Saibal K. Biswas

Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method

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 cigarette smoke alter chromatin remodeling but differentially regulate NF-κB activation and proinflammatory cytokine release in alveolar epithelial cells

Oxidative stress is implicated in lung inflammation due to its effect on proinflammatory gene transcription. Changes in gene transcription depend on chromatin remodeling and the relative activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Alterations in the nuclear histone acetylation:deacetylation balance may result in uncontrolled transcription of specific proinflammatory genes. We studied the effect of hydrogen peroxide (H2O2) and cigarette smoke condensate (CSC) on histone acetylation:deacetylation in human alveolar epithelial cells (A549). H2O2 and CSC significantly increased acetylation of histone H4 proteins and were associated with decreased HDAC activity and HDAC2 levels in A549 cells. Also, the decreased HDAC2 activity was due to protein modification by aldehydes and nitric oxide products. Pretreatment of A549 cells with N‐acetyl‐L‐cysteine attenuated the oxidant‐mediated reduction in HDAC activity. Treatment of A549 cells with CSC did not cause nuclear factor‐κB (NF‐κB) activation or expression and release of either interleukin (IL)‐8 or IL‐6. However, H2O2, tumor necrosis factor‐α (TNF‐α), and IL‐1β significantly increased NF‐κB activation and expression of IL‐8 compared with control cells. Interestingly, CSC dose dependently inhibited TNF‐α‐ and IL‐ 1β‐mediated NF‐κB activation and IL‐8 expression. Thus, H2O2 and CSC enhance acetylation of histone proteins and decrease histone deacetylase activity but differentially regulate proinflammatory cytokine release in alveolar epithelial cells.

Environmental toxicity, redox signaling and lung inflammation: the role of glutathione.

Glutathione (gamma-glutamyl-cysteinyl-glycine, GSH) is the most abundant intracellular antioxidant thiol and is central to redox defense during oxidative stress. GSH metabolism is tightly regulated and has been implicated in redox signaling and also in protection against environmental oxidant-mediated injury. Changes in the ratio of the reduced and disulfide form (GSH/GSSG) can affect signaling pathways that participate in a broad array of physiological responses from cell proliferation, autophagy and apoptosis to gene expression that involve H(2)O(2) as a second messenger. Oxidative stress due to oxidant/antioxidant imbalance and also due to environmental oxidants is an important component during inflammation and respiratory diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, and asthma. It is known to activate multiple stress kinase pathways and redox-sensitive transcription factors such as Nrf2, NF-kappaB and AP-1, which differentially regulate the genes for pro-inflammatory cytokines as well as the protective antioxidant genes. Understanding the regulatory mechanisms for the induction of antioxidants, such as GSH, versus pro-inflammatory mediators at sites of oxidant-directed injuries may allow for the development of novel therapies which will allow pharmacological manipulation of GSH synthesis during inflammation and oxidative injury. This article features the current knowledge about the role of GSH in redox signaling, GSH biosynthesis and particularly the regulation of transcription factor Nrf2 by GSH and downstream signaling during oxidative stress and inflammation in various pulmonary diseases. We also discussed the current therapeutic clinical trials using GSH and other thiol compounds, such as N-acetyl-l-cysteine, fudosteine, carbocysteine, erdosteine in environment-induced airways disease.

Curcumin Restores Corticosteroid Function in Monocytes Exposed to Oxidants by Maintaining HDAC2

Oxidative stress as a result of cigarette smoking is an important etiologic factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), a chronic steroid-insensitive inflammatory disease of the airways. Histone deacetylase-2 (HDAC2), a critical component of the corticosteroid anti-inflammatory action, is impaired in lungs of patients with COPD and correlates with disease severity. We demonstrate here that curcumin (diferuloylmethane), a dietary polyphenol, at nanomolar concentrations specifically restores cigarette smoke extract (CSE)- or oxidative stress-impaired HDAC2 activity and corticosteroid efficacy in vitro with an EC(50) of approximately 30 nM and 200 nM, respectively. CSE caused a reduction in HDAC2 protein expression that was restored by curcumin. This decrease in HDAC2 protein expression was reversed by curcumin even in the presence of cycloheximide, a protein synthesis inhibitor. The proteasomal inhibitor, MG132, also blocked CSE-induced HDAC2 degradation, increasing the levels of ubiquitinated HDAC2. Biochemical and gene chip analysis indicated that curcumin at concentrations up to 1 muM propagates its effect via antioxidant-independent mechanisms associated with the phosphorylation-ubiquitin-proteasome pathway. Thus curcumin acts at a post-translational level by maintaining both HDAC2 activity and expression, thereby reversing steroid insensitivity induced by either CSE or oxidative stress in monocytes. Curcumin may therefore have potential to reverse steroid resistance, which is common in patients with COPD and asthma.

Non-invasive biomarkers of oxidative stress: reproducibility and methodological issues

Abstract Oxidative stress is the hallmark of various chronic inflammatory lung diseases. Increased concentrations of reactive oxygen species (ROS) in the lungs of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. Traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples or to examine the affected compartments, to the patients discomfort. As a consequence, there is a need for less or non-invasive approaches to measure oxidative stress. The collection of exhaled breath condensate (EBC) has recently emerged as a non-invasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. The biomarkers of oxidative stress such as H2O2, F2-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species have been successfully measured in EBC. The reproducibility, sensitivity and specificity of the methodologies used in the measurements of EBC oxidative stress biomarkers are discussed. Oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. EBC is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. It is expected that EBC can be exploited to detect specific levels of biomarkers and monitor disease severity in response to appropriate prescribed therapy/treatment.

Ergothioneine inhibits oxidative stress- and TNF-α-induced NF-κB activation and interleukin-8 release in alveolar epithelial cells

Oxidants and inflammatory mediators such as tumour necrosis factor-alpha (TNF-alpha) activate transcription factors such as NF-kappa B. Interleukin-8 (IL-8) is a ubiquitous inflammatory chemokine that mediates a multitude of inflammatory events in the lung. Ergothioneine is a naturally occurring thiol compound, which possesses antioxidant property. The aim of this study was to determine whether ergothioneine can inhibit the hydrogen peroxide (H(2)O(2))- and TNF-alpha-mediated activation of NF-kappa B and the release of IL-8 in human alveolar epithelial cells (A549). Treatment of A549 cells with H(2)O(2) (100 microM) and TNF-alpha (10 ng/ml) significantly increased NF-kappa B activation using a reporter assay. Ergothioneine inhibited both H(2)O(2)- and TNF-alpha-mediated activation of NF-kappa B. Both H(2)O(2) and TNF-alpha significantly increased IL-8 release, which was inhibited by pre-treatment of A549 cells with ergothioneine compared to the control untreated cells. Ergothioneine also abolished the transcriptional activation of IL-8 in an IL-8-chloramphenicol acetyltransferase (CAT) reporter system, transfected into A549 cells. This indicates a molecular mechanism for the anti-inflammatory effects of ergothioneine.

Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert neuroprotection by modulating the antioxidant system in rat hippocampal slices subjected to oxygen glucose deprivation

BackgroundThe major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. However, their mechanism of action was largely unknown. We therefore selected these herbs for the present study to test their neuroprotective ability and the associated mechanism in rat hippocampal slices subjected to oxygen-glucose deprivation (OGD).MethodsHippocampal Slices were subjected to OGD (oxygen glucose deprivation) and divided into 3 groups: control, OGD and OGD + drug treated. Cytosolic Cu-Zn superoxide dismutase (Cu-Zn SOD), reduced glutathione (GSH), glutathione peroxidase (GPx), nitric oxide (NO) was measured as nitrite (NO2) in the supernatant and protein assays were performed in the respective groups at various time intervals. EPR was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O2.-), hydroxyl radicals (. OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-NO and Sin-1 respectively. RT-PCR was performed for the three groups for GCLC, iNOS, Cu-Zn SOD and GAPDH gene expression.ResultsAll the three herbs were effective in elevating the GSH levels, expression of the gamma-glutamylcysteine ligase and Cu-Zn SOD genes. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. In addition all the three herbs significantly diminished the expression of iNOS gene after 48 hours which plays a major role in neuronal injury during hypoxia/ischemia.ConclusionsRC, FC and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression level and may be an effective therapeutic tool against ischemic brain damage.

Pharmacological and dietary antioxidant therapies for chronic obstructive pulmonary disease.

The progression and exacerbations of chronic obstructive pulmonary disease (COPD) are intimately associated with tobacco smoke/biomass fuel-induced oxidative and aldehyde/carbonyl stress. Alterations in redox signaling proinflammatory kinases and transcription factors, steroid resistance, unfolded protein response, mucus hypersecretion, extracellular matrix remodeling, autophagy/apoptosis, epigenetic changes, cellular senescence/aging, endothelial dysfunction, autoimmunity, and skeletal muscle dysfunction are some of the pathological hallmarks of COPD. In light of the above it would be prudent to target systemic and local oxidative stress with agents that can modulate the antioxidants/ redox system or by boosting the endogenous levels of antioxidants for the treatment and management of COPD. Identification of various antioxidant agents, such as thiol molecules (glutathione and mucolytic drugs, such as N-acetyl-L-cysteine, N-acystelyn, erdosteine, fudosteine, ergothioneine, and carbocysteine lysine salt), dietary natural product-derived polyphenols and other compounds (curcumin, resveratrol, green tea catechins, quercetin sulforaphane, lycopene, acai, alpha-lipoic acid, tocotrienols, and apocynin) have made it possible to modulate various biochemical aspects of COPD. Various researches and clinical trials have revealed that these antioxidants can detoxify free radicals and oxidants, control expression of redox and glutathione biosynthesis genes, chromatin remodeling, and ultimately inflammatory gene expression. In addition, modulation of cigarette smoke-induced oxidative stress and related cellular changes have also been reported to be effected by synthetic molecules. This includes specific spin traps like α-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a superoxide dismutase mimetic M40419, lipid peroxidation and protein carbonylation blockers/inhibitors, such as edaravone and lazaroids/tirilazad, myeloperoxidase inhibitors, as well as specialized pro-resolving mediators/inflammatory resolving lipid mediators, omega-3 fatty acids, vitamin D, and hydrogen sulfide. According to various studies it appears that the administration of multiple antioxidants could be a more effective mode used in the treatment of COPD. In this review, various pharmacological and dietary approaches to enhance lung antioxidant levels and beneficial effects of antioxidant therapeutics in treating or intervening the progression of COPD have been discussed.

Modulation of steroid activity in chronic inflammation: a novel anti-inflammatory role for curcumin.

The expression of NF-kappaB (NF-kappaB)-dependent pro-inflammatory genes in response to oxidative stress is regulated by the acetylation-deacetylation status of histones bound to the DNA. It has been suggested that in severe asthma and in chronic obstructive pulmonary disease (COPD) patients, oxidative stress not only activates the NF-kappaB pathway but also alters the histone acetylation and deacetylation balance via post-translational modification of histone deacetylases (HDACs). Corticosteroids have been one of the major modes of therapy against various chronic respiratory diseases such as asthma and COPD. Failure of corticosteroids to ameliorate such disease conditions has been attributed to their inability to either recruit HDAC2 or to the presence of an oxidatively modified HDAC2 in asthmatics and COPD subjects. Naturally occurring polyphenols such as curcumin and resveratrol have been increasingly considered as safer nutraceuticals. Curcumin is a polyphenol present in the spice turmeric, which can directly scavenge free radicals such as superoxide anion and nitric oxide and modulate important signaling pathways mediated via NF-kappaB and mitogen-activated protein kinase pathways. Polyphenols also down-regulate expression of pro-inflammatory mediators, matrix metalloproteinases, adhesion molecules, and growth factor receptor genes and they up-regulate HDAC2 in the lung. Thus, curcumin may be a potential antioxidant and anti-inflammatory therapeutic agent against chronic inflammatory lung diseases.