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.

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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-kappaB (NF-kappaB) activation or expression and release of either interleukin (IL)-8 or IL-6. However, H2O2, tumor necrosis factor-alpha (TNF-alpha), and IL-1beta significantly increased NF-kappaB activation and expression of IL-8 compared with control cells. Interestingly, CSC dose dependently inhibited TNF-alpha- and IL-1beta-mediated NF-kappaB 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.