Endocrine‐disrupting potentials of equine estrogens equilin, equilenin, and their metabolites, in the medaka Oryzias latipes: in silico and DNA microarray studies

Abstract

Although several previous studies have demonstrated the presence of equine estrogens in the aquatic environment, limited data are currently available on the endocrine-disrupting potentials in fish and the risks they pose to aquatic organisms. To investigate the interactions of major equine estrogens equilin (Eq) and equilenin (Eqn), as well as their metabolites 17α-dihydroequilin, 17β-dihydroequilin, 17α-dihydroequilenin and 17β-dihydroequilenin, with the estrogen receptor α (ERα) of medaka (Oryzias latipes), a three-dimensional model of the ligand-binding domain (LBD) of ERα was built in silico, and docking simulations were performed. The docking simulation analysis indicated that the interaction of 17β-dihydroequilenin with the ERα LBD is the most potent, followed by those of 17α-dihydroequilin and 17β-dihydroequilin, whereas those of Eq and Eqn were least potent. We further analyzed gene expression profiles in the livers of male medaka exposed to Eq and Eqn. A DNA microarray representing 6000 genes revealed that 24-h exposure to Eq and Eqn (100 ng/L) upregulated the expression of 6 and 34 genes in the livers of males, respectively. Genes upregulated by Eq included the estrogenic biomarker genes vitellogenins and choriogenins, suggesting the estrogenic potential of Eq. In contrast, Eqn exposure upregulated several cancer-related genes, such as mediator complex subunit 16 and RAS oncogene family members, suggesting a carcinogenic potential for Eqn. These results suggest that equine estrogens may have not only endocrine-disrupting potentials via the ERα signaling pathway but also carcinogenic potency in male medaka.

In vivo and in silico analyses of estrogenic potential of bisphenol analogs in medaka (Oryzias latipes) and common carp (Cyprinus carpio)

“…Table 2 summarizes the key amino acid residues in the hydrogen-bonding interactions of the medaka/carp ERα-BP complexes. Among these amino acids, glutamic acid commonly interacted with the three BPs in both medaka and carp ERα, as well as our previous study using the medaka ERα and equine estrogens (Uchida et al, 2015). Similar observation was also reported in the docking simulation analysis between the rainbow trout ERα and perfluoroalkyl acids (Benninghoff et al, 2011).…”

“…The interaction potential of BPC (U-dock: À 77.27 kcal/mol) for the medaka ERα LBD was the most potent (stable), followed by BPAF ( À 71.47 kcal/mol) and BPA ( À 48.03 kcal/mol) ( Table 2). By comparing these data with E2 (U-dock: À 59.50 kcal/mol) which was previously reported by Uchida et al (2015), BPC and BPAF showed potent binding potentials for the medaka ERα LBD. Our in vivo study also showed that BPC and BPAF were at least 10-100 times more potent than BPA for upregulating hepatic Vtg and Chg genes ( Fig.…”

“…The homology model for the medaka/carp ERα LBD and the in silico analysis of the interaction potential between BPs and the medaka/carp ERα LBD were performed using the Molecular Operation Environment (MOE) program (Chemical Computing Group Inc., Montreal, QB, Canada), as described previously (Dau et al, 2013;Uchida et al, 2015). The crystal structures of the human ERα LBDs were obtained from the Protein Data Bank (PDB).…”

Gene expression analyses of vitellogenin, choriogenin and estrogen receptor subtypes in the livers of male medaka (Oryzias latipes) exposed to equine estrogens

“…The male medaka (approximately 6 months after hatching) was exposed to 17β‐E 2 and six equine estrogens according to the previously reported method (Ishibashi et al, ; Yamauchi et al, ). The exposure concentrations of these compounds were selected on the basis of the previously reported studies (Yamaguchi et al, ; Tyler et al, ; Uchida et al, ). Briefly, fish ( n = 6 per group) were exposed to each of the following conditions: 17β‐E 2 (nominal concentration: 1, 10, 30, 100 and 300 ng l −1 ), Eq (1, 10, 30 and 100 ng l −1 ) and other equine estrogens (1, 10, 30, 100 and 300 ng l −1 ) in 1‐l glass beakers for 3 days at 25 ± 1 °C.…”

“…With another study they also reported the significant induction of plasma Vtg synthesis in rainbow trout ( O. mykiss ) and in carp ( Cyprinus carpio ), both of the fish species had been exposed to Eqn and 17β‐Eqn for 21 days, suggesting in vivo estrogenic potencies of these compounds in roach as well. Furthermore, our recent study demonstrated the interaction potential of six equine estrogens with the ligand‐binding domain (LBD) of medaka ERα in silico , and a DNA microarray analysis revealed up‐regulation of estrogen‐responsive genes such as Vtgs and Chgs in the liver of male medaka that were exposed to 100 ng l −1 each of Eq and Eqn (Uchida et al, ). However, no comprehensive data are yet available regarding the estrogenic potentials and risks of these equine estrogens to aquatic organisms.…”