Chemicals inducing acute irritant contact dermatitis mobilize intracellular calcium in human keratinocytes Matthieu Raoux a,1,2 , Nathalie Azorin a,2 , Cécile Colomban b , Stéphanie Rivoire b , Thierry Merrot c , Patrick Delmas a , Marcel Crest a,⇑ a Université de la Méditerranée, Centre National de la Recherche Scientifique UMR 6231, Marseille, France b Eurofins/ATS, Actimart, Aix-en-Provence, France c Université de la Méditerranée, Assistance Publique Hôpitaux de Marseille (APHM), Marseille, France article info Article history: Received 4 July 2011 Accepted 2 August 2012 Available online 10 August 2012 Keywords: Chemical irritant In vitro irritation model Irritant contact dermatitis Skin Keratinocyte Calcium signaling abstract Intracellular Ca 2+ increase is a common feature of multiple cellular pathways associated with receptor and channel activation, mediator secretion and gene regulation. We investigated the possibility of using this Ca 2+ signal as a biomarker for a reaction to chemical irritants of normal human keratinocytes (NHK) in submerged primary cell culture. We tested 14 referenced chemical compounds classified as strong (seven), weak (four) or non- (three) irritants in acute irritant contact dermatitis. We found that the strong irritant compounds tested at 20–40 mM induced an intracellular Ca 2+ increase measurable by spectroflu- orimetry in an automated test. Weak and non-irritant compounds however did not increase intracellular Ca 2+ concentration. We further investigated the mechanisms by which the amine heptylamine, classified as a R34 corrosive compound, increases intracellular Ca 2+ . Heptylamine (20 mM) induced an ATP release that persisted in the absence of intra- and extra-cellular Ca 2+ . In addition, we found that this ATP activates NHK purinergic receptors that subsequently cause the increase in intracellular Ca 2+ from sarcoplasmic reticular stores. We conclude that measuring the intracellular Ca 2+ concentration in NHK is a suitable and easy way of determining any potential reaction to soluble chemical compounds. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The epidermis, being the topmost living layer of the skin, is con- stantly exposed to external injury from such as mechanical stimu- lation, chemical irritants and noxious temperatures. Contact dermatitis is one of the most frequent skin diseases. It comprises a major portion of occupational dermatoses in industrialized soci- eties, resulting in considerable social and economic implications. It can be divided into irritant contact dermatitis and allergic contact dermatitis depending on the production or not of specific antibod- ies. Irritant contact dermatitis is defined as a localized inflamma- tion of the skin caused by contact with toxic compounds such as metals, cleaning solutions, detergents, cosmetics, industrial chem- icals and latex rubber. Depending on the time course of the skin reaction, irritation may be classified as acute, delayed or chronic i.e. developing slowly after exposure (Chew and Maibach, 2003). Historically, the irritation index during acute irritant dermatitis due to a single exposure to a chemical has been determined in vivo using the Draize skin irritation test on rabbits (Draize et al., 1944). As a measure of acute irritancy potential, each chem- ical is given a score based on erythema and edema grade (Bagley et al., 1996). Despite the universal acceptance of this assay, the correlation between animal and human irritancy has come under question since, for some cases, chemicals have been misclassified using in vivo rabbit data (York et al., 1996). In view of this as well as eth- ical concerns, the 7th amendment to the European Cosmetics Directive stimulated the development of alternative tests for the assessment of the potential toxicological effects of substances. 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.08.010 Abbreviations: ATP, adenosine triphosphate; [Ca 2+ ] i , intracellular calcium concentration; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGTA, ethyleneglycol-bis(b-aminoethyl)- N,N,N 0 ,N 0 -tetraacetoxymethyl ester; F/F0, normalized ratio of the Fluo-4 AM fluorescence (F) to the basal fluorescence (F0); HBSS, Hanks’ Balanced Salt Solution; IL1a, interleukine 1a; IL8, interleukine 8; NHK, normal human keratinocytes; NRR, neutral red release; PGE2, prostaglandin 2; PLC, phospholipase C; TNFa, tumor necrosis factor a; DMIPA, dimethylisopropylamine; PPADS, pyridoxal-phosphate6- azophenyl-2 0 -4 0 -disulfonate; SLS, sodium lauryl sulfate; SEM, standard error of the mean. ⇑ Corresponding author. Address: Université de la Méditerranée, CNRS UMR 6231, Centre de Recherche en Neurobiologie-Neurophysiologie de Marseille, Faculté de Médecine, 51 Bd Pierre Dramard, CS80011, 13344 Marseille, Cedex 20, France. Tel.: +33 491 69 89 75; fax: +33 491 69 89 77. E-mail address: marcel.crest@univmed.fr (M. Crest). 1 Present address: Institut Européen de Chimie-Biologie, Centre National de la Recherche Scientifique UMR 5248, Université de Bordeaux, Bordeaux, France 2 These authors contributed equally to this work. Toxicology in Vitro 27 (2013) 402–408 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit