Dose–response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol

Abstract

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss, were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as "expression signatures". Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that diffierent doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose. KeywordsRainbow trout; cDNA microarray; EstrogensThe field of toxicogenomics holds great promise for environmental and aquatic toxicology. Specific applications of microarrays in ecotoxicological studies include the identification of contaminants through analysis of expression patterns measured via microarrays in test organism exposed to previously uncharacterized contaminant mixtures, the identification of chemical mode of action based on the mRNA expression patterns, and the development of tools to assess endpoints for chronic exposure to mixtures that are tied to population-level consequences (Miracle and Ankley, 2005). However, the baseline transcriptomic response in organisms exposed to model toxicants must be characterized before this technology can be meaningfully applied to contaminated field sites. The objective of this study is to determine the relationship between gene expression profile and toxicant dose for the synthetic estrogen, ethynylestradiol (EE2).Methods for exposure and EE2 kinetics are described in more detail in Skillman et al. (in press), while RNA preparation and labeling, microarray hybridization and data analysis are described in more detail in Hook et al. (2006). We used isogenic fish in this study to minimize the confounding effects of individual variability. Male isogenic Rainbow Trout (Oncorhynchus mykiss) were exposed to EE2 at nominal concentrations of 10, 50 and 100 ng/L EE2 (actual *Corresponding author., E-mail address: Sharon.Hook@pnl.gov (S.E. Hook). NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript concentrations were 8, 33, and 115 ng/L EE2) via methanol drip (methanol concentration was less than 0.02%) for seven days and compared to methanol only controls. Fish were euthanized, then RNA...

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“…The effect of estrogen on gapdh and g6pd expression observed here likely results from their involvement in metabolism, since estrogens, like other sex steroids, are well known to have roles in the regulation of the metabolic processes associated with altered energy demands during gonad development and reproduction in fish [45][46][47]. Moreover, several other metabolic enzymes are also known to be controlled by sex steroids including estrogens in fish [27,46,48].…”

“…A wide range of 'housekeeping' genes have been used as internal controls in expression profiling following exposure of fish to estrogens, including bactin (e.g. 17β-estradiol [E 2 ] [24,25]; EE 2 [26][27][28]; bisphenol A [BPA] [26]; nonylphenol [NP] [26]; alpha-zearalenol [24]), 16S rRNA Expression of the candidate 'housekeeping' genes in fathead minnow liver following exposure to 10 ng EE 2 /L Figure 1 Expression of the candidate 'housekeeping' genes in fathead minnow liver following exposure to 10 ng EE 2 /L.…”

Effects of atrazine on endocrinology and physiology in juvenile barramundi, Lates calcarifer (Bloch)

“…In the third control group, 450 mL EE2 stock solution was added to each tank to obtain a 100 ng/L nominal concentration per tank [18]. In the first 2 control groups, no solution was added in the blank group, and 450 mL of 100% EtOH was added to each tank in the carrier group.…”

“…In the first 2 control groups, no solution was added in the blank group, and 450 mL of 100% EtOH was added to each tank in the carrier group. In the third control group, 450 mL EE2 stock solution was added to each tank to obtain a 100 ng/L nominal concentration per tank [18]. In the 5 experimental groups, we added 9 mL, 45 mL, and 450 mL of a 500 mg/mL atrazine stock solution, and 225 mL and 450 mL of a 10 000 mg/mL atrazine stock solution, respectively, to 5 tanks each to obtain nominal concentrations of 0.1 mg/L, 0.5 mg/L, 5 mg/L, 50 mg/L, and 100 mg/L per treatment group.…”

Detecting genome-wide gene transcription profiles associated with high pollution burden in the critically endangered European eel

“…Understanding the effects of chronic exposure to environmental pollutants on the transcriptome is crucial to safeguard the adaptive potential of natural populations under heavy anthropogenic pressure (Cheung and Spielman, 2002;Hoffmann and Willi, 2008;Oleksiak, 2008). Since gene transcription is often altered as a result of toxicant exposure (Thomas et al, 2001;Hamadeh et al, 2002;Oleksiak, 2008), quantifying gene transcription levels during exposure to pollutants has the potential for early detection of compromised health (decreased fitness and survival), which has been used for improved conservation measures in several fish species, including rainbow trout Oncorhynchus mykiss (Hook et al, 2006), barbel Barbus graellsii (Quiros et al, 2007), the well-established model species Atlantic killifish Fundulus heteroclitus (Oleksiak, 2008) or the Mediterranean killifish Aphanius fasciatus (Kessabi et al, 2010).…”