Use of IL-8 release and p38 MAPK activation in THP-1 cells to identify allergens and to assess their potency in vitro M. Mitjans a , V. Galbiati b , L. Lucchi b , B. Viviani b , M. Marinovich b , C.L. Galli b , E. Corsini b, * a Dpt. Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Spain b Laboratory of Toxicology, Department of Pharmacological Sciences, Università degli Studi di Milano, Italy article info Article history: Received 13 January 2010 Accepted 3 June 2010 Available online 9 June 2010 Keywords: Hpersensitivity Dndritic cells Cytokines In vitro toxicology Alternative method Allergen potency abstract The local lymph node assay (LLNA) has been developed to assess skin sensitization, and based on the EC3 value, it can also be used to evaluate allergen potency. Therefore, in the development of in vitro alterna- tives to the LLNA assay, one should not only consider the hazard identification but also the possibility to classify allergens relatively to their potency. We have recently described a selective release of interleukin-8 (IL-8) by chemical allergens in THP-1 cell line, and identified the activation of p38 mitogen-activated protein kinase (p38 MAPK) as a common pathway. Therefore, the purpose of this study was to expand the number of chemicals tested and to investigate whether IL-8 production and p38 MAPK activation can be used to classify allergens according to their potency. THP-1 cells were exposed to the contact allergens (p-benzoquinone, 2-aminophenol, isoeugenol, diethyl maleate, citral and imidazolidinyl urea), selected according to their potency in the LLNA, and to lactic acid and propylene glycol as non-sensitizers. p38 MAPK activation was evaluated 5–15 min after treatment by FACS analysis, while IL-8 release was assed by ELISA following 24 h of incubation. p38 MAPK was activated by all contact allergens, including the pro-apten isoeugenol, whereas IL-8 release was significantly increased after stimulation with all allergens tested, except for isoeugenol. The failure of isoeugenol may be due to decrease in the stability of IL-8 mRNA. Irritants exposure, as expected, failed to induce both p38 MAPK activation and IL-8 release. A significant correlation between IL-8 release and the LLNA EC 3 was found (Pearson correlation r = 0.743, p = 0.0036, n = 12). On the contrary, the activation of p38 MAPK showed no significant correla- tion between LLNA data and vigor of p38 MAPK activation. Overall, data presented confirm our previous observations and reveal IL-8 as potential tool not only to identify sensitizers, with the exception of pro-haptens, but also to classify them according to their potency, while p38 MAPK activation allows the identification of all sensitizers, including pro-haptens, but was not useful for potency classification. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction The possibility that chemicals used in the workplace and in con- sumer products might cause skin sensitization is major concern. Chemical allergens are small molecules able to form a sensitizing complex once they bound to proteins (Corsini and Kimber, 2007). One of the most frequent manifestations of chemical allergy is con- tact hypersensitivity, which can have serious impact on the quality of life and represents a common occupational health. For this rea- son, evaluation of the potential of a compound to cause allergic contact dermatitis has to be integral part of the development of consumer products and their ingredients. To date the identification and evaluation of unknown sensitiz- ers relies on animal testing, mainly by the local lymph node assay (LLNA) (Kimber et al., 1986), as no validated alternative exists. The additional testing of chemicals for their allergenic potential required by the new EU-legislation on chemicals (REACH, EU, 2006) is expected to significantly increase the use of animals. It has been estimated that skin sensitization testing is among those effects that would require large numbers of animals. Conversely, the 7th amendment to the cosmetics directive (Directive 76/768/ EEC) completely bans all animal testing for cosmetic ingredients for all human-health related effects by 2009 for all endpoints except the repeated-dose toxicity endpoints for which the market- ing ban deadline is 2013. Therefore, in the screening of drugs, cosmetics and other chemicals for human use, it is very important, 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.06.001 * Corresponding author. Address: Laboratory of Toxicology, Department of Pharmacological Sciences, Università degli Studi di Milano, Faculty of Pharmacy, Via Balzaretti 9, 20133 Milano, Italy. Tel.: +39 02 50318368; fax: +39 02 50318284. E-mail address: emanuela.corsini@unimi.it (E. Corsini). Toxicology in Vitro 24 (2010) 1803–1809 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit