Comment[ArrayExpressAccession] E-GEOD-27621 Public Release Date 2011-06-04 Investigation Title Dose dependent response of liver gene expression and lipoperoxidation to dietary long chain omega 3 PUFA Comment[Submitted Name] Dose dependent response of liver gene expression and lipoperoxidation to dietary long chain omega 3 PUFA Experiment Description The cardioprotective effects of long chain (LC) 3PUFA can be achieved at the gene expression level, notably in liver. However, the complexity of biological pathways modulations and the nature of the bioactive molecules are still under investigation. The present study aimed to investigate the dose-response effects of LC 3PUFA on the production of peroxidated metabolites and on global gene expression in liver. The intake of LC ω3PUFA increased, in a dose-dependent manner, their incorporation in liver phospholipids but also the hepatic production of 4-HHE. Pathways related to inflammation were dose-dependently associated with the 3 groups but Group 2 was rather associated with inflammatory effects while Group 3 was anti-inflammatory. LC ω3PUFA had no effect on PPAR-controlled genes. However, they modified, in a dose-dependent manner, the expression of major genes related to lipoprotein metabolism (LDLR, VLDLR, INSIG1 and MTTP), possibly through the FXR signaling pathway. In conclusion, the effect of LC ω3PUFA is dependent on the dose possibly because of the production of peroxidated metabolites such as 4-HHE. New-Zealand white rabbits were fed (7 wk) a high cholesterol diet and received by daily oral gavages either oleic acid rich oil or a mixture of oils providing 0.1% (Group 1), 0.5% (Group 2) or 1% (Group 3) of energy as docosahexaenoic acid. Specific peroxidated metabolite issued from LC 3PUFA (4-hydroxyhexenal or 4-HHE) were measured by GC/MS/MS and transcription profiling was conducted in liver. Differentially expressed genes were identified using Bioconductor (Moderated p<0.05, Fold Change>1.20) and clustered into pathways (Ingenuity Pathway Analysis 7.0). Date of Experiment Term Source Name EFO Term Source Version Term Source File http://bar.ebi.ac.uk:8080/trac/browser/branches/curator/ExperimentalFactorOntology/ExFactorInOWL/currentrelease/eforelease/efo.owl Person Last Name McCulloch Gladine Person First Name Alan Cecile Person Mid Initials Francis Person Email alan.mcculloch@agresearch.co.nz Person Affiliation AgResearch NZ Person Phone 64 3 4899080 Person Fax Person Address AgResearch NZ, Invermay, Puddle Alley, Mosgiel, New Zealand Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE27621-1 P-GSE27621-13 P-GSE27621-9 P-GSE27621-7 P-GSE27621-4 P-GSE27621-6 P-GSE27621-3 P-GSE27621-8 P-GSE27621-5 P-GSE27621-15 P-GSE27621-14 P-GSE27621-17 P-GSE27621-2 P-GSE27621-12 P-GSE27621-16 P-GSE27621-10 P-GSE27621-11 Protocol Description ID_REF =
VALUE = Normalised log2 ratio (Ch1/Ch2)
ch1_sig_mean = Raw Channel 1 Foreground mean intensity
CH1_BKD_MEAN = Raw Channel 1 Background mean intensity
ch2_sig_mean = Raw Channel 2 Foreground mean intensity
CH2_BKD_MEAN = Raw Channel 2 Background mean intensity
gisfeatnonunifol = Green dye, outlier as Foreground not uniform
risfeatpopnol = Red dye, population outlier – differs to other spots of the same type
risfeatnonunifol = Red dye, outlier as Foreground not uniform
gisfeatpopnol = Green dye, population outlier – differs to other spots of the same type The fluorescently labelled cRNA was hybridized using the Agilent gene expression hybridization kit (5188-5242-A) following the manufacturer’s instructions. Briefly, 825 ng cyanine 3-labelled, linearly amplified cRNA were mixed with 825 ng cyanine 5-labelled, linearly amplified cRNA. The mix was loaded onto the Agilent Rabbit Gene Expression Microarrays (4x44k) following a loop design and hybridization proceeded in a hybridization oven set to 65°C for 17 hours. Then, the slides were washed in solutions I, II and III (Agilent Technologies) and air-dried. Diets fed for 7 weeks. B : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.5% of energy as DHA The diet abbreviations T, A, B, C used in the sample records refer to the following : A : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.1% of energy as DHA New-Zealand white rabbits were fed (7 wk) a high cholesterol diet and received by daily oral gavages either oleic acid rich oil or a mixture of oils providing 0.1% (Group 1), 0.5% (Group 2) or 1% (Group 3) of energy as docosahexaenoic acid. C : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 1% of energy as DHA T : cholesterol rich diet + oleic acid rich oil Differentially expressed genes were identified using Bioconductor (Moderated p<0.05, Fold Change>1.20) and clustered into pathways (Ingenuity Pathway Analysis 7.0). Slides were scanned immediately after washing using the Agilent DNA Microarray Scanner (G2565AA/G2565BA), at photomultiplier tube voltages red and green. Spot identification and quantification were performed using Agilent Feature Extraction Software 7.0. B : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.5% of energy as DHA T : cholesterol rich diet + oleic acid rich oil A : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 0.1% of energy as DHA C : cholesterol rich diet + mix of oleic acid rich oil and tuna oil providing 1% of energy as DHA Total RNA from liver tissue was isolated using the Norgen RNA Purification kit (Norgen Bioteck Corporation, Ontario, Canada) according to the manufacturer’s instruction. RNA was quantified with a Nanodrop ND-1000 spectrophotometer (nanoDrop Technologies, Wilmington, Del., USA), and RNA integrity assessed with a RNA 6000 Nano Labchip kit using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA). Only total RNA with an OD 260/230 ratio > 1.7 and a RNA integrity number > 8 was used for microarray hybridization. RNA samples were labelled using the Agilent Quick-Amp Labelling kit (5190-0424) according to the manufacturer’s instructions. Briefly, 500 ng of purified total RNA from each sample was amplified and reverse transcribed in vitro to cDNA using the T7-polymerase, which was subsequently labelled with either cyanine 3-CTP or cyanine 5-CTP dyes (5188-1170-P, Agilent Technologies, Wilmington, USA). The Nanodrop ND-1000 was used to monitor the yield of amplification and the dye incorporation; all samples had a yield > 825ng cRNA and a specific activity > 8.0 pmol Cy3 or Cy5 per ug cRNA. Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization grow grow grow grow grow grow grow feature_extraction image_aquisition specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE27621 Comment[GEOLastUpdateDate] 2011-06-03 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2011-06-03 Comment[ArrayExpressSubmissionDate] 2011-03-01 SDRF File E-GEOD-27621.sdrf.txt