Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Molecular Impact of Conventional and Electronic Cigarettes on Pulmonary Surfactant.

The alveolar epithelium is covered by a non-cellular layer consisting of an aqueous hypophase topped by pulmonary surfactant, a lipo-protein mixture with surface-active properties. Exposure to cigarette smoke (CS) affects lung physiology and is linked to the development of several diseases. The macroscopic effects of CS are determined by several types of cell and molecular dysfunction, which, among other consequences, lead to surfactant alterations. The purpose of this review is to summarize the published studies aimed at uncovering the effects of CS on both the lipid and protein constituents of surfactant, discussing the molecular mechanisms involved in surfactant homeostasis that are altered by CS. Although surfactant homeostasis has been the topic of several studies and some molecular pathways can be deduced from an analysis of the literature, it remains evident that many aspects of the mechanisms of action of CS on surfactant homeostasis deserve further investigation.

How to Increase Cellular Glutathione.

Glutathione (GSH) has special antioxidant properties due to its high intracellular concentration, ubiquity, and high reactivity towards electrophiles of the sulfhydryl group of its cysteine moiety. In most diseases where oxidative stress is thought to play a pathogenic role, GSH concentration is significantly reduced, making cells more susceptible to oxidative damage. Therefore, there is a growing interest in determining the best method(s) to increase cellular glutathione for both disease prevention and treatment. This review summarizes the major strategies for successfully increasing cellular GSH stores. These include GSH itself, its derivatives, NRf-2 activators, cysteine prodrugs, foods, and special diets. The possible mechanisms by which these molecules can act as GSH boosters, their related pharmacokinetic issues, and their advantages and disadvantages are discussed.

Antioxidants in smokers.

Cigarette smoke (CS) is likely the most common preventable cause of human morbidity and mortality worldwide. Consequently, inexpensive interventional strategies for preventing CS-related diseases would positively impact health systems. Inhaled CS is a powerful inflammatory stimulus and produces a shift in the normal balance between antioxidants and oxidants, inducing oxidative stress in both the respiratory system and throughout the body. This enduring and systemic pro-oxidative state within the body is reflected by increased levels of oxidative stress and inflammation biomarkers seen in smokers. Smokers might benefit from consuming antioxidant supplements, or a diet rich in fruit and vegetables, which can reduce the CS-related oxidative stress. This review provides an overview of the plasma profile of antioxidants observable in smokers and examines the heterogeneous literature to elucidate and discuss the effectiveness of interventional strategies based on antioxidant supplements or an antioxidant-rich diet to improve the health of smokers. An antioxidant-rich diet can provide an easy-to-implement and cost-effective preventative strategy to reduce the risk of CS-related diseases, thus being one of the simplest ways for smokers to stay in good health for as long as possible. The health benefits attributable to the intake of antioxidants have been observed predominantly when these have been consumed within their natural food matrices in an optimal antioxidant-rich diet, while these preventive effects are rarely achieved with the intake of individual antioxidants, even at high doses.

Preliminary experience on the use of sucrosomial iron in hemodialysis: focus on safety, hemoglobin maintenance and oxidative stress.

PURPOSE: Iron is usually administered in hemodialysis patients by parenteral route, as oral absorption is poor due to high hepcidin levels. However, administrations of intravenous iron and iron overload are associated with high oxidative stress and systemic inflammation that can affect patient survival. With this study, we evaluated an alternative type of oral iron for the treatment of anemia in hemodialysis patients. The formulation consists in ferric pyrophosphate covered by phospholipids plus sucrose ester of fatty acid matrix, named sucrosomial iron, whose absorption is not influenced by hepcidin. METHODS: Twenty-four (24) patients undergoing chronic hemodialysis switched iron supplementation from intravenous (ferric gluconate 62.5 mg weekly) to oral (sucrosomial iron, 90 mg weekly in 3 administrations of 30 mg) route for 3 months. Classical anemia, iron metabolism, inflammation and nutritional biomarkers were monitored, as well as biomarkers of oxidative stress, such as protein-bound di-tyrosines, protein carbonylation, advanced oxidation protein products and protein thiols. RESULTS: Over the 3 months, hemoglobin values remained stable, as the values of hematocrit and mean corpuscular volume. In parallel, other anemia parameters dropped, including ferritin, transferrin saturation and serum iron. On the other side, nutritional biomarkers, such as total proteins and transferrin, increased significantly during the time frame. We also observed a significant decrease in white blood cells as well as a non-significant reduction in C-reactive protein and some oxidative stress biomarkers, such as protein carbonyls and di-tyrosines. CONCLUSION: Our study demonstrates that a therapy with sucrosomial iron in hemodialysis patients is safe and can maintain stable hemoglobin levels in a three-month period with a possible beneficial effect on oxidative stress parameters. However, the reduction of ferritin and transferrin saturation suggests that a weekly dosage of 90 mg is not sufficient in hemodialysis patients in the long time to maintain hemoglobin.

Measurement of S-glutathionylated proteins by HPLC.

S-glutathionylated proteins (GSSP), i.e., protein-mixed disulfides with glutathione (GSH), are considered a suitable biomarker of oxidative stress. In fact, they occur within cells at low level and their concentration increases markedly under pro-oxidant conditions. Plasma is something different, since it is physiologically rich in S-thiolated proteins (RSSP), i.e., protein-mixed disulfides with various types of low molecular mass thiols (LMM-SH). However, albumin, which is largely the most abundant plasma protein, possesses a cysteine residue at position 34 that is mostly reduced (about 60%) under physiological conditions, but easily involved in the formation of additional RSSP in the presence of oxidants. The quantification of GSSP requires special attention to sample handling, since their level can be overestimated as a result of artefactual oxidation of GSH. We have developed the present protocol to avoid this methodological problem. Samples should be treated as soon as possible after their collection with the alkylating agent N-ethylmaleimide that masks -SH groups and prevents their oxidation. The GSH released from mixed disulfides by reduction with dithiothreitol is then labeled with the fluorescent probe monobromobimane and quantified by HPLC. The method can be applied to many different biological samples, comprising blood components, red blood cell plasma membrane, cultured cells, and solid organs from animal models.

Anethole Dithiolethione Increases Glutathione in Kidney by Inhibiting γ-Glutamyltranspeptidase: Biochemical Interpretation and Pharmacological Consequences.

AIMS: Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. RESULTS: Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. CONCLUSION: s. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.

Cigarette smoke and glutathione: Focus on in vitro cell models.

Cigarette smoke (CS) is one of the most important preventable risk factors for the development of respiratory diseases, cardiovascular diseases, stroke, and various types of cancer. Due to its high intracellular concentration and central role in maintaining the cellular redox state, glutathione (GSH) is one of the key players in several enzymatic and non-enzymatic reactions necessary for protecting cells against CS-induced oxidative stress. A plethora of in vitro cell models have been used over the years to assess the effects of CS on intracellular GSH and its disulphide forms, i.e. glutathione disulphide (GSSG) and S-glutathionylated proteins. In this review, we described the effects of cell exposure to CS on cellular GSH and formation of its oxidized forms and adducts (GSH-conjugates). We also discussed the limitations and relevance of in vitro cell models of exposure to CS and critically assessed the congruence between smokers and in vitro cell models. What emerges clearly is that results obtained in vitro should be interpreted with extreme caution, bearing in mind the limitations of the specific cell model used. Despite this, in vitro cell models remain important tools in the assessment of CS-induced oxidative damage.

Membrane Skeletal Protein S-Glutathionylation in Human Red Blood Cells as Index of Oxidative Stress.

Glutathione (GSH) is one of the most well-studied biomarkers of oxidative stress. Under oxidizing conditions, GSH is transformed into its disulfide forms, glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG), which are considered to be reliable biomarkers of oxidative stress. In red blood cells (RBCs), the main targets of S-glutathionylation are hemoglobin and membrane-associated skeletal proteins, but S-glutathionylated hemoglobin (HbSSG) has been more thoroughly studied as a biomarker of oxidative stress than S-glutahionylated RBC membrane skeletal proteins. Here, we have investigated whether and how all these biomarkers are altered in human RBCs treated with a slow and cyclically intermittent flux of the oxidant tert-butyl hydroperoxide. To this aim, a new device for sample treatment and collection was developed. During and at the end of the treatment, GSH, GSSG, and PSSG (discriminating between HbSSG and membrane PSSG) were measured by the use of spectrophotometer (for GSSG) and HPLC (for GSH, HbSSG, and membrane PSSG). The main results of our study are as follows: (i) GSH decreased and GSSG increased, but only in the presence of the oxidant, and recovered their initial values at the end of the infusion; (ii) the increase in total PSSG concentration was lower than that of GSSG, but it kept on throughout the experiments; (iii) membrane skeletal proteins did not recover their initial values, whereas HbSSG levels recovered their initial values similarly to GSH and GSSG; (d) membrane skeletal PSSG were more stable and also more abundant than HbSSG. Western blot analysis indicated spectrin, ankyrin, and bands 3, 4.1, and 4.2 as the proteins most susceptible to S-glutathionylation in RBC membrane. These results suggest that S-glutathionylated membrane skeletal proteins can be considered as a suitable biomarker of oxidative stress. Mostly when the oxidant insult is slight and intermittent, PSSG in RBC membranes are worth measuring in addition to GSSG by virtue of their greater stability.

Protein carbonylation in human bronchial epithelial cells exposed to cigarette smoke extract.

Cigarette smoke is a well-established exogenous risk factor containing toxic reactive molecules able to induce oxidative stress, which in turn contributes to smoking-related diseases, including cardiovascular, pulmonary, and oral cavity diseases. We investigated the effects of cigarette smoke extract on human bronchial epithelial cells. Cells were exposed to various concentrations (2.5-5-10-20%) of cigarette smoke extract for 1, 3, and 24 h. Carbonylation was assessed by 2,4-dinitrophenylhydrazine using both immunocytochemical and Western immunoblotting assays. Cigarette smoke induced increasing protein carbonylation in a concentration-dependent manner. The main carbonylated proteins were identified by means of two-dimensional electrophoresis coupled to MALDI-TOF mass spectrometry analysis and database search (redox proteomics). We demonstrated that exposure of bronchial cells to cigarette smoke extract induces carbonylation of a large number of proteins distributed throughout the cell. Proteins undergoing carbonylation are involved in primary metabolic processes, such as protein and lipid metabolism and metabolite and energy production as well as in fundamental cellular processes, such as cell cycle and chromosome segregation, thus confirming that reactive carbonyl species contained in cigarette smoke markedly alter cell homeostasis and functions.

N-acetylcysteine ethyl ester as GSH enhancer in human primary endothelial cells: A comparative study with other drugs.

Several drugs are currently in use as glutathione (GSH) enhancers in clinical, pre-clinical and experimental research. Here we compare the ability of N-acetylcysteine (NAC), 2-oxothiazolidine-4-carboxylic acid (OTC), glutathione ethyl ester (GSH-EE) and N-acetylcysteine ethyl ester (NACET) to increase the intracellular concentration of GSH using primary human umbilical vein endothelial cells (HUVEC) as in vitro model. Our experiments highlighted that NACET is largely the most efficient molecule in increasing the intracellular levels of GSH, cysteine, and γ-glutamylcysteine. This is because NACET is lipophilic and can freely cross plasma membrane but, inside the cell, it is de-esterified to the more hydrophilic NAC, which, in turn, is trapped into the cell and slowly transformed into cysteine. The higher availability of cysteine is matched by an increase in GSH synthesis, cysteine availability being the rate limiting step for this reaction. Surprisingly, the increase in GSH concentration was not linear but peaked at 0.5 mM NACET and gradually decreased when cells were treated with higher concentrations of NACET. We demonstrated that this puzzling ceiling effect was due to the fact that NAC released from NACET turned out to be a competitive inhibitor of the enzyme glutamate-cysteine ligase, with a Ki value of 3.2 mM. By using a cell culture medium lacking of cysteine and methionine, we could demonstrate that the slight increase in intracellular levels of cysteine and GSH induced by NAC in HUVEC grown in standard medium was due to the reduction of the cystine present in the medium itself there rather than to the action of NAC as Cys pro-drug. This fact may explain why NAC works well as GSH enhancer at very high concentrations in pre-clinical and in vitro studies, whereas it failed in most clinical trials.

Determination of protein thiolation index (PTI) as a biomarker of oxidative stress in human serum.

We have introduced protein thiolation index (PTI), i.e. the molar ratio of the sum of all low molecular mass thiols bound to plasma proteins to protein free cysteinyl residues, as a sensitive biomarker of oxidative stress. According to the original procedure its determination requires a rapid separation of plasma and a specific treatment of samples to stabilize thiols. Here we demonstrate that samples can be collected without use of any anticoagulant to prevent blood clotting and without any stabilization of thiols too. This simplification of the determination of PTI makes its analysis more feasible also in routine clinical laboratories.

Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells.

Glutathione (GSH) is the major non-protein thiol in humans and other mammals, which is present in millimolar concentrations within cells, but at much lower concentrations in the blood plasma. GSH and GSH-related enzymes act both to prevent oxidative damage and to detoxify electrophiles. Under oxidative stress, two GSH molecules become linked by a disulphide bridge to form glutathione disulphide (GSSG). Therefore, assessment of the GSH/GSSG ratio may provide an estimation of cellular redox metabolism. Current evidence resulting from studies in human blood, solid tissues, and cultured cells suggests that GSH also plays a prominent role in protein redox regulation via S -glutathionylation, i.e., the conjugation of GSH to reactive protein cysteine residues. A number of methodologies that enable quantitative analysis of GSH/GSSG ratio and S-glutathionylated proteins (PSSG), as well as identification and visualization of PSSG in tissue sections or cultured cells are currently available. Here, we have considered the main methodologies applied for GSH, GSSG and PSSG detection in biological samples. This review paper provides an up-to-date critical overview of the application of the most relevant analytical, morphological, and proteomics approaches to detect and analyse GSH, GSSG and PSSG in mammalian samples as well as discusses their current limitations.

Protein Carbonylation in Human Smokers and Mammalian Models of Exposure to Cigarette Smoke: Focus on Redox Proteomic Studies.

SIGNIFICANCE: Oxidative stress is one mechanism whereby tobacco smoking affects human health, as reflected by increased levels of several biomarkers of oxidative stress/damage isolated from tissues and biological fluids of active and passive smokers. Many investigations of cigarette smoke (CS)-induced oxidative stress/damage have been carried out in mammalian animal and cellular models of exposure to CS. Animal models allow the investigation of many parameters that are similar to those measured in human smokers. In vitro cell models may provide new information on molecular and functional differences between cells of smokers and nonsmokers. Recent Advances: Over the past decade or so, a growing number of researches highlighted that CS induces protein carbonylation in different tissues and body fluids of smokers as well as in in vivo and in vitro models of exposure to CS. CRITICAL ISSUES: We review recent findings on protein carbonylation in smokers and models thereof, focusing on redox proteomic studies. We also discuss the relevance and limitations of these models of exposure to CS and critically assess the congruence between the smoker's condition and laboratory models. FUTURE DIRECTIONS: The identification of protein targets is crucial for understanding the mechanism(s) by which carbonylated proteins accumulate and potentially affect cellular functions. Recent progress in redox proteomics allows the enrichment, identification, and characterization of specific oxidative protein modifications, including carbonylation. Therefore, redox proteomics can be a powerful tool to gain new insights into the onset and/or progression of CS-related diseases and to develop strategies to prevent and/or treat them. Antioxid. Redox Signal. 26, 406-426.

Dietary flavonoids advance timing of moult but do not affect redox status of juvenile blackbirds (Turdus merula).

Flavonoids are the most abundant plant polyphenols, widely occurring in fruits and berries, and show a strong antioxidant activity in vitro Studies of avian species feeding on berries suggest that dietary flavonoids have health-promoting effects and may enhance the expression of melanin-based plumage traits. These effects are probably mediated by the antioxidant activity of flavonoids. However, the effect of dietary flavonoids on oxidative status has never been investigated in any bird species. We analysed the effects of dietary flavonoids on blood non-enzymatic antioxidants and protein oxidative damage of juvenile European blackbirds (Turdus merula). In addition, we analysed the effects of flavonoid-enriched diet on body condition and on the timing of moult from juvenile to adult plumage. Dietary flavonoids did not significantly affect redox status but significantly advanced the onset of moult, hastening plumage development. Moulting birds showed higher protein oxidative damage compared with those that had not yet started moulting. The probability of initiating moult after 40 days of dietary treatment was higher for birds with low circulating levels of oxidizing agents and high glutathione concentration. The metabolization of flavonoids could have altered their redox potential, resulting in no net effects on redox status. However, flavonoid consumption before and during moult may contribute to enhance plumage development. Moreover, our findings suggest that moulting feathers may result in redox imbalance. Given their effect on moult and growth of melanin-rich feathers, fruit flavonoids may have contributed to shape plant fruiting time in relation to fruit consumption preferences by birds.

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room.

Glutathione (GSH) is the most abundant low-molecular-mass thiol within cells and one of the major antioxidant compounds in body fluids. Under pro-oxidant conditions, two GSH molecules donate one electron each and are converted into glutathione disulfide (GSSG). The GSH/GSSG molar ratio is considered a powerful index of oxidative stress and disease risk. Despite high interest in GSH/GSSG titration as measures of thiol redox balance, no broad agreement has yet been reached as to the best pre-analytical and analytical methods for the quantitation of these molecules in biological samples. Consequently, measured concentrations of GSH and GSSG and calculated GSH/GSSG molar ratios vary widely among laboratories. Here, we describe in detail the main analytical and pre-analytical problems related to the artificial oxidation of the sulfhydryl (SH) group of GSH that occur during sample manipulation. We underline how this aspect has been neglected for long time after its first description more than fifty years ago. Finally, selected reliable procedures and methods to measure GSH and GSSG in biological samples are discussed.

Identification of dityrosine cross-linked sites in oxidized human serum albumin.

Reactive oxygen species (ROS) can oxidize virtually all cellular components. In proteins cysteine, methionine, tryptophan, and tyrosine residues are most prone to oxidation and their oxidized forms are thus considered as biomarkers of oxidative protein damages. Ultraviolet radiation and some endogenous ROS can produce tyrosine radicals reacting with other tyrosine residues yielding intra- or intermolecular cross-links in proteins. These 3,3'-dityrosines can be quantified by their characteristic fluorescence, but analytical methods to identify the modification sites in proteins are still missing. Although mass spectrometry (MS) is routinely used to map other post-translational modifications, the analysis of dityrosines is challenged by simultaneous fragmentations of both cross-linked peptide chains producing complex tandem mass spectra. Additionally, the fragmentation patterns differ from linear peptides. Here, we studied the fragmentation behavior of dityrosine cross-linked peptides obtained by incubating three peptides (AAVYHHFISDGVR, TEVSSNHVLIYLDK, and LVAYYTLIGASGQR) with horseradish peroxidase in the presence of hydrogen peroxide. Homo- and hetero-dimerization via dityrosine was monitored by fluorescence spectroscopy and MS. The fragmentation characteristics of dityrosine-linked peptides were studied on an ESI-LTQ-Orbitrap-MS using collision induced dissociation, which allowed localizing the cross-linked positions and provided generic rules to identify this oxidative modification. When human serum albumin oxidized with 50-fold molar excess of HOCl in phosphate buffer saline was analyzed by nanoRPC-ESI-MS/MS, an automatic database search considering all possible (in-silico generated) tyrosine-containing peptides as dynamic modifications revealed four different types of oxidatively modified tyrosine residues including dityrosines linking ten different Tyr residues. The automatic database search was confirmed by manual interpretation of each tandem mass spectrum.

Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures.

The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 µM concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 µM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.

Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients.

The role of oxidative stress in patients with end stage renal disease (ESRD), which occurs at significantly higher levels than in the general population, is often underestimated in clinical practice. Emerging evidence highlights the strong correlation of oxidative stress with chronic inflammation and cardiovascular disease, which are highly prevalent in most patients on maintenance haemodialysis (HD) and are a major risk factor for mortality in this population. In this study, total plasma thiols and plasma S-thiolated proteins were measured in patients with ESRD, before and after a regular HD session, and compared to age-matched healthy subjects. We found a significant decrease in the level of total plasma thiols and, conversely, a significant increase in the level of S-thiolated proteins in these patients. In most patients, post-HD plasma level of total thiols did not differ from the one in healthy subjects, whereas plasma level of S-thiolated proteins was lower in HD patients than in age-matched healthy controls. This suggests that a single HD session restores plasma thiol redox status and re-establishes the antioxidant capacity of plasma thiols. Additionally, we determined protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to S-thiolated plasma proteins and protein free cysteinyl residues. Patients with ESRD had a significantly higher PTI compared to age-matched healthy subjects and HD was associated with a decrease in PTI to normal, or lower than normal, levels. Although this study is limited in size, our results suggest that PTI is a useful indicator of thiol-specific oxidative stress in patients with ESRD on maintenance HD. This study also emphasizes that PTI determination is a cheap and simple tool suitable for large-scale clinical studies that could be used for routine screening of thiol-specific oxidative stress.

Cigarette smoke induces alterations in the drug-binding properties of human serum albumin.

Albumin is the most abundant plasma protein and serves as a transport and depot protein for numerous endogenous and exogenous compounds. Earlier we had shown that cigarette smoke induces carbonylation of human serum albumin (HSA) and alters its redox state. Here, the effect of whole-phase cigarette smoke on HSA ligand-binding properties was evaluated by equilibrium dialysis and size-exclusion HPLC or tryptophan fluorescence. The binding of salicylic acid and naproxen to cigarette smoke-oxidized HSA resulted to be impaired, unlike that of curcumin and genistein, chosen as representative ligands. Binding of the hydrophobic fluorescent probe 4,4'-bis(1-anilino-8-naphtalenesulfonic acid) (bis-ANS), intrinsic tryptophan fluorescence, and susceptibility to enzymatic proteolysis revealed slight changes in albumin conformation. These findings suggest that cigarette smoke-induced modifications of HSA may affect the binding, transport and bioavailability of specific ligands in smokers.