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

Pulp and paper mill effluents have been shown to affect reproductive and endocrine functions in fish. We investigated the potential of β-sitosterol, a plant sterol found in relatively large quantities in most pulp and paper mill effluents, to contribute to these effects. The actions of β-sitosterol were investigated using a series of in vitro and in vivo assays based on different levels of cellular organization in rainbow trout using the environmental estrogen nonylphenol (NP) and 17β-estradiol (E2) as references. In a receptor binding assay, β-sitosterol was found to have a lower affinity for the rainbow trout hepatic estrogen receptor (ER) than NP and E2. In a whole cell assay using primary cultures of hepatocytes, β-sitosterol was found to induce the production of vitellogenin (Vtg), an estrogen dependent process. The relative potencies estimated from the receptor binding assay for β-sitosterol and Np (<1×10−5, 2.5×10−4) predicted within approximately an order of magnitude their potencies in the hepatocyte assay (5×10−5, 1.6×10−3). The estrogenic actions of β-sitosterol and NP were confirmed in a whole animal assay, where Vtg was induced in sexually immature rainbow trout exposed to waterborne β-sitosterol, NP and E2 for 21 days. NP and β-sitosterol induced a much lower production of Vtg than E2. Plasma testosterone, pregnenolone and total cholesterol levels were reduced by β-sitosterol but not by E2 and NP, implying that this effect was independent of its estrogenic activity. This study has shown that all three assays (receptor binding, hepatocyte Vtg production, in vivo Vtg production) confirmed the estrogenicity of β-sitosterol in trout. The in vivo assay was significant in demonstrating other effects not related to the estrogenicity of β-sitosterol and suggests there are multiple mechanisms through which β-sitosterol can modulate the endocrine system of trout.