Enhanced protein glutathiolation and oxidative stress in cigarette smokers

Abstract

There are many functional assays of oxidative damage to DNA, protein, and lipids but few reliable markers of chronic oxidative stress. The glutathiolation of proteins at key Cys residues is considered an important redox-sensitive, posttranslational signaling mechanism in the regulation of critical cellular functions. To determine whether protein bound glutathione (GSSP) is a sensitive indicator of oxidative stress, red blood cell and plasma concentrations were measured and compared between smokers and nonsmokers. In a community-based study conducted in Westchester County, New York, USA, blood samples were obtained from 354 cigarette smokers and 97 never smokers. The mean concentration of blood GSSP (μmol/L) was 32% higher in cigarette smokers and 43% higher when standardized by hemoglobin concentrations (p < .01). Plasma GSSP levels were also 20% higher in smokers than in nonsmokers (p < .001). The relationship was dose-dependent, with blood GSSP levels significantly correlated with cigarettes smoked per day, plasma cotinine, and plasma thiocyanate (r values ranged from .25 to .40). In smokers, there were no significant differences in GSSP and GSH levels by GSTM1 or GSTM3 genotype. Intraindividual variation in blood samples, as determined by taking serial samples over a 2-week period, was low (CV = 12.1%, n = 8). GSSP levels are stable over time but increase in response to the abundant free radicals in cigarette smoke. These findings support the use of GSSP as a sensitive biomarker of oxidative stress.

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.