Differing Species Responsiveness of Estrogenic Contaminants in Fish Is Conferred by the Ligand Binding Domain of the Estrogen Receptor

Abstract

Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17β (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼ 10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol A and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species.

Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecology

“…Although there were differences between the fish species studied for some of the interactions of pharmaceutical oestrogens with ESR1, far greater interspecies variation has been shown for other less potent environmental oestrogens. For example, ESR1 transactivation EC 50 s for 4-nonylphenol and 4-octylphenol are 30 times and 23 times lower, respectively, in common carp compared with medaka [37]. The much smaller 2.4-fold difference in the EC 50 for E2 (and 4.3-fold for EE2) between the fish species studied, likely reflects the crucial roles of this natural oestrogen (and synthetic mimic) in a wide range of tissues, functions and processes in fish [66,67].…”

“…Full-coding regions for ESR1 in each species were cloned and transfected into separate HEK293 cell lines and ESR1 receptor trans-activation assays were conducted as described in [37].…”

“…The greater interspecies variation in EC 50 s and fold-activation for DES (up to 5.7-fold variation) could therefore not easily be explained by the variations in sequence similarity in the LBD (electronic supplementary material, table S4). Instead, the influence of cofactors, differences in promoter sequences and amino acid residues in other domains, such as the DNA binding domain [37,68,69], may better account for the observed differences in ESR1 activation.…”

“…The level of protein sequence similarity between the human drug target and the fish orthologue (especially for the ligand binding domain; LBD) may enable better predictions regarding the species responsiveness to pharmaceutical exposure [6]. However, currently, there are few experimental data comparing drug target responsiveness in fish, other than for the oestrogen receptor a (ESR1), and these indicate that LBD sequence similarity is not always predictive of receptor activation and therefore physiological response [37].…”

Characterization of evolutionary trend in squamate estrogen receptor sensitivity

“…3A; Table 2) (Katsu et al, 2008a, Katsu et al, 2008b, Katsu et al, 2010aand Katsu et al, 2010bMiyagawa et al, 2014 andNaidoo et al, 2008 (Miyagawa et al, 2014). Hence, multiple sequence alignment of the LBD was conducted with the investigated tetrapod ERα orthologs, with inclusion of human ERα-LBD as well (Fig.…”