Biological impact of high-dose nicotine pouch extracts on oral epithelial homeostasis: an in vitro assessment of oxidative stress, cell death, and TP53/EGFR gene expression

Background Nicotine pouches release nicotine and other soluble formulation constituents directly at the oral mucosal surface, but the acute cellular effects of high-strength commercial products remain incompletely characterized. Objective To compare the acute toxicity of aqueous extracts prepared from products labeled 20 mg and 30 mg nicotine per pouch in primary human oral keratinocytes (HOKs), using complementary functional, biochemical, molecular, flow cytometric, and ultrastructural endpoints. Methods Primary HOKs were exposed for 24 h to aqueous extracts of the two products. Concentration-response cytotoxicity and half-maximal inhibitory concentrations (IC₅₀) were determined by MTT assay. Each extract was then tested at its own MTT-defined IC₅₀. Cellular morphology was examined microscopically; CAT and SOD activities and MDA and NO levels were measured to characterize redox status; cell-death populations were quantified by Annexin V-FITC/propidium iodide flow cytometry; TP53 and EGFR transcripts were assessed by quantitative real-time PCR; and ultrastructural alterations were examined by transmission electron microscopy. Results Both extracts reduced MTT-derived viability as extract concentration increased. The 30 mg product extract had a lower IC₅₀ than the 20 mg product extract (49.6% versus 83.2% of the stock extract), indicating greater cytotoxic potency. At the selected exposure concentrations, both extracts reduced CAT and SOD activities, increased MDA and NO levels, decreased the viable cell fraction, and produced predominantly necrotic cell death. TP53 mRNA increased to 2.31-fold and 3.16-fold of control levels after exposure to the 20 mg and 30 mg extracts, respectively, whereas EGFR mRNA decreased to 0.62-fold and 0.47-fold. Electron microscopy showed mitochondrial swelling, cristae disruption, chromatin condensation, cytoplasmic vacuolization, and plasma membrane injury, with more extensive alterations after exposure to the 30 mg product extract. Conclusion Commercial high-strength nicotine pouch extracts induce acute oxidative, molecular, and structural injury in primary oral keratinocytes. The 30 mg product extract showed greater cytotoxic potency, but the use of whole-product extracts means that the observed effects cannot be attributed to nicotine alone.