Mirjana Djukic

Protective role of glutathione reductase in paraquat induced neurotoxicity

Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic pathways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was on the role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathione reductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ. The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n = 8) and four experimental groups (n = 24). All tested compounds were administered intrastriatally (i.s.) in one single dose. The following parameters of oxidative status were measured in the striatum, hippocampus and cortex, at 30 min, 24 h and 7 days post treatment: superoxide anion radical (O₂·⁻), nitrate (NO₃⁻), malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form (GSSG) and glutathione peroxidase (GPx). Results obtained from the intact and the sham operated groups were not statistically different, confirming that invasive i.s. route of administration would not influence the reliability of results. Also, similar pattern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapid spatial spreading of oxidative stress. Mortality of the animals (10%), within 24h, along with symptoms of Parkinsonism, after awakening from anesthesia for 2-3 h, were observed in the PQ group, only. Increased levels of O₂·⁻, NO₃⁻ and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were measured at 30 min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measured parameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In the GR+PQ group, markedly low activity of GPx and low content of NO₃⁻ (in striatum and cortex) were measured during whole experiment, while increase value was observed only for O₂·⁻, at 7th days. We concluded that oxidative/nitrosative stress and excitotoxicity are the most important events since the early stage of PQ induced neurotoxicity. Based on the ratio GSSG/GSH, the oxidation of GSH to GSSG is probably dominant way of GHS depletion and main reason for reduced antioxidative defense against PQ harmful oxidative effect. The GR pre-treatment resulted in the absence of Parkinsons disease-like symptoms and mortality of the rats. Additionally, oxidative/nitrosative stress did not developed, as well as almost diminished metabolism of the VBRs at 24th hours (as has been documented in the PQ group) did not occurred in the GR+PQ, suggesting a neuroprotective role for the GR in PQ induced neurotoxicity.

Structural Re-arrangement and Peroxidase Activation of Cytochrome c by Anionic Analogues of Vitamin E, Tocopherol Succinate and Tocopherol Phosphate

Background: An anionic phospholipid, cardiolipin, converts cytochrome c into a peroxidase. Results: Anionic tocopherol derivatives, tocopherol succinate and tocopherol phosphate, similarly to cardiolipin, unfold cytpchrome c and stimulate its peroxidase activity. Conclusion: Peroxidase activation of cytochrome c by tocopherol analogues is one of their pharmacological mechanisms. Significance: Peroxidase activation of cytochrome c may induce apoptosis and contribute to anti-cancer properties of α-tocopherol succinate. Cytochrome c is a multifunctional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cytochrome c into a peroxidase, we investigated two hydrophobic anionic analogues of vitamin E, α-tocopherol succinate (α-TOS) and α-tocopherol phosphate (α-TOP), as potential inducers of peroxidase activity of cytochrome c. NMR studies and computational modeling indicate that they interact with cytochrome c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met80) bond associated with unfolding of cytochrome c. We found that α-TOS and α-TOP stimulate peroxidase activity of cytochrome c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with α-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (mito-VES), was more efficient in inducing H2O2-dependent apoptosis in mouse embryonic cytochrome c+/+ cells than in cytochrome c−/− cells. Essential for execution of the apoptotic program peroxidase activation of cytochrome c by α-TOS may contribute to its known anti-cancer pharmacological activity.

A subpopulation that may correspond to granulocytic myeloid-derived suppressor cells reflects the clinical stage and progression of cutaneous melanoma

Seventy-eight melanoma patients and 10 healthy individuals were examined. Follow-up examinations of all melanoma patients were performed regularly every three months. Myeloid-derived suppressor cells (MDSC) were defined as lineage negative (CD3(-), CD19(-), CD56(-)), HLA-DR(-/low), CD11b(+) and CD33(+). Classification of granulocytic (GrMDSC) and monocytic (MoMDSC) subsets was based on the CD15 and CD14 expression, respectively. Unlike the MoMDSC, that were present in 60% of healthy controls and 15% of melanoma patients, the GrMDSC were present in all examined participants, and the melanoma patients were found to have statistically higher frequencies compared with healthy controls. Accordingly, we kept focused on GrMDSC frequencies in relation to the melanoma stages and course of the disease. The GrMDSC values are highest in stage IV melanoma patients, with statistical significance compared with stages IA, IB, IIA and IIB. Patients with progression had statistically higher GrMDSC counts comparing with those with stable disease (P = 0.0079). Patients who had progression-free interval (PFI) < 12 months showed significantly higher GrMDSC values compared with those with PFI > 12 months (P = 0.0333). GrMDSC showed significant negative correlation with PFI intervals (P = 0.0095). The GrMDSC subset was predominant in all our patients. We confirmed that GrMDSC do accumulate early in the peripheral blood of melanoma patients and their frequencies correlate narrowly with the clinical stage and the spread of the disease. The increase in GrMDSC frequencies correlates well with a progressive disease and could be considered a potential predictive biomarker of high-risk melanoma cases that are more likely to have a shorter PFI.

Antimycobacterial activity of novel hydrazide-hydrazone derivatives with 2H-chromene and coumarin scaffold.

This study reports the synthesis of new 2H-chromene or coumarin based acylhydrazones, which were evaluated for their in vitro antimycobacterial activity against reference strain Mycobacterium tuberculosis H37Rv and compared to the first-line antituberculosis drugs, isoniazid (INH) and ethambutol (EMB). The most active compounds 7m (MIC 0.13μM), 7o (MIC 0.15μM) and 7k (MIC 0.17μM) demonstrated antimycobacterial activity at submicromolar concentration level and remarkably minimal associated cytotoxicity in the human embryonic kidney cell line HEK-293T. Structure-activity relationship for this class of compounds has been established.

In vitro antioxidant activity of thiazolidinone derivatives of 1,3-thiazole and 1,3,4-thiadiazole

The initial steps in preclinical drug developing research concern the synthesis of new compounds for specific therapeutic use which needs to be confirmed by in vitro and then in vivo testing. Nine thiazolidinone derivatives (numerically labeled 1-9) classified as follows: 1,3-thiazole-based compounds (1 and 2); 1,3,4-thiadiazole based compounds (3 and 4); substituted 5-benzylideno-2-adamantylthiazol[3,2-b][1,2,4]triazol-6(5H)ones (5-8); and an ethylaminothiazole-based chalcone (9), were tested for antioxidant activity (AOA) by using three in vitro assays: DPPH (1,1-diphenyl-2-picrylhydrazyl scavenging capacity test); FRAP (ferric reducing antioxidant power test); and TBARS (thiobarbituric acid reactive substances test). Compounds 1-4 and 9 in particular are newly synthesized compounds. Also, traditional antioxidants Vitamins E and C and α-lipoic acid (α-LA) were tested. The results of DPPH testing: Vitamin C 94.35%, Vitamin E 2.99% and α-LA 1.57%; compounds: 4 33.98%; 2 18.73%; 1 15.62%; 5 6.59%; 3 4.99%; 6-9 demonstrated almost no AOA. The results of TBARS testing (% of LPO inhibition): Vitamin C 62.32%; Vitamin E 36.29%; α-LA 51.36%; compounds: 1 62.11%; 5 66.71%; 9 60.93%; 4, 6 and 7 demonstrated ∼50%; 3 and 8 displayed ∼38%; 2 23.51%. By FRAP method, Vitamins E and C showed equal AOA, ∼100%, unlike α-LA (no AOA), and AOA of the tested compounds (expressed as a fraction of the AOA of Vitamin C) were: 2 and 4-75%; 8, 3 and 1-45%; 5-7 and 9-27%. Different red-ox reaction principles between these assays dictate different AOA outcomes for a single compound. Vitamin C appeared to be the superior antioxidant out of the traditional antioxidants; and compound 4 was superior to other tested thiazolidinone derivatives. Vitamin C appeared to be the superior antioxidant out of the traditional antioxidants; and compound 4 was superior to other tested thiazolidinone derivatives. Phenyl-functionalized benzylidene, amino-carbonyl functional domains and chelating ligand properties of the thiazolidinone derivatives correlated with AOA.

The Role of Oxidative Stress in the Clinical Manifestations of Childhood Asthma

ABSTRACT Introduction: The significance of oxidative stress in pathogenesis of childhood asthma was recognized, but its role in the clinical manifestations of disease is still unclear. Materials and Methods: The study was conducted in 96 asthmatic children. The urinary biomarker of oxidative stress, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG/creatinine) was determined by using HPLC-MS/MS. ELISA was performed to measure myeloperoxidase (MPO) and Cu,Zn- superoxide dismutase (Cu,Zn-SOD) in serum. Results: Logistic regression analysis revealed that female gender, tobacco smoke exposure, and increased 8-oxodG/creatinine were associated with risk for intermittent asthma, while the positive allergy test and increased Cu,Zn-SOD were associated with eczema in asthmatic children. Higher MPO (p = 0.033), and percent of granulocytes (p = 0.030) were found in severe persistent asthma in comparison to intermittent or mild persistent asthma. Conclusion: The main findings that TSE-induced oxidative stress is a risk for intermittent asthma and eczema may be clinically significant for the disease prevention and therapeutic improvements.

Disulfiram moderately restores impaired hepatic redox status of rats subchronically exposed to cadmium

Abstract Examination of cadmium (Cd) toxicity and disulfiram (DSF) effect on liver was focused on oxidative stress (OS), bioelements status, morphological and functional changes. Male Wistar rats were intraperitoneally treated with 1 mg CdCl2/kg BW/day; orally with 178.5 mg DSF/kg BW/day for 1, 3, 10 and 21 days; and co-exposed from 22nd to 42nd day. The co-exposure nearly restored previously suppressed total superoxide dismutase (SOD), catalase (CAT) and increased glutathione peroxidase (GPx) activities; increased previously reduced glutathione reductase (GR) and total glutathione-S-transferase (GST) activities; reduced previously increased superoxide anion radical (O2·−) and malondialdehyde (MDA) levels; increased zinc (Zn) and iron (Fe), and decreased copper (Cu) (yet above control value), while magnesium (Mg) was not affected; and decreased serum alanine aminotransferases (ALT) levels. Histopathological examination showed signs of inflammation process as previously demonstrated by exposure to Cd. Overall, we ascertained partial liver redox status improvement, compared with the formerly Cd-induced impact. Graphical Abstract

The simple isocratic HPLC—UV method for the simultaneous determination of reduced and oxidized glutathione in animal tissue

The aim of our work was to optimize and apply simple high-performance liquid chromatography method with ultraviolet detection (HPLC—UV) for simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in biological matrix (specifically, the rat liver tissue was used herein), since the ratio between oxidized and reduced glutathione forms (GSSG—GSH) has been recognized as an important biological marker of oxidatively depleted GSH in oxidative stress (OS)-associated diseases and poisonings. An isocratic chromatographic separation of GSH and GSSG (2.8 min and 6.3 min, respectively) was performed with the mobile phase consisted of sodium perchlorate solution (pH adjusted to 2.8) at flow rate of 1 mL min−1, detection set at 215 nm, and column temperature of 40 °C. The method offers short run time, linearity in the range of 0.01—200 μM concentration for both compounds (R2 = 1), low limits of detection and quantification (GSH: 0.18 μM and 0.56 μM, GSSG: 0.52 μM and 1.58 μM, respectively), precision...

High Interleukin 27 Production is Associated with Early Clinical Stage and Localized Disease in Patients with Melanoma

Summary Background: The immune response in patients with melanoma is an important focus of research due to the tumor’s resistance and immunotherapy possibilities. IL-27 is one of the cytokines with antitumor properties. The role of IL-27 in the pathogenesis of melanoma is still unclear. The aim of this study was to examine the association between serum IL-27 levels and the clinical parameters of melanoma patients. Methods: The IL-27 concentration was determined by commercial ELISA in serum samples from melanoma patients (n=72) and healthy control subjects (n=44). Patients were classified according to AJCC clinical stage, TNM stage, the length of progression-free interval (PFI) and the extent of the disease (localized or widespread). Results: Average IL-27 values were increased in patients with early stages of melanoma compared to patients with terminal stages and control values. The highest IL-27 concentration was found in stage IIa. Patients in stages III and IV had significantly lower values of IL-27 compared to control. Patients with localized melanoma and shorter PFI had insignificantly increased IL-27 levels compared to patients with widespread disease and longer PFI. Patients with metastatic disease and stage TNM4 had significantly lower average IL-27 values compared to control. Patients with high production of IL-27 (>1000 pg/mL) were most numerous in IIa AJCC stage, with initial tumor size TNM2 and in the group of patients with localized disease. Conclusions: High levels of IL-27 in patients with melanoma are associated with the initial stages and localized disease.

Influence of Dental Restorations on Oxidative Stress in Gingival Crevicular Fluid

Biocompatibility of dental materials (DM) can be evaluated by gingival crevicular fluid (GCF) oxidative stress (OS) status. The goal of the study was to ascertain influence of dental caries degree, teeth position, and type and amount of applied DM on GCF OS profile. For this purpose, we tested six DMs that were sealed in one session: amalgam (Amg), composites: Tetric EvoCeram and Beautifil (BF), phosphate cement—zinc phosphate and polycarboxylate cements—zinc polycarboxylate cements, and glass ionomer cement (GIC). The study included 88 dental outpatients. Follow-up was scheduled at 7th and 30th day. Oxidative stress parameters (malondialdehyde (MDA) and glutathione (GSH) levels and total superoxide dismutase (tSOD) activity) were measured before (0th day) and after the treatment (7th and 30th day) in GCF. Control teeth were mirror-positioned healthy teeth. The DM accomplished the following effects (listed in descending order): increase of GSH in GCF was realized by ZPoC > BF > GIC > Amg; tSOD activity increase by ZPoC > BF > Amg; and MDA decrease by ZPoC > ZPhC > Amg > TEC. Dental caries provokes insignificant rise of OS in GCF. ZPoC and ZPhC showed the highest antioxidant effect, contrary to GIC. Restorations with antioxidant properties may reduce gum diseases initiated by caries lesion, what is of great clinical relevance in dentistry.