Endocrine-disrupting effects of cattle feedlot effluent on an aquatic sentinel species, the fathead minnow - Research

Environmental Health Perspectives, March, 2004 by Edward F. Orlando, Alan S. Kolok, Gerry A. Binzcik, Jennifer L. Gates, Megan k Horton, Christy S. Lambright, L. Earl Gray, Jr., Ana M. Soto, Louis J. Guilette, Jr.

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CV-1 AR-dependent transcriptional activation assays. We assessed androgenicity in seven replicate experiments (with duplicates of each replicate). Androgenicity was defined as the ability of FLE or dihydrotestosterone (DHT, Sigma Chemical Co.) to induce AR-dependent luciferase gene expression in a transfected CV-1 cell line. In every sample and in all seven replicates, FLE induced AR-dependent luciferase gene expression. The data presented in Figure 4 are expressed as fold induction over the control medium (without FLE) and compared with the positive control of 1 nM DHT (near maximal concentration in terms of its ability to induce luciferase expression), FLE and DHT each exhibited significantly higher androgen activity than did media (p < 0.0001, for each treatment vs. medium control). DHT-and FLE-induced responses were not significantly different (p = 0.35) from each other.

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Discussion

To our knowledge, this is the first study to document endocrine disruption in fish exposed to FLE. Wild fish collected below a feedlot exhibited altered reproductive biology, including decreased T synthesis, altered head morphometrics, and smaller testis size in males and decreased E:A ratio in female fish. We did not observe overt characteristics in either male or female fish suggesting environmental exposure to estrogens. With an in vitro assay using cells transfected with hAR, we detected potent androgenic responses from the FLE. Taken together, our morphologic, endocrinologic, and in vino gene activation assay data suggest two hypotheses: a) there is an androgenic substance(s) in the FLE and/or b) there is a mixture of endocrine-active substances that alter the hypothalamic-pituitary-gonadal axis. Further support for the hypothesis that androgens are present in the FLE comes from observations of androgenic activity (Soto et al. 2004). However, Soto et al. (2004) also found estrogenic activity in FLE using the MCF-7 cell in vitro E-SCREEN assay, suggesting that there could be a complex mixture of natural and pharmaceutical compounds in the effluent.

Our data clearly demonstrate androgenic activity from water obtained below feedlots. However, it does not identify the causal agents. Androgenic activity could be due to natural androgens found in fecal material or androgenic pharmaceuticals used in growth implants (Meyer 2001). Natural androgens have relatively short half-lives in feces and in the open water of retaining ponds (Meyer 2001). In contrast, recent studies demonstrate that metabolites of synthetic androgens (e.g., trenbolone-[beta] from trenbolone acetate) used in growth implants have longer half-lives. Approximately 27.5% of the initial concentration of trenbolone-[beta] was still present in manure piles 4.5 months after deposition (Schiffer et al. 2001). Natural steroids appear to be rapidly degraded, with half-lives measured on the order of days to hours. No literature could be found regarding the relative persistence of zeranol or melengestrol in feed-lot retaining ponds, however.