Masculinization of Female Mosquitofish in Kraft Mill Effluent-Contaminated Fenholloway River Water Is Associated with Androgen Receptor Agonist Activity

Abstract

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 in Florida 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, from 3 sites downstream from the mill, and from another reference site on the Econfina River, also in Florida, where there is no paper 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 vs. 28.1 +/- 0.9 [p < 0.001]) in the Econfina River vs. the Fenholloway River, respectively). In a COS whole cell-binding assay, all 3 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 was inhibited by about 50% by coadministration of hydroxyflutamide (1 microM), 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 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-translocalization assay. PME bound hAR and induced translocalization of AR into the nucleus. 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.

Aromatase activity in the ovary and brain of the eastern mosquitofish (Gambusia holbrooki) exposed to paper mill effluent.

“…Recently, it was reported that androstenedione was present after a chemical characterization of the effluent from a paper mill plant in Florida (50). Using a number of in vitro receptor binding and gene expression assays, our laboratory in collaboration with others has not found sufficient androstenedione concentration in Fenholloway River water samples to support the reported significant androgenic actions of this effluent, however (15).…”

“…A wide range of chemicals were shown to have an affinity for estrogen receptors from various species (12). In addition to the estrogenic or antiestrogenic nature of chemical contaminants, data are currently available documenting antiandrogenic (8,9), androgenic (13)(14)(15), antiprogestogenic (16), and antithyroidal actions (17)(18)(19). The mechanisms behind these actions appear varied, with receptor binding only one of a number of possible responses.…”

Screening of multiple hormonal activities in surface water and sediment from the Pearl River system, South China, using effect‐directed in vitro bioassays

“…Surface water and sediment samples were collected from the Liuxi, Shijing, and Zhujiang rivers of the Pearl River system in the dry season (December [17][18]2007) and the wet season (September [10][11][12]2008). In total, 15 sampling sites along the three rivers were selected (Fig.…”

“…Male fish feminization in receiving waters has been related to residual natural and synthetic estrogens from municipal STP effluents [8,9]. Effluents from pulp and paper mills displayed androgenic activity in female fish, leading to masculinization or sex reversal of female fish [10,11]. Very recently, screening assays revealed that androgenic activity also exists widely in industrial and hospital wastewaters, municipal STP effluents, and even in surface water in association with estrogen, progesterone, and glucocorticoid receptor activities [12,13].…”

Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment

“…The increasing number of reports on intersex organisms found in global aquatic ecosystems has raised concerns regarding the occurrence and persistence of steroid hormones in the environment (Safe, 2004;Wise et al, 2011). Several studies have reported the masculinization of freshwater wildlife exposed to androgens in polluted rivers (Howell et al, 1980;Bortone et al, 1989;Parks et al, 2001;Orlando et al, 2004). Furthermore, steroids were identified as pheromones and adversely affect fish behavior even at extremely low concentrations (Kolodziej et al, 2003).…”