Time and concentration dependent transcriptome signatures in the ZFE of diruon, diclofenac and naproxen

Time and concentration dependent transcriptome signatures in the ZFE of diruon, diclofenac and naproxen Keywords: Expression profiling by array Wild-type adult zebrafish, originally received from OBI petshop (Leipzig, Germany) were kept in 120L fish tanks containing carbon-filtered tap water at 26°C under a 12h:12h dark-light cycle. Eggs were collected approximately 1 hour after light onset and inspected using a light microscope. Fertilized eggs were incubated at 26°C, while unfertilized eggs were discarded. For lethality experiments, which were conducted to find adequate exposure concentrations for transcriptome experiments, nine embryos per replicate were distributed into three 7.5ml glas vials, each containing 6ml exposure or control medium. Vials were sealed and incubated in a climate chamber shaking (Edmund Bühler SM-30 Control, 26°Celsius, 75 rpm, 12h:12h light:dark) till 96 hours post fertilization (hpf). At 48 hpf, 72 hpf and 96 hpf sublethal and lethal effects were recorded. For transcriptome experiments 20 embryos per replicate were transfered to two 20ml GC-glas vials at 24 hpf. A volume of 18ml of exposure medium was added to each vial and vials were sealed and incubated in a climate chamber (same settings as above) until sampling. Embryos were exposed to a range of 5 different concentrations of toxicant and RNA was extracted at 6 different timepoints. Exposure medium for all experiments was prepared by dissolving the substance in freshly prepared ISO-water (pH 7.4, oxygenized) (Diclofenac, Naproxen) or in 0.1 % methanol as solubilising agent (Diuron). Time points of RNA-extraction during the exposure were 3, 6, 12, 24, 48, and 72 hour post exposure start. The exposure concentrations were phenotypically anchored to the LC at 96 hpf/72 hpe. The LC25, modeled from experimental observations served as highest and the LC0.5 as lowest exposure concentration with 6 equal dilution steps in between, with dilution steps 1,2,4 and 6 chosen for exposure.