| To understand the underlying mechanisms for how methoxychlor (MXC) exerts its deleterious effects to the endocrine system, male largemouth bass (LMB) were treated with 2.5, 10, or 25 mg/kg MXC and compared to fish pair-treated with 1 mg/kg E2 and vehicle control. The fish were euthanized 24, 48, or 72 h following treatment. The liver and testes were then assayed for changes in expression of the three LMB estrogen receptors (ERs) in both tissues, as well as vitellogenin (Vtg) 1 in the liver. Significant increases in gene expression were seen for each of the genes measured by 24 h, and returned to the level of vehicle control by 72 hours.; After finding that the expression of the ERs was up-regulated by each of the treatments in vivo, the ERs were used in transient transfection assays in HepG2 cells to determine the capacity of E2, MXC, and two demethylated MXC metabolites (OH-MXC and HPTE) to stimulate activity from the ERs. In addition, the transactivation of the receptors by nonylphenol to drive transcription through a reporter construct containing a 2x-repeat of the estrogen-response element (ERE) from the Xenopus vitellogenin A2 promoter was performed. Each of the ERs was stimulated by E2 with approximately equal EC50 values (approximately 115 nM), although ERalpha stimulated the greatest magnitude of activity. Nonylphenol was able to stimulate activity from ERalpha and ERbetab, but not from ERbetaa. All three ERs were stimulated by MXC and its two metabolites. The order of the potency series based on EC 50 values was HPTE>OH-MXC>MXC for ERalpha and ERbetab, while the reverse was found for ERbetaa. As expected, E2 was a more potent and efficacious agonist for LMB ERalpha, than were any of the MXC compounds. When the cells were transfected with ERbetaa, and then treated with ligand, E2 was also the most potent and efficacious agonist. Interestingly, for ERbetab, E2 was also the most potent agonist, but MXC itself was the most efficacious agonist. When used in combination with E2, MXC was able to stimulate greater activity from each of the ERs. HPTE, on the other hand, acted to suppress E2-stimulated activity from ERalpha, had no effect on ERbetaa, and further stimulated activity from ERbetab. The differences among the LMB ERs continued to be apparent with treatment with the ER antagonists ICI 182,780 and ZM 189,154. ICI was able to eliminate all activity of ERalpha, and had partial antagonistic effect on ERbetab, while it was unable to significantly decrease activity from ERbetaa. On the other hand, ZM was able to decrease the level of activity to that of vehicle with all three ERs.; Taken together, it is clear that MXC is able to act as a disruptor of ER-mediated signaling in the LMB in vivo. In addition, the work with transient transfections provided evidence that the three LMB ERs each have different functions based on ligand sensitivities. The duplicated ERbetaa isotype is not just a redundant receptor, but likely has some of its own activities, and possibly regulates a set of genes that is different from those regulated by ERalpha and ERbetab isotypes. |
