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

Abstract

BackgroundThe 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.ResultsA 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).ConclusionsCoupling 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.

Exposure to Paper Mill Effluent at a Site in North Central Florida Elicits Molecular-Level Changes in Gene Expression Indicative of Progesterone and Androgen Exposure

“…The Low RNA Input Amplification Kit for one color labeling (Cy3) (Agilent, Santa Clara, USA) was used to synthesize cRNA using procedures previously described [16]. Purified cRNA concentrations and specific activities were determined by Nanodrop (ThermoScientific, Waltham, USA) with a specific activity >8 as the cut-off for further processing [16]. Hybridization of 600 ng cRNA and slide scanning was conducted as previously described [16].…”

“…Purified cRNA concentrations and specific activities were determined by Nanodrop (ThermoScientific, Waltham, USA) with a specific activity >8 as the cut-off for further processing [16]. Hybridization of 600 ng cRNA and slide scanning was conducted as previously described [16]. The microarray data set (GSE49238) was submitted to the Gene Expression Omnibus (GEO) database, with all reports and deposits made according to the Minimum Information About a Microarray Experiment (MIAME) guidelines [21].…”

“…All hepatic gene expression results were log-transformed before statistical analyses. Statistical analysis of shh was conducted by the delta delta Ct method as previously described [16]. A Student's t-test was used (after confirming normality and homogeneity of data distributions) to determine if there was a statistically significant difference between anal fin elongation, bone segment numbers, and gene expression levels between the Fenholloway River and the Econfina River.…”

“…However, better insights into the mechanisms of action of chemicals in paper mill effluent can be obtained by including molecular endpoints in these analyses [13]. mRNA levels of bone growth factor sonic hedgehog ( shh ) [14], [15] and steroidogenic enzyme 17β-hydroxysteroid dehydrogenase 3 ( 17βhsd3 ) are potential biomarkers of androgen exposure in G. holbrooki [16], [17]. In fish, vitellogenin ( vtg ) and zona pellucida glycoproteins ( zp ) are crucial components of oocyte quality [18] and decreases in their expression often occur during androgen exposure [15], [19], which can possibly lead to low-quality eggs.…”

“…Transcriptomics experiments of fish exposed to androgenic chemicals have revealed large impacts on the biological processes of metabolism [16], [19] so focusing on the liver is of relevance for evaluating the androgenicity of paper mill effluents. In addition, in vitro methods in the lab can be used to help screen samples of unknown chemical contaminants for their ability to activate the transcription of genes via nuclear receptors.…”