Estrogenic effects of environmental chemicals: An interspecies comparison

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Abstract

The development of various in vitro screening methods has led to identification of novel estrogenic chemicals of natural and anthropogenic origin. In this study, the (anti)estrogenic potential of several environmental chemicals were compared in an array of in vitro test systems comprising: (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and rainbow trout (Oncorhynchus mykiss) (rtER), (ii) a proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes. The results showed substantial differences in assay sensitivity for potent estrogens like 17β-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER  rtER  rtVtg). Chemicals like 4-n-nonylphenol and bisphenol A had higher relative binding affinity to the hER, whereas 4-t-butylphenol and 4-n-butylphenol showed highest affinity to the rtER. Zearalenone and the novel estrogen 4-t-butylhexanol displayed a considerable higher relative potency in the E-SCREEN than the rtVtg assay, whereas alkylphenols and the novel estrogen mimic 4-t-butyl-nitrobenzene were most potent in fish cells. Correlation analysis of data from the test systems suggest that interspecies differences is largely due to inter-assay variation of the ER-dependent cellular responses, whereas binding to the ER are fairly similar in the two species tested.

Introduction

Environmental estrogens, or estrogen mimics, have been suspected of modulating the endocrine system through multiple mechanisms of action and may potentially affect natural growth, development and reproduction in wildlife and humans (Colborn et al., 1993). The environmental estrogens, which may be of both natural and anthropogenic origin, have been found in all major compartments of the environment and the potential impact on ecosystem health has elicited considerable concern within the scientific, regulatory and public community. Many of the effects of these environmental estrogens are mediated through activation of the estrogen receptor (ER), which functions as a ligand-dependent transcriptions factor. The ER, has a variable N-terminal region (A/B-domain), a highly conserved central DNA binding region (C-domain), a variable hinge region (D-domain), a moderately conserved ligand-binding region (E-domain) and a poorly conserved short sequence at the carboxy-end (F-domain) (Pakdel et al., 1989). In recent years, presence of ERs have been documented in various vertebrates including fish, amphibians, reptiles, birds, and mammals and multiple forms of ER has been demonstrated in some species like fish and humans (see Hawkins et al., 2000 for details). Although the ER from closely related species exhibits similar binding affinities for endogenous and exogenous estrogens (Tollefsen et al., 2002), large differences in estrogen binding have been demonstrated for the ER from divergent species (Matthews et al., 2000, Matthews et al., 2001, Harris et al., 2002). For instance, the ERs derived from rainbow trout (rtER) has a highly divergent amino acid sequence within its ligand binding region (E-domain), with similarity of 60% when compared to the human ERα (hER) (Pakdel et al., 1989). This introduces the possibility of differential ligand-binding preferences and/or affinities for both endogenous and exogenous estrogens in species like fish and humans.

Several in vitro systems have been developed and utilised for screening chemicals for their estrogenic properties (Andersen et al., 1999, Gutendorf and Westendorf, 2001). These screening methods, which are derived from native or modified cells systems from various species, have been shown to display an inter-assay variation to estrogen mimics (Andersen et al., 1999, Andersson et al., 1999, Coldham et al., 1997, Fang et al., 2000, Gutendorf and Westendorf, 2001). The aim of this study was to compare the ER affinity and the (anti)estrogenic potencies of a range of estrogen mimics in commonly used test systems derived from fish and humans. Frequently used test protocols were adopted in this study. These systems include (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and the rainbow trout (Oncorhynchus mykiss) liver (rtER), (ii) a estrogen dependent proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes.

Section snippets

Chemicals

The test chemicals: 17β-estradiol (E2), diethylstilbestrol (DES), bisphenol A (BPA) and zearalenone (ZEN) were all from Sigma (St. Louis, MI, USA), 4-n-butylphenol (C4n) was from TCI (Tokyo, Japan) and 4-t-octylphenol (C8t) was obtained from Aldrich (Milwaukee, WI, USA). 4-n-nonylphenol (C9n), 4-t-butylphenol (C4t), cis/trans 4-t-butylcyclohexanol (C4-Cyclo), 4-n-butyl-chlorobenzene (C4–Cl), 4-t-butylnitrobenzene (C4–NO2) and 4-n-butylaniline (C4–NH2) were all supplied by Lancaster,

Binding to the hER and rtER

All chemicals tested had detectable affinity to the crudely isolated ERs (Fig. 1, Fig. 2). When comparing IC50 values, E2, DES, ZEN, BPA, C8t and C9n had higher affinity to the hER than to the rtER (Table 1). However, the alkylphenols C4t and C4n had the highest affinity to the rtER. The relative binding affinity (RBA) values for DES, ZEN and C8t obtained for the two receptor populations were quite similar, whereas RBA values for BPA and C9n were 54 and 21 times higher for hER compared to rtER,

Discussion

Binding of ligand to ER is most often the initial event in a series of activating steps leading to an estrogen specific response. As seen in the present work the ER from humans and fish have the ability to accommodate the binding of several structurally different compounds including E2, DES, ZEN, BPA, alkylphenols and non-phenolic compounds. Some of the well-known estrogenic chemicals like E2, DES and ZEN were highly potent binders in both systems, whereas others like BPA and the alkylphenols

Acknowledgements

This study was supported by grant from the Research Council of Norway and Norsk Hydro (Norway). We are grateful to Dr. A. M. Soto for kindly providing us with MCF-7 cells and Dr. T. Hutchinson for providing the antiestrogen ZM189.154.

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