Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Effects of Combined Persistant Organic Pollutants on Global Gene Expression in Human HepaRG Cells

ABSTRACT: The exposure to and contamination by Persistent Organic Pollutants (POPs), which include pesticides used worldwide and polyaromatic hydrocarbons, is detrimental to human health and diverse ecosystems. Although most mechanistic studies have focused on single compounds, living organisms are exposed to multiple environmental xenobiotics, simultaneously, throughout their lives. The experimental evidence useful for assessing the effects of exposure to pollutant mixtures is scarce. We investigated the effects of exposure to a combination of two POPs, which employ different xenosensors, on global gene expression in a human hepatocyte cell model, HepaRG. Whole genome microarrays were used to investigate the effects on the HepaRG transcriptome following exposure to the combination of POPs as compared to each compound individually. Differentiated HepaRG cells were treated with either 2,3,7,8 tetrachlorodibenzo-p-dioxin, alpha-endosulfan (an organochlorine pesticide), the mixture or the DMSO vehicle for 30 hours after which RNA was extracted for hybridization on Affymetrix whole human genome microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Lawrence Aggerbeck

PROVIDER: E-GEOD-46874 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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Effects of Combined Persistant Organic Pollutants on Global Gene Expression in Human HepaRG Cells

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