Expression of p53, Ki-67, Sox-2, Oct-4, and Nanog in the tongue and trachea of swiss mice exposed to narghile smoke

The objective of the study is to investigate the modulation of neoplastic stem cell markers (SOX-2, OCT-4, and Nanog) in tissues exposed exclusively to narghile (hookah) smoke. The research aims to clarify whether this exposure triggers early cellular changes associated with stemness, which are closely linked to tumor development and growth.

To achieve this, a descriptive in vivo experimental study was conducted using a full-body exposure system. The sample consisted of 60 female Swiss mice, approximately two months old and weighing 25 grams, randomly divided into six groups of 10 animals each. The groups included one control group, exposed only to ambient air, and five experimental groups exposed to narghile smoke for periods of 7, 15, 30, 60, and 90 days. The exposure protocol involved placing the animals in a sealed glass chamber for daily 30-minute sessions, configured to approximate human breathing patterns during narghile use (35 mL of smoke every two seconds, followed by 58 seconds of pure air). Tongue exposure occurred indirectly through the deposition of smoke components on the animals' fur and subsequent self-grooming.

At the end of the respective exposure periods, the animals were euthanized to harvest the tongue and trachea. These tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 3 μm for laboratory analysis. Histopathological evaluation was performed using Hematoxylin and Eosin (H&E) staining based on the World Health Organization dysplasia classification. Immunohistochemistry was employed to assess the presence of the markers, with immunopositivity determined by brown nuclear staining in epithelial cells. Samples were evaluated under light microscopy at standardized equidistant points—six on the tongue and four on the trachea—and images were analyzed using ImageJ software by a blinded, pre-calibrated examiner. Finally, statistical analysis was conducted using SPSS software with non-parametric tests; the Kruskal-Wallis test compared immunopositivity between groups, and Spearman’s correlation coefficient assessed protein expression levels, with significance set at $p < 0.05$.