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IN VITRO ANDROGENIC ACTIVITY OF KRAFT MILL EFFLUENT IS ASSOCIATED WITH MASCULINIZATION OF FEMALE FISH
Citation:
Lambright, C R., L. G. Parks, E. Orlando, L. J. Guillette Jr., G Ankley, AND L. E. Gray Jr. IN VITRO ANDROGENIC ACTIVITY OF KRAFT MILL EFFLUENT IS ASSOCIATED WITH MASCULINIZATION OF FEMALE FISH. Presented at Society of Toxicology, San Francisco, CA, March 25 - 29, 2001.
Description:
In Vitro Androgenic Activity of Kraft Mill Effluent is Associated with Masculinization of Female Fish. Lambright, CS 1 , Parks, LG 1, Orlando, E 2, Guillette, LJ, Jr.2, Ankley, G 3, Gray, LE, Jr.1 , 1USEPA, NHEERL, RTP, NC, 2 University of Florida, Dept. of Zoology, Gainesville FL, 3 USEPA, MED, Duluth, MN. Female mosquito fish (Gambusia holbrooki) in the Fenholloway River downstream from Kraft Mill Effluent (KME) outflow display masculinization of the anal fin. The current investigation was designed to determine if KME from this river displayed androgenic activity in vitro. We examined water from 3 sites downstream from the Kraft mill (sites 1,2,3; site 1 is closest to outflow), one upstream (site 4) and another from the Econfina River, FL (site 5), on which there is no Kraft mill. Sites 1-3 (40 ml concentrated by C18 solid phase extraction (SPE) eluted with 100% methanol) competed with a synthetic androgen for binding to the human androgen receptor (hAR). Unextracted water from sites 1-3 were also found to induce AR-dependent transcriptional activation in CV-1 cells (cotransfected with pCMVhAR and MMTV luciferase reporter) with 15.7 (similar to 1nM DHT), 4.6, and 3.8 fold induction. The androgenic activity of KME was inhibited by hydroxyflutamide (1microM). Water samples from site 1 were separated by eluting fractions from the column using a series of methanol concentrations ( 25, 50, 75, 80, 85, 90, 95 and 100%) in order to identify which fraction(s) displayed androgenic activity. Activity was assessed using transcriptional activation in CV-1 cells and showed a 2.5 and 3.4 fold induction in the 75 and 80 % fractions, respectively. Further studies are ongoing to identify the androgenic component(s) in the KME. While our initial data were from site water collected in May 1999, additional site water samples collected in June 2000 also displayed androgenic activity, demonstrating the continued presence of the androgenic contaminant(s). In summary, we have identified androgenic activity in water from the Fenholloway River which is associated with masculinization of female fish.
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