Detection of Estrogen- and Dioxin-like Activity in Pulp and Paper Mill Black Liquor and Effluent Using in vitro Recombinant Receptor/Reporter Gene Assays

Abstract

In vitro recombinant receptor/reporter gene assays were used to examine pulp and paper mill black liquor and effluent for estrogenic, dioxin-like, and antiestrogenic activities. Using MCF-7 cells transiently transfected with a Gal4-estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc), it was estimated that black liquor contains 4 ±2 ppb estrogen equivalents, while negligible estrogenic activity was observed in a methanol-extracted pulp and paper mill effluent fraction (MF). A dioxin response element (DRE)-regulated luciferase reporter gene (pGudLuc1.1) transiently transfected into Hepa1c1c7 wild-type cells exhibited a dose-dependent increase in luciferase activity following treatment with 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), black liquor, and MF. Based on the dose-response curves, black liquor and MF contain 10 ± 4 ppb and 20 ± 6 ppt TCDD equivalents, respectively. Moreover, MF exhibited significant AhR-mediated antiestrogenic activity. These results demonstrate the utility of these bioassays and suggest that the effects observed in fish exposed to pulp and paper mill effluent may be due to unidentified ER and AhR ligands not detected by conventional chemical analysis due to the lack of appropriate standards.