Microarray analysis in the zebrafish (Danio rerio) liver and telencephalon after exposure to low concentration of 17alpha-ethinylestradiol
Introduction
The pharmaceutical estrogen 17alpha-ethinylestradiol (EE2) is used widely as the active ingredient in birth control pills and hormone replacement therapy. EE2 is detectable in the final effluent of some municipal wastewater treatment plants at concentrations approaching 40 ng/L (∼0.2 nM) (Ternes et al., 1999, Yin et al., 2002). Even at low concentrations, EE2 as well as other estrogenic compounds, can elicit biological effects in fish including plasma vitellogenin (Vtg) induction (Sumpter and Jobling, 1995, Islinger et al., 2003, Miracle et al., 2006), reduction in gonad size (Parrott and Blunt, 2005), impairment of reproductive behaviours (Robinson et al., 2003), and decreased fecundity and reproductive success (Balch et al., 2004). It is also important to note that exposure in the aquatic environment is not only to a single compound and there exists a complex mixture of chemicals at a given time. In particular, xenoestrogens may have significant additive effects, resulting in detrimental physiological effects in aquatic animals (Brian et al., 2005, Heneweer et al., 2005).
Zebrafish (Danio rerio) are increasingly being used to study the effects of chemicals and pharmaceuticals in the environment (Fraysse et al., 2006, van der Ven et al., 2005, van der Ven et al., 2006). Male and female zebrafish are sensitive to low concentrations of waterborne EE2. For example, adult zebrafish exposed to >10 ng/L EE2 show significant elevations in plasma vitellogenin and decreased spawning capability due to reduced gonad size after short exposures to EE2 (Van den Belt et al., 2001, Versonnen and Janssen, 2004). Negative effects of estrogenic exposure on zebrafish reproductive success, for example, the loss of functional testis in multi-generation studies has also been observed (Nash et al., 2004).
Gene expression profiling using microarrays are a powerful and increasingly important molecular tool for identifying responsive genes that may serve as biomarkers of estrogenic pharmaceutical exposures in fish (van der Ven et al., 2005, van der Ven et al., 2006, Martyniuk et al., 2006). Hoffmann et al. (2006) recently profiled hepatic gene expression in female zebrafish exposed to 15, 40, and 100 ng/L over a 7-day exposure to EE2, identifying E2 responsive genes involved in biological processes such as cell division, lipid and vitamin metabolism, hormone production, and sterol biosynthesis. The present study investigated the response of the transcriptome in the male zebrafish liver to a waterborne exposure to an environmentally relevant dose of 10 ng/L EE2 for a longer 21-day exposure period. We chose a lower dose comparable with the study to Hoffmann et al. (2006) but used male zebrafish instead of females. We also profiled the genomic response in the telencephalon of the brain which contains the preoptic area, the major site for neuroendocrine control of reproduction. Although brain derived cDNA microarrays for teleost fish are now developed (van der Ven et al., 2005, Martyniuk et al., 2006), there are still few studies that investigate the effects of pharmaceuticals found in the environment on brain function. Using somatic gene transfer of an estrogen response element-driven luciferase construct, Trudeau et al. (2005) demonstrated that gene transcription in the brain is modified significantly by estrogenic exposures. However, studying the effects of waterborne estrogen on brain function remains a challenge. The teleost brain has a very high capacity for endogenous estrogen production because of high aromatase activity (Callard et al., 2001). Therefore, it remains to be determined if a low environmental level of E2 would differentially affect brain versus liver.
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Animals and experimental design
Adult male zebrafish were supplied by a local pet shop and maintained in 34 L H2O in aquaria at a constant temperature of 28 °C, 14 h:10 h light:dark cycle, and standard diet (Westerfield, 1994). Experimental animals were staged according to hours post-fertilization (Kimmel et al., 1995). Adult males were sorted from females according to size and body shape. Male adult zebrafish were exposed to a nominal concentration of 10 ng/L EE2 in 0.001% EtOH for a period of 21 days. Control male fish received
Microarray analysis and gene categorization: liver and telencephalon
Table 2A shows the list of genes that we identified as being significantly regulated in the liver after EE2 treatment. Genes are grouped according to clusters of expression patterns. There were four clusters (A–D) that showed a consistent pattern of expression changes across microarray slides for example, two clusters contained known E2 responsive genes such as vtg1, vtg3, and esr1. Table 2B lists the genes in the telencephalon that microarray analysis identified as consistently changing across
Functional categorization of genes using GO Slim terms
Our analysis identified few altered genes that were identical between the liver and the telencephalon. Examples of genes being altered in both liver and telencephalon includes pituitary adenylate cyclase-activating peptide (PACAP) which was induced whereas developmental receptor tyrosine kinase showed a reduction. Beta-2-microglobulin, a cell structural protein, showed a decrease in steady state mRNA abundance in the liver but an increase in the telencephalon, demonstrating that there can be
Conclusions
Here we show that, although there is largely a different genomic response to EE2 between the liver and the telencephalon, there are estrogen responsive genes identified that are involved in common biological processes and molecular functions. Currently, it is difficult to comment on sex specific regulation of genes between males and females due to the differences in the length of the exposure and dose of EE2 between this study on males and that of Hoffmann et al. (2006) on females. However,
Acknowledgements
We would like to thank Bill Fletcher for assistance with animal care, Louise Coverdale for assistance with microarray data analysis, and Jenn Watt for zebrafish dissections. This research was supported by an Ontario Graduate Scholarship to CJM and JTP, and NSERC Strategic and Discovery Grants to VLT and ME.
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