A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol

Vandeputte, Caroline; Guizon, Isabelle; Genestie-Denis, I; Vannier, Bernard; Lorenzon, Giocondo

doi:10.1007/bf00755791

Cited by 318 publications

(177 citation statements)

References 11 publications

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“…Measurement of GSH and GSSG-After having incubated the cells under the experimental conditions described under "Results," intracellular and extracellular GSH and GSSG were measured spectrophotometrically as described (22) and expressed as pmol/mg of cellular proteins.…”

Section: Methodsmentioning

confidence: 99%

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Riganti

Gazzano

Polimeni

et al. 2004

Journal of Biological Chemistry

178

133

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Diphenyleneiodonium (DPI) and the structurally related compound diphenyliodonium (DIP) are widely used as inhibitors of flavoenzymes, particularly NADPH oxidase. Here we report further evidence that DPI and DIP are not specific flavin binders. A 3-h incubation of N11 glial cells with DPI significantly inhibited in a dosedependent way both the pentose phosphate pathway and the tricarboxylic acid cycle. In parallel, we observed a dose-dependent increase of reactive oxygen species generation and lipoperoxidation and increased leakage of lactate dehydrogenase activity in the extracellular medium. The glutathione/glutathione disulfide ratio decreased, whereas the efflux of glutathione out of the cells increased. This suggests that DPI causes an augmented oxidative stress and exerts a cytotoxic effect in N11 cells. Indeed, the cells were protected from these events when loaded with glutathione. Similar results were observed using DIP instead of DPI and also in other cell types. We suggest that the DPI-elicited inhibition of the pentose phosphate pathway and tricarboxylic acid cycle may be mediated by the blockade of several NAD(P)-dependent enzymes, such as glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase. In light of these results, we think that some effects of DPI or DIP in in vitro and in vivo experimental models should be interpreted with caution. Diphenyleneiodonium (DPI) 1 and the structurally related compound diphenyliodonium (DIP) are widely used as uncompetitive inhibitors of flavoenzymes. Firstly identified as a hypoglycemic agent able to block gluconeogenesis and respiration in rat liver (1), DPI has been subsequently shown to inhibit the activity of NADH:ubiquinone oxidoreductase (2, 3), NADPH oxidase (4 -6), nitric-oxide synthase (7), xanthine oxidase (6), and NADPH cytochrome P450 oxidoreductase (8). DPI and other iodonium derivatives have been shown to react via a radical mechanism, whereby an electron is abstracted from FAD or FMN to form a radical, which then adds back to the flavin to form covalent, phenylated adducts (9). Also, heme groups, such as the heme b of NADPH oxidase, have been found to react with DPI and DIP (10).In most experimental works of the last years, DPI or DIP have been used as inhibitors of NADPH oxidase. NADPH oxidases are a group of plasma membrane-associated enzymes found in a variety of cells of mesodermal origin. The most thoroughly studied is the leukocyte isoform, which catalyzes the production of superoxide (O 2 . ) by the one-electron reduction of oxygen, using NADPH as the reducing agent (11). The O 2 .generated by NADPH oxidase serves as the starting material for the production of a vast assortment of reactive oxidants used by phagocytes to kill invading microorganisms or tumor cells (11). A low activity NADPH oxidase is present in a variety of nonphagocytic cells, wherein this enzyme is a source of second messengers. It has been postulated, for instance, that the O 2 . generated by the aorta functions as a blood ...

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Section: Methodsmentioning

confidence: 99%

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Riganti

Gazzano

Polimeni

et al. 2004

Journal of Biological Chemistry

178

133

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“…The concentration of GSH in sperm was determined as described by Vandeputte et al (1994) using a Total Glutathione Quantification Kit (Dojindo Laboratories, Kumamoto, Japan). Before and after the incubation for capacitation, the washed sperm (5.3!10 7 cells) were homogenized in 0.5 ml of 5% sulfosalicylic acid and centrifuged at 8000 g for 10 min at 4 8C.…”

Section: Determination Of Ros and Gsh Concentrationsmentioning

confidence: 99%

Porcine sperm capacitation involves tyrosine phosphorylation and activation of aldose reductase

Katoh¹,

Takebayashi²,

Kikuchi³

et al. 2014

Reproduction

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Mammalian sperm must be activated in the tubal isthmus through capacitation to induce the acrosome reaction and subsequent fertilization. Although the molecular mechanisms involved in capacitation have yet to be fully elucidated, increased concentrations of reactive oxygen species (ROS) and the extent of tyrosine phosphorylation of proteins have been suggested to play central roles in the completion of capacitation. In this study, aldose reductase was for the first time identified as one of the tyrosine-phosphorylated proteins involved in the capacitation of porcine cauda epididymal sperm. Both tyrosine phosphorylation and activity of aldose reductase associated with the particulate fraction of sperm cells were significantly enhanced during capacitation. Alrestatin, a membranepermeable and specific inhibitor of aldose reductase, plays a role in the inhibition of aldose reductase activity, elevation of intracellular levels of ROS, and induction of hyperactivated motility, all at similar dose dependencies. Alrestatin canceled both the increase in the tyrosine phosphorylation of aldose reductase and the decrease in the glutathione levels in sperm-induced during capacitation. The hyperactivated motility was induced to a higher extent in the presence of glucose than in the presence of fructose. These results indicate that aldose reductase plays an important role in induction of hyperactivation and capacitation of sperm through the elevation of ROS in sperm cells. Furthermore, aldose reductase was shown to be added to sperm during transit through the epididymis, suggesting that aldose reductase is one of the key proteins that support the functional maturation of sperm.

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“…Total glutathione and oxidized glutathione levels were determined by a microtiter plate assay according to the method of Vandeputte et al (47). Reduced glutathione (GSH) levels were determined by subtracting oxidized glutathione from total glutathione and were expressed as nanomoles of GSH per milligram of protein.…”

Section: Glutathione Content Of Treated Dendritic Cellsmentioning

confidence: 99%

Sulfamethoxazole and Its Metabolite Nitroso Sulfamethoxazole Stimulate Dendritic Cell Costimulatory Signaling

Sanderson

Naisbitt

Farrell

et al. 2007

The Journal of Immunology

117

107

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Different signals in addition to the antigenic signal are required to initiate an immunological reaction. In the context of sulfamethoxazole allergy, the Ag is thought to be derived from its toxic nitroso metabolite, but little is known about the costimulatory signals, including those associated with dendritic cell maturation. In this study, we demonstrate increased CD40 expression, but not CD80, CD83, or CD86, with dendritic cell surfaces exposed to sulfamethoxazole (250–500 μM) and the protein-reactive metabolite nitroso sulfamethoxazole (1–10 μM). Increased CD40 expression was not associated with apoptosis or necrosis, or glutathione depletion. Covalently modified intracellular proteins were detected when sulfamethoxazole was incubated with dendritic cells. Importantly, the enzyme inhibitor 1-aminobenzotriazole prevented the increase in CD40 expression with sulfamethoxazole, but not with nitroso sulfamethoxazole or LPS. The enzymes CYP2C9, CYP2C8, and myeloperoxidase catalyzed the conversion of sulfamethoxazole to sulfamethoxazole hydroxylamine. Myeloperoxidase was expressed at high levels in dendritic cells. Nitroso sulfamethoxazole immunogenicity was inhibited in mice with a blocking anti-CD40L Ab. In addition, when a primary nitroso sulfamethoxazole-specific T cell response using drug-naive human cells was generated, the magnitude of the response was enhanced when cultures were exposed to a stimulatory anti-CD40 Ab. Finally, increased CD40 expression was 5-fold higher on nitroso sulfamethoxazole-treated dendritic cells from an HIV-positive allergic patient compared with volunteers. These data provide evidence of a link between localized metabolism, dendritic cell activation, and drug immunogenicity.

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A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated cells: performance study of a new miniaturized protocol

Cited by 318 publications

References 11 publications

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Porcine sperm capacitation involves tyrosine phosphorylation and activation of aldose reductase

Sulfamethoxazole and Its Metabolite Nitroso Sulfamethoxazole Stimulate Dendritic Cell Costimulatory Signaling

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