Periodontal disease affects oral cancer progression in a surrogate animal model for tobacco exposure

Abstract

For decades, the link between poor oral hygiene and the increased prevalence of oral cancer has been suggested. Most recently, emerging evidence has suggested that chronic inflammatory diseases from the oral cavity (e.g., periodontal disease), to some extent, play a role in the development of oral squamous cell carcinoma (OSCC). The present study aimed to explore the direct impact of biofilm‑induced periodontitis in the carcinogenesis process using a tobacco surrogate animal model for oral cancer. A total of 42 Wistar rats were distributed into four experimental groups: Control group, periodontitis (Perio) group, 4‑nitroquinoline 1‑oxide (4‑NQO) group and 4NQO/Perio group. Periodontitis was stimulated by placing a ligature subgingivally, while oral carcinogenesis was induced by systemic administration of 4NQO in the drinking water for 20 weeks. It was observed that the Perio, 4NQO and 4NQO/Perio groups presented with significantly higher alveolar bone loss compared with that in the control group. Furthermore, all groups receiving 4NQO developed lesions on the dorsal surface of the tongue; however, the 4NQO/Perio group presented larger lesions compared with the 4NQO group. There was also a modest overall increase in the number of epithelial dysplasia and OSCC lesions in the 4NQO/Perio group. Notably, abnormal focal activation of cellular differentiation (cytokeratin 10‑positive cells) that extended near the basal cell layer of the mucosa was observed in rats receiving 4NQO alone, but was absent in rats receiving 4NQO and presenting with periodontal disease. Altogether, the presence of periodontitis combined with 4NQO administration augmented tumor size in the current rat model and tampered with the protective mechanisms of the cellular differentiation of epithelial cells.

Keywords: 4‑nitroquinoline‑1‑oxide; cell differentiation; head and neck cancer; ligature; periodontitis.

Figure 1

Study methodological aspects. (A) Study flowchart according to experimental groups. All groups were acclimated for 2 weeks. Periodontitis was induced by ligature in the Perio and 4NQO/Perio groups 2 weeks before 4NQO exposure. The 4NQO and 4NQO/Perio groups were exposed to 4NQO carcinogen in the drinking water for 20 weeks. (B) Weight of rats in experimental groups every 4 weeks during the 20 weeks of 4NQO exposure (^**P<0.01). (C) Solution consumption of rats in different groups every 4 weeks during the 20 weeks of 4NQO exposure. Data are presented as the mean ± SEM (^*P<0.05 and ^**P<0.01). (D) Representative images of lesion size determination through software analysis of altered areas in the tongues of the rats (scale bar in millimeters). (E) Representative images of morphometric evaluation of bone loss. The distance between the cementoenamel junction and the alveolar bone crest was computed at 5 points in the second upper molar. (F) Representative histological section from a tongue receiving 4NQO stained with hematoxylin and eosin. (G) Representative images of epithelial dysplasia and invasive carcinoma from tongue. Cases presenting basal membrane invasion (indicated by arrows) were considered carcinomas. Perio, periodontitis; 4NQO, 4-nitroquinoline 1-oxide; SCC, squamous cell carcinoma.

Figure 2

Morphometric alveolar bone loss evaluation. (A) Mean ± SEM level of alveolar bone loss in the ligature-induced periodontitis sites in each experimental group (^*P<0.05, ^**P<0.01 and ^****P<0.0001). (B) Representative images of alveolar bone loss in the Perio, 4NQO, and 4NQO/Perio groups. The 4NQO/Perio group exhibited the largest distance between the cementoenamel junction and the alveolar bone crest. Perio, periodontitis; 4NQO, 4-nitroquinoline 1-oxide.

Figure 3

Analysis of lesion size in 4NQO-treated groups. (A) Mean ± SEM level of lesion surface area in each experimental group (^***P<0.001), and individual tumor surface area in the 4NQO and 4NQO/Perio groups. (B) Representative images of tongue lesions and digital quantification in the 4NQO and 4NQO/Perio groups. The 4NQO/Perio exhibited the largest area. Moreover, a more heterogeneous lesion was observed, with large exophytic and verrucous regions, while the 4NQO group showed a more flat and homogeneous lesion (scale bar in millimeters). Perio, periodontitis; 4NQO, 4-nitroquinoline 1-oxide.

Figure 4

Histopathological analysis. (A) Percentage of cases diagnosed as epithelial dysplasia and carcinoma according in each experimental group. (B) Representative case showing microscopic findings in the 4NQO group. A carcinoma was present at the posterior portion of the tongue. Note the invasive characteristics of the dorsal carcinoma along local tissue destruction depicted by hematoxylin and eosin staining (inserts). (C) Representative example of 4NQO-treated rats depicting focal activation of cellular differentiation within the dorsal tongue mucosa. Epithelial cells exhibiting enhanced eosinophilic cytoplasm were present next to the basal layer of the mucosa (arrows). Perio, periodontitis; 4NQO, 4-nitroquinoline 1-oxide; OSCC, oral squamous cell carcinoma.

Figure 5

Cell differentiation analysis. (A) CK10 basal layer expression by fields in each experimental group (^****P<0.0001). (B) Representative images of CK10 immunostaining (red) merged with Hoechst 33342 (blue) in the 4NQO and 4NQO/Perio groups. The 4NQO group contains CK10-positive cells within the basal cells layer (white dotted line). By contrast, the 4NQO/Perio group lacks the expression of CK10 in the basal epithelial layers and just expresses CK10 on the upper layers of the epithelial layer of the mucosa. Perio, periodontitis; 4NQO, 4-nitroquinoline 1-oxide; CK10, cytokeratin 10.

Figure 6

Diagram depicting increased cellular differentiation on the oral mucosa exposed to 4NQO and the presence of a smaller tumor area on the tongue of the rats. The presence of periodontal disease associated with the administration of 4NQO resulted in reduced cellular differentiation of the oral mucosa and the presence of larger tumor areas in the tongue of rats. Fig. 6 was created with BioRender.com. 4NQO, 4-nitroquinoline 1-oxide; CK10, cytokeratin 10.