Potencies of estrogenic compounds in in vitro screening assays and in life cycle tests with zebrafish in vivo

Abstract

The objective of this study was to compare the estrogenic potency of environmental estrogens at two testing tiers: at the initial level of in vitro screening assays, and at the level of definitive fish reproduction tests in vivo. The in vitro tests included a recombinant yeast estrogen receptor (ER) assay, a competitive radioreceptor assay using the hepatic ER of carp (Cyprinus carpio), and assays on vitellogenin induction in cultured hepatocytes of rainbow trout (Oncorhynchus mykiss) and carp. In vivo, full life cycle tests with zebrafish (Danio rerio) were perfomed, using fertilization success as estrogen-sensitive reproductive endpoint. The test compounds included the natural estrogen 17β-estradiol (E2) (only applied in the in vitro assays); the synthetic estrogen ethynylestradiol (EE2); and two xenoestrogens, 4-tert-octylphenol (OP) and bisphenol A (BPA). Among the in vitro assays, differences were observed in the relative ranking of the test substances, and in the absolute sensitivity (EC50 values), although the interassay differences of EC50 values were within one order of magnitude. The in vivo activity of the test compounds was not accurately predicted by the in vitro assays, with respect to neither sensitivity nor ranking. The in vitro assays tended to overestimate the relative potency of the xenoestrogens; i.e. the ratio between the activity of the reference compound, EE2, and that of the test compound. The best prediction of the in vivo fish test results was obtained from the recombinant yeast assay.

Introduction

It is now widely accepted that there are a number of natural and man-made substances in the aquatic environment that have the ability to interfere with the endocrine system of animals (e.g., Tyler et al., 1998; Vos et al., 2000). Among the endocrine-active compounds (EACs), primary concerns at present are for substances with estrogenic activity. Both field and laboratory studies have provided evidence that exposure to environmental estrogens can lead to the modulation or disruption of development and reproduction (Colborn et al., 1993; Danzo, 1998; Tyler et al., 1998). Environmental estrogens constitute a diverse group of chemicals, including natural and synthetic estrogens [e.g., 17β-estradiol (E2), 17α-ethynylestradiol], natural substances (e.g., wood-derived phytoestrogens), and xenobiotics with estrogenic activity (e.g., nonylphenol) (Nimrod and Benson, 1996; Sumpter et al., 1996; Vos et al., 2000). Although environmental estrogens do not necessarily share structural resemblance to the prototypical estrogen, E2, they are able to act as agonists or antagonists of the estrogen receptor (ER), and thus to modulate the endocrine pathways via a receptor-mediated process (Gillesby and Zacharewski, 1998; Sonnenschein and Soto, 1998; Blair et al., 2000).

Concern about the presence of EACs in the environment has triggered research on practical and validated screening assays and tests that are able to detect substances with hormonal activity, and/or to evaluate adverse effects of EACs on development and reproduction. Currently, hierarchical testing strategies, in which the initial tier may include in vitro screening assays, are considered to be most appropriate for hazard assessment of EACs (EMWAT, 1997; Ashby, 2000; Fenner-Crisp et al., 2000; Huet, 2000). A number of in vitro assays on estrogenic potency have been developed that exploit the proposed receptor-mediated mechanism of action for rational identification of alleged environmental estrogens, and for priority setting by determining their relative potencies in comparison to the prototypical hormone E2 (Zacharewski, 1997). The current challenge is to assess the effects of environmental estrogens in the intact organism, and it is an open question how to calibrate and validate results from in vitro assays in relation to developmental and reproductive performance of the animal in vivo (Zacharewski, 1997; Ashby, 2000). In order to further evaluate the utility and role of in vitro assays in the hazard assessment process, comparative in vitro/in vivo studies need to be undertaken (Zacharewski (1997), Zacharewsk (1998)).

The objective of this study was to compare the absolute and relative estrogenic potencies of environmental estrogens at two testing tiers: at the initial level of in vitro screening assays, and at the level of in vivo fish reproduction tests. Contrary to previous in vitro/in vivo comparisons, the in vivo test selected in this study is not an in vivo screening test such as the induction of vitellogenin (VTG) in exposed fish, but a definitive full life cycle test that allows the determination of estrogenic effects on fish development and reproduction. As test compounds, we used the synthetic estrogen ethynylestradiol (EE2), and two xenoestrogens, 4-tert-octylphenol (OP) and bisphenol A (BPA). In the in vitro assays, E2 was also used. The estrogenic potencies of the test compounds were examined in the in vitro screens based on the endpoints “ER binding” and “activation of ER-regulated genes”: a recombinant yeast ER assay, a radioreceptor assay with the hepatic estrogen receptor of carp, Cyprinus carpio, and vitellogenin induction in cultured hepatocytes of rainbow trout (Oncorhynchus mykiss) and carp. Further, the effects of the test compounds on fish reproduction in vivo were determined in a full life cycle test with zebrafish, Danio rerio, and the results were compared to the in vitro data.