Project description:Compounds with direct immunotoxic properties, including metals, mycotoxins, agricultural pesticides and industrial chemicals, form potential human health risks due to exposure through food, drinking water, and the environment. Insights into the mechanisms of action are currently lacking for the majority of these direct immunotoxicants. Therefore, the present work aimed to gain insights into the molecular mechanisms underlying direct immunotoxicity. To this end, we assessed in vitro the effects of 31 test compounds on the transcriptome of the human Jurkat T cell line. These compounds included direct immunotoxicants, immunosuppressive drugs with different mode of actions, and non-immunotoxic control chemicals. Pathway analysis of the microarray data allowed us to identify canonical pathways and Gene Ontology processes that were transcriptionally regulated in common by immunotoxicants (i) with structural similarities, such as the tributyltins TBTC and TBTO that activated the retinoic acid / X receptor (RAR / RXR) signaling pathway, and (ii) without structural similarities, such as As2O3, DBTC, diazinon, MeHg, ochratoxin A, S9 treated ochratoxin A, S9 treated cyclophosphamide, and S9 treated benzo[a]pyrene, that activated unfolded protein response, and FTY720, lindane, and propanil, that activated the cholesterol biosynthesis pathway. In addition, processes uniquely affected by individual immunotoxicants were identified, such as the induction of Notch receptor signaling and the down regulation of acute phase response genes by ochratoxin A. These findings were validated by quantitative Real-Time PCR (Q-RT-PCR) analysis of genes involved in these processes. Our study indicated that diverse modes of action are involved in direct immunotoxicity and that a set of pathways or genes, rather than one single gene can be used to screen compounds for direct immunotoxicity. We used microarrays to study the gene expression changes in Jurkat cells upon exposure to various immunotoxicants and identified distinct pathways related with chemical-induced direct immunotoxicity. Jurkat cells were exposed to 31 compounds. The exposure experiments were done on four different days, with four different passages of Jurkat cells. Therefore, for each compound, we have four biological replicates. DMSO was used as the carrier control for all the samples. In total, 128 samples (including four untreated cell samples) were analyzed and will be uploaded. All the data were normalized by the average of the carrier control (DMSO).

Project description:Compounds with direct immunotoxic properties, including metals, mycotoxins, agricultural pesticides and industrial chemicals, form potential human health risks due to exposure through food, drinking water, and the environment. Insights into the mechanisms of action are currently lacking for the majority of these direct immunotoxicants. Therefore, the present work aimed to gain insights into the molecular mechanisms underlying direct immunotoxicity. To this end, we assessed in vitro the effects of 31 test compounds on the transcriptome of the human Jurkat T cell line. These compounds included direct immunotoxicants, immunosuppressive drugs with different mode of actions, and non-immunotoxic control chemicals. Pathway analysis of the microarray data allowed us to identify canonical pathways and Gene Ontology processes that were transcriptionally regulated in common by immunotoxicants (i) with structural similarities, such as the tributyltins TBTC and TBTO that activated the retinoic acid / X receptor (RAR / RXR) signaling pathway, and (ii) without structural similarities, such as As2O3, DBTC, diazinon, MeHg, ochratoxin A, S9 treated ochratoxin A, S9 treated cyclophosphamide, and S9 treated benzo[a]pyrene, that activated unfolded protein response, and FTY720, lindane, and propanil, that activated the cholesterol biosynthesis pathway. In addition, processes uniquely affected by individual immunotoxicants were identified, such as the induction of Notch receptor signaling and the down regulation of acute phase response genes by ochratoxin A. These findings were validated by quantitative Real-Time PCR (Q-RT-PCR) analysis of genes involved in these processes. Our study indicated that diverse modes of action are involved in direct immunotoxicity and that a set of pathways or genes, rather than one single gene can be used to screen compounds for direct immunotoxicity. We used microarrays to study the gene expression changes in Jurkat cells upon exposure to various immunotoxicants and identified distinct pathways related with chemical-induced direct immunotoxicity.

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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Transcriptomic profile indicative of immunotoxic exposure; in vitro studies in peripheral blood mononuclear cells

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