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Heat shock proteins as biomarkers of lung oxidant stress

Institution: University of California, Davis
Investigator(s): Kurt Williams, D.V.M.
Award Cycle: 2000 (Cycle 9) Grant #: 9DT-0204 Award: $24,338
Subject Area: Pulmonary Disease
Award Type: Dissertation Awards
Abstracts

Initial Award Abstract
Tobacco smoke is comprised of many different compounds that are potentially harmful to the lung. As a matter of fact both the smoke taken in by smokers, as well as the second-hand smoke that others in the vicinity are exposed to, contains over 4000 potentially toxic agents. One of the major ways that many of these materials can cause injury is by oxidant damage. Oxidant damage occurs when oxygen atoms in the cell, either free and on their own (or as part of another molecule), become reactive and interact with many other molecules in the cells. The first line of defense for cells is to “soak” up this damaging oxygen by allowing it to react with an abundant cellular molecule without harming the cell. Glutathione (GSH) is such an abundant molecule found in cells. Heat shock proteins (Hsps) are another important group of proteins in cells that are capable of repairing damage to the cell from oxidants if GSH is unable to prevent this injury. Hsps have also been found to have the potential to protect both individual cells and the intact lung from injury if they can be produced prior to exposure to potentially damaging conditions. Levels of GSH in tissues are important in the initial protection of animals from oxidant injury, and Hsps seem to be associated with this protective attempt. It would be valuable if we could use the easily detectable presence of Hsps as biomarkers of evidence of tobacco oxidant exposure in the lung. The smallest airways of the lung, the terminal bronchioles, are the last portion of the lung that inhaled materials pass through before the air, and materials in it, reach the region of gas exchange. A common cell type in the terminal bronchiole, the Clara cell, is capable of taking pollutants in tobacco smoke or other forms of air pollution and converting them into another, potentially more toxic material in an attempt to rid the body of them. Naphthalene (NA) is an example of a very common toxin in tobacco smoke that the Clara cell tries to break down, but instead creates other compounds that can cause oxidant injury in the lung. Because of this we will use NA to recreate a potential scenario in the lung that mimics the changes that may occur after tobacco use. This will address a priority of TRDRP, the issue of biomarkers in lung injury after exposure to an important constituent found in tobacco smoke. Additionally we expect to gain considerable knowledge about how the lung protects itself from tobacco constituent-induced lung injury. Our aim is to provide, through these studies, some insight into how tobacco smoke interacts with two of the critical protective measures taken by the lung; further we will attempt to identify a valuable biomarker of oxidant stress and tissue injury after exposure to an abundant tobacco smoke constituent.

Final Report
In the past year I have been able to identify a sensitive indicator of biochemical tissue alterations in the lungs of mice exposed to a common component of tobacco smoke. Tobacco smoke is comprised of many different compounds that are potentially harmful to the lung. Both the smoke taken in by smokers, as well as the second hand smoke that others in the vicinity are exposed to contains over 4000 potentially toxic agents. One of the major ways that many of these materials can cause injury is by oxidant damage. Oxidant damage occurs when oxygen atoms in the cell; either free and on their own or as part of another molecule, become reactive and interact with many other molecules in cells. The first line of defense for cells is to "soak" up this damaging oxygen by allowing it to react with an abundant cellular molecule without harming the cell. Glutathione (GSH) is such an abundant molecule found in cells. When GSH reacts with these forms of oxygen, the cell is said to be experiencing an oxidant stress. Oxidant stress is a common, and often harmful, condition in many cell types and is felt to be an important part of the damage to the lungs caused by smoking. I attempted to identify a reliable "biomarker" of this oxidant stress by analyzing for increased production of Hsp 72, a member of the heat shock protein (Hsp) family. Hsps are important to cells because of their role in producing proteins needed by cells, as well as for their ability to repair proteins damaged during cell injury. Additionally there is evidence that the induction of Hsp production in cells is tied closely to the levels of GSH in the cells. We used a common constituent of tobacco smoke, naphthalene (NA), in mice to investigate whether or not Hsp 72 might be a sensitive indicator of oxidant stress in the lung. NA causes a well-characterized depletion of GSH in the airway epithelium of the lung. The depletion of GSH in the distal airways is associated with significant damage to the Clara cells of the epithelium; interestingly GSH is also markedly depleted in the more proximal airway epithelium without causing significant cell damage. I determined that the depletion of GSH at both airway levels is associated with induction of Hsp 72. 1 then followed this work by depleting the lung of GSH with a non-toxic GSH depletor, diethyl maleate (DEM). DEM causes profound depletion of GSH in the proximal and distal airways of treated mice without creating significant cell injury. Hsp 72 is induced significantly in both the proximal and distal airway epithelial cells after this non-toxic oxidant stress. In conclusion, it appears that Hsp 72 expression is a sensitive indicator of oxidant stress in the airway epithelium and may be valuable in continued investigations into the role,that oxidant stress plays in the cell injury caused by tobacco smoke.
Publications

Glutathione depletion is associated with airway epithelial F-Actin alterations
Periodical: American Journal of Respiratory and Critical Care Medicine Index Medicus:
Authors: Williams KJ, Phimister AJ, Fanucchi MV, Plopper CG ART
Yr: 2001 Vol: 163 Nbr: 5 Abs: A842 Pg:

Glutathione depletion initiates a stress response in Clara cells
Periodical: American Journal of Respiratory and Critical Care Medicine Index Medicus:
Authors: Phimister AJ, Williams KJ, Plopper CG ABS
Yr: 2001 Vol: 163 Nbr: 5 Abs: A843 Pg: