The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5'-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The glutathione disulfide (GSSG) formed can be recycled to GSH by glutathione reductase in the presence of NADPH. The assay is composed of two parts: the preparation of cell cytosolic/tissue extracts and the detection of total glutathione (GSH and GSSG). The method is simple, convenient, sensitive and accurate. The lowest detection for GSH and GSSG is 0.103 nM in a 96-well plate. This method is rapid and the whole procedure takes no longer than 15 min including reagent preparation. The method can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell extracts and can be extended for drug discovery/pharmacology and toxicology protocols to study the effects of drugs and toxic compounds on glutathione metabolism.

Oxidative stress and inflammatory response are the key events in the pathogenesis of chronic airway diseases. The consumption of electronic cigarettes (e-cigs) with a variety of e-liquids/e-juices is alarmingly increasing without the unrealized potential harmful health effects. We hypothesized that electronic nicotine delivery systems (ENDS)/e-cigs pose health concerns due to oxidative toxicity and inflammatory response in lung cells exposed to their aerosols. The aerosols produced by vaporizing ENDS e-liquids exhibit oxidant reactivity suggesting oxidants or reactive oxygen species (OX/ROS) may be inhaled directly into the lung during a “vaping” session. These OX/ROS are generated through activation of the heating element which is affected by heating element status (new versus used), and occurs during the process of e-liquid vaporization. Unvaporized e-liquids were oxidative in a manner dependent on flavor additives, while flavors containing sweet or fruit flavors were stronger oxidizers than tobacco flavors. In light of OX/ROS generated in ENDS e-liquids and aerosols, the effects of ENDS aerosols on tissues and cells of the lung were measured. Exposure of human airway epithelial cells (H292) in an air-liquid interface to ENDS aerosols from a popular device resulted in increased secretion of inflammatory cytokines, such as IL-6 and IL-8. Furthermore, human lung fibroblasts exhibited stress and morphological change in response to treatment with ENDS/e-liquids. These cells also secrete increased IL-8 in response to a cinnamon flavored e-liquid and are susceptible to loss of cell viability by ENDS e-liquids. Finally, exposure of wild type C57BL/6J mice to aerosols produced from a popular e-cig increase pro-inflammatory cytokines and diminished lung glutathione levels which are critical in maintaining cellular redox balance. Thus, exposure to e-cig aerosols/juices incurs measurable oxidative and inflammatory responses in lung cells and tissues that could lead to unrealized health consequences.

Reactive oxygen species, either directly or via the formation of lipid peroxidation products, may play a role in enhancing inflammation through the activation of stress kinases (cJun activated kinase, extracellular signal-regulated kinase, p38) and redox-sensitive transcription factors, such as nuclear factor (NF)-kB and activator protein-1. This results in increased expression of a battery of distinct pro-inflammatory mediators. Oxidative stress activates NF-kBmediated transcription of pro-inflammatory mediators either through activation of its activating inhibitor of kB-a kinase or the enhanced recruitment and activation of transcriptional coactivators. Enhanced NF-kB-co-activator complex formation results in targeted increases in histone modifications, such as acetylation leading to inflammatory gene expression.Emerging evidence suggests the glutathione redox couple may entail dynamic regulation of protein function by reversible disulphide bond formation on kinases, phosphatases and transcription factors. Oxidative stress also inhibits histone deacetylase activity and in doing so further enhances inflammatory gene expression and may attenuate glucocorticoid sensitivity.The antioxidant/anti-inflammatory effects of thiol molecules (glutathione, N-acetyl-L-cysteine and N-acystelyn, erdosteine), dietary polyphenols (curcumin-diferuloylmethane, cathechins/ quercetin and reserveratol), specific spin traps, such as a-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (extracellular superoxide dismutase (SOD) mimetic, SOD mimetic M40419 and SOD, and catalase manganic salen compound, eukarion-8), porphyrins (AEOL 10150 and AEOL 10113) and theophylline have all been shown to play a role in either controlling NF-kB activation or affecting histone modifications with subsequent effects on inflammatory gene expression in lung epithelial cells.Thus, oxidative stress regulates both key signal transduction pathways and histone modifications involved in lung inflammation. Various approaches to enhance lung antioxidant capacity and clinical trials of antioxidant compounds in chronic obstructive pulmonary disease are also discussed.

No abstract

Inflammatory lung diseases are characterized by chronic inflammation and oxidant/antioxidant imbalance, a major cause of cell damage. The development of an oxidant/antioxidant imbalance in lung inflammation may activate redox‐sensitive transcription factors such as nuclear factor‐κB, and activator protein‐1 (AP‐1), which regulate the genes for pro‐inflammatory mediators and protective antioxidant genes. Glutathione (GSH), a ubiquitous tripeptide thiol, is a vital intra‐ and extracellular protective antioxidant against oxidative/nitrosative stresses, which plays a key role in the control of pro‐inflammatory processes in the lungs. Recent findings have suggested that GSH is important in immune modulation, remodelling of the extracellular matrix, apoptosis and mitochondrial respiration. The rate‐limiting enzyme in GSH synthesis is γ‐glutamylcysteine synthetase (γ‐GCS). The human γ‐GCS heavy and light subunits are regulated by AP‐1 and antioxidant response elements and are modulated by oxidants, phenolic antioxidants, growth factors, and inflammatory and anti‐inflammatory agents in lung cells. Alterations in alveolar and lung GSH metabolism are widely recognized as a central feature of many inflammatory lung diseases such as idiopathic pulmonary fibrosis, acute respiratory distress syndrome, cystic fibrosis and asthma. The imbalance and/or genetic variation in antioxidant γ‐GCS and pro‐inflammatory versus antioxidant genes in response to oxidative stress and inflammation in some individuals may render them more susceptible to lung inflammation. Knowledge of the mechanisms of GSH regulation and balance between the release and expression of pro‐ and anti‐inflammatory mediators could lead to the development of novel therapies based on the pharmacological manipulation of the production as well as gene transfer of this important antioxidant in lung inflammation and injury. This review describes the redox control and involvement of nuclear factor‐κB and activator protein‐1 in the regulation of cellular glutathione and γ‐glutamylcysteine synthetase under conditions of oxidative stress and inflammation, the role of glutathione in oxidant‐mediated susceptibility/tolerance, γ‐glutamylcysteine synthetase genetic susceptibility and the potential therapeutic role of glutathione and its precursors in protecting against lung oxidant stress, inflammation and injury.