382 Introduction Cigarette smoke is one of the ive leading risk factors for mortality in the world (World Health Organization, 2009) and smoking related lung cancer is the main cause of deaths from cancer in both sexes in the United States (Jemal et al., 2009). his habit is particularly popular in Western low- and middle-income countries, with about 1 billion males and 250 million females smoking in the world (Domagala-Kulawik, 2008). Mainstream smoke represents a well-known cause of airway (for review see Brody & Steiling, 2011) and oral tissue damage (Proia et al., 2006; Steiling et al., 2008; Warnakulasuriya et al., 2010). Among the irst target organs of smoke inhalation histopathological changes of the nasal mucosa were recently reported (Calsina et al., 2002; Hamm et al., 2007; Moimaz et al., 2009; Wan et al., 2009), while scanty evidence is available about the early efects on the cigarette combustion chamber, i.e. the human oral cavity. Extensive studies focused on the role of smoke in head and neck cancer (Mirbod & Ahing, 2000; Gandini et al., 2008; Curado & Hashibe, 2009; Zygogianni et al., 2011) showing a direct link between cigarette smoke chemical composition and molecular changes such as DNA adducts formation (Schwartz et al., RESEARCH ARTICLE Acute efects of cigarette smoke on three-dimensional cultures of normal human oral mucosa Alice Gualerzi 1 , Michele Sciarabba 2 , Gianluca Tartaglia 1 , Chiarella Sforza 1 , and Elena Donetti 1 1 Dipartimento di Morfologia Umana e Scienze Biomediche – Città Studi and 2 Dipartimento di Informatica e Comunicazione, Università degli Studi di Milano, Milan, Italy Abstract Context: Human oral mucosa is the combustion chamber of cigarette, but scanty evidence is available about the early smoke effects. Objective: The present work aimed at evaluating from a morphological point of view whole smoke early effects on epithelial intercellular adhesion and keratinocyte terminal differentiation in a three-dimensional model of human oral mucosa. Materials and methods: Biopsies of keratinized oral mucosa of healthy nonsmoking women (n = 5) were collected. After culturing in a Transwell system, one fragment of each biopsy was exposed to the smoke of one single cigarette; the remnant represented the internal control. The distribution of epithelial differentiation markers (keratin-10, K10, and keratin-14, K14, for suprabasal and basal cells respectively), desmosomes (desmoglein-1, desmoglein-3), tight junctions (occludin), adherens junctions (E-cadherin, β-catenin), and apoptotic cells (p53, caspase 3) were evaluated by immunofluorescence. Results: Quantitative analysis of K14 immunolabeling revealed an overexpression in the suprabasal layers as early as 3 h after smoke exposure, without impairment of the epithelial junctional apparatus and apoptosis induction. Discussion and conclusion: These results suggested that the first significant response to cigarette smoke came from the basal and suprabasal layers of the human oral epithelium. The considered model maintained the three-dimensional arrangement of the human mucosa in the oral cavity and mimicked the inhalation/exhalation cycle during the exposure to cigarette smoke, offering a good possibility to extrapolate the reported observations to humans. Keywords: Keratins, organotypic cultures, desmosomes, adherens junctions, tight junctions Address for Correspondence: Prof. Elena Donetti, Dipartimento di Morfologia Umana e Scienze Biomediche – Città Studi, Università degli Studi di Milano, via Mangiagalli, 31–20133 Milan, Italy. Tel: +390250315400. Fax: +390250315387. E-mail: elena.donetti@unimi.it (Received 16 February 2012; revised 15 March 2012; accepted 21 March 2012) Inhalation Toxicology, 2012; 24(6): 382–389 © 2012 Informa Healthcare USA, Inc. ISSN 0895-8378 print/ISSN 1091-7691 online DOI: 10.3109/08958378.2012.679367 Inhalation Toxicology Downloaded from informahealthcare.com by Biblioteca Alberto Malliani on 05/08/12 For personal use only.