Oxidative damage in human gingival fibroblasts exposed to cigarette smoke

Graziano Colombo, Isabella Dalle-Donne, Marica Orioli, Daniela Giustarini, Ranieri Rossi, Marco Clerici, Luca Regazzoni, Giancarlo Aldini, Aldo Milzani, D. Allan Butterfield, Nicoletta Gagliano

Research output: Contribution to journalArticlepeer-review

72 Scopus citations


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.

Original languageEnglish
Pages (from-to)1584-1596
Number of pages13
JournalFree Radical Biology and Medicine
Issue number9
StatePublished - May 1 2012

Bibliographical note

Funding Information:
This research was supported by funds provided by PUR 2009 (Programma dell'Università per la Ricerca), Università degli Studi di Milano.


  • Cigarette smoke
  • Free radicals
  • GSH-α,β-unsaturated aldehyde adducts
  • Human gingival fibroblasts
  • Protein carbonylation
  • Protein thiols
  • Redox proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)


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