Oral carcinogenesis is a multifactorial and intricate process including the expanding incidence of genetic changes that promote the inhibitory or excitatory activities of tumor oncogenes and gene suppressors and impair the histophysiology of cell division, differentiation, and cell death [1], [2], [3]. Multiple genetic, molecular, and immunological modifications are caused in normal oral mucosa by the interaction of environmental factors such as the synergistic effects of alcohol and tobacco, and hereditary predispositions [4], [5], [6]. Thus, understanding the genetic, immunological, and molecular background of oral cancer may be of help for the development of preventive medications and new treatment modalities and may enhance patient prognosis.

The process of carcinogenesis can be investigated using cell lines derived from patients with carcinoma, transgenic animals, or animal models of chemical carcinogenesis [3]. Because the chemical components of tobacco and alcohol have been shown to be a potential cause of most mouth cancers in humans, they have been selected as the principal chemical agents used in animal models of carcinogenesis [7], [8]. Among the chemical carcinogens used, 4NQO is the preferred agent for the development of experimental oral carcinogenesis [8], [9], [10].

4NQO is a synthetic carcinogen that can be applied as topic application or systemic administration [9], [10]. This substance has been considered to be the best carcinogen currently available for producing oral carcinogenesis in rats [9]. It has been shown that 4NQO forms DNA adducts, leading to the replacement of adenosine by guanosine, thus inducing intracellular oxidative stress [9]. Histologically, changes in the oral epithelium ranging from hyperplasia to mild, moderate, and severe dysplasia can be observed before the formation of invasive carcinoma [9].

However, because 4NQO is a highly toxic substance that must be administered for a long time to have its carcinogenic effects on animals, its use in fundamental research can be difficult [10]. As a result, during the experimental procedures, adverse effects and animal losses may occur, and the ideal dosage must balance unwanted effects with efficient tumor development. In this sense, the aim of this study was to propose a new protocol using 4-NQO for oral carcinogenesis studies.