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

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 17?-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?? 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 ?g/L of TB (a potent anabolic androgen) revealed 229 genes upregulated and 279 downregulated by TB (one-way ANOVA, p < 0.05, FDR ? = 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 17?-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.

SUBMITTER: Brockmeier EK

PROVIDER: S-EPMC3852779 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Brockmeier Erica K EK Yu Fahong F Amador David Moraga DM Bargar Timothy A TA Denslow Nancy D ND

BMC genomics 20130928

<h4>Background</h4>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 o ...[more]

PMID: 24074126

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Project description:Masculinized female Eastern Mosquitofish (Gambusia holbrooki) have resided downstream of paper mills in Florida since the 1980's. The potential impacts of this effluent on the mosquitofish endocrine system are unknown. The objective of this study was to evaluate gene expression patterns of endocrine system genes and global gene expression patterns in female G. holbrooki from a paper mill-impacted site. Masculinized female G. holbrooki were collected from a paper mill-impacted site (Fenholloway River) and from a reference site (Econfina River) and microarray analysis in livers was conducted. Hepatic microarray analysis revealed an increase in the expression of metabolic genes at the Fenholloway, with similarities in individual genes and biological processes compared to G. holbrooki exposed to androgens. These data indicate G. holbrooki from the Fenholloway may be impacted by a mixture of endocrine-active chemicals, including androgens. During the summer of 2012, G. holbrooki were captured from one site downstream of the Buckeye Pulp and Paper Mill (Taylor County, Perry, FL, USA) on the Fenholloway River (GPS coordinates: N 30 058.341’, W 83 588.569’) and one site in the Econfina conservation area (GPS coordinates: N 30 08.549', W 83 51.962') . Only sexually mature G. holbrooki (females > 15cm standard length and with the presence of the gravid spot near the vent) were collected. A 1/8 mesh seine was used for sample collection. Female G. holbrooki were transferred to 5 gallon aerated buckets filled with site water and were processed at the site immediately after collection. Fish were anesthetized using Tricaine-S (Western Chemical, Ferndale, USA) and sacrificed via spinal transection. Oocyte development was assessed upon dissection and livers were removed and stored in RNAlater (Qiagen, Hilden, Germany) overnight at 4 C before storage at -80 C. RNA was isolated from the livers using TRIzol (Invitrogen, Grand Island, USA), hydrated using RNAsecure (Ambion, Grand Island, USA), and DNase treated using the Turbo DNA-free kit (Ambion, Grand Island, USA). Four oocyte-development stage-matched RNA samples per treatment were evaluated for RNA integrity using the 2100 BioAnalyzer (Agilent, Santa Clara, USA). The range of RIN values was 7.8-8.9.

Dataset Information

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

Publications

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

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