In Vitro and in Vivo Effects of 17β-Trenbolone: A Feedlot Effluent Contaminant

Vickie S. Wilson 0 Christy Lambright 0 Joe Ostby 0 L. E. Gray 0 Jr. 0 0 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 - 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, 17 -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 androgendependent 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 The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. 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 the mention of trade names or commercial products constitute endorsement or recommendation for use. 1 To whom correspondence should be addressed. Fax: (919) 541-4017. E-mail: . androgen both in vitro and in vivo and, in contrast to other reports, can induce developmental abnormalities in the fetus. Key Words: 17 -trenbolone; environmental androgen; feedlot contaminant; in vivo; in vitro; in utero screen. Most of the chemicals identified as endocrine disruptors display weak hormonal activity as an unintended side effect. In contrast, there are a variety of other chemicals that have been developed to intentionally alter the endocrine system. These synthetic hormones are used for medical purposes or as growth promoters in farm animals. There is growing concern that these synthetic hormones are making their way into surface water and even ground water via animal and human wastes. Public concern initially focused on the synthetic components of oral contraceptives and their discharge from sewage treatment facilities after their incomplete removal during the treatment process (Ternes et al., 1999). These chemicals have high physiological activity at low concentrations and sewage treatment effluents have been shown to induce vitellogenin production, an estrogen-inducible protein normally expressed only in females, in male oviparous fish (Purdom et al., 1994). Thus there is evidence that wildlife is impacted by effluents containing synthetic steroids, while the potential impact on human populations remains uncertain. Concerns are not limited to sewage treatment effluents, however. Recently, both in vitro androgenic activity in feedlot effluent samples (Gray et al., 2001; Jegou et al., 2001) and reproductive alterations in fish living downstream from animal feedlot operations (Jegou et al., 2001) and in water dosed with low levels of 17 -trenbolone in the lab (Ankley et al., in preparation) have been reported. The anabolic steroid, trenbolone acetate (TBA; 17 -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, 17 -trenbolone (TB; 17 -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 17 -isomer (TB), respectively (Schiffer et al., 2001). Although TBA has been used in cattle feedlot operations for several decades, the affinity of 17 - 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 abili (...truncated)