Effect of cadmium on the interaction of 17beta-estradiol with the rainbow trout estrogen receptor

The widely reported negative effects of xenoestrogens on the endocrine system of aquatic organisms gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that not only organic chemicals but also certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol (E2) and lead to estrogen receptor activation. While the effects of cadmium on the endocrine system and its potential to harm living organisms are no longer in doubt, the exact mode of action is still essentially unknown. In the present study we utilized the rainbow trout ER alpha ligand binding domain (rtER-LBD) fused to glutathione-S-transferase, to study noncovalent interactions between cadmium and the rtER-LBD. ICP-MS data showed that the Cd uptake by the rtER-LBD was strongly pH-dependent. Previous results showing that Cd shields Cys residues of the rtER-LBD against chemical modification, and competitive binding experiments reported here provide insights into the specificity of the interaction of cadmium with the ER hormone binding cavity. It could, for instance, be shown that most of the cadmium adsorbed to the protein could be released into solution either under denaturing conditions, or by stripping from the protein surface using EDTA at physiological conditions. Competitive binding experiments using radio-labeled estradiol showed that, in contrast to previously published data, E2 has an affinity an order of magnitude higher for the ER than for Cd. ICP-MS experiments showed that, despite its higher affinity, increasing E2 concentrations were unable to replace Cd from the rtER-LBD that had been preequilibrated with Cd. These findings were independently confirmed by the [3H]-E2 binding assay. At the same time both ICP-MS and the [3H]-E2 binding assay showed that increasing Cd concentrations not only lead to a decrease in the specific estradiol binding, but also to the release of E2 from the preequilibrated rtER-LBD. The gradual release of [3H]-E2 from the rtER-LBD following incubation with increasing Cd concentrations indicates either direct competition for the same binding site or Cd-induced conformational changes resulting in the release of estradiol.