We examined the effects of reference tobacco preparations on DNA damage in human oral cavity cells. The oral squamous cell carcinoma cell line (101A), normal human gingival epithelial cells (HGEC), and human gingival fibroblasts (HGF) were treated with total particulate matter from 3R4F cigarettes (TPM), 2S3 smokeless tobacco extracted with complete artificial saliva (ST/CAS), or nicotine alone (NIC). Cells were treated for 24 hours with TPM at respective EC50 doses (13.7, 8.6, or 17.2 µg/ml of equi-nicotine units, as determined in previous experiments), or the doses with equi-nicotine units for ST/CAS. Also, cells were exposed to a high dose of ST/CAS (565.3 µg/ml of equi-nicotine units). DNA damage in exposed cells was assessed by alkaline Comet assays and immunofluorescence staining for the damage-specific protein ɣ-H2AX.

Both assays showed that only TPM caused readily detectable DNA breaks in exposed cells whereas ST/CAS or NIC did not; only the high dose of ST/CAS caused some weakly measurable DNA damage. Intriguingly, the malignant 101A cells were more susceptible to DNA damage than the normal HGEC and HGF cells.

These studies demonstrate that combusted tobacco products can cause substantial DNA damage in normal and malignant oral cavity cells, whereas non-combusted ST/CAS, or NIC alone, exert no detectable or only minimal DNA damage after 24 hour of exposure. The data will assist in evaluating relative genotoxic and other harmful effects of different categories of tobacco products on oral cavity cells. Such knowledge may help to further understand the involvement of combusted versus non-combusted tobacco products in the etiology of oral cancers.