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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, SepĂșlveda 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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Related in: MedlinePlus

Comparison of overall gene regulation. (A) 5 ng EE2/L and (B) a mixture of 5 ng EE2/L and 100 ng ZM/L as determined by the 22 K array. The genes were ordered according to their expression level in the EE2 treatment (p < 0.01) and represent median expression values of the four arrays for each condition.
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Figure 3: Comparison of overall gene regulation. (A) 5 ng EE2/L and (B) a mixture of 5 ng EE2/L and 100 ng ZM/L as determined by the 22 K array. The genes were ordered according to their expression level in the EE2 treatment (p < 0.01) and represent median expression values of the four arrays for each condition.

Mentions: Based on these initial results, we conducted a second study, this time using 5 ng EE2/L and a mixture of 100 ng ZM/L with 5 ng EE2/L (a ratio of 20:1; Figure 2B). For this analysis, we used a newer 22,000 gene array that had subsequently become available. Exposure to 5 ng EE2/L increased plasma VTG (Figure 1C), while the 20-fold excess of ZM in the mixture did not affect this increase. A group of 173 genes was altered (p < 0.01) after exposure to either 5 ng EE2/L or to the mixture of 5 ng EE2/L and 100 ng ZM/L (Figure 3). These changes are plotted in order of their degree of expression change for EE2 (Figure 3A), with 83 genes up-regulated and 90 genes down-regulated. Keeping the same order of genes, their fold-expression is plotted for the mixture (Figure 3B). It is clear from this graph that while ZM blocks the EE2 effects for some genes, it does not do so for all. There also appears to be a few genes in the middle of this distribution that are significantly altered only by the mixture and not by EE2 alone.


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, SepĂșlveda MS, Villeneuve DL, Perkins EJ, Ankley GT, Denslow ND - BMC Genomics (2009)

Comparison of overall gene regulation. (A) 5 ng EE2/L and (B) a mixture of 5 ng EE2/L and 100 ng ZM/L as determined by the 22 K array. The genes were ordered according to their expression level in the EE2 treatment (p < 0.01) and represent median expression values of the four arrays for each condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Comparison of overall gene regulation. (A) 5 ng EE2/L and (B) a mixture of 5 ng EE2/L and 100 ng ZM/L as determined by the 22 K array. The genes were ordered according to their expression level in the EE2 treatment (p < 0.01) and represent median expression values of the four arrays for each condition.
Mentions: Based on these initial results, we conducted a second study, this time using 5 ng EE2/L and a mixture of 100 ng ZM/L with 5 ng EE2/L (a ratio of 20:1; Figure 2B). For this analysis, we used a newer 22,000 gene array that had subsequently become available. Exposure to 5 ng EE2/L increased plasma VTG (Figure 1C), while the 20-fold excess of ZM in the mixture did not affect this increase. A group of 173 genes was altered (p < 0.01) after exposure to either 5 ng EE2/L or to the mixture of 5 ng EE2/L and 100 ng ZM/L (Figure 3). These changes are plotted in order of their degree of expression change for EE2 (Figure 3A), with 83 genes up-regulated and 90 genes down-regulated. Keeping the same order of genes, their fold-expression is plotted for the mixture (Figure 3B). It is clear from this graph that while ZM blocks the EE2 effects for some genes, it does not do so for all. There also appears to be a few genes in the middle of this distribution that are significantly altered only by the mixture and not by EE2 alone.

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