Elsevier

Aquatic Toxicology

Volume 60, Issues 1–2, 2 October 2002, Pages 101-110
Aquatic Toxicology

A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro

https://doi.org/10.1016/S0166-445X(01)00276-4Get rights and content

Abstract

Five natural, pharmaceutical, or xenobiotic chemicals [17β-estradiol (E2), ethynylestradiol (EE2), diethystilbestrol (DES), methoxychlor (MXC), nonylphenol (NP)] were tested in two in vitro assays [yeast estrogen screen (YES), MCF-7 breast tumor cell proliferation (E-Screen)], and compared with previously reported results from two in vivo male sheepshead minnow vitellogenin (VTG) production studies. The purpose of this investigation was to determine how accurately the two in vitro assays predicted responses observed in live animals. EC50 values for all five chemicals were approximately one order of magnitude less sensitive in the YES assay than in the MCF-7 assay. Based on the EC50 values, DES was 1.1 (YES) to 2.5 (MCF-7) times more potent in these receptor binding assays than was E2, while EE2 was slightly less potent than E2 in the YES assay (0.7) and nearly twice as potent (1.9) as E2 in the MCF-7 assay. EE2 and DES were of approximately equal potency in the 13-day sheepshead minnow VTG production bioassay. Both MXC and NP were 107 times less potent than E2 in the YES assay, MXC was 105 times less estrogenic than E2 in the MCF-7 assay, while both were approximately 100 times less potent than E2 in the live animal bioassay. The in vitro tests were substantially less sensitive (at least 1000 times) than the sheepshead minnow VTG assay for estimating estrogenic potency of the two xenobiotic chemicals, which suggests that in vitro-based, large-scale screening programs could potentially result in many false negative evaluations.

Introduction

A wide range of natural, pharmaceutical and synthetic chemicals released into the environment are estrogenic (Purdom et al., 1994, White et al., 1994, Blackburn and Waldock, 1995, Field and Reed, 1996, Folmar et al., 1996, Folmar et al., 2001, Nimrod and Benson, 1996, Bennie et al., 1997, Harries et al., 1997). These chemicals enter aquatic ecosystems through both point (sewage treatment, pulp mill and industrial effluent) and non-point (urban and agricultural runoff) sources and include natural and pharmaceutical chemicals excreted in the urine of humans and domestic livestock, organochlorine pesticides, polychlorinated biphenyls, polynuclear aromatic hydrocarbons, dioxins, plasticizers and and surfactants.

In 1996, Congress mandated the US Environmental Protection Agency (EPA) to develop and implement screening evaluations for potential endocrine-disrupting chemicals [Public Laws 104–182 (Safe Drinking Water Amendments) and 104–170 (Food Quality Protection Act)] with emphasis on estrogen-mimicking chemicals. To meet those Congressional requirements, the EPA organized the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to recommend specific screening and testing protocols to evaluate the effects of endocrine disrupting chemicals on human and environmental health.

There have been numerous studies evaluating a wide variety of chemicals for their activities in several in vitro estrogen responsive assays (Soto et al., 1991, Soto et al., 1995, Jobling and Sumpter, 1993, Jobling et al., 1995, White et al., 1994, Arnold et al., 1996, Routledge and Sumpter, 1996, Shelby et al., 1996, Coldham et al., 1997, Desbrow et al., 1998, Anderson et al., 1999). The oldest, most widely used in vivo assay for estrogenic activity is the mouse uterotrophic assay. This assay is sensitive and can account for additive activity of multiple estrogens, however, exposure to androgens or progestins can also produce positive results (Kupfer, 1988). In fish, the most commonly measured in vivo responses to estrogen exposure are, (1) developmental abnormalities of the gonads (Gimeno et al., 1996, Jobling et al., 1996, Gray and Metcalfe, 1997, Panter et al., 1998, Metcalfe et al., 2000, Metcalfe et al., 2001); and (2) the upregulation and expression of vitellogenin (VTG) and zona radiata genes in males (Emmersen et al., 1979, Jobling and Sumpter, 1993, Purdom et al., 1994, White et al., 1994, Folmar et al., 1996, Folmar et al., 2000, Hyllner et al., 1991, Oppen-Berntsen et al., 1992, Larsson et al., 1994, Arukwe et al., 1997, Arukwe et al., 1998, Denslow et al., 1997, Murata et al., 1997; Celius and Walther, 1998a, Celius and Walther, 1998b, Knudsen et al., 1998, Hemmer et al., 2001).

Other comparative studies have been conducted with mammalian systems (Anderson et al., 1999) to provide EDSTAC with information on the comparability of certain in vitro and in vivo assays for evaluating estrogenic potency. Here, we have tested five chemicals in the yeast-estrogen screen (YES) (Arnold et al., 1996) and MCF-7 breast tumor cell proliferation (E-Screen) (Soto et al., 1995), then compared those results with the sheepshead minnow (Cyprinodon variegatus) VTG expression assay (Folmar et al., 2000, Hemmer et al., 2001). The predominant vertebrate estrogen (17β-estradiol), two pharmaceutical estrogens [17β-ethynylestradiol (EE2) and diethylstilbestrol (DES)], a pesticide (methoxychlor, MXC) and a widely used surfactant (nonylphenol, NP) common to domestic sewage were selected for comparisons.

Section snippets

Chemicals

The chemicals used in the in vitro and in vivo assays were from the same production lot and obtained from the following sources: 17β-estradiol (E2) [1,3,5[10]-estratriene-3,17β-diol], 98.0% purity, 17β-EE2 [17β-ethynyl-1,3,5[10]-estriene-3,17β-diol], 98.0% purity, DES [3,4-bis[4-hydroxyphenyl]-3-hexene], 99.0% purity, from Sigma (St Louis, MO); p-NP [para-NP], 96.4% purity, from Schenectady International (Schenectady, NY); and MXC [1,1′-(2,2,2-trichloroethylidiene)-bis(4-methoxybenzene)], 99.0%

YES assay

The calculated EC50 values and 95% confidence intervals (C.I.) for each chemical tested are presented in Table 2. While the two pharmaceutical estrogens showed responses within the same order of magnitude as the native ligand E2, the two xenobiotic chemicals, MXC and NP demonstrated approximately 106–107 less affinity for the estrogen receptor (ER), respectively. Of the two pharmaceuticals tested, DES showed the greatest apparent affinity for the ER with a relative potency 1.1 times that of E2 (

Discussion

The two in vitro assays in our evaluations were selected, because, they are widely used and are under consideration to become part of EDSTAC's screening program for estrogenic chemicals. We followed standard protocols to determine EC50 concentrations for the five test chemicals in both assays. We compared those in vitro results with evaluations from an in vivo SHM flow-through VTG production assay which was recently developed in our laboratories. Estradiol, EE2 and DES were evaluated in one

Conclusion

In vitro assays can rank chemicals within a given test, but they cannot be extrapolated or predict whether the test chemicals will maintain the same order of potency in a live animal bioassay. This and previous studies have clearly demonstrated that in vitro assessments for estrogenicity, particularly for chemicals which require metabolic activation or are capable of substantial bioaccumulation, underestimate the responses observed with in vivo testing. Additionally, the in vitro assays do not

Acknowledgements

The mention of commercial trade names does not constitute endorsement by the US Government. This study was partially supported by EPA Cooperative Agreement CR826357 to NDD.

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