Application of mechanism -specific in vitro bioassays to address questions in environmental toxicology.

摘要

This dissertation presents four studies centered around the application of mechanism-specific in vitro bioassays in the field of environmental toxicology. The first study used in vitro bioassays to screen and rank 11 chemicals based on their affinity for the estrogen receptor (ER) and ability to elicit an estrogenic response in vitro. Six of the eleven chemicals tested were able to displace tritiated 17β-estradiol from the ER. Their rank order of affinity was 17β-estradiol (E2) > coumestrol > 17β-ethynyl estradiol (EE2) > nonylphenol (NP) > octylphenol (OP) > bisphenol A (BPA). The rank order of potency for inducing reporter gene expression in the MCF-7-luc in vitro bioassay was E2 > EE2 > NP > OP > coumestrol > atrazine > BPA. ER binding and MCF-7-luc gene expression results were compared to generate hypotheses regarding potential mechanisms of action for the compounds analyzed. The second study focused on the development and characterization of a recombinant rainbow trout cell line (RLT 2.0)-based assay for assessing dioxin-like potency. Methods were adapted for a 96-well microplate format, and assay specific relative potencies (REPs) were derived for a number of halogenated aromatic hydrocarbons. Overall, the rank order of RLT 2.0-derived REPs was comparable to those generated based on other fish and mammalian bioassays, both in vitro and in vivo. A sensitivity analysis was also conducted to estimate the uncertainty associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ) calculated using the REPs generated. Result indicated that variability in the RLT 2.0-derived REPs could yield up to 10-fold uncertainty in RLT 2.0-based TEQ estimates. The third study focused on the current debate surrounding the derivation, use, and misuse of in vitro bioassaybased REP estimates. A systematic approach for deriving REP estimates from non-ideal in vitro bioassay results was presented and demonstrated using example data sets. The final study was designed to examine the relevance of current in vitro models for predicting estrogenic potencies in fish. Sexually mature male common carp (Cyprinus carpio) were exposed to 4-nonylphenol. Potential indirect mechanisms of action involving the modulation of plasma steroid concentrations were examined. The study detected no treatment related increases in concentrations of plasma E2, testosterone, or vitellogenin, despite measurable levels of NP in the plasma and tissues of exposed carp. Unexpectedly high variability among fish, and plasma E2 and VTG concentrations below method detection limits limited the ability to resolve effects, however. The lack of a detectable estrogenic effect in vivo hindered the ability to calibrate the known in vitro potency of NP to its potency for producing estrogenic effects in sexually mature male carp. The lack of estrogenic response also raised questions regarding the utility of estimating plasma or tissue concentrations of 17β-estradiol equivalents as a means of predicting the potential for estrogenic effects in vivo . Overall, the studies described in this dissertation exemplify research which is being done to develop and establish the utility of mechanism-specific in vitro bioassays as tools for environmental toxicology research and risk characterization.