Comment[ArrayExpressAccession] E-GEOD-48990 MAGE-TAB Version 1.1 Public Release Date 2014-09-01 Investigation Title Evaluation of the robustness of classification of carcinogen-modified transcriptomic responses in HepaRG cells and the interlaboratory reproducibility of the model Comment[Submitted Name] Evaluation of the robustness of classification of carcinogen-modified transcriptomic responses in HepaRG cells and the interlaboratory reproducibility of the model Experiment Description Efforts to develop alternatives which can at least partially replace some of the currently used in vivo tests are ongoing. The recently ended FP6 European project carcinoGENOMICS had the goal to use the combination of toxicogenomics and in vitro cell culture models for identification of genotoxic- and non-genotoxic carcinogen-specific gene signatures. In this study is presented a part of the outcome of the project and in particular the performance of the gene classifier derived after exposure of the HepaRG cell line to prototypical hepatocarcinogens. Upon analyzing the data at a gene and a pathway level by using diverse biostatistical approaches, a clear-cut separation of the genotoxic from the non-genotoxic hepatocarcinogens and non-carcinogens was achieved (up to 88% correct prediction). The most characteristic pathway for genotoxic exposure was DNA damage. Further to show the robustness of the HepaRG model, the interlaboratory reproducibility of 3 blindly tested compounds was assessed. The results showed between 20% and 35% reproducibility. The subsequent classification of the 3 blindly tested compounds resulted in correct prediction of the genotoxicant, whereas the other two compounds were misclassified. In conclusion, the combination of transcriptomics and HepaRG in vitro cell model provides a solid basis for the detection of the genotoxic potential of unknown chemicals. HepaRG cells were exposed to 15 selected compounds for 72 hours, i.e. 5 GTX (N-nitrosomorpholine, NMP; Hydroquinone, HQO; Hydrazine dihydrochloride, HHC; 2-acetylaminofluorene, TAF; 2-amino-3-methylimidazo(4,5-f)quinoline, AMQ), 5 NGTX (Fumonisin B1, FMB; Cyclosporine A, CsA; Acetamide, ACE; Diethylhexyl Phthalate, DHP; Ethanol, ETH), 5 NC (4-acetylaminofluorene, FAF; D,L-Menthol, DLM; Benzoin, BEN; Benzyl Alcohol, BEA; Triclosan, TRI). The IC10 concentrations (reducing cell viability by 10%) were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT test) (number of replicates n=3). Further RNA samples were generated by exposure of the HepaRG cells to the MTT-determined IC10 for a period of 24h and 72h. Complementary RNA targets were prepared and hybridized according to the manufacturer's procedures on high-density oligonucleotide microarrays (i.e. Affymetrix U133 Plus 2.0 GeneChip). Finally,alaysis was performed at a gene and pathway level. In the analysis, control (DMSO) samples were re-normalized with several sample groups and new processed data were created for each analysis. Thus, some of the Samples in this Series represent a re-analysis of other Samples and CEL files are identical. 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 Doktorova Doktorova Yildirimman Ceelen Vilardell Vanhaecke Vinken Ates Heymans Gmuender Bort Corvi Phrakonkham Li Mouchet Chesne van Delft Kleinjans Castell Herwig Rogiers Person First Name Tatyana Tatyana Reha Liesbeth Mireia Tamara Mathieu Gamze Anja Hans Roque Raffaella Pascal Ruoya Nicolas Christophe Joost Jos Jose Ralf Vera Person Mid Initials Y Person Email geo@ncbi.nlm.nih.gov Person Affiliation VUB Person Address FAFY, VUB, Laarbeeklaan 103, building G, Brussels, Belgium, Belgium Person Roles submitter Protocol Name P-GSE48990-1 P-GSE48990-5 P-GSE48990-6 P-GSE48990-2 P-GSE48990-3 P-GSE48990-4 P-GSE48990-7 Protocol Description The Affymetrix IDs were alternatively remapped to the alternative IDs from Brainarray (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/CDF_download.asp), thus the ensemb version 61 was used (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/14.1.0/ensg.download/HGU133Plus2_Hs_ENSG_14.1.0.zip). All samples were further normalized by GC-RMA algorithm. As the number of controls were less than the number of treatments, the same controls were used several to normalize data from several treatments. ID_REF = VALUE = GC-RMA We used standard Affymetrix labeling protocols We used standard Affymetrix hybridisation rotocols The cells were individually exposed to 15 prototypical compounds for 24h and 72 hours at IC10 concentrations, i.e. 5 GTX (N-nitrosomorpholine, NMP, IC10=0.216mM; Hydroquinone, HQO, IC10=0.15mM; Hydrazine dihydrochloride, HHC, IC10=0.86mM; 2-acetylaminofluorene, TAF, IC10=0.0408mM; 2-amino-3-methylimidazo(4,5-f)quinoline, AMQ, IC10=0.003mM), 5 NGTX (Fumonisin B1, FMB, IC10=2.4mM; Cyclosporine A, CsA, IC10=0.0026mM; Acetamide, ACE, IC10=5.9; Diethylhexyl Phthalate, DHP, IC10=10mM; Ethanol, ETH, IC10=10mM), 5 NC (4-acetylaminofluorene, FAF, IC10=0.22; D,L-Menthol, DLM, IC10=1.2mM; Benzoin, BEN, IC10=0.345mM; Benzyl Alcohol, BEA, IC10=6.5mM; Triclosan, TRI, IC10=0.022mM). The human hepatoma-derived HepaRG cells (Biopredic International, France) were cultivated as previously described by A. Guillouzo et al. 168 (2007) 66-73. and P. Gripon et al 99 (2002) 15655-15660. At day 13 of cultivation, dimethylsulfoxide (DMSO)-containing medium (2%) was added for 7 days . At day 19 exposure with the selected compounds was initiated. Samples for RNA isolation were collected after 24 and 72 hours of exposure. Minimum three independent biological experiments were conducted for each compound. The total RNA extraction (RNA extraction kit, Qiagen), including a DNase digestion step, was done according to the manufacturer’s instructions. We used standard Affymetrix scanning parameters Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name TREATMENT REPLICATE Experimental Factor Type treatment replicate Comment[SecondaryAccession] GSE48990 Comment[GEOReleaseDate] 2014-09-01 Comment[ArrayExpressSubmissionDate] 2013-07-17 Comment[GEOLastUpdateDate] 2014-09-02 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-48990.sdrf.txt