Increased Volume of the Calbindin D28k-Labeled Sexually Dimorphic Hypothalamus in Genistein and Nonylphenol-Treated Male Rats

A. C. Scallet*,1, R. L. Divine{dagger}, R. R. Newbold{ddagger} and K. B. Delclos§

* Division of Neurotoxicology National Center for Toxicological Research/FDA, Jefferson, Arkansas 72079; {dagger} Charles River Laboratories, Jefferson, Arkansas 72079; {ddagger} Environmental Toxicology Program, National Institute for Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709; and § Division of Biochemical Toxicology, National Center for Toxicological Research/FDA, Jefferson, Arkansas 72079

Received July 16, 2004; accepted September 7, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The adult rat brain develops through an interplay of neuronal proliferation and programmed cell death. Steroid hormones and growth factors may alter the balance between these competing processes. "Endocrine disrupters" (EDs) may also alter brain development, by mimicry or modulation of endogenous hormone systems. Under control conditions, the sexually dimorphic nucleus (SDN) of the medial preoptic hypothalamus becomes larger in adult males than females, but its final volume may also reflect the hormonal conditions prevailing during development. Two EDs that have recently been studied in protocols involving lifespan exposures are the phytoestrogen genistein and the weakly estrogenic compound para-nonylphenol, which is used in the production of many surfactants and plastics. Experimental dietary exposure of adult female rats to genistein or p-nonylphenol began 28 days prior to their mating at concentrations of 5 ppm, 100 ppm, and 500 ppm for genistein or 25 ppm, 200 ppm, and 750 ppm for p-nonylphenol. Exposure of the offspring continued throughout gestation and lactation, as well as in their chow after weaning, until they were sacrificed at 140 days of age for immunohistochemical labeling of the calbindin D28k-labeled subdivision of the SDN: the CALB-SDN. Both genistein and nonylphenol were found to increase the volume of the CALB-SDN in male rats (p's < 0.01), but not in female rats.

Key Words: medial preoptic nucleus; hypothalamus; 3-D reconstruction; endocrine disrupters; estrogens; sex differences; calcium binding proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endocrine disrupters (EDs) are chemicals found in the environment that mimic or modulate the physiological effects of endogenous hormones and that may contribute to adverse effects observed in wildlife and humans (National Research Council, 1999Go). The National Toxicology Program has established an interagency agreement between the National Institute of Environmental Health Sciences and the National Center for Toxicological Research designed to evaluate the effects of a range of doses of endocrine disrupters on hormone-sensitive tissues over multiple generations in Sprague-Dawley rats. Among the compounds selected were genistein and p-nonylphenol. Genistein is a phytoestrogen found in many soy products consumed by humans (Doerge et al., 2000aGo, 2001Go; Strauss et al., 1998Go; Wu et al., 2004Go), while p-nonylphenol is an intermediate used in the production of alkylphenol ethoxylate surfactants and plastics that can be released into the environment upon breakdown of these products (Doerge et al., 2002Go).

Measurements of the anatomical development of hormonally sensitive and sexually dimorphic brain structures in rats, such as the SDN (Bleier et al., 1982Go; Gorski, 1985Go), may be useful as biomarkers of the effects of exposure to endocrine disrupters. It is known that SDN structure may be altered following perinatal exposure to either estrogens or androgens; the androgens, however, must first be aromatized to estrogens in order to affect the rat SDN (Dohler, 1991Go). An irreversible syndrome then follows estrogen exposure during a critical perinatal period. The typical pattern of development of ER-containing neurons of the SDN is altered, and a distinctive pattern of neurobehavioral changes takes place (Davis et al., 1995Go, 1996bGo).

Ordinarily, the female rat hypothalamus appears to develop according to an orchestrated removal of a number of its SDN neurons by programmed cell death, i.e., apoptosis (Davis et al., 1996aGo). Suspension or reduction of this process of programmed cell death, such as may occur with exposure to estrogen or testosterone, results in the male pattern of hypothalamic development. However, females usually have little testosterone and their endogenous estrogen is excluded from the brain due to its forming a complex with the circulating steroid binding protein, {alpha}-fetoprotein (Milligan et al., 1998Go). Thus their adult SDNs achieve the characteristically smaller female pattern during development.

The male rat hypothalamus maintains a large population of SDN neurons throughout development. Since their circulating testosterone is not bound to {alpha}-fetoprotein, it has access to the hypothalamus where a cytochrome P450 enzyme, CYP19 (Abdelgadir et al., 1997Go; Yamada-Mouri et al., 1995Go), also known as "aromatase," converts the testosterone to estrogen (Roselli et al., 1997Go). In the ordinary course of events for male rats, their hypothalamic neuronal ERs then remain liganded with estrogen produced by the aromatization of their circulating testosterone. These liganded ERs form dimers that bind to nuclear estrogen response elements (EREs), which can then promote the synthesis of nerve growth factor(s) and/or their "trk-A" family (tyrosine kinase) receptors, which result in the down regulation of apoptosis (Gibbs, 1998Go; Toran-Allerand, 1996Go). The result is the characteristically larger adult male rat SDN.

Under more extraordinary or experimental circumstances, the structure of the genetic female hypothalamus may become "masculinized" (Faber and Hughes, 1992Go; Lewis et al., 2003Go). A masculinized female SDN may result from perinatal exposure to testosterone (which is aromatized to estradiol), by excessive levels of estrogen (more than can be bound by circulating {alpha}-fetoprotein), or by moderate amounts of any estrogenic compound that fails to bind to the protective {alpha}-fetoprotein.

The occurrence of a "feminized" SDN in a genetic male may likewise occur: insufficient testosterone (as a source of estrogen) may be available, aromatase activity may be suppressed, or estrogen receptors may be absent or blocked by an antagonist (Amin et al., 2000Go; Ogawa et al., 1997Go; Resko and Roselli, 1997Go).

Finally, "hypermasculinized" genetic males, both behaviorally and neurologically, have been observed to occur when excessive testosterone or estrogenic compounds are present during development in songbirds (Mathews et al., 1988Go; Mathews and Arnold, 1991Go; Schlinger and Arnold, 1991Go). Under such circumstances, apoptosis may be even more completely suppressed than in control males that have been exposed only to their own endogenous source of testosterone during development (Davis et al., 1996aGo; Levy et al., 1995Go). However, studies of estrogenic or androgenic compounds in male rats have not usually observed an enlargement of the SDN (Faber and Hughes, 1991Go; Nagao et al., 1999Go; Register et al., 1995Go).

Changes in the reproductive function and behavior of rats who have undergone masculinization or feminization of the SDN have also been reported (Ogawa et al., 1997Go; Rhees et al., 1999Go). Masculinization of genetic female rats results in delayed and more difficult induction of sexual receptivity (lordosis). Feminization of genetic male rats may slow or prevent them from mounting a stimulus female rat or increase the probability that they will exhibit the female pattern of lordosis after appropriate hormonal priming. Less is known about functional changes in adult hypermasculinized male rats.

Toxicological studies of endocrine disrupters have attempted to utilize measurements of the SDN as biomarkers of developmental exposure, with variable results. In female rats, injections of 500 or 1000 µg/day genistein sc during postnatal days 1–10 increased the SDN volume while there were no significant effects on males (Faber and Hughes, 1991Go, 1993Go). With prenatal (GD 16–20) administration of 5000 µg/day genistein to the dam, a decrease in SDN volume in females was observed (Levy et al., 1995Go). A study of prenatal exposure through the maternal diet beginning on GD 7, followed by postnatal lactational exposure of the offspring and continued exposure after weaning until PND 50 reported that both genistein and nonylphenol decreased SDN volumes in males, but was without effect in females (Ferguson et al., 2000bGo; Slikker et al., 2001Go). Genistein at 40 mg/kg, but not 4 mg/kg, given sc on PND 1–7 as well as po from PND 8–21 increased the female, but not the male, SDN size (Lewis et al., 2003Go). This study, using a partially overlapping period of developmental exposure and the same route of genistein administration at least for PND 1–7, revealed an increase in female SDN volume, as previously reported (Faber and Hughes, 1991Go, 1993Go). Dietary exposure of dams to up to 1000 ppm of genistein (Masutomi et al., 2003Go) and up to 3000 ppm of bisphenol-A and nonylphenol (Takagi et al., 2004Go) from GD 15 through postnatal day 10 was without effect on the SDNs of either males or females. Exposure to up to 320 mg/kg/day of bisphenol-A po from GD 11 through PND 20 was also without effect on the volume of the SDN (Kwon et al., 2000Go), as was bisphenol-A given at a dose of 300 µg/kg sc from postnatal days 1–5 (Nagao et al., 1999Go). These studies may be inconclusive due to wide differences between protocols in the developmental timing and route of administration of the compounds as reviewed above. In addition, measurement of the SDN volume is technically difficult, as is apparent from the wide variability reported both within and between laboratories, as previously reviewed (Meredith et al., 2001Go).

Recently, immunohistochemical labeling of calbindin D28k was shown to identify a subpopulation of neurons within the SDN, called the CALB-SDN, that could be measured with less variability than Nissl-stained SDN neurons (Lephart et al., 1998Go; Sickel and McCarthy, 2000Go). The calbindin-labeled neuronal population, like Nissl-stained SDN neurons, increased in volume when exposed to perinatal testosterone or estradiol, and proceeded through early development at about the same rate.

Since SDN volume has often been used as a biomarker of estradiol or ED exposure (Faber and Hughes, 1991Go, 1993Go; Faber et al., 1993Go; Ferguson et al., 2000bGo; Kwon et al., 2000Go; Masutomi et al., 2003Go; Nagao et al., 1999Go; Register et al., 1995Go; Slikker et al., 2001Go; Vancutsem and Roessler, 1997Go), we thought the volume of the calbindin D28k immunoreactive SDN (CALB-SDN) neurons (Taylor et al., 1999Go; Sickel and McCarthy, 2000Go) might also comprise a suitable biomarker of exposure to estrogenic endocrine disruptors. For this purpose, a desirable method of measuring calbindin immunoreactive SDN volume should provide reproducible, accurate, and unbiased measurements suitable for statistical analysis, with sufficient ease and rapidity so that a large number of brains can be processed. We developed an approach utilizing the methodology of 3-D reconstruction to provide quantitative estimates of the volume of the CALB-SDN in experimental animals (Meredith et al., 2001Go). As a test of the practical application of these methods, we measured the CALB-SDN of rats following lifetime dietary exposure to either genistein or para-nonylphenol.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and groups. All animal handling and treatment procedures were approved by the NCTR Institutional Animal Care and Use Committee. Female adult Sprague-Dawley rats were date-mated in the NCTR breeding colony. Experimental dietary exposure to genistein (10 control litters and 10 litters per dose group) or p-nonylphenol (10 control litters and 10 litters per dose group) began by adding the endocrine disrupters to the dams' chow 28 days prior to mating. The chow contained either control levels of genistein or p-nonylphenol (less than 1 ppm), or were supplemented to final concentrations of 5 ppm, 100 ppm, or 500 ppm for genistein or 25 ppm, 200 ppm, or 750 ppm for p-nonylphenol. Offspring were culled to four males and four females per litter on postnatal day 2 (PND 2), and the dams remained on the experimental diets throughout lactation, whereupon the offspring were continued on the same diets until they were sacrificed (gonadally intact) at 140 days of age (PND 140). All female rats were cycling at this age, but the stage of estrus was not taken into account in the neurohistochemical analyses of the animals. One male and one female rat from each litter were designated for 3-D reconstruction of the CALB-SDN: no two same-sexed rats of a treatment group were from the same litter. The AALAC-approved vivarium was maintained under controlled environmental conditions (temperature 22°C, relative humidity 50%, 12 h light:dark cycle with lights out at 1800 h); rats were housed in plexiglas cages with hardwood chips for bedding. Water and experimental rat diets were available ad libitum.

Chemical Exposures
Control diet. The basal (control) diet was irradiated 5K96 meal that contains 3.13 kcal/g, 20% 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 were replaced by casein and soy oil was replaced by corn oil. 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 (Doerge et al., 2000bGo).

Genistein. 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.

P-nonylphenol. Nonylphenol, obtained from Schenectady International (Schenectady, NY), consisted of 95% branched side chain isomers of 4-NP and 5% of branched side chain isomers of 2-NP as determined using 1H- and 13C-NMR.

Choice of doses. Complete details regarding feed consumption and body weights from a related dose range-finding study have been published elsewhere (Delclos et al., 2001Go). The doses of genistein used in the present study covered the dose range used in previous studies (Delclos et al., 2001Go; Ferguson et al., 2002Go; Flynn et al., 2000aGo,bGo) so that the CALB-SDN results could be compared to other known effects of genistein. The doses of p-nonylphenol employed here were the same as doses used in previous studies of other endpoints (Doerge et al., 2002Go; Ferguson et al., 2002Go).

Treatment groups. Forty dams were exposed to genistein and forty dams to p-nonylphenol in their chow beginning 28 days prior to being mated. Four groups of male rats (n = 10) and four groups of female rats (n = 5) from independently dosed litters were then continuously exposed either to genistein (control, 5 ppm, 100 ppm, or 500 ppm) or p-nonylphenol (control, 25 ppm, 200 ppm, or 750 ppm) added to their dam's chow throughout gestation and lactation and to their own chow from the time of weaning until sacrifice at PND 140. Fewer female rats per group were designated for CALB-SDN measurements because our previous work (Ferguson et al., 2000bGo; Slikker et al., 2001Go) had indicated that the effects of genistein were restricted to males.

Tissue preparation. Brain tissue was collected from 120 gonadally intact Sprague-Dawley rats sacrificed by asphyxiation at 140 days of age. Tissues were postfixed in 4% formaldehyde at 22°C until further processing. These rat brains were obtained from the F1 generation of multigenerational studies conducted at NCTR, and additional details of the experiments are available (Ferguson et al., 2002Go; Flynn et al., 2000bGo).

The brains were placed in a stainless steel coronal brain matrix (Ted Pella, Inc., Redding, CA) and the slab containing the sexually dimorphic region of the hypothalamus (Paxinos and Watson, 1986Go) was removed using a single-edged razor blade. The resulting slab of tissue was then mounted on a rubber block and immersed in phosphate buffer for sectioning with a vibrating microtome.

Tissue sectioning and staining. Briefly, forty µm thick serial sections were cut using a vibrating microtome (The Vibratome Company, St. Louis, MO). Sections were rinsed three times for 5 min each in 0.1 M phosphate buffer (pH 7.4). The sections were then kept free-floating overnight in 24-well baskets at 5°C in rabbit anti-calbindin D28k primary antisera (Sigma Chemical Company, St. Louis, MO) diluted 1:500 in antibody diluent (Scallet, 1995Go; Scallet et al., 1988Go). The next day they were rinsed in 0.1 M phosphate buffer and incubated for 1 h in goat anti-rabbit secondary antisera (Jackson Immuno Research, West Grove, PA) diluted 1:100 in antibody diluent. Finally, they were rinsed again in buffer and incubated for 1 h in rabbit peroxidase/anti-peroxidase complex (Jackson ImmunoResearch, West Grove, PA) diluted 1:100 in buffer. They were then rinsed again and stained for 10 min using diaminobenzidine (DAB, Sigma Chemical Company, St. Louis, MO) as substrate for the peroxidase. The DAB creates a brown stain where the primary antisera was localized. Finally, the sections were mounted on glass slides and coverslipped with Permount.

Three-dimensional reconstruction. Briefly, the imaging system was calibrated by using a certified measurement standard which was digitally photomicrographed using each microscope objective (Meredith et al., 2001Go). Then the known length (in mm) of the line on the measurement standard was divided by the number of pixels (individual data points forming the digital image) corresponding to that length. From the number of mm per pixel, the volume of a "voxel" (cubical pixel) can then be determined by cubing the length of a single pixel. With the computer mouse, outlines were drawn around the immunolabeled calbindin-positive cells in each section (see Fig. 1). The tracings were then filled to facilitate the three-dimensional reconstruction. A computer generated three-dimensional rendering containing the calbindin-positive cells was then created from a stack of about 4–6 sequential sections. Volume measurements of the calbindin-positive neurons within the SDN were obtained as the number of voxels, times the size of each voxel in cubic microns, that were contained within the surface rendered over the calbindin-positive cell group outlined within the SDN (see Figs. 1e and 1f). To validate this method, a set of 10 different animals with obscured identifications was reconstructed by two independent observers (A.C.S. and B.L.D.), with the resulting inter-rater correlation coefficient of r = 0.949, p < 0.0001.



View larger version (129K):
[in this window]
[in a new window]
 
FIG. 1. Three-dimensional reconstructions of the CALB-SDN of a male and female control rat (each 140 days old). (a) A micrograph of calbindin-immunopositive neurons in the anterior medial basal hypothalamus near the wall of the third ventricle in a section from a male control rat (x10). (b) A micrograph of calbindin-immunopositive neurons in the anterior medial basal hypothalamus near the wall of the third ventricle in a section from a female control rat (x10). (c) Shows an outline around the CALB-SDN drawn using a mouse and the MCID Elite Version 6 computer program on the control male section (x10). (d) Shows an outline around the CALB-SDN drawn on the control female section (x10). (e) Shows the resultant 3-D reconstruction of the control male CALB-SDN, while (f) shows the 3-D reconstruction from the control female CALB-SDN. The computer program fits a surface or "skin" over the series of adjacent sections taken through the CALB-SDN and the volume of each rat's CALB-SDN can be obtained as the total number of voxels contained within the rendered surface times the volume of each voxel (known from the calibration steps).

 
Statistical analysis. Two-way analyses of variance (SigmaStat, Cary, NC) were performed on the volume data from each experiment (genistein and nonylphenol), with dose of the endocrine disrupter (four levels) and sex (two levels) as factors. In the presence of a significant Main or Interaction effect by ANOVA, Fisher's Least Significant Difference (LSD) post hoc tests comparing individual means were carried out. Results were considered significant at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Variability of Calb-SDN Volume Measurements in Controls
The variance in male control animals of the present study was about 9.84 µm3 x 106, while the variance for control females was about 1.47 µm3 x 106.

Genistein
Genistein exposure caused a significant increase in the volume occupied by the calbindin-immunopositive neurons within the SDN (F(3,52) = 4.3, p < 0.01). There was also a significant difference between the sexes (F(1,52) = 62.2, p < 0.001). Although the interaction between genistein-treatment and sex was not statistically significant (F(3,52) = 2.00, p > 0.10), individual post-hoc tests (see Fig. 2) indicated that the effects of genistein exposure were restricted to the males. The CALB-SDNs of the genistein-treated female groups were not significantly different in volume from their female control group (all p's > 0.10).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 2. The effects of lifetime dietary exposure to genistein on the volume of the CALB-SDN. A dose-related increase occurred in the males (*indicates p < 0.01 greater than the male control group), but the females, though significantly smaller in volume than the males (p < 0.001), showed no effect of treatment with genistein (p > 0.10).

 
Nonylphenol
Nonylphenol exposure did not significantly increase the volume occupied by the calbindin-immunopositive neurons within the SDN (F(3,52) = 1.9, p > 0.10). However, there was a significant difference between the sexes (F(1,52) = 45.5, p < 0.001). Although the interaction between genistein-treatment and sex was not statistically significant (F(3,52) = 1.2, p > 0.10), individual post-hoc tests (see Fig. 3) indicated that the 25 ppm and 200 ppm groups of males were significantly larger than the male controls (p's < 0.01). The CALB-SDN volume of the 750 ppm group was not different from controls (p > 0.10), suggesting an "inverted U" shaped dose-response relationship. The CALB-SDNs of the nonylphenol-treated female groups were not significantly different in volume from their female control group (all p's > 0.10).



View larger version (23K):
[in this window]
[in a new window]
 
FIG. 3. The effects of lifetime exposure to p-nonylphenol on the volume of the CALB-SDN. An inverted-U pattern of increase occurred in the males (*indicates p < 0.01 greater than the male control group), but the females, although significantly smaller in volume than the males (p < 0.001), showed no effect of treatment with p-nonylphenol (p > 0.10).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Visual Representation and Measurement of the CALB-SDN Portion of the Hypothalamus
We have delineated the calbindin-immunopositive neurons within the anterior ventral hypothalamic area of both male and female rats (Fig. 1). In addition to visualizing these structures three-dimensionally, the volume of the CALB-SDN was measured based on the computer's summation of the total number of voxels contained in the rendered object times the calibrated volume of each voxel. There was a clear sexual dimorphism in the computed CALB-SDN volumes: male CALB-SDNs were {approx} 3 times larger than their female counterparts in both experiments. This ratio is similar to what has typically been reported for the Nissl-stained rat SDN in previous studies (Meredith et al., 2001Go). Moreover, the variance that we measured in the present study for the volume of the CALB-SDN of male control rats (9.84 µm3 x 106) was smaller than the variances for the Nissl-SDN that were reported in six out of the nine studies reviewed previously (Meredith et al., 2001Go). Similarly, the variance of the mean of the CALB-SDN measured in our female rats (1.47 µm3 x 106) was smaller than the variances for the Nissl-SDN, but only in three out of the seven studies reviewed. Future studies will be required to determine whether the means for SDN volume as determined using the CALB-SDN methodology are a more accurate or reproducible biomarker of ED exposure than measurement of the Nissl-stained SDN.

Genistein
In the present study, there is a rather clear enlargement of the CALB-SDN in male rats with increasing lifetime dietary exposure to genistein. Given the estrogenic properties of genistein, this observation may correspond to a previous report of increased calbindin content of the medial basal hypothalamic preoptic area (MBH-POA) of prepubertal male rats treated with estrogen, as measured by Western blots (Stuart et al., 2001Go). However, it fails to correspond to the observation that, in the adult male rat, phytoestrogen exposure decreased calbindin content in the MBH-POA by Western blot (Lephart et al., 2000Go). These observations taken together may indicate that in life-long exposures of male rats to estrogenic compounds, the perinatal exposure "trumps" the adult exposure and exerts the ultimate controlling influence on the MBH-POA content of calbindin. It indicates that the occurrence of a "hypermasculinized" rat may be expected with lifetime exposure to genistein, such as might be expected from excessive activation of hypothalamic estrogen receptors during the perinatal period.

P-Nonylphenol
As with genistein, p-nonylphenol appeared to increase the size of the CALB-SDN, at least at the intermediate doses used here. There was a suggestion of a plateau or reduction of activity at the highest dose of p-nonylphenol (750 ppm). Taken together with the genistein data, it appears that lifetime dietary exposure to endocrine disrupters with estrogenic activity may result in an enlarged CALB-SDN and a "hypermasculinized" male. The functional consequences of these effects are uncertain, as neither compound was observed to effect sexually dimorphic behaviors such as open field or running wheel activity (Ferguson et al., 2000aGo; Flynn et al., 2000aGo), nor did they alter play behavior (Ferguson et al., 2000aGo; Flynn et al., 2000aGo) or the nursing behavior of dam/offsping dyads (Flynn et al., 2000bGo). The most consistent functional effect of these endocrine disrupters was an increased intake of sodium chloride solutions (Ferguson et al., 2000aGo; Flynn et al., 2000aGo), probably mediated by increased hypothalamic vasopressin (Scallet et al., 2003Go), but these effects occurred in both males and females and thus are unlikely to be related to the "hypermasculinization" effect of estrogenic endocrine disrupters on the male CALB-SDN. It appears from a careful review of the literature that variations in the precise timing of the exposure to an ED, e.g., pregestational (which may influence the capacity of the maternal binding proteins), gestational, perinatal, prepubertal, or adult) and the various routes of administration (SC, PO, lactational, through the chow) may have contributed to the variability of results obtained. Very few studies have employed exactly the same exposure procedures. Perhaps the CALB-SDN measurement procedures will help contribute to resolving these differences.


    ACKNOWLEDGMENTS
 
The advice, assistance, and comments of Drs. Dan Doerge, William Allaben, Sherry Ferguson, Sarah James, and Katherine Flynn (NCTR) as well as John Bucher (NIEHS, National Toxicology Program) and John Meredith (Schering-Plough Corp.) are greatly appreciated.


    NOTES
 
Support for this research was provided by an Interagency agreement between NIEHS, National Toxicology Program, Research Triangle Park, NC 27709 and the National Center for Toxicological Research, USFDA. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

1 To whom correspondence should be addressed at National Center for Toxicological Research, HFT-132, 3900 NCTR Drive, Jefferson, AR 72079. Fax: (870) 543-7745. E-mail: ascallet{at}nctr.fda.gov.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abdelgadir, S. E., Roselli, C. E., Choate, J. V., and Resko, J. A. (1997). Distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains. Biol. Reprod. 57, 772–777.[Abstract]

Amin, S., Moore, R. W., Peterson, R. E., and Schantz, S. L. (2000). Gestational and lactational exposure to TCDD or coplanar PCBs alters adult expression of saccharin preference behavior in female rats. Neurotoxicol. Teratol. 22, 675–682.[CrossRef][ISI][Medline]

Bleier, R., Byne, W., and Siggelkow, I. (1982). Cytoarchitectonic sexual dimorphisms of the medial preoptic and anterior hypothalamic areas in guinea pig, rat, hamster, and mouse. J. Comp. Neurol. 212, 118–130.[ISI][Medline]

Davis, E. C., Popper, P., and Gorski, R. A. (1996a). The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area. Brain Res. 734, 10–18.[CrossRef][ISI][Medline]

Davis, E. C., Shryne, J. E., and Gorski, R. A. (1995). A revised critical period for the sexual differentiation of the sexually dimorphic nucleus of the preoptic area in the rat. Neuroendocrinology 62, 579–585.[ISI][Medline]

Davis, E. C., Shryne, J. E., and Gorski, R. A. (1996b). Structural sexual dimorphisms in the anteroventral periventricular nucleus of the rat hypothalamus are sensitive to gonadal steroids perinatally, but develop peripubertally. Neuroendocrinology 63, 142–148.[ISI][Medline]

Delclos, K. B., Bucci, T. J., Lomax, L. G., Latendresse, J. R., Warbritton, A., Weis, C. C., and Newbold, R. R. (2001). Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats. Reprod. Toxicol. 15, 647–663.[CrossRef][ISI][Medline]

Doerge, D. R., Chang, H. C., Churchwell, M. I., and Holder, C. L. (2000a). Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry. Drug Metab. Dispos. 28, 298–307.[Abstract/Free Full Text]

Doerge, D. R., Churchwell, M. I., Chang, H. C., Newbold, R. R., and Delclos, K. B. (2001). Placental transfer of the soy isoflavone genistein following dietary and gavage administration to Sprague Dawley rats. Reprod. Toxicol. 15, 105–110.[CrossRef][ISI][Medline]

Doerge, D. R., Churchwell, M. I., and Delclos, K. B. (2000b). On-line sample preparation using restricted-access media in the analysis of the soy isoflavones, genistein and daidzein, in rat serum using liquid chromatography electrospray mass spectrometry. Rapid Commun. Mass Spectrom. 14, 673–678.[CrossRef][ISI][Medline]

Doerge, D. R., Twaddle, N. C., Churchwell, M. I., Chang, H. C., Newbold, R. R., and Delclos, K. B. (2002). Mass spectrometric determination of p-nonylphenol metabolism and disposition following oral administration to Sprague-Dawley rats. Reprod. Toxicol. 16, 45–56.[CrossRef][ISI][Medline]

Dohler, K. D. (1991). The pre- and postnatal influence of hormones and neurotransmitters on sexual differentiation of the mammalian hypothalamus. [Review]. Int. Rev. Cytol. 131, 1–57.[Medline]

Faber, K. A., Ayyash, L., Dixon, S., and Hughes, C. L., Jr. (1993). Effect of neonatal diethylstilbestrol exposure on volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus and pituitary responsiveness to gonadotropin-releasing hormone in female rats of known anogenital distance at birth. Biol. Reprod. 48, 947–951.[Abstract]

Faber, K. A., and Hughes, C. L., Jr. (1991). The effect of neonatal exposure to diethylstilbestrol, genistein, and zearalenone on pituitary responsiveness and sexually dimorphic nucleus volume in the castrated adult rat. Biol. Reprod. 45, 649–653.[Abstract]

Faber, K. A., and Hughes, C. L., Jr. (1992). Anogenital distance at birth as a predictor of volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus and pituitary responsiveness in castrated adult rats. Biol. Reprod. 46, 101–104.[Abstract]

Faber, K. A., and Hughes, C. L., Jr. (1993). Dose-response characteristics of neonatal exposure to genistein on pituitary responsiveness to gonadotropin releasing hormone and volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in postpubertal castrated female rats. Reprod. Toxicol. 7, 35–39.[CrossRef][ISI][Medline]

Ferguson, S. A., Flynn, K. M., Delclos, K. B., and Newbold, R. R. (2000a). Maternal and offspring toxicity but few sexually dimorphic behavioral alterations result from nonylphenol exposure. Neurotoxicol. Teratol. 22, 583–591.[CrossRef][ISI][Medline]

Ferguson, S. A., Flynn, K. M., Delclos, K. B., Newbold, R. R., and Gough, B. J. (2002). Effects of lifelong dietary exposure to genistein or nonylphenol on amphetamine-stimulated striatal dopamine release in male and female rats. Neurotoxicol. Teratol. 24, 37–45.[CrossRef][ISI][Medline]

Ferguson, S. A., Scallet, A. C., Flynn, K. M., Meredith, J. M., and Schwetz, B. A. (2000b). Developmental neurotoxicity of endocrine disrupters: Focus on estrogens. Neurotoxicology 21, 947–956.[ISI][Medline]

Flynn, K. M., Ferguson, S. A., Delclos, K. B., and Newbold, R. R. (2000a). Effects of genistein exposure on sexually dimorphic behaviors in rats. Toxicol. Sci. 55, 311–319.[Abstract/Free Full Text]

Flynn, K. M., Ferguson, S. A., Delclos, K. B., and Newbold, R. R. (2000b). Multigenerational exposure to dietary genistein has no severe effects on nursing behavior in rats. Neurotoxicology 21, 997-1001.[ISI][Medline]

Gibbs, R. B. (1998). Levels of trkA and BDNF mRNA, but not NGF mRNA, fluctuate across the estrous cycle and increase in response to acute hormone replacement. Brain Res. 787, 259–268.[CrossRef][ISI][Medline]

Gorski, R. A. (1985). The 13th J. A. F. Stevenson memorial lecture. Sexual differentiation of the brain: possible mechanisms and implications. Can. J. Physiol. Pharmacol. 63, 577–594.[ISI][Medline]

Kwon, S., Stedman, D. B., Elswick, B. A., Cattley, R. C., and Welsch, F. (2000). Pubertal development and reproductive functions of Crl:CD BR Sprague-Dawley rats exposed to bisphenol A during prenatal and postnatal development. Toxicol. Sci. 55, 399–406.[Abstract/Free Full Text]

Lephart, E. D., Taylor, H., Jacobson, N. A., and Watson, M. A. (1998). Calretinin and calbindin-D28K in male rats during postnatal development. Neurobiol. Aging 19, 253–257.[CrossRef][ISI][Medline]

Lephart, E. D., Thompson, J. M., Setchell, K. D., Adlercreutz, H., and Weber, K. S. (2000). Phytoestrogens decrease brain calcium-binding proteins but do not alter hypothalamic androgen metabolizing enzymes in adult male rats. Brain Res. 859, 123–131.[CrossRef][ISI][Medline]

Levy, J. R., Faber, K. A., Ayyash, L., and Hughes, C. L., Jr. (1995). The effect of prenatal exposure to the phytoestrogen genistein on sexual differentiation in rats. Proc. Soc. Exp. Biol. Med. 208, 60–66.[Abstract]

Lewis, R. W., Brooks, N., Milburn, G. M., Soames, A., Stone, S., Hall, M., and Ashby, J. (2003). The effects of the phytoestrogen genistein on the postnatal development of the rat. Toxicol. Sci. 71, 74–83.[Abstract/Free Full Text]

Masutomi, N., Shibutani, M., Takagi, H., Uneyama, C., Takahashi, N., and Hirose, M. (2003). Impact of dietary exposure to methoxychlor, genistein, or diisononyl phthalate during the perinatal period on the development of the rat endocrine/reproductive systems in later life. Toxicology 192, 149–170.[CrossRef][ISI][Medline]

Mathews, G. A., and Arnold, A. P. (1991). Tamoxifen's effects on the zebra finch song system are estrogenic, not antiestrogenic. J. Neurobiol. 22, 957–969.[ISI][Medline]

Mathews, G. A., Brenowitz, E. A., and Arnold, A. P. (1988). Paradoxical hypermasculinization of the zebra finch song system by an antiestrogen. Horm. Behav. 22, 540–551.[ISI][Medline]

Meredith, J. M., Bennett, C., and Scallet, A. C. (2001). A practical three-dimensional reconstruction method to measure the volume of the sexually-dimorphic central nucleus of the medial preoptic area (MPOC) of the rat hypothalamus. J. Neurosci. Methods 104, 113–121.[CrossRef][ISI][Medline]

Milligan, S. R., Khan, O., and Nash, M. (1998). Competitive binding of xenobiotic oestrogens to rat alpha-fetoprotein and to sex steroid binding proteins in human and rainbow trout (Oncorhynchus mykiss) plasma. Gen. Comp. Endocrinol. 112, 89–95.[CrossRef][ISI][Medline]

Nagao, T., Saito, Y., Usumi, K., Kuwagata, M., and Imai, K. (1999). Reproductive function in rats exposed neonatally to bisphenol A and estradiol benzoate. Reprod. Toxicol. 13, 303–311.[CrossRef][ISI][Medline]

National Research Council (1999). Hormonally Active Agents in the Environment. National Academy Press, Washington, D.C.

Ogawa, S., Lubahn, D. B., Korach, K. S., and Pfaff, D. W. (1997). Behavioral effects of estrogen receptor gene disruption in male mice. Proc. Nat. Acad. Sci. U.S.A. 94, 1476–1481.[Abstract/Free Full Text]

Paxinos, G., and Watson, C. (1986). The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, Orlando, FL.

Register, B., Bethel, M. A., Thompson, N., Walmer, D., Blohm, P., Ayyash, L., and Hughes, C., Jr. (1995). The effect of neonatal exposure to diethylstilbestrol, coumestrol, and beta-sitosterol on pituitary responsiveness and sexually dimorphic nucleus volume in the castrated adult rat. Exp. Biol. Med. 208, 72–77.[Abstract]

Resko, J. A., and Roselli, C. E. (1997). Prenatal hormones organize sex differences of the neuroendocrine reproductive system: Observations on guinea pigs and nonhuman primates. Cell. Mol. Neurobiol. 17, 627–648.[CrossRef][ISI][Medline]

Rhees, R. W., Al-Saleh, H. N., Kinghorn, E. W., Fleming, D. E., and Lephart, E. D. (1999). Relationship between sexual behavior and sexually dimorphic structures in the anterior hypothalamus in control and prenatally stressed male rats. Brain Res. Bull. 50, 193–199.[CrossRef][ISI][Medline]

Roselli, C. E., Abdelgadir, S. E., and Resko, J. A. (1997). Regulation of aromatase gene expression in the adult rat brain. Brain Res. Bull. 44, 351–357.[CrossRef][ISI][Medline]

Scallet, A. C. (1995). Quantitative morphometry for neurotoxicity assessment. In Neurotoxicology: Approaches and Methods (L. W. Chang, and W. J. Slikker, Eds.), pp. 99–132. Academic Press, San Diego, CA.

Scallet, A. C., Lipe, G. W., Ali, S. F., Holson, R. R., Frith, C. H., and Slikker, W., Jr. (1988). Neuropathological evaluation by combined immunohistochemistry and degeneration-specific methods: application to methylenedioxymethamphetamine. Neurotoxicology 9, 529–537.[ISI][Medline]

Scallet, A. C., Wofford, M., Meredith, J. C., Allaben, W. T., and Ferguson, S. A. (2003). Dietary exposure to genistein increases vasopressin but does not alter beta-endorphin in the rat hypothalamus. Toxicol. Sci. 72, 296–300.[Abstract/Free Full Text]

Schlinger, B. A., and Arnold, A. P. (1991). Androgen effects on the development of the zebra finch song system. Brain Res. 561, 99–105.[CrossRef][ISI][Medline]

Sickel, M. J., and McCarthy, M. M. (2000). Calbindin-D28k immunoreactivity is a marker for a subdivision of the sexually dimorphic nucleus of the preoptic area of the rat: Developmental profile and gonadal steroid modulation. J. Neuroendocrinol. 12, 397–402.[CrossRef][ISI][Medline]

Slikker, W., Jr., Scallet, A. C., Doerge, D. R., and Ferguson, S. A. (2001). Gender-based differences in rats after chronic dietary exposure to genistein. Int. J. Toxicol. 20, 175–179.[CrossRef][ISI][Medline]

Strauss, L., Makela, S., Joshi, S., Huhtaniemi, I., and Santti, R. (1998). Genistein exerts estrogen-like effects in male mouse reproductive tract. Mol. Cell. Endocrinol. 144, 83–93.[CrossRef][ISI][Medline]

Stuart, E. B., Thompson, J. M., Rhees, R. W., and Lephart, E. D. (2001). Steroid hormone influence on brain calbindin-D(28K) in male prepubertal and ovariectomized rats. Brain Res. Dev. Brain Res. 129, 125–133.[ISI][Medline]

Takagi, H., Shibutani, M., Masutomi, N., Uneyama, C., Takahashi, N., Mitsumori, K., and Hirose, M. (2004). Lack of maternal dietary exposure effects of bisphenol A and nonylphenol during the critical period for brain sexual differentiation on the reproductive/endocrine systems in later life. Arch. Toxicol. 78, 97–105.[CrossRef][ISI][Medline]

Taylor, H., Quintero, E. M., Iacopino, A. M., and Lephart, E. D. (1999). Phytoestrogens alter hypothalamic calbindin-D28k levels during prenatal development. Brain Res. Dev. Brain Res. 114, 277–281.[ISI][Medline]

Toran-Allerand, C. D. (1996). Mechanisms of estrogen action during neural development: Mediation by interactions with the neurotrophins and their receptors? J. Steroid Biochem. Mol. Biol. 56, 169–178.[CrossRef][ISI][Medline]

Vancutsem, P. M., and Roessler, M. L. (1997). Neonatal treatment with tamoxifen causes immediate alterations of the sexually dimorphic nucleus of the preoptic area and medial preoptic area in male rats. Teratology 56, 220–228.[CrossRef][ISI][Medline]

Wu, A. H., Yu, M. C., Tseng, C. C., Twaddle, N. C., and Doerge, D. R. (2004). Plasma isoflavone levels versus self-reported soy isoflavone levels in Asian-American women in Los Angeles County. Carcinogenesis 25, 77–81.[Abstract/Free Full Text]

Yamada-Mouri, N., Hirata, S., Hayashi, M., and Kato, J. (1995). Analysis of the expression and the first exon of aromatase mRNA in monkey brain. J. Steroid Biochem. Mol. Biol. 55, 17–23.[CrossRef][ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
82/2/570    most recent
kfh297v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Disclaimer
Request Permissions
Google Scholar
Articles by Scallet, A. C.
Articles by Delclos, K. B.
PubMed
PubMed Citation
Articles by Scallet, A. C.
Articles by Delclos, K. B.