Tooth bleaching has been used for a long time in dentistry and is currently one of the most requested dental treatments to obtain an aesthetic smile (Sossai et al., 2011). For tooth bleaching of vital teeth, hydrogen peroxide (H2O₂), carbamide peroxide (CH6N2O3) or hydrogen peroxide (H2O₂) combined with ozone are used in different concentrations (Rezende et al., 2014, Al-Omiri et al., 2018). However, it is common for patients undergoing bleaching of vital teeth to report sensitivity or discomfort during treatment, which can reach rates of up to 87% (Cerqueira et al., 2013). A systematic review comparing different bleaching products, use of desensitizing products, activation of bleaching by light, occurrence, duration, incidence, severity, and risk factors for bleaching sensitivity after tooth bleaching pointed to unclear mechanisms such as reversible pulpitis, dehydration/osmotic gradient, hydrodynamic alterations in dentin, activation of TRPA1 receptors and substance P (Kielbassa et al., 2015). This study was unable to consolidate the mechanisms involved in sensitivity after tooth bleaching and claimed for well-designed studies investigating these mechanisms.

When bleaching gels come into contact with tooth structures, they promote the release of reactive oxygen species, which cleave the chromophore molecules present in the dentin, transforming them into smaller molecules and therefore bleaching the teeth (Da Silva et al., 2020). This damage occurs because hydrogen peroxide (H₂O₂) releases reactive oxygen species (ROS), which react with organic and inorganic molecules. ROS can reach the dental pulp due to their low molecular weight, and the cytopathological effects are mutation, enzyme inactivation, protein degradation, and cell apoptosis or tissue necrosis (Carminatti et al. 2015).

ROS generate oxidative stress and are associated with the induction of inflammation in the body as an immune response through cell signaling and toxicity to pathogens. The exacerbated oxidative stress can induce to the dental tissues the reduction of pulp regeneration capacity, leading to necrosis, pain, reduction of proliferation and cellular metabolism (Cintra et al., 2013, Soares et al., 2015; Souza et al. 2010), and dentin sensitivity is common after the performance of tooth bleaching (Dietrich et al., 2021).

Nitric oxide (NO) is a free radical in the intracellular environment whose presence or absence regulates various important physiological responses, including vessel dilation, respiration, cell migration, immune response, and apoptosis (Muntané et al. 2010). Its action in immunoregulation is present in the inflammatory process as well as in the mechanisms of autoimmunity (Lima-Júnior et al., 2012). NO is produced by a family of enzymes nitric oxide synthase (NOS) from the conversion of L-arginine into L-citrulline in the presence of O₂ and the required enzymatic co-factors such as NADPH (Nicotinamide Adenine Dinucleotide Phosphate), FAD (Flavin Dinucleotide) and BH4 (4-hydrobiopterin) (Forstermann et al. 2012).

NO synthesis occurs from the activation of the enzyme NOsynthase (NOS), which is constituted of two isoforms: the constitutive isoform (cNOS) and the inducible isoform (iNOS) (Förstermann et al. 2012). This isoform can be expressed in a wide variety of cell types, including macrophages, lymphocytes, neutrophils, eosinophils, Kupffer cells, hepatocytes, and endothelial cells, and iNOS is capable of releasing large amounts of NO for prolonged periods, which can produce exaggerated effects, resulting in toxic responses to the body (Davis et al., 2013).

Given that aminoguanidine (AG) is a specific inhibitor of iNOS that also acts by competition with the existing L-arginine binding site on the heme group of the enzyme and the NG-nitro-L-arginine-methyl ester (L-NAME) and has been identified as a potent inhibitor of all isoforms (Bogdan, 2015), the present study aimed to evaluate the influence of iNOS on nociception and orofacial discomfort associated with tooth bleaching treatment with hydrogen peroxide (H₂O₂). The null hypothesis (H0) of this study is that iNOS do not play role of in orofacial nociception/discomfort after dental tooth bleaching with hydrogen peroxide (H₂O₂).