Abstract
Current screening methods for direct immunotoxic chemicals are mainly based on general toxicity studies with rodents. The present study aimed to identify transcriptome-based functional classifiers that can eventually be exploited for the development of in vitro screening assays for direct immunotoxicity. To this end, a toxicogenomics approach was applied in which gene expression changes in human Jurkat lymphoblastic T cells were investigated in response to a wide range of compounds, including direct immunotoxicants, immunosuppressive drugs, and non-immunotoxic control chemicals. On the basis of DNA microarray data previously obtained by the exposure of Jurkat cells to 31 test compounds (Shao et al. in Toxicol Sci 135(2):328–346, 2013), we identified a set of 93 genes, of which 80 were significantly regulated (|numerical ratio| ≥1.62) by at least three compounds and the other 13 genes were significantly regulated by either one single compound or compound class. A total of 28 most differentially regulated genes were selected for qRT-PCR verification using a training set of 44 compounds consisting of the above-mentioned 31 compounds (23 immunotoxic and 8 non-immunotoxic) and 13 additional immunotoxicants. Good correlation between the results of microarray and qRT-PCR (Pearson’s correlation, R ≥ 0.69) was found for 27 out of the 28 genes. Redundancy analysis of these 27 potential classifiers led to a final set of 25 genes. To assess the performance of these genes, Jurkat cells were exposed to 20 additional compounds (external verification set) followed by qRT-PCR. The classifier set of 25 genes gave a good performance in the external verification: accuracy 85 %, true positive rate (sensitivity) 88 %, and true negative rate (specificity) 67 %. Furthermore, on the basis of the gene ontology annotation of the 25 classifier genes, the immunotoxicants examined in this study could be categorized into distinct functional subclasses. In conclusion, we have identified and validated classifier genes that can be used for the development of an in vitro assay for the identification and initial characterization of hazards for direct immunotoxicity of chemicals and drugs. This assay promises to complement animal-free toxicity testing approaches within the field of direct immunotoxicity.
Similar content being viewed by others
References
Archibald K, Coleman R, Foster C (2011) Open letter to UK Prime Minister David Cameron and Health Secretary Andrew Lansley on safety of medicines. The Lancet 377:1915
Baken KA, Pennings JLA, Jonker MJ, Schaap MM, de Vries A, van Steeg H, Breit TM, van Loveren H (2008) Overlapping gene expression profiles of model compounds provide opportunities for immunotoxicity screening. Toxicol Appl Pharmacol 226:46–59
Bouvier d’Yvoire M, Bremer S, Casati S, Ceridono M, Coecke S, Corvi R, Eskes C, Gribaldo L, Griesinger C, Knaut H, Linge JP, Roi A, Zuang V, Balls M, Combes RD, Bhogal N (2012) ECVAM and new technologies for toxicity testing. Springer, New York, pp 154–180
Carlson EA, Li Y, Zelikoff JT (2004) Benzo[a]pyrene-induced immunotoxicity in Japanese medaka (Oryzias latipes): relationship between lymphoid CYP1A activity and humoral immune suppression. Toxicol Appl Pharmacol 201:40–52
Chan M, Chan MW, Loh TW, Law HY, Yoon CS, Than SS, Chua JM, Wong CY, Yong WS, Yap YS, Ho GH, Ang P, Lee ASG (2011) Evaluation of nanofluidics technology for high-throughput SNP genotyping in a clinical setting. J Mol Diagn 13:305–312
Daaka Y, Friedman H, Klein TW (1996) Cannabinoid receptor proteins are increased in Jurkat, human T-cell line after mitogen activation. J Pharmacol Exp Ther 276:776–783
Davis DAP, Archuleta MM, Born JL, Knize MG, Felton JS, Burchiel SW (1994) Inhibition of humoral immunity and mitogen responsiveness of lymphoid cells following oral administration of the heterocyclic food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to B6C3F1 mice. Fundam Appl Toxicol 23:81–86
De Jong WH, Van Loveren H (2007) Screening of xenobiotics for direct immunotoxicity in an animal study. Methods 41:3–8
Delcuve G, Khan D, Davie J (2012) Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors. Clin Epigenet 4:5
Ekhart C, Rodenhuis S, Beijnen J, Huitema A (2009) Carbamazepine induces bioactivation of cyclophosphamide and thiotepa. Cancer Chemother Pharmacol 63:543–547
EMA (2000) Note for guidance on repeated dose toxicity, CPMP/SWP/1042/99
EPA (1998) Health effects test guidelines, Immunotoxicity, OPPTS 870. 7800
Ezendam J, Staedtler F, Pennings J, Vandebriel RJ, Pieters R, Boffetta P, Harleman JH, Vos JG (2004) Toxicogenomics of subchronic hexachlorobenzene exposure in Brown Norway rats. Environ Health Perspect 112:782–791
FDA (2002) Guidance for industry: immunotoxicology evaluation of investigational new drugs
Fernández-Riejos P, Goberna R, Sánchez-Margalet V (2008) Leptin promotes cell survival and activates Jurkat T lymphocytes by stimulation of mitogen-activated protein kinase. Clin Exp Immunol 151:505–518
Fischer AM, Mercer JC, Iyer A, Ragin MJ, August A (2004) Regulation of CXC chemokine receptor 4-mediated migration by the Tec family tyrosine kinase ITK. J Biol Chem 279:29816–29820
Hochstenbach K, van Leeuwen DM, Gmuender H, Stølevik SB, Nygaard UC, Løvik M, Granum B, Namork E, van Delft JHM, van Loveren H (2010) Transcriptomic profile indicative of immunotoxic exposure. In vitro studies in peripheral blood mononuclear cells. Toxicol Sci 118:19–30
Hollants S, Redeker EJW, Matthijs G (2012) Microfluidic amplification as a tool for massive parallel sequencing of the familial hypercholesterolemia genes. Clin Chem 58:717–724
Horne MC, Donaldson KL, Goolsby GL, Tran D, Mulheisen M, Hell JW, Wahl AF (1997) Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest. J Biol Chem 272:12650–12661
Institóris L, Siroki O, Dési I, Lesznyák J, Serényi P, Szekeres É, Petri I (1998) Extension of the protocol of OECD guideline 407 (28-day repeated dose oral toxicity test in the rat) to detect potential immunotoxicity of chemicals. Hum Exp Toxicol 17:206–211
Jang J, Simon V, Feddersen R, Rakhshan F, Schultz D, Zschunke M, Lingle W, Kolbert C, Jen J (2011) Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays. BMC Genomics 12:144
Jones-Mason Mary E, Zhao X, Kappes D, Lasorella A, Iavarone A, Zhuang Y (2012) E protein transcription factors are required for the development of CD4+ lineage T cells. Immunity 36:348–361
Katika MR, Hendriksen PJM, van Loveren H, Peijnenburg A (2011) Exposure of Jurkat cells to bis (tri-n-butyltin) oxide (TBTO) induces transcriptomics changes indicative for ER- and oxidative stress, T cell activation and apoptosis. Toxicol Appl Pharmacol 254:311–322
Katika MR, Hendriksen PJM, Shao J, van Loveren H, Peijnenburg A (2012) Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): new mechanistic insights. Toxicol Appl Pharmacol 264:51–64
Kawabata TT, Evans EW (2012) Development of immunotoxicity testing strategies for immunomodulatory drugs. Toxicol Pathol 40:288–293
Kim HJ, Kim JY, Park YY, Choi HS (2003) Synergistic activation of the human orphan nuclear receptor SHP gene promoter by basic helix–loop–helix protein E2A and orphan nuclear receptor SF-1. Nucleic Acids Res 31:6860–6872
Lankveld DPK, Loveren H, Baken KA, Vandebriel RJ, Dietert RR (2010) In vitro testing for direct immunotoxicity: state of the art immunotoxicity testing. In: Walker JM (ed) Immunotoxicity Testing. Humana Press, New York, pp 401–423
Luebke RW, Holsapple MP, Ladics GS, Luster MI, Selgrade M, Smialowicz RJ, Woolhiser MR, Germolec DR (2006) Immunotoxicogenomics: the potential of genomics technology in the immunotoxicity risk assessment process. Toxicol Sci 94:22–27
Makar RS, Lipsky PE, Cuthbert JA (1994) Non-sterol regulation of low density lipoprotein receptor gene expression in T cells. J Lipid Res 35:1888–1895
Manderville RA (2005) A case for the genotoxicity of ochratoxin A by bioactivation and covalent DNA adduction. Chem Res Toxicol 18:1091–1097
Morrison L (2010) Basic principles of fluorescence and energy transfer applied to Real-Time PCR. Mol Biotechnol 44:168–176
Nagai F, Hiyoshi Y, Sugimachi K, Tamura H-O (2002) Cytochrome P450 (CYP) expression in human myeloblastic and lymphoid cell lines. Biol Pharm Bull 25:383–385
Oh S-Y, Balch C, Cliff R, Sharma B, Boermans H, Swamy HVLN, Quinton VM, Karrow N (2013) Exposure to Penicillium mycotoxins alters gene expression of enzymes involved in the epigenetic regulation of bovine macrophages (BoMacs). Mycotoxin Res 29(4):235–243
Perel P, Robert I, Sena E, Wheble P, Briscoe C, Sandercock P, Macleod M, Mignini L, Jayaram P, Khan K (2007) Comparison of treatment effects between animal experiments and clinical trials: systematic review. BMJ 334:197–200
Ramsdell H, Parkinson A, Eddy A, Eaton D (1991) Bioactivation of aflatoxin B1 by human liver microsomes: role of cytochrome P450 IIIA enzymes. Toxicol Appl Pharmacol 108:436–447
Reif DM, Sypa M, Lock EF, Wright FA, Wilson A, Cathey T, Judson RR, Rusyn I (2013) ToxPi GUI: an interactive visualization tool for transparent integration of data from diverse sources of evidence. Bioinformatics 29:402–403
Shao J, Katika MR, Schmeits PCJ, Hendriksen PJM, van Loveren H, Peijnenburg AACM, Volger OL (2013) Toxicogenomics-based identification of mechanisms for direct immunotoxicity. Toxicol Sci 135(2):328–346
Tugwood JD, Hollins LE, Cockerill MJ (2003) Genomics and the search for novel biomarkers in toxicology. Biomarkers 8:79–92
Wang H-C, Perry SS, Sun X-H (2009) Id1 attenuates notch signaling and impairs T-cell commitment by elevating Deltex1 expression. Mol Cell Biol 29:4640–4652
Waters MD, Fostel JM (2004) Toxicogenomics and systems toxicology: aims and prospects. Nat Rev Genet 5:936–948
Zheng W, Wang H, Xue L, Zhang Z, Tong T (2004) Regulation of cellular senescence and p16INK4a expression by Id1 and E47 proteins in human diploid fibroblast. J Biol Chem 279:31524–31532
Zimmermann M, Arachchige-Don AS, Donaldson MS, Dallapiazza RF, Cowan CE, Horne MC (2012) Elevated cyclin G2 expression intersects with DNA damage checkpoint signaling and is required for a potent G2/M checkpoint arrest response to doxorubicin. J Biol Chem 287:22838–22853
Acknowledgments
This study was financially supported by the Netherlands Genomics Initiative, the Netherlands Toxicogenomics Centre (Grant 05060510).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Shao, J., Berger, L.F., Hendriksen, P.J.M. et al. Transcriptome-based functional classifiers for direct immunotoxicity. Arch Toxicol 88, 673–689 (2014). https://doi.org/10.1007/s00204-013-1179-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-013-1179-1