Abstract
Estrogenic chemicals are major contaminants of surface waters and can threaten the sustainability of natural fish populations. Characterization of the global molecular mechanisms of toxicity of environmental contaminants has been conducted primarily in model species rather than species with limited existing transcriptomic or genomic sequence information. We aimed to investigate the global mechanisms of toxicity of an endocrine disrupting chemical of environmental concern [17β-estradiol (E2)] using high-throughput RNA sequencing (RNA-Seq) in an environmentally relevant species, brown trout (Salmo trutta). We exposed mature males to measured concentrations of 1.94, 18.06, and 34.38 ng E2/l for 4 days and sequenced three individual liver samples per treatment using an Illumina HiSeq 2500 platform. Exposure to 34.4 ng E2/L resulted in 2,113 differentially regulated transcripts (FDR < 0.05). Functional analysis revealed upregulation of processes associated with vitellogenesis, including lipid metabolism, cellular proliferation, and ribosome biogenesis, together with a downregulation of carbohydrate metabolism. Using real-time quantitative PCR, we validated the expression of eight target genes and identified significant differences in the regulation of several known estrogen-responsive transcripts in fish exposed to the lower treatment concentrations (including esr1 and zp2.5). We successfully used RNA-Seq to identify highly conserved responses to estrogen and also identified some estrogen-responsive transcripts that have been less well characterized, including nots and tgm2l. These results demonstrate the potential application of RNA-Seq as a valuable tool for assessing mechanistic effects of pollutants in ecologically relevant species for which little genomic information is available.
Highlights
THE MAJOR ENDOGENOUS ESTROGEN in vertebrates, 17-estradiol (E2), is a significant contributor to the estrogenic contamination of surface waters, and E2 equivalent concentrations (EEQs) of up to 10 ng/l have been reported in rivers worldwide [10, 16, 26]
Transcriptomic approaches have been employed to characterize both the normal endogenous effects of estrogen signaling in females, and the effects of exposure to a number of estrogenic chemicals in male and juvenile fish using microarrays (e.g., 4, 18, 30, 34) and high-throughput sequencing (RNASAGE) [60]
The mean condition factors, hepatosomatic index (HSI), and gonadosomatic index (GSI) were, respectively, 1.16 Ϯ 0.01, 1.11 Ϯ 0.03, and 3.95 Ϯ 0.32 for mature males, 1.24 Ϯ 0.12, 0.85 Ϯ 0.04, and 0.06 Ϯ 0.01 for immature males, and 1.11 Ϯ 0.02, 1.00 Ϯ 0.03, and 0.31 Ϯ 0.02 for immature females, and there were no significant differences for these parameters between treatment groups
Summary
THE MAJOR ENDOGENOUS ESTROGEN in vertebrates, 17-estradiol (E2), is a significant contributor to the estrogenic contamination of surface waters, and E2 equivalent concentrations (EEQs) of up to 10 ng/l have been reported in rivers worldwide [10, 16, 26]. Transcriptomic approaches have been employed to characterize both the normal endogenous effects of estrogen signaling in females, and the effects of exposure to a number of estrogenic chemicals in male and juvenile fish using microarrays (e.g., 4, 18, 30, 34) and high-throughput sequencing (RNASAGE) [60] These studies have reported extensive transcriptional changes, reflecting the diverse range of genes and processes regulated by estrogens, including a number of broadly conserved pathways. A major advantage of this technique is that it can be used to conduct unbiased, global mechanistic analysis in any species of interest without a requirement for prior sequence information In this project, we employed RNA-Seq on an Illumina HiSeq 2500 platform to characterize the global hepatic transcriptomic responses of sexually mature male brown trout following exposure to E2, including at environmentally relevant concentration. By investigating the response to an estrogen, we aimed to discuss the suitability of RNA-Seq to identify a conserved mechanistic
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