Amphibians as a model to study endocrine disruptors: I. Environmental pollution and estrogen receptor binding

Many chemicals released into the environment without toxicological risks ha ve the capacities to disrupt the function of endocrine systems. These endoc rine disrupters disturb normal endocrine mechanisms and have been observed in nearly all classes of vertebrates. The aim of this research is to develo p a comprehensive model to study endocrine disruption using the amphibian X enopus laevis. The assessment of estrogenic potencies of endocrine disrupte rs includes several levels of investigation: (I) binding to liver estrogen receptor, (II) estrogenic activity in vitro by inducing vitellogenin synthe sis in primary cultured hepatocytes, and (III) in vivo effects on sexual de velopment caused by exposure of larvae. The present paper is focused on the first part by establishing a radioreceptorassay for [H-3]17 beta-estradiol ([H-3]E2) binding using liver cytosol fraction. In order to get optimum bi nding conditions we performed kinetic, saturation, and competitive displace ment experiments. Association of [H-3]E2 to estrogen receptor revealed that maximum specific binding is achieved between 18 and 48 h of incubation. Sc atchard analyses of saturation experiments resulted in a homogenous saturab le population of estrogen receptors having no significant differences of bi nding parameters between both sexes. The values of K-d (dissociation consta nt) in males and females were 22.4 +/- 6.0 and 15.0 +/- 2.8 nM (mean +/- S. E.M.; n = 5), respectively, while corresponding B-max (maximum binding capa city) revealed 89 +/- 46 and 136 +/- 46 fmol [H-3]E2/mg protein. The specif icity of estrogen receptors as shown by competitive displacement experiment s demonstrated receptors being highly specific just for estrogens, but not for other endogenous steroids having the following ranking of binding affin ities: E2 > estrone > dehydroepiandrosterone > aldosterone greater than or equal to testosterone greater than or equal to corticosterone greater than or equal to progesterone. The affinity ranking of environmental chemicals c ompared to E2 was: E2 > tetrachlorbiphenyl > diethylphthalate > 2,2-bis-(4- hydroxyphenyl)-propan (bisphenol A)greater than or equal to 4-nonylphenol g reater than or equal to 3-t-butyl-4-hydroxyanisale greater than or equal to 4-octylphenol > dichlor-diphenyl-trichlor-ethan (4,4'-DDT). Analyses of fi ve sewage effluents for displacement of [H-3]E2 binding resulted in three s amples displacing more than 50% of specific binding at their original conce ntration. Taken together the established radioreceptorassay for [H-3]E2 bin ding in Xenopus laevis liver cytosol is useful to screen estrogen receptor binding of pure compounds or complex mixtures of them, which is the prerequ isite for causing either estrogenic or antiestrogenic effects. (C) 1999 Els evier Science B.V. All rights reserved.