Analyses of transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models
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ABSTRACT:
ORGANISM(S): Rattus norvegicus Homo sapiens
PROVIDER: GSE40117 | GEO | 2013/02/20
SECONDARY ACCESSION(S): PRJNA173075
REPOSITORIES: GEO
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Project description:For assessing the cancer-causing potential for humans of a chemical compound, the conventional approach is the use of the 2-year rodent carcinogenicity bioassay, thus alternatives such as in vitro toxicogenomics are highly desired. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically-stabilized cultures of primary rat hepatocytes, the human hepatoma-derived HepaRG and HepG2 cell lines and the human embryonic stem cell-derived hepatocyte-like cells hES-Heps are examined and compared. The global gene expression profiles of five commonly used liver-based in vitro systems are investigated, namely conventional cultures of primary rat hepatocytes (HepsC), Trichostatin A (TSA)-stabilized cultures of primary rat hepatocytes (HepsT), human embryonic stem cell-derived hepatocyte-like cells (hES-Hep), HepG2 and HepaRG cells. These models are exposed to 15 prototypical compounds which have been carefully chosen and belong to 3 toxic classes i.e. (i) genotoxic (GTX) (aflatoxin B1, AFB1; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK; 2-nitrofluorene, 2NF; benzo(a)pyrene, BaP; cyclophosphamide, CYCLO), (ii) non-genotoxic (NGTX) (methapyrilene hydrochloride, MPH; piperonylbutoxide , PIPB; Wy-14643, WYE; phenobarbital sodium, SPB; 12-O-tetradecanoylphorbol-13-acetate, TPA) carcinogens and (iii) non-carcinogens (NC) (nifedipine, NIF; clonidine, CND; D-mannitol, MAN; tolbutamide, TOL; diclofenac sodium, SDF). For the gene- and pathway- based analysis, the raw microarray data was re-annotated to Ensembl version 61 genome and Gene Chip Robust Multi-array Average (GC-RMA) normalized (Dai et al. 2005). This resulted in 11,187 and 18,919 probe sets for the rat and the human models, respectively. For the classification analysis, the raw microarray data was annotated using the chip description files from Affymetrix and then GC-RMA normalized. In order to eliminate batch effects, data from the different studies were half-z normalized. Half-z-normalization adjusts the logarithmic expression values of transcripts within a group such that each transcript has zero mean.
2013-02-20 | E-GEOD-40117 | biostudies-arrayexpress
Project description:The prediction of possible carcinogenicity of chemicals for humans represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk at only limited reliability while simultaneously being expensive and long-lasting. In order to seek for alternatives, in the present study, the ability of a transcriptomics-based primary mouse hepatocyte model to classify carcinogens by their modes of action was evaluated. As it is obvious that exposure will induce a cascade of gene expression modifications, in particular, the influence of exposure time in vitro on discriminating genotoxic (GTX) carcinogens from non-genotoxic (NGTX) carcinogens class prediction was investigated. Primary mouse hepatocytes from male C57Bl6 mice were treated for 12, 24, 36 and 48 h with two GTX and two NGTX carcinogens. For the purpose of validation, two additional GTX compounds were studied after incubation periods of 24 and 48 h. Immunostaining of ?H2AX foci was applied to phenotypically verify DNA damage. Whole genome gene expression modifications were analyzed by means of Affymetrix mouse genome 430 2.0 microarrays. Datasets were normalized using RMA and differentially expressed genes were selected for assessing class prediction. The ?H2AX assay confirmed significant induction of DNA damage after treatment with GTX compounds, whereas NGTX compounds showed no activity beyond background levels. Discrimination of GTX and NGTX carcinogens by Prediction Analysis of Microarray (PAM) was validated and resulted in a perfect classification. The present study shows that gene expression profiling in primary mouse hepatocytes is promising for discriminating between GTX and NGTX compounds and that this classification improves with increasing treatment period.
2010-06-09 | E-MEXP-2209 | biostudies-arrayexpress
Project description:Several studies have successfully detected hepatocarcinogenicity in rats based on gene expression data. Here, we constructed a model for detecting non-genotoxic (NGTX) hepatocarcinogens and predicted their MOAs in rats. Piperonyl butoxide (PBO) and Dammar resin (DAM) are NGTX hepatocarcinogens. Gene expression data in the liver treated by PBO and DAM was used for validation of constructed model.
2015-07-29 | GSE71413 | GEO
Project 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.
2014-09-01 | E-GEOD-48990 | biostudies-arrayexpress
Project description:At present, substantial efforts are focused on the development of in vitro assays coupled with M-bM-^@M-^]omicsM-bM-^@M-^] technologies for the identification of carcinogenic substances as an alternative to the classical 2-year rodent carcinogenicity bioassay. A prerequisite for the eventual regulatory acceptance of such assays, however, is the in vivo relevance of the observed in vitro findings. In the current study, hepatocarcinogen-induced gene expression profiles generated after exposure of conventional cultures of primary rat hepatocytes to three non-genotoxic carcinogens (methapyrilene hydrochloride, piperonyl butoxide and Wy-14643), three genotoxic carcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene), and two non-carcinogens (nifedipine and clonidine) are compared with previously obtained in vivo data after oral administration for up to 14 days of the same hepatocarcinogens to rats.
2012-04-23 | E-GEOD-36524 | biostudies-arrayexpress
Project description:Direct comparison of the hepatoma cell lines HepG2 and HepaRG using whole genome gene expression analysis before and after exposure to the GTX carcinogens AFB1 and BaP and the NGTX carcinogens CsA, E2 and TCDD for 12 and 48 h.
2010-04-15 | E-MEXP-2458 | biostudies-arrayexpress
Project description: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.
2016-06-28 | GSE72081 | GEO
Project description: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.
2016-06-28 | GSE72014 | GEO
Project description:Liver tumors in rodents are frequently induced by non-genotoxic carcinogens. These hepatocarcinogens generally activate hepatic nuclear receptors (e.g., CAR and PXR), resulting in a cascade of signals causing modifications in the expression of genes responsible for several processes involved in carcinogenesis. Evaluation of the carcinogenic potential of chemicals is a regulatory requirement but is time-consuming and expensive. Consequently, several short-term in vivo and in vitro approaches, using molecular tools, have been proposed as predictive models for non-genotoxic hepatocarcinogens. The objective of our study was to discriminate between chemicals that are either non-genotoxic hepatocarcinogens or merely hepatotoxicants and also between CAR and PXR modulators on the basis of their gene expression profiles. Thus, we treated rats for seven days with the hepatoxicants, diclofenac and diazepam, or with several CAR and PXR modulators, which were mainly hepatocarcinogens. Different hepatic gene expression profiles were obtained not only between the hepatotoxicants and the non-genotoxic hepatocarcinogens but also between the CAR activators phenobarbital, phenytoin and 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene which were grouped together, and the two PXR activators pregnenolone 16α-carbonitrile and clotrimazole. Diethylstilbestrol had an expression profile that was quite distinct from the other PXR activators, suggesting that this compound is certainly not a classic PXR modulator. Moreover, some differences were observed between phenytoin (not considered as a hepatocarcinogen), and the other two CAR activators. Our data therefore indicate that discrimination is possible between hepatocarcinogens and hepatotoxicants, between CAR and PXR modulators and also between compounds within the same class of modulators using a short-term transcriptomic approach.
2013-07-31 | GSE39350 | GEO
Project description:There is no accurate and well-validated short-term test for non-genotoxic carcinogens, necessitating an expensive two year rodent bioassay before a risk assessment can begin. We have developed a short-term in vivo rat assay that predicts whether non-genotoxic chemicals are likely to induce hepatic tumors based on transcript profiles in the liver. Using a large independent test set, assay accuracy was found to be superior to existing pathological and genomic markers. Comparison of the test chemical's signature profile to reference carcinogens of known mechanism can also identify a potential mode(s) of action, allowing an early assessment of human cancer risk. Guidelines for commercial use: http://www.iconixbiosciences.com/guidelineCommUse.pdf Keywords: dose response, time course, compound treatment Treatment of male Sprague-Dawley rats with 147 non-genotoxic compounds at various doses and durations, in biological triplicate, along with vehicle-matched control animals. Liver samples were assayed for gene expression. Hepatocarcinogenic and non-carcinogenic compounds were included. A classifier for carcinogenicity was built on a training set of 25 carcinogens and 75 noncarcinogens (randomly selected compounds, maximum tolerated dose, 5 day timepoints), and tested on the remaining 47 compounds at various timepoints. A total of 990 samples were hybridized to single-channel CodeLink RU1 arrays. Biological triplicates were combined with matched control samples to calculate log ratios.
2007-06-22 | E-GEOD-8251 | biostudies-arrayexpress