Radical problems: Cigarette smoke attacks proteins in oral tissue

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  • Published: May 15, 2012
  • Author: Steve Down
  • Channels: Proteomics & Genomics / Proteomics
thumbnail image: Radical problems: Cigarette smoke attacks proteins in oral tissue

Cigarette smoke cocktail

The reactive aldehydes present in cigarette smoke have a rapid and toxic effect on human oral fibroblasts, oxidising key proteins and reducing their cellular levels, according to a transatlantic study.


 

The latest report by the Surgeon General in the US on the effects of smoke inhalation, published in 2010, reported that there are more than 7000 chemicals in cigarette smoke. Hundreds of these are toxic and about 70 are known to be carcinogenic. Smokers and non-smokers are all exposed to this vast range of compounds which are present in mainstream smoke, emitted from the end of the cigarette, and sidestream smoke, emitted from the cigarette between puffs.

Many of these components can cause immediate damage to the body, including reactive oxygen species and reactive nitrogen species. It is believed that oxidative stress caused by the free radicals is one of the primary routes for diseases caused by tobacco smoke, and the first point of attack is within the mouth. For this reason, a team of scientists from Italy and the USA has been examining the effects of smoke on oral tissue.

Isabella Dalle-Donne and colleagues from the University of Milan, the University of Siena, and the University of Kentucky, Lexington, specifically targeted the effects of smoke on fibroblasts extracted from the gingiva (the soft tissue around the base of the teeth) of a healthy subject. Exposure to smoke can inhibit the functions of fibroblasts such as adhesion, growth, proliferation and the generation of extracellular matrix.

They were particularly interested in the formation of carbonyl groups in fibroblast-related proteins produced by reaction with unsaturated aldehydes in the smoke, like acrolein (2,3-propenal) and crotonaldehyde (2-butenal). These reactive compounds are present in the saliva of healthy subjects but are about ten-fold more abundant in that of smokers.

Search for protein carbonyls

The researchers used a smoking machine fitted with a syringe to deliver mainstream cigarette smoke to the fibroblasts in a culture flask for a specific number of puffs from one to twelve, the latter corresponding to one whole cigarette. The fibroblast cells were then subjected to a raft of tests for protein carbonylation.

First of all, a fluorescent dye assay was used to measure the amount of reactive oxygen species produced in the cells. Then the degree of protein carbonylation was measured by immunocytochemistry following derivatisation with 2,4-dintrophenylhydrazine.

A further test for protein carbonylation and S-glutathionylation was carried out by SDS-PAGE and Western blotting and the affected proteins were separated by 2D gel electrophoresis for enzymatic digestion with trypsin and identification by MALDI mass spectrometry.

Specific adducts of acrolein and crotonaldehyde with the thiol groups of intracellular glutathione were measured by a specially developed LC/MS/MS method with electrospray ionisation and multiple reaction monitoring.

Oxidative damage in the mouth

Exposure of the fibroblasts to cigarette smoke resulted in an immediate effect, with cell viability beginning to drop away after just half a puff and falling to 71% after 12 puffs. Phase contrast microscopy showed that the affected cells shrank, becoming more distorted as exposure increased.

Dalle-Donne suggested that this change was at least partly caused by the observed increase in reactive oxygen species and the rise in protein carbonylation. A total of 21 proteins were found to be carbonylated but nine of them were also moderated in control fibroblasts, although to a lesser extent. They included proteins involved in various cellular processes.

For instance, actin and some actin-binding proteins like cofilin-1 were carbonylated, causing disruption to the actin cytoskeleton and inhibiting processes which rely on actin such as tissue remodelling and wound repair. Elongation factor 1alpha is also associated with the reorganisation of F-actin. Other carbonylated proteins were involved with energy metabolism, like alpha-enolase, glycveraldehyde 3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase.

In parallel to these changes in protein carbonylation, protein thiols were reversibly oxidised in a dose-dependent manner by the cigarette smoke. One particular thiol, glutathione, is a key component in the cell antioxidant defence and its depletion leaves cells open to damage. Loss of glutathione could be a major reason why oxidation and carbonylation occur in the mouth during smoking.

The mass spectrometric studies confirmed that the thiols were removed from the fibroblast cells by adduct formation with acrolein and crotonaldehyde, although the adducts did not account for the total loss of glutathione. So, other factors must be at play, perhaps involving different aldehydes known to be present in the smoke, like formaldehyde and acetaldehyde.

The researchers were careful to point out that these results were gathered in vitro and it should not be assumed that the same processes occur in vivo. However, it appears that the inbuilt response to cigarette smoke cannot prevent protein oxidation and carbonylation.

The results could be used to develop pharmacological strategies for reducing or preventing smoke-related tissue damage that target the aldehydes, although, as the team declared "the best antioxidant would be to give up smoking."

Related Links

Free Radical Biology and Medicine 2012, 52, 1584-1596: "Oxidative damage in human gingival fibroblasts exposed to cigarette smoke"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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