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MASCULINIZATION OF FEMALE MOSQUITOFISH IN KRAFT MILL EFFLUENT-CONTAMINATED FENHOLLOWAY RIVER WATER IS ASSOCIATED WITH ANDROGEN RECEPTOR AGONIST ACTIVITY
Citation:
Parks, L. G., C R. Lambright, E. Orlando, L. J. Guillette Jr., G T. Ankley, AND L E. Gray Jr. MASCULINIZATION OF FEMALE MOSQUITOFISH IN KRAFT MILL EFFLUENT-CONTAMINATED FENHOLLOWAY RIVER WATER IS ASSOCIATED WITH ANDROGEN RECEPTOR AGONIST ACTIVITY. TOXICOLOGICAL SCIENCES 62(2):257-267, (2001).
Description:
Female mosquitofish (Gambusia affinis holbrooki) downstream from Kraft paper mills in Florida display masculinization of the anal fin, an androgen-dependent trait. The current investigation was designed to determine if water contaminated with pulp mill effluent (PME) from the Fenholloway River FL displayed androgenic activity in vitro and to relate this activity to the reproductive status of female mosquitofish taken from this river. We tested water samples for androgenic activity from a reference site upstream of a Kraft pulp and paper mill on the Fenholloway River, FL, from three sites downstream from the mill, and from another reference site on the Econfina River, FL, where there is no Kraft mill. We also examined anal fin ray morphology in mosquitofish from these rivers for evidence of masculinization. Eighty percent of the female mosquitofish from the Fenholloway River were partially masculinized while another 10% were completely masculinized based upon the numbers of segments in the longest anal fin ray (18.0 ?0.4 versus 28.1 ?0.9 (p <0.001), in Econfina River versus Fenholloway River, respectively). In a COS whole-cell binding assay, all three PME samples displayed affinity for human androgen receptor (hAR) (p < 0.001). In addition, PME induced androgen-dependent gene expression in CV-1 cells (cotransfected with pCMV hAR and MMTV luciferase reporter) which could be inhibited by about 50% by coadministration of hydroxyflutamide (1 mM) an AR antagonist. Water samples collected upstream of the Kraft mill or from the Econfina River did not bind hAR or induce luciferase expression. When CV-1 cells were transfected with hGR rather than hAR, PME failed to significantly induce MMTV-luciferase expression. Further evidence of the androgenicity was observed using a COS cell AR nuclear translocalization assay. PME bound hAR and induced translocalization of AR into the nucleus while, in contrast, AR remained perinuclear when treated with water from the control sites (indicating the absence of an AR ligand). Interestingly, PME also displayed "testosterone-like" immunoreactivity in a testosterone radioimmunoassay, whereas water from the reference sites did not. In summary, water collected downstream of the Kraft mill on the Fenholloway River contains unidentified androgenic substances whose presence is associated with masculinization of female mosquitofish.