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Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen.

Garcia-Reyero N, Kroll KJ, Liu L, Orlando EF, Watanabe KH, Sepúlveda MS, Villeneuve DL, Perkins EJ, Ankley GT, Denslow ND - BMC Genomics (2009)

Bottom Line: Aquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others.In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17alpha-ethinylestradiol (EE2)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE(2)/L with 100 ng ZM 189,154/L.Steroidogenesis was down-regulated by EE(2) as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA. natalia@ccmsi.us

ABSTRACT

Background: Aquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others. In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17alpha-ethinylestradiol (EE2)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE(2)/L with 100 ng ZM 189,154/L. We analyzed gene expression changes in the gonad, as well as hormone and vitellogenin plasma levels.

Results: Steroidogenesis was down-regulated by EE(2) as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway. Microarray analysis of testis of fathead minnows treated with 5 ng EE(2)/L or with the mixture of 5 ng EE(2)/L and 100 ng ZM 189,154/L indicated that some of the genes whose expression was changed by EE(2) were blocked by ZM 189,154, while others were either not blocked or enhanced by the mixture, generating two distinct expression patterns. Gene ontology and pathway analysis programs were used to determine categories of genes for each expression pattern.

Conclusion: Our results suggest that response to estrogens occurs via multiple mechanisms, including canonical binding to soluble estrogen receptors, membrane estrogen receptors, and other mechanisms that are not blocked by pure antiestrogens.

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Q-PCR analysis of mRNAs for proteins involved in steroidogenesis. Q-PCR results are expressed as fold difference compared to control. Panel A, StAR; Panel B, P450scc; Panel C, CYP17; Panel D, Inhibin; Panel E, CYP19; Panel F, 3β-HSD; Panel G, 11β-HSD; Panel H, model for steroidogenesis. Green boxes refer to mRNAs that are significantly decreased by the treatment in accordance with the Q-PCR graphs illustrated within the panels. Yellow boxes refer to mRNAs that are not significantly changed by the treatments. FHM were treated with TEG, 2, 5, 10 or 50 ng EE2/L. StAR, steroidogenic acute regulatory protein, P450scc, Cytochrome P450 side chain cleavage enzyme, CYP17, Cytochrome P450 17, CYP 19, gonadal aromatase, inhibin, hydroxysteroid dehydrogenases including 3β-HSD and 11β-HSD.
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Figure 5: Q-PCR analysis of mRNAs for proteins involved in steroidogenesis. Q-PCR results are expressed as fold difference compared to control. Panel A, StAR; Panel B, P450scc; Panel C, CYP17; Panel D, Inhibin; Panel E, CYP19; Panel F, 3β-HSD; Panel G, 11β-HSD; Panel H, model for steroidogenesis. Green boxes refer to mRNAs that are significantly decreased by the treatment in accordance with the Q-PCR graphs illustrated within the panels. Yellow boxes refer to mRNAs that are not significantly changed by the treatments. FHM were treated with TEG, 2, 5, 10 or 50 ng EE2/L. StAR, steroidogenic acute regulatory protein, P450scc, Cytochrome P450 side chain cleavage enzyme, CYP17, Cytochrome P450 17, CYP 19, gonadal aromatase, inhibin, hydroxysteroid dehydrogenases including 3β-HSD and 11β-HSD.

Mentions: Genes that were tested by Q-PCR (Figure 5) were used to both validate the arrays and to focus on genes whose protein products are involved in steroidogenesis and were expected to be affected by EE2 [42]. Of the genes tested, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17α hydroxylase, 17,20 lyase (CYP17) and inhibin were significantly down-regulated by 2 to 50 ng EE2/L. Genes for hydroxysteroid dehydrogenases (HSDs) 3β-HSD and 11β-HSD and cytochrome P450 aromatase A-isoform (CYP19A) were not significantly altered, but 11β-HSD and CYP19A showed a downward tendency.


Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen.

Garcia-Reyero N, Kroll KJ, Liu L, Orlando EF, Watanabe KH, Sepúlveda MS, Villeneuve DL, Perkins EJ, Ankley GT, Denslow ND - BMC Genomics (2009)

Q-PCR analysis of mRNAs for proteins involved in steroidogenesis. Q-PCR results are expressed as fold difference compared to control. Panel A, StAR; Panel B, P450scc; Panel C, CYP17; Panel D, Inhibin; Panel E, CYP19; Panel F, 3β-HSD; Panel G, 11β-HSD; Panel H, model for steroidogenesis. Green boxes refer to mRNAs that are significantly decreased by the treatment in accordance with the Q-PCR graphs illustrated within the panels. Yellow boxes refer to mRNAs that are not significantly changed by the treatments. FHM were treated with TEG, 2, 5, 10 or 50 ng EE2/L. StAR, steroidogenic acute regulatory protein, P450scc, Cytochrome P450 side chain cleavage enzyme, CYP17, Cytochrome P450 17, CYP 19, gonadal aromatase, inhibin, hydroxysteroid dehydrogenases including 3β-HSD and 11β-HSD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713996&req=5

Figure 5: Q-PCR analysis of mRNAs for proteins involved in steroidogenesis. Q-PCR results are expressed as fold difference compared to control. Panel A, StAR; Panel B, P450scc; Panel C, CYP17; Panel D, Inhibin; Panel E, CYP19; Panel F, 3β-HSD; Panel G, 11β-HSD; Panel H, model for steroidogenesis. Green boxes refer to mRNAs that are significantly decreased by the treatment in accordance with the Q-PCR graphs illustrated within the panels. Yellow boxes refer to mRNAs that are not significantly changed by the treatments. FHM were treated with TEG, 2, 5, 10 or 50 ng EE2/L. StAR, steroidogenic acute regulatory protein, P450scc, Cytochrome P450 side chain cleavage enzyme, CYP17, Cytochrome P450 17, CYP 19, gonadal aromatase, inhibin, hydroxysteroid dehydrogenases including 3β-HSD and 11β-HSD.
Mentions: Genes that were tested by Q-PCR (Figure 5) were used to both validate the arrays and to focus on genes whose protein products are involved in steroidogenesis and were expected to be affected by EE2 [42]. Of the genes tested, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17α hydroxylase, 17,20 lyase (CYP17) and inhibin were significantly down-regulated by 2 to 50 ng EE2/L. Genes for hydroxysteroid dehydrogenases (HSDs) 3β-HSD and 11β-HSD and cytochrome P450 aromatase A-isoform (CYP19A) were not significantly altered, but 11β-HSD and CYP19A showed a downward tendency.

Bottom Line: Aquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others.In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17alpha-ethinylestradiol (EE2)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE(2)/L with 100 ng ZM 189,154/L.Steroidogenesis was down-regulated by EE(2) as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA. natalia@ccmsi.us

ABSTRACT

Background: Aquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others. In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17alpha-ethinylestradiol (EE2)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE(2)/L with 100 ng ZM 189,154/L. We analyzed gene expression changes in the gonad, as well as hormone and vitellogenin plasma levels.

Results: Steroidogenesis was down-regulated by EE(2) as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway. Microarray analysis of testis of fathead minnows treated with 5 ng EE(2)/L or with the mixture of 5 ng EE(2)/L and 100 ng ZM 189,154/L indicated that some of the genes whose expression was changed by EE(2) were blocked by ZM 189,154, while others were either not blocked or enhanced by the mixture, generating two distinct expression patterns. Gene ontology and pathway analysis programs were used to determine categories of genes for each expression pattern.

Conclusion: Our results suggest that response to estrogens occurs via multiple mechanisms, including canonical binding to soluble estrogen receptors, membrane estrogen receptors, and other mechanisms that are not blocked by pure antiestrogens.

Show MeSH
Related in: MedlinePlus