Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females) aged 17 to 58 years. Twenty one (53.84%) of the patients presented a slow acetylatingphenotype and 18(46.16%) a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD) acti vity was decreased in 5(23.80%) slow acetylators and in 4(22.22%) fast acetylators. Glutathione reductase activity was decreased in 14 (66.66%) slow acetylators and in 12 (66.66%) fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p < 0.02). Analysis of the resultspermitted us to conclude that serum sulfadoxin levels are related to the acetylatorphenotype. Furthermore, sulfadoxin levels were always above 50 µg/ml, a value considered therapeutic. Glutathione reductase deficiency observed in 66% of patients may be related to the intestinal malabsorption of nutrients, among them riboflavin, a FAD precursor vitamin, inpatients with paracoceidioidomycosis.

Monitoring antibiotics in the environment. Study of Quinoxaline derivatives bioactivity

Dissertação para obtenção do Grau de Doutor em Química Sustentável

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

Mecanismos apoptóticos desencadenados por vitamina D y drogas que deplecionan glutatión en células de cáncer de mama. Apoptotic mechanisms triggered by vitamin D and glutathione depleting drugs in breast cancer cells.

El cáncer de mama es una de las neoplasias más frecuentes de nuestro medio. El calcitriol y sus análogos son una alternativa nueva al uso convencional de antiestrógenos como quimioterapia. Sin embargo, los efectos hipercalcemiantes, secundarios a su aplicación, constituyen una limitación para su uso. Este proyecto está orientado al conocimiento de las bases moleculares antiproliferativas del uso del calcitriol en forma conjunta con drogas que deplecionan glutatión (GSH) tales como menadiona (MEN) y DL-butionina-S,R-sulfoximina (BSO). La hipótesis que se sostiene es que MEN y BSO, al disminuir el contenido de GSH, generan estrés oxidativo el cual puede potenciar el efecto antineoplásico del calcitriol, permitiendo lograr un mayor efecto antiproliferativo con dosis menores del secoesteroide, evitándose los efectos hipercalcemiantes. El objetivo general de este proyecto es dilucidar los mecanismos moleculares de apoptosis desencadenados por calcitriol (D) y/o drogas que deplecionan GSH (MEN o BSO) sobre las células de cáncer de mama MCF-7 en cultivo. Para ello, se tratarán células MCF-7 con concentraciones variables de D (en ausencia y presencia de MEN ó BSO) a diferentes tiempos. Se medirá proliferación celular mediante las técnicas de incorpororación de bromodeoxiuridina y de violeta de cristal. Se analizará el ciclo celular por medio de técnicas de citometría de flujo. Se determinará la participación tanto de la vía intrínseca como de la vía extrínseca de apoptosis. El contenido de GSH y la medición de las actividades del sistema antioxidante se llevará a cabo con técnicas espectrofotométricas. La expresión proteica de diversas caspasas se analizará por Western blots y la expresión génica por transcriptasa reversa-reacción en cadena de la polimerasa. Además, se desarrollarán artificialmente tumores de mama en ratas y se aplicará el tratamiento combinado midiéndose el efecto antitumoral mediante análisis histológicos. Se espera que el tratamiento combinado inhiba la proliferación de las células MCF-7, a través de incremento en la producción de especies reactivas derivadas del oxígeno involucrando la participación de las principales vías apoptóticas, extrínseca e intrínseca. En consecuencia, habría desrregulación de la función mitocondrial. Las defensas antioxidantes podrían estar alteradas. De ocurrir así, el tamaño de los tumores de mama desarrollados experimentalmente y tratados con el tratamiento combinado, estaría disminuido. La importancia de este estudio consiste en la exploración de una nueva estrategia terapéutica para el tratamiento de cáncer de mama.

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Rational design of an organometallic glutathione transferase inhibitor.

Double trouble: A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor (see picture). The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

Induction of Glutathione S-transferase Activity in Triatoma infestans

Several synthetic pesticides and allelochemicals used to treat Triatoma infestans adults by topic application showed some degree of cytosolic glutathione S-transferase (GST) induction. General inducers of detoxication systems such as phenobarbital and 3-methylcholantrene topically applied on T. infestans resulted in no GST induction. Meanwhile, general insecticide synergist such as piperonyl butoxide (160 mg/insect) increased the GST-activity in the range of 120-140%. Insects injected with reduced glutathione (300 mg/insect) presented at the forth day elevated GST activity

Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

A dendritic cell vaccine pulsed with autologous hypochlorous Acid-oxidized ovarian cancer lysate primes effective broad antitumor immunity: from bench to bedside.

PURPOSE: Whole tumor lysates are promising antigen sources for dendritic cell (DC) therapy as they contain many relevant immunogenic epitopes to help prevent tumor escape. Two common methods of tumor lysate preparations are freeze-thaw processing and UVB irradiation to induce necrosis and apoptosis, respectively. Hypochlorous acid (HOCl) oxidation is a new method for inducing primary necrosis and enhancing the immunogenicity of tumor cells. EXPERIMENTAL DESIGN: We compared the ability of DCs to engulf three different tumor lysate preparations, produce T-helper 1 (TH1)-priming cytokines and chemokines, stimulate mixed leukocyte reactions (MLR), and finally elicit T-cell responses capable of controlling tumor growth in vivo. RESULTS: We showed that DCs engulfed HOCl-oxidized lysate most efficiently stimulated robust MLRs, and elicited strong tumor-specific IFN-γ secretions in autologous T cells. These DCs produced the highest levels of TH1-priming cytokines and chemokines, including interleukin (IL)-12. Mice vaccinated with HOCl-oxidized ID8-ova lysate-pulsed DCs developed T-cell responses that effectively controlled tumor growth. Safety, immunogenicity of autologous DCs pulsed with HOCl-oxidized autologous tumor lysate (OCDC vaccine), clinical efficacy, and progression-free survival (PFS) were evaluated in a pilot study of five subjects with recurrent ovarian cancer. OCDC vaccination produced few grade 1 toxicities and elicited potent T-cell responses against known ovarian tumor antigens. Circulating regulatory T cells and serum IL-10 were also reduced. Two subjects experienced durable PFS of 24 months or more after OCDC. CONCLUSIONS: This is the first study showing the potential efficacy of a DC vaccine pulsed with HOCl-oxidized tumor lysate, a novel approach in preparing DC vaccine that is potentially applicable to many cancers. Clin Cancer Res; 19(17); 4801-15. ©2013 AACR.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

Effects of dietary protein type on oxidized cholesterol-induced alteration in age-related modulation of lipid metabolism and indices of immune function in rats.

Exogenous oxidized cholesterol disturbs both lipid metabolism and immune functions. Therefore, it may perturb these modulations with ageing. Effects of the dietary protein type on oxidized cholesterol-induced modulations of age-related changes in lipid metabolism and immune function was examined using differently aged (4 weeks versus 8 months) male Sprague-Dawley rats when casein, soybean protein or milk whey protein isolate (WPI) was the dietary protein source, respectively. The rats were given one of the three proteins in diet containing 0.2% oxidized cholesterols mixture. Soybean protein, as compared with the other two proteins, significantly lowered both the serum thiobarbituric acid reactive substances value and cholesterol, whereas it elevated the ratio of high density lipoprotein-cholesterol/cholesterol in young rats, but not in adult. Moreover, soybean protein, but not casein and WPI, suppressed the elevation of Delta6 desaturation indices of phospholipids in both liver and spleen, particularly in young. On the other hand, WPI, compared to the other two proteins, inhibited the leukotriene B4 production of spleen, irrespective of age. Soybean protein reduced the ratio of CD4(+)/CD8(+) T-cells in splenic lymphocytes. Therefore, the levels of immunoglobulin (Ig)A, IgE and IgG in serum were lowered in rats given soybean protein in both age groups except for IgA in adult, although these observations were not shown in rats given other proteins. Thus, various perturbations of lipid metabolism and immune function caused by oxidized cholesterol were modified depending on the type of dietary protein. The moderation by soybean protein on the change of lipid metabolism seems to be susceptible in young rats whose homeostatic ability is immature. These observations may be exerted through both the promotion of oxidized cholesterol excretion to feces and the change of hormonal release, while WPI may suppress the disturbance of immune function by oxidized cholesterol in both ages. This alleviation may be associated with a large amount of lactoglobulin in WPI. These results thus showed a possibility that oxidized cholesterol-induced perturbations of age-related changes of lipid metabolism and immune function can be moderated by both the selection and combination of dietary protein.

The role of platelet and plasma markers of antioxidant status and oxidative stress in thrombocytopenia among patients with vivax malaria

Malaria remains an important health problem in tropical countries like Brazil. Thrombocytopenia is the most common hematological disturbance seen in malarial infection. Oxidative stress (OS) has been implicated as a possible mediator of thrombocytopenia in patients with malaria. This study aimed to investigate the role of OS in the thrombocytopenia of Plasmodium vivax malaria through the measurement of oxidant and antioxidant biochemical markers in plasma and in isolated platelets. Eighty-six patients with P. vivax malaria were enrolled. Blood samples were analyzed for total antioxidant and oxidant status, albumin, total protein, uric acid, zinc, magnesium, bilirubin, total thiols, glutathione peroxidase (GPx), malondialdehyde (MDA), antibodies against mildly oxidized low-density lipoproteins (LDL-/nLDL ratio) and nitrite/nitrate levels in blood plasma and GPx and MDA in isolated platelets. Plasma MDA levels were higher in thrombocytopenic (TCP) (median 3.47; range 1.55-12.90 µmol/L) compared with the non-thrombocytopenic (NTCP) patients (median 2.57; range 1.95-8.60 µmol/L). Moreover, the LDL-/nLDL autoantibody ratio was lower in TCP (median 3.0; range 1.5-14.8) than in NTCP patients (median 4.0; range 1.9-35.5). Finally, GPx and MDA were higher in the platelets of TPC patients. These results suggest that oxidative damage of platelets might be important in the pathogenesis of thrombocytopenia found in P. vivax malaria as indicated by alterations of GPx and MDA.