Changes in Androgen-Mediated Reproductive Development in Male Rat Offspring Following Exposure to a Single Oral Dose of Flutamide at Differe
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《毒物学科学杂志》
Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
ABSTRACT
Previous studies have indicated that the androgen receptor antagonist, flutamide, can produce a suite of reproductive malformations in the male rat when orally administered daily on gestation days (GD) 12–21. The objective of this study was to investigate the gestation time dependence for the induction of these malformations to establish a robust animal model for future studies of gene expression related to specific malformations. Groups of timed-pregnant Sprague-Dawley rats (GD 0 = day of mating) were administered flutamide as a single gavage dose (50 mg/kg) on GD 16, 17, 18, or 19 with 10 dams per group. Control animals (5 dams per time per group) were administered corn oil vehicle (2 ml/kg). Dams were allowed to litter, and their adult male offspring were killed at postnatal day (PND) 100 ± 10. Anogenital distance was measured at PND 1 and 100. Areolae were scored at PND 13, and permanent nipples evaluated at PND 100. No reproductive tract malformations were found in control male offspring. In the treated groups, malformations were noted following exposure at every GD, although the incidence of specific malformations varied by GD. At GD 16, the highest incidence was noted for permanent nipples (46% pups, 60% litters), epispadias (12% pups, 30% litters), and missing epididymal components (5% pups, 20% litters). The highest incidences for hypospadias (58% pups, 80% litters), vaginal pouch (49% pups, 70% litters), cleft prepuce (29% pups, 60% litters), and missing prostate lobes (12% pups, 60% litters) were noted at GD 17. At GD 18 the highest incidence of malformations noted were epispadias (5% pups, 30% litters), reduced prostate size (32% pups, 90% litters), and abnormal kidneys (3% pups, 30% litters) and bladders (7% pups, 30% litters), while on GD 19 70% of the litters had animals with abnormal seminal vesicles. Testicular and epididymal morphological changes were noted at all GDs and were consistent with the gross observations and peaked in incidence and severity on GD17. The major discrepancy between this study and previous multiple-dose studies was in the very few numbers of animals presenting with cryptorchidism (only one each on GDs 16 and 17), suggesting that exposure over multiple days may be required to induce this malformation. Thus, a single gestational exposure of flutamide induced numerous reproductive tract malformations consistent with previously reports following multiple exposures, with the timing of the exposure producing marked tissue selectivity in the response noted in adult offspring.
Key Words: flutamide; gestational exposure; endocrine; disruptor; antiandiogen.
INTRODUCTION
In the human population it has been proposed that interference with testicular function during in utero development can lead to a variety of human reproductive abnormalities including cryptorchidism, hypospadias, testicular cancer, and decreases in semen parameters characterized by a testicular dysgenesis syndrome (Skakkebaek et al., 2001). Cryptorchidism and hypospadias are the most common human birth defects based on incidence data from a variety of countries (Boisen et al., 2004; Brucker-Davis et al., 2003; Manson and Carr, 2003; Norgil Damgaard et al., 2002).
Antiandrogens have the potential to perturb male reproductive development and function in humans and experimental animals. They can act via a variety of mechanisms, including decreased androgen synthesis, disturbance of the pituitary–gonadal axis, and blockade of the androgen receptor (AR) (Baskin et al., 2001; Gray et al., 1994, 1999; LeBlanc et al., 1997; McIntyre et al., 2001, 2002; Mylchreest et al., 1998). Environmental antiandrogens have recently gained much attention in the lay and scientific literature for their ability to disrupt reproductive development when male offspring are exposed during gestation. Endocrine-active agents such as di(n-butyl) phthalate (DBP), linuron, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), and vinclozolin are readily found in the environment and have been shown to disrupt the androgen-dependent development of male rat pups born to dams exposed to these agents during pregnancy (Gray et al., 1999). Different reproductive phenotypes can result from antiandrogens that share a common mode of action as well as those that act via different pharmacological mechanisms (Gray et al., 1999; McIntyre et al., 2001, 2002; Mylchreest et al., 1998).
In the rat and human, fetal androgen production during gestation is required for normal male sexual differentiation (Schardein, 1993). Testosterone (T) is necessary for proper development of the testes as well as differentiation of the Wolffian ducts into the epididymides, vasa deferentia, and seminal vesicles, whereas dihydrotestosterone (DHT), locally produced from T by 5-reductase, stimulates normal differentiation and development of the genital tubercle and urogenital sinus into the prostate and external genitalia (Berman et al., 1995; Clark et al., 1993; Imperato-McGinley et al., 1992; Kassim et al., 1997; Veyssiere et al., 1982). Flutamide (4'-nitro-3'-trifluoromethyl-isobutyranilide) is a potent, nonsteroidal androgen receptor antagonist that has been used therapeutically to treat prostate cancer and as a tool to study the role of modulating androgen signaling during male reproductive development (Cain et al., 1995; Chung and Ferland-Raymond, 1975; Imperato-McGinley et al., 1992; Kassim et al., 1997; McIntyre et al., 2001; Rivas et al., 2002).
In early, limited dose-response studies, flutamide has been shown to demasculinize male rat pups whose dams had been injected subcutaneously with 100–300 mg/kg/d during gestation (Imperato-McGinley et al., 1992). When pregnant rat dams were exposed to flutamide through the period of sexual differentiation (gestation days (GD) 12–21) using the oral route (and dose levels from 6.25 to 50 mg/kg/d), significant adverse effects were noted in the male offspring on reproductive development mediated by both T (on the testes and Wolffian duct) and DHT on the prostate, external genitalia, retention of nipples, and permanent changes in anogenital distance (AGD) (McIntyre et al., 2001). In general, the effects of flutamide on the development of the reproductive organ systems that are DHT-mediated occurred at lower dose levels than those mediated by testosterone (McIntyre et al., 2001).
The objective of the current study was to determine the time-dependent response of flutamide on the development of the rat male reproductive system based on our previous dose-response studies where treatment was throughout sexual differentiation. Employing a dosing regimen throughout the period of reproductive development produced a suite of malformations that has proved useful in predicting tissue selectivity and the range of reproductive tissues under androgen control for their normal development and differentiation. However, this is not a useful model in understanding the downstream consequences of AR inhibition on the production of specific malformations in different organ systems in the developing male reproductive tract. The overall aim of this study was to determine if specific malformations of the various organ systems under androgen control could be separated by employing a single dose of flutamide given at different days of gestation during this critical window of reproductive development. It was anticipated that the establishment of a robust, single-dose, animal model for the induction of male reproductive malformations could then be employed in future studies to relate changes in gene expression in specific fetal tissues at defined gestational ages to the induction of the various flutamide-induced malformations.
MATERIALS AND METHODS
Animals.
All animals were housed in polycarbonate cages in the animal facility with a 12-h light-dark cycle and controlled environmental facilities. They were fed NIH #07 certified feed (Ziegler Bros., Inc., Gardners, PA) and deionized water. Both food and water were available ad libitum throughout the experiments. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee and conducted in strict accordance with NIH animal care and use guidelines (National Research Council's Guide for Care and Use of Laboratory Animals, 1996, National Academy of Science). Time-mated, 8- to 10-week-old, nulliparous CRL:CD (SD)Br rats were obtained from Charles River Laboratories Inc. (Raleigh, NC) on gestation day (GD) 0. GD 0 was defined as the day that sperm was found in the vagina of the mated female. Animal allocation to treatment groups was done by body weight randomization to ensure unbiased weight distribution among groups. Individual dams and offspring were housed together until weaning (postnatal day [PND] 21), at which time animals were group-housed, up to four per cage, by sex and treatment. After PND 60, all male littermates were transferred, 2–3 per cage, until necropsy. Female offspring were euthanized by CO2 asphyxiation on PND 21 and were not subjected to detailed postmortem examination.
Treatment.
Sperm-positive animals, 5 dams for the control group and 10 dams for the flutamide-treated groups, were gavaged (0800–1030) on GD 16, 17, 18, or 19 with either corn oil (2 ml/kg) or flutamide (dosing solution formulations prepared and analyzed by Battelle, Columbus, OH) at 50 mg/kg (2 ml/kg). This dose level was based on our previous studies indicating that this dose level, when given daily for 10 days, was without maternal toxicity, but induced an array of male reproductive tract malformations at high incidence (McIntyre et al., 2001). Dams were examined daily for clinical signs of toxicity. Dam body weights were recorded daily during dosing and weekly during lactation.
Androgen-dependent reproductive end points.
On the day following delivery, which was considered to be postnatal day (PND) 1, pups were counted and examined for signs of clinical toxicity. Pups were uniquely identified by foot tattoo, and the AGD was measured using digital calipers. The AGD for all pups was measured by an individual investigator who was unaware of animal treatment. Definitive sex of offspring was determined after weaning. Pup litter weights (by sex and litter) and individual pup body weights after weaning were collected weekly. On PND 13, male pups were inspected by a single investigator who was unaware of animal treatment for the presence and number of areolae, nipples, or both. No distinction was made between the retention of an areola or nipple on PND 13.
Necropsy of dams.
Pups were weaned on PND 21, and dams were euthanized by CO2 asphyxiation.
Necropsy of F1 animals.
Sexually mature (PND 95–105) male rats were euthanized by decapitation, and trunk blood was collected and stored for potential hormone analysis. Following blood collection, the ventral surface of the animal was shaved for counting the number of nipples, and the AGD measured with a digital caliper. The external genitalia, including the scrotum, prepuce, and penis, were visually inspected. Gross internal examination of the reproductive tract included inspection of the testes, epididymides, vasa deferentia, prostate, seminal vesicles, and coagulating glands. Additionally, the liver, and kidneys were grossly examined. Body and organ weights (epididymides, testes, prostate, seminal vesicles and coagulating glands [with fluid], liver and kidneys) were collected. The testes and epididymides were fixed in Bouin's fixative, processed, paraffin-embedded, sectioned (5 μm), and stained with hematoxylin and eosin.
Statistical analysis.
Statistical analyses were conducted using JMP (version 5.0.1, SAS Institute, Cary, NC). Pup data was analyzed both individually and nested by dam to yield litter means. Either ANOVA or ANCOVA was used to test for significance of treatment effects, and the covariates are defined in the figure and table legends. The incidence of specific reproductive malformations, presence of nipples, and testicular and epididymal histological findings were compared to corresponding control using Fisher's exact test. A p value of <0.05 was considered to be statistically significant.
RESULTS
There were no significant effects on fertility indices or body weights of the dams or pups following treatment with single doses of flutamide (data not shown). There was no effect on the sex ratio of the F1 offspring. Measurement of anogenital distance (AGD) on PND 1, when covaried by body weight and nested by litter, indicated a significant difference between each flutamide-treated group and the corresponding age-matched controls on all days of exposure (Table 1). Similar statistically significant effects were noted at PND 100, indicating that the changes in androgen-dependent growth of the perineum were permanent (Table 1).
Nipples and areolae at PND 13 were not observed in any control male. In the flutamide-exposed males, the highest incidence was seen after exposure on gestation day 16, where almost 90% of adult male offspring exhibited at least one nipple/areola. A high incidence was also seen following treatment on gestation day 17 (69%), but only 14% of the pups had areolae following GD-18 exposure, and none were noted after exposure on GD 19 (Table 1). Not all areolae became permanent nipples at PND 100 after a single dose of flutamide. Permanent nipples in adult animals were only found in male offspring treated on GDs 16 and 17, with the mean count for the animals exposed on GD 16 twice that on GD 17 of exposure (Table 1).
Malformation Incidence
No reproductive-tract malformations were noted in control animals. The incidence of specific malformations following treatment with flutamide on each of the gestational exposure days is shown in Figures 1, 2, and 3. Malformations were grouped by effects on the testis and Wolffian ducts (Fig. 1) on the external genitalia (Fig. 2), and the prostate, bladder, and kidneys (Fig. 3). Animals with missing epididymal components were always related to the presence of a small testis (but not vice versa). These specific epididymal malformations were only noted with exposures on GDs 16 and 17. The incidence of small testes and missing epididymal components was highest on GD 16. Abnormal seminal vesicles (normally characterized by missing or greatly reduced lobes) was noted at all gestational days of exposure and reached almost a 20% incidence (60% of litters) on GD 17, with a similar response on GDs 18 and 19 (Fig. 1). The incidence of males with prostate malformations (either with missing lobes or greatly reduced in size) peaked on GD 17, but was still present on GD 18. Animals with reduced prostate size were also noted following GD-19 exposure (but not with missing lobes) that were statistically significantly different from control. A low incidence of bladder malformations, characterized by distension and the presence of calculi was noted following dosing on GDs 18 and 19 (Fig. 3). Hydronephrosis was noted in a small number of animals (<5%) following exposure on GDs 17 and 18. Surprisingly, only two animals exhibited cryptorchidism (both unilateral) in this study, one each following treatment on GDs 16 and 17.
The highest incidences of malformations observed were those associated with the development of the external genitalia. The most severe manifestation of flutamide exposure was observed when a hypospadias was accompanied by a vaginal pouch and cleft prepuce, only found in combination following GD-17 exposure, when almost 70% of the offspring (80% of the litters) possessed a hypospadias. Hypospadias was noted at much lower incidence following GD-18 exposure. Epispadias was also observed, peaking in incidence on GD-16 exposure when approximately 12% of the offspring (30% of the litters) exhibited this malformation.
Testicular and Epididymal Histopathology
Abnormal morphology was noted in the testes of male offspring following every gestational day of flutamide exposure (Fig. 4). These effects ranged from complete seminiferous tubular atrophy to more subtle effects on spermatogenesis involving a maturation arrest. The presence of epididymal malformations with missing components was always accompanied by complete testicular atrophy. The more severe testicular lesions with a hypoplastic epididymis were also associated with a decreased presence of sperm in the epididymis. There was a good concordance between the gross observations on these two organs and the associated histological pattern. Testicular changes in the control animals were either not present or very slight, generally affecting only 1 to 3 seminiferous tubules. The incidence of specific changes to the testis and epididymis are shown in Table 2.
Following GD-16 exposure, severe testicular atrophy (Fig. 4B) was only associated with a missing or hypoplastic epididymis, no other changes were noted that could be ascribed to treatment. After GD-17 exposure, similar findings were noted, although the incidence of testicular and epididymal changes were higher. At this exposure time there were also a small number of animals that showed testicular morphological changes ranging from Sertoli cell only (SCO) tubules to those where the most mature germ cell present were spermatocytes (Fig. 4C). If the epididymis was malformed, marked seminiferous tubular atrophy was noted (Fig. 4D). At GD-18 exposure, testicular effects were much less severe, and many seminiferous tubules had a normal appearance, with only small areas that contained few germ cells (Fig. 4F) or where spermatocytes were the most mature germ cell present (Fig. 4G). Following treatment at gestation day 19, very few animals had abnormal testicular changes, and where these were noted, they were less severe than those observed at earlier times, with a few tubules exhibiting spermatogenic arrest (Figs. 4G and 4H).
Changes in histological epididymal morphology closely followed the gross observations and were reflective of the changes noted in the testis. In those animals that had a hypoplastic epididymis on GD-17 exposure, the epididymis was essentially devoid of sperm, and the tubules were smaller in size and had a "thickened" appearance of the epithelium (Figs. 5B and 5C) versus controls (Fig. 5A), where abundant sperm were present. Similarly, in GD 18–treated animals, abnormal epididymal morphology was apparent in the hypoplastic epididymides, which also frequently contained cellular debris in the lumina (Fig. 5D). At GD-19 exposure, only two animals exhibited abnormal epididymal morphology (Fig. 5E), but mature sperm were present in the epididymis, consistent with the pattern of change noted in the testis. Thus, treatment on GD 17 appeared to be the time when both severity and incidence peaked for changes in epididymal and testicular morphology.
DISCUSSION
Cryptorchidism and hypospadias are the most common malformations noted in live born human males, and some concern has been raised on the potential that environmental exposure to endocrine active agents and particularly antiandrogens may contribute to the induction of this disease (Boisen et al., 2004; Brucker-Davis et al., 2003; Manson and Carr, 2003; Norgil Damgaard et al., 2002). The employment of a potent androgen receptor antagonist like flutamide provides a useful tool to investigate the role of androgens in the induction of these and other reproductive tract malformations that may be informative in our further studies of environmentally important agents with antiandrogenic action.
This study demonstrated that a single dose of flutamide to a pregnant rat is sufficient to produce a range of reproductive tract malformations in male offspring in the absence of toxicity to the dam. The range of these malformations was similar to those observed previously in our multiple-dose oral studies (GD 12–21) given throughout the period of sexual differentiation in the rat (McIntyre et al., 2001). Moreover, the specificity of the reproductive tract malformations induced in utero by flutamide exposure was dependent on the timing of the exposure during gestation. Thus, with exposure at GD 16 the peak incidence of malformations were noted as small testes, missing epididymides, retained nipples, and epispadias. At GD-17 exposure the malformations showing peak incidence were small epididymides, hypospadias, vaginal pouch, cleft prepuce, and missing prostate lobes. Abnormal seminal vesicles were noted at similar incidence with treatment from GDs 17 through 19. The observation of abnormal morphology of the bladder was only seen after exposure at GDs 18 (highest) and 19, whereas the kidney malformations were seen at low incidence (<5%) following treatment on each of GDs 16, 17, and 18. Permanent changes in the androgen-dependent growth of the perineum (measured by AGD) did not show any time-dependent changes, with similar, significant reductions noted with exposures at all gestational ages.
Testicular pathology was noted following exposure on all gestational days, although the most severe manifestations were seen with GD-17 exposure. These changes were dependent upon (a) the presence of an epididymal malformation, if this was severely malformed (missing) or with only remnants of the organ present it lead to complete seminiferous tubular atrophy with tubules exhibiting Sertoli cell only morphology; and (b) the timing of the gestational exposure that can still result in testicular pathology with a complete (but frequently hypoplastic) epididymis.
The most interesting data discrepancy was in the marked lack of induction of cryptorchidism observed in the current study (only two animals, one each following treatment on GDs 16 and 17) compared to the multiple dose study where this was one of the more common observations noted at 50 mg/kg/d (McIntyre et al., 2001). The last stages of normal descent of the testes has been shown to be dependent on DHT and T (George, 1989; Imperato-McGinley et al., 1992). This large reduction in the effect of a single dose of flutamide on the induction of cryptorchidism compared to a multiple dose regimen could be due to a number of reasons, the most obvious being that perhaps a higher dose level would be required to elicit this malformation (although almost 50% of the animals in the multiple dose study exhibited cryptorchidism at 6.25 mg/kg/d) or, perhaps more likely, that the induction of this malformation requires exposure over a more prolonged period of gestation. Flutamide and its major metabolite hydroxyflutamide have a half life of <8 h in the rat (Xu and Li, 1998, 1999), and thus most of the agent would be cleared within the 24-h period between oral dosing on a daily basis, supporting the notion that the critical window for androgen-dependent testicular descent may be over multiple days of gestation.
In common with many classic teratogens, treatment with a single dose of flutamide on different gestational days changed the pattern of malformations noted in the adult offspring. This separation of specific malformations should enable more precise questions to be posed with regard to the mechanisms that result in these malformations at the gene level. Our future experiments will be directed at using the data obtained from this model and examining specific fetal tissues, such as the testis, epididymis, urogenital sinus, and genital tubercle at specific gestational days after flutamide treatment to examine some of the detailed gene expression changes that would be downstream from the pharmacological inhibition of the androgen receptor. This should enable a clearer insight into the biology for the induction of some of these malformations that have high prevalence in the normal human population.
ACKNOWLEDGMENTS
The authors would like to thank Eric Haskins, Willie Purdie, and Dr. Christina Carruthers for their excellent technical assistance and Drs. Michael Shelby and Earl Gray for critical review of the manuscript.
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ABSTRACT
Previous studies have indicated that the androgen receptor antagonist, flutamide, can produce a suite of reproductive malformations in the male rat when orally administered daily on gestation days (GD) 12–21. The objective of this study was to investigate the gestation time dependence for the induction of these malformations to establish a robust animal model for future studies of gene expression related to specific malformations. Groups of timed-pregnant Sprague-Dawley rats (GD 0 = day of mating) were administered flutamide as a single gavage dose (50 mg/kg) on GD 16, 17, 18, or 19 with 10 dams per group. Control animals (5 dams per time per group) were administered corn oil vehicle (2 ml/kg). Dams were allowed to litter, and their adult male offspring were killed at postnatal day (PND) 100 ± 10. Anogenital distance was measured at PND 1 and 100. Areolae were scored at PND 13, and permanent nipples evaluated at PND 100. No reproductive tract malformations were found in control male offspring. In the treated groups, malformations were noted following exposure at every GD, although the incidence of specific malformations varied by GD. At GD 16, the highest incidence was noted for permanent nipples (46% pups, 60% litters), epispadias (12% pups, 30% litters), and missing epididymal components (5% pups, 20% litters). The highest incidences for hypospadias (58% pups, 80% litters), vaginal pouch (49% pups, 70% litters), cleft prepuce (29% pups, 60% litters), and missing prostate lobes (12% pups, 60% litters) were noted at GD 17. At GD 18 the highest incidence of malformations noted were epispadias (5% pups, 30% litters), reduced prostate size (32% pups, 90% litters), and abnormal kidneys (3% pups, 30% litters) and bladders (7% pups, 30% litters), while on GD 19 70% of the litters had animals with abnormal seminal vesicles. Testicular and epididymal morphological changes were noted at all GDs and were consistent with the gross observations and peaked in incidence and severity on GD17. The major discrepancy between this study and previous multiple-dose studies was in the very few numbers of animals presenting with cryptorchidism (only one each on GDs 16 and 17), suggesting that exposure over multiple days may be required to induce this malformation. Thus, a single gestational exposure of flutamide induced numerous reproductive tract malformations consistent with previously reports following multiple exposures, with the timing of the exposure producing marked tissue selectivity in the response noted in adult offspring.
Key Words: flutamide; gestational exposure; endocrine; disruptor; antiandiogen.
INTRODUCTION
In the human population it has been proposed that interference with testicular function during in utero development can lead to a variety of human reproductive abnormalities including cryptorchidism, hypospadias, testicular cancer, and decreases in semen parameters characterized by a testicular dysgenesis syndrome (Skakkebaek et al., 2001). Cryptorchidism and hypospadias are the most common human birth defects based on incidence data from a variety of countries (Boisen et al., 2004; Brucker-Davis et al., 2003; Manson and Carr, 2003; Norgil Damgaard et al., 2002).
Antiandrogens have the potential to perturb male reproductive development and function in humans and experimental animals. They can act via a variety of mechanisms, including decreased androgen synthesis, disturbance of the pituitary–gonadal axis, and blockade of the androgen receptor (AR) (Baskin et al., 2001; Gray et al., 1994, 1999; LeBlanc et al., 1997; McIntyre et al., 2001, 2002; Mylchreest et al., 1998). Environmental antiandrogens have recently gained much attention in the lay and scientific literature for their ability to disrupt reproductive development when male offspring are exposed during gestation. Endocrine-active agents such as di(n-butyl) phthalate (DBP), linuron, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), and vinclozolin are readily found in the environment and have been shown to disrupt the androgen-dependent development of male rat pups born to dams exposed to these agents during pregnancy (Gray et al., 1999). Different reproductive phenotypes can result from antiandrogens that share a common mode of action as well as those that act via different pharmacological mechanisms (Gray et al., 1999; McIntyre et al., 2001, 2002; Mylchreest et al., 1998).
In the rat and human, fetal androgen production during gestation is required for normal male sexual differentiation (Schardein, 1993). Testosterone (T) is necessary for proper development of the testes as well as differentiation of the Wolffian ducts into the epididymides, vasa deferentia, and seminal vesicles, whereas dihydrotestosterone (DHT), locally produced from T by 5-reductase, stimulates normal differentiation and development of the genital tubercle and urogenital sinus into the prostate and external genitalia (Berman et al., 1995; Clark et al., 1993; Imperato-McGinley et al., 1992; Kassim et al., 1997; Veyssiere et al., 1982). Flutamide (4'-nitro-3'-trifluoromethyl-isobutyranilide) is a potent, nonsteroidal androgen receptor antagonist that has been used therapeutically to treat prostate cancer and as a tool to study the role of modulating androgen signaling during male reproductive development (Cain et al., 1995; Chung and Ferland-Raymond, 1975; Imperato-McGinley et al., 1992; Kassim et al., 1997; McIntyre et al., 2001; Rivas et al., 2002).
In early, limited dose-response studies, flutamide has been shown to demasculinize male rat pups whose dams had been injected subcutaneously with 100–300 mg/kg/d during gestation (Imperato-McGinley et al., 1992). When pregnant rat dams were exposed to flutamide through the period of sexual differentiation (gestation days (GD) 12–21) using the oral route (and dose levels from 6.25 to 50 mg/kg/d), significant adverse effects were noted in the male offspring on reproductive development mediated by both T (on the testes and Wolffian duct) and DHT on the prostate, external genitalia, retention of nipples, and permanent changes in anogenital distance (AGD) (McIntyre et al., 2001). In general, the effects of flutamide on the development of the reproductive organ systems that are DHT-mediated occurred at lower dose levels than those mediated by testosterone (McIntyre et al., 2001).
The objective of the current study was to determine the time-dependent response of flutamide on the development of the rat male reproductive system based on our previous dose-response studies where treatment was throughout sexual differentiation. Employing a dosing regimen throughout the period of reproductive development produced a suite of malformations that has proved useful in predicting tissue selectivity and the range of reproductive tissues under androgen control for their normal development and differentiation. However, this is not a useful model in understanding the downstream consequences of AR inhibition on the production of specific malformations in different organ systems in the developing male reproductive tract. The overall aim of this study was to determine if specific malformations of the various organ systems under androgen control could be separated by employing a single dose of flutamide given at different days of gestation during this critical window of reproductive development. It was anticipated that the establishment of a robust, single-dose, animal model for the induction of male reproductive malformations could then be employed in future studies to relate changes in gene expression in specific fetal tissues at defined gestational ages to the induction of the various flutamide-induced malformations.
MATERIALS AND METHODS
Animals.
All animals were housed in polycarbonate cages in the animal facility with a 12-h light-dark cycle and controlled environmental facilities. They were fed NIH #07 certified feed (Ziegler Bros., Inc., Gardners, PA) and deionized water. Both food and water were available ad libitum throughout the experiments. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee and conducted in strict accordance with NIH animal care and use guidelines (National Research Council's Guide for Care and Use of Laboratory Animals, 1996, National Academy of Science). Time-mated, 8- to 10-week-old, nulliparous CRL:CD (SD)Br rats were obtained from Charles River Laboratories Inc. (Raleigh, NC) on gestation day (GD) 0. GD 0 was defined as the day that sperm was found in the vagina of the mated female. Animal allocation to treatment groups was done by body weight randomization to ensure unbiased weight distribution among groups. Individual dams and offspring were housed together until weaning (postnatal day [PND] 21), at which time animals were group-housed, up to four per cage, by sex and treatment. After PND 60, all male littermates were transferred, 2–3 per cage, until necropsy. Female offspring were euthanized by CO2 asphyxiation on PND 21 and were not subjected to detailed postmortem examination.
Treatment.
Sperm-positive animals, 5 dams for the control group and 10 dams for the flutamide-treated groups, were gavaged (0800–1030) on GD 16, 17, 18, or 19 with either corn oil (2 ml/kg) or flutamide (dosing solution formulations prepared and analyzed by Battelle, Columbus, OH) at 50 mg/kg (2 ml/kg). This dose level was based on our previous studies indicating that this dose level, when given daily for 10 days, was without maternal toxicity, but induced an array of male reproductive tract malformations at high incidence (McIntyre et al., 2001). Dams were examined daily for clinical signs of toxicity. Dam body weights were recorded daily during dosing and weekly during lactation.
Androgen-dependent reproductive end points.
On the day following delivery, which was considered to be postnatal day (PND) 1, pups were counted and examined for signs of clinical toxicity. Pups were uniquely identified by foot tattoo, and the AGD was measured using digital calipers. The AGD for all pups was measured by an individual investigator who was unaware of animal treatment. Definitive sex of offspring was determined after weaning. Pup litter weights (by sex and litter) and individual pup body weights after weaning were collected weekly. On PND 13, male pups were inspected by a single investigator who was unaware of animal treatment for the presence and number of areolae, nipples, or both. No distinction was made between the retention of an areola or nipple on PND 13.
Necropsy of dams.
Pups were weaned on PND 21, and dams were euthanized by CO2 asphyxiation.
Necropsy of F1 animals.
Sexually mature (PND 95–105) male rats were euthanized by decapitation, and trunk blood was collected and stored for potential hormone analysis. Following blood collection, the ventral surface of the animal was shaved for counting the number of nipples, and the AGD measured with a digital caliper. The external genitalia, including the scrotum, prepuce, and penis, were visually inspected. Gross internal examination of the reproductive tract included inspection of the testes, epididymides, vasa deferentia, prostate, seminal vesicles, and coagulating glands. Additionally, the liver, and kidneys were grossly examined. Body and organ weights (epididymides, testes, prostate, seminal vesicles and coagulating glands [with fluid], liver and kidneys) were collected. The testes and epididymides were fixed in Bouin's fixative, processed, paraffin-embedded, sectioned (5 μm), and stained with hematoxylin and eosin.
Statistical analysis.
Statistical analyses were conducted using JMP (version 5.0.1, SAS Institute, Cary, NC). Pup data was analyzed both individually and nested by dam to yield litter means. Either ANOVA or ANCOVA was used to test for significance of treatment effects, and the covariates are defined in the figure and table legends. The incidence of specific reproductive malformations, presence of nipples, and testicular and epididymal histological findings were compared to corresponding control using Fisher's exact test. A p value of <0.05 was considered to be statistically significant.
RESULTS
There were no significant effects on fertility indices or body weights of the dams or pups following treatment with single doses of flutamide (data not shown). There was no effect on the sex ratio of the F1 offspring. Measurement of anogenital distance (AGD) on PND 1, when covaried by body weight and nested by litter, indicated a significant difference between each flutamide-treated group and the corresponding age-matched controls on all days of exposure (Table 1). Similar statistically significant effects were noted at PND 100, indicating that the changes in androgen-dependent growth of the perineum were permanent (Table 1).
Nipples and areolae at PND 13 were not observed in any control male. In the flutamide-exposed males, the highest incidence was seen after exposure on gestation day 16, where almost 90% of adult male offspring exhibited at least one nipple/areola. A high incidence was also seen following treatment on gestation day 17 (69%), but only 14% of the pups had areolae following GD-18 exposure, and none were noted after exposure on GD 19 (Table 1). Not all areolae became permanent nipples at PND 100 after a single dose of flutamide. Permanent nipples in adult animals were only found in male offspring treated on GDs 16 and 17, with the mean count for the animals exposed on GD 16 twice that on GD 17 of exposure (Table 1).
Malformation Incidence
No reproductive-tract malformations were noted in control animals. The incidence of specific malformations following treatment with flutamide on each of the gestational exposure days is shown in Figures 1, 2, and 3. Malformations were grouped by effects on the testis and Wolffian ducts (Fig. 1) on the external genitalia (Fig. 2), and the prostate, bladder, and kidneys (Fig. 3). Animals with missing epididymal components were always related to the presence of a small testis (but not vice versa). These specific epididymal malformations were only noted with exposures on GDs 16 and 17. The incidence of small testes and missing epididymal components was highest on GD 16. Abnormal seminal vesicles (normally characterized by missing or greatly reduced lobes) was noted at all gestational days of exposure and reached almost a 20% incidence (60% of litters) on GD 17, with a similar response on GDs 18 and 19 (Fig. 1). The incidence of males with prostate malformations (either with missing lobes or greatly reduced in size) peaked on GD 17, but was still present on GD 18. Animals with reduced prostate size were also noted following GD-19 exposure (but not with missing lobes) that were statistically significantly different from control. A low incidence of bladder malformations, characterized by distension and the presence of calculi was noted following dosing on GDs 18 and 19 (Fig. 3). Hydronephrosis was noted in a small number of animals (<5%) following exposure on GDs 17 and 18. Surprisingly, only two animals exhibited cryptorchidism (both unilateral) in this study, one each following treatment on GDs 16 and 17.
The highest incidences of malformations observed were those associated with the development of the external genitalia. The most severe manifestation of flutamide exposure was observed when a hypospadias was accompanied by a vaginal pouch and cleft prepuce, only found in combination following GD-17 exposure, when almost 70% of the offspring (80% of the litters) possessed a hypospadias. Hypospadias was noted at much lower incidence following GD-18 exposure. Epispadias was also observed, peaking in incidence on GD-16 exposure when approximately 12% of the offspring (30% of the litters) exhibited this malformation.
Testicular and Epididymal Histopathology
Abnormal morphology was noted in the testes of male offspring following every gestational day of flutamide exposure (Fig. 4). These effects ranged from complete seminiferous tubular atrophy to more subtle effects on spermatogenesis involving a maturation arrest. The presence of epididymal malformations with missing components was always accompanied by complete testicular atrophy. The more severe testicular lesions with a hypoplastic epididymis were also associated with a decreased presence of sperm in the epididymis. There was a good concordance between the gross observations on these two organs and the associated histological pattern. Testicular changes in the control animals were either not present or very slight, generally affecting only 1 to 3 seminiferous tubules. The incidence of specific changes to the testis and epididymis are shown in Table 2.
Following GD-16 exposure, severe testicular atrophy (Fig. 4B) was only associated with a missing or hypoplastic epididymis, no other changes were noted that could be ascribed to treatment. After GD-17 exposure, similar findings were noted, although the incidence of testicular and epididymal changes were higher. At this exposure time there were also a small number of animals that showed testicular morphological changes ranging from Sertoli cell only (SCO) tubules to those where the most mature germ cell present were spermatocytes (Fig. 4C). If the epididymis was malformed, marked seminiferous tubular atrophy was noted (Fig. 4D). At GD-18 exposure, testicular effects were much less severe, and many seminiferous tubules had a normal appearance, with only small areas that contained few germ cells (Fig. 4F) or where spermatocytes were the most mature germ cell present (Fig. 4G). Following treatment at gestation day 19, very few animals had abnormal testicular changes, and where these were noted, they were less severe than those observed at earlier times, with a few tubules exhibiting spermatogenic arrest (Figs. 4G and 4H).
Changes in histological epididymal morphology closely followed the gross observations and were reflective of the changes noted in the testis. In those animals that had a hypoplastic epididymis on GD-17 exposure, the epididymis was essentially devoid of sperm, and the tubules were smaller in size and had a "thickened" appearance of the epithelium (Figs. 5B and 5C) versus controls (Fig. 5A), where abundant sperm were present. Similarly, in GD 18–treated animals, abnormal epididymal morphology was apparent in the hypoplastic epididymides, which also frequently contained cellular debris in the lumina (Fig. 5D). At GD-19 exposure, only two animals exhibited abnormal epididymal morphology (Fig. 5E), but mature sperm were present in the epididymis, consistent with the pattern of change noted in the testis. Thus, treatment on GD 17 appeared to be the time when both severity and incidence peaked for changes in epididymal and testicular morphology.
DISCUSSION
Cryptorchidism and hypospadias are the most common malformations noted in live born human males, and some concern has been raised on the potential that environmental exposure to endocrine active agents and particularly antiandrogens may contribute to the induction of this disease (Boisen et al., 2004; Brucker-Davis et al., 2003; Manson and Carr, 2003; Norgil Damgaard et al., 2002). The employment of a potent androgen receptor antagonist like flutamide provides a useful tool to investigate the role of androgens in the induction of these and other reproductive tract malformations that may be informative in our further studies of environmentally important agents with antiandrogenic action.
This study demonstrated that a single dose of flutamide to a pregnant rat is sufficient to produce a range of reproductive tract malformations in male offspring in the absence of toxicity to the dam. The range of these malformations was similar to those observed previously in our multiple-dose oral studies (GD 12–21) given throughout the period of sexual differentiation in the rat (McIntyre et al., 2001). Moreover, the specificity of the reproductive tract malformations induced in utero by flutamide exposure was dependent on the timing of the exposure during gestation. Thus, with exposure at GD 16 the peak incidence of malformations were noted as small testes, missing epididymides, retained nipples, and epispadias. At GD-17 exposure the malformations showing peak incidence were small epididymides, hypospadias, vaginal pouch, cleft prepuce, and missing prostate lobes. Abnormal seminal vesicles were noted at similar incidence with treatment from GDs 17 through 19. The observation of abnormal morphology of the bladder was only seen after exposure at GDs 18 (highest) and 19, whereas the kidney malformations were seen at low incidence (<5%) following treatment on each of GDs 16, 17, and 18. Permanent changes in the androgen-dependent growth of the perineum (measured by AGD) did not show any time-dependent changes, with similar, significant reductions noted with exposures at all gestational ages.
Testicular pathology was noted following exposure on all gestational days, although the most severe manifestations were seen with GD-17 exposure. These changes were dependent upon (a) the presence of an epididymal malformation, if this was severely malformed (missing) or with only remnants of the organ present it lead to complete seminiferous tubular atrophy with tubules exhibiting Sertoli cell only morphology; and (b) the timing of the gestational exposure that can still result in testicular pathology with a complete (but frequently hypoplastic) epididymis.
The most interesting data discrepancy was in the marked lack of induction of cryptorchidism observed in the current study (only two animals, one each following treatment on GDs 16 and 17) compared to the multiple dose study where this was one of the more common observations noted at 50 mg/kg/d (McIntyre et al., 2001). The last stages of normal descent of the testes has been shown to be dependent on DHT and T (George, 1989; Imperato-McGinley et al., 1992). This large reduction in the effect of a single dose of flutamide on the induction of cryptorchidism compared to a multiple dose regimen could be due to a number of reasons, the most obvious being that perhaps a higher dose level would be required to elicit this malformation (although almost 50% of the animals in the multiple dose study exhibited cryptorchidism at 6.25 mg/kg/d) or, perhaps more likely, that the induction of this malformation requires exposure over a more prolonged period of gestation. Flutamide and its major metabolite hydroxyflutamide have a half life of <8 h in the rat (Xu and Li, 1998, 1999), and thus most of the agent would be cleared within the 24-h period between oral dosing on a daily basis, supporting the notion that the critical window for androgen-dependent testicular descent may be over multiple days of gestation.
In common with many classic teratogens, treatment with a single dose of flutamide on different gestational days changed the pattern of malformations noted in the adult offspring. This separation of specific malformations should enable more precise questions to be posed with regard to the mechanisms that result in these malformations at the gene level. Our future experiments will be directed at using the data obtained from this model and examining specific fetal tissues, such as the testis, epididymis, urogenital sinus, and genital tubercle at specific gestational days after flutamide treatment to examine some of the detailed gene expression changes that would be downstream from the pharmacological inhibition of the androgen receptor. This should enable a clearer insight into the biology for the induction of some of these malformations that have high prevalence in the normal human population.
ACKNOWLEDGMENTS
The authors would like to thank Eric Haskins, Willie Purdie, and Dr. Christina Carruthers for their excellent technical assistance and Drs. Michael Shelby and Earl Gray for critical review of the manuscript.
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