An oral injury is thought to presage the development of mucosal lesions that are common in moist smokeless tobacco (MST) users. The abrasion or mechanical stress caused by direct contact of MST with the oral mucosa may contribute to this injury by causing transient disruptions in the cell membrane. In order to test this hypothesis, we developed an in vitro exposure system that directly exposes cells to reference MST on a rocking platform to simulate the abrasion that might be experienced in the oral cavity when using MST. Using this treatment paradigm, we monitored plasma membrane disruption as a measure of cell wounding caused by direct interaction of the tobacco material itself with monolayer cultures of Het-1A immortalized human esophageal cells as a potential contributor to the injury process. We found that a washed reference MST preparation, in which MST-associated chemicals were removed but the tobacco material retained, causes cell wounding as indicated by the uptake through plasma membrane disruptions of a fluorescent marker normally impermeable to the cell. Having established that non-chemical properties of MST cause cell wounding, subsequent experiments revealed that cell wounding during simultaneous exposure to an aqueous MST-extract result in greater than additive cell death when compared to treatment with washed MST or MST-extract alone. Furthermore, we found that the high levels of free calcium found in MST-extract appear to be playing an important role. Taken together, these results indicate that MST-induced oral injury may result from a combined interaction of physical disruption of the plasma membrane by the tobacco material itself and the adverse effects of MST chemical constituents, notably high levels of calcium, that gain entry to the cell by way of MST-induced cell wounding.
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