tissue: liver gender: female treatment group: H1 treatment: exposed to high dose (10 μg L-1) carbamazepine (CBZ)
Treatment protocol
Pooled livers of adult female zebrafish exposed to high dose (10 μg L-1) carbamazepine (CBZ), dosed every three days for six weeks after a 90% system water change-out. RNA sampled at the end of the six week exposure. Hepatic transcriptional responses in zebrafish to four common, environmentally relevant pharmaceuticals (ACE, CBZ, GEM, VEN), a mixture of the pharmaceuticals (MIX), and dilutions of wastewater effluent (WWE) were determined after a 6 week chronic exposure to a low (0.5 μg L-1 or 5% effluent) or high (10 μg L-1 or 10% effluent) concentration. The final nominal concentration of each pharmaceutical in exposure tanks was 0 (control), 0.5 µg L-1 (low dose) or 10 µg L-1 (high dose), with the pharmaceutical mixture (MIX) including equal concentrations of each pharmaceutical (ACE, VEN, GEM, CBZ) at the same two doses (0.5 or 10 µg L-1). The control tank for experiments with gemfibrozil (GEM), carbamazepine (CBZ), and mixture (MIX) had a final concentration of DMSO below 0.004% to account for solvent use in experimental tanks. Waste water effluent (WWE) exposure was conducted using final effluent collected twice per week and diluted to 5% (low dose) or 25% (high dose) using system water. The wastewater treatment plant used secondary treatment with activated sludge technology, as previously described. Effluent was stored in a dark, cold room for no more than 3 days after collection and was diluted just prior to water change-outs. Triplicate tanks for each treatment group housed 50 adult zebrafish each. Tanks were dosed every three days for six weeks after a 90% system water change-out. Venlafaxine (VEN) and acetaminophen (ACE) exposures were run together with shared controls (water only) while GEM and the MIX exposures were run together with shared controls (DMSO solvent control). CBZ and WWE effluent exposures were run separately with their own set of controls (DMSO solvent control for CBZ, water control for wastewater effluent).
Growth protocol
Adult wild-type zebrafish were purchased from DAP International (Toronto, ON) and housed in aquaria at a density of 4 fish per liter in 1:1 sex ratio. All aquaria were maintained at 28°C, pH 7-8, dissolved oxygen ≥87%, and conductivity at 470 µS in a partially re-circulating system. Fish were fed twice per day with tropical fish flake food and once per day with live, adult brine shrimp. Zebrafish were kept on a light cycle of 14h day / 10h night.
Extracted molecule
total RNA
Extraction protocol
After 6 weeks of exposure, zebrafish were sacrificed, weighed and tissues (GI tract with livers) were placed into RNAlater, topped up to 5-10 mL RNAlater per gram tissue. Tubes were incubated overnight at 4 °C to allow for tissues to be saturated with RNAlater and all tissues were then stored at -80 °C in RNAlater. Prior to RNA extraction, samples were thawed on ice and livers were stripped from the GI tract in RNAlater. Livers from 10-20 fish per gender liver were pooled to a maximum 100 mg of tissue and kept in RNAlater and on ice until RNA extractions. Immediately prior to extraction, RNAlater was diluted 50% with molecular water and total RNA was then extracted using100 mg of liver tissue in 1 ml of TRIzol reagent. Tissues were homogenized on ice in TRIzol for 10-30 seconds using an OMNI GLH homogenizer, following the manufacturer’s protocol. The RNA was reconstituted in molecular biology grade water, quantified spectrophotometrically, and absorbance at 260 and 280 nm was used as a measure of relative purity and quantity. The RNA samples were further analyzed for structural integrity using an Agilent 2100 Bioanalyzer with the Agilent RNA 6000 Nano Kit.
Label
Cy3
Label protocol
500 ug total RNA was Cy3 labeled using the Agilent Low Input Quick Amp Labeling kit, following the manufacturer's instructions.
Hybridization protocol
1.65 ug Cy3 labeled cDNA hybridized to a customized Agilent zebrafish single-channel 4 x 44K V2 microarray by the University Health Network Microarray Centre (Toronto, Canada).
Scan protocol
Hybridization results were scanned using an Agilent DNA Micorarray Scanner.
Description
Gene expression in pooled livers of adult female zebrafish exposed to high dose (10 μg L-1) carbamazepine (CBZ), replicate 1.
Data processing
Raw array data obtained from the University Health Network Microarray Centre were extracted using Agilent's feature extraction software using background detrending (spatial and multiplicative). Prior to normalization, Cy3 values below 5 were set to 5. The data were then normalized using the non-linear scaling method based on rank invariant probes. After normalization but before statistical analyses, probes not significantly above background in all microarrays were removed.
