Unknown,Transcriptomics,Genomics,Proteomics

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Hepatic transcriptome analysis of male and female Eastern Mosquitofish (Gambusia holbrooki) exposed to a progestin and anti-progesterone reveals the mode of action of endocrine disruption of synthetic steroids in an aquatic organism

ABSTRACT: Major classes of hormone mimics that have been studied include environmental estrogens and androgens, but recent studies have also demonstrated the significant impacts of natural and synthetic progesterones in the environment. The objective of this study was to evaluate the molecular and physiological impacts of progestin, anti-progestin, and mixture exposures in the Eastern Mosquitofish (G. holbrooki). By comparison of gene expression profiles and modulated biological processes in the three groups, it was determined that mifepristone acts more as a progestin than as an anti-progestin, as has also been demonstrated in other species of fish. This work contributes to the overall knowledge of the impacts of this class of chemical contaminats on aquatic organisms, which are a sentinel species for pollutants as aquatic ecosystems often become a reservoir for anthropogenic contaminants. G. holbrooki adult males and females were exposed to one of the following conditions: vehicle control (ethanol), 100 ng/L of levonorgestrel, 100 ng/L of mifepristone, or a mixture of both levonorgestrel and mifepristone. All exposures were conducted for 48 hours with water changed and chemicals renewed daily. Fish were anesthetized using 100 mg/L Benzocaine (Ethyl 4-aminobenzoate). 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 8.2-9.6

ORGANISM(S): Gambusia holbrooki

SUBMITTER: Erica Brockmeier

PROVIDER: E-GEOD-55518 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:The Eastern Mosquitofish (Gambusia holbrooki) is a non-model species with the potential for development of biomarkers of endocrine disrutpting chemical (EDC) exposure due to its androgen-driven secondary sexual characteristics. While G. holbrooki have been utilized in the field as bioindicator organisms EDC exposure in areas impacted by pulp and paper mills, the lack of molecular tools and general understanding of how G. holbrooki are impacted by androgen exposure hinder the use of this organism as a widespread tool for the evaluation of these chemicals in the environment. While traditional gene-by-gene approaches have provided a list of genes that may be appropriate for developing into biomarkers of androgen exposure, a more inclusive method could provide more rapid and expansive determination of genes that can be developed into biomarkers of androgen exposure. The objective of this study is toutilize a mosquitofish microarray to determine potential biomarkers of chronic androgen exposure. The specific aim was to determine genes that may be developed into biomarkers of chronic androgen exposure in hepatic tissues using 17M-NM-2-trenbolone (TB) exposed adult female G. holbrooki by microarray analysis. Liver tissues from a 14-day exposure of female G. holbrooki to 1 M-BM-5g/L of the potent androgen receptor agonist 17M-NM-2-trenbolone (17M-NM-2-hydroxyestra-4,9,11-trien-3-one; abbreviated as TB) [Brockmeier 2013] were used as the first sample for microarray analysis using the custom G. holbrooki microarray to determine genes that were significantly up or down-regulated during a chronic androgen agonist exposure. RNA was isolated from the livers as previously described 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 8.3-8.9.

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Dataset Information

Hepatic transcriptome analysis of male and female Eastern Mosquitofish (Gambusia holbrooki) exposed to a progestin and anti-progesterone reveals the mode of action of endocrine disruption of synthetic steroids in an aquatic organism

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