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IN VITRO CONFIRMATION OF ANDROGENIC ACTIVITY IN KRAFT MILL EFFLUENT WHICH IS ASSOCIATED WITH MASCULINIZED FEMALE MOSQUITOFISH FORP
Citation:
Parks, L G., C. S. Lambright, E. Orlando, L. Guillette Jr., G T. Ankley, AND L E. Gray Jr. IN VITRO CONFIRMATION OF ANDROGENIC ACTIVITY IN KRAFT MILL EFFLUENT WHICH IS ASSOCIATED WITH MASCULINIZED FEMALE MOSQUITOFISH FORP. TOXICOLOGICAL SCIENCES 62:257-267, (2001).
Description:
Female mosquitofish downstream from Kraft paper mills in Florida display masculinization of the anal fin, an androgen-dependent response. This effect can be introduced in the laboratory with exposure to either paper mill effluent (PME) or to androgenic drugs. Hence, it has been hypothesized that alterations in anal fin morphology in female mosquitofish in these rivers results from the presence of androgenic substances in PME. The current investigation was designed to determine if water contaminated with PME from the Fenholloway River displayed androgenic activity in vitro and to relate this activity to the reproductive status of female mosquitofish taken from the river. In the present study, we examined river water from five sites, including one upstream and three downstream from a Kraft mill on the Fenholloway River and another site from the Econfina River, FL, on which there is no Kraft mill. We also evaluated anal fin ray morphlogy, an androgen-dependent sexually-dimorphic trait, in mosquitofish from these rivers. Based upon the numbers of segments in the longest anal fin ray, eighty percent of the female mosquitofish from the Fenholloway River were partially masculinized while another ten percent were completely masculinized. The numbers of segments in the longest ray of the anal fin was increased from 18. +/- 0.4 to 28.1 +/- 0.9 (p <0.001), in Econfina versus Fenholloway River female mosquito fish respectively. In vitro, all three PME samples displayed affinity for human androgen receptor (hAR) (p <0.001) in a binding assay using COS cells transiently transfected with the hAR. This activity was not displayed by water collected upstream of the Kraft mill or from the Econfina River. As hyphothesized, PME induced androgen-dependent gene expression in CV-1 cells (contransfected with CMV hAR and MMTV luciferase reporter). The androgenic activity displayed by PME was equivalent to 0.1 nM dihydrotestosterone, the positive control. The fact that this activity was inhibited by hydroxyflutamide (1 uM) demonstrartes that it was an AR-specific response and not due to the binding of PME to the glucocorticoil receptor (hGR). Furthermore, when CV-1 cells were transfected with hGR rather than hAR, PME did not induce MMTV-luciferase expression. Interestingly, PME displayed "testosterone-like" immunoreactivity in a testosterone radioimmunoassay, whereas water from the control sites did not. Further evidence of the androgenicity of the PME samples was observed using COS cells in which PME bound hAR and induced translocalization of perinuclear AR into the nucleus. In contrast, AR remained perinuclear, indicating the absence of an AR ligand when treated with water from the control sites. In summary, water collected downstream of the Kraft paper mill on the Fenholloway River contains unidentified androgenic substances whose presence is associated with masculinization of female fish.