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.

Figure 1

MXC-stimulated transactivation of the LMB ERs. HepG2 cells were transfected with each ER along with a reporter luciferase construct driven by a 2x tandem repeat of the Vtg A2 ERE. Data are means of the fold-change in luciferase activity versus vehicle (DMSO) control +/- SE from three independent experiments (n=3 per experiment). A) Activation of ERα. B) Activation of ERβa. Inset is a re-plot of the data on a different scale to better visualize the dose response. C) Activation of ERβb. Differences between activities for MXC concentrations were determined by Duncan’s multiple range test.

Figure 2

Representative image of the TLC plates run following treatment of HepG2 cells with [¹⁴C]-MXC. The cells were treated with a concentration of 10 μM [¹⁴C]-MXC for 48 h. Following treatment the culture medium was collected and the cells were lysed and both the culture medium and cell lysates were extracted with water-saturated ethylacetate. The organic phase was dried the residue was dissolved in methanol and spotted onto the TLC plates. The metabolites were separated with a mobile phase of n-heptane:diethylether (1:1). OH-MXC was found in both the cellular and medium phases while HPTE was only found in the medium.

Figure 3

OH-MXC-stimulated transactivation of LMB ERs. HepG2 cells were treated following transfection. Data are means of the fold-change in luciferase activity versus vehicle control +/- SE from three independent experiments (n=3 per experiment). A) Activation of ERα. B) Activation of ERβa. Inset is a re-plot of the data on a different scale to better visualize the dose response. C) Activation of ERβb. Differences between activities for OH-MXC concentrations were determined by Duncan’s multiple range test.

Figure 4

HPTE-stimulated transactivation of the LMB ERs. Data are means of the fold-change in luciferase activity versus vehicle control +/- SE from three independent experiments (n=3 per experiment) using HepG2 cells. A) Activation of ERα. B) Activation of ERβa. Inset is a re-plot of the data on a different scale to better visualize the dose response. C) Activation of ERβb. Differences between activities for HPTE concentrations were determined by Duncan’s multiple range test.

Figure 5

MXC and E₂ cotreatment effect on ER activation in HepG2 cells. Following transfection, HepG2 cells were treated as described in materials and methods. Data are means of the fold-change in luciferase activity versus vehicle alone +/- SE from three independent experiments (n=3 per experiment). A) Additive effects on ERα. B) Additive effects on ERβa. C) Additive effects on ERβb. Differences between treatment groups were determined by Duncan’s multiple range test.

Figure 6

HPTE and E₂ cotreatment of HepG2 cells with HPTE and E₂. Data are means of the fold-change in luciferase activity versus vehicle alone +/- SE from three independent experiments (n=3 per experiment). A) Antagonistic effects on ERα. B) Effects on ERβa. C) Additive effects on ERβb. Differences between treatment group were determined by Duncan’s multiple range test.