding protein calbindin, as well as high voltage-activated calcium channel subunit genes.
Using immunohistochemical staining methods, Levin and Sawchenko observed the same set of neuropeptides described by Glasgow et al. (above), plus an additional blood pressure-regulating peptide, angiotensin II (Levin and Sawchenko, 1993
). Their observations suggested that CRH, CCK, and vasopressin all showed evidence of enhanced expression after treatment with 17ß estradiol. Using in situ hybridization in the 17ß estradiol-treated rhesus monkey hypothalamus, Roy et al.(1999)
showed that the CRH message was increased in the magnocellular neurons, but that there was no change in vasopressin mRNA amounts. Such an observation of unchanged mRNA concentration, however, remains consistent with the general findings reported above of increased vasopressin peptide, since the amount of vasopressin mRNA may not be rate-limiting for vasopressin synthesis.
Estradiol is not the only estrogenic compound previously shown to alter hypothalamic magnocellular peptides. Diethylstilbesterol (DES, 70 mg/day (sc) for 2 days increased OT but decreased met-enkephalin in magnocellular neurons, as measured by RIA (Schriefer, 1991). Vasopressin was also elevated, by about 10%, but the effect was not statistically reliable. Term-pregnant rats (like the DES-treated rats) had elevated OT and decreased met-enkephalin levels. However, the term-pregnant rats also showed statistically significant increase in both vasopressin and dynorphin levels. Schriefer (1991)
also reported mRNA measurements consistent with their radioimmunoassay results. These findings suggested the possibility of different, but overlapping, actions of DES compared to the effects of endogenous steroids elevated by pregnancy. Thus estrogenic compounds are clearly important factors regulating hypothalamic neurosecretion, which in turn may influence neuroreproductive function.
Limited neuropeptide data is available from studies of nonsteroidal estrogenic compounds given at various doses by different routes of administration. Our present data indicates that chronic dietary exposure to 1250 ppm of genistein is sufficient to increase hypothalamic vasopressin. Such changes in vasopressin might have functional consequences for the regulation of blood pressure, fluid intake, and/or reproductive functions (He et al., 1999). Moreover, the dose-response function for vasopressin elevation seen in the present study is nearly identical to the dose-response relationship by which genistein increases rodents preference for drinking salt water over tap water (Flynn et al., 2000
). In that study, as in the present one, only the 1250-ppm genistein group showed a significant difference from controls. Moreover, the percent increase in salt preference compared to control reported by Flynn et al.(2000)
was similar to the percent increase in vasopressin levels reported here (see Fig. 2
). Neuropeptide analysis, in conjunction with functional/behavioral measures, appears to be a useful tool for better characterizing the neurotoxicology of estrogenic endocrine disruptor compounds.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Department of Microbiology, Forensic Science Program, 406 Kaul Human Genetics Building, University of AlabamaBirmingham, Birmingham, AL 35205.
3 Present address: Schering-Plough Research Institute, Lafayette, NJ 07848.
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