Masculinization of female mosquitofish in kraft mill effluent-contaminatedFenholloway River water is associated with androgen receptor agonist activity

Female mosquitofish (Gambusia affinis holbrooki) downstream from Kraft pape r mills in Florida display masculinization of the anal fin, an androgen-de pendent trait. The current investigation was designed to determine if water contaminated with pulp-mill effluent (PME) from the Fenholloway River in F lorida displayed androgenic activity in vitro and to relate this activity t o 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, from 3 sites down stream from the mill, and from another reference site on the Econfina. Rive r, also in Florida, where there is no paper mill. We also examined anal fin ray morphology in mosquitofish from these rivers for evidence of masculini zation. Eighty percent of the female mosquitofish from the Fenholloway Rive r were partially masculinized while another 10% were completely masculinize d, based upon the numbers of segments in the longest anal fin ray (18.0 +/- 0.4 vs. 28.1 +/- 0.9 [p < 0.001]) in the Econfina River vs. the Fenhollowa y River, respectively). In a COS whole cell-binding assay, all 3 PME sample s displayed affinity for human androgen receptor (hAR) (p < 0.001). In addi tion, PME induced androgen-dependent gene expression in CV-1 cells (cotrans fected with pCMV hAR and MMTV luciferase reporter), which was inhibited by about 50% by coadministration of hydroxyflutamide (1 muM), an AR antagonist . Water samples collected upstream of the Kraft mill or from the Econfina R iver did not bind hAR or induce luciferase expression. When CV-1 cells were transfected with human glucocorticoid receptor (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-translocaliz ation assay. PME bound hAR and induced translocalization of AR into the nuc leus. 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 radi oimmunoassay, whereas water from the reference sites did not. In summary, w ater collected downstream of the Kraft mill on the Fenholloway River contai ns unidentified androgenic substances whose presence is associated with mas culinization of female mosquitofish.