Molecular basis for endocrine disruptor - estrogen receptor interactions: a species comparison. TR Zacharewski.

Despite the evolutionary conservation of the activities of estrogen and the functions of the ER, there is considerable variation in the amino acid sequence of the estrogen receptor ligand binding domain (ER LBD) between species. It has been hypothesized that this variation in ER LBD may lead to differences in endocrine disruptor-estrogen receptor interactions, and therefore the use of a single surrogate species to screen and prioritize endocrine disruptors may be inappropriate. In order to investigate potential differences in species-specific sensitivities to non-traditional ER ligands, a comparative study was undertaken in which endogenous estrogens, pharmaceuticals, phytoestrogens, mycotoxins, polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), hydroxylated PCBs, Aroclors, pesticides and industrial chemicals were examined for their ability to compete with tritiated 17b -estradiol (E2) for binding to partially purified bacterially expressed fusion proteins using a semi-high throughput competitive binding assay. The fusion proteins consisted of ER LDB (domains D, E and F) of human (a ), mouse (a ), chicken, green anole (Anolis Carolinensis) and rainbow trout (Onchorhynkiss mykiss) linked to the glutathione-S-transferase (GST) protein. Saturation ligand-binding analysis of GST-hERa def (human), GST-mERa def (mouse), GST-cERdef (chicken), GST-aERdef (green anole) and GST-rtERdef (rainbow trout) fusion proteins revealed a single high affinity binding component for E2 with dissociation constants (K_d) ranging from of 0.3 to 0.9 nM. Although, the ERs from the different species exhibited similar binding preferences and binding affinities for many of the compounds examined, several differences in absolute and relative binding affinities were observed. The ranking of the potency of the xenobiotics and phytoestrogens varied among species with no predictable patterns and many examples of species-specific ligand preference. For example, a -zearalenol (IC₅₀ = 1.5 ± 0.7 x 10^-9 M) competed for binding with greater affinity than E2 for GST-rtERdef which was in contrast to all other GST-ERdef fusion proteins examined. However, the phytoestrogen coumestrol (IC₅₀ = 1.6 ± 0.3 x 10^-6 M) bound with lowest affinity to GST-rtERdef, while genistein (IC₅₀ = 3.0 ± 2.0 x 10^-8 M) bound with greatest affinity with GST-aERdef. Differences in the relative binding affinity for a -zearalenol between human and rainbow trout ERs were further examined using computational methods. Coordinates from crystal structures of the human ERa E domain complexed with E2, diethylstilbestrol, hydroxytamoxifen and raloxifene were used to generate a homology model of the rainbow trout ligand binding pocket. GOLD and AutoDock computer aided ligand docking software were then used to predict binding modes and residue contacts for a -zearalenol interactions with the human and rainbow trout binding pocket. In both models, a -zearalenol was found to utilize a similar binding mode when compared to E2 but formed unique contacts. However, a -zearalenol was predicted to be rotated 180^o when bound in the rainbow trout ER ligand binding pocket. The results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic substances and that these differences may be due to the variability in the amino acid sequence within the ER ligand binding among species. These studies suggest that the use of a single surrogate model may not be suitable for the prediction of estrogenic responses in all species.