ABSTRACT: This study explores the mechanisms of cardiotoxicity of the three widespread model polycyclic aromatic hydrocarbons retene, pyrene and phenanthrene in the rainbow trout (Oncorhyncus mykiss) early life stages. Newly hatched larvae were exposed to each individual compound at sublethal doses: DMSO (control group, 0.001%), retene (RET, 32 µg.L-1), pyrene (PYR, 32 µg.L-1) and phenanthrene (PHE, 100 µg.L-1). Changes in the cardiac transcriptome were assessed after 1, 3, 7 and 14 days of exposure using custom Salmo salar microarrays. Total RNA was extracted from the heart samples using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) following the manufacturer’s instructions. A total input amount of 220 ng of RNA per sample was amplified and labeled with the Cy3 dye using the one-color Agilent Low Input Quick Amp Labeling Kit (product number 5190-2305). A subsample equivalent to 1650 ng of Cy3-labeled cRNA was hybridized to Agilent 4x44k custom arrays (Salmo salar Salgeno-1 array, design ID 082522) using the Agilent Gene Expression Hybridization Kit (product number 5188-5242) and incubated overnight at 65°C. Hybridization washes were performed the following day using the Agilent Gene Expression Wash Buffer Kit (product number 5188-5327). Array readings were performed using an Agilent SureScan Microarray Scanner. The gene expression data was processed and analyzed with the help of the bioinformatics package described in Krasnov et al. (2011). The mean intensities of all arrays were normalized and the individual values of each feature were divided by the mean values of all samples. Significantly differentially expressed genes (DEGs) were determined by difference from their respective controls (Student’s t-test, p ≤ 0.05). The data was expressed as log2 of the fold changes (log2-FC) compared to the respective control. Reference: Krasnov, A., Timmerhaus, G., Afanasyev, S., Jørgensen, S.M., 2011. Development and assessment of oligonucleotide microarrays for Atlantic salmon (Salmo salar L.). Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 6, 31-38.
