Utility of in vitro test methods to assess the activity of xenoestrogens in fish

Abstract

The results of the present study have demonstrated the utility of an estrogen receptor (ER) competitive ligand-binding assay, a hepatocyte vitellogenin (VTG) induction bioassay, and an ER reporter gene bioassay in characterizing the activity of model estrogen agonists (17beta-estradiol [E2], ethynylestradiol, and nonylphenol) and antagonists (tamoxifen and ZM 189154) in rainbow trout (Oncorhynchus mykiss). The in vitro results were validated against in vivo trout waterborne exposures to E2 and tamoxifen. The results showed that all three in vitro assays were capable of detecting the hormonal activities of the selected model compounds in a dose-dependent manner, with the exception of nonylphenol in the ER reporter gene bioassay. However, the relative potency rankings of the model compounds were not consistent between these assays, which suggests that the relative potencies obtained within assays may have limited predictive value between assays. Discrepancies in potencies most likely can be attributed to the different levels of cellular organization in each assay. In addition to model compounds, we also evaluated the responses of the ER-binding assay and the hepatocyte VTG induction bioassay to complex mixtures associated with endocrine effects in fish, specifically extracts of pulp mill effluent. Of the 14 pulp mill effluent extracts tested, only six showed activity in both assays, whereas the remaining eight samples showed activity in only one of the two assays. The hepatocyte VTG induction bioassay consistently showed that the pulp mill effluent extracts were antiestrogenic, which to our knowledge has not been reported in previous studies. Collectively, these results suggest that a combination of in vitro assays that depend on differing endpoints is required to identify potential xenoestrogens and to characterize their modes of action.