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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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Related in: MedlinePlus

Effect of MAA on estrogen-mediated endogenous gene expression in vitro and in vivo. (A) MCF-7 cells were pretreated with either vehicle [ethanol (EtOH)] or 5 mM MAA for 2 hr and then treated for 18 hr with either vehicle or 1 nM E2. The expression of endogenous estrogen-responsive genes was analyzed by real-time PCR. Data represent the average fold over control (± SE) of duplicate samples from at least three independent experiments. (B) Mice were pretreated for 30 min with either saline or 400 mg/kg MAA and then treated with either vehicle or 1 μg/kg E2 for 2 hr. Uteri were collected and estrogen-responsive gene expression was analyzed by real-time PCR. Data represent the average fold over control (± SE) obtained from three mice per treatment.#p < 0.01 compared with 1 nM E2 alone.
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f3-ehp-117-1702: Effect of MAA on estrogen-mediated endogenous gene expression in vitro and in vivo. (A) MCF-7 cells were pretreated with either vehicle [ethanol (EtOH)] or 5 mM MAA for 2 hr and then treated for 18 hr with either vehicle or 1 nM E2. The expression of endogenous estrogen-responsive genes was analyzed by real-time PCR. Data represent the average fold over control (± SE) of duplicate samples from at least three independent experiments. (B) Mice were pretreated for 30 min with either saline or 400 mg/kg MAA and then treated with either vehicle or 1 μg/kg E2 for 2 hr. Uteri were collected and estrogen-responsive gene expression was analyzed by real-time PCR. Data represent the average fold over control (± SE) obtained from three mice per treatment.#p < 0.01 compared with 1 nM E2 alone.

Mentions: We performed further experiments to determine if decreased ERα expression after MAA treatment resulted in disrupted ERα-mediated signaling. Toward this end, we treated MCF-7 cells with either 1 nM E2, 5 mM MAA, or 1 nM E2 plus 5 mM MAA for 24 hr, and evaluated estrogen-regulated gene expression by real-time PCR. As shown in Figure 3A, the expression of pS2, MYC, GREB1, SPUVE, and MCM3 was potentiated by E2 treatment; however, pretreatment with 5 mM MAA attenuated the estrogen-induced responses. Taken together, these data show that MAA attenuates endogenous ER signaling, resulting in disruption of estrogen-modulated endogenous gene expression in MCF-7 cells.


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 estrogen-mediated endogenous gene expression in vitro and in vivo. (A) MCF-7 cells were pretreated with either vehicle [ethanol (EtOH)] or 5 mM MAA for 2 hr and then treated for 18 hr with either vehicle or 1 nM E2. The expression of endogenous estrogen-responsive genes was analyzed by real-time PCR. Data represent the average fold over control (± SE) of duplicate samples from at least three independent experiments. (B) Mice were pretreated for 30 min with either saline or 400 mg/kg MAA and then treated with either vehicle or 1 μg/kg E2 for 2 hr. Uteri were collected and estrogen-responsive gene expression was analyzed by real-time PCR. Data represent the average fold over control (± SE) obtained from three mice per treatment.#p < 0.01 compared with 1 nM E2 alone.
© Copyright Policy - public-domain
Related In: Results  -  Collection

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

f3-ehp-117-1702: Effect of MAA on estrogen-mediated endogenous gene expression in vitro and in vivo. (A) MCF-7 cells were pretreated with either vehicle [ethanol (EtOH)] or 5 mM MAA for 2 hr and then treated for 18 hr with either vehicle or 1 nM E2. The expression of endogenous estrogen-responsive genes was analyzed by real-time PCR. Data represent the average fold over control (± SE) of duplicate samples from at least three independent experiments. (B) Mice were pretreated for 30 min with either saline or 400 mg/kg MAA and then treated with either vehicle or 1 μg/kg E2 for 2 hr. Uteri were collected and estrogen-responsive gene expression was analyzed by real-time PCR. Data represent the average fold over control (± SE) obtained from three mice per treatment.#p < 0.01 compared with 1 nM E2 alone.
Mentions: We performed further experiments to determine if decreased ERα expression after MAA treatment resulted in disrupted ERα-mediated signaling. Toward this end, we treated MCF-7 cells with either 1 nM E2, 5 mM MAA, or 1 nM E2 plus 5 mM MAA for 24 hr, and evaluated estrogen-regulated gene expression by real-time PCR. As shown in Figure 3A, the expression of pS2, MYC, GREB1, SPUVE, and MCM3 was potentiated by E2 treatment; however, pretreatment with 5 mM MAA attenuated the estrogen-induced responses. Taken together, these data show that MAA attenuates endogenous ER signaling, resulting in disruption of estrogen-modulated endogenous gene expression in MCF-7 cells.

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