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The short-chain fatty acid methoxyacetic acid disrupts endogenous estrogen receptor-alpha-mediated signaling.

Henley DV, Mueller S, Korach KS - Environ. Health Perspect. (2009)

Bottom Line: This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter.In contrast to its effects on exogenous ER, MAA decreases endogenous ERalpha expression and attenuates E(2)-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus.These results illustrate the importance of careful experimental design and analysis when assessing the potential endocrine-disrupting properties of a compound to ensure biological responses are in concordance with in vitro analyses.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.

ABSTRACT

Background: Ethylene glycol monomethyl ether (EGME) exposure is associated with impaired reproductive function. The primary metabolite of EGME is methoxyacetic acid (MAA), a short-chain fatty acid that inhibits histone deacetylase activity and alters gene expression.

Objective: Because estrogen signaling is necessary for normal reproductive function and modulates gene expression, the estrogen-signaling pathway is a likely target for MAA; however, little is known about the effects of MAA in this regard.

Methods: We evaluated the mechanistic effects of MAA on estrogen receptor (ER) expression and estrogen signaling using in vitro and in vivo model systems.

Results: MAA potentiates 17beta-estradiol (E(2)) stimulation of an estrogen-responsive reporter plasmid in HeLa cells transiently transfected with either a human ERalpha or ERbeta expression vector containing a cytomegalovirus (CMV) promoter. This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter. In contrast to its effects on exogenous ER, MAA decreases endogenous ERalpha expression and attenuates E(2)-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus.

Conclusions: These results illustrate the importance of careful experimental design and analysis when assessing the potential endocrine-disrupting properties of a compound to ensure biological responses are in concordance with in vitro analyses. Given the established role of the ER in normal reproductive function, the effects of MAA on the endogenous ER reported here are consistent with the reproductive abnormalities observed after EGME exposure and suggest that these toxicities may be due, at least in part, to attenuation of endogenous ER-mediated signaling.

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Effect of MAA on endogenous ERα expression in vitro and in vivo. (A) MCF-7 cells were treated for 24 hr with either vehicle [ethanol (EtOH)] or increasing concentrations of MAA, and ERα protein expression was assessed by Western blot. Data are representative of results from three independent experiments. (B) MCF-7 cells were treated for 24 hr with either vehicle or 5 mM MAA, and ERα mRNA levels were measured by real-time PCR. Data represent the average fold over control (± SE) obtained from duplicate samples in four independent experiments. (C) Uteri were collected from mice treated for 2.5 hr with either saline or 400 mg/kg MAA, and real-time PCR was performed to determine the levels of ERα mRNA in each sample. Data are plotted as fold over control (± SE) and represent the average values obtained from three mice per treatment.##p < 0.01 compared with vehicle control.
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f2-ehp-117-1702: Effect of MAA on endogenous ERα expression in vitro and in vivo. (A) MCF-7 cells were treated for 24 hr with either vehicle [ethanol (EtOH)] or increasing concentrations of MAA, and ERα protein expression was assessed by Western blot. Data are representative of results from three independent experiments. (B) MCF-7 cells were treated for 24 hr with either vehicle or 5 mM MAA, and ERα mRNA levels were measured by real-time PCR. Data represent the average fold over control (± SE) obtained from duplicate samples in four independent experiments. (C) Uteri were collected from mice treated for 2.5 hr with either saline or 400 mg/kg MAA, and real-time PCR was performed to determine the levels of ERα mRNA in each sample. Data are plotted as fold over control (± SE) and represent the average values obtained from three mice per treatment.##p < 0.01 compared with vehicle control.

Mentions: MAA-induced transactivation of the CMV promoter complicates the interpretation of data obtained from in vitro experimental systems incorporating exogenous ER. Therefore, we performed experiments to examine the effect of MAA on the endogenous expression of ERα in MCF-7 cells. MCF-7 cells were treated with increasing concentrations of MAA, and endogenous ERα protein expression was detected by Western blot. We observed a concentration-dependent decrease in endogenous ERα protein expression, with maximal decreases occurring after treatment with 20 mM MAA, the highest concentration tested in these experiments (Figure 2A). To determine if the decrease in ERα protein levels corresponded with decreased steady-state levels of ERα mRNA, MCF-7 cells were treated with 5 mM MAA for 24 hr, and ERα expression was analyzed by real-time PCR. Treatment with 5 mM MAA decreased the expression of ERα mRNA by ~ 50% relative to vehicle controls (Figure 2B), indicating that the decreased protein expression is due, at least in part, to diminished levels of ERα mRNA.


The short-chain fatty acid methoxyacetic acid disrupts endogenous estrogen receptor-alpha-mediated signaling.

Henley DV, Mueller S, Korach KS - Environ. Health Perspect. (2009)

Effect of MAA on endogenous ERα expression in vitro and in vivo. (A) MCF-7 cells were treated for 24 hr with either vehicle [ethanol (EtOH)] or increasing concentrations of MAA, and ERα protein expression was assessed by Western blot. Data are representative of results from three independent experiments. (B) MCF-7 cells were treated for 24 hr with either vehicle or 5 mM MAA, and ERα mRNA levels were measured by real-time PCR. Data represent the average fold over control (± SE) obtained from duplicate samples in four independent experiments. (C) Uteri were collected from mice treated for 2.5 hr with either saline or 400 mg/kg MAA, and real-time PCR was performed to determine the levels of ERα mRNA in each sample. Data are plotted as fold over control (± SE) and represent the average values obtained from three mice per treatment.##p < 0.01 compared with vehicle control.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2801194&req=5

f2-ehp-117-1702: Effect of MAA on endogenous ERα expression in vitro and in vivo. (A) MCF-7 cells were treated for 24 hr with either vehicle [ethanol (EtOH)] or increasing concentrations of MAA, and ERα protein expression was assessed by Western blot. Data are representative of results from three independent experiments. (B) MCF-7 cells were treated for 24 hr with either vehicle or 5 mM MAA, and ERα mRNA levels were measured by real-time PCR. Data represent the average fold over control (± SE) obtained from duplicate samples in four independent experiments. (C) Uteri were collected from mice treated for 2.5 hr with either saline or 400 mg/kg MAA, and real-time PCR was performed to determine the levels of ERα mRNA in each sample. Data are plotted as fold over control (± SE) and represent the average values obtained from three mice per treatment.##p < 0.01 compared with vehicle control.
Mentions: MAA-induced transactivation of the CMV promoter complicates the interpretation of data obtained from in vitro experimental systems incorporating exogenous ER. Therefore, we performed experiments to examine the effect of MAA on the endogenous expression of ERα in MCF-7 cells. MCF-7 cells were treated with increasing concentrations of MAA, and endogenous ERα protein expression was detected by Western blot. We observed a concentration-dependent decrease in endogenous ERα protein expression, with maximal decreases occurring after treatment with 20 mM MAA, the highest concentration tested in these experiments (Figure 2A). To determine if the decrease in ERα protein levels corresponded with decreased steady-state levels of ERα mRNA, MCF-7 cells were treated with 5 mM MAA for 24 hr, and ERα expression was analyzed by real-time PCR. Treatment with 5 mM MAA decreased the expression of ERα mRNA by ~ 50% relative to vehicle controls (Figure 2B), indicating that the decreased protein expression is due, at least in part, to diminished levels of ERα mRNA.

Bottom Line: This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter.In contrast to its effects on exogenous ER, MAA decreases endogenous ERalpha expression and attenuates E(2)-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus.These results illustrate the importance of careful experimental design and analysis when assessing the potential endocrine-disrupting properties of a compound to ensure biological responses are in concordance with in vitro analyses.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.

ABSTRACT

Background: Ethylene glycol monomethyl ether (EGME) exposure is associated with impaired reproductive function. The primary metabolite of EGME is methoxyacetic acid (MAA), a short-chain fatty acid that inhibits histone deacetylase activity and alters gene expression.

Objective: Because estrogen signaling is necessary for normal reproductive function and modulates gene expression, the estrogen-signaling pathway is a likely target for MAA; however, little is known about the effects of MAA in this regard.

Methods: We evaluated the mechanistic effects of MAA on estrogen receptor (ER) expression and estrogen signaling using in vitro and in vivo model systems.

Results: MAA potentiates 17beta-estradiol (E(2)) stimulation of an estrogen-responsive reporter plasmid in HeLa cells transiently transfected with either a human ERalpha or ERbeta expression vector containing a cytomegalovirus (CMV) promoter. This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter. In contrast to its effects on exogenous ER, MAA decreases endogenous ERalpha expression and attenuates E(2)-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus.

Conclusions: These results illustrate the importance of careful experimental design and analysis when assessing the potential endocrine-disrupting properties of a compound to ensure biological responses are in concordance with in vitro analyses. Given the established role of the ER in normal reproductive function, the effects of MAA on the endogenous ER reported here are consistent with the reproductive abnormalities observed after EGME exposure and suggest that these toxicities may be due, at least in part, to attenuation of endogenous ER-mediated signaling.

Show MeSH
Related in: MedlinePlus