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

• Published in: BMC genomics 2013-09, Vol.14 (1), p.660-660, Article 660
• Main Authors: Brockmeier, Erica K, Yu, Fahong, Amador, David Moraga, Bargar, Timothy A, Denslow, Nancy D
• Format: Article
• Language: English
• Subjects: Academic libraries; Androgens; Androgens - pharmacology; Animal behavior; Animals; Biological markers; Biomarkers; Biomarkers - metabolism; Chemicals; Cluster Analysis; Culicidae; Cyprinodontiformes - genetics; DNA microarrays; Endocrine disruptors; Environmental Exposure; Female; Females; Fishes; Freshwater; Gambusia holbrooki; Gene expression; Gene Expression Regulation - drug effects; Gene Library; Genetic aspects; Genomic libraries; Male; Males; Oligonucleotide Array Sequence Analysis - methods; Pulp & paper mills; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Signal Transduction - drug effects; Signal Transduction - genetics; Standard deviation; Studies; Trenbolone Acetate - pharmacology; Zebrafish
• ISSN: 1471-2164; 1471-2164
• DOI: 10.1186/1471-2164-14-660

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. 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). 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.