Use of a series of homologous in vitro and in vivo assays to evaluate the endocrine modulating actions of beta-sitosterol in rainbow trout

Pulp and paper mill effluents have been shown to affect reproductive and en docrine functions in fish. We investigated the potential of beta-sitosterol , a plant sterol found in relatively large quantities in most pulp and pape r mill effluents, to contribute to these effects. The actions of beta-sitos terol were investigated using a series of in vitro and in vivo assays based on different levels of cellular organization in rainbow trout using the en vironmental estrogen nonylphenol (NP) and 17 beta-estradiol (E-2) as refere nces. In a receptor binding assay, beta-silosterol was found to have a lowe r affinity for the rainbow trout hepatic estrogen receptor (ER) than NP and E-2. In a whole cell assay using primary cultures of hepatocytes, beta-sit osterol was found to induce the production of vitellogenin (Vtg), an estrog en dependent process. The relative potencies estimated from the receptor bi nding assay for beta-sitosterol and Np (< 1 x 10(-5), 2.5 x 10(-4)) predict ed within approximately an order of magnitude their potencies in the hepato cyte assay (5 x 10(-5), 1.6 x 10(-3)). The estrogenic actions of beta-sitos terol and NP were confirmed in a whole animal assay, where Vtg was induced in sexually immature rainbow trout exposed to waterborne beta-sitosterol, N P and E-2 for 21 days. NP and beta-sitosterol induced a much lower producti on of Vtg than E-2. Plasma testosterone, pregnenolone and total cholesterol levels were reduced by beta-sitosterol but not by E-2 and NP, implying tha t this effect was independent of its estrogenic activity. This study has sh own that all three assays (receptor binding, hepatocyte Vtg production, in vivo Vtg production) confirmed the estrogenicity of beta-sitosterol in trou t. The in vivo assay was significant in demonstrating other effects not rel ated to the estrogenicity of beta-sitosterol and suggests there are multipl e mechanisms through which beta-sitosterol can modulate the endocrine syste m of trout. (C} 1998 Elsevier Science B.V. All rights reserved.