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Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish (Gambusia holbrooki).

Brockmeier EK, Yu F, Amador DM, Bargar TA, Denslow ND - BMC Genomics (2013)

Bottom Line: The expression levels of 17β-hydroxysteroid dehydrogenase 3 and zona pellucida glycoprotein 2 were validated by quantitative polymerase chain reaction (qPCR) (Student's t-test, p < 0.05).Future studies using this array will enhance knowledge of the biology and toxicological response of this species.This work provides a foundation of molecular knowledge and tools that can be used to delve further into understanding the biology of G. holbrooki and how this organism can be used as a bioindicator organism for endocrine disrupting pollutants in the environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, 2187 Mowry Road, P,O, Box 110885, 32611 Gainesville, FL, USA. ndenslow@ufl.edu.

ABSTRACT

Background: The eastern mosquitofish (Gambusia holbrooki) has the potential to become a bioindicator organism of endocrine disrupting chemicals (EDCs) due to its androgen-driven secondary sexual characteristics. However, the lack of molecular information on G. holbrooki hinders its use as a bioindicator coupled with biomarker data. While traditional gene-by-gene approaches provide insight for biomarker development, a holistic analysis would provide more rapid and expansive determination of potential biomarkers. The objective of this study was to develop and utilize a mosquitofish microarray to determine potential biomarkers of subchronic androgen exposure. To achieve this objective, two specific aims were developed: 1) Sequence a G. holbrooki cDNA library, and 2) Use microarray analysis to determine genes that are differentially regulated by subchronic androgen exposure in hepatic tissues of 17β-trenbolone (TB) exposed adult female G. holbrooki.

Results: A normalized library of multiple organs of male and female G. holbrooki was prepared and sequenced by the Illumina GA IIx and Roche 454 XLR70. Over 30,000 genes with e-value ≤ 10⁻⁴ were annotated and 14,758 of these genes were selected for inclusion on the microarray. Hepatic microarray analysis of adult female G. holbrooki exposed to the vehicle control or 1 μg/L of TB (a potent anabolic androgen) revealed 229 genes upregulated and 279 downregulated by TB (one-way ANOVA, p < 0.05, FDR α = 0.05, fold change > 1.5 and < -1.5). Fifteen gene ontology biological processes were enriched by TB exposure (Fisher's Exact Test, p < 0.05). The expression levels of 17β-hydroxysteroid dehydrogenase 3 and zona pellucida glycoprotein 2 were validated by quantitative polymerase chain reaction (qPCR) (Student's t-test, p < 0.05).

Conclusions: Coupling microarray data with phenotypic changes driven by androgen exposure in mosquitofish is key for developing this organism into a bioindicator for EDCs. Future studies using this array will enhance knowledge of the biology and toxicological response of this species. This work provides a foundation of molecular knowledge and tools that can be used to delve further into understanding the biology of G. holbrooki and how this organism can be used as a bioindicator organism for endocrine disrupting pollutants in the environment.

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Pathway analysis of hepatic genes differentially regulated by 17β-trenbolone: Impacts on metabolic pathways. Connections between significantly up or down-regulated genes in the liver and enriched Biological Processes from human homolog data were visualized. The red color represents up-regulation by TB and blue color represents down-regulation by TB compared to the samples exposed to the vehicle control. The intensity of the color is correlated with the degree of differential regulation. Red solid lines represent positive regulation of gene expression, blue solid lines represent negative regulation of gene expression, and grey solid lines represent unknown direction of regulation. Oval shapes represent individual genes and boxes represent GO Biological Processes. Gene abbreviations: COX2: cyclooxygenase II; CYBA: cytochrome b-245, alpha polypeptide; CYBB: cytochrome b-245, beta polypeptide; CYB5A: cytochrome b5 type A (microsomal); CYC1: cytochrome c-1;CYCS: cytochrome c, somatic; CYGB: cytoglobin; CYP(number)(letter)(number) for all entries: cytochrome P450, family number, subfamily letter, polypeptide variant number; DOCK2: dedicator of cytokinesis 2; EPX: eosinophil peroxidase; GUCY1B3: guanylate cyclase 1, soluble, beta 3;MB: myoglobin; MPO: myeloperoxidase; NOS1: nitric oxide synthase 1; NOX5: NADPH oxidase, EF-hand calcium binding domain 5; PRDX1: peroxiredoxin 1; SDHC: succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa; TDO2: tryptophan 2,3-dioxygenase.
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Figure 2: Pathway analysis of hepatic genes differentially regulated by 17β-trenbolone: Impacts on metabolic pathways. Connections between significantly up or down-regulated genes in the liver and enriched Biological Processes from human homolog data were visualized. The red color represents up-regulation by TB and blue color represents down-regulation by TB compared to the samples exposed to the vehicle control. The intensity of the color is correlated with the degree of differential regulation. Red solid lines represent positive regulation of gene expression, blue solid lines represent negative regulation of gene expression, and grey solid lines represent unknown direction of regulation. Oval shapes represent individual genes and boxes represent GO Biological Processes. Gene abbreviations: COX2: cyclooxygenase II; CYBA: cytochrome b-245, alpha polypeptide; CYBB: cytochrome b-245, beta polypeptide; CYB5A: cytochrome b5 type A (microsomal); CYC1: cytochrome c-1;CYCS: cytochrome c, somatic; CYGB: cytoglobin; CYP(number)(letter)(number) for all entries: cytochrome P450, family number, subfamily letter, polypeptide variant number; DOCK2: dedicator of cytokinesis 2; EPX: eosinophil peroxidase; GUCY1B3: guanylate cyclase 1, soluble, beta 3;MB: myoglobin; MPO: myeloperoxidase; NOS1: nitric oxide synthase 1; NOX5: NADPH oxidase, EF-hand calcium binding domain 5; PRDX1: peroxiredoxin 1; SDHC: succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa; TDO2: tryptophan 2,3-dioxygenase.

Mentions: Figure 2 illustrates the impacts of TB exposure on processes linked to metabolism and biosynthesis using PathwayStudioTM, providing further support for the results of the Fisher’s exact test. A significant increase in the processes of cholesterol metabolism, steroid metabolism, and respiratory chain are correlated with an increase in the expression of genes linked to these processes, including many cytochrome P450 enzyme subtypes (Figure 2).


Custom microarray construction and analysis for determining potential biomarkers of subchronic androgen exposure in the Eastern Mosquitofish (Gambusia holbrooki).

Brockmeier EK, Yu F, Amador DM, Bargar TA, Denslow ND - BMC Genomics (2013)

Pathway analysis of hepatic genes differentially regulated by 17β-trenbolone: Impacts on metabolic pathways. Connections between significantly up or down-regulated genes in the liver and enriched Biological Processes from human homolog data were visualized. The red color represents up-regulation by TB and blue color represents down-regulation by TB compared to the samples exposed to the vehicle control. The intensity of the color is correlated with the degree of differential regulation. Red solid lines represent positive regulation of gene expression, blue solid lines represent negative regulation of gene expression, and grey solid lines represent unknown direction of regulation. Oval shapes represent individual genes and boxes represent GO Biological Processes. Gene abbreviations: COX2: cyclooxygenase II; CYBA: cytochrome b-245, alpha polypeptide; CYBB: cytochrome b-245, beta polypeptide; CYB5A: cytochrome b5 type A (microsomal); CYC1: cytochrome c-1;CYCS: cytochrome c, somatic; CYGB: cytoglobin; CYP(number)(letter)(number) for all entries: cytochrome P450, family number, subfamily letter, polypeptide variant number; DOCK2: dedicator of cytokinesis 2; EPX: eosinophil peroxidase; GUCY1B3: guanylate cyclase 1, soluble, beta 3;MB: myoglobin; MPO: myeloperoxidase; NOS1: nitric oxide synthase 1; NOX5: NADPH oxidase, EF-hand calcium binding domain 5; PRDX1: peroxiredoxin 1; SDHC: succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa; TDO2: tryptophan 2,3-dioxygenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3852779&req=5

Figure 2: Pathway analysis of hepatic genes differentially regulated by 17β-trenbolone: Impacts on metabolic pathways. Connections between significantly up or down-regulated genes in the liver and enriched Biological Processes from human homolog data were visualized. The red color represents up-regulation by TB and blue color represents down-regulation by TB compared to the samples exposed to the vehicle control. The intensity of the color is correlated with the degree of differential regulation. Red solid lines represent positive regulation of gene expression, blue solid lines represent negative regulation of gene expression, and grey solid lines represent unknown direction of regulation. Oval shapes represent individual genes and boxes represent GO Biological Processes. Gene abbreviations: COX2: cyclooxygenase II; CYBA: cytochrome b-245, alpha polypeptide; CYBB: cytochrome b-245, beta polypeptide; CYB5A: cytochrome b5 type A (microsomal); CYC1: cytochrome c-1;CYCS: cytochrome c, somatic; CYGB: cytoglobin; CYP(number)(letter)(number) for all entries: cytochrome P450, family number, subfamily letter, polypeptide variant number; DOCK2: dedicator of cytokinesis 2; EPX: eosinophil peroxidase; GUCY1B3: guanylate cyclase 1, soluble, beta 3;MB: myoglobin; MPO: myeloperoxidase; NOS1: nitric oxide synthase 1; NOX5: NADPH oxidase, EF-hand calcium binding domain 5; PRDX1: peroxiredoxin 1; SDHC: succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa; TDO2: tryptophan 2,3-dioxygenase.
Mentions: Figure 2 illustrates the impacts of TB exposure on processes linked to metabolism and biosynthesis using PathwayStudioTM, providing further support for the results of the Fisher’s exact test. A significant increase in the processes of cholesterol metabolism, steroid metabolism, and respiratory chain are correlated with an increase in the expression of genes linked to these processes, including many cytochrome P450 enzyme subtypes (Figure 2).

Bottom Line: The expression levels of 17β-hydroxysteroid dehydrogenase 3 and zona pellucida glycoprotein 2 were validated by quantitative polymerase chain reaction (qPCR) (Student's t-test, p < 0.05).Future studies using this array will enhance knowledge of the biology and toxicological response of this species.This work provides a foundation of molecular knowledge and tools that can be used to delve further into understanding the biology of G. holbrooki and how this organism can be used as a bioindicator organism for endocrine disrupting pollutants in the environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, 2187 Mowry Road, P,O, Box 110885, 32611 Gainesville, FL, USA. ndenslow@ufl.edu.

ABSTRACT

Background: The eastern mosquitofish (Gambusia holbrooki) has the potential to become a bioindicator organism of endocrine disrupting chemicals (EDCs) due to its androgen-driven secondary sexual characteristics. However, the lack of molecular information on G. holbrooki hinders its use as a bioindicator coupled with biomarker data. While traditional gene-by-gene approaches provide insight for biomarker development, a holistic analysis would provide more rapid and expansive determination of potential biomarkers. The objective of this study was to develop and utilize a mosquitofish microarray to determine potential biomarkers of subchronic androgen exposure. To achieve this objective, two specific aims were developed: 1) Sequence a G. holbrooki cDNA library, and 2) Use microarray analysis to determine genes that are differentially regulated by subchronic androgen exposure in hepatic tissues of 17β-trenbolone (TB) exposed adult female G. holbrooki.

Results: A normalized library of multiple organs of male and female G. holbrooki was prepared and sequenced by the Illumina GA IIx and Roche 454 XLR70. Over 30,000 genes with e-value ≤ 10⁻⁴ were annotated and 14,758 of these genes were selected for inclusion on the microarray. Hepatic microarray analysis of adult female G. holbrooki exposed to the vehicle control or 1 μg/L of TB (a potent anabolic androgen) revealed 229 genes upregulated and 279 downregulated by TB (one-way ANOVA, p < 0.05, FDR α = 0.05, fold change > 1.5 and < -1.5). Fifteen gene ontology biological processes were enriched by TB exposure (Fisher's Exact Test, p < 0.05). The expression levels of 17β-hydroxysteroid dehydrogenase 3 and zona pellucida glycoprotein 2 were validated by quantitative polymerase chain reaction (qPCR) (Student's t-test, p < 0.05).

Conclusions: Coupling microarray data with phenotypic changes driven by androgen exposure in mosquitofish is key for developing this organism into a bioindicator for EDCs. Future studies using this array will enhance knowledge of the biology and toxicological response of this species. This work provides a foundation of molecular knowledge and tools that can be used to delve further into understanding the biology of G. holbrooki and how this organism can be used as a bioindicator organism for endocrine disrupting pollutants in the environment.

Show MeSH
Related in: MedlinePlus