title: Oxidative damage in human gingival fibroblasts exposed to cigarette smoke.
abstract: 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.
authors.affiliation: Department of Biology, Università degli Studi di Milano, I-20133 Milan, Italy.
authors.forename: Graziano
authors.initials: G
authors.lastname: Colombo
authors.forename: Isabella
authors.initials: I
authors.lastname: Dalle-Donne
authors.forename: Marica
authors.initials: M
authors.lastname: Orioli
authors.forename: Daniela
authors.initials: D
authors.lastname: Giustarini
authors.forename: Ranieri
authors.initials: R
authors.lastname: Rossi
authors.forename: Marco
authors.initials: M
authors.lastname: Clerici
authors.forename: Luca
authors.initials: L
authors.lastname: Regazzoni
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Aldini
authors.forename: Aldo
authors.initials: A
authors.lastname: Milzani
authors.forename: D Allan
authors.initials: DA
authors.lastname: Butterfield
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Gagliano
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005881:Gingiva / Q000166:cytology / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D018384:Oxidative Stress; D010316:Particle Size; D017382:Reactive Oxygen Species / Q000378:metabolism; D012906:Smoke; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D017382:Reactive Oxygen Species; D012906:Smoke; D005978:Glutathione