title: Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity.
abstract: The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing to correctly predict the in vivo genotoxic and carcinogenic properties of test compounds. Toxicogenomics, i.e. mRNA-profiling, has been proven successful in improving the prediction of genotoxicity in vivo and the understanding of underlying mechanisms. Recently, microRNAs have been discovered as post-transcriptional regulators of mRNAs. It is thus hypothesised that using microRNA response-patterns may further improve current prediction methods. This study aimed at predicting genotoxicity and non-genotoxic carcinogenicity in vivo, by comparing microRNA- and mRNA-based profiles, using a frequently applied in vitro liver model and exposing this to a range of well-chosen prototypical carcinogens. Primary mouse hepatocytes (PMH) were treated for 24 and 48h with 21 chemical compounds [genotoxins (GTX) vs. non-genotoxins (NGTX) and non-genotoxic carcinogens (NGTX-C) versus non-carcinogens (NC)]. MicroRNA and mRNA expression changes were analysed by means of Exiqon and Affymetrix microarray-platforms, respectively. Classification was performed by using Prediction Analysis for Microarrays (PAM). Compounds were randomly assigned to training and validation sets (repeated 10 times). Before prediction analysis, pre-selection of microRNAs and mRNAs was performed by using a leave-one-out t-test. No microRNAs could be identified that accurately predicted genotoxicity or non-genotoxic carcinogenicity in vivo. However, mRNAs could be detected which appeared reliable in predicting genotoxicity in vivo after 24h (7 genes) and 48h (2 genes) of exposure (accuracy: 90% and 93%, sensitivity: 65% and 75%, specificity: 100% and 100%). Tributylinoxide and para-Cresidine were misclassified. Also, mRNAs were identified capable of classifying NGTX-C after 24h (5 genes) as well as after 48h (3 genes) of treatment (accuracy: 78% and 88%, sensitivity: 83% and 83%, specificity: 75% and 93%). Wy-14,643, phenobarbital and ampicillin trihydrate were misclassified. We conclude that genotoxicity and non-genotoxic carcinogenicity probably cannot be accurately predicted based on microRNA profiles. Overall, transcript-based prediction analyses appeared to clearly outperform microRNA-based analyses.
authors.affiliation: Department of Toxicogenomics, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229ER Maastricht, Netherlands and Netherlands Toxicogenomics Centre (NTC), Universiteitssingel 40, 6229ER Maastricht, Netherlands linda.rieswijk@maastrichtuniversity.nl.
authors.forename: Linda
authors.initials: L
authors.lastname: Rieswijk
authors.affiliation: Department of Toxicogenomics, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229ER Maastricht, Netherlands and.
authors.forename: Karen J J
authors.initials: KJ
authors.lastname: Brauers
authors.affiliation: Department of Toxicogenomics, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229ER Maastricht, Netherlands and Netherlands Toxicogenomics Centre (NTC), Universiteitssingel 40, 6229ER Maastricht, Netherlands.
authors.forename: Maarten L J
authors.initials: ML
authors.lastname: Coonen
authors.affiliation: Department of Toxicogenomics, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229ER Maastricht, Netherlands and Netherlands Toxicogenomics Centre (NTC), Universiteitssingel 40, 6229ER Maastricht, Netherlands.
authors.forename: Danyel G J
authors.initials: DG
authors.lastname: Jennen
authors.affiliation: Department of Toxicogenomics, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229ER Maastricht, Netherlands and.
authors.forename: Simone G J
authors.initials: SG
authors.lastname: van Breda
authors.affiliation: Department of Toxicogenomics, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229ER Maastricht, Netherlands and Netherlands Toxicogenomics Centre (NTC), Universiteitssingel 40, 6229ER Maastricht, Netherlands.
authors.forename: Jos C S
authors.initials: JC
authors.lastname: Kleinjans
mesh_terms: D000818:Animals; D002273:Carcinogens / Q000494:pharmacology / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000187:drug effects* / Q000235:genetics; D009152:Mutagenicity Tests / Q000379:methods; D011506:Proteins / Q000187:drug effects* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012680:Sensitivity and Specificity; D043922:Toxicogenetics / Q000379:methods*; D059467:Transcriptome
chemical_list: D002273:Carcinogens; D035683:MicroRNAs; D011506:Proteins; D012333:RNA, Messenger