Identification of metabolites of trenbolone acetate in androgenic runoff from a beef feedlot

Abstract

Little is known concerning the potential ecological effects of hormonally active substances associated with discharges from animal feeding operations. Trenbolone acetate is a synthetic anabolic steroid that is widely used in the United States to promote growth of beef cattle. Metabolites of trenbolone acetate include the stereoisomers 17alpha- and 17beta-trenbolone, both of which are stable in animal wastes and are relatively potent androgens in fish and mammals. Our purpose in this study was to evaluate the occurrence of 17alpha- and 17beta-trenbolone in a beef cattle feedlot discharge and in river water upstream and downstream from the discharge. In conjunction with that effort, we measured in vitro androgenic activity of the discharge using CV-1 cells that had been transiently cotransfected with human androgen receptor and reporter gene constructs. Samples were collected on nine different occasions during 2002 and 2003. Whole-water samples from the discharge caused a significant androgenic response in the CV-1 cells and contained detectable concentrations of 17alpha- and 17beta-trenbolone. Further work is needed to ascertain the degree to which synthetic androgens such as trenbolone contribute to androgenic activity of feedlot discharges.

Figure 1

Androgenic activity in CV-1 cells of (A) 17α-trenbolone, and (B) 17β-trenbolone. Variability bars (± SE) reflect duplicate determinations, expressed as fold over media. The trend lines on A and B are a polynomial regression of the second order. *Samples that have statistically significant androgenic activity compared with the media control.

Figure 2

Androgenic activity in CV-1 cells of upstream, downstream, and discharge samples expressed relative to media. Only a discharge sample was collected on D, and only upstream and downstream samples were collected on C. Pooled data are from samples A, B, F, and G, for which the CV-1 assays were conducted in triplicate. Variability bars for these samples reflect the SE of individual replicate analyses, while for the pooled sample set, the bars indicate variability of all data for samples A, B, F, and G. The mean (± SE) DHT positive controls from all assays are also shown with pooled data. *Samples that have statistically significant androgenic activity compared with the media control.

Figure 3

Concentrations (nanograms per liter) of 17α-trenbolone (A), and 17β-trenbolone (B) in upstream, downstream, and discharge samples as determined by HPLC.

Figure 4

GC-MS trace of 17α- and 17β-trenbolone standards (A), 17α- and 17β-trenbolone in fortified discharge sample E (B), and discharge sample E (C).