scholarly journals Direct Determination of Glutathione S-transferase and Glucose-6-phosphate Dehydrogenase Activities in Cells Cultured in Microtitre Plates as Biomarkers for Oxidative Stress

1998 ◽  
Vol 26 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Concepción García-Alfonso ◽  
Guillermo Repetto ◽  
Pilar Sanz ◽  
Manuel Repetto ◽  
Juan López-Barea
Keyword(s):  
Oxidative Stress ◽  
Direct Determination ◽  
Cell Number ◽  
Glutathione S Transferase ◽  
Enzymatic Assays ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Gst Activity ◽  
In Cells

The enzymes glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G-6PDH) are implicated in the defence against oxidative stress. GST is mainly involved in the conjugation of electrophilic compounds with glutathione (GSH), although some of its isoenzymes display peroxidase activity. G-6PDH and glutathione reductase regenerate NADPH and GSH, respectively, to restore the reduced intracellular redox status following oxidative stress. Enzymatic assays for GST and G-6PDH were adapted and optimised to permit the direct in vitro determination of the effects of toxicants which induce oxidative stress in cells on microtitre plates, thereby avoiding the need to prepare cell-free extracts. To optimise the conditions of the enzymatic assays, GST activity’ was measured at substrate concentrations of 1–3mM GSH and 1–3mM 1-chloro-2,4-dinitrobenzene, while G-6PDH activity was measured at 7.5–37.5mM glucose-6-phosphate and 55–275mM NADP. Both enzymatic activities were directly proportional to cell number up to a density of 1 x 105 cells/well. The effects on GST and G-6PDH activities of three toxicants which induce oxidative stress — paraquat, iron (II) chloride and iron (III) chloride — were compared in cultured Vero cells to validate the new assays. Specific GST activity increased to 145% and 171% compared to the controls in cells treated with 5mM paraquat and 5mM iron (II) chloride, respectively, but was inhibited after exposure to 25mM iron (III) chloride. Specific G-6PDH activity increased to 136% compared to the control after exposure to 5mM paraquat, but was inhibited in cells exposed to 5mM iron (II) chloride and 25mM iron (III) chloride.

scholarly journals Direct Determination of Glutathione Reductase in Cells Cultured in Microtitre Plates as a Biomarker for Oxidative Stress

1995 ◽  
Vol 23 (4) ◽  
pp. 531-538
Author(s):  
Concepción García-Alfonso ◽  
Pilar Sanz ◽  
Guillermo Repetto ◽  
Manuel Repetto ◽  
Juan López-Barea
Keyword(s):  
Oxidative Stress ◽  
Glutathione Reductase ◽  
Potassium Phosphate ◽  
Direct Determination ◽  
Vero Cells ◽  
Control Activity ◽  
Wide Range ◽  
Cells Cultured ◽  
In Cells

— A new method was developed for the direct determination of glutathione reductase (GOR) activity in Vero cells cultured in microtitre plates, avoiding cell-free extract preparation. The cells in each well were washed twice with phosphate-buffered saline, lysed with Triton X-100, and assayed in 0.1M potassium phosphate, pH 7.0. After subtracting oxidase activity, which increased with NADPH concentration, the net GOR activity was similar at different oxidised glutathione (GSSG) and NADPH concentrations, thus confirming enzyme saturation. The optimised GOR assay used 2.5mM GSSG and 0.12mM NADPH; 5mM EDTA was also added to prevent the enzyme from redox inactivation. The GOR activity was directly proportional to the number of cells per well for a wide range of cell densities, thus supporting the assay's validity for use with cultured cells. The effects on GOR activity of three chemicals which induce oxidative stress, namely, paraquat, iron (II) chloride and iron (III) chloride, were examined to validate the assay under experimental conditions. The specific enzymatic activity increased to 357% of untreated control activity in 5mM paraquat-treated cells, and to 407% of control activity in cells exposed to 7.5mM iron (II) chloride. By contrast, activity decreased to 56% of control activity in cells exposed to 5mM iron (III) chloride. In conclusion, the changes in GOR activity detected in Vero cells confirm that the new assay is suitable for routine in vitro screening of toxicants capable of inducing oxidative stress.

MiR-485-5p Promotes Neuron Survival through Mediating Rac1/Notch2 Signaling Pathway after Cerebral Ischemia/Reperfusion

2020 ◽  
Vol 17 (3) ◽  
pp. 259-266 ◽  
Author(s):  
Xuan Chen ◽  
Sumei Zhang ◽  
Peipei Shi ◽  
Yangli Su ◽  
Dong Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Therapeutic Option ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Small Gtpase ◽  
Luciferase Reporter ◽  
Pathological Feature ◽  
In Cells

Objective: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury. Methods: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR-485- 5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using the MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p. Results: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485-5p expression, whereas miR- 485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR-485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury. Conclusion: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling suggest that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.

Chemoprotective Effects of Propolis on Aflatoxin B1-Induced Hepatotoxicity in Rats: Oxidative Damage and Hepatotoxicity by Modulating TP53, Oxidative Stress

2020 ◽  
Vol 17 (3) ◽  
pp. 191-199
Author(s):  
Seval Yilmaz ◽  
Fatih Mehmet Kandemir ◽  
Emre Kaya ◽  
Mustafa Ozkaraca
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Oxidative Damage ◽  
Aflatoxin B1 ◽  
Tp53 Gene ◽  
Control Group ◽  
Glutathione S Transferase ◽  
Gene Expressions ◽  
Cat Gene ◽  
Gst Activity

Objective: This study aimed to detect hepatic oxidative damage caused by aflatoxin B1 (AFB1), as well as to examine how propolis protects against hepatotoxic effects of AFB1. Method: Rats were split into four groups as control group, AFB1 group, propolis group, AFB1+ propolis group. Results: There was significant increase in malondialdehyde (MDA) level and tumor suppressor protein (TP53) gene expression, Glutathione (GSH) level, Catalase (CAT) activity, CAT gene expression decreased in AFB1 group in blood. MDA level and Glutathione-S-Transferase (GST) activity, GST and TP53 gene expressions increased in AFB1 group, whereas GSH level and CAT activity alongside CAT gene expression decreased in liver. AFB1+propolis group showed significant decrease in MDA level, GST activity, TP53 and GST gene expressions, GSH level and CAT activity and CAT gene expression increased in liver compared to AFB1 group. Conclusion: These results suggest that propolis may potentially be natural agent that prevents AFB1- induced oxidative stress and hepatotoxicity.

scholarly journals Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T

2021 ◽  
Vol 22 (8) ◽  
pp. 4009
Author(s):  
Maik Liedtke ◽  
Christin Völkner ◽  
Alexandra V. Jürs ◽  
Franziska Peter ◽  
Michael Rabenstein ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transmembrane Protein ◽  
Hereditary Disease ◽  
Cholesterol Homeostasis ◽  
Patient Specific ◽  
Differentiated Cells ◽  
Npc1 Gene ◽  
Niemann Pick

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.

scholarly journals Oxidative Stress Induction of DJ-1 Protein in Reactive Astrocytes Scavenges Free Radicals and Reduces Cell Injury

2009 ◽  
Vol 2 (1) ◽  
pp. 36-42 ◽  
Author(s):  
Takashi Yanagida ◽  
Jun Tsushima ◽  
Yoshihisa Kitamura ◽  
Daijiro Yanagisawa ◽  
Kazuyuki Takata ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
Primary Cultures ◽  
Artery Occlusion ◽  
Reactive Astrocytes ◽  
Glutathione S Transferase ◽  
Triphenyltetrazolium Chloride ◽  
Ischemic Region

Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Under cerebral ischemia/reperfusion-induced oxidative conditions, astrocytes accumulate and activate in the ischemic region. DJ-1 has recently been shown to be a sensor of oxidative stress in living cells. However, the function of astrocytic DJ-1 is still unknown. In the present study, to clarify the effect of astrocytic DJ-1 protein under massive oxidative insult, we used a focal ischemic rat model that had been subjected to middle cerebral artery occlusion (MCAO) and reperfusion. We then investigated changes in the distribution of DJ-1 in astrocytes, DJ-1 release from cultured astrocytes, and the effects of recombinant DJ-1 protein on hydrogen peroxide (H2O2)-induced death in normal and DJ-1-knockdown SH-SY5Y cells and on in vitro scavenging of hydroxyl radicals (•OH) by electron spin resonance spectrometry. At 24 h after 2-h MCAO and reperfusion, an infarct lesion was markedly observed using magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining. In addition, reactive astrocytes enhanced DJ-1 expression in the penumbral zone of the ischemic core and that DJ-1 protein was extracellularly released from astrocytes by H2O2 in in vitro primary cultures. Although DJ-1-knockdown SH-SY5Y cells were markedly vulnerable to oxidative stress, treatment with glutathione S-transferase-tagged recombinant human DJ-1 protein (GST-DJ-1) significantly inhibited H2O2-induced cell death. In addition, GST-DJ-1 protein directly scavenged•OH. These results suggest that oxidative stress induces the release of astrocytic DJ-1 protein, which may contribute to astrocyte-mediated neuroprotection.

scholarly journals Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 787
Author(s):  
Enrique García-Pérez ◽  
Dojin Ryu ◽  
Hwa-Young Kim ◽  
Hae Dun Kim ◽  
Hyun Jung Lee
Keyword(s):  
Oxidative Stress ◽  
Ochratoxin A ◽  
Cell Lines ◽  
Time Dependent ◽  
Mrna Levels ◽  
Dependent Manner ◽  
In Vitro System ◽  
Glutathione Peroxidase 1 ◽  
Glucose 6 Phosphate Dehydrogenase

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

scholarly journals Comparative Phytochemical, Antioxidant and Haemostatic Studies of Preparations from Selected Vegetables from Cucurbitaceae Family

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4326
Author(s):  
Agata Rolnik ◽  
Iwona Kowalska ◽  
Agata Soluch ◽  
Anna Stochmal ◽  
Beata Olas
Keyword(s):  
Oxidative Stress ◽  
Thrombin Time ◽  
Phytochemical Composition ◽  
Significant Interest ◽  
Phytochemical Compounds ◽  
Potential Use ◽  
Insight Into ◽  
Cucurbitaceae Family

The aim of this study was to provide detailed insight into the chemical composition and activity of five cucurbit vegetable preparations (pumpkin, zucchini, cucumber, white and yellow pattypan squash), each containing various phytochemical compounds with potential use against oxidative stress induced by the hydroxyl radical donors in human plasma in vitro. We studied the antiradical capacity of vegetable preparations using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. As oxidative stress may induce changes in hemostasis, our aim included the determination of their effect on three selected hemostatic parameters of plasma, which are three coagulation times: PT (prothrombin time), APTT (activated partial thromboplastin time) and TT (thrombin time). However, none of used vegetable preparations changed APTT, PT or TT compared to the control. The phytochemical composition of the tested preparations was determined by UPLC-ESI-QTOF-MS. In our in vitro experiments, while all five tested preparations had antioxidant potential, the preparation from yellow pattypan squash showed the strongest potential. All cucurbit vegetable preparations inhibited lipid peroxidation. Only zucchini did not have an effect on protein carbonylation and only yellow pattypan squash inhibited thiol oxidation. The antioxidant activity of cucurbits appears to have triggered significant interest in multiple applications, including CVDs (cardiovascular diseases) associated with oxidative stress, which can be treated by supplementation based on these vegetables.

scholarly journals Determination of activity of antioxidants in human subjects

1999 ◽  
Vol 58 (4) ◽  
pp. 1015-1024 ◽  
Author(s):  
Garry G. Duthie
Keyword(s):  
Oxidative Stress ◽  
Human Subjects ◽  
Control Schemes ◽  
Total Antioxidant ◽  
Plasma Measurements ◽  
Determination Of Activity ◽  
Nutritional Antioxidants

Evidence from biochemical and animal models suggests that nutritional antioxidants should inhibit the development of diseases such as CHD and certain cancers. This evidence is not clearly corroborated by intervention studies in human subjects, due, in part, to inadequacies in current analytical methodologies. Althoughin vitroassays can give useful information on the attributes required by a compound to act as an antioxidant, results may have little nutritional relevance due to limited bioavailability. The determination of antioxidants in blood is often used as a measure of antioxidant statusin vivo, but may not necessarily reflect concentrations in target tissues where oxidative stress is greatest. In addition, the accumulation of antioxidants in selective tissues may not be apparent from plasma measurements. Participation in quality-control schemes for antioxidant determination by HPLC allows inter-laboratory comparison of results. Moderation of indices of oxidative damage to lipids, proteins and DNA can provide information on the effectiveness of compounds as nutritional antioxidants. However, most current methods of assessing oxidative stress are subject to confounding factors of non-oxidative origin. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. They give different results, should never be used in isolation, and results should be interpreted with caution. Until more is known about the activity and metabolic fate of antioxidants, caution should be exercised in the consumption of large amounts of commercially-available antioxidant preparations.

177 ANTHOCYANIN ISOLATED FROM PURPLE SWEET POTATO IMPROVES DEVELOPMENT AND REDUCES OXIDATIVE STRESS OF BOVINE PRE-IMPLANTATION EMBRYOS EXPOSED TO HEAT SHOCK

2006 ◽  
Vol 18 (2) ◽  
pp. 196
Author(s):  
M. Sakatani ◽  
I. Suda ◽  
T. Oki ◽  
S.-I. Kobayashi ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Sweet Potato ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Cell Stage ◽  
Intracellular Ros ◽  
Purple Sweet Potato

Development of cleavage-stage pre-implantation embryos is disrupted by exposure to heat shock. Heat shock also increases intracellular reactive oxygen species (ROS) in pre-implantation embryos. Therefore, reduction of intracellular ROS levels might improve the development of heat-shocked embryos. Recently the antioxidative activities of polyphenols have been widely reported to reduce the oxidative stress. In this study, we investigated the effect of purple sweet potato anthocyanin, a kind of polyphenol that is a strong ROS scavenger, on development and intracellular redox status of bovine pre-implantation embryos exposed to heat shock. Experiment 1: In vitro-produced 8-16-cell-stage embryos on Day 2 after fertilization were exposed to 41.5�C for 6 h in CR1aa containing 0, 0.1, 1, and 10 �g/mL anthocyanin at 5% CO2, 5% O2, and 90% N2. After heat shock, embryos were cultured at 38.5�C at 5% CO2, 5% O2 until Day 8. On Day 8, the proportion of embryos developing to the blastocyst stage was evaluated. Blastocyst total cell number and the ratio between inner cell mass and tropheoderm were evaluated by differential staining. The experiment was replicated five times with more than 70 embryos used in each treatment. Experiment 2: Heat shock treatment of in vitro-produced 8-16-cell-stage embryos was carried out as described in experiment 1. After heat shock, intracellular ROS and glutathione (GSH) levels were measured in individual 8-16 cell stage embryos with fluorescent probes (22,72-dichlorodihydrofluorescein diacetate for ROS and CellTracker" Blue (Invitrogen Japan K. K., Tokyo, Japan) for GSH). The fluorescence emissions of each treatment were normalized to those of 8-16 cell stage embryos cultured at 38.5�C without anthocyanin to obtain the relative fluorescence emission. This experiment was replicated four times. Embryos treated with heat stress without anthocyanin (0 �g/mL) showed low development (14.6 � 3.6%) and blastocyst total cell number (88.2 � 9.4). However, embryos treated with 0.1 �g/mL anthocyanin improved development (31.7 � 4.5%, P < 0.05) and increased the total cell number (96.5 � 11.3). The higher concentrations of anthocyanin (1 and 10 �g/mL) did not affect development and cell number. The intracellular ROS levels in heat-shocked embryos were significantly reduced by all concentrations of anthocyanin (P < 0.05). In addition, anthocyanin increased GSH levels at all doses tested (P < 0.05). These results indicate that an appropriate concentration of anthocyanin improves development by regulating intracellular redox balance in bovine embryos exposed to heat shock.

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