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.

Biological mechanism of cell oxidative stress and death during short-term exposure to nano CuO.

It is well known that copper oxide nanoparticles (CuO NPs) are heavily toxic on in vitro systems. In human alveolar epithelial cells, the mechanism of toxicity is mostly related to oxidative insults, coming from intracellularly dissolved copper ions, finally leading to apoptotic or autophagic cell death. Our hypothesis is based on possible early oxidative events coming from specific NP surface reactivity able to undermine the cell integrity and to drive cell to death, independently from Lysosomal-Enhanced Trojan Horse mechanism. Two types of CuO NPs, with different oxidative potential, were selected and tested on A549 cells for 1 h and 3 h at 10, 25, 50 and 100 µg/ml. Cells were then analyzed for viability and oxidative change of the proteome. Oxidative by-products were localized by immunocytochemistry and cell-NP interactions characterized by confocal and electron microscopy techniques. The results show that CuO NPs induced oxidative changes soon after 1 h exposure as revealed by the increase in protein carbonylation and reduced-protein-thiol oxidation. In parallel, cell viability significantly decreased, as shown by MTT assay. Such effects were higher for CuO NPs with more crystalline defects and with higher ROS production than for fully crystalline NPs. At these exposure times, although NPs efficiently interacted with cell surface and were taken up by small endocytic vesicles, no ion dissolution was visible inside the lysosomal compartment and no effects were produced by extracellularly dissolved copper ions. In conclusion, a specific NP surface-dependent oxidative cell injury was demonstrated. More detailed studies are required to understand which targets precociously react with CuO NPs, but these results introduce new paradigms for the toxicity of the metal-based NPs, beyond the Lysosomal-Enhanced Trojan horse-related mechanism, and open-up new opportunities to investigate the interactions and effects at the bio-interface for designing safer as well as more effective CuO-based biocides.

Effects of the uremic toxin indoxyl sulphate on human microvascular endothelial cells.

Indoxyl sulphate (IS) is a uremic toxin accumulating in the plasma of chronic kidney disease (CKD) patients. IS accumulation induces side effects in the kidneys, bones and cardiovascular system. Most studies assessed IS effects on cell lines by testing higher concentrations than those measured in CKD patients. Differently, we exposed a human microvascular endothelial cell line (HMEC-1) to the IS concentrations measured in the plasma of healthy subjects (physiological) or CKD patients (pathological). Pathological concentrations reduced cell proliferation rate but did not increase long-term oxidative stress level. Indeed, total protein thiols decreased only after 24 h of exposure in parallel with an increased Nrf-2 protein expression. IS induced actin cytoskeleton rearrangement with formation of stress fibres. Proteomic analysis supported this hypothesis as many deregulated proteins are related to actin filaments organization or involved in the endothelial to mesenchymal transition. Interestingly, two proteins directly linked to cardiovascular diseases (CVD) in in vitro and in vivo studies underwent deregulation: COP9 signalosome complex subunit 9 and thrombomodulin. Future experiments will be needed to investigate the role of these proteins and the signalling pathways in which they are involved to clarify the possible link between CKD and CVD.

Blood Thiol Redox State in Chronic Kidney Disease.

Thiols (sulfhydryl groups) are effective antioxidants that can preserve the correct structure of proteins, and can protect cells and tissues from damage induced by oxidative stress. Abnormal levels of thiols have been measured in the blood of patients with moderate-to-severe chronic kidney disease (CKD) compared to healthy subjects, as well as in end-stage renal disease (ESRD) patients on haemodialysis or peritoneal dialysis. The levels of protein thiols (a measure of the endogenous antioxidant capacity inversely related to protein oxidation) and S-thiolated proteins (mixed disulphides of protein thiols and low molecular mass thiols), and the protein thiolation index (the molar ratio of the S-thiolated proteins to free protein thiols in plasma) have been investigated in the plasma or red blood cells of CKD and ESRD patients as possible biomarkers of oxidative stress. This type of minimally invasive analysis provides valuable information on the redox status of the less-easily accessible tissues and organs, and of the whole organism. This review provides an overview of reversible modifications in protein thiols in the setting of CKD and renal replacement therapy. The evidence suggests that protein thiols, S-thiolated proteins, and the protein thiolation index are promising biomarkers of reversible oxidative stress that could be included in the routine monitoring of CKD and ESRD patients.

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.

Effects of Physiological and Pathological Urea Concentrations on Human Microvascular Endothelial Cells.

Urea is the uremic toxin accumulating with the highest concentration in the plasma of chronic kidney disease (CKD) patients, not being completely cleared by dialysis. Urea accumulation is reported to exert direct and indirect side effects on the gastrointestinal tract, kidneys, adipocytes, and cardiovascular system (CVS), although its pathogenicity is still questioned since studies evaluating its side effects lack homogeneity. Here, we investigated the effects of physiological and pathological urea concentrations on a human endothelial cell line from the microcirculation (Human Microvascular Endothelial Cells-1, HMEC-1). Urea (5 g/L) caused a reduction in the proliferation rate after 72 h of exposure and appeared to be a potential endothelial-to-mesenchymal transition (EndMT) stimulus. Moreover, urea induced actin filament rearrangement, a significant increase in matrix metalloproteinases 2 (MMP-2) expression in the medium, and a significant up- or down-regulation of other EndMT biomarkers (keratin, fibrillin-2, and collagen IV), as highlighted by differential proteomic analysis. Among proteins whose expression was found to be significantly dysregulated following exposure of HMEC-1 to urea, dimethylarginine dimethylaminohydrolase (DDAH) and vasorin turned out to be down-regulated. Both proteins have been directly linked to cardiovascular diseases (CVD) by in vitro and in vivo studies. Future experiments will be needed to deepen their role and investigate the signaling pathways in which they are involved to clarify the possible link between CKD and CVD.

Protein thiolation index in microvolumes of plasma.

Protein Thiolation Index (PTI) has been recently proposed as a new biomarker of oxidative stress. It is calculated by measuring both free thiols and S-thiolated proteins in plasma with the assumption that this redox ratio is altered by a pro-oxidant stimulus. Here the original protocol was modified and adapted to the use of microvolumes of blood collected by finger prick and down to 3 µl blood was shown to be the lowest volume suitable for this kind of analysis. The new procedure was used to evaluate both the circadian rhythm and the annual fluctuations of PTI in healthy humans.

In vitro copper oxide nanoparticle toxicity on intestinal barrier.

The use of CuO nanoparticles (NPs) has increased greatly and their potential effects on human health need to be investigated. Differentiated Caco-2 cells were treated from the apical (Ap) and the basolateral (Bl) compartment with different concentrations (0, 10, 50 and 100 µg/mL) of commercial or sonochemically synthesized (sono) CuO NPs. Sono NPs were prepared in ethanol (CuOe) or in water (CuOw), obtaining CuO NPs differing in size and shape. The effects on the Caco-2 cell barrier were assessed via transepithelial electrical resistance (TEER) evaluation just before and after 1, 2 and 24 hours of exposure and through the analysis of cytokine release and biomarkers of oxidative damage to proteins after 24 hours. Sono CuOe and CuOw NPs induced a TEER decrease with a dose-dependent pattern after Bl exposure. Conversely, TEER values were not affected by the Ap exposure to commercial CuO NPs and, concerning the Bl exposure, only the lowest concentration tested (10 µg/mL) caused a TEER decrease after 24 hours of exposure. An increased release of interleukin-8 was induced by sono CuO NPs after the Ap exposure to 100 µg/mL and by sono and commercial CuO after the Bl exposure to all the concentrations. No effects of commercial and sono CuO NPs on interleukin-6 (with the only exception of 100 µg/mL Bl commercial CuO) and tumor necrosis factor-α release were observed. Ap treatment with commercial and CuOw NPs was able to induce significant alterations on specific biomarkers of protein oxidative damage (protein sulfhydryl group oxidation and protein carbonylation).

Plasma Protein Carbonyls as Biomarkers of Oxidative Stress in Chronic Kidney Disease, Dialysis, and Transplantation.

Accumulating evidence indicates that oxidative stress plays a role in the pathophysiology of chronic kidney disease (CKD) and its progression; during renal replacement therapy, oxidative stress-derived oxidative damage also contributes to the development of CKD systemic complications, such as cardiovascular disease, hypertension, atherosclerosis, inflammation, anaemia, and impaired host defence. The main mechanism underlying these events is the retention of uremic toxins, which act as a substrate for oxidative processes and elicit the activation of inflammatory pathways targeting endothelial and immune cells. Due to the growing worldwide spread of CKD, there is an overwhelming need to find oxidative damage biomarkers that are easy to measure in biological fluids of subjects with CKD and patients undergoing renal replacement therapy (haemodialysis, peritoneal dialysis, and kidney transplantation), in order to overcome limitations of invasive monitoring of CKD progression. Several studies investigated biomarkers of protein oxidative damage in CKD, including plasma protein carbonyls (PCO), the most frequently used biomarker of protein damage. This review provides an up-to-date overview on advances concerning the correlation between plasma protein carbonylation in CKD progression (from stage 1 to stage 5) and the possibility that haemodialysis, peritoneal dialysis, and kidney transplantation improve plasma PCO levels. Despite the fact that the role of plasma PCO in CKD is often underestimated in clinical practice, emerging evidence highlights that plasma PCO can serve as good biomarkers of oxidative stress in CKD and substitutive therapies. Whether plasma PCO levels merely serve as biomarkers of CKD-related oxidative stress or whether they are associated with the pathogenesis of CKD complications deserves further evaluation.

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.

Advanced oxidation protein products in nondiabetic end stage renal disease patients on maintenance haemodialysis.

In chronic kidney disease (CKD), the impairment of the excretory function leads to elevation in the blood concentrations of urea, creatinine, and various protein metabolic products. Advanced oxidation protein products (AOPP), along with protein carbonyls, protein-bound di-tyrosines and S-thiolated proteins, are considered biomarkers of oxidative stress in end-stage renal disease (ESRD) patients on maintenance haemodialysis (HD). In this study, we evaluated the correlations between plasma levels of AOPP (measured by size exclusion/gel filtration high performance liquid chromatography) and those of protein-bound di-tyrosines, protein carbonyls, albumin and fibrinogen in 50 nondiabetic ESRD patients on maintenance HD. Considering that AOPP could represent the bridge between oxidative stress and inflammation, having been identified as proinflammatory mediators, we also evaluated the association between AOPP levels, C-reactive protein concentration and white blood cells count. Finally, we assessed the associations between plasma level of AOPP and serum concentrations of creatinine and urea, both of which showed a strong dependence on the chronological age of haemodialysed patients. Taken together, our results confirm the robust relationship between uraemia and oxidative stress, especially when measured as biomarkers of severe protein oxidative damage (e.g. plasma AOPP).

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.

Cytotoxic and proinflammatory responses induced by ZnO nanoparticles in in vitro intestinal barrier.

ZnO nanoparticles (NPs) are widely used nowadays, thus the gastrointestinal exposure to ZnO NPs is likely to be relevant and the effects on the intestinal barrier should be investigated. Polarized Caco-2 cells were exposed from the apical (Ap) and basolateral (Bl) compartments to increasing concentrations (0, 10, 50 and 100 µg/mL) of sonochemical (sono) and commercial ZnO NPs. The transepithelial electrical resistance (TEER), cell viability, proinflammatory cytokine release and presence of protein oxidative damage were evaluated after exposure. TEER was not significantly affected by Ap exposure to either sono or commercial ZnO NPs at any tested concentrations. After Bl exposure to sono ZnO NPs (all the concentrations) and to 100 µg/mL of commercial ZnO NPs TEER was decreased (P < 0.05). Ap and Bl exposure to 100 µg/mL sono ZnO NPs and Ap exposure to 50 µg/mL commercial ZnO NPs induced a significant (P < 0.05) release of interleukin-6. A significant (P < 0.05) release of interleukin-8 was observed after Ap exposure to ZnO NPs at 100 µg/mL and after Bl exposure to sono ZnO NPs at 100 µg/mL. Ap or Bl exposure to sono or commercial ZnO NPs did not affect tumour necrosis factor-alpha secretion or protein sulphydryl oxidation. In conclusion, the ZnO NP exposure from the Ap compartment appeared almost safe, while the exposure through the basal compartment appeared to be more hazardous and the different NP size and crystallinity seem to affect the mode of action, but further studies are necessary to elucidate better these toxicity mechanisms.

Sulforaphane Cannot Protect Human Fibroblasts From Repeated, Short and Sublethal Treatments with Hydrogen Peroxide.

A delicate balance of reactive oxygen species (ROS) exists inside the cell: when the mechanisms that control the level of ROS fail, the cell is in an oxidative stress state, a condition that can accelerate aging processes. To contrast the pro-aging effect of ROS, the supplementation of antioxidants has been recently proposed. Sulforaphane (SFN) is an isothiocyanate isolated from Brassica plants that has been shown to modulate many critical factors inside the cells helping to counteract aging processes. In the present work, we exposed human dermal fibroblast to short, sublethal and repeated treatments with hydrogen peroxide for eight days, without or in combination with low concentration of SFN. Hydrogen peroxide treatments did not affect the oxidative status of the cells, without any significant change of the intracellular ROS levels or the number of mitochondria or thiols in total proteins. However, our regime promoted cell cycle progression and cell viability, increased the anti-apoptotic factor survivin and increased DNA damage, measured as number of foci positive for γ -H2AX. On the other hand, the treatment with SFN alone seemed to exert a protective effect, increasing the level of p53, which can block the expansion of possible DNA damaged cells. However, continued exposure to SFN at this concentration could not protect the cells from stress induced by hydrogen peroxide.

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.