In utero exposure to the environmental androgen trenbolone masculinizes female Sprague–Dawley rats

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Toxicology Letters 174 (2007) 31–41

In utero exposure to the environmental androgen trenbolone masculinizes female Sprague–Dawley rats A.K. Hotchkiss a,c , J. Furr a , E.A. Makynen b , G.T. Ankley b , L.E. Gray Jr. a,∗ a

b

Reproductive Toxicology Division, Endocrinology Branch, MD 72, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA Mid-continent Division, Toxic Effects Characterization Research Branch, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, 6201 Congdon Boulevard Duluth, MN 55804, USA c USEPA/NCSU Cooperative Training agreement, Raleigh, NC 27695, USA Received 30 June 2007; received in revised form 20 August 2007; accepted 20 August 2007 Available online 25 August 2007

Abstract Recently, the occurrence of environmental contaminants with androgenic activity has been described from pulp and paper mill effluents and beef feedlot discharges. A synthetic androgen associated with beef production is trenbolone acetate, which is used to promote growth in cattle. A primary metabolite, 17␤ Trenbolone (TB), has been characterized as a potent androgen in both in vitro and in vivo studies with rats. The current study was designed to characterize the permanent morphological and functional consequences of prenatal TB exposure on female rats compared with those produced in an earlier study with testosterone propionate (TP). Female rat offspring were exposed to 0 mg/day, 0.1 mg/day, 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day TB on gestational days 14–19. The 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day TB groups displayed increases in neonatal anogenital distance (AGD) which persisted in the high dose group. Puberty was delayed in the high dose group and there were increased incidences of external genital malformations and the presence of male prostatic tissue in the 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day groups. These changes were associated with amniotic fluid concentrations of TB that compare favorably with concentrations known to be active in both in vitro systems and in fish. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Trenbolone; EDC; Androgen; Masculinization; Female; Reproductive development

1. Introduction Since the 1990s, there has been a rising scientific and regulatory interest in environmental chemicals capable of interfering with the endocrine systems of wildlife species and humans (Colborn and Clement, 1992). Although initial research has focused mostly on environ-

∗ Corresponding author. Tel.: +1 919 541 7750; fax: +1 919 541 4017. E-mail address: [email protected] (L.E. Gray Jr.).

mental estrogens, androgenic activity has been described in water from pulp and paper mills and concentrated animal feed operations in the U.S. and Europe (Orlando et al., 2004; Parks et al., 2001; Radl et al., 2005; Durhan et al., 2006; Ellis et al., 2003). Suspected androgenic chemicals associated with pulp and paper mill effluents bound to the androgen receptor (AR) and induced androgendependent gene expression in vitro (van den Heuvel et al., 2006; Parks et al., 2001; Larsson and Forlin, 2002). In addition, female mosquito fish (Gambusia holbrooki) collected from contaminated rivers were masculinized (Parks et al., 2001).

0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2007.08.008

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Recently, the anabolic steroid trenbolone acetate, used extensively as a growth promoter for farm animals, has been added to an expanding list of environmental androgens (Purdom et al., 1994; Wilson et al., 2002; Jegou et al., 2001; Ankley et al., 2003). The biologically active forms of this androgenic chemical can be comparatively persistent, with a half-life of approximately 260 days (Schiffer et al., 2001). A recent study examining cattle feedlot discharge showed a high level of androgenicity, and wild-caught fathead minnows (Pimephales promelas) from a pond adjacent to the site displayed morphological (50% reduction in testis weight) and endocrine (abnormal testosterone/estradiol ratios) alterations as compared to fish from an uncontaminated site (Orlando et al., 2004; Ankley et al., 2003). One androgenic metabolite of trenbolone acetate, 17␤ trenbolone (TB) or simply trenbolone, is as potent in vitro and in vivo as are the most potent natural and synthetic androgens (Wilson et al., 2002). Other metabolites include the biologically-active 17␣ trenbolone and the inactive metabolite, trendione. In vitro experiments, TB binds human and fish androgen receptors with high affinity and induces androgen-dependent gene expression in MDA-KB2 cells at concentrations similar to those for dihydrotestosterone (DHT). In vivo androgenicity testing using the Hershberger (castrate-immature male rat) assay shows that TB is as potent as testosterone propionate (TP) in inducing growth of the androgendependent levator ani-bulbocavernosus muscles (LABC) (Wilson et al., 2002). However, in certain tissues, such as the ventral prostate and seminal vesicle, TB (sc) is less effective than equivalent doses of TP in stimulating growth in the immature-castrate male rat. This tissuespecific response is potentially due to the fact that TB, unlike testosterone (T), does not appear to be activated to a more potent androgen by 5␣ reductase, an enzyme present in high concentrations in tissue such as the ventral prostate and seminal vesicle, but not in the LABC (Wilson et al., 2002; Sundaram et al., 1995; Toth and Zakar, 1986). Hence, TB is described as having anabolic activity (effect on muscles) equivalent to T, but less androgenic (effect on prostate) activity. These chemical properties are not unique to TB and have been seen with many other synthetic C-19 norandrogens. Prenatal exposure of female rodents to exogenous androgens results in both physiological and behavioral masculinization although the effects vary with the timing of exposure (Wolf et al., 2002; Greene et al., 1939; Huffman and Hendricks, 1981; Slob et al., 1983; Rhees et al., 1997; Hotchkiss et al., 2007; Swanson et al., 1965). Sexual differentiation of the reproductive tract is most sensitive to disruption from days 14–19 in

the rat, whereas behavioral sex differentiation is later during perinatal life. The external and internal morphological tissues masculinized by prenatal androgen exposure have been extensively described in rodents. These include the anogenital distance (AGD) and the prepubertal nipple/areolae number. AGD is defined as the distance between the genital papilla and the anus; male rodents have AGDs that are approximately twice those of females (Vandenbergh and Huggett, 1995; Gray et al., 1999). Areolae (areolas) are dark areas surrounding the nipple bud and are indicative of adult nipples. Female rats typically have 12 nipples whereas males have none. Both of these biomarkers are altered with prenatal exposure to androgens or antiandrogens in females and males, respectively (Gray et al., 1999). Internally, normal development of the Wolffian duct derivatives (sex accessory tissues and epididymis) and gubernacular ligaments are influenced by androgens. In this study, our objective was to characterize the effects of prenatal exposure to trenbolone on female rat reproductive development and measure the serum and amniotic fluid concentrations of the active androgen in order to relate internal exposure levels of TB to the severity of the effects. Effects assessed included changes in biomarkers of prenatal androgen exposure (AGD and areolas), onset of puberty, reproductive potential, and malformations in adult females. 2. Methods 2.1. Animals Pregnant Sprague–Dawley rats (Charles River Breeding Laboratory, Raleigh, NC) were shipped on the day after mating and housed individually in clear plastic cages (20 cm × 25 cm × 47 cm) with laboratory grade pine shavings as bedding (Northeastern Products, Warrensburg, NY). The day after mating was designated day 1 of gestation. Animals were provided Purina Rat Chow (5008 during pregnancy and lactation and 5001 as juveniles and adults) and filtered (5 microns) water, ad libitum, in a room with a 14:10 h (light/dark, lights off at 11:00 a.m. EST) photoperiod and temperature of 20–22 ◦ C with a relative humidity of 45–55%. On gestational day 13 (GD 13) animals were weight ranked and assigned randomly to treatment in a manner that provided similar means in body weights for the different treatment groups. Pregnant rats were injected with laboratory-grade corn oil (CAS # 8001-30-7, Sigma, lot # 70K0127) or 17␤ trenbolone (TB) (CAS # 10161-33-8, Sigma, lot # 60K16611; purity ≥98%) subcutaneously (sc) from GD 14–19 at 0 mg/day, 0.1 mg/day, 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day in 0.1 ml of corn oil. We elected to use this route of exposure and this dosing regime so we could compare the results of this study to those previously reported from this laboratory using TP

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(Hotchkiss et al., 2007). For experiment 1, only the 0 mg/day, 0.5 mg/day, and 2.0 mg/day dose groups were used. These studies were conducted under a protocol that had been approved by the National Health and Environmental Effects Research Laboratory institutional animal care and use committee. 2.2. Experiment 1—serum and amniotic fluid trenbolone concentrations In the first experiment, pregnant females (n = 4–7 females/dose) were anesthetized by exposure to CO2 and euthanized by decapitation between the hours of 08:00 and 11:00 EST on GD 19 about 1–2 h after the last dose was administered. Maternal serum was collected and frozen at −70 ◦ C for later chemical analysis. Fetuses were then removed from the uterus and placed on ice. Amniotic fluid and maternal serum were collected and frozen on dry ice and stored in a −70 ◦ C freezer until analysis. Samples were analyzed for 17␤ trenbolone, 17␣ trenbolone, and trendione using a modification of the method described by Ankley et al. (2003). Amniotic fluid and maternal serum were extracted with acetonitrile and analyzed by injection onto an Agilent 1100 high-pressure liquid chromatograph (HPLC) with an Alltech Nucleosil column (Deerfield, Il, USA) and a fluorescence detector; excitation and emission wavelengths were 359 nm and 458 nm, respectively. An aliquot (200 ␮l) of the extract was injected into the HPLC, and the column was eluted (at 35 ◦ C) using a gradient program starting at 35% methanol in water and ramping up to 93% methanol in water at a flow rate of 0.9 ml/min. An external standard method of quantitation was used with a six-point linear calibration curve. Quality assurance analyses (matrix blanks, matrix spikes, and duplicate samples of amniotic fluid and serum) were conducted with each sample set. The agreement (mean ± S.D.) among duplicate samples of amniotic fluid and serum were 85 ± 12.2 (n = 3) and 89 ± 15.8 (n = 2), respectively. No 17␤ trenbolone, 17␣ trenbolone, or trendione were detected in control samples or the blanks; the detection limit was 0.2 ng/ml for the amniotic fluid analysis and 2.0 ng/ml for serum. 2.3. Experiment 2—postnatal effects of prenatal trenbolone exposure in female rats 2.3.1. Maternal dosing On gestational day 13 (GD 13) animals were weight ranked and assigned randomly to treatment in a manner that provided similar means in body weights for the different treatment groups (N = 6 females/dose) and treated as described above. 2.3.2. Neonatal and pubertal data On postnatal day 2 (PND 2) (morning after delivery was defined as PND 1) pups were sexed, body weight recorded, and AGD measurements taken by an observer blinded to treatment using a dissecting microscope with an ocular micrometer (15×). The anogenital distance was defined as the distance

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between the base of the genital papilla and the rostral end of the anal opening. At PND 13, animals were reweighed and examined for presence or absence of areolae (dark areas lacking hair found in the region of the developing nipple bud). The AGD and areolae data, previously reported by Wilson et al. (2002), are also shown here so they can be compared to the data collected herein in adults. This enables us to determine the ability of the neonatal AGD and infant areolae to predict permanent effects adults. On PND 23, female animals were weighed and AGD was measured with calipers to the nearest 0.1 mm. On PND 24 female animals were weaned and housed with 2–3 littermates. The onset of puberty was assessed in females by monitoring animals daily for vaginal opening (VO) (normally an indicator of puberty in rats) from PND 29–45. Animals were weighed daily until VO was achieved. Treated females without a vaginal orifice were not included in data analysis for age at VO. 2.3.3. Mating and lactation performance At approximately 4 months of age, two females per litter from the 0 mg/day, 0.1 mg/day, 0.5 mg/day, and 1.0 mg/day treatment groups were randomly selected for mating with nonlittermate control males. Pairs were then checked for sperm presence in vaginal smears for 7 days following pairing. At PND 6 the percent of litters surviving was noted. Lactation ability also was assessed in females that were exposed to TB in utero during the light phase of the animal’s activity cycle, when they are normally nursing. This was done to see if TB reduced the functional capacity of the mammary glands, thereby resulting in decreased pup survival and/or retarded growth. Briefly, pups were removed for 6 h from the lactating dams on PND 14 to allow pups to fully digest stomach contents but without being stressed by maternal deprivation. At the end of this period, pups were counted and weighed by sex and then reintroduced to the dams and allowed to nurse for 2 h and then reweighed by sex (i.e., the weight gained is approximately equal to the amount suckled during this 2 h period). Nine to ten females were examined in the 0, 0.1 and 0.5 dose groups (only one female at 1 mg/day had a viable litter at this age). 2.3.4. Necropsy At approximately 4 months of age, all 2.0 mg/day TB-dosed females and a random sample of control females were collected for necropsy. This was to prevent distress from hydrometrocolpos, an effect seen in females exposed to this dose of TP previously. All other dose groups were collected at approximately 15 months of age after mating trials. During necropsy, females were weighed, anesthetized with CO2 and decapitated. The ventral surface of each animal was shaved and the number of permanent nipples was counted. External malformations such as lack of vaginal opening, presence of cleft phallus, hypospadias, or vaginal thread were noted. Finally, females were examined internally for presence of male-like structures such as LABC muscles, ventral prostate, bulbourethral glands, or seminal vesicles.

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Table 1 Fetal 17␤ trenbolone and trendione concentrations for female offspring prenatally exposed to 0 mg/day, 0.5 mg/day, or 2.0 mg/day 17␤ trenbolone (TB) through maternal administration

Values are means (standard errors of the mean). Note: An (p < 0.05).

*

in a shaded cell indicates that the value differs significantly from the control value

2.3.5. Correlation analyses for AGD at weaning In addition to correlation analysis on AGD data for individual animals, data were separated into categories for graphical purposes. AGDs were separated into 6 different categories spanning the entire range of values, with category 1 being a weaning AGD of 9 mm and smaller, category 2 = 9.0–10.0 mm, category 3 = 10.0–11.0 mm, category 4 = 11.0–12.0 mm, category 5 = 12.0–13.0 mm, category 6 = 13.0 mm and larger. 2.3.6. Statistical analysis All analyses for dose effects were done using litter means, and values in the tables are litter-based. As there was, in general, a significant correlation between neonatal body weight and AGD, body weight was used as a covariate in the analysis of AGD in all analyses. Analysis and statistics were calculated by analysis of variance using PROC GLM Statistical Analysis Software (SAS version 6.08, Cary, N.C.) on the EPA RTP IBM mainframe. If overall analysis of variance was significant (p < 0.05), a LSMEANS post hoc test was then used to further investigate differences between groups. Since neonatal body weights of the female pups displayed a dose-related “trend” towards a decline that was not significant by ANOVA, a linear trend analysis was conducted on these data. In addition, correlation analyses were done using the data from individual animals and the PROC CORR option on SAS that included all females from all treatments for each dose of TB.

3. Results 3.1. Experiment 1—serum and amniotic fluid trenbolone concentrations No trenbolone or metabolites were detected in the maternal serum or amniotic fluid of control rats. Both the 0.5 and 2.0 dose groups displayed a dose-

dependent elevation of TB and trendione in both compartments (Table 1). The highest concentrations of TB were observed in the maternal serum, with amniotic fluid concentrations approximately 11-fold lower in any given treatment. Trendione concentrations were highest in the amniotic fluid with maternal serum concentrations approximately 4-fold lower in any given treatment. No 17␣ trenbolone was detected in any of the samples. 3.2. Experiment 2—postnatal effects of prenatal trenbolone exposure in female rats 3.2.1. Maternal and pregnancy P0 data Trenbolone treatment significantly reduced body weight gain in pregnant females at 1.0 mg and 2.0 mg TB/rat/day (Table 2). Implantation numbers, litter size, and pup mortality in treated litters were not significantly different from controls (Table 2). Pup survival was lower than typically experienced in our laboratory. Routine monitoring of these animals did not identify infectious disease and we do not believe that this confounds our results. 3.2.2. Neonatal and infant F1female rat data Overall, analysis of variance indicated that body weight at PND 2 in female pups was not reduced by TB treatment. However, there was a significant trend for reduced body weights for in the 2 mg/day dose group (p < 0.02, by t-test or linear trend analysis (Table 2)). Neonatal AGD and areolae data for these females were previously reported in Wilson et al. (2002) and this study is a continuation of that study. For comparative

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Table 2 Maternal and litter size data after prenatal maternal exposure to trenbolone (TB) from gestational day (GD) 14–19

Values are means (standard errors of the mean). Note: Neonatal mortality is defined as the difference between observed implantation number and number of male and female pups on postnatal day 1; Juvenile mortality is defined as the difference between observed male and female pups on PND 2 and the number of pups present at weaning. An * in a shaded cell indicates that the value differs significantly from the control value (p < 0.05). TB-treatment induced significant reductions in maternal weight gain and female pup weight at 2 days of age.

purposes, the neonatal and infant data are presented in Figs. 1 and 2 along with the weanling AGD and adult nipple data. Although TB treatment significantly increased the AGD in females, it was still possible to distinguish males from females in the experiment at birth. Prenatal exposure to TB decreased the number of areolae in F1 females at 2 mg/kg/day (Fig. 2) and some of the areolae were faint in females in the 0.5 mg/kg/day and 1 mg/kg/day dose groups (data not shown). 3.2.3. Postweaning and adult data in F1 Females At weaning, the mean AGD was significantly increased by 24.2% in 2.0 mg/day trenbolone-treated females. In addition, in the lower treatments increasing AGDs were observed with increasing dose of TB, although these lower values were not significantly different from control (Fig. 1). Body weights at this age were not affected by treatment. Analysis of AGD results with

body weights revealed that body weight at this age was weakly, though not significantly correlated with AGD (r = 0.14, p > 0.08). There was a significant delay in onset of VO in the 2.0 mg/day group by approximately 2 days (Table 3). Onset of VO appeared to be independent of any effect on body weights, as weights of the females at VO were greater in the 1.0 mg/day and 2.0 mg/day treatment groups, than the controls (Table 3). The age at puberty could not be determined in females lacking a lower vagina. 3.2.4. Mating, fecundity and lactation by F1 females Females prenatally exposed to TB at 1.0 mg/day and below did not display a significant reduction in pregnancy rates (Table 4). However, F2 neonates in this dose group had increased mortality rates as indicated by reduced percentages of litters surviving to PNDs 1

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Fig. 2. Numbers of areolae/nipples in infant F1 female rats are compared to the numbers of nipples observed at necropsy of the adult females. Pregnant rats were treated from gestational day 14–19 to oil, 0.1 mg/day, 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day TB. The infantile data were previously reported by Wilson et al. (2002). * Significantly reduced numbers of nipples versus control female (p < 0.05).

Fig. 1. Neonatal and weaning anogenital distance for F1 female rats exposed from gestational day 14–19 to oil, 0.1 mg/day, 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day TB. Neonatal data were previously reported in Wilson et al. (2002). *Significantly longer than control AGD (p < 0.05).

and 6 such that only one of ten females had viable pups by PND 14. Females with viable litters on PND 14 did not display any significant deficits in milk production (data

not shown) and the treated females did not use fewer nipples to nurse their pups (data not shown). Further, F2 offspring from TB-exposed females were not lighter than controls at PND 23, immediately before weaning (Table 3). 3.2.5. Adult F1 female necropsy data: external quantitative measurements Animals treated with 2.0 mg TB/day had decreased numbers of nipples (Fig. 2) and two of 21 F1 females in the 1.0 mg/kg/day dose group had reduced nipple numbers (6 and 9 nipples each).

Table 3 Weaning and pubertal endpoints for F1 female rats prenatally exposed to trenbolone (TB) from GD 14–19

Values are means (standard errors of the mean). Note: AGD: distance from anus to genital papilla at weaning. An * in a shaded cell indicates that the value differs significantly from the control value (p < 0.05). N = 5–6 litters per treatment. TB-treatment induced significant increases in weaning AGD, age at vaginal opening, and body weight.

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Table 4 Fertility of F1 females exposed in utero to 0 mg/day, 0.1 mg/day, 0.5 mg/day, or 1.0 mg/day trenbolone (TB) from GD 14–19

Values are means (standard errors of the mean). Note: An (p < 0.05).

*

in a shaded cell indicates that the value differs significantly from the control value

3.2.6. Adult F1 female necropsy data: external and internal malformations Dose-dependent malformations of the genital area were observed including absence of VO, and presence of male-like prostatic tissue. The incidence of cleft phallus was significantly increased by TB treatment; however, the 1.0 mg/day TB dose displayed the highest incidence (Fig. 3). Finally, the high (2.0 mg/day) dose group displayed hydrometrocolpos and absent lower vagina. When these malformations are grouped into external (missing nipples, absent vaginal opening,

cleft phallus, and vaginal thread), internal (absent lower vagina, presence of ventral prostate, presence of seminal vesicle), and total malformations (external + internal malformations), a dose-dependent response is apparent (Fig. 3). 3.2.7. Correlation of early developmental parameters with permanent effects Overall, individual female AGD at weaning correlated with the incidence of total malformations among treatments (r = 0.39; p < 0.0001; Fig. 4), numbers of nipples at necropsy (r = −0.55, p < 0.0001), and external (r = 0.45; p < 0.0001), and internal (r = 0.43; p < 0.0001) malformations. 3.2.8. Relationship between amniotic fluid TB concentration and reproductive malformations Fig. 5 shows the relationship among dose of trenbolone administered to the dam, TB concentration in amniotic fluid, neonatal AGD, and adult reproductive malformations. Increasing exposure to TB in the amniotic fluid was associated with increased neonatal AGD and adult reproductive malformations. 4. Discussion

Fig. 3. Malformations for adult female rats exposed from gestational day 14–19 to 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day TB. All values above 35% are significantly increased above control values and an * indicates the lone value below 35% that also is significantly increased (p < 0.05). Note: External malformations = absence of any nipples, absent vaginal opening, cleft phallus and vaginal thread. Internal malformations = absent lower vagina, presence of ventral prostate. Total malformations = external + internal malformations.

In this study, in utero exposure to nM concentrations of TB had permanent effects on androgen-sensitive endpoints in female rats. Specifically, prenatal exposure to TB resulted in alterations to the early developmental biomarkers of androgenicity (AGD and areolae), known to be predictive of masculinzed adult female rat phenotypes. Further, exposure to this androgenic drug caused a significant delay in puberty and an increase

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Fig. 4. Correlation of weaning anogenital distance with permanent effects in female rats exposed from gestational day 14–19 to oil, 0.1 mg/day, 0.5 mg/day, 1.0 mg/day, or 2.0 mg/day TB. A * indicates value is significantly different (p < 0.05) from control. Overall, weaning anogenital distance correlated with malformations across treatments. Adult nipple numbers (r = −0.55, p < 0.0001), external (r = 0.45; p < 0.0001), internal (r = 0.43; p < 0.0001), and total malformations (r = 0.39; p < 0.0001) correlated with weaning AGD. AGDs were separated into 6 different categories spanning the entire range of values, with category 1 being a weaning AGD of 9 mm and smaller, category 2 = 9.0–10.0 mm, category 3 = 10.0–11.0 mm, category 4 = 11.0–12.0 mm, category 5 = 12.0–13.0 mm, category 6 = 13.0 mm and larger. Note: internal malformations = absent lower vagina, presence of ventral prostate. External malformations = cleft phallus, no vaginal opening, vaginal thread, and/or absence of any nipples. Total malformations include internal malformations + external malformations.

Fig. 5. Relationships among dose of trenbolone administered to the dam, TB concentration in amniotic fluid, neonatal AGD, and adult reproductive malformations. Note: Internal malformations: absent lower vagina, presence of ventral prostate, presence of seminal vesicle. External malformations: cleft phallus, no vaginal opening, vaginal thread, and/or absence of any nipples.

in neonatal mortality. Finally, F1 females displayed dose-related malformations consistent with prenatal exposure to androgen-active chemicals. These malformations included both external (cleft phallus, absent vaginal opening) and internal (prostatic tissue present, absent lower vagina) malformations. The highest doses of TB used in the current study also reduced maternal weight gain during pregnancy and the high dose of TB reduced F1 female pup weight at birth. Maternal administration of TB resulted in detectable concentrations of the androgen and TB metabolites in the maternal serum and amniotic fluid of the developing fetus. Therefore, it is clear from this study, that trenbolone is capable of crossing through placenta and into the amniotic fluid. However, the concentration of the biologically-active TB observed in maternal serum was approximately 11-fold higher than in the amniotic fluid. In fact, the concentrations of TB in amniotic fluid (18.8 nM in the 0.5 mg/day group and 56.2 nM in the 2.0 mg/day group) are higher than the estimated TB Ki (1.3 nM) for the androgen receptor (AR) (Wilson et al., 2002; Chawnshang et al., 1988), suggesting that a high

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percentage of AR are likely occupied by TB in the fetal tissues (assuming that other fetal tissues have concentrations similar to the amniotic fluid). Further, both neonatal AGD and the number of adult reproductive malformations increased with the associated increase exposure to TB in amniotic fluid (Fig. 5). Recent studies have focused on quantifying the concentrations of endocrinedisrupting chemicals in the amniotic compartment (e.g., phthalates) (Calafat et al., 2006), but to our knowledge this is the first study that relating fetal TB concentrations with adverse developmental effects. These data are important because there are concerns about the effects of trenbolone in humans and potential exposure to the fetus. However, TB residue concentrations are low in domestic animals treated with trenbolone, generally in the ppt range (Hendricks et al., 2001). A recent study reported that declining sperm counts in young men are associated with increasing maternal meat consumption, and it has been suggested that hormone residues in beef might be associated with this alteration (Swan et al., 2007). However, one would expect that daughters would be more affected by in utero androgen (TB) exposure than males based upon rat studies and studies of children born with congenital adrenal hyperplasia (Berenbaum, 1999). Future rodent studies should focus on relevant oral administration of TB at levels producing lower amniotic fluid concentrations than those used here in order to identify a NOAEL. It is interesting to note that the amniotic fluid concentrations obtained in this study compare favorably to measured test concentrations observed in a fathead minnow study (Ankley et al., 2003). Specifically, in the fathead minnow, Ankley et al. detected a LOAEL for reproductive effects at water concentrations of 0.027 ppb TB. The concentration of TB in the lowest treatment group here was approximately 5 ppb, or well above the LOAEL for fathead minnows. Further, Ankley et al. (2003) commented that the binding affinity for the fathead androgen receptor compares favorably with that of the human and rat androgen receptors. (Ankley et al., 2003; Wilson et al., 2002; Wilson et al., 2004). Taken together, the in vitro binding data and in vivo data in mammals and fish suggest comparable cross species sensitivity to TB and support the validity of cross-species extrapolation relative to EDCs such as TB. Wilson et al. (2002) reported that prenatal exposure to trenbolone resulted in an increase in AGD and a decrease in the number of normally formed areolae in infant F1 female rats. These effects were permanent in the high dose group and a few females in the 1.0 mg/kg/day group display reduced numbers of nipples at necropsy. Previous

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studies have shown that these early alterations accurately predict adult internal and external malformations in both males and females (Hotchkiss et al., 2004; Gray et al., 1999; Faber and Hughes, 1992; Bowman et al., 2003; McIntyre et al., 2001; Hotchkiss et al., 2007). Since pups were uniquely identified in the current study at weaning rather than at birth, we could not correlate either neonatal AGD or juvenile areolae numbers with the effects seen later in life; however, we found that AGD at weaning was correlated with adult alterations and provided an accurate predictor of an adult masculinized female rat phenotype. The sensitivity and ease with which both of these biomarkers can be measured and their highly predictive nature support their inclusion in test guidelines studies of EDCs. The delay in the onset of puberty as measured by the age at vaginal opening in the 2.0 mg TB/day group (in those females that had a lower vaginal canal) may indicate that TB masculinized components of the neuroendocrine hypothalamic-pituitary axis. If so, this effect occurred in spite of the fact that our dosing regime did not include perinatal development during which the hypothalamus is most sensitive to androgenic disruption. Further, lower doses showed a non-significant trend towards a dose response for delayed puberty. Altered onset of puberty due to prenatal/neonatal androgens has been observed in mice (Vandenbergh and Huggett, 1995; Ryan and Vandenbergh, 2002), rats (Rhees et al., 1997), and gerbils (Clark et al., 1993). Prenatal exposure to TB did not significantly decrease the fertility in the 1.0 mg/day treatment group. However, pup mortality was markedly increased on both PND 1 and 6 in this dose group. TB may have caused neonatal mortality by any one of several mechanisms or by a combination of these factors. TB may have altered maternal behavior either by general systemic maternal toxicity or by direct hormonal effects on these behaviors. It is also possible that TB reduced milk production in those F1 dams unable to sustain pups after birth since we evaluated this endpoint only in dams with viable litters at 14 days of age, well after the mortality had occurred. Each hypothesis is biologically plausible since prenatal exposure to androgens is known to adversely reduce maternal body weight gain during pregnancy, maternal care of pups (Juarez et al., 1998) and mammary gland differentiation. As discussed above, the profile of malformations seen in female rats exposed in utero to TB is very similar to that produced by similar exposure to testosterone propionate (TP). However, TB herein appeared to be slightly less potent than does TP (Hotchkiss et al., 2007) in masculinizing female rats in utero. The similarity in the

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profiles of masculinization displayed by female rat offspring exposed in utero to TB or TP is interesting given that TB, unlike testosterone, is a C19 norandrogen that is apparently not activated to a more potent androgen by 5␣-reductase in some androgen-dependent tissues in adult animals. However, the data presented here and elsewhere (Hotchkiss and Nelson, 2007; Wilson et al., 2002), show that TB is capable of significantly affecting both the testosterone-dependent tissues, as well as the DHT-dependent tissues. In conclusion, prenatal exposure to TB, a major metabolite of the growth promoter trenbolone acetate that is implanted in several million beef cattle per year in the US, results in androgenized adult female rats at nM concentrations. Concentrations of TB similar to those that masculinized female rats in utero in the current study also are extremely active in vitro (AR binding) and they effectively masculinize female fish (Ankley et al., 2003). In rats, this masculinization is manifest in internal and external malformations of female reproductive tissues as well as changes in onset of puberty and reproductive potential. Further characterization of the doses producing the adverse effects of TB in utero is warranted given the adverse effects seen herein after in utero exposure to relatively high doses of TB and the concerns by some international trade groups about the presence of hormones in US beef. Disclosure The research described in the article has been reviewed by the National Health and Environmental Effects Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Acknowledgements The authors would like to thank JS Ostby and Dr. CJ Wolf for expert technical assistance. Funding was provided by the USEPA/NCSU Cooperative Training agreement CT826512010 (for AKH). References Ankley, G.T., Jensen, K.M., Makynen, E.A., Kahl, M.D., Korte, J.J., Hornung, M.W., Henry, T.R., Denny, J.S., Leino, R.L., Wilson, V.S., Cardon, M.C., Hartig, P.C., Gray, L.E., 2003. Effects of the androgenic growth promoter 17-beta-trenbolone on fecundity and

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