Smokeless Tobacco And Superoxide Anion Production In Human Oral Keratinocytes

Superoxide anion production by NHOK after treatment with various concentrations of STE and/or antioxidants was measured according to our previously published procedure.40 Human oral keratinocytes were treated with 0 to 300^g/ml of STE for 24h. The cells (1x106cells/ml) were incubated at 37°C for 15 min in a reaction mixture containing 0.05mM cytochrome c (Type III, Sigma). The reactions were stopped by placing the reaction tubes in an ice bath. The mixtures were centrifuged for 10 min at 1500g at 4°C, and the absorbance values of the supernatant fractions were measured at 550nm.40 To verify that the reactive oxygen species were produced in response to STE, cytochrome c reduction was measured. This assay is a specific test for the production of superoxide anion. Following treatment of the NHOK with 100, 200, and 300^g/ml of

STE, 1.6-, 2.0-, and 2.7-fold increases in superoxide anion production were observed. Preincubation of the cells with vitamin C (75pM), vitamin E (75pM), a combination of vitamins C plus E (75 pM each), and grape seed proanthocyanidin extract (GSPE) (100pg/ml) for 4h decreased STE (300pg/ml)-induced cytochrome c reduction by 37, 39, 44, and 48%, respectively, as compared with the STE-treated cells.28

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