* Endocrinology Branch, MD 72, Reproductive Toxicology Division, Endocrinology Branch, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and
Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695
Received November 3, 1999; accepted January 10, 2000
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ABSTRACT |
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Key Words: vinclozolin; antiandrogen; androgen receptor; male reproductive development; critical period; levator ani; hypospadias; anogenital distance.
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INTRODUCTION |
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Vinclozolin, or 3-(3,5-dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione, is a fungicide used on fruits, vegetables, turfgrass, and ornamental plants (U.S. EPA, 1998). Antiandrogenic effects of vinclozolin exposure have been found in our laboratory and by others in several mammalian species such as the rabbit, mouse, rat, and dog (U.S. EPA, 1993
). Vinclozolin degrades in the environment or is metabolized in the body to several metabolites, two of which (M1 and M2) have been shown to competitively inhibit the androgen receptor (AR) in vitro (Kelce et al., 1994
; Wong et al. 1995
). In vivo, vinclozolin inhibits AR-dependent gene expression (Kelce et al., 1997
) and produces a spectrum of anatomical defects. Administration of 50 and 100 mg/kg vinclozolin to rats on gestational day (GD) 14 through postnatal day (PND) 3 resulted in effects in the male offspring similar to those caused by flutamide, a well known AR antagonist. These effects include reduced anogenital distance (AGD); persistent nipples; cleft phallus; hypospadias; reduced weights of the ventral prostate, seminal vesicles and epididymis; and reduced sperm counts (Gray et al., 1994
; 1999a
). In addition, peripubertal administration of 100 mg/kg vinclozolin to male rats delays puberty and reduces weights of androgen-dependent tissues including the ventral prostate, seminal vesicles, and epididymis (Monosson et al., 1999
). Doses as low as 3 to 12.5 mg/kg administered perinatally reduce AGD and ventral prostate weight and induce areolas in males (Gray et al., 1999a
). These responses in male offspring to low doses of vinclozolin emphasize the sensitivity of the developing fetus and pup to vinclozolin.
The heightened susceptibility of the fetus to antiandrogenic insult can be explained by the onset of AR expression during sexual differentiation, and the fact that normal AR function is essential for male reproductive tract differentiation. AR appear first in the rat testes at GD 14 (Bentvelsen et al., 1995) or GD 15 (You et al., 1998
) and in the mesenchyme of the reproductive tract nearest the testes at GD 14 in the rat (Bentvelsen, et al., 1995
) and at GD 12 in the mouse (Cooke et al., 1991
; Prins and Birch, 1995
). AR first appear in the mesenchyme of the murine mammary anlage at about GD 12 (Wasner et al., 1983
) and the rat mammary anlage is responsive to androgens at the time the fetal testes produce testosterone (Imagawa et al., 1994
), which is GD 14 (Picon, 1976
). The appearance of AR along the reproductive tract is also coincident with the production of testosterone (T) by the testes and the onset of sexual differentiation of the tract (Wilson et al., 1995
). Sexual differentiation is dependent on the androgens T or dihydrotestosterone (DHT), and on AR function. The role of androgens in male sexual development is made evident from human genetic and animal studies. Human males with mutations of the AR display androgen insensitivity syndrome, in which males have testes but display female-like external genitalia and breast development with variable development of the internal male reproductive tract (Quigley et al., 1995
). Males lacking 5
-reductase, which converts T to DHT, also display female-like genitalia but have normal Wolffian duct structures (Wilson et al., 1981). Similar results have been obtained in animal studies using antiandrogens that either inhibit the AR, such as flutamide or vinclozolin, or inhibit the 5
-reductase conversion of T to DHT, as with finasteride. The development of the Wolffian duct system, which includes the epididymides, vas deferens, and seminal vesicles, is much more affected by an AR inhibitor, flutamide, than by a 5
-reductase inhibitor, finasteride (Imperato-McGinley et al., 1992
), which illustrates T dependency in the Wolffian duct system. Testicular descent (Husmann and McPhaul, 1991a
; Imperato-McGinley et al., 1992
; Spencer et al., 1991
) and levator ani weight (Breedlove and Arnold, 1983
; Tobin and Joubert, 1991
; van der Schoot, 1992
) also appear to be mediated by T. In contrast, perinatal finasteride exposure did not affect the epididymis or vas deferens, and the incidence of undescended testes was much lower, although finasteride treatment greatly reduced the prostate weight, prevented closure of the penile folds as evidenced by a cleft phallus, and allowed for development of nipples and a vaginal pouch (Clark et al., 1993
; Imperato-McGinley et al., 1992
), which reflects a dependency of the urogenital sinus, perineum and mammary anlage on DHT. Finasteride or flutamide treatment also induce hypospadias, microphallus, and preputial adhesion in rodents and monkeys (Prahalada et al., 1997
; Silversides et al., 1995
).
We hypothesize that the development of any of these structures can be most effectively altered by an AR inhibitor such as vinclozolin at the time of onset of differentiation of each structure and coincident appearance of AR in the mesenchyme. In amendment of Paracelsus' statement "The right dose differentiates a poison from a remedy" (Gallo and Doull, 1993), dose alone does not determine the poison. The timing, duration and dose of the antiandrogenic agent used will influence the severity and spectrum of effects. The objectives of this study were to establish the critical period(s) during fetal development within which male offspring are most sensitive to the effects of vinclozolin on the reproductive organs, and to characterize the spectrum and level of antiandrogenic effects obtained from these dosing regimes for use in future studies. We dosed several groups of rats in separate 2-day time frames during gestation with high concentrations of vinclozolin (400 mg/kg) in order to identify the critical time to dose. We then extended the dose period and lowered the concentration of the dose in order to find a dose regimen that would significantly affect all of the offspring.
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METHODS AND MATERIALS |
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Extended Window Study (Experiment 2)
Methods were the same as for the above experiment except for the following: (1) the experiment was performed on one set of animals; (2) dams were dosed with 200 mg/kg bw vinclozolin (n = 10), 400 mg/kg bw vinclozolin (n = 10), or corn oil vehicle (control; n = 10) on GD 1419; (3) male offspring were weaned on PND 22; (4) males were sacrificed on PNDs 107128 (n = 105); (5) testis and epididymis were not weighed but were observed for abnormalities; and (6) serum was collected for radioimmunoassay of T levels by a method described previously (Kelce et al., 1991).
Statistics
Data were analyzed on a litter-means basis using one-way ANOVA (Experiment 2), or 2-way ANOVA when the experiment was performed in 2 blocks, representing 2 sets of dams and their offspring (Experiment 1), using general linear models procedure PROC GLM on SAS for Windows 95TM version 3.0.554 (Cary, NC). AGD and organ weights were analyzed with body weight as a covariate. When general differences (p < 0.05 in ANOVA) for treatment effects were found, specific differences between groups were analyzed with a 2-tailed t test, using least-square means. Data given in percentages (areola, nipple and malformation data) were analyzed after arcsin transformation of litter means, in order to correct for heterogeneity of variance typical of percentage data. Fisher's Exact test was also performed on categorical data (malformations, vaginal pouch, and ectopic/undescended testes) on an individual basis.
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RESULTS |
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Nipples.
Since untreated male rat pups can present areolas early in life but do not retain nipples, it was important to monitor the presence of nipples in adulthood. Nipples were observed on adult male offspring in the GD 1415, GD 1617, and GD 1819 groups only. The percentage of males with nipples was significant in the GD 1617 and GD 1819 groups, while the highest incidence appeared in the GD 1617 group. The number of nipples per animal was also highest in the GD 1617 group (Fig. 1, Table 2
).
Malformations of genitalia.
Malformations of the external genitalia included cleft prepuce, incomplete preputial separation, cleft phallus with accompanying exposed os, and hypospadias. These malformations were observed only in the GD 1415, GD 1617, and GD 1819 groups. One animal from the GD 1617 group displayed ectopic testis (Table 2). The incidence of malformations was significant only in the GD 1617 group, by both ANOVA (p < 0.005) and Fisher's Exact test (12/24 individuals, vs. 0/17 individuals in control group; p < 0.001; Fig. 1
; Table 2
).
Body weight.
Body weight was not significantly affected in any dose group (p > 0.85; Table 3).
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Experiment 2
Dam and pre-weaning pup data.
No dams presented dystocia or delivered late. Dam weight gain through the dosing period (GD 14GD 19) was slightly reduced by vinclozolin treatment at 400 mg/kg (p < 0.05; Table 4). Litter size and pup weight on PND 1 and pup survival to PND 13 and to weaning were not significantly reduced by vinclozolin treatment (Table 4
).
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Nipples.
No adult control male had nipples while 100% of the treated males had nipples. The number of nipples per animal, out of possible 12 nipples, was 9.5 in the 200 mg/kg group and 9.6 in the 400 mg/kg group, both significantly different from controls (Table 5).
Malformations of the genitalia.
While no malformations were observed in the control group, virtually all animals in the vinclozolin-treated groups displayed at least one malformation of the reproductive tract, and this treatment effect was significant (Table 5). Malformations included cleft prepuce, cleft phallus accompanied by exposed os and hypospadias, vaginal pouch, and ectopic and undescended testes. The degree to which each of these malformations manifested varied between individuals, and the incidence and severity of some malformations were higher in the 400 mg/kg than in the 200 mg/kg dose group. The incidence of severe vaginal pouch increased in a dose-dependent fashion and was significant in both the 200 mg/kg and 400 mg/kg groups by Fisher's Exact test (Fig. 2
) and in the 400 mg/kg group by ANOVA (F[2,18] = 7.3; p < 0.005). Ectopic or undescended testes occurred only in the 400 mg/kg group and was significant by Fisher's Exact test (Fig. 2
). The abnormal testicular position found in this study was always near the inguinal region as opposed to the kidney area, but was outside the scrotum in a suprainguinal position either immediately rostral to the inguinal ring in the abdomen (undescended) or outside the abdominal muscle wall in the abdominal or pubic area immediately under the skin (ectopic).
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Organ weights.
Vinclozolin treatment at 200 and 400 mg/kg significantly reduced the weights of the seminal vesicles, ventral prostate and LA/BC by p < 0.0001 and there was no significant difference between the 200 and 400 mg/kg groups (Table 5). Also, ventral prostate agenesis occurred in one animal in the 200 mg/kg group and in one animal in the 400 mg/kg group. Significant reductions were independent of body weight as found by covariate analysis.
Serum T-levels.
Testosterone levels were not significantly altered by vinclozolin treatment (p > 0.22; Table 5).
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DISCUSSION |
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The endpoints most affected by 2-day dosing include AGD, presence of areolas and permanent nipples, weight of the levator ani/bulbocavernosus muscles, and malformations of the external genitalia. These results not only show the critical 2 day period for sensitivity of sexual development to vinclozolin, but show that AGD, nipple retention, and levator ani/bulbocavernosus weight are sensitive indicators of antiandrogenicity. We also found that a 200 mg vinclozolin/kg dam body weight, administered from GD 14 through GD 19, is sufficient to significantly affect the reproductive development of every male rat fetus. In addition, a dose of 400 mg/kg vinclozolin administered in this time period is required to significantly induce ectopic or undescended testes. The fact that serum T levels in the adult were unaltered by prenatal vinclozolin treatment at a time when androgen-dependent organs were reduced indicates that the decreased organ weights are not due to a decreased T level, but to an organizational effect during sexual differentiation.
The effects observed in this study can be related to the timing of appearance of AR in the male reproductive tract, and illustrate the dose-dependent response of the tissues in the genital region.
The sensitive periods found with 2-day AR inhibition by vinclozolin correlate with the onset of appearance of ARs in the mesenchyme of each tissue rather than the epithelium, as described above. Appearance of ARs in the mesenchyme directs the development of reproductive tissues (Cunha et al., 1992). Morphological development in the seminal vesicle, the prostate, and the Wolffian duct begin after AR appear in the mesenchyme and before AR appear in the epithelial portion of these tissues. Epithelial proliferation and cytodifferentiation do not occur until testosterone binds mesenchymal cells nearest to the epithelial layer (reviewed by Cunha et al., 1992). Furthermore, mammary glands of the fetal mouse contain ARs in the mesenchyme surrounding the epithelial buds and not in the epithelium at the time of its responsiveness to testosterone and outgrowth of the primordial bud (Wasner et al., 1983
).
AGD has been shown to be a sensitive indicator of antiandrogenicity. Doses as low as 3 mg/kg vinclozolin administered perinatally reduced the AGD on PND 2 in male rats (Gray et al., 1999a). AGD is used commonly as an indicator of antiandrogenicity and has been found by many to be sensitive with various types of antiandrogens (Clark et al., 1993
; Ema et al., 1998
; Gray et al., 1999b
; Mylchreest et al., 1998
; Ostby et al., 1999
; You et al., 1998
). In the current study, AGD proved a sensitive endpoint by its ability to be significantly reduced with only a 2-day administration of vinclozolin, and by the significance of these reductions. Also, the temporal effect on AGD in our study correlates with the appearance of AR in the mesenchyme of the affected tissue. The urogenital tubercle, folds, and swellings, which give rise to the phallus and perineum, first express mesenchymal ARs in the rat on GD 14 (Bentvelsen et al., 1995
). AGD, a measurement in the perineal region, was first reduced with administration of vinclozolin on GD 1415, and was further reduced on GD 1617. The perineum on GD 1819 still appeared responsive, since AGD was slightly reduced on GD 1819.
The appearance of nipples has also been reported in studies on antiandrogens (Gray et al., 1999b; Kelce et al., 1994
; Mylchreest et al., 1998
; You et al., 1998
) as a definitive indicator of antiandrogenicity. Nipple retention was a sensitive endpoint to vinclozolin toxicity in this study, shown by its inducibility at several 2-day dosing periods and the great significance of the effect. Although many studies report only thoracic nipples, we observed nipples at every point along the "milk line," or all 12 nipples. Male adult rats do not normally display nipples, and the presence of nipples indicates an interference of androgen action in this tissue. In the male rodent fetus, the mesenchyme of the mammary anlage contain ARs and respond to the rise in circulating testosterone, resulting in the condensation of the mesenchyme and disintegration and rupture of the epithelial stalk leading to the external skin (Topper et al., 1980). This response is to DHT rather than to T since 5
-reductase inhibition results in the presence of nipples in the male rat (Imperato-McGinley et al., 1992
). Most investigation of mammary gland and nipple development has been conducted in the mouse where the onset of appearance of AR in the mammary anlage is on GD 12 (Wasner et al., 1983
), which translates to GD 14
in the rat (Butler and Juurlink, 1987
). Also, condensation of the mesenchyme of the mammary gland occurs at the time of testosterone production, or GD 14 in the rat. This time point correlates with the effects on nipple development in our study. Nipples were present in male adults after exposure to vinclozolin at GD 1415 and increased in number, percent males affected, and significance at GD 1617, when testosterone concentrations rise systemically.
Another significant finding is the sensitivity of the levator ani/bulbocavernosus muscle group to vinclozolin. The levator ani (LA) is sexually dimorphic at birth in rats and humans, which illustrates that the fetal period is the time of susceptibility to antiandrogenic insult. At GD 22 in the rat, the number of muscle units (MUs) is greater in the male than in the female (Tobin and Joubert, 1991) and the LA is larger in weight and cross sectional area in males (Jordan et al., 1997
). The male rat LA muscle at PND 7 is highly immunoreactive to testosterone and contains a high number of AR (Jordan et al., 1997
). This response is directly to testosterone and not DHT, as there is no 5
-reductase activity in this muscle. The onset of expression of AR in the fetal rat LA and reactivity of the fetal LA to androgens or antiandrogens has not been studied. We found the timing of effects in the LA/BC to be similar to that found in the nearby external genitalia. The LA/BC weight was significantly reduced at GD 1415, and the reduction was even more significant at GD 1617, while GD 1819 dosing still resulted in a significantly reduced weight. These results provide indirect evidence that AR appear in the levator ani/bulbocavernosus muscles in the rat on GD 14 and increase in number and/or activity by GD 1617.
In the current study, ventral prostate weight was less sensitive to vinclozolin than the above endpoints. The prostate is derived form the DHT-dependent urogenital sinus (UGS). ARs first appear in the UGS in the rat on GD 16 but greatly increase in expression by GD 18 (Bentvelsen et al., 1995; Hayward et al., 1996
), when rapid prostate development begins. In our study, the effect occurred with GD 1819 dosing, which correlates with the rise in AR and rapid proliferation of cells in the prostate rather than at the onset of appearance of AR, and it is then that AR may be more functional.
The external genitalia displayed abnormalities in response to vinclozolin exposure in this study, consistent with the effects observed in other studies, and the temporal response to vinclozolin correlated with the appearance of ARs in the mesenchyme of the tissue. The mesenchyme of the external genitalia express AR on GD 14 and expression increases thereafter (Bentvelsen et al., 1995). These AR directly affect development of the penis since AR continue to be present in the penis of the immature and adult rat and are responsive to T (Rajfer et al., 1980
). The incidence of malformations of the external genitalia occurred to a significant degree with GD 1617 dosing. This response also occurs on treatment with the 5
-reductase inhibitor, finasteride, and it is most sensitive at this same gestational time point (Clark et al., 1990). As observed in the ventral prostate, the effect on the external genitalia correlated with the rise in AR rather than in the onset of appearance of AR.
The timing of appearance of AR in the Wolffian duct system could not be directly correlated with effects, since a large portion of the Wolffian-derived structures was unaffected by 2-day vinclozolin dosing. Bentvelsen et al. (rat; 1995) and Cooke et al. (mouse; 1991) both observed a correlation between the onset of appearance of AR and the distance from the testes in the male reproductive tract: the more proximal to the testes, the earlier AR appeared in the mesenchyme. In the Wolffian duct system of the rat, AR appear first in the efferent ductules and epididymides around GD 14 (Bentvelsen et al., 1995) or GD 15 (You and Sar, 1998
), in the vas on GD 19 or 20, and in the seminal vesicles on GD 21 (Bentvelsen et al., 1995
). Although vinclozolin administration included these time points, no effects were observed in the testes, epididymides, or seminal vesicles. Gray et al. (1994) observed that the Wolffian duct appeared more resistant to insult by vinclozolin, and Imperato-McGinley et al. (1992) found that much higher doses of flutamide were required to affect the testes, epididymides, and vas deferens.
Most of the aforementioned effects did not become more significant as the dose and duration of dosing were increased. Some effects, ectopic/undescended testes and vaginal pouch, were observed only when both the dose and duration of dosing were increased.
Ectopic and undescended testes were observed in our study to a significant degree only with GD 1419 administration of vinclozolin and only in the 400 mg/kg group. Thus, this T-dependent process (Imperato-McGinley et al., 1992) appears to be less sensitive to antiandrogenic insult than other events studied here. Testicular descent has been described in 2 stages, transabdominal and transscrotal, and both these events may be under androgenic control. However, factors such as intraabdominal pressure, suppression of the cranial suspensory ligament, neural control, and hormones other than androgens may play roles in testicular descent (Levy and Husmann, 1995
). The gubernaculum is at least partially responsible. ARs are present in the gubernaculum (Husmann and McPhaul, 1991b
) and flutamide administered on GDs 15 to 17 prevented gubernaculum cord regression in the rat (Cain et al., 1995
). The abnormal testicular position found in this study was always near the inguinal region, i.e., the testes had accomplished transabdominal migration while transinguinal migration was interfered with. It appeared more specifically that the positioning of the gubernaculum was in the abdominal wall rather than in the scrotum. Anomalies such as these have been described in rodents after antiandrogenic treatment (Neuman et al., 1970; van der Schoot, 1992
). Also, inguinal hernias were produced in wallabies treated with flutamide during sexual development (Lucas et al., 1997
).
The fact that the presence of a vaginal orifice was observed only with extended dosing in Experiment 2 and that the severity of this malformation increased with dose illustrates both the dose-dependency of this tissue on androgen action and its relative insensitivity to antiandrogenic insult. This finding is consistent with those of other AR inhibitors. The AR inhibitor flutamide demasculinized the external genitalia more completely than did the 5-reductase inhibitor finasteride (Imperato-McGinley et al., 1992
). Vaginal orifices appeared in rats after 12-day prenatal dosing using 10 mg flutamide per dam (van der Schoot, 1992
), which is comparable in potency to the dosing regime used in our Experiment 2. Although 25100 mg/kg/day of procymidone, a fungicide and AR inhibitor, significantly affected AGD and nipple formation, vaginal pouch was significantly affected only at 200 mg/kg from GD 14PND 3 (Ostby et al., 1999
). Also, while AGD was decreased and nipples were induced, vaginal pouches were not observed in male offspring exposed perinatally to p,p'-DDE (You et al., 1998
), nor in males treated with various antiandrogens that displayed hypospadias (Gray et al., 1999b
).
Extending the dosing period to include gestational days 14 through 19 did not increase the significance of the effect in the more sensitive endpoints (i.e., AGD, nipples, LA/BC weight), even at half the dose. However, this dosing regimen increased the significance of other effects and induced still others, including the reduced weight of the Wolffian duct-derived seminal vesicle. This is not to say other Wolffian duct derivatives were not affected. The epididymis, which was not weighed, could have been affected with the extended dose regime in our study, since administration of vinclozolin at 200 mg/kg from GD 14 through PND 4 reduced the weight of the epididymis (Gray et al., 1999b). The greater overall effect in androgen-dependent target tissues with the extended dosing period may show that effects are dependent upon the cumulative dose, or area under the curve (AUC), in addition to the timing or concentration of the dose alone (e.g., 6 days of 200 mg, or 1200 mg > 2 days of 400 mg, or 800 mg). In support of this theory, raising the dose level to 400 mg/kg vinclozolin for 6 days further affected development of the male genitalia. The effects may also be dependent on clearance of vinclozolin and its metabolites, requiring a continuous dose of the reversible AR-inhibitor over the course of a developmental event to ensure an effect in that organ. Since dosing on GD 1213, a stage immediately before sexual differentiation and expression of AR, did not affect any tissues, clearance of vinclozolin and its metabolites from the rat may take place within a day. In light of these two considerations, some antiandrogenic effects found in Experiment 2 may actually be more significant if the dosing period is extended, or even moved to a later time frame.
This study illustrates the sensitivity of the fetus to vinclozolin as evident in the significance and spectrum of the effects. These effects and the timing of the effects are consistent with other studies (Clark et al., 1990; Husmann and McPhaul, 1991a; Silversides et al., 1995
). The tissues affected by vinclozolin are most sensitive at or soon after the time they are acquiring AR in the mesenchyme, and are further sensitive to the dose and duration of dosing during this sensitive period. By the time ARs are no longer located in the mesenchyme but rather primarily in the epithelium, malformations cannot be induced. Indeed, studies in which neonates are dosed with high levels of EDCs are unable to induce gross malformations (Prins, 1995). The information obtained with the current study is invaluable to further investigation into the mechanisms of vinclozolin-induced antiandrogenicity in the developing fetus.
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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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