U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD-72, Research Triangle Park, North Carolina 27711
Received July 18, 2002; accepted September 10, 2002
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ABSTRACT |
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Key Words: 17ß-trenbolone; environmental androgen; feedlot contaminant; in vivo; in vitro; in utero screen.
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INTRODUCTION |
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The anabolic steroid, trenbolone acetate (TBA; 17ß-hydroxy-estra-4,9,11-trien-3-one-17-acetate), is a growth promoter used in cattle in the U.S. and Canada either alone or in combination with an estrogenic compound. After absorption, TBA is hydrolyzed to the active androgen, 17ß-trenbolone (TB; 17ß-hydroxy-estra-4,9,11-trien-3-one). A portion of the active androgen, TB, is excreted by the cattle along with its metabolites, primarily, 17-trenbolone and triendione (Pottier et al., 1981
). TB, along with its metabolites, has been identified in liquid and solid waste from cattle, and studies conducted on stored liquid cattle waste indicated half-lives of 267 and 257 days for the 17
-isomer (
-TB) and 17ß-isomer (TB), respectively (Schiffer et al., 2001
).
Although TBA has been used in cattle feedlot operations for several decades, the affinity of 17ß- and 17-trenbolone for the human androgen receptor (AR) was only recently reported (Bauer et al., 2000
). It has also been reported that TB exhibits additional endocrine activities that distinguish it from androgens like testosterone and dihydrotestosterone (DHT) by displaying potent antiglucocorticoid activity in vivo (Danhaive and Rousseau, 1988
), which may explain its effects on adrenal gland morphology and function (Sillence and Rodway, 1990
; Thomas and Rodway, 1983
). This steroid also has high affinity for the bovine progestin receptor (Bauer et al., 2000
; Meyer and Rapp, 1985
). While the in vivo effects of TBA have been extensively examined in teratology studies, multigenerational studies, and the Hershberger assay, much of this data is unpublished, having been conducted in industry laboratories, and only brief summaries are available on the internet from the World Health Organization (WHO) Joint FAO/WHO Expert Committee on Food Additives (www.inchem.org/documents/jecfa/jecmono/v25je08.htm). In this regard, TBA has been reported to be "nonteratogenic" because it failed to produce malformations in several teratology and multigenerational studies.
The purpose of this study was to examine the potency of TB in both in vitro and in vivo screening assays for androgenic activity. Initially, the ability of TB to bind to the AR was confirmed and its ability to alter AR-dependent gene transcription in the MDA-kb2 cell line was examined. This cell line contains endogenous AR and has been stably transfected with an MMTV-luciferase reporter (Wilson et al., 2002). Via immunofluorescence, we examined the abililty of TB to induce ligand-dependent translocation of the AR into the nucleus of COS cells. Using a transient transfection system, we also examined the ability of TB to either activate or reduce dexamethasone-induced transcriptional activation through the glucocorticoid receptor (GR). The dose dependent effects of TB and testosterone propionate (TP) on androgen-dependent tissues were examined in vivo in castrated-immature male rats using the Hershberger assay. In contrast to testosterone, TB is a C-19 norandrogen and likely has different endocrine activities. Unlike testosterone, C-19 norandrogens cannot be aromatized, some cannot undergo 5-
reduction, and others are inactivated by 5-
reduction. Conversely, testosterone can both be aromatized and is activated to the more potent androgen, DHT, by 5-
reductase. In vivo, the goal was first to determine if TB displayed androgenic effects on all androgen (testosterone and DHT)-dependent tissues to the same degree as testosterone propionate when administered by sc injection. In addition, a second trenbolone study was conducted in which the animals were dosed with TB by gavage in order to compare the potency of trenbolone administered by oral dosing to the activity seen after sc administration. Finally, we wanted to examine the effects of TB in a short-term in utero screening assay we have developed as a screen for chemicals with AR agonist or antagonist activities. In this assay, chemicals with androgenic activity administered to the pregnant dam from gestational day (GD) 14 to 19 increase anogenital distance (AGD) of female offspring at birth and inhibit the display of nipples in infant female rats. If these effects are noted, all offspring from the screening study are retained for further investigation. The effects of TB were compared to comparable doses of TP administered in utero in a similar study in our laboratory (Wolf et al., 2002
).
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MATERIALS AND METHODS |
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Binding assay procedures were modified from previously described protocols (Lambright et al., 2000). Briefly, ventral prostate cytosol was diluted with ice-cold low-salt TEDG buffer to a protein concentration of 1.2 mg per 300 µl. Increasing concentrations of TB (0.1, 0.316, 1.0, 3.16, 10, 31.6, and 100 nM) were incubated on a rotary mixer at 4°C overnight (20 h) in the presence of 1 nM [3H]R1881 and 10 µM triamcinolone acetonide (binds and blocks progesterone and glucocorticoid receptors). Inert R1881 (100 X) was added to assess nonspecific binding. After incubation, radioligand bound and free receptors were separated using 500 µl of 60% hydroxyapatite (HAP) slurry in 50 mM Tris buffer. Samples were washed 3 times with 50 mM Tris (centrifuged at 600 x g) to assure complete removal of unbound ligand. Receptor bound ligand was recovered using 2 ml ethanol. Counts were determined using liquid scintillation counting.
COS whole-cell hAR binding assay.
This assay was used to evaluate the ability of TB to compete with 1 nM [3H]R1881 for binding to the human androgen receptor (hAR). COS cells (monkey kidney cells, ATCC) were transiently transfected with the hAR expression vector pCMVhAR as described by Wong et al.(1995). COS cells were plated at 200,000 cells/well in 12-well plates and transfected with 1 µg of pCMVhAR using diethylaminoethyl dextran. Twenty-four h later, cells were exposed to 1 nM [3H]R1881 in the presence and absence of unlabeled TB at concentrations ranging from 0.1 nM to 10 µM (two wells per concentration) and incubated for 2 h at 37°C. Nonspecific binding was determined by adding a 100-fold molar excess of unlabeled R1881. After incubation, cells were washed with phosphate-buffered saline and lysed in 200 µl ZAP (0.13 M ethyldimethylhexadecylammonium bromide with 3% glacial acetic acid). Radioactivity of the lysate was determined by liquid scintillation counting. Reported data are the mean of four replicate assays.
AR-dependent gene transcriptional activation assay in MDA-kb2 stable cell line.
The ability of TB to activate AR-mediated gene transcription was evaluated using MDA-kb2 cells stably transformed with the pMMTV.neo.luc reporter gene construct. Cells were maintained in L-15 media (Gibco BRL) supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C without CO2. AR-dependent transcriptional activation assays were conducted as previously described (Wilson et al., 2002). Briefly, cells were plated at 1 x 104 cells per well in 100 µl media in 96-well luminometer plates (Costar) and allowed to attach. Cells were then dosed with fresh media containing increasing concentration of TB, TB plus 1 µM hydroxyflutamide (OHF), or DHT. Each plate also contained vehicle (ethanol) control wells and 1.0 nM DHT and DHT/plus 1 µM OHF as agonist and antagonist controls, respectively. Cells were incubated in a humidified atmosphere overnight at 37°C without CO2. After incubation, cells were washed once with phosphate buffered saline at room temperature and then lysed by the addition of 25 µl of lysis buffer (Ligand Pharmaceuticals). Luciferase activity was determined using an MLX microtiter plate luminometer (Dynex, Chantilly, VA) and quantified as relative light units (RLU). Data are the mean of three replicate assays (four wells per treatment per assay).
GR dependent transcriptional activation assay in CV-1 cells.
The ability of TB to either activate GR-mediated transcription or to inhibit dexamethasone -induced GR activity, was examined as previously described using CV-1 cells cotransfected with 1 µg of pCMVhGR and 5 µg MMTV-luciferase reporter (Parks et al., 2001). Briefly, cells were plated at 200,000 cells/60 mm dish and transfected using 5 µl Fugene reagent (Boehringer Mannheim) plus 95 µl serum-free medium per dish using the manufacturers protocol. Twenty-four h after transfection, duplicate wells were treated with either vehicle only, 1 nM Dex only, or increasing concentrations of TB both with and without Dex cotreatment in Dulbeccos Modified Eagle Medium (DMEM) with 5% Dextran coated charcoal stripped fetal bovine serum (DCC FBS). After 24 h of exposure to test chemicals, cells were washed and harvested with 0.5 ml cell lysis buffer. Luciferase activity was detected using 0.05 ml of cell lysate, and relative light units were determined using a Monolight 2010 luminometer (Analytical Laboratories).
Immunocytochemistry in COS cells.
The following experiments were conducted to visualize, by immunofluorescence, TB-induced nuclear translocation of hAR in COS cells. Two-chambered slides (Nunc) were seeded with 100,000 cells/chamber in 2 ml DMEM (Gibco) supplemented with 10% FBS (Hyclone) after which cells were transfected with 0.5 µg pCMVhAR. Following transfection, medium was replaced with 2 ml DMEM containing 5% dextran-coated charcoal stripped FBS (Hyclone) plus 1 pM, 1 nM, or 1 µM TB, 100 nM DHT (positive control) or vehicle only (negative control), and incubated for 24 h at 37°C with 5% CO2. Cells were then washed once with Dulbeccos phosphate buffered saline (DPBS), allowed to dry for 45 min at room temperature, fixed for 10 min with 95% ethanol (20°C), and then blocked with 5% BSA (Sigma, in DPBS). Cells were incubated overnight with primary AR antibody (Affinity Bioreagents, 1:1000) at 4°C. The following day, cells were washed once with DPBS and then incubated with fluorescently labeled secondary antibody (Molecular Probes) for 30 min at room temperature. To visualize the nuclei, cells were counterstained with DAPI (Sigma), a DNA stain, mounted with fluoromount (Electron Microscopy Sciences) and examined using a Nikon Eclipse E800 microscope at magnification x20. The location of the AR was visually classified in a blinded manner as either nuclear, perinuclear, or both from eight or nine randomly selected fields from each slide by each of two independent observers. The percent nuclear staining was quantified for each field and the resultant data used for statistical analysis.
In Vivo
Hershberger assay.
Castrated-immature Sprague-Dawley (SD) rats were shipped from Charles River Breeding Laboratory (Raleigh, NC) the day after surgery and housed in groups of two or three per cage in clear plastic cages (20 x 25 x 47 cm) with heat-treated (to eliminate resins that induce liver enzymes) laboratory-grade pine shavings (Northeastern Products, Warrensburg, NY) as bedding. Animals were maintained on Purina Rat Chow (5001) and filtered tap water ad libitum. They were kept in a room with a 14:10 h photo-period (light/dark [L/D]; lights off at 1100 h EST), a temperature of 2022°C, and a relative humidity of 4050%.
The purpose of the first study was to determine if TB displayed androgenic effects on all androgen (testosterone and DHT)-dependent tissues to the same degree as TP when administered by sc injection in the castrate-immature male rat. A second TB study was conducted in which the animals were dosed with TB by gavage in order to compare the potency of TB administered by po dosing to the activity seen after sc administration. The Hershberger assay used herein was adapted from Hershberger et al.(1953), and was similar to that recommended by the Organization for Economic Co-operation and Development (OECD) for their interlaboratory prevalidation studies of this assay.
Immature SD male rats were castrated at 4142 days of age, received at 4243 days of age, and allowed to acclimate for 813 days (8 days in the TP study, 13 days in the two TB studies), at which time rats were randomly assigned to treatment groups in a manner that provided each group with similar mean initial body weights. In the TP study, castrate-immature male rats (n = 4 per group) were injected daily with TP (Sigma, Cat. #T 1875, CAS #57-85-2, Lot #98H0566) at 0, 12.5, 25, 50, 100, or 200 µg per rat per day for 10 days and then necropsied. Doses were administered sc on a mg/rat rather than a mg/kg body weight basis so we could directly compare the potency of TP in the Hershberger assay to the developmental effects of TP administered to the dam, since in the past most such studies have dosed rats in this manner (Green et al., 1939). In addition, as shown in the results herein and from other studies, sample sizes of 34 are adequate to detect the anabolic effects of potent androgenic substances in this assay. In the sc TB study, rats were injected sc with the vehicle only (0.1 ml corn oil; n = 6), 50 µg TP/0.1 ml corn oil (n = 6) or 50, 100, or 200 µg TB/0.1 ml corn oil (n = 3/group) for 10 consecutive days from 5665 days of age (Vehicle = corn oil, Sigma, Cat. #C 8267, CAS #8001-30-7, Lot #89H0149; TP, Sigma, Cat #T 1875, CAS #57-85-2, Lot #98H0566; TB, Sigma, Cat #T 3925, CAS #10161-33-8, Lot #40K0596, purity 98%). In the oral TB study, rats were dosed by gavage with either the vehicle only (2.5 ml corn oil/kg), 0.1, 1, 10, or 50 mg TB/kg/2.5 ml corn oil (n = 3/group) for 10 consecutive days from 5665 days of age.
On the day after the last treatment, males were anesthetized using carbon dioxide, euthanized by exsanguination, necropsied, and tissues weighed. The androgen-dependent tissues evaluated included the seminal vesicle plus coagulating gland (including fluid; SVCG), ventral prostate (VP), paired Cowpers glands, levator ani plus bulbocavernosus muscles (LABC), and the glans penis. In addition, body, liver, kidney, and adrenal glands were also weighed. The latter tissues also contain AR and are affected by androgens to some degree, albeit less than the sex accessory tissues. In the TP study, males were anaesthetized with Halothane at 5758 days of age and serum collected for measurement of testosterone via RIA. In the two TB studies, males were necropsied at 66 days of age.
Short-term in utero androgen-screening assay.
Timed-pregnant SD rats were shipped to our laboratory on GD 23 and then housed individually in clear polycarbonate cages. Animals were provided Purina Rat Chow (5008) and filtered (5 microns) water ad libitum in a room with a 14:10 h (L/D) photo-period (lights off at 1100 h EST) and temperature of 2022°C with relative humidly 4050%. Thirty pregnant rats (six rats/dose group) were dosed sc with either vehicle alone at 0.1 ml/rat/day or 0.1, 0.5, 1, or 2 mg TB/rat/0.1 ml corn oil from GD 1419. Dams were assigned to treatments in a manner that provided similar means and variances in body weight before dosing was initiated. Maternal weight during treatment was monitored throughout the dosing period. The number of pups was determined at birth and at 2 days of age (the day after birth = day 1) when individual pup body weights and AGD were measured. AGD was measured in a blind manner using a dissecting microscope with an ocular reticle calibrated with a 1 mm stage (0.01 mm divisions) micrometer at 1.5x. At 13 days of age all pups were weighed and the ventral surface examined (blind as to treatment) and the number of nipples counted. For female pups, which normally have 12 nipples, the nipples were counted after being scored as normal, faint, or absent. As obvious effects on AGD and areola numbers were noted in this study, all F1 offspring were retained for long-term evaluation based upon these triggers. These studies will be ongoing for several months as males are being retained until puberty and females until after puberty into adulthood and mated.
Statistical analysis.
In these studies, in vitro data was analyzed using PROC GLM, SAS version 6.08, on the U.S. EPA IBM mainframe computer. Data collected from in vitro binding and transcriptional activation assays were from at least three independent experiments with two or more replicates per experiment. In transcriptional activation assays, relative light units were converted to fold induction over media controls for each replicate for statistical analysis. Fold data was analyzed in a GLM model that included the concentration and replicates. For the immunocytochemistry, data were collected from two independent experiments (each in duplicate) where eight to nine fields from each slide were evaluated by two separate individuals blinded as to treatment. Stained cells in each field were examined and the location of the AR classified as either nuclear, perinuclear, or both. The percent of cells with only nuclear staining in each field was calculated and treatment effects compared by ANOVA. In the Hershberger assay, body and organ weight and serum hormone data were also analyzed using PROC GLM the SAS version 6.08 on the U.S. EPA IBM mainframe. Sex accessory tissue and serum testosterone data were log10 transformed prior to analysis to correct for heterogeneity of variance, the SD being proportional to the means. The regression models for organ weights included body weight at necropsy as a covariant. Statistically significant effects (p < 0.05, F-statistic) were examined using the LSMEANS procedure on SAS (two-tailed t-test) to compare the controls (castrate group without TP) to the TP- and TB-treated groups. In the in utero screening study, maternal weight change and litter sizes were analyzed as above, while AGD length, pup weight, and the numbers of normal nipples were analyzed using litter means for each sex.
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RESULTS |
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In Vivo, Hershberger Assay
In the sc TP study, androgen-dependent tissue weights (LABC, VP, SVCG, and glans penis) were significantly increased at all dosage levels, including the lowest dose of 12.5 µg TP/rat/day (Table 1). TP treatment in this study produced serum T levels and seminal vesicle and ventral prostate weights that ranged from subphysiological in the lower dosage groups to values in the 200 µg/rat/day normal for uncastrated control animals of this age (Monosson et al., 1999
). Adrenal weights were reduced (linear regression analysis) by TP treatment in this study, while body weight gain was significantly enhanced by TP administration.
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DISCUSSION |
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The toxicity of TB in the environment recently became an issue when it was recognized that this chemical and one of its metabolites, 17-trenbolone, which is a weaker androgen in vitro, are excreted into feedlots at concentrations that might be expected to be physiologically active (Schiffer et al., 2001
). In fact, Ankley et al. (in preparation) recently reported that environmentally relevant concentrations of TB in the low ng/l (ppt) range masculinized female fathead minnows in the laboratory and reduced fecundity in the fish. Here, we show that low ppt (nM and below) concentrations of TB also stimulate gene expression in vitro via the human AR. To date, regulatory agencies have not conducted an environmental risk assessment of this compound; in spite of the fact that, as shown here and by Ankley et al. (in preparation), ppt concentrations are active in vitro and in vivo in two vertebrate classes. Schiffer et al.(2001)
found concentrations of TB ranging from 5 to 75 ng/g and from 22 to 49 times higher levels of 17
-trenbolone in the manure canal. In addition, it has been shown that feedlot effluent from a concentrated animal feedlot operation (CAFO) displays a high level of androgenic activity (Gray et al., 2001
; Jegou et al., 2001
). Furthermore, altered endocrine physiology was detected in fathead minnows collected from streams near this CAFO (Jegou et al., 2001
). While there are no data on the concentrations of TB in biota from different trophic levels around these CAFO sites, one would speculate that fish would be at greater risk, absorbing the chemical from the water across the gills, than would mammals that might ingest contaminated fish or water. The potential risk to lower vertebrates like amphibians, reptiles, and birds at the CAFO site, is too speculative to comment on at this time due to lack of data. Our hypothesis that mammals would be at less risk after oral ingestion of TB is based upon our results from the two Hershberger assays which demonstrate that TB was about 80100 fold less effective via the oral route than via injection in the Hershberger assay. Similar results were cited by the WHO from unpublished Hershberger assay studies (Escuret and Bas, 1978
; Schroder, 1971a
,b
). Such speculation, however, should be confirmed by data on this point because long-term dietary TBA treatment has adverse effects on reproduction at µg/kg/day dosage levels (Hunter et al., 1976
, 1981
, 1982
). Although the oral route was less effective than was sc injection, trenbolone acetate (TBA) and TB have been shown to disrupt the reproductive system of humans, pigs, mice, rats, and other mammalian species at relatively low dosage levels when administered orally (Hess, 1983
, 1984
; Hunter et al., 1981
, 1982
; Kruskemper et al., 1967
, Lopez-Bote et al., 1994
).
The developmental and reproductive effects of TBA were extensively studied in the 1970s and 1980s (Trenbolone acetate: WHO Food Additives Series 25). However, none of these studies are published and only brief summaries of the results are available from WHO documents. In those studies, reproductive effects were seen in multigenerational studies using rats at doses ranging from 0.518 ppm in the diet. However, those studies did not report any effects that were clearly related to alterations of sexual differentiation. It is possible that some developmental effects were missed because they involve nonstandard endpoints (e.g., looking for the ventral prostate in females or reduced nipple numbers) or confused with direct effects on the adult because treatments were continued throughout life. Furthermore, several teratology studies cited in this review failed to observe any malformations in TBA or TB-treated fetuses and measurement of AGD in males also revealed no effect. In our study, it is clear that sc TB administration does induce malformations and it is likely that oral TB also will be "teratogenic," albeit at higher dosage levels.
In summary, this study confirms that in vitro TB is a high affinity ligand for both the rat and human AR and also induces AR-dependent gene expression with a potency equal to or greater than DHT. Along with its AR agonist activity, TB also acted as a GR antagonist in vitro. In the castrated immature rat, TB displays selective androgenic activity as compared to testosterone, affecting tissues that lack 5-reductase more than those with this enzyme. Conversely, administration of TB during the critical period of sexual differentiation increased AGD and attenuated nipple formation in female offspring, both of which are DHT-dependent tissues. These types of malformations have been shown to be indicators of more serious reproductive malformations later in life after exposure during sexual differentiation. Given the extensive use of TB in certain types of livestock feeding operations, its persistence in the environment, and the fact that it does induce reproductive malformations, further studies would be warranted. Similar to the problems with estrogenic effects seen with steroidal estogens found is sewage effluents, one would predict that fish residing downstream of feedlot operations where TB is used would be masculinized. In addition, as relatively little is known about the fate and tranport of TB in such systems, it seems reasonable that an ecological risk assessment should be conducted.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (919) 541-4017. E-mail: wilson.vickie{at}epa.gov.
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