H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides.

Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs), we have studied the phosphorylation of H2AX (H2AXgamma).

Determination of site-specificity of S-glutathionylated cellular proteins.

Redox modification by S-glutathionylation is an expanding field within cell signalling research. However, the methods available for analysis of S-glutathionylated proteins in complex mixtures are not sufficiently accurate to specifically and in a high-throughput manner on a structural level establish the effects of S-glutathionylation on the individual proteins. A method has been developed for rapid identification of the S-glutathionylation sites of proteins in diamide-treated ECV304 cells, through tagging of deglutathionylated proteins with a cysteine-reactive biotin-affinity tag, trypsinisation, avidin-affinity purification of tagged peptides, and subsequent analysis by liquid chromatography and quadrupole time-of-flight tandem mass spectrometry.

Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA.

Human A549 lung epithelial cells were challenged with 18O-labeled hydrogen peroxide ([18O]-H2O2), the total RNA and DNA extracted in parallel, and analyzed for 18O-labeled 8-oxo-7,8-dihydroguanosine ([18O]-8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine ([18O]-8-oxodGuo) respectively, using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-MS/MS). [18O]-H2O2 exposure resulted in dose-response formation of both [18O]-8-oxoGuo and [18O]-8-oxodGuo and 18O-labeling of guanine in RNA was 14-25 times more common than in DNA. Kinetics of formation and subsequent removal of oxidized nucleic acids adducts were also monitored up to 24 h.

Biological stress responses to radio frequency electromagnetic radiation: are mobile phones really so (heat) shocking?

Cells phenotypically adapt to alterations in their intra- and extracellular environment via organised alterations to gene and protein expression. Many chemical and physical stimuli are known to drive such responses, including the induction of oxidative stress and heat shock. Increasing use of mobile telephones in our society, has brought focus on the potential for radio frequency (microwave) electromagnetic radiation to elicit biological stress responses, in association with potentially detrimental effects of this to human health.

Cyclodextrin-derived diorganyl tellurides as glutathione peroxidase mimics and inhibitors of thioredoxin reductase and cancer cell growth.

Water-soluble diorganyl tellurides of the alkyl aryl or dialkyl type were prepared by treatment of mono-6-tosyl-beta-cyclodextrin with sodium alkanetellurolates or arenetellurolates or sodium telluride. The novel cyclodextrin-derived organotelluriums were evaluated for their capacity to catalyze the reduction of hydrogen peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide in the presence of glutathione, NADPH, and GSSG-reductase (coupled reductase assay). Cyclodextrins 4d and 4e, carrying 4-(N,N-dimethylamino)phenyltelluro and n-butyltelluro groups, respectively, were the most efficient glutathione peroxidase mimics.

Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells.

Fluorescence microscopy of A549 cells stained with a glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the peri-nuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol-reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)-dependent inhibition of GSH synthesis.

Protein S-glutathionylation correlates to selective stress gene expression and cytoprotection.

During situations of oxidative stress phenotypic adaptation to altered redox state is achieved by changes in expression of selected genes. The mechanisms regulating this may involve reversible S-glutathionylation of cellular proteins. In this study we compared and contrasted changes in gene expression patterns in human type II lung epithelial A549 cells and human endothelial ECV304 cells in correlation to glutathione oxidation and the formation of glutathione-protein mixed disulphides, after exposure to subtoxic levels of hydrogen peroxide, formed in the medium by addition of glucose oxidase, or the thiol oxidant diamide.

Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis.

Redox modification of proteins is proposed to play a central role in regulating cellular function. However, high-throughput techniques for the analysis of the redox status of individual proteins in complex mixtures are lacking. The aim was thus to develop a suitable technique to rapidly identify proteins undergoing oxidation of critical thiols by S-glutathionylation.

S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase: role of thiol oxidation and catalysis by glutaredoxin.

The findings in this article illustrate the complexity residing in the regulation of reversible S-glutathionylation of proteins, such as GAPDH. This is clearly reflected in the design of suitable experimental approaches designed to cope with the interaction of several redox-dependent factors. Clear interactions are demonstrated between oxidative modification of GAPDH and its subsequent S-glutathionylation.

Differentiation-specific alterations to glutathione synthesis in and hormonally stimulated release from human skeletal muscle cells.

Muscle atrophy and cachexia are associated with many human diseases. These catabolic states are often associated with the loss of glutathione (GSH), which is thought to contribute to the induction of oxidative stress within the muscle. Glutathione synthesis and secretary characteristics were studied in human skeletal muscle myoblasts and myotube-like cells derived from the myoblasts by growth factor restriction.

Novel Water-Soluble Diorganyl Tellurides with Thiol Peroxidase and Antioxidant Activity.

Novel water-soluble diaryl tellurides, alkyl aryl tellurides, and dialkyl tellurides carrying sulfopropyl groups were prepared and found to possess potent peroxide decomposing and chain-breaking antioxidative capacity. The dilithium, disodium, dipotassium, and bis-tetramethylammonium salts of bis(4-hydroxyphenyl) telluride (4) were treated with 2.3 equiv of 1,3-propanesultone in aqueous tert-butyl alcohol to give the corresponding salts 5 of bis-O-sulfopropylated diaryl telluride.