Comment[ArrayExpressAccession] E-GEOD-41901 MAGE-TAB Version 1.1 Public Release Date 2012-10-30 Investigation Title Comparison of the effects of Tris (2-ethylhexyl) trimellitate (TOTM) on gene expression associated with testicular maldevelopment (TMD) in male rat foetal testes by transcription profiling Comment[Submitted Name] Comparison of the effects of Tris (2-ethylhexyl) trimellitate (TOTM) on gene expression associated with testicular maldevelopment (TMD) in male rat foetal testes by transcription profiling Experiment Description The purpose of this study is to assess the potential of TOTM to induce testicular maldevelopment in the rat. We studied the effects of TOTM on the expression of genes in pathways involved in steriodogenesis and testes development. Certain phthalate esters have previously been shown to be involved in the induction of rat testicular mal-development (TMD) through effects on the expression of genes in pathways involved in steroidogenesis and testes development. In order to assess the effects of a potential alternate plasticizer, tris(2-ethylhexyl)trimellitate (TOTM), rats were exposed daily, in utero, to TOTM in order to assess the potential of this compound to induce developmental effects on the fetal testes. Pregnant rats were exposed between gestational day 12 and 19 and fetal testes RNA was analysed using whole genome microarrays. The effects of TOTM on the expression of genes in pathways involved in steroidogenesis and testes development were examined. The effects of TOTM were also compared with di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)phthalate (MEHP), an active metabolite of DEHP, and 2-ethylhexanol (2-EH), which were used as positive and negative controls, respectively. MEHP & DEHP (500mg/kg) caused a repression of genes in TMD pathways involved in cholesterol synthesis and transport (HMGCS, HMGCR, StAR, SCARB1, FDFT1, FDPS), steroidogenesis (Cyp11a, HSD3B1, SC4MOL) and testes development (INSL3, INHA). 2-EH caused minor repression of some of the genes in the TMD pathway. This was rationalised on the basis that 2-EH, a DEHP metabolite, is also a weak PPARĪ± agonist. It has been shown that in utero treatment with DBP will repress the genes from fetal testes involved in steroidogenesis and that this effect is associated with direct DBP-mediated binding of PPARĪ± to the promoters of these genes (Plummer et. al. 2010). TOTM did not cause a significant repression of genes in the TMD pathway. Based on these data, it is highly unlikely that TOTM will cause testicular dysgenesis in rats. Rats were exposed to TOTM in utero by the administration of daily oral gavage doses to pregnant dams between gestational day 12 and 19. The foetal testes were obtained by micro-dissection and prepared to facilitate the isolation of RNA, which was subsequently analysed using whole rat genome microarrays. 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 Chamberlain Plummer Chamberlain Farrar Elcombe Elcombe Quinney Person First Name Mark Simon Mark David Clifford Christopher Joanne Person Mid Initials Peter Person Email markchamberlain@cxrbiosciences.com Person Affiliation CXR Biosciences Person Phone 01382432163 Person Fax 01382432153 Person Address MCT, CXR Biosciences, 2 James Lindsay Place, Dundee, Tayside, United Kingdom Person Roles submitter Protocol Name P-GSE41901-1 P-GSE41901-6 P-GSE41901-5 P-GSE41901-2 P-GSE41901-3 P-GSE41901-4 P-GSE41901-7 Protocol Description ID_REF = VALUE = normalized P-value = Arrays were scanned using an Agilent Microarray Scanner according to CXR Method Sheet 'Microarray Hybridisation Scanning (1x44k, 4x44k and 8x15k)' Agilent 4x44K Whole Rat Genolme Oligo Microarray slides (G4131F) were hybridised, washed and then scanned. Pregnant dams were treated daily with vehicle (corn oil), TOTM, DEHP, EHO, MEHP at a dose of 500mg/kg by oral gavage on gestation days 12 to 19 inclusive. Pools of foetal testes from a minimum of three pups per litter (6 foetal testes) were disrupted using a qiashredder column and purified using Rneasy mini columns according to the CXR method entitled 'Method for Isolation of RNA from Foetal Testes for use in Microarray Analysis' (Plummer et al. 2007) Total RNA 700ng was labelled prior to microarray hybridisation using the Agilent Quick Amp Labelling Kit One Colour (Agilent# 5190-0442), according to the CXR Method Sheet entitled 'Transcroptional profiling using standard Agilent one-colour protocol'. Genes that are significantly expressed above background on individual arrays are identified using Agilent Feature Extraction software. This process involves deletion control and background subtraction and calculation of a p value reflecting the probability that there is no difference in intensity for a given feature to that of background using a two sided T test. The P value calculation is based on an error calculated using an Agilent error model (Inc, A. t. (2007). Agilent Feature Extraction Software v9.5 User Guide. Agilent Publication number g4460-90005, 1-250.). The 'significantly_altered_genes.txt' file content (available on Series records): In order to identify significantly altered genes, relative to the reference control RNA sample, in the treated samples Rosetta Resolver software is used to calculate a p value based on an error weighted mean of the data for the treated samples. Rosetta software builds ratio experiments from one colour microarrays using data from the test array sets and the reference (control) array set. The reference control array data is used as the denominator (D) in thisratio such that up-regulation or down-regulation of genes in the test set (T) is expressed relative to the reference set (T/D). The Rosetta error model [PMID 16522673] calculates both the propagated (technical) and scattered (biological) error and combines these two errors to make a more reliable error estimation. The p value allows an estimation of the confidence that a given feature/gene is not significantly different (the null hypothesis) relative to the reference. A p value of less than 0.01 is considered sufficient to reject the null hypothesis. Protocol Type bioassay_data_transformation image_aquisition hybridization specified_biomaterial_action nucleic_acid_extraction labeling feature_extraction Experimental Factor Name TREATED DAILY WITH Experimental Factor Type treated daily with Comment[SecondaryAccession] GSE41901 Comment[GEOReleaseDate] 2012-10-30 Comment[ArrayExpressSubmissionDate] 2012-10-29 Comment[GEOLastUpdateDate] 2012-10-31 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE41901_significantly_altered_genes.txt SDRF File E-GEOD-41901.sdrf.txt