Oxidative damage in human gingival fibroblasts exposed to cigarette smoke

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• 作者：Graziano Colombo^a ; ¹ ; Isabella Dalle-Donne^a ; ¹ ; ^{quack@unimi.it} ; Marica Orioli^b ; Daniela Giustarini^c ; Ranieri Rossi^c ; Marco Clerici^a ; ^d ; Luca Regazzoni^b ; Giancarlo Aldini^b ; Aldo Milzani^a ; D. Allan Butterfield^e ; ^f ; ^g ; Nicoletta Gagliano^d
• 关键词：biotin&ndash ; HPDP ; N-(6-(biotinamido)hexyl)-3&prime ; -(2&prime ; -pyridyldithio)propionamide ; DAPI ; 4&prime ; 6-diamidino-2-phenylindole ; DCF ; 2&prime ; 7&prime ; -dichlorofluorescein ; DCFH-DA ; 2&prime ; 7&prime ; -dichlorodihydrofluorescein diacetate ; DMEM ; Dulbec
• 刊名：Free Radical Biology and Medicine
• 出版年：2012
• 期刊代码：105_08915849
• 类别：med
• 出版时间：1 May, 2012
• 卷：52
• 期：9
• 页码：1584-1596
• 文件大小：1065 K

摘要

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鈥?7鈥?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.