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Expression of intracellular HSP70 is associated with cytoprotective effects against a wide range of stressful stimuli, such as inflammation, oxidative stress, hypoxia, endotoxins, infections, and fever. This cytoprotective effect is mainly attributed to their ability to stabilize protein structures through chaperone-like reversible interactions. HSP70 was recently detected in the extracellular medium, and its presence in serum is commonly associated with pathological situations, where it exerts modulatory effects on cells of the immune system. Previously, we have described the relationship between serum HSP70 levels, oxidant status, and clinical outcome of septic patients; the group of patients with higher prooxidant status and higher serum HSP70 had also higher mortality. To investigate the possible association between oxidized HSP70 and cytoprotection or cell death, we incubated RAW 264.7 macrophages with oxidized HSP70 and evaluated nitrite production, cell proliferation, cell viability, TNF-α release, and phagocytic activity. We also evaluated structural modifications caused by oxidation in purified HSP70. Oxidation of HSP70 altered its protein structure; besides, the modulatory effect of oxidized HSP70 on RAW264.7 cells was different from that of native HSP70. Macrophages treated with oxidized HSP70 presented lower proliferation and viability, lower phagocytic activity, and lower TNF-α release. These results indicate that oxidation of extracellular HSP70 modified its signaling properties, causing alterations on its modulatory effects on macrophage function and viability.

As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 μM) or therapeutic (5, 10 or 20 μM). Retinol at 10 and 20 μM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 μM), SB203580 (10 μM) or siRNA to either p38α (MAPK14) or p38β (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 μg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by retinol is mediated by the redox-dependent activation of p38/NF-kB signaling pathway.

In this study, we developed curcumin-loaded lipid-core nanocapsules (C-LNCs) in an attempt to improve the antiglioma activity of this polyphenol. C-LNC showed nanotechnological properties such as nanometric mean size (196 nm), 100% encapsulation efficiency, polydispersity index below 0.1, and negative zeta potential. The in vitro release assays demonstrated a controlled release of curcumin from lipid-core nanocapsules. In C6 and U251MG gliomas, C-LNC promoted a biphasic delivery of curcumin: the first peak occurred early in the treatment (1-3h), whereas the onset of the second phase occurred after 48 h. In C6 cells, the cytotoxicity of C-LNC was comparable to non-encapsulated curcumin only after 96 h, whereas C-LNCs were more cytotoxic than non-encapsulated curcumin after 24h of incubation in U251MG. Induction of G2/M arrest and autophagy were observed in C-LNC as well as in free-curcumin treatments. In rats bearing C6 gliomas, C-LNC (1.5mg/kg/day, i.p.) decreased the tumor size and malignance and prolonged animal survival when compared to same dose of non-encapsulated drug. In addition, serum markers of tissue toxicity and histological parameters were not altered. Considered overall, the data suggest that the nanoencapsulation of curcumin in LNC is an important strategy to improve its pharmacological efficacy in the treatment of gliomas.

Previous studies have linked oxidative stress with aging and aging-related processes, including menopause. Abnormalities in the redox state similar to those observed in menopausal women can be modeled experimentally with rat ovariectomy. The aim of the present study was to investigate the effects of vitamin A (retinol palmitate) supplementation (500 or 1,500 IU kg(-1) day(-1) for 30 days) on behavioral parameters and brain redox profile in ovariectomized (OVX) and sham-operated rats. Ovariectomy caused pronounced uterine atrophy and decreased locomotor/exploratory activity. Moreover, we found increased hypothalamic and frontal cortex superoxide dismutase/catalase (SOD/CAT) ratio and decreased hippocampal thiol content, accompanied by increased frontal cortex lipid oxidative damage (TBARS) in OVX rats. Vitamin A at 1,500 IUkg(-1) day(-1) decreased exploratory behavior and decreased total hippocampal thiol content in sham-operated rats, increased hippocampal SOD/CAT ratio and decreased total antioxidant potential in the hippocampus of both sham and OVX groups, and increased cortical TBARS levels in OVX rats. Thus, vitamin A may induce a pro-oxidant state in discrete brain regions of sham-operated and OVX rats. These results suggest some caution regarding the use of high doses of vitamin A supplementation during menopause.

Previous studies suggested that curcumin is a potential agent against glioblastomas (GBMs). However, the in vivo efficacy of curcumin in gliomas remains not established. In this work, we examined the mechanisms underlying apoptosis, selectivity, efficacy and safety of curcumin from in vitro (U138MG, U87, U373 and C6 cell lines) and in vivo (C6 implants) models of GBM. In vitro, curcumin markedly inhibited proliferation and migration and induced cell death in liquid and soft agar models of GBM growth. Curcumin effects occurred irrespective of the p53 and PTEN mutational status of the cells. Interestingly, curcumin did not affect viability of primary astrocytes, suggesting that curcumin selectivity targeted transformed cells. In U138MG and C6 cells, curcumin decreased the constitutive activation of PI3K/Akt and NFkappaB survival pathways, down-regulated the antiapoptotic NFkappaB-regulated protein bcl-xl and induced mitochondrial dysfunction as a prelude to apoptosis. Cells developed an early G2/M cell cycle arrest followed by sub-G1 apoptosis and apoptotic bodies formation. Caspase-3 activation occurred in the p53-normal cell type C6, but not in the p53-mutant U138MG. Besides its apoptotic effect, curcumin also synergized with the chemotherapeutics cisplatin and doxorubicin to enhance GBM cells death. In C6-implanted rats, intraperitoneal curcumin (50 mg kg(-1) d(-1)) decreased brain tumors in 9/11 (81.8%) animals against 0/11 (0%) in the vehicle-treated group. Importantly, no evidence of tissue (transaminases, creatinine and alkaline phosphatase), metabolic (cholesterol and glucose), oxidative or hematological toxicity was observed. In summary, data presented here suggest curcumin as a potential agent for therapy of GBMs.

Cellular and molecular mechanisms related to lung cancer have been extensively studied in recent years, but the availability of effective treatments is still scarce. Hecogenin acetate, a natural saponin presenting a wide spectrum of reported pharmacological activities, has been previously evaluated for its anticancer/antiproliferative activity in some in vivo and in vitro models. Here, we investigated the effects of hecogenin acetate in a human lung cancer cell line. A549 non-small lung cancer cells were exposed to different concentrations of hecogenin acetate and reactive species production, ERK1/2 activation, matrix metalloproteinase expression, cell cycle arrest and cell senescence parameters were evaluated. Hecogenin acetate significantly inhibited increase in intracellular reactive species production induced by H 2 O 2 . In addition, hecogenin acetate blocked ERK1/2 phosphorylation and inhibited the increase in MMP-2 caused by H 2 O 2 . Treatment with hecogenin acetate induced G 0 /G 1 -phase arrest at two concentrations (75 and 100 µM, 74% and 84.3% respectively), and increased the staining of senescence-associated β -galactosidase positive cells. These data indicate that hecogenin acetate is able to exert anti-cancer effects by modulating reactive species production, inducing cell cycle arrest and senescence and also modulating ERK1/2 phosphorylation and MMP-2 production.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:40Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:32:38Z : No. of bitstreams: 1 2-s2.0-84878680462.pdf: 16796911 bytes, checksum: da5683aaa10a345e105b8e5b6372639e (MD5) Made available in DSpace on 2014-05-27T11:29:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-12 Scope. To elucidate the morphological and biochemical in vitro effects exerted by caffeine, taurine, and guarana, alone or in combination, since they are major components in energy drinks (EDs). Methods and Results. On human neuronal SH-SY5Y cells, caffeine (0.125-2 mg/mL), taurine (1-16 mg/mL), and guarana (3.125-50 mg/mL) showed concentration-dependent nonenzymatic antioxidant potential, decreased the basal levels of free radical generation, and reduced both superoxide dismutase (SOD) and catalase (CAT) activities, especially when combined together. However, guarana-treated cells developed signs of neurite degeneration in the form of swellings at various segments in a beaded or pearl chain-like appearance and fragmentation of such neurites at concentrations ranging from 12.5 to 50 mg/mL. Swellings, but not neuritic fragmentation, were detected when cells were treated with 0.5 mg/mL (or higher doses) of caffeine, concentrations that are present in EDs. Cells treated with guarana also showed qualitative signs of apoptosis, including membrane blebbing, cell shrinkage, and cleaved caspase-3 positivity. Flow cytometric analysis confirmed that cells treated with 12.5-50 mg/mL of guarana and its combinations with caffeine and/or taurine underwent apoptosis. Conclusion. Excessive removal of intracellular reactive oxygen species, to nonphysiological levels (or antioxidative stress), could be a cause of in vitro toxicity induced by these drugs. © 2013 Fares Zeidán-Chuliá et al. Department of Biochemistry Institute of Basic Health Sciences Federal University of Rio Grande Do sul (UFRGS), 90035-003 Porto Alegre, RS Departamento de Física e Biofísica Instituto de Biociências de Botucatu Universidade Estadual Paulista (UNESP), 18618-970 Botucatu, SP Departamento de Química Centro de Ciências Naturais e Exatas (CCNE) Universidade Federal de Santa Maria (UFSM), 97105-900 Santa Maria, RS Department of Psychiatry and Behavioral Neurosciences McMaster University, Hamilton, ON L8P 3B6 Departamento de Física e Biofísica Instituto de Biociências de Botucatu Universidade Estadual Paulista (UNESP), 18618-970 Botucatu, SP

Objective. To evaluate antinocicpetive and redox properties of the monoterpenes (+)-camphene,p-cymene, and geranyl acetate inin vivoandin vitroexperimental models.Methods. Evaluation of thein vitroantioxidant activity of (+)-camphene,p-cymene, and geranyl acetate using different free radical-generating systems and evaluation of antinociceptive actions by acetic acid-induced writhing and formalin-induced nociception tests in mice.Results.p-Cymene has the strongest antinociceptive effect, but (+)-camphene and geranyl acetate also present significant activity at high doses (200 mg/kg). (+)-Camphene had the strongest antioxidant effectin vitroat TBARS and TRAP/TAR assays and also had the highest scavenging activities against different free radicals, such as hydroxyl and superoxide radicals. Sodium nitroprussiate-derived NO production was enhanced by (+)-camphene. Geranyl acetate andp-cymene also presented some antioxidant effects, but with a varying profile according the free radical-generating system studied.Conclusion. (+)-Camphene,p-cymene, and geranyl acetate may present pharmacological properties related to inflammation and pain-related processes, being potentially useful for development of new therapeutic strategies, with limited possibilities forp-cymene and geranyl acetate.

This study evaluated the oxidative stress through enzymatic and nonenzymatic biomarkers in diabetic patients with and without hypertension and prediabetics. The SOD and CAT (in erythrocytes) and GPx (in plasma) enzymatic activities, plasma levels of lipid peroxidation, and total thiols were measured in the blood of 55 subjects with type 2 diabetes and 38 subjects without diabetes (9 pre-diabetics and 29 controls) aged 40–86 years. The total SOD activity and the lipid peroxidation were higher in diabetics compared to nondiabetics. In stratified groups, the total SOD activity was different for the hypertensive diabetics compared to the prediabetics and normotensive controls. Lipid peroxidation was significantly higher in both groups of diabetics (hypertensive and normotensive) compared to prediabetic groups and hypertensive and normotensive controls. There was no significant difference in the CAT and GPx activities, as well as in the concentration of total thiols in the groups studied. Present data strongly suggest the involvement of oxidative stress in the pathophysiology of diabetes, revealing that the increased lipid peroxidation has a close relationship with high glucose levels, as observed by the fasting glucose and HbA1c levels. The results evidence the correlation between lipid peroxidation and DM, irrespective of the presence of hypertension.

The outcome of sepsis occurs due to influence of environmental and genetic factors besides genes variants whose expression support its outcome or not. Oxidative stress is associated to the pathogenicity of sepsis, occurring when there is a reactive species overproduction associated with inflammation. The aim of this study was to characterize the cellular redox status of human peripheral blood mononuclear cells (PBMCs) with either −9Ala (AA) or −9Val (VV) SOD2 genotypes and evaluate their response to oxidative stress induced by lipopolysaccharide (LPS). The PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers for each allele) and the following assays were performed: antioxidant enzyme activities (superoxide dismutase; catalase; glutathione peroxidase), total radical-trapping antioxidant parameter, non-enzymatic antioxidant capacity (total antioxidant reactivity), and quantification of conjugated dienes (lipid peroxidation). At basal conditions (i.e., not stimulated by LPS), cells from 47C allele carriers showed higher activities of CAT and SOD, as well as higher TAR compared to 47T allele. However, when 47CC cells were challenged with LPS, we observed a higher shift toward a pro-oxidant state compared to 47TT cells. The CAT activity and lipid peroxidation were increased in cells with both alleles, but SOD activity increased significantly only in 47TT cells. These results demonstrate that SOD2 polymorphisms are associated with different cellular redox environments at both basal and LPS-stimulated states, and identification of this polymorphism may be important for a better understanding of pro-inflammatory conditions.

Ethnopharmacological relevance The study was aimed at evaluating medicinal and therapeutic potentials of two Lycopodiaceae species, Lycopodium clavatum (L.) and Lycopodium thyoides (Humb. & Bonpl. ex Willd), both used in South American folk medicine for central nervous system conditions. Alkaloid extracts were evaluated for chemical characterization, acetylcholinesterase and antioxidant activities. Materials and methods The alkaloid extracts obtained by alkaline extraction were determined for each species by GC/MS examination. The evaluation of the anticholinesterase and the antioxidant activities of the extracts were tested by determining in vitro and ex vivo models. Effects on acetylcholinesterase (AChE) were tested in vitro using rat brain homogenates and ex vivo after a single administration (25, 10 and 1 mg/kg i.p.) of the alkaloid extracts in mice. The in vitro antioxidant effects were tested for the 2-deoxyribose degradation, nitric oxide (NO) interaction, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity and total reactive antioxidant potential (TRAP). After an acute administration (25 and 10 mg/kg i.p.) of the extracts in middle-aged (12 months) mice, the antioxidant effects were estimated through the thiobarbituric acid reactive substances test (TBARS), and the antioxidant enzymes activities for catalase (CAT) and superoxide dismutase (SOD) were measured. Results AChE activity was inhibited in vitro by the alkaloid-enriched extracts of both Lycopodium species in a dose and time-dependent manner in rat cortex, striatum and hippocampus. A significant inhibition was also observed in areas of the brain after acute administration of extracts, as well as decreased lipid peroxidation and increased CAT activity in the cortex, hippocampus and cerebellum. A moderate antioxidant activity was observed in vitro for the extracts. Chemically, the main alkaloids found for the two species were lycopodine and acetyldihidrolycopodine. Conclusion This study showed that the biological properties of the folk medicinal plants Lycopodium clavatum and Lycopodium thyoides include AChE inhibitory activity and antioxidant effects, two possible mechanisms of action in Alzheimer's related processes. Fil: Konrath, Eduardo Luis. Universidade Federal do Rio Grande do Sul; Brasil Fil: Neves, Bruna Medeiros. Universidade Federal do Rio Grande do Sul. Departamento de Bioquímica; Brasil Fil: Lunardi, Paula Santana. Universidade Federal do Rio Grande do Sul. Departamento de Bioquímica; Brasil Fil: Passos, Carolina Dos Santos. Universidade Federal do Rio Grande do Sul; Brasil Fil: Simões Pires, André. Universidade Federal do Rio Grande do Sul. Departamento de Bioquímica; Brasil Fil: Ortega, María Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Gonçalves, Carlos Alberto. Universidade Federal do Rio Grande do Sul; Brasil Fil: Cabrera, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Fonseca Moreira, José Cláudio. Universidade Federal do Rio Grande do Sul. Departamento de Bioquímica; Brasil Fil: Henriques, Amélia T.. Universidad Federal do Rio Grande do Sul; Brasil

Though, it is quite well-known how retinoic acid (RA) is able to induce neuritogenesis in different in vitro models, the putative role exerted by reactive oxygen species (ROS) during this process still need to be further studied. For such purpose, we used a neuronal-like cell line (SH-SY5Y cells) in order to investigate whether the antioxidant Trolox (a hydrophilic analog of alpha-tocopherol) could have any effect on the number of RA-induced neurites, and how significant changes in cellular redox homeostasis may affect the cellular endogenous expression of tyrosine hydroxylase (TH). Our results show a significant enhancement of RA (10 μM)-induced neuritogenesis and TH endogenous expression, when cells were co-treated with Trolox (100 μM) for 7 days. Moreover, this effect was associated with an improvement in cellular viability. The mechanism seems to mainly involve PI3 K/Akt rather than MEK signaling pathway. Therefore, our data demonstrate that concomitant decreases in basal reactive oxygen species (ROS) production could exert a positive effect on the neuritogenic process of RA-treated SH-SY5Y cells.

Vitamin A is important for both development and maintenance of adult brain homeostasis. However, excessive vitamin A exposure has been linked to cognitive impairments and may induce congenital defects, including neuronal malformations. Recently, we demonstrated that vitamin A supplementation is able to alter behavioral parameters and induce a pro-oxidant state in hippocampus and striatum of adult male rat. Thus, the aim of the present work was to investigate the effects of vitamin A supplementation in pregnant and nursing rats on maternal and offspring striatum and hippocampus. Wistar female rats (7 per group) were orally supplemented with retinyl palmitate (2500, 12,500 and 25,000IU/kg/day) or saline (control) throughout pregnancy and nursing. Homing test was performed at postnatal days (PND) 5 and 10 for offspring, while open field test (OFT) was carried out at PND19 and 20 for dams and offspring, respectively. Redox parameters were evaluated at PND21 for both. Vitamin A supplementation during pregnancy and nursing increased superoxide dismutase/catalase (SOD/CAT) ratio and oxidative damage in maternal and offspring striatum and hippocampus. Additionally, supplementation induced behavioral alterations. In conclusion, we suggest some caution regarding vitamin A intake during pregnancy and breastfeeding, since oxidative stress can disturb several biological phenomena, including neuronal signaling and neurotransmission, which may induce several behavioral deficits.