ABSTRACT: Effects of bisphenol A (BPA) on ovarian transcript profiles as well as targeted endpoints with endocrine/reproductive relevance were examined in two fish species, fathead minnow (Pimephales promelas) and zebrafish (Danio rerio), exposed in parallel using matched experimental designs. Four days of waterborne exposure to 10 µg BPA caused significant vitellogenin induction in both species. However, zebrafish were less sensitive to effects on hepatic gene expression and steroid production than fathead minnow and the magnitude of vitellogenin induction was more modest. The concentration-response at the ovarian transcriptome level was non-monotonic and violated assumptions that underlie proposed methods for estimating hazard thresholds from transcriptomic results. However, the non-monotonic profile was consistent among species and there were nominal similarities in the functions associated with the differentially expressed genes, suggesting potential activation of common pathway perturbation motifs in both species. Overall, the results provide an effective case study for considering the potential application of ecotoxicogenomics to ecological risk assessments and provide novel comparative data regarding effects of BPA in fish. Fish were exposed to 0, 0.01, 0.1, 1.0, 10, or 100 µg BPA/L delivered in a continuous flow (45 ml/min) of sand filtered, UV treated, Lake Superior Water, without the use of carrier solvents. The design employed six replicate tanks per treatment. Three of the replicates were loaded with three male and three female zebrafish per tank, while the remaining three replicates were loaded with three male and three female fathead minnows. Addition of fish to the exposure tanks was staggered by replicate such that all sampling could be completed within 60 min of the desired 96 h exposure duration. After 96 h of exposure, fish were anesthetized in a buffered solution of tricaine methanesulfonate (MS-222; Finquel; Argent, Redmond, WA, USA) and weighed. Blood was collected from the caudal vasculature using microhematocrit tubes, centrifuged to separate the plasma, and plasma was stored at -80oC until analyzed. Liver, gonad, and brain tissues (including pituitary gland) were snap frozen in liquid nitrogen and stored at -80oC until extracted. Total RNA was isolated from ovary tissue of both fathead minnow and zebrafish using RNeasy kits (Qiagen, Valencia, CA, USA). RNA quality was evaluated using an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE, USA) and RNA was quantified spectrophotometrically (Nanodrop). Total RNA samples were stored at -80oC until used for microarray analyses. Ovarian transcripts from 32 fathead minnows (n=5 per treatment, except for control and 1.0 µg BPA/L, n=6) were analyzed using a custom, 15,000 gene microarray (GEO Platform Accession GPL9248) purchased from Agilent Technologies (Palo Alto, CA, USA). Ovarian transcripts from 34 zebrafish (n=6 per treatment, except 0.01 and 0.1 µg BPA/L, n=5) were analyzed using a commercial 44,000 feature microarray (Agilent design 019161; GEO Platform Accession GPL6457). The same hybridization and scanning procedures were used for both platforms. Briefly, microarrays were hybridized following the manufacturer’s guidelines for One-Color Microarray-Based Gene Expression Analysis (version 5.7; Agilent). Hybridizations were conducted with 1 µg of total RNA. Complementary DNA synthesis, cRNA labeling, amplification, and hybridizations were performed following the manufacturer’s kits and protocols (Quick Amp labeling kit; Agilent). Scanning was conducted at 5 µm resolution using an Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc., Concord, Ontario, Canada). Data were extracted from microarray array images using Agilent Feature Extraction Software (Agilent; Palo Alto, CA, USA).
