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ENDOCRINE TOXICOLOGY
In Vitro and in Vivo Effects of 17ß-Trenbolone: A Feedlot Effluent Contaminant
Vickie S. Wilson1, Christy Lambright, Joe Ostby and L. E. Gray, Jr.
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
ABSTRACT
Concern has arisen regarding the presence and persistence of trenbolone in the environment. Trenbolone acetate is an anabolic steroid used to promote growth in beef cattle. It is hydrolyzed to the active compound, 17ß-trenbolone (TB), which is also one of the metabolites excreted by cattle. Reproductive alterations have been reported in fish living in waters receiving cattle feedlot effluent, and in vitro androgenic activity displayed by feedlot effluent samples has been related to these effects. In the current study, the androgenic potency of TB was examined both in vitro and in short-term in vivo assays. TB was a high affinity ligand for the androgen receptor (AR), with an IC50 of about 4 nM in rat ventral prostate cytosol and about 33 nM in cells transfected with the human AR when competed with 1 nM [3H]R1881. TB induced AR-dependent gene expression in MDA-kb2 cells with a potency equal to or greater than dihydrotestosterone. In immunocytochemistry experiments with the human AR, concentrations as low as 1 pM significantly induced androgen-dependent translocation of the AR into the cell nucleus. TB also displayed antiglucocorticoid activity in vitro, inhibiting dexamethasone-induced transcriptional activity, and reduced adrenal gland size in vivo. In the Hershberger assay (in vivo), TB was as potent as testosterone propionate in tissues that lack 5 -reductase but less effective at increasing weight of tissues with this enzyme. Such tissue specificity was anticipated because other C-19 norsteroidal androgens display a similar profile in this assay. Subcutaneous TB treatment was about 50- to 100-fold more effective in stimulating growth of androgen-dependent tissues than was oral treatment. In our in utero screening assay, maternal TB administration increased AGD and attenuated the display of nipples in female offspring in a dose-related manner, similar to the published effects of testosterone propionate. Previous studies have documented that these types of malformations in newborn and infant rats are not only permanent effects but are also highly correlated with serious reproductive malformations as adults. In summary, TB is a potent environmental androgen both in vitro and in vivo and, in contrast to other reports, can induce developmental abnormalities in the fetus.
Key Words: 17ß-trenbolone; environmental androgen; feedlot contaminant; in vivo; in vitro; in utero screen.
DISCUSSION
In the current study, we demonstrated in vitro that TB is a high affinity AR ligand that induces AR-dependent gene transcription with potency equal to or greater than DHT. We also found that this steroid is active in vivo both in the Hershberger assay and during sexual differentiation. In the castrated-immature male rat, TB displays tissue-selective androgenic activity, exhibiting potency similar to TP in stimulating growth of the LABC muscles, while in contrast, tissues high in 5 -reductase activity such as the VP and seminal vesicles were stimulated only slightly as compared to TP. Such tissue-specific androgenicity is not unusual; for example, this has been described for 7 -methyl-19-nortestosterone, which cannot be metabolized by 5 -reductase, and 19-nortestosterone, which is inactivated by 5 -reductase (Sundaram et al., 1995 ; Toth and Zakar, 1986 ). In contrast to the ventral prostate and seminal vesicles, the effect of androgens in the LABC muscles is normally mediated via testosterone rather than conversion of testosterone to DHT. It is interesting to note, however, that maternal administration of TB during the critical period of sexual differentiation had marked effects on both AGD length and nipple regression, which are DHT-dependent processes. In the present study, TB administration during this period increased AGD and caused complete atrophy of nipple anlagen in female offspring. In comparison to the effects of TP administered at identical dosages levels during the same period of gestation, TB was about half as potent as TP in inducing malformations in these androgen-dependent tissues. Studies in our laboratory (Gray et al., 1999 ; Hotchkiss et al., submitted manuscript; Wolf et al., 2002 ) and in other laboratories (McIntyre et al., 2001 , 2002 ) have documented that AGD and nipple formation in newborn and infant male and female rats are not only permanent effects but also are highly correlated with other serious reproductive malformations. With this in mind, we have developed a short-term in utero protocol to screen for androgens and antiandrogens using AGD and infant nipple formation as triggers. Reduced AGD at birth and increased infant nipple formation in male rats at birth is indicative of antiandrogenic activity and triggers an extensive evaluation of all F1 male offspring after puberty. On the other hand, as seen here, increased AGD in newborn female rats and reduced nipple formation triggers an extensive evaluation of all F1 female offspring after puberty. Evaluation of F1 offspring from this study is still ongoing and will determine what other adverse effects are associated with administration of TB during sexual differentiation. Based upon previous studies with TP, we anticipate retained male sex accessory tissues, vaginal agenesis, and hydrometrocolpus in addition to other effects (Wolf et al., 2002 ).
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 80–100 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 0.5–18 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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