Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid.
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ABSTRACT:
SUBMITTER: Vissers MC
PROVIDER: S-EPMC1136744 | biostudies-other | 1995 Apr
REPOSITORIES: biostudies-other
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Project description:Neutrophils, when stimulated, generate reactive oxygen species including myeloperoxidase-derived HOCl. There is an associated decrease in reduced glutathione (GSH) concentration. We have shown that neutrophil GSH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell lysis, was not reversible, and was accompanied by the loss of an equivalent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with 35S-labelled cells indicated that much of the GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the reaction of HOCl and pure GSH. The properties of this compound are consistent with an intramolecular sulphonamide. Neutrophils stimulated with PMA lost 30-40% of their GSH and a similar proportion of protein thiols. Little glutathione disulphide was formed and the products were the same as seen with HOCl-treated cells. From the results and studies with inhibitors and scavengers, we conclude that HOCl was responsible for the GSH loss. Propargylglycine and buthionine sulphoximine, inhibitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of GSH synthesis. The lack of thiol selectivity by HOCl and irreversibility of oxidation means that GSH will provide limited antioxidant protection for thiol enzymes in stimulated neutrophils.
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Project description:Reduced glutathione (GSH) is one of the most preferred biological substrates of myeloperoxidase-derived hypochlorous acid and is a likely target for neutrophil oxidants. We have used HPLC to show that the oxidation of GSH by hypochlorous acid gives two major, stable products in addition to glutathione disulphide (GSSG). The most prevalent product lacks free amine and thiol groups, and was shown by electrospray MS to have a molecular mass of 337 Da. This corresponds to GSH with a gain of two oxygen atoms and a loss of two hydrogen atoms, and is consistent with the product being an internal sulphonamide. The other novel product has a molecular mass of 644 Da, and has amine groups but no free thiols. These properties are consistent with it being glutathione thiolsulphonate. Whereas GSSG in the cell is recycled enzymically, formation of these higher oxidation products is likely to be irreversible. Hypochlorous acid, therefore, could compromise the cell by depleting GSH. The putative sulphonamide may be unique for oxidation by hypochlorous acid and thus provide a useful marker of neutrophil oxidant activity.
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Project description:Hypochlorous acid (HClO) is produced by white blood cells to defend against injury and bacteria. However, as one of the reactive oxygen species, high intracellular HClO concentration could lead to chronic diseases that affect the cardiovascular and nervous systems. To monitor HClO concentrations in bio-samples, the fluorescent probe is preferred to have: a) absorbability in the far-red window with reduced light-toxicity and improved tissue penetration depth, b) ratiometric feature for accurate analysis. In this study, we reported a far-red ratiometric HClO fluorescence probe based on BODIPY chromophore and aldoxime sensing group. Not only the color change of the probe solution can be detected by naked eyes, but also the emission ratios (I645/I670) showed a significant increase upon the introduction of HClO. More importantly, the feasibility of HClO monitoring in bio-samples was demonstrated in vitro using a confocal microscope.
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Project description:We report the development of a new colorimetric probe (L-ol) for investigations of the redox process regulated by hypochlorous acid (HOCl) and glutathione (GSH). The HOCl/GSH redox-switching cycle process was investigated in detail by UV-vis absorption spectroscopy, colorimetric analysis assay and high-resolution mass spectrometry (HRMS). The switchable absorbance responses were attributed to the HOCl-induced oxidation of the p-methoxyphenol unit to the benzoquinone derivative (L-one) and sequential reduction of L-one to hydroquinone (L-ol') by GSH. In phosphate-buffered saline (PBS) buffer, the absorbance of L-ol at 619 nm underwent a remarkable bathochromic-shift, accompanied by a color change from pale yellow to blue in the presence of HOCl. With further addition of GSH, the absorbance of L-one exclusively recovered to the original level. Meanwhile, the blue-colored solution returned to the naive pale yellow color in the presence of GSH. The detection limits for HOCl and GSH were calculated to be 6.3 and 96 nM according to the IUPAC criteria. Furthermore, L-ol-loaded chromatography plates have been prepared and successfully applied to visualize and quantitatively analyze HOCl in several natural waters.
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Project description:DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported to undergo preferential oxidation of the cysteine residue at position 106 (Cys-106) under oxidative stress; however, details of the molecular mechanisms are not well known. In the present study, mechanisms of DJ-1 oxidation induced by 6-hydroxydopamine (6-OHDA) were investigated by using SH-SY5Y cells. The treatment of these cells with 6-OHDA caused an obvious acidic spot sift of DJ-1 due to its oxidation. However, when catalase, which is an hydrogen peroxide (H(2)O(2))-removing enzyme, was added during the treatment, it failed to prevent the oxidation induced by 6-OHDA, suggesting that electrophilic p-quinone formed from 6-OHDA, but not H(2)O(2), was responsible for the DJ-1 oxidation. Benzoquinone, another electrophilic p-quinone, also induced DJ-1 oxidation. The intracellular glutathione (GSH) levels were significantly decreased by 6-OHDA, irrespective of the presence or absence of catalase. The inhibition of GSH synthesis by buthionine sulfoximine resulted in a decrease in GSH levels and enhancement of DJ-1 oxidation. The pretreatment of cells with N-acetyl-cysteine prevented the loss of intracellular GSH and subsequently DJ-1 oxidation induced by 6-OHDA. Collectively, these results suggest that electrophilic p-quinone formed from 6-OHDA induces DJ-1 oxidation by decreasing intracellular GSH.
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Project description:Circadian (∼24 hour) clocks are fundamentally important for coordinated physiology in organisms as diverse as cyanobacteria and humans. All current models of the molecular circadian clockwork in eukaryotic cells are based on transcription-translation feedback loops. Non-transcriptional mechanisms in the clockwork have been difficult to study in mammalian systems. We circumvented these problems by developing novel assays using human red blood cells, which have no nucleus (or DNA) and therefore cannot perform transcription. Our results show that transcription is not required for circadian oscillations in humans, and that non-transcriptional events seem to be sufficient to sustain cellular circadian rhythms. Using red blood cells, we found that peroxiredoxins, highly conserved antioxidant proteins, undergo ∼24-hour redox cycles, which persist for many days under constant conditions (that is, in the absence of external cues). Moreover, these rhythms are entrainable (that is, tunable by environmental stimuli) and temperature-compensated, both key features of circadian rhythms. We anticipate that our findings will facilitate more sophisticated cellular clock models, highlighting the interdependency of transcriptional and non-transcriptional oscillations in potentially all eukaryotic cells.
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