* Endocrinology Branch, MD-72, U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, Research Triangle Park, North Carolina 27711; and
CIIT, Endocrine, Reproductive, and Developmental Toxicology Program, Research Triangle Park, North Carolina
Received May 26, 2000; accepted August 11, 2000
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ABSTRACT |
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Key Words: antiandrogen; phthalate; DEHP; testosterone; testis; sexual differentiation; endocrine disrupters.
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INTRODUCTION |
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It was initially proposed that some phthalates (i.e., dibutyl phthalate [DBP] and butylbenzyl phthalate [BBP], but not DEHP) were estrogenic (Sharpe et al., 1995), based upon in vitro data (Harris et al., 1997
; Jobling et al. 1995
). However, none of the phthalates that are active in vitro display such estrogenic activity in vivo (Gray et al., 1999
; Zacharewski et al., 1998
). This in vivo activity was assessed using assays sensitive to estrogens, including the uterotropic assay, the timing of vaginal opening, induction of female sexual behavior, and vaginal cornification.
This does not mean, however, that the PEs are not endocrine disrupters. Mylchreest et al. (1998, 1999) and Gray et al. (1999) hypothesized that DBP and DEHP altered fetal development via an antiandrogenic mechanism. This hypothesis is based upon the observation that reproductive tract malformations in androgen-dependent tissues in male rat offspring are similar to effects of antiandrogens such as vinclozolin, procymidone, and flutamide. For example, in utero exposure to dibutyl phthalate (DBP) (Gray et al., 1999; Mylchreest et al., 1999
; Wine and Chapin, 1999
), DEHP (Arcadi et al., 1998
; Gray et al., 1999
), butylbenzyl phthalate (BBP) and di-isononyl phthalate (DINP) (Gray et al., 2000
), have been shown to disrupt differentiation of androgen-dependent tissues in male rat offspring. These alterations include hypospadias and vaginal pouch formation, alterations in androgen-dependent processes (ie., testis descent, retained nipples), and malformations (i.e., ventral prostate, seminal vesicle, levator ani plus bulbocavernosus muscles, gubernacular cord, and the epididymis). While these observations demonstrate that PEs alter differentiation of androgen-dependent tissues during fetal life, the mechanism by which these effects occur has not been determined.
In contrast to the above effects seen in transgenerational studies, standard developmental toxicity tests of DEHP have not detected any of the aforementioned reproductive malformations (Narotsky et al., 1995; Thomas and Thomas, 1984
; Tyl et al., 1988
). In order to evaluate the potential of DEHP to adversely affect sensitive populations such as developing fetuses and children, mechanism of action studies need to be conducted to determine how alterations of the fetal hormonal milieu, as well as other effects, result in reproductive malformations in the offspring.
The purpose of this study was to elucidate the alterations in testicular testosterone production and reproductive development with DEHP exposure during late gestation and neonatal life. The current study used a battery of in vitro, ex vivo, and in vivo assays to examine two hypotheses: (1) DEHP and its major metabolite mono-ethylhexyl phthalate (MEHP) bind to the human androgen receptor (hAR), and (2) testicular testosterone (T) production and levels of T in the fetus are altered by maternal perinatal DEHP exposure. This paper, along with similar studies on DBP (Foster et al., 1999) strengthen the characterization of PEs as antiandrogens and contributes to the body of mechanistic information that will enhance our ability to extrapolate the developmental reproductive toxicity and subsequent malformations of PEs in rats to humans and other species.
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MATERIALS AND METHODS |
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In Vivo Transgenerational Study in the Rat
General methods.
Pregnant Sprague-Dawley rats (Charles River Breeding Laboratory, Raleigh, NC) of approximately 90 days of age were mated (mating confirmed by sperm presence in vaginal smears) and shipped on the day after mating. Dams were housed individually in clear polycarbonate cages (20 x 25 x 47 cm) with laboratory-grade pine shavings as bedding (Northeastern Products, Warrensburg, NY). The day after mating is designated gestation day (GD) 1, and the day after birth is designated postnatal day (PND) 1. Animals were provided Purina Rat Chow (5008) and filtered (5 microns) water, ad libitum, in a room with a 14:10 h (light/dark cycle, lights off at 11:00 A.M.) photoperiod and temperature of 2022°C, with a relative humidity of 4555%. Durham, NC municipal water was used for drinking water and was tested monthly for pseudomonas and every 4 months for a suite of chemicals including pesticides and heavy metals.
Treatment administration.
Pregnant rats were randomly assigned to treatment groups on GD 14 in a manner that provided similar body weight means and distribution. Dams were dosed daily in the morning by gavage from GD 14 through the morning of necropsy (GD 17,-18,-20, or postnatal day (PND)-2) with vehicle or 750 mg DEHP/kg/d (Aldrich, lot # 48H3537, CAS # 117817) in 2.5 µl of corn oil/g body weight (Sigma- cat # C 8267, lot # 107H1649, CAS # 8001307). In previous studies adult offspring from this treatment regimen had a high incidence of reproductive tract malformations. Thus, this dosing regime was chosen to increase the chances of detecting endocrine alterations during the early stages of late gestation and just after birth.
Experimental design.
This study consisted of 2 experimental blocks. Block 1, 11 controls and 11 treated dams: GD 17 (n = 4), GD 18 (n = 4) and PND 2 (n = 3); block 2, 6 controls and 6 treated dams: GD 20 (n = 4) and PND 2 (n = 2). As there were no significant block differences, data for PND 2 are presented as one study. Anogenital distance (AGD) and body weights were measured on all male and female offspring on PND 2, prior to necropsy. AGD was measured in a blinded fashion, using a dissecting scope with an ocular micrometer (magnification x15).
Dissections.
On gestational days 17, 18, and 20, dams were anesthetized using carbon dioxide and euthanized by decapitation. Dams were sacrificed in a random fashion, alternating between control and treated, and dissections for all ages were conducted within a 4-h period in the morning. Fetuses were removed, anesthetized on ice, and necropsied using a dissecting scope. Neonatal pups were euthanized by decapitation. One testis from 23 males per litter from GD17 to GD20 was incubated in media to determine T production, and the other testis was frozen whole for determination of T levels (n = 11 to 12 per treatment). After removal of fetal testes, the carcass was frozen and kept at 20°C until extracted for determination of T levels (n = 18 to 20 per treatment). On PND 2, one testis each from 2 to 3 males per litter were incubated in medium to determine T production (n = 11 to 14 per treatment) and the other testis was frozen whole for determination of T levels (n = 1214 per treatment).
Ex vivo testicular T production.
Testes (with torn tunica) were incubated in either 400 µl (GD17, 18, 20) or 1 ml (PND 2) of M-199 medium (Hazleton Biologics, Inc., St. Lenexa, KS) plus 10% steroid-stripped serum (charcoal/dextran-treated fetal bovine serum, Hyclone Laboratories, Logan, UT) for 3 h in a 34°C incubator on a rocker (GD 17, 18, 20) or in a 34°C water bath (PND 2). At the end of the incubation, the medium was removed and placed in a microcentrifuge tube, frozen on dry ice, and stored at 20 °C for T radioimmunoassay (RIA) analysis.
Testicular T extraction.
Each testis was placed in a 12 x 75 mm glass test tube with 100 µl of distilled water. One ml (0.5 ml, 2x) of ethyl ether (99% pure) was added and the testis was crushed using a plastic pestle. The homogenized sample was placed in an acetone/dry ice bath until the aqueous portion was frozen. The ethyl ether fraction was poured off into a clean 12 x 75 mm glass test tube and the 2 extractions were pooled and evaporated to dryness in a fume hood overnight. Tubes were parafilm-sealed and kept at room temperature until RIA analysis for T, no longer than 2 weeks.
Whole body T extraction.
The fetus was weighed (testes already removed), placed in a 15-ml Falcon tube (Becton Dickinson, Lincoln Park, NJ) to which was added 0.5 ml (on GD 17, 18, 20) or 2 ml (PND 2) of double-distilled deionized water and homogenized using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). After homogenization, ethyl ether (2 x 2 ml) was added, samples vortexed for at least 30 s, and centrifuged at 2,200 rpm (1000 x g) for 10 min. After centrifugation, the sample was placed in an acetone/dry ice bath until the aqueous portion was frozen. The ethyl ether fraction was poured off into a clean 12 x 75 mm glass test tube and allowed to evaporate in a fume hood overnight. The extraction efficiency of radiolabeled T from whole body homogenate was between 65% and 68%. Tubes were parafilm-sealed and kept at room temperature until RIA analysis for T, no longer than 2 weeks.
RIA analysis for T.
Testosterone levels in media and from fetal tissue extracts were measured by RIA using a Coat-a-Count total T kit, according to specifications (Diagnostic Products Corporation, Los Angeles, CA). Tubes containing dried fetal extract were resuspended in 70 µl of zero standard, vortexed for 30 s, and a 50-µl aliquot assayed for T. Testosterone cross-reactivity with 5-dihydrotestosterone was 7.9%. The interassay coefficient of variation was 7%. Extracted testicular tissues were assayed for T by RIA, as previously described by Cochran et al., 1981; Ewing et al., 1984; Schanbacher and Ewing, 1975. Briefly, dried testes extracts were resuspended in 300 µl of phosphate-buffered saline with 1% gelatin (PBSG), vortexed for 30 s, and incubated for 10 min in a 45°C water bath. Six hundred µl of PBSG containing T antibody (1:10,000) and tritiated T (10,000 DPM/100 µl, 1 mCi/ml, DuPontNEN) was added to this suspension. Data are presented as litter means for (1) T production as ng T produced per testis per 3 h, (2) testicular T levels as ng T per testis, and (3) for the fetal carcass as both total T (ng T per fetus) and T concentration (ng T/g fetus).
Testis histology.
Testes from PND 2 male rats were fixed with 5% glutaraldehyde in 0.05 M collidine containing 0.1 M sucrose (collidine buffer) overnight at 4°C (n = 4 control and 6 DEHP males). Tissues were then rinsed x2 with collidine buffer and post-fixed for 1 to 2 h, depending on the size of the tissue, in 1% aqueous osmium tetroxide in 0.05 M collidine buffer. Dehydration of samples progressed through 15-min incubations in 70, 80, and 95% ethanol, followed by 2 15-min incubations in 100% ethanol, and with a final rinse in propylene oxide at room temperature. Final fixation continued with propylene oxide:EPON incubation for one h, followed by overnight incubation in 100% EPON (poly/Bed 812) at 4°C. Tissues for light microscopy were then embedded in capsules, allowed to harden, sectioned (1 µ), stained with aqueous toluidine blue, and photographed on a Vanox light microscope. Sections were evaluated at the light microscopic level in a treatment-blinded fashion.
For histochemical localization of 3ß-HSD activity, testes were removed, placed in OTC compound (Miles, Inc. Elkhart, IN), frozen immediately in liquid nitrogen, and stored at 80°C until sectioned. Testicular samples were taken on GD 20 from surplus males, to evaluate 3ß-HSD staining from testes of 4 control males (from 3 litters) and 5 males (3 litters) in the DEHP-treated group. In addition, given the robust differences between the 2 treatment groups in 3ß HSD staining from GD 20 fetal testes, testes also were taken from neonatal males on PND 3 from a parallel study treated identically with DEHP (companion paper by Gray et al., 2000) for 3ß-HSD staining (for control: 1 from 3 litters and for DEHP treated: 1 from 3 litters).
Testes were sectioned and stained as previously described by Payne, 1980. Briefly, cryostat sections (7 µm) of the testis were cut, placed on a glass slide, and stored at 20°C until staining. At the time of 3ß-HSD staining, slides were held at room temperature for 10 to 15 min. Stain was applied and slides were incubated for 1 h at 37°C under humid conditions, to decrease evaporation of the staining solution. The solution contained 1 mg/ml etiocholanolone (epiandrosterone-5ß-androstan-3ß-ol-17-one), 3.3 mg/ml nitroblue tetrazolium salt, 1% triton x100, and 1.05 mg/ml NAD+ in Dulbecco's phosphate-buffered saline (D-PBS). Stained sections were washed with distilled water, fixed with 5% formalin containing 5% sucrose, and cover slipped with glycerol/D-PBS (50:50, v/v). Photomicrographs of these testicular cross sections from GD 20 and PND 3 (7 control and 8 treated) were examined by 10 individuals in a treatment-blinded fashion to determine if the intensity of LC staining differed between the control and treated groups.
Statistical analysis.
Data were analyzed by analysis of variance (ANOVA) using Proc GLM (Statistical Analysis System [SAS version 6.08], SAS Institute, Inc., Cary, NC) using litter means where appropriate. When statistically significant effects (p < 0.05, F statistic) were detected in the overall ANOVA model, control means were compared to treated groups using the LSMEANS procedure on SAS (a 2-tailed t-test). Endocrine endpoints (testis T, T production, and fetal T) were log-transformed to correct for heterogeneity of variance. AGD data were analyzed for a treatment effect by sex using litter-mean values corrected for body weight by analysis of covariance. Incidences of testicular histological alterations were statistically evaluated by Fisher's exact test, using individual scores.
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RESULTS |
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Anogenital distance (AGD).
AGD at 2 days of age, adjusted for body weight by analysis of covariance, was significantly reduced in males from the DEHP group, measuring 3.35 ± 0.12 mm (mean ± SE) in the control versus 2.14 ± 0.10 mm in treated males. AGD was not reduced in treated females as compared to females from the control group (Fig. 4). These results were assessed using litter means from 5 litters per treatment with 28 to 32 observations for males (210 pups per litter) and 23 to 33 observations for females (3 to 8 pups per litter).
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Maternal toxicity.
During 11 days of dosing, from GD 14 to PND 2 (24 days post conception), maternal weight of treated dams was significantly altered compared to control dams on gestational days 16, 17, and 18 (Fig. 7A). Maternal weight gain of treated dams compared to controls was significantly reduced on gestational days 17, 18, and 20 as well as on PND 2 (Fig. 7B
). As dams were sacrificed on different days, subsequent results were assessed using a subset of the original dams: each group used 17, 13, 9, and 5 dams for GDs 17, 18, 19 and 20, and GD 21 to PND 2, respectively. The number of live pups was not reduced by maternal DEHP treatment (Table 1
) when examined by gestational day or overall (14.0 pups versus 12.8, NS, pooled across gestational days).
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DISCUSSION |
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Testicular T and T production peaked in our control male rats on GD 20, similar to previous study results assessing whole fetal T levels (El-Gehani et al., 1998a,b
; Weisz and Ward, 1980
). However, DEHP-treated male fetuses displayed only a marginal increase in testicular T and T production from GD 17 to GD 20 (Fig. 2
), with levels that were near female tissue levels (Parks, unpublished). Adult male offspring of dams, dosed identically to doses in this study, possessed malformations of androgen-dependent tissues including severe hypospadias, vaginal pouch formation, cryptorchidism, and nipple retention (Gray et al., 1999
, 2000
). To our knowledge, results from this study are the first to indicate that the antiandrogenic effects in utero of DEHP correlate with reductions in tissue T levels and T production during a critical period of sexual differentiation in the male rat fetus.
Along with the reductions in tissue T levels in male rats, the AGD of our DEHP-exposed males was significantly (after adjustment for body weight by analysis of covariance) reduced at 2 days of age (Fig. 4). While body weight was reduced in both male and female neonates, AGD was reduced only in males (down 36%). AGD is a sexual dimorphism that results from the sex difference in fetal androgen (DHT) levels (Rhees et al., 1997
). This result indicates that perinatal DEHP treatment inhibited the effects of dihydrotestosterone on the differentiation of this sexually dimorphic endpoint.
It is important to note that dramatic alterations in T synthesis and tissue T levels were initially seen at GD 17, whereas testis and body weights were only markedly affected in neonatal but not fetal life (Fig. 3). Furthermore, while the effects of low testosterone levels on the reproductive tract are permanent, body weight was only transiently affected, such that differences in body weights between control and DEHP-treated male rat offspring were absent later in life (Gray et al., 1999
). Thus, it is clear that the effects of DEHP on T-production and sexual differentiation do not result from systemic or general fetotoxicity, as none is evident at GDs 17, 18, or 20 (Fig. 3
).
During fetal and neonatal life, the "fetal" Leydig cells (LC) produce T. At approximately two weeks after birth, the fetal LCs regress and are thought to be replaced by progenitors to the "adult" LC (Hardy et al., 1990). The effect of DEHP on Leydig cell T production appears to be transient and may be unique to the fetal LC, because T levels are not consistently reduced when these offspring reach maturity (Gray et al., 2000
). The cellular and molecular differences between these LC lineages may account for the enhanced sensitivity of the fetal LC, compared to the adult LC, to DEHP-induced reductions in T production. On the other hand, altered fetal LC function could result from altered Sertoli-cell paracrine factors that regulate LC differentiation and T production. While one might expect that a reduction in LC numbers would be concurrent with reduced T levels, this does not appear to be the case. LC numbers appeared to be increased, rather than decreased, in DEHP-treated testes as compared to controls, and DEHP-treatment altered the pattern of fetal LC organization (Figs. 5 and 6
). In exposed males, on PND 2 there were areas of LC hyperplasia in the testicular interstitium, whereas the LCs of control animals were arranged in smaller aggregates that were more uniformly distributed through the interstitial region of the testis. Even though T production of the testes was reduced, 3ß HSD activity in the LCs of DEHP-treated males was increased, rather than decreased. On GD 20 and PND 3, LC staining for 3ß HSD was uniformly dispersed in small clusters in control testes, in contrast to the DEHP-exposed testes in which there was an apparent increase in areas of the interstitium of the testis staining for 3ß HSD, indicating an apparent increase in the numbers of LCs as compared to controls.
The apparent increase in LC number may be a compensatory response to the decreased levels of testosterone production or altered paracrine Sertoli cell secretions. In the pubertal and neonatal rat, DEHP exposure directly affects Sertoli cell function (Dostal et al., 1988; Li et al., 1998
). The Sertoli cell secretes paracrine factors that are necessary for the stimulation and maintenance of testosterone synthesis and normal fetal LC function. Hence, altered Sertoli cell function could direct LCs to proliferate instead of to differentiate and produce testosterone (Sharpe, 1993
). On the other hand, LC clustering could prevent the cells from receiving the appropriate paracrine factors from the Sertoli cells. However, as none of the exposed males displayed retained Mullerian-duct derivatives, the secretion of at least one Sertoli cell hormone, Mullerian inhibiting substance (MIS), was not significantly altered by DEHP treatment. It is likely that the effect of DEHP on T production reflects the direct action of MEHP on the testis, as opposed to an alteration of hypothalamic-pituitary function, because initiation of testosterone synthesis by the fetal rat testis does not appear to be regulated by pituitary luteinizing hormone (LH) secretion (El-Gehani et al., 1998b
). Histological examination of the testes also revealed alterations in the seminiferous cords within which the Sertoli cells reside. Gonocytes within the seminiferous cords were more numerous in the treated animals and some of these cells contained multiple nuclei. Although the changes in testicular histology are dramatic and rather novel because they have only been described in GD 20 and PND 2 males, it is uncertain if these alterations provide clues about the direct mode of action of DEHP in the fetal male or if they are merely one of the many indirect effects of DEHP treatment. Examination of testicular histology earlier in fetal life, especially at the onset rather than at the end of sexual differentiation, will reveal the true significance of these alterations.
Based upon results of the current study, we hypothesize that DEHP, or a metabolite, reduces T production either by directly acting on the LCs to reduce T synthesis, or by interfering with Sertoli-cell paracrine factors that regulate LC differentiation and function. Regardless of the mechanism, if the LCs in exposed males continued to divide rather than differentiate for only a brief period of sexual differentiation, this could delay the onset of LC T production and lead to malformations of the reproductive tract, external genitalia, and other androgen-dependent tissues (nipples and levator ani muscle).
The fact that the phthalates are not AR antagonists but act as antiandrogens during fetal life by reducing T levels, probably explains why the profiles of effects induced by DEHP (Gray et al., 1999) or DBP (Mylchreest et al., 1998
, 1999
) differ from those seen after in utero treatment with vinclozolin (Gray et al., 1999
) and procymidone (Ostby et al., 1999
) which act as AR antagonists. DEHP and DBP induce more severe effects on the T-dependent tissues like the epididymis than do the AR antagonists (Gray et al., 1999
, 2000
; Mylchreest et al., 1998
, 1999
).
Determining the mechanism of testicular effects of perinatal DEHP exposure in the rat, and the dosage levels that produce such effects, are essential steps in extrapolating these effects to humans. While some of the effects of DEHP, acting via peroxisome proliferating mechanisms, have been dismissed as irrelevant to human health (Koop and Juberg, 1999), peroxisome proliferator-activated receptor alpha (PPAR
) is not required for PE-induced testis and kidney lesions, as indicated by the observation that DEHP treatment induces pathology in these organs in PPAR
-knockout mice (Ward et al., 1998
).
It is clear that the testicular effects of PEs are not limited to rodent species. PEs have been reported to cause reproductive abnormalities following in utero exposure in rabbits (Higuchi et al., 1999), guinea pigs (Gray et al., 1982
), ferrets (Lake et al., 1976
), and frogs (Higuchi et al., 1999
). In addition, the lack of sensitivity of the hamster testis to PEs is often overstated (Koop and Juberg, 1999
). Testicular lesions are displayed in the testes of hamsters treated with DEHP, with even more severe testicular lesions with MEHP treatment of male hamsters (Gray et al., 1982
).
Not only are these effects seen in several different species, but they are seen at relatively low dosage levels. Arcadi et al. (1998) reported that in utero DEHP exposure at a concentration as low as 3 mg/kg/day induced testicular alterations in the rat offspring such as decreased testes weight and delayed appearance or absence of elongated spermatids. Poon et al. (1997) observed that administration of DEHP at about 37 mg/kg/day caused effects on the Sertoli cells of the testis, which they described as adverse (selected as the NOAEL by the European Commission [EC], 1998). Without including exposures from other routes or sources, the EC estimated a margin of exposure (MOE) for DEHP of 19 from infant mouthing of toys alone, which is about 5-fold lower than a more acceptable MOE of 100 (Faustman and Omenn, 1996). In this regard, the EC has proposed banning the use of several phthalates, including DEHP, in mouthing toys (ENS, 1999
). While there are many sources of DEHP, maternal and neonatal dialysis treatments and blood transfusions during development can result in high serum MEHP and DEHP levels. A comprehensive assessment of the risk of PEs will need to include the most sensitive life stages, along with an assessment of aggregate and combined phthalate exposure levels.
It is noteworthy that the developmental effects of the PEs in rodents include testicular cancer, hypospadias, undescended testes, and permanently reduced sperm production, a list that resembles the reported adverse trends in human male reproductive health that is frequently associated with exposure to environmental contaminants (Toppari, 1998). Since human serum contains MEHP after DEHP exposure, we should be concerned about the ability of this metabolite to alter human reproductive function by affecting testicular function during development. While only a few studies have evaluated PE exposure in nonhuman primates (Rhodes et al., 1986; Kurata et al., 1998
), none of these reported testicular effects from DEHP treatment. However, these studies were designed to investigate the effects of PE exposures during adult life and did not include exposure during the most sensitive stages of life, which appears to be from reproductive-tract differentiation in utero until puberty.
The results of this study contribute to understanding of the mechanism of action of PEs by identifying alterations in testicular steroid production with in utero DEHP exposure. These data also emphasize the need for additional mechanistic information to decrease uncertainty in extrapolation of the effects of PEs from rats to humans. The more we know about the cellular and molecular mechanism of action of DEHP in the fetal rodent testis, the greater certainty we will have concerning the likelihood that the effects are relevant to human development. Since both the process of steroidogenesis and the role of androgens in sexual differentiation are highly conserved among all mammalian species, the effects seen here are not likely to be unique to rodents. In summary, our results demonstrate that (1) inhibition of testicular testosterone production to female levels in the male during sexual differentiation is likely the direct cause of the malformations in the androgen-dependent tissues of male rat offspring, and (2) these malformations appear to result from an AR-independent mechanism of antiandrogenicity.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (919) 541-4017. E-mail: gray.earl{at}epa.gov.
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