Use of a microplate reader in an assay of glutathione reductase using 5,5′-dithiobis(2-nitrobenzoic acid)

Cribb, Alastair E.; Leeder, J. Steven; Spielberg, Stephen P.

doi:10.1016/0003-2697(89)90188-7

Cited by 267 publications

(121 citation statements)

References 3 publications

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“…Eel cholinesterase activity detected was considered as true AChE, as was previously characterised (Ferenczy et al 1997). GR (EC 1.6.4.2) activity was assayed by the method of Cribb et al (1989) Smith et al (1988). A calibration curve of known activities of purified enzymes was used on every 96-well plate to avoid miscalculations that stem from an ill-defined path length.…”

Section: Methodsmentioning

confidence: 99%

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Peña-Llopis¹,

Ferrando²,

Jb³

2003

Dis. Aquat. Org.

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Organophosphate (OP) pesticides are widely used as antiparasitic chemicals in finfish aquaculture. However, current antidotes cannot be applied to treat intoxicated fish. We showed in previous studies the importance of glutathione (GSH) metabolism in pesticide resistance of the European eel Anguilla anguilla L. The present work studied the effects of the antioxidant and glutathione pro-drug N-acetyl-L-cysteine (NAC) on the recovery of European eels exposed for 96 h to a sublethal concentration (0.17 mg l -1 ; 20% of its 96 h LC 50 ) of the OP pesticide dichlorvos (2, 2-dichlorovinyl dimethyl phosphate; DDVP). This insecticide and acaricide decreased muscular GSH content and increased oxidised glutathione (GSSG), lowering the GSH:GSSG ratio, which is indicative of a condition of oxidative stress. Acetylcholinesterase (AChE) and glutathione reductase (GR) activities in the brain, which were biomarkers of neurotoxicity and oxidative stress, respectively, were also highly inhibited. Recovery in a 0.5 mM (81.6 mg l -1 ) NAC concentration ameliorated muscular GSH depletion, GSH:GSSG ratio, and the inhibition of brain AChE and GR activities. Hence, this is the first evidence of improved recovery of organophosphate-poisoned fish by bath treatments.

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Section: Methodsmentioning

confidence: 99%

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Peña-Llopis¹,

Ferrando²,

Jb³

2003

Dis. Aquat. Org.

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“…To quantify the GPx activity, we measured NADPH oxidation by reading absorbance at a wavelength of 340 nm. We quantified GR activity following Cribb et al (1989). We measured the change in absorbance at 340 nm due to NADPH oxidation, as described in the GPx assay.…”

Section: Oxidative Stressmentioning

confidence: 99%

Physiological Stress Responses in Amphibian Larvae to Multiple Stressors Reveal Marked Anthropogenic Effects even below Lethal Levels

Burraco

Gómez-Mestre

2016

Physiological and Biochemical Zoology

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Natural and anthropogenic disturbances cause profound alterations in organisms, inducing physiological adjustments to avoid, reduce, or remedy the impact of disturbances. In vertebrates, the stress response is regulated via neuroendocrine pathways, including the hypothalamic-pituitary-interrenal axis that regulates the secretion of glucocorticoids. Glucocorticoids have cascading effects on multiple physiological pathways, affecting the metabolic rate, reactive oxygen species production, or immune system. Determining the extent to which natural and anthropogenic environmental factors induce stress responses in vertebrates is of great importance in ecology and conservation biology. Here we study the physiological stress response in spadefoot toad tadpoles (Pelobates cultripes) against three levels of a series of natural and anthropogenic stressors common to many aquatic systems: salinity (0, 6, and 9 ppt), herbicide (0, 1, and 2 mg/L acid equivalent of glyphosate), water acidity (pH 4.5, 7.0, and 9.5), predators (absent, native, and invasive), and temperature (217, 257, and 297C). The physiological stress response was assessed examining corticosterone levels, standard metabolic rate, activity of antioxidant enzymes, oxidative cellular damage in lipids, and immunological status. We found that common stressors substantially altered the physiological state of tadpoles. In particular, salinity and herbicides cause dramatic physiological changes in tadpoles. Moreover, tadpoles reduced corticosterone levels in the presence of natural predators but did not do so against invasive predators, indicating a lack of innate recognition. Corticosterone and the antioxidant enzyme glutathione reductase were the most sensitive parameters to stress in this study. Anthropogenic perturbations of aquatic systems pose serious threats to larval amphibians even at nonlethal concentrations, judging from the marked physiological stress responses generated, and reveal the importance of incorporating physiological information onto conservation, ecological, and evolutionary studies.

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“…There was a linear relationship between rate of increase in the absorbance at 405 nm and GR activity from 2 to 40 mU/mL of GR in the reaction buffer. 28 …”

Section: Glutathione Reductase Activity Assaymentioning

confidence: 99%

Fluid Shear Stress Attenuates Hydrogen Peroxide–Induced c-Jun NH ₂ -Terminal Kinase Activation via a Glutathione Reductase–Mediated Mechanism

Hojo

Saito

Tanimoto

et al. 2002

Circulation Research

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Abstract-c-Jun NH 2 -terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 mol/L hydrogen peroxide (H 2 O 2 ) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3Ϯ1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stressϭ12 dyne/cm 2 ) for 10 minutes significantly decreased H 2 O 2 -induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H 2 O 2 -induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells. Key Words: oxidative stress Ⅲ endothelial cell Ⅲ signal transduction A growing body of evidence indicates that reactive oxygen species (ROS) are involved in cardiovascular diseases such as atherosclerosis, 1 reperfusion injury, 2 heart failure, 3-5 and hypertension. 6,7 Previous studies have clarified that ROS are important regulators of signaling events in pathological cardiovascular condition. 8,9 Among cellular signals, members of mitogen-activated protein (MAP) kinases play critical roles in cellular proliferation, inflammation, and apoptosis. 10 -12 We have demonstrated that hydrogen peroxide (H 2 O 2 ) activates MAP kinases in cultured cells derived from vascular tissue. [13][14][15] c-Jun NH 2 -terminal kinase (JNK) is activated by a number of cellular stimuli including proinflammatory cytokines and ROS. 16,17 It is speculated that JNK plays an important role in proatherogenic signal events through phosphorylation of c-Jun, activation of AP-1, and stimulation of proinflammatory gene expression such as ICAM-1. Our previous studies have demonstrated that laminar fluid shear stress (flow) inhibits cytokine-induced JNK activation in vascular endothelial cells. 18,19 These results are consistent with the concept that flow exerts an atheroprotective effect against inflammatory cytokines. 20 -23 Cardiovascular risk factors such as smoking, hypercholesterolemia, diabetes, and hypertension have been shown to share a common...

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Use of a microplate reader in an assay of glutathione reductase using 5,5′-dithiobis(2-nitrobenzoic acid)

Cited by 267 publications

References 3 publications

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Physiological Stress Responses in Amphibian Larvae to Multiple Stressors Reveal Marked Anthropogenic Effects even below Lethal Levels

Fluid Shear Stress Attenuates Hydrogen Peroxide–Induced c-Jun NH ₂ -Terminal Kinase Activation via a Glutathione Reductase–Mediated Mechanism

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Use of a microplate reader in an assay of glutathione reductase using 5,5′-dithiobis(2-nitrobenzoic acid)

Cited by 267 publications

References 3 publications

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Physiological Stress Responses in Amphibian Larvae to Multiple Stressors Reveal Marked Anthropogenic Effects even below Lethal Levels

Fluid Shear Stress Attenuates Hydrogen Peroxide–Induced c-Jun NH 2 -Terminal Kinase Activation via a Glutathione Reductase–Mediated Mechanism

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Fluid Shear Stress Attenuates Hydrogen Peroxide–Induced c-Jun NH ₂ -Terminal Kinase Activation via a Glutathione Reductase–Mediated Mechanism