Cigarette smoke, a complex mixture of over 7000 chemicals, contains many components capable of eliciting oxidative stress, which may induce smoking-related disorders, including oral cavity diseases. In this study, we investigated the effects of whole (mainstream) cigarette smoke on human gingival fibroblasts (HGFs). Cells were exposed to various puffs (0.5-12) of whole cigarette smoke and oxidative stress was assessed by 2',7'-dichlorofluorescein fluorescence. The extent of protein carbonylation was determined by use of 2,4-dinitrophenylhydrazine with both immunocytochemical and Western immunoblotting assays. Cigarette smoke-induced protein carbonylation exhibited a puff-dependent increase. The main carbonylated proteins were identified by means of two-dimensional electrophoresis and MALDI-TOF mass spectrometry (redox proteomics). We demonstrated that exposure of HGFs to cigarette smoke decreased cellular protein thiols and rapidly depleted intracellular glutathione (GSH), with a minimal increase in the intracellular levels of glutathione disulfide and S-glutathionylated proteins, as well as total glutathione levels. Mass spectrometric analyses showed that total GSH consumption is due to the export by the cells of GSH-acrolein and GSH-crotonaldehyde adducts. GSH depletion could be a mechanism for cigarette smoke-induced cytotoxicity and could be correlated with the reduced reparative and regenerative activity of gingival and periodontal tissues previously reported in smokers.
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