LuSens: Shedding Light on Skin Sensitization

  • Tzutzuy RamirezEmail author
  • Annette Mehling
  • Robert Landsiedel


Skin sensitizers or their metabolites are predominantly electrophilic molecules and have the capacity to induce the Nrf2 signalling pathway. Therefore, they trigger the expression of genes under the control of the antioxidant response element (ARE), most of which are detoxifying enzymes and other protective proteins. In the meantime extensive evidence is available to demonstrate that the activation of the Nrf2 pathway provides valuable information for identification of skin sensitizers. The activation of keratinocytes is the second key event of skin sensitization adverse outcome pathway described by the OECD, and the LuSens assay is used to generate information to cover this key event. The LuSens assay is based on a human keratinocyte reporter cell line harbouring the ARE of the NADPH:quinone oxidoreductase 1 (nqo1) gene from the rat and the reporter gene of luciferase. In the presence of skin sensitizers, luciferase is overexpressed indicating direct activation of the nqo1-ARE. The LuSens assay has demonstrated good proficiency to detect skin sensitizers, good reproducibility, as well as its interchangeability with the KeratinoSens™ assay. Moreover, studies have also demonstrated its use as part of a testing battery, resulting in accuracy similar to that obtained with the in vivo gold standard, the local lymph node assay. In this context, the LuSens assay is a major contribution to the replacement of in vivo experiments for skin sensitization.


  1. 1.
    Bruckner AL, Weston WL, Morelli JG. Does sensitization to contact allergens begin in infancy? Pediatrics. 2000;105(1):e3.CrossRefPubMedGoogle Scholar
  2. 2.
    Thyssen JP, Linneberg A, Menné T, Johansen JD. The epidemiology of contact allergy in the general population–prevalence and main findings. Contact Dermatitis. 2007;57(5):287–99.CrossRefPubMedGoogle Scholar
  3. 3.
    Lunder T, Kansky A. Increase in contact allergy to fragrances: patch-test results 1989–1998. Contact Dermatitis. 2000;43(2):107–9.CrossRefPubMedGoogle Scholar
  4. 4.
    Nguyen SH, Dang TP, MacPherson C, Maibach H, Maibach HI. Prevalence of patch test results from 1970 to 2002 in a multi-centre population in North America (NACDG). Contact Dermatitis. 2008;58(2):101–6.CrossRefPubMedGoogle Scholar
  5. 5.
    Peiser M, Tralau T, Heidler J, Api AM, Arts JH, Basketter DA, English J, Diepgen TL, Fuhlbrigge RC, Gaspari AA, Johansen JD, Karlberg AT, Kimber I, Lepoittevin J-P, Liebsch M, Maibach HI, Martin SF, Merk HF, Platzek T, Rustemeyer T, Schnuch A, Vandebriel RJ, White IR, Luch A. Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany. Cell Mol Life Sci. 2012;69(5):763–81.CrossRefPubMedGoogle Scholar
  6. 6.
    OECD. OECD TG 429. Skin sensitization, Local Lymph Node Assay. 2010.Google Scholar
  7. 7.
    OECD. OECD test guidelines (TG) 442A. Skin Sensitization: Local Lymph Node Assay: DA. 2010.Google Scholar
  8. 8.
    OECD. OECD test guidelines (TG) 442B. Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA. 2010.Google Scholar
  9. 9.
    OECD. OECD TG 406. Skin Sensitization. 1992.Google Scholar
  10. 10.
    EC. Reach; Regulation No 1907/2006 of the European parliament and of the council of 18 December 2006 concerning the Registration, Evaluation, authorisation and restriction of chemicals (REACH), establishing a European chemicals agency, amending directive 1999/45/EC and repealing Council regulation (EEC) no 793/93 and commission regulation (EC) no 1488/94 as well as Council directive 76/769/EEC and commission directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. Off J Eur Union Legis. 2006; 396:1–849.Google Scholar
  11. 11.
    ECHA-14-A-07-EN. The use of alternatives to testing on animals for the REACH regulation second report under article 117(3) of the REACH regulation. 2014. ED-04-14-483-EN-N - ISBN: 978–92–9244-593-5. DOI:  10.2823/22471. 2 June 2014.
  12. 12.
    EU Regulation 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products. Official Journal of the European Union L 342/59-209. 22.12.2009.Google Scholar
  13. 13.
    Ladics GS, Fry J, Goodman R, Herouet-Guicheney C, Hoffmann-Sommergruber K, Madsen CB, Penninks A, Pomés A, Roggen EL, Smit J, Wal JM. Allergic sensitization: screening methods. Clin Transl Allergy. 2014;4(1):13.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Mehling A, Eriksson T, Eltze T, Kolle S, Ramirez T, Teubner W, van Ravenzwaay B, Landsiedel R. Non-animal test methods for predicting skin sensitization potentials. Arch Toxicol. 2012;86:1273–95.CrossRefPubMedGoogle Scholar
  15. 15.
    OECD. ENV/JM/MONO(2012)10/PART1. The adverse outcome pathway for skin Sensitisation Initiated by Covalent binding to proteins PART 1: scientific evidence. Series on testing and assessment no.168. 2012.Google Scholar
  16. 16.
    OECD. ENV/JM/MONO(2012)10/PART2. The adverse outcome pathway for skin Sensitisation Initiated by Covalent binding to proteins part 2: use of the AOP to develop chemical categories and integrated assessment and testing approaches. Series on testing and assessment no.168. 2012.Google Scholar
  17. 17.
    Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin J-P, Natsch A, Ovigne JM, Rovida C, Sakaguchi H, Schultz T. Chemical reactivity measurement and the predicitve identification of skin sensitisers. The report and recommendations of ECVAM workshop 64. Altern Lab Anim. 2008;36(2):215–42.PubMedGoogle Scholar
  18. 18.
    Gallucci S, Matzinger P. Danger signals: SOS to the immune system. Curr Opin Immunol. 2001;13(1):114–9.CrossRefPubMedGoogle Scholar
  19. 19.
    McFadden JP, Basketter DA. Contact allergy, irritancy and danger. Contact Dermatitis. 2000;42(3):123–7.CrossRefPubMedGoogle Scholar
  20. 20.
    Natsch A, Emter R. Skin sensitizers induce antioxidant response element dependent genes: application to the in vitro testing of the sensitization potential of chemicals. Toxicol Sci. 2008;102(1):110–9.CrossRefPubMedGoogle Scholar
  21. 21.
    Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991;9:271–96. ReviewCrossRefPubMedGoogle Scholar
  22. 22.
    Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature. 2007;449(7161):419–26.CrossRefPubMedGoogle Scholar
  23. 23.
    Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392(6673):245–52.CrossRefPubMedGoogle Scholar
  24. 24.
    Ade N, Leon F, Pallardy M, Peiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I, Ourlin JC. HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol Sci. 2009;107(2):451–60.CrossRefPubMedGoogle Scholar
  25. 25.
    Natsch A. The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers--functional relevance and a hypothesis on innate reactions to skin sensitizers. Toxicol Sci. 2010;113(2):284–92.CrossRefPubMedGoogle Scholar
  26. 26.
    Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, Igarashi K, Yamamoto M. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol. 2004;24(16):7130–9.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Emter R, Ellis G, Natsch A. Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro. Toxicol Appl Pharmacol. 2010;245(3):281–90.CrossRefPubMedGoogle Scholar
  28. 28.
    Motohashi H, Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med. 2004;10(11):549–57.CrossRefGoogle Scholar
  29. 29.
    Emter R, Natsch A. Skin sensitizers induce antioxidant response element dependent genes: application to the in vitro testing of the sensitization potential of chemicals. Toxicol Sci. 2008;102(1):110–9.CrossRefPubMedGoogle Scholar
  30. 30.
    Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D, van Ravenzwaay B, Landsiedel R. LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol In Vitro. 2014;28(8):1482–97.CrossRefPubMedGoogle Scholar
  31. 31.
    Migdal C, Botton J, El Ali Z, Azoury ME, Guldemann J, Giménez-Arnau E, Lepoittevin J-P, Kerdine-Römer S, Pallardy M. Reactivity of chemical sensitizers toward amino acids in cellulo plays a role in the activation of the Nrf2-ARE pathway in human monocyte dendritic cells and the THP-1 cell line. Toxicol Sci. 2013;133(2):259–74.CrossRefPubMedGoogle Scholar
  32. 32.
    Mehling A, Beissert S. Dendritic cells under investigation in autoimmune disease. Crit Rev Biochem Mol Biol. 2003;38(1):1–21.CrossRefPubMedGoogle Scholar
  33. 33.
    OECD. OECD TG 442D: in vitro skin Sensitisation: ARE-Nrf2 luciferase test method. 2015.Google Scholar
  34. 34.
    Vandebriel RJ, Pennings JL, Baken KA, Pronk TE, Boorsma A, Gottschalk R, Van Loveren H. Keratinocyte gene expression profiles discriminate sensitizing and irritating compounds. Toxicol Sci. 2010;117(1):81–9.CrossRefPubMedGoogle Scholar
  35. 35.
    Wasserman WW, Fahl WE. Comprehensive analysis of proteins which interact with the antioxidant responsive element: correlation of ARE-BP-1 with the chemoprotective induction response. Arch Biochem Biophys. 1997;344(2):387–96.CrossRefPubMedGoogle Scholar
  36. 36.
    Cooper JA, Saracci R, Cole P. Describing the validity of carcinogen screening-tests. Br J Cancer. 1979;39(1):87–9.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Urbisch D, Mehling A, Guth K, Ramirez T, Honarvar N, Kolle S, Landsiedel R, Jaworska J, Kern PS, Gerberick F, Natsch A, Emter R, Ashikaga T, Miyazawa M, Sakaguchi H. Assessing skin sensitization hazard in mice and men using non-animal test methods. Regul Toxicol Pharmacol. 2014; pii: S0273-2300(14)00309–2.Google Scholar
  38. 38.
    Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortaner S, Gribaldo L, Halder M, Hoffmann S, Roi AJ, Prieto P, Sabbioni E, Scott L, Worth A, Zuang V. A modular approach to the ECVAM principles on test validity. Altern Lab Anim. 2004;32:467–72. Scholar
  39. 39.
    OECD. Guidance document on the validation and international acceptance of new or updated test methods for hazard assessment, Series on testing and assessment, no. 34. Paris: OECD; 2005.Google Scholar
  40. 40.
    OECD. OECD TG 442C: in Chemico skin Sensitisation: direct peptide reactivity assay (DPRA). 2015.Google Scholar
  41. 41.
    Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol. 2012;63(3):489–504.CrossRefPubMedGoogle Scholar
  42. 42.
    Natsch A, Ryan CA, Foertsch L, Emter R, Jaworska J, Gerberick F, Kern P. A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation. J Appl Toxicol. 2013;33(11):1337–52.PubMedPubMedCentralGoogle Scholar
  43. 43.
    ESAC (2016). ESAC opinion on the BASF-coordinated Performance Standards-based validation of the LuSens test method for skin sensitisation testing. Available at: []
  44. 44.
    OECD. OECD TG 442D: in Chemico skin Sensitisation: ARE-Nrf2 Luciferase Test Method. 2015Google Scholar
  45. 45.
    Ramirez T, Stein N, Aumann A, Remus T, Edwards A, Norman KG, Ryan C, Bader JE, Fehr M, Burleson F, Foertsch L, Wang X, Gerberick F, Beilstein P, Hoffmann S, Mehling A, van Ravenzwaay B, Landsiedel R. Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers. Toxicol in Vitro. 2016;32:278–286CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Tzutzuy Ramirez
    • 1
    Email author
  • Annette Mehling
    • 2
  • Robert Landsiedel
    • 1
  1. 1.BASF SE, Experimental Toxicology and EcologyLudwigshafenGermany
  2. 2.BASF Personal Care and Nutrition GmbHDüsseldorfGermany

Personalised recommendations