Enhanced protein glutathiolation and oxidative stress in cigarette smokers
Introduction
Glutathione (GSH) is an abundant tripeptide that protects against oxidative stress and damage in nearly all cells and tissues [1], [2]. GSH is the major intracellular antioxidant and functions by scavenging free radicals, detoxifying lipid peroxides via glutathione peroxidase, and conjugating reactive electrophilic toxicants and carcinogens. In addition, GSH is involved in numerous other cellular pathways including protein and DNA synthesis, DNA repair, and immune surveillance. GSH is oxidized to its disulfide form (GSSG), but is subsequently reduced back to GSH by GSH reductase. An alternative pathway for GSSG metabolism is protein glutathiolation (also referred to as glutathionylation) where thiol–disulfide exchange occurs with Cys residues in proteins to form protein-mixed disulfides (GSSP). The formation of GSSP within cells can be substantial and reach 200 μM in certain tissues [3].
There is considerable evidence that glutathiolation represents an important redox-sensitive regulator of cellular activities [4], [5]. Glutathiolation alters the function of critical cellular proteins involved in cancer development and other diseases. These proteins include c-Jun [6], spectrin [7], protein kinase C [8], ubiquitin conjugating enzymes [9], carbonic anhydrase III [10], H-ras [11], NF-κB [12], and thioredoxin [13]. Glutathiolation has also been implicated as a protective mechanism against irreversible oxidation of critical sulfhydryl groups on proteins during periods of intense oxidative stress [14], [15]. Because GSSP formation occurs by oxidation and can affect the function of proteins, its levels may indicate the degree of oxidative stress within cells and tissues. Further, the blood concentration of GSSP has been proposed as a marker of oxidative stress in humans [3], [16], [17]. While GSSP levels in blood are high compared with other tissues, ranging from 0.05 to 0.32 μmol/ml in healthy adults [3], [18], little is known regarding the regulation of these levels in response to oxidative stress.
There remains a critical need to develop accurate and reliable measures of oxidative stress for use in clinical investigations because of its potential role in chronic disease development and aging [19]. The major cause of chronic oxidative stress in humans is exposure to free radicals in cigarette smoke. Cigarette smoke free radicals are considered an important cause of atherosclerosis and cancer [20], [21]. Yet direct evidence for a role of oxidative stress is lacking in part because biological markers are not responsive to the subtle and chronic changes in overall oxidative stress levels, and provide information only on specific forms of oxidative damage. The difference between biomarkers of oxidative stress and oxidative damage, while perhaps not widely recognized, is important since recent concepts on oxidative stress have emphasized the regulation of key cellular activities through pathways that do not involve direct and irreversible damage of macromolecules [22]. New methods are needed to measure oxidative stress and its possible effects on cellular mechanisms such as cell proliferation and apoptosis. Further, with the possible exception of f2-isoprostanes, a biomarker of lipid peroxidation, commonly used biological markers of lipid peroxidation and oxidative DNA damage are often unreliable indicators of disease because of a lack of stability, artifactual formation during sample processing or storage, or a lack of mechanistic involvement in the disease process [23], [24].
In the current study, we determined whether blood GSSP is a valid and sensitive indicator of oxidative stress by comparing levels in cigarette smokers and nonsmokers.
Section snippets
Methods
The current analysis was conducted using data obtained from a community-based study of 354 current smokers in lower Westchester County, New York, USA and of 97 nonsmokers employed at the host institute. The methods and details of the study recruitment are described elsewhere [25]. In brief, current smokers were recruited using fliers, public service announcements, assistance from community and church leaders, and other methods. Trained personnel conducted interviews using a structured
Results
The mean intraindividual variation of blood GSH and GSSP was 9.4 and 12.1%, respectively. The interindividual CV of GSH and GSSP was 21.1 and 34.8%, respectively, in the subjects.
Table 1 depicts characteristics of the 354 community-based subjects and the 97 nonsmokers. Approximately half were men, with mean ages of 43 in nonsmokers and 30 in smokers. About 5% of nonsmokers and 50% of smokers were non-Hispanic blacks. The average number of cigarettes smoked per day was 18 in the smoking group.
Discussion
These findings provide compelling evidence that blood GSSP is an indicator of oxidative stress, and that abundant free radicals in cigarette smoke cause increases in blood GSSP concentrations. The amount of protein that is glutathiolated in the blood of smokers was high (ranging from 0.05 to 0.38 mmol/l), 34 to 43% higher than those observed in nonsmokers. A dose–response relationship was apparent between GSSP levels and tobacco smoke measurements such as cigarettes smoked per day, blood
Acknowledgements
This work was supported by NIH Grants CA68384 and CA17613.
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