Oxidative damage in human gingival fibroblasts exposed to cigarette smoke

Free Radic Biol Med. 2012 May 1;52(9):1584-96. doi: 10.1016/j.freeradbiomed.2012.02.030. Epub 2012 Mar 1.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Chromatography, Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gingiva / cytology
  • Gingiva / metabolism*
  • Glutathione / metabolism
  • Humans
  • Immunohistochemistry
  • Oxidative Stress*
  • Particle Size
  • Reactive Oxygen Species / metabolism
  • Smoke*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tandem Mass Spectrometry

Substances

  • Reactive Oxygen Species
  • Smoke
  • Glutathione