Advertisement

In Vitro Dendritic Cell-Based Test for Skin Sensitizers Identification and Potency Estimation

  • Bruno Miguel Neves
  • João Demétrio Martins
  • Isabel Ferreira
  • Ana Silva
  • Maria Teresa Cruz
Chapter

Abstract

Ethical and economic concerns and the recent European legislative framework triggered intensive research efforts towards the development and validation of alternative methods. We developed an in vitro predictive test to assess the skin sensitizing potential of chemicals. The test was based on the analysis and integration of intracellular signalling pathways evoked by chemicals concomitantly with gene expression modulation in skin-derived dendritic cells. In a first approach, cells were treated with four known sensitizers and two non-sensitizers, and the effects on the expression of 20 candidate genes and on the activation of several signalling pathways were analysed by real-time RT-PCR and Western blot, respectively. The genes Trxr1, Hmox1, Nqo1 and Cxcl10 and the signalling pathways p38 MAPK and JNK were identified as good predictor variables and used to construct a dichotomous classifier. For validation of the model, a large number of chemicals were afterwards analysed in a blinded assay. From the total 18 compounds tested, 17 were correctly classified, representing a concordance of 94%, with a sensitivity of 92% and a specificity of 100%. Additionally, we also analysed the feasibility to predict the sensitizer’s potency using in vitro-generated data and several in silico-calculated descriptors. A strong correlation with LLNA EC3 values was obtained (Pearson correlation coefficient r = 0.85, p < 0.001, n = 12). Overall, our results indicate that the analysis of proposed gene and signalling pathway signatures in a mouse skin-derived dendritic cell line represents a valuable model to be integrated in a future in vitro test platform.

Notes

Acknowledgements

This work is funded by FEDER funds through the Operational Programme Competitiveness Factors—COMPETE, “ToxVitroTesting”, Quadro de Referência Estratégico Nacional (QREN)—Empresas em co-promoção project ref.: 38977 and national funds by FCT—Foundation for Science and Technology under the project grant number PTDC/SAU-OSM/099762/2008 and strategic project UID/NEU/04539/2013. João Demétrio Martins had a FCT grant number SFRH/BD/73065/2010.

References

  1. 1.
    Basketter D, Alépée N, Casati S, et al. Skin sensitisation-moving forward with non-animal testing strategies for regulatory purposes in the EU. Regul Toxicol Pharmacol. 2013;67:531–5. doi: 10.1016/j.yrtph.2013.10.002.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bauch C, Kolle SN, Ramirez T, et al. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol. 2012;63:489–504. doi: 10.1016/j.yrtph.2012.05.013.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Rovida C. Integrated Testing Strategies (ITS) for safety assessment. ALTEX. 2014;32:25–40. doi: 10.14573/altex.1411011.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Reisinger K, Hoffmann S, Alépée N, et al. Systematic evaluation of non-animal test methods for skin sensitisation safety assessment. Toxicol In Vitro. 2015;29:259–70. doi: 10.1016/j.tiv.2014.10.018.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    OECD. OECD guideline for the testing of chemicals No. 168, part 1. In: The adverse outcome pathway for skin sensitization initiated by covalent binding proteins. Paris: Organisation for Economic Cooperation and Development; 2012. Accessible at: http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en.Google Scholar
  6. 6.
    OECD. OECD guideline for the testing of chemicals No. 168, part 2. In: The adverse outcome pathway for skin sensitisation initated by covalent binding to proteins. Paris: Organisation for Economic Cooperation and Development; 2012. Accessible at: http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2012)10/part2&doclanguage=en.Google Scholar
  7. 7.
    Roggen EL. In vitro approaches for detection of chemical sensitization. Basic Clin Pharmacol Toxicol. 2014;115:32–40. doi: 10.1111/bcpt.12202.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Neves BM, Gonçalo M, Figueiredo A, et al. Signal transduction profile of chemical sensitisers in dendritic cells: an endpoint to be included in a cell-based in vitro alternative approach to hazard identification? Toxicol Appl Pharmacol. 2011;250:87–95. doi: 10.1016/j.taap.2010.10.003.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Neves BM, Rosa SC, Martins JD, et al. Development of an in vitro dendritic cell-based test for skin sensitizer identification. Chem Res Toxicol. 2013;26:368–78. doi: 10.1021/tx300472d.CrossRefPubMedGoogle Scholar
  10. 10.
    Casati S, Aeby P, Kimber I, et al. Selection of chemicals for the development and evaluation of in vitro methods for skin sensitisation testing. Altern Lab Anim. 2009;37:305–12.PubMedGoogle Scholar
  11. 11.
    Gerberick GF, Vassallo JD, Bailey RE, et al. Development of a peptide reactivity assay for screening contact allergens. Toxicol Sci. 2004;81:332–43. doi: 10.1093/toxsci/kfh213.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Loveless SE, Ladies GS, Gerberick GF, et al. Further evaluation of the local lymph node assay in the final phase of an international collaborative trial. Toxicology. 1996;108:141–52. doi: 10.1016/0300-483X(95)03279-O.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Dos Santos GG, Spiekstra SW, Sampat-Sardjoepersad SC, et al. A potential in vitro epidermal equivalent assay to determine sensitizer potency. Toxicol In Vitro. 2011;25:347–57. doi: 10.1016/j.tiv.2010.10.008.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lambrechts N, Vanheel H, Nelissen I, et al. Assessment of chemical skin-sensitizing potency by an in vitro assay based on human dendritic cells. Toxicol Sci. 2010;116:122–9. doi: 10.1093/toxsci/kfq108.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Gamerdinger K, Moulon C, Karp DR, et al. A new type of metal recognition by human T cells: contact residues for peptide-independent bridging of T cell receptor and major histocompatibility complex by nickel. J Exp Med. 2003;197:1345–53. doi: 10.1084/jem.20030121.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lu L, Vollmer J, Moulon C, et al. Components of the ligand for a Ni++ reactive human T cell clone. J Exp Med. 2003;197:567–74. doi: 10.1084/jem.20021762.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Basketter DA, Alépée N, Ashikaga T, et al. Categorization of chemicals according to their relative human skin sensitizing potency. Dermatitis. 2014;25:11–21. doi: 10.1097/DER.0000000000000003.CrossRefPubMedGoogle Scholar
  18. 18.
    Girolomoni G, Lutz MB, Pastore S, et al. Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin. Eur J Immunol. 1995;25:2163–9. doi: 10.1002/eji.1830250807.CrossRefPubMedGoogle Scholar
  19. 19.
    Hulette BC, Ryan CA, Gildea LA, Gerberick GF. Relationship of CD86 surface marker expression and cytotoxicity on dendritic cells exposed to chemical allergen. Toxicol Appl Pharmacol. 2005;209:159–66. doi: 10.1016/j.taap.2005.03.019.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Neves BM, Cruz MT, Francisco V, et al. Differential roles of PI3-Kinase, MAPKs and NF-κB on the manipulation of dendritic cell Th1/Th2 cytokine/chemokine polarizing profile. Mol Immunol. 2009;46:2481–92. doi: 10.1016/j.molimm.2009.05.021.CrossRefPubMedGoogle Scholar
  22. 22.
    Wilschut A, ten Berge WF, Robinson PJ, McKone TE. Estimating skin permeation. The validation of five mathematical skin permeation models. Chemosphere. 1995;30:1275–96. doi: 10.1016/0045-6535(95)00023-2.CrossRefPubMedGoogle Scholar
  23. 23.
    Chipinda I, Ajibola RO, Morakinyo MK, et al. Rapid and simple kinetics screening assay for electrophilic dermal sensitizers using nitrobenzenethiol. Chem Res Toxicol. 2010;23:918–25. doi: 10.1021/tx100003w.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Parr RG, Szentpály L, Liu S. Electrophilicity index. J Am Chem Soc. 1999;121:1922–4. doi: 10.1021/ja983494x.CrossRefGoogle Scholar
  25. 25.
    Enoch SJ, Roberts DW, Cronin MTD. Mechanistic category formation for the prediction of respiratory sensitization. Chem Res Toxicol. 2010;23:1547–55. doi: 10.1021/tx100218h.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Bruno Miguel Neves
    • 1
    • 2
  • João Demétrio Martins
    • 1
    • 2
  • Isabel Ferreira
    • 1
    • 2
  • Ana Silva
    • 1
  • Maria Teresa Cruz
    • 1
    • 2
  1. 1.Centre for Neuroscience and Cell Biology, University of CoimbraCoimbraPortugal
  2. 2.Faculty of Pharmacy, Center for Neuroscience and Cell BiologyUniversity of CoimbraCoimbraPortugal

Personalised recommendations