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In vitro effect of liver carcinogens aflatoxin B1 and methapyrilene on transcriptomic profiles in human liver HepaRG cells

ABSTRACT: Rapidly increasing number of man-made chemicals urges the development of reliable time- and cost-effective approaches for the carcinogen detection and identification. Considering this, the utility of high throughput microarray gene expression profiling for the identification of genotoxic and non-genotoxic carcinogens in vitro was investigated. Human terminally differentiated hepatic HepaRG cells were treated with model liver carcinogens, genotoxic carcinogen aflatoxin B1 (AFB1) and non-genotoxic carcinogen methapyrilene, at IC10 and IC25 concentrations for 72 hours, and transcriptomic profiles were determined. Treatment of HepaRG cells with IC10 and IC25 concentrations of AFB1 resulted in altered expression of 538 and 3033 genes (p-value ≤0.01 and fold change ≥2.0), respectively, and treatment of HepaRG cells with methapyrilene at the IC10 and IC25 concentrations altered the expression of 1255 and 1861 genes, respectively. Pathway analysis of transcriptomic signatures in HepaRG cells treated with minimally cytotoxic IC10 concentrations of AFB1 and methapyrilene demonstrated a strong enrichment in genes involved in key carcinogen-associated pathways, including receptor-mediated effects, detoxification response, cell death and apoptosis, cell proliferation and survival, oxidative stress and inflammation. Importantly, DNA damage and repair, cell cycle progression, and cell cycle checkpoint control pathways were uniquely activated in AFB1-treated HepaRG cells, whereas receptor-mediated signaling detoxification response pathway was predominantly altered in methapyrilene-treated HepaRG cells. In summary, high throughput microarray gene expression approach identifies specific carcinogen-exposure-associated transcriptomic responses and identifies affected molecular pathways, and categorize pathways associated with carcinogen exposure in a short-term in vitro test.

ORGANISM(S): Homo sapiens

PROVIDER: GSE87028 | GEO | 2016/09/17

SECONDARY ACCESSION(S): PRJNA343246

REPOSITORIES: GEO

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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.

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In vitro effect of liver carcinogens aflatoxin B1 and methapyrilene on transcriptomic profiles in human liver HepaRG cells

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