Ian A. Cotgreave

N-acetylcysteine: pharmacological considerations and experimental and clinical applications.

The diversity of application of the thiol drug NAC in both the experimental setting, as a tool for the study of the mechanisms and consequences of oxidative stress, and the clinical setting, as a therapeutic agent, clearly reflects the central role played by the redox chemistries of the group XVI elements, oxygen and sulfur, in biology. As our understanding of such redox processes increases, particularly their roles in specific pathophysiological processes, new avenues will open for the use of NAC in the clinical setting. As a drug, NAC represents perhaps the ideal xenobiotic, capable of directly entering endogenous biochemical processes as a result of its own metabolism. Thus, it is hoped that the experience gained with this unique agent will help in future efforts to design antioxidants and chemoprotective principles which are able to more accurately utilize endogenous biochemical processes for cell- or tissue-specific therapy.

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. The method is based on the specific reduction of mixed disulfides by glutaredoxin, their reaction with N-ethylmaleimide-biotin, affinity purification of tagged proteins, and identification by proteomic analysis. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These include protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. Comparisons of the patterns of S-glutathionylated proteins extracted from cells undergoing diamide-induced oxidative stress and during constitutive metabolism reveal both common protein substrates and substrates failing to undergo enhanced S-glutathionylation during oxidative stress. The ability to chemically tag, select, and identify S-glutathionylated proteins, particularly during constitutive metabolism, will greatly enhance efforts to establish posttranslational redox modification of cellular proteins as an important biochemical control mechanism in coordinating cellular function.

Methodologies for the application of monobromobimane to the simultaneous analysis of soluble and protein thiol components of biological systems.

A series of simple methodologies for the determination of the redox status of low molecular weight and protein thiols in biological systems is described. Based centrally upon the use of monobromobimane, we describe a standard in situ derivatisation procedure simultaneously resulting in maximal recovery of both free, reduced low molecular weight and bromobimane accessible protein thiols as their corresponding bimane adducts from intact biological systems. Test systems include isolated and cultured cells, tissue homogenates and body fluids such as blood plasma. Quantitation of the bimane adducts of cysteine and glutathione is achieved by reversed phase high performance liquid chromatography, whereas quantitation of the corresponding adducts of protein thiols is achieved by fluorescence spectroscopy following protein precipitation. Full validation data for quantitative estimates are described. Additionally we have coupled these procedures to prederivatization denaturation treatments of biological protein samples in order to quantitate pools of protein thiols which are inaccessible to bromobimane in samples of native protein. We have also coupled these procedures with prederivatization reductions of biological systems under study with dithiothreitol, rendering simultaneously both oxidized low molecular weight thiols and oxidized protein thiols accessible to derivatisation with monobromobimane. Thus, we have obtained quantitative determinations of cysteine and glutathione present in mixed disulfides with protein and in soluble low molecular weight disulfides and estimates of intraprotein disulfides in a number of test biological systems.

Lung Protection by a Thiol-Containing Antioxidant: N-Acetylcysteine

N-acetylcysteine (NAC) is a thiol-containing compound which nonenzymatically interacts and detoxifies reactive electrophiles and free radicals. NAC was shown to effectively protect human bronchial fibroblasts against the toxic effects of tobacco smoke condensates and the isolated perfused lung against the glutathione (GSH)-depleting effect of tobacco smoke. NAC was also shown to reduce the reactive oxygen intermediate hydrogen peroxide (H2O2) and protect against the toxic effects of H2O2. In vivo studies, however, demonstrated that NAC when administered orally has very low bioavailability due to rapid metabolism to GSH among other metabolites. Thus, even though NAC is very effective in protecting cells of different origins from the toxicity of reactive components in tobacco smoke and reactive oxygen species, a direct scavenging effect by NAC in vivo, particularly when administered orally, does not seem likely. The bioavailability of NAC itself is very low when given this route. A more relevant mechanism in vivo for any protective effect NAC may exert against toxic species may be due to NAC acting as a precursor of GSH and facilitating its biosynthesis. GSH will then serve as the protective agent and detoxify reactive species both enzymatically and nonenzymatically.

Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli

Abstract Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c–mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.

Skeletal muscle glutathione is depleted in critically ill patients.

OBJECTIVE To investigate the concentrations of reduced and total glutathione in relation to the muscle free amino acid pattern in critically ill patients and matched healthy controls. DESIGN Prospective case control. SETTING University hospital intensive care unit (ICU). PATIENTS Eleven critically ill patients in the intensive care unit were studied after a stay of at least 4 days. Eleven age- and gender-matched metabolically healthy patients undergoing elective surgical procedures served as controls. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Reduced and total glutathione concentrations were determined in skeletal muscle, in plasma, and in whole blood, together with muscle free amino acid concentrations. In the ICU group, reduced and total glutathione values were 57% and 62%, respectively, of the values seen in the control group (p < .001). In addition, a decreased ratio between reduced and total glutathione compared with the controls was seen (0.80 as compared with 0.91, p < .001). The glutamine concentration in skeletal muscle in the ICU group was 72% lower compared with that value seen in healthy controls (p < .001). Correlations were found between the concentrations of glutamine and the total muscle glutathione (r2 = .46, p < .001), as well as between glutamine and the ratio of reduced and total glutathione (r2 = .45, p < .001) in skeletal muscle, suggesting that the redox status of glutathione and the glutamine status of the tissue are related. CONCLUSIONS Critical illness is associated with alterations in muscle glutathione metabolism. The muscle-reduced glutathione concentrations decrease and, in addition, the ratio between reduced and total glutathione decreases, indicating a situation of oxidative stress in this tissue. This decrease may impair the defense of muscle against oxygen free radicals and influence amino acid transport, thus contributing to the loss of balance between protein synthesis and protein degradation that is characteristic of protein catabolism.

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

Abstract 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. The A549 showed slow turnover rates of adducts in RNA and DNA giving half-lives of approximately 12.5 h for [18O]-8-oxoGuo in RNA and 20.7 h for [18O]-8-oxodGuo in DNA, respectively.

Studies on the anti-inflammatory activity of Ebselen: Ebselen interferes with granulocyte oxidative burst by dual inhibition of NADPH oxidase and protein kinase C?

Ebselen (PZ51, 2-phenyl-1,2-benzoisoselenazol-3-(2H)-one) was shown to be an inhibitor of human granulocyte oxidative burst stimulated by phorbol myristate acetate (IC50 25 microM). Estimation of the primary oxygen metabolites of the burst was complicated by the redox chemistry of Ebselen. Ebselen inhibited NADPH-stimulated superoxide generation by a partially purified NADPH oxidase preparation with an IC50 of 0.5-1.0 microM. Ebselen was also shown to inhibit the activity of partially purified Ca2+- and phospholipid-dependent protein kinase C (IC50 ca. 0.5 microM). Phorbol ester-stimulated phosphorylation of protein in intact cells was inhibited by Ebselen (IC50 ca. 50 microM). These pharmacodynamic properties of Ebselen are discussed in terms of its anti-inflammatory activity in vivo. The findings are also discussed in terms of Ebselens known ability to interact with sulfhydryl components of cells, particularly critical thiol components of the enzymes studied.

[48] N-acetylcysteine

Publisher Summary N-Acetylcysteine (NAC) is a thiol-containing compound that has been used in clinical practice for several years. Historically, NAC was introduced for the treatment of congestive and obstructive lung diseases; primarily those associated with hypersecretion of mucus, for example, chronic bronchitis and cystic fibrosis. NAC has also been used as the drug of choice in the treatment of paracetamol intoxication. Currently, NAC and its applications include use in the treatment of pulmonary oxygen toxicity, adult respiratory distress syndrome (ARDS), and, potentially, cases of human immunodeficiency virus (HIV-I) infections. This diversity of pharmacological applications of NAC is because of the multifaceted chemical properties of the cysteinyl thiol of the molecule. These include its nucleophilicity and redox activity, providing scavenger and antioxidant properties and its ability to undergo transhydrogenation or thiol-disulfide exchange (TDE) reactions with other thiol redox couples. Advances in the clinical use of NAC have stemmed mainly from the development of suitable analytical techniques for the analysis of NAC and potential metabolites in biological systems. These techniques are being used to determine the human pharmacokinetic behavior of NAC and its metabolic disposition. This chapter describes the analytical concepts that are currently available for the determination of NAC and metabolites in biological fluids. It also presents the available data on the disposition of NAC in humans and discusses the biological properties of NAC. It then discusses some future possibilities of NAC in clinical practice.

A genetic polymorphism in connexin 37 as a prognostic marker for atherosclerotic plaque development

Abstract. Boerma M, Forsberg L, van Zeijl L, Morgenstern R, de Faire U, Lemne C, Erlinge D, Thulin T, Hong Y, Cotgreave IA (Karolinska Institute and Karolinska Hospital, Stockholm; Lund University, Lund, Sweden. Washington University School of Medicine, MO, USA). A genetic polymorphism in connexin 37 as a prognostic marker for atherosclerotic plaque development. J Intern Med 1999; 246: 211–218.