1 2 Applicability of a keratinocyte gene signature to predict skin sensitizing potential 3 Jochem W. van der Veen a,b,⇑ Q1 , Tessa E. Pronk a,b , Henk van Loveren a,b , Janine Ezendam b 4 a Department of Toxicogenomics, Maastricht University, P.O. Box 616, NL-6200 MD Maastricht, The Netherlands 5 b Laboratory for Health Protection Research (GBO), National Institute for Public Health and the Environment (RIVM), P.O. Box 1, NL-3720BA Bilthoven, The Netherlands 6 7 9 article info 10 Article history: 11 Received 21 May 2012 12 Accepted 17 August 2012 13 Available online xxxx 14 Keywords: 15 Skin sensitization 16 Alternative methods 17 In vitro 18 Keratinocytes 19 Toxicogenomics 20 21 abstract 22 There is a need to replace animal tests for the identification of skin sensitizers and currently many alter- 23 native assays are being developed that have very promising results. In this study a gene signature capable 24 of very accurate identification of sensitizers was established in the HaCaT human keratinocyte cell line. 25 This signature was evaluated in a separate study using six chemicals that are either local lymph node 26 (LLNA) false-positive or false-negative chemicals in addition to nine sensitizers and four non-sensitizers. 27 Similar studies do not apply these more difficult to classify chemicals, which show the true potential for 28 human predictions of an assay. Although the gene signature has improved prediction accuracy compared 29 to the LLNA, the misclassified compounds were comparable between the two assays. Gene profiling also 30 showed a sensitizer specific response of the Nrf2-keap1 and Toll-like receptor signaling pathways. After 31 exposure to non-sensitizing chemicals that induce either of the pathways the signature misclassified all 32 Nrf2-inducers, while the Toll-like receptor ligands were correctly classified. In conclusion, we confirm 33 that keratinocyte based prediction assays may provide essential information on the properties of com- 34 pounds. Furthermore, chemical selection is critical for assessment of the performance of in vitro alterna- 35 tive assays. 36 Ó 2012 Elsevier Ltd. All rights reserved. 37 38 39 1. Introduction 40 Allergic contact dermatitis is a delayed-type IV hypersensitivity 41 reaction that can be induced after skin contact with chemical hap- 42 tens. It is a common occupational and consumer health problem 43 which develops through a series of immunological events caused 44 by repeated contact with compounds that have skin sensitizing po- 45 tential (Kimber et al., 2002). The current methods for assessing the 46 sensitizing potential of chemicals are the Local Lymph Node Assay 47 (LLNA) or guinea pig tests (Guinea Pig Maximization Test (GPMT) 48 or Buehler test) (Gerberick et al., 2007; Kimber et al., 1994). There 49 is great demand for validated non-animal alternatives to replace 50 these animal tests, due to the ban on animal testing described in 51 the 7th amendment to the European Union Cosmetics Directive. 52 In addition, the REACH (Registration, Evaluation, and Authorization 53 of Chemicals) regulation requires that the safety of a large amount 54 of chemicals has to be assessed and it stimulates the use of alter- 55 native test methods. These legislative changes, combined with eth- 56 ical issues and societal acceptance towards animal use in toxicity 57 testing, drive further development of alternative test methods. 58 Although much progress has been made for assessing skin sensitiz- 59 ing potential, no alternative test methods have been validated yet. 60 In recent years, it has become clear that a combination of methods 61 in a testing strategy will be required for correct identification of 62 sensitizers, rather than a single test (Vandebriel and van Loveren, 63 2010). 64 Many of current cell based alternative assays for skin sensitiza- 65 tion use either keratinocytes or dendritic cells. Read-outs are either 66 changes in gene regulation in these cells (Arkusz et al., 2010; 67 Johansson et al., 2011; Natsch, 2009; Vandebriel et al., 2010), pro- 68 duction of cytokines, such as IL-18 in keratinocytes (Corsini et al., 69 2009) or upregulation of cell surface markers, including CD86 70 and CD54 on dendritic cells (Aeby et al., 2004; Ashikaga et al., 71 2006; Sakaguchi et al., 2006; Schreiner et al., 2008) exposed to sen- 72 sitizers. The prediction accuracy of these assays range between 71% 73 and 99% (Bauch et al., 2011). 74 In the present study the focus is on the predictive power and 75 the driving pathways involved in the initial response of keratino- 76 cytes (KCs). KCs are abundantly present in the skin and play an 77 important role in the initial stages of skin sensitization as they 78 are the first cells to come into contact with chemicals. In addition, 79 KCs are able to secrete several pro-inflammatory mediators and 80 metabolize prohaptens into protein-reactive haptens (Jowsey 81 et al., 2006; Martin et al., 2011; Vandebriel and van Loveren, 82 2010). More recently, it has been proposed that KCs generate ‘dan- 83 ger’ signals in response to skin sensitizers that trigger the innate 84 immune system through TLR activation (Martin et al., 2011; 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.08.023 ⇑ Corresponding author at: Q2 Laboratory for Health Protection Research (GBO), National Institute for Public Health and the Environment (RIVM), P.O. Box 1, NL- 3720BA Bilthoven, The Netherlands. E-mail addresses: jochem.van.der.veen@rivm.nl (J.W. van der Veen), Tessa .Pronk@rivm.nl (T.E. Pronk), Henk.van.Loveren@rivm.nl (H. van Loveren), Janine .Ezendam@rivm.nl (J. Ezendam). Toxicology in Vitro xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit TIV 2934 No. of Pages 9, Model 5G 31 August 2012 Please cite this article in press as: van der Veen, J.W., et al. Applicability of a keratinocyte gene signature to predict skin sensitizing potential. Toxicol. in Vitro (2012), http://dx.doi.org/10.1016/j.tiv.2012.08.023