ct-id: 110066 agent: BPA dose: 10 µg BPA/L tissue: ovary rep(tank): ZF 2 take down date: 2010-01-14 gender: F FISH wet wt (g): 0.94 gonad wt (g): 0.2236 plasma vtg (mg/ml): 10.5 array rep: E slide: 25191610974_1-4 array: 1.4
Treatment protocol
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. Following approximately 36 h of chemical (or control water) delivery to condition the tanks (20 L aquaria filled with 10 L of exposure water) and exposure apparatus, exposures were initiated by loading fish into the conditioned tanks. 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. Zebrafish were loaded and sampled one day prior to loading and sampling the fathead minnows so that three of the four exposure days for each species directly overlapped. 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.
Growth protocol
Sexually mature adult fathead minnows (5-6 month old) and zebrafish (ab wild-type strain; 6-7 month old) were obtained from an on-site aquatic culture facility at the US EPA Mid-Continent Ecology Division, Duluth, MN, USA. Both species were held under a 16:8 light:dark photoperiod and fed brine shrimp to satiation twice daily. However, fathead minnows were fed frozen adult brine shrimp (San Francisco Bay Brand, Newark, CA, USA) while zebrafish were fed newly hatched (< 24 h old) brine shrimp nauplii (BioMarine Brand artemia cysts; Hawthorne, CA, USA). Fish survival, water temperature (mean ± SD; 25.0±0.3; both species), and dissolved oxygen (mean ± SD; zebrafish 6.7±0.3 mg/L; fathead 7.0±0.2) were monitored daily over the duration of the exposure and did not vary significantly between treatment groups or species.
Extracted molecule
total RNA
Extraction protocol
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.
Label
cy3
Label protocol
cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturer's kits and protocols (Quick Amp Labeling Kit; Agilent, Palo Alto, CA)
Hybridization protocol
The Agilent one-color microarray hybridization protocol (One-Color Microarray-Based Gene Expression Analysis, version 5.7, Agilent Technologies, Palo Alto, CA) was used for microarray hybridizations following the manufacturer’s protocol and recommendations.
Scan protocol
An Axon GenePix® 4000B Microarray Scanner (Molecular Devices Inc.) was used to scan microarray images at 5 μm resolution
Description
BPA_NOTEL_96h_10_E
Data processing
Microarray image processing and data pre-processing were performed Agilent's Feature Extraction software v 9.5 following the manufacturer’s protocol and recommendations. Fastlo normalization was implemented in R 2.10.1
