# Electronic Cigarettes' Toxicity: From Periodontal Disease to Oral Cancer
## 2 Effects of Electronic Cigarettes on the Oral Cavity
### DNA Damage and Genotoxic Mechanism  
Genotoxicity and DNA damage are linked to the malignant transformation of normal cells. [PERSON] et al. demonstrated that electronic cigarettes affect periodontal tissue by inhibiting the proliferation of human gingival fibroblasts through apoptotic mechanisms. Cell proliferation is a key process for tissue repair. Cell apoptosis was investigated by DNA fragmentation using the TUNEL assay. Exposure to e-vapor condensate also led to altered fibroblasts' morphology. These effects were also visible for nicotine-free e-liquids, pointing to the conclusion that other compounds, besides nicotine, present a toxic potential to the cells [36]. [PERSON] et al. also studied the viability and proliferation of human periodontal fibroblasts exposed to e-liquids, and they observed that the highest reduction in the proliferation rate was observed for menthol flavored liquids, pointing to the cytotoxic potential of some additives [37]. In another study, [PERSON] et al. conclusively linked e-cigarettes to DNA breakage, regardless of nicotine concentration. The accumulation of double-strand breaks is suggestive of the carcinogenic potential of e-cigarettes. Increased rates of G1 and G2 arrest were observed among exposed cells, reducing the intervention of homologous recombination, a higher-fidelity double-strand breaks repair mechanism primarily active in the S phase [38]. One assay used for the noninvasive assessment of chromosomal instability, genomic damage, and oral cell death is the buccal micronucleus cytome assay, indicating an elevated risk of carcinogenesis. Micronuclei, extra-nuclear structures produced during cell division containing chromosomes fragments, are important for chromosomal aberrations biomonitoring, both structural or numerical, while binucleation is related to defects in cytokinesis [39]. Different studies demonstrated the genotoxic potential of nicotine. Nicotine-induced micronuclei formation in human gingival fibroblasts and in human primary parotid gland cells. A higher frequency of micronuclei can be associated with a higher risk of cancer [40; 41]. However, in a cytological study that assessed the prevalence of micronuclei in oral cavity cells, there were no significant alterations in the micronuclei distribution in e-cigarette users in comparison to nonsmokers [33]. Nicotine exposed cells presented increased comet tail length and \(\gamma\)-H2 AX foci, signs of increased DNA strand breaks [2]. Nicotine can induce the over-expression of human telomerase reverse transcriptase mRNA in oral keratinocytes, which may play a role in the progression and malignancy of oral submuc fibrosis. This aspect is important because approximately 7-13% of patients with oral submuc fibrosis will eventually progress to oral squamous cell carcinoma [42]. Deregulations of the cell cycle are considered a fundamental hallmark of cancer progression. In oral carcinoma, several deregulations in cell cycle regulatory protein are frequently seen, and they stimulate carcinogenesis. The abrogation of cell cycle regulatory proteins such as retinoblastoma tumor suppressor protein (pRB), cyclin D1, cyclin-dependent kinase (CDKs), and CDK inhibitors (p12 WAF1/CIP1, p27 KIP1, and p16 INK4a) occurs in response to stress (both extracellular and intracellular) and DNA damage [13]. When the regulation of genes and associated molecular pathways involved in crucial cellular functions (growth control and differentiation) were analyzed using RNA-sequencing, a significant number of aberrantly expressed transcripts were identified in e-cigarette smokers. The Wnt/Ca\({}^{+}\) pathway and the Rho family GTPases signaling pathway were the most affected. In e-cigarette users the activators of the Wnt/Ca\({}^{+}\) signaling pathway (the tumor suppressor WNT5A gene and the fizzled receptor FDZ7 gene) were down-regulated, this causing the inhibition of downstream effectors of the cascade.