Stimulation of transactivation of the largemouth bass estrogen receptors alpha, beta-a, and beta-b by methoxychlor and its mono- and bis-demethylated metabolites in HepG2 cells

Abstract

The purpose of this study was to determine the mechanisms by which the pesticide, methoxychlor (MXC), acts as an environmental endocrine disruptor through interaction with the three largemouth bass (Micropterus salmoides) estrogen receptors (ERs) alpha, betaa, and betab. MXC is a less-environmentally persistent analog of DDT that behaves as a weak estrogen. Using transient transfection assays in HepG2 cells, we have previously shown that each receptor is responsive to the endogenous ligand 17beta-estradiol (E(2)) in a dose-dependent manner. The parent compound, MXC, showed dose-dependent stimulation of transcriptional activation through all three ERs. In addition to the parent molecule, each of the metabolites was also estrogenic with all three ERs. The order of potency for ERalpha and ERbetab was HPTE>OH-MXC>MXC, while the opposite order was seen for ERbetaa. HepG2 cells did not substantially metabolize MXC to the active metabolites, thus the activity of MXC was not due to metabolism. When examining the effects of increasing concentrations of MXC at a fixed concentration of E(2), all three ERs show increased activity compared to that with E(2) alone, showing that the effects of MXC and E(2) are additive. However, when this experiment was repeated with increasing concentrations of HPTE at a fixed concentration of E(2), the activity of ERalpha was decreased, that of ERbetab was increased, while that of ERbetaa was unaffected compared to E(2) alone. These experiments suggest that HPTE functions as an E(2) antagonist with ERalpha, an E(2) agonist with ERbetab and does not perturb E(2) stimulation of ERbetaa. While it is clear the ERbeta subtypes are the products of different genes (due to a gene duplication in teleosts) the differences in their responses to MXC and its metabolites indicate that their functions diverge, both in their in vivo molecular response to E(2), as well as in their interaction with endocrine disrupting compounds found in the wild.

Methoxychlor affects multiple hormone signaling pathways in the largemouth bass (Micropterus salmoides) liver

“…Unique gene expression profiles from other weak xenoestrogens compared to E 2 have been previously been characterized in teleost fish (Benninghoff and Williams, 2008;Larkin et al, 2002; Moens et al, 2007; Sabo-Attwood et al, 2007). There is evidence to suggest that MXC has estrogenic, anti-estrogenic, and also anti-androgenic activities (Blum et al, 2008b; Eroschenko et al, 1996, 2000; Folmar et al, 2002; Larkin et al, 2003; Schlenk et al, 1998) and in the present study, MXC alters transcript levels associated with multiple receptor-mediated signaling pathways.…”

“…Therefore, it is plausible that 1) AR transcription is directly increasing as a result of MXC acting as an androgen or 2) as a result of a complex temporal signaling cascade stemming from MXC binding estrogen receptors, which is a more likely scenario based on the weight of evidence from previous studies. In particular, given the knowledge that the MXC metabolite HPTE is a partial agonist/antagonist of LMB ESR1 (Blum et al, 2008b), binding of HPTE to ESR1 could theoretically result in decreased transcription of PELP1/MNAR, c-FOS, or other ESR1 targets, as observed in this study. This could alter the signaling events between esr1 and ar expression, leading to the increased ar transcription observed after 48 hours.…”

“…Both gene expression data and transient transfection experiments with LMB ERs in HepG2 cells suggests that MXC has genomic effects in LMB that parallel endogenous estrogens as well as other xenoestrogens (Blum et al, 2008 a,b; Sabo-Attwood et al, 2007). This is based upon data demonstrating that MXC and two of its metabolites, OH-MXC and HPTE, are agonists of LMB ERs driving them to activate gene transcription.…”

“…While it is clear that MXC negatively impacts fish reproduction (Ankley et al, 2001; Schlenk et al, 1998) and MXC and/or its metabolites interact with the binding site of fish ERs (Nimrod and Benson, 1997; Blum et al 2008b), the multiple modes of action by which MXC exerts its effect in fish is unclear. For example, MXC may differ from E 2 in that it is both weakly estrogenic and is also capable of competitive inhibition of more potent ER activators.…”

Environmental Chemicals Targeting Estrogen Signaling Pathways

“…The bisphenol metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) (Fig. Nevertheless, an in vitro study in a human liver carcinoma cell line Hep G2 cells demonstrated that parent methoxychlor and its metabolites were all estrogenic through the ERs (Blum et al, 2008). HPTE selectively antagonizes E2-mediated ERβ activation (Gaido et al, 1999).…”