The role of the TP53 gene during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide E.M. Minicucci a , D.A. Ribeiro b,n , G.N. da Silva c , M.I.M.C. Pardini d , J.C. Montovani e , D.M.F. Salvadori c a Department of Dermatology and Radiotherapy, S ~ ao Paulo State University, UNESP, SP, Brazil b Department of Biosciences, Federal University of S ~ ao Paulo, UNIFESP, SP, Brazil c Department of Pathology, S ~ ao Paulo State University, UNESP, SP, Brazil d Department of Medical Clinics, S ~ ao Paulo State University, UNESP, SP, Brazil e Department of Ophthalmology and Otorhinolaryngology, S ~ ao Paulo State University, UNESP, SP, Brazil article info Article history: Received 19 May 2009 Accepted 24 March 2010 Keywords: Tp53 Rat tongue mucosa Oral squamous cell carcinoma 4-Nitroquinoline 1-oxide Direct DNA-sequencing abstract The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The aim of the present study was to investigate the expression of p53 by immunohistochemistry and examine the DNA sequence of exons 5, 6, 7, and 8 of Tp53 for mutations during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). A total of 30 male Wistar rats were treated with 4-nitroquinoline 1-oxide in their drinking water for 4, 12, and 20 weeks at a dose of 50 ppm. Ten animals were used as negative controls. No histopathological changes in the tongue epithelia were observed in the control group or in the treatment group after 4 weeks of 4NQO. Following 12 weeks of treatment, hyperplasia as well as epithelial dysplasia was found in both mild and moderate forms. At 20 weeks, moderate and/or severe oral dysplasia and squamous cell carcinoma of the tongue were found, and the majority of animals had squamous cell carcinoma. The levels of p53 protein were increased (p o0.05) in pre-neoplastic lesions and in squamous cell carcinomas in some of the tumor cells in squamous cell carcinomas. No mutations were found in any of the exons that were evaluated after the 4-, 12-, or 20-week treatments. Taken together, our results suggest that p53 expression may be an important event in the malignant conversion, whereas Tp53 mutations are not involved in the multi-step tongue carcinogenesis of Wistar rats induced by 4NQO. & 2010 Elsevier GmbH. All rights reserved. 1. Introduction Oral squamous cell carcinoma is a common neoplasm world- wide, and it is characterized by poor prognosis and a low survival rate (Pereira et al., 2007). Despite the abundant research data in cell and molecular biology and the advances in oncology and surgery, the mortality and morbidity rates in oral cancer patients remain unchanged (Xi and Grandis, 2003). Although histopathological hallmarks can provide useful information, such indirect surrogates invariably cannot be observed from the limited information provided by pathological sections. Consequently, biomarkers that can be used to characterize tumor behavior and predict outcome will have an important role in optimizing the prevention as well as the therapeutic plan in patients with oral neoplasms (Kashiwazaki et al., 2008). In this regard, a great deal of enthusiasm has been shown for the examination of the precise pathobiological mechan- isms that are involved in oral tumorigenesis in order to identify reliable biomarkers for squamous cell carcinomas of the tongue. The most frequently used animal models in this line of research are cancer in the hamster that is induced by fat-soluble 7,12 dimethylbenzanthracene (DMBA) and cancer in the rat that is induced by water-soluble 4-nitroquinoline 1-oxide (4NQO). Since one of the most important routes of oral carcinogenesis is through liquid containing water-soluble carcinogens, 4NQO is suitable for examining the role of xenobiotics in experimental oral carcinogen- esis (Tanaka et al., 2002). Based on the multi-step process of carcinogenesis, which is characterized by initiation, promotion, and tumor progression, chronic administration of 4NQO in drinking water stimulates rat tongue carcinogenesis in a manner similar to its human counterpart (Nishimura, 1999; Ribeiro et al., 2005; 2007; Kitakawa et al., 2006; Silva et al., 2007). The human Tp53 gene has been reported to be a tumor suppressor gene. It is located on human chromosome 17 and rodent chromosome 10 (Canzian et al., 1996). The Tp53 gene encodes a nuclear phosphoprotein, called the p53 protein (May and May, 1999). This protein consists of 393 amino acids and contains four domains: an amino-terminal transactivation domain, a core DNA-binding domain, a carboxy-terminal tetra- merization domain, and a regulatory domain. Each domain has different functions (Nylander et al., 2000; Bullock and Fersht, Contents lists available at ScienceDirect journal homepage: www.elsevier.de/etp Experimental and Toxicologic Pathology 0940-2993/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.03.009 n Correspondence to: Departamento de Biociˆ encias, Av. Ana Costa, 95, Vila Mathias, Santos–SP, Brazil, Zip code: 11060-001. Tel.: +55 13 32218058; fax: + 55 13 32232592. E-mail addresses: daribeiro@unifesp.br, daribeiro@pesquisador.cnpq.br (D.A. Ribeiro). Experimental and Toxicologic Pathology 63 (2011) 483–489