Preclinical Characterization of Engineered Nanoparticles Intended for Cancer Therapeutics
Mansoor M. Amiji in Nanotechnology for Cancer Therapy, 2006
Biomarkers of nanoparticle-induced oxidative stress measured in our laboratory include ROS, lipid peroxidation products, and GSH/GSSG ratio. The fluorescent dichlorodihydroflourescein (DCFH) assay is used for measurement of ROS, such as hydrogen peroxide.106 DCFH-DA is a ROS probe that undergoes intracellular deacetylation, followed by ROS-mediated oxidation to a fluorescent species, with excitation 485 nm and emission 530 nm. DCFH-DA can be used to measure ROS generation in the cytoplasm and cellular organelles, such as the mitochondria. The thiobarbituric acid reactive substances (TBARS) assay is used for measurement of lipid peroxidation products, such as lipid hydroperoxides and aldehydes. A molondialdehyde (MDA) standard curve is used for quantitation. MDA, a lipid peroxidation product, combines with thiobarbituric acid in a 1:2 ratio to form a fluorescent adduct, that is measured at 521 nm (excitation) and 552 nm (emission). TBARS are expressed as MDA equivalents.107 The dithionitrobenzene (DTNB) assay is used for evaluation of glutathione homeostasis. In the DTNB assay, reduced GSH interacts with 5,5′ -dithiobis(2-nitrobenzoic acid) (DTNB) to form the colored product 2-nitro-5-thiobenzoic acid, which is measured at 415 nm, and GSSG. GSSG is then reduced by glutathione reductase to form reduced GSH, which is again measured by the preceding method. Pretreatment with thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate, prevents GSH measurement, resulting in measurement of GSSG alone.108
Glutathione
Ruth G. Alscher, John L. Hess in Antioxidants in Higher Plants, 2017
Enzymatic and HPLC methods for the determination of GSH have recently been reviewed by Anderson11 and Fahey.12 Enzymatic determination in a coupled assay system containing glutathione reductase and dithiobis(2-nitrobenzoic acid) (DTNB) is a convenient, inexpensive method which is highly sensitive and specific. After removal of GSH from extracts by 2-vinylpyridine, the same assay can be used to measure GSSG. It should be cautioned, however, that this assay shows twofold higher reaction rates with hGSH than with GSH, which can lead to erroneous results when investigating hGSH-containing plants.2 A second widely used method is HPLC separation with fluorometric detection after derivatization of thiols with monobromobimane. The method has the advantage of simultaneously measuring other low-molecular weight thiols, such as cysteine, γ-glutamylcysteine, or cys-teinylglycine, and has been adapted for the separation of hGSH from GSH.2 However, the determination of GSSG by this method is less reliable, since it can only be measured as the difference in GSH content before and after dithiothreitol (DTT) reduction of extracts. GSH can be up to 95% of the total glutathione pool;6 the reduction of GSSG will, therefore, only slightly increase the value obtained for GSH.
Gastric Lipase
Margit Hamosh in Lingual and Gastric Lipases: Their Role in Fat Digestion, 2020
The number of free sulfhydryl groups, their reactivity, and their role in the expression of gastric lipase activity was studied recently with purified preparations of both human,123 and rabbit gastric lipase.124 Both enzymes are reported to have only one sulfhydryl group which is essential for lipolytic activity. Thus, in contrast to porcine pancreatic lipase, which has six disulfide bridges,125 gastric lipase has only one disulfide bridge, a configuration that may explain the latter enzyme’s higher sensitivity to surface denaturation than that of pancreatic lipase. Kinetics of the interaction with DTNB (a hydrophylic sulfhydryl reagent) and with C12- TNB (a hydrophobic sulfhydryl reagent) indicate that the immediate area surrounding the sulfhydryl group in gastric lipase may be more hydrophobic than that of porcine pancreatic lipase.123 The rapid inactivation of lipolytic activity following modification of the sulfhydryl group indicates that in gastric lipase this group is “essential” for catalytic activity123 (Figures 31 and 32).
Onopordopicrin from the new genus Shangwua as a novel thioredoxin reductase inhibitor to induce oxidative stress-mediated tumor cell apoptosis
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Junmin Zhang, Zai-Qin Zheng, Qianhe Xu, Ya Li, Kun Gao, Jianguo Fang
Dimethyl sulfoxide (DMSO), Dulbecco′s modified Eagle’s medium (DMEM), G418, puromycin, 2′, 7′-dichlorfluorescein diacetate (DCFH-DA), Hoechst 33342, dihydroethidium (DHE), insulin (from bovine), DL-dithiothreitol (DTT), N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), and 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) were obtained from Sigma-Aldrich (St. Louis, USA). Penicillin, MTT and streptomycin were products of Amresco (Solon, OH). NADPH was purchased from Roche (Mannheim, Germany). Fetal bovine serum (FBS) was a product of Sijiqing (Hangzhou, China). DTNB (5,5′-dithiobis-2-nitrobenzoic acid) was a product of J&K Scientific (Beijing, China). Bovine serum albumin (BSA), Trypan blue, phenylmethylsulfonyl fluoride (PMSF), and Na3VO4 were products of Beyotime (Nantong, China). The propidium iodide (PI) and Annexin V-FITC apoptosis assay kit was a product of Zoman Biotech (Beijing, China). HPLC grade methanol and acetonitrile were products of Merck (Darmstadt, Germany). All other chemicals used were of analytic grade.
The effect of desloratadine on ischemia reperfusion induced oxidative and inflammatory renal injury in rats
Published in Renal Failure, 2020
Huseyin Kocaturk, Fevzi Bedir, Mehmet Sefa Altay, Ebubekir Bakan, Bahadir Suleyman, Gulce Naz Yazici, Mukadder Sunar, Zeynep Suleyman, Halis Suleyman
The DTNB [5,5′-Dithiobis (2-nitrobenzoic acid)] in the assessment environment is a disulphide chromogen and is easily reduced by sulfhydryl group compounds. The resultant yellow color was spectrophotometrically assessed at 412 nm[13]. Homogenates were centrifuged for 10 min at 12,000 g and the supernatants were used in the determination of the amount of MDA. 250 μl measuring buffer (200 mM Tris–HCl, pH = 8.2 involving 0.2 mM EDTA), 500 μl supernatant, 100 μl 5,5′-Dithio-bis (2-nitrobenzoic acid) (DTNB) and 7900 μl methanol were vortexed into capped test tubes through pipette. The mixture was incubated for 30 min at 37 °C and then, spectrophotometric analysis was conducted. The amounts of resultant yellow color were measured using 3 mL quartz cuvettes at 412 nm. GSH amounts of the samples were determined by using the standard graphics formed with the GSH stock solution which was previously prepared considering the dilution coefficients.
Nω-nitro-L-arginine, a nitric oxide synthase inhibitor, attenuates nickel-induced neurotoxicity
Published in Drug and Chemical Toxicology, 2022
Omamuyovwi M. Ijomone, Oritoke M. Aluko, Comfort O. A. Okoh, Azubuike P. Ebokaiwe
GSH-Px activity was measured according to protocols developed by Rotruck et al. (1973). The assay is performed in solution of sodium phosphate buffer, 10 mM sodium azide, 4 mM GSH, 2.5 mM H2O2, and brain homogenate. Following a 3 min incubation, 0.5 mL of 10% trichloroacetic acid (TCA) was added to solution. Thereafter, residual glutathione content was assessed by the addition of 4mL disodium hydrogen phosphate (0.3 M) solution and 1 mL of the Ellman’s (DTNB) reagent (Thermo Scientific, Waltham, MA) to the supernatant. Activity of enzyme is indicated as units/mg of protein. GST was assessed using of Habig et al. (1974) protocols, with the substrate, 1-Chloro-2,4-dinitrobenzene (CDNB). The assay was performed in a solution of 100 mmol/L phosphate buffer (pH 6.5), 30 mmol/L of CDNB, and 20 μL of brain homogenates. Enzyme activity is indicated as nmoles of CDNB/GSH conjugate generated in 1 min/mg of protein via an extinction coefficient of 9.6 L/(mmol/cm). GSH was assessed using Jollow et al. (1974) protocols. The assay was performed in a solution of 4% sulfosalicylic acid, brain homogenates, and later 4.5 ml of DTNB. Values are indicated in μg/mg protein.
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