Immunohistochemical expression of H3K9ac and H3K27ac in epithelial tongue lesions of wild-type and Nos2-knockout mice treated with 4NQO.

Abstract

Introduction: Nitric oxide is an important regulator of the epigenetic landscape of homeostatic and pathological cell states, in which post-translational modifications of epigenome-modulating enzymes are the best described mechanism. In several types of cancer, nitric oxide acts either by promoting or by inhibiting tumor growth. The synthesis of nitric oxide depends on three enzymes of the same family, called nitric oxide synthases: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). Neuronal isoforms are classified as constitutive enzymes due to their continuous and calcium and calmodulin-dependent activity, synthesizing low and transient concentrations of nitric oxide. In contrast, iNOS is an inducible and calcium-independent isoform, whose nitric oxide synthesis is continuous and characterized by high concentrations, usually in pathological processes, such as during the inflammatory response and carcinogenesis. In recent years, the literature has shown the role of nitric oxide and nitric oxide synthases in the direct maintenance of the epigenetic “landscape”, acting through several mechanisms, including post-translational modifications of histones, DNA methylation and regulation of microRNAs. Objective: The aim of this study is to analyze the immunohistochemical expression of epigenetic markers related to histone acetylation, namely, H3K9ac (acetylation of lysine 9 in histone 3) and H3K27ac (acetylation of lysine 27 in histone 3), in different stages of the process of oral carcinogenesis induced by 4-nitroquinoline N-oxide (4NQO) in wild Nos2+/+ and knockout mice for the iNOS enzyme (Nos2-/-). Materials and methods: Our experimental unit consisted of male Mus musculus mice of the C57Bl/6J strains (wild type, Nos2+/+), wild animals that express the gene for inducible nitric oxide synthase (iNOS) and B6.129P2-Nos2tm1Lau/J ( knockout, Nos2-/-), which are genetically modified mice that do not express the iNOS gene. They were treated with 4NQO in drinking water at 50μg/mL for 16 weeks and then observed for 8 weeks. The tongues underwent histopathological analysis for the classification of dysplastic and neoplastic lesions, as well as immunohistochemical assay for H3K9ac and H3K27ac expression. The antigen-antibody reaction was analyzed using the quickscore method (QS). The experimental procedures were approved by the Committee on Ethics in Animal Experimentation (CEUA-UFU, registration nº 100/18). Results: It was observed that both histone acetylation markers were expressed in normal epithelium. QS values ​​were higher in moderate dysplasia of Nos2-/- mice (p=0.025) compared to Nos2+/+, and mild dysplasia had lower values ​​for H3K9ac when compared to moderate and severe dysplasia in Nos2-/ group - (p=0.015). H3K27ac increased significantly from normal mucosa to mild dysplasia in Nos2+/+ mice (p=0.007). Furthermore, Nos2+/+ mice had a higher number of H3K27ac-positive mild dysplasias compared to Nos2-/- (p=0.023). Conclusion: The histone H3 acetylation pattern varies significantly during the process of oral carcinogenesis in a murine model, especially when the epithelial lining of the tongue becomes dysplastic. Considering the differences in marker expression between wild-type and knockout animals, we can conclude that such epigenetic modifications may be mediated by inducible nitric oxide synthase.