Comment[ArrayExpressAccession] E-GEOD-43675 MAGE-TAB Version 1.1 Public Release Date 2013-10-01 Investigation Title Effect of waterborne exposure to carcinogenic PAHs on adult zebrafish hepatic transcriptome Comment[Submitted Name] Effect of waterborne exposure to carcinogenic PAHs on adult zebrafish hepatic transcriptome Experiment Description Several environmental pollutants, especially organic compounds such as polycyclic aromatic hydrocarbons (PAHs) are potent carcinogenic chemicals. Exposure of different fish species to PAHs causes a high prevalence and variety of tumor lesions. To identify and compare the mode of action (MOA) of different model carcinogenic PAHs, a transcriptomic study was carried out in adult zebrafish (Danio rerio) after exposure to 0.3 mg/l of benzo(a)pyrene (B(a)P) or to 7,12-dimethylbenz(a)anthracene (DMBA), both of them dissolved in dimethyl sulfoxide (DMSO; final concentration of 0.01%). Samples for microarray analysis were taken after 1 and 2 weeks of exposure. The changes in the mRNA transcription levels over the corresponding controls were compared among treatments. Correspondence analysis was able to discriminate among treatments; factor 1 separated both sampling times while factor 2 separated the compounds. A total number of 3043 transcripts was shown to be differentially regulated in at least one of the treatments. B(a)P treatment produced regulation of sequences related to GO categories associated to cell cycle and cell division whereas DMBA regulated GO terms related to oxidation/reduction responses, responses to chemicals and response to bacterium. Both compounds were able to alter many xenobiotic metabolism related genes, especially upregulating the transcription of phase I metabolism-related genes such as cytochrome P450 members (cyp1a or cyp1b), and many cancer-related genes such as the Jun B proto-oncogene (junb). Overall, these results indicated that the exposure to both PAHs was able to differentially alter the transcription levels of genes involved in both the detoxification metabolism and the cell-cycle control, including different tumor-supressor genes and oncogens. These alterations have been often related to the appearance of tumor lesions. Zebrafish were waterborne exposed to 0.3ppm BaP or DMBA for 1 and 2 weeks. After 1 and 2 weeks hepatic samples were collected from each treatment group as well as from the control group. 3 biological replicates have been collected per exposure group and time point. Each biological replicate is a pool of 5 livers. Samples from the different treatments were hybridized in an n+2 (n = 9) A-optimal loop design. Additionally, these two designs were linked by two extra hybridizations to enable comparison between time points Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Vicario Vicario-Parés Vergauwen Knapen Blust Cajaraville Orbea Person First Name Unai Unai Lucia Dries Ronny Miren Amaia Person Mid Initials P Person Email unai.vicario@ehu.es Person Affiliation University of the Basque Country (UPV/EHU) Person Address Zoology and Animal Cell Biology, University of the Basque Country (UPV/EHU), Barrio Sarriena SN, Leioa, Bizkaia, Spain Person Roles submitter Protocol Name P-GSE43675-4 P-GSE43675-5 P-GSE43675-1 P-GSE43675-2 P-GSE43675-3 P-GSE43675-6 Protocol Description Fluorescently labelled cRNA was constructed starting from total RNA extracts, following Agilent's two-colour microarray-based gene expression analysis protocol (version 5.7, http://www.agilent.com) using the Quick Amp kit (Agilent Technologies, Diegem, Belgium). Samples from the different treatments were hybridized in two n+2 (n = 9) A-optimal loop design. Additionally, these two designs were linked by two extra hybridizations to enable comparison between time points. 825 ng of both Cy3 and Cy5 labelled cRNA was applied onto every microarray according to the hybridization design. Microarray slides were incubated at 65 °C for 17 h in a rotating Agilent hybridization chamber. Slides were subsequently washed with Agilent wash buffers and acetonitrile and finally submersed in stabilization and drying solution (Agilent Technologies) to prevent ozone-induced Cy5-degradation. 3 months old fish were exposed to nominal concentrations of 0.3 mg/l B(a)P (Sigma-Aldrich, St. Louis, USA) or 0.3 mg/l DMBA (Sigma-Aldrich) for 2 weeks at a density of 1.375 fish/l in 50 l tanks using one tank per experimental group. During the experiment, 20% of the water in each tank was renewed and the corresponding amount of test solution was added twice a week. DMSO (Sigma-Aldrich) was used as a carrier for the exposure at a final concentration of 0.01%, being present in the control group as well as in the exposure groups. Samples from each treatment were collected after 1 and 2 weeks of exposure.From each treatment at each sampling time 3 biological pseudoreplicates consisting of pools of 5 livers each were prepared. Fish were sacrificed, after euthanasia by overdose of MS-222 (tricaine methane-sulfonate, Sigma-Aldrich), and livers were dissected, immersed in RNA Later ® (Sigma-Aldrich), immediately frozen in liquid nitrogen, and stored at -80ºC until RNA extraction. Adult zebrafish (Danio rerio; AB Tubingen) were raised and maintained at 27 ± 1 ºC with a 14-hour light / 10-hour dark cycle in 100 l tanks at a density of 1.5 fish/l. Tank water was prepared by conditioning osmotic water with marine basic salt (Sera Gmbh, Heinsberg, Germany) and KH/pH plus (Sera) up to 600 µS and pH 7.4, before mechanical filtering (1 µm) and sterilization by ultraviolet light. Water aeration and biological filtration was achieved by an airlift pump in each tank. Residual metabolites were measured using Sera GmbH ammonium, nitrite and nitrates kit, maintaining water at 0-0.5 mg/L; 0-0.5 mg/L and 5-10 mg/L respectively. When the highest concentration values were surpassed water was partially replaced. Fish were fed ad libitum with live Artemia nauplii (INVE, Dendermonde, Belgium) and commercial dry food, Microgran (Sera). Total RNA extraction was performed following the TRIzol® extraction method (Invitrogen Life-Technologies, Merelbeke, Belgium). Microarrays were scanned using a Genepix Personal 4100A confocal scanner (Axon Instruments, Union City, CA, USA) at a resolution of 5 μm. The photomultiplier tube voltages for separate wavelengths were adjusted to obtain an overall green/red ratio of one. Images were processed using GenePix Pro 4.1 software (Axon Instruments) for spot identification and quantification of the fluorescent signal intensities. Protocol Type labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TREATMENT EXPOSURE TIME Experimental Factor Type treatment exposure time Comment[SecondaryAccession] GSE43675 Comment[GEOReleaseDate] 2013-10-01 Comment[ArrayExpressSubmissionDate] 2013-01-23 Comment[GEOLastUpdateDate] 2013-10-02 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE43675_LogBaPExpvsCo_2week.txt Comment[AdditionalFile:Data2] GSE43675_LogBaPexpvsCo_1week.txt Comment[AdditionalFile:Data3] GSE43675_LogDMBAExpvsCo_1week.txt Comment[AdditionalFile:Data4] GSE43675_LogDMBAExpvsCo_2week.txt Comment[AdditionalFile:Data5] GSE43675_matrix_log2FC_4contrasts.txt SDRF File E-GEOD-43675.sdrf.txt