Cerebral White Matter Oxidation and Nitrosylation in Young Rodents With Kaolin-Induced Hydrocephalus

Hydrocephalus is associated with reduced blood flow in periventricular white matter. To investigate hypoxic and oxidative damage in the brains of rats with hydrocephalus, kaolin was injected into the cisterna magna of newborn 7- and 21-day-old Sprague-Dawley rats, and ventricle size was assessed by magnetic resonance imaging at 7, 21, and 42 days of age. In-situ evidence of hypoxia in periventricular capillaries and glial cells was shown by pimonidazole hydrochloride binding. Biochemical assay of thiobarbituric acid reaction and immunohistochemical detection of malondialdehyde and 4-hydroxy-2-nonenal indicated the presence of lipid peroxidation in white matter. Biochemical assay of nitrite indicated increased nitric oxide production. Nitrotyrosine immunohistochemistry showed nitrosylated proteins in white matter reactive microglia and astrocytes. Activities of the antioxidant enzymes catalase and glutathione peroxidase were not increased, and altered hypoxia-inducible factor 1 alpha was not detected by quantitative reverse transcription-polymerase chain reaction. Cerebral vascular endothelial growth factor expression determined by quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay was not changed, but vascular endothelial growth factor immunoreactivity was increased in reactive astrocytes of hydrocephalic white matter. To determine if nitric oxide synthase is involved in the pathogenesis, we induced hydrocephalus in 7-day-old wild-type and neuronal nitric oxide synthase-deficient mice. At 7 days, the wild-type and mutant mice exhibited equally severe ventriculomegaly and no behavioral differences, although increased glial fibrillary acidic protein was less in the mutant mice. We conclude that hypoxia, via peroxidation and nitrosylation, contributes to brain changes in young rodents with hydrocephalus and that compensatory mechanisms are negligible.

Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress

Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 mu M CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFN gamma through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPAR gamma receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2 alpha, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2 alpha induced by LPS/IFN gamma. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the 'oligoprotective' effects of CBD during inflammation. Cell Death and Disease (2012) 3, e331; doi:10.1038/cddis.2012.71; published online 28 June 2012

Inhibition of Macrophage Oxidative Stress Prevents the Reduction of ABCA-1 Transporter Induced by Advanced Glycated Albumin

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.

Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Partial Replacement of omega-6 Fatty Acids With Medium-Chain Triglycerides, but Not Olive Oil, Improves Colon Cytokine Response and Damage in Experimental Colitis

Background: Soybean oil is rich in omega-6 fatty acids, which are associated with higher incidence and more severe cases of inflammatory bowel diseases. The authors evaluated whether partial replacement of soybean oil by medium-chain triglycerides (MCTs) or olive oil influenced the incidence and severity of experimental ulcerative colitis by using different parenteral lipid emulsions (LEs). Methods: Wistar rats (n = 40) were randomized to receive parenteral infusion of the following LE: 100% soybean oil (SO), 50% MCT mixed with 50% soybean oil (MCT/SO), 80% olive oil mixed with 20% soybean oil (OO/SO), or saline (CC). After 72 hours of infusion, acetic acid experimental colitis was induced. After 24 hours, colon histology and cytokine expression were analyzed. Results: SO was not significantly associated with overall tissue damage. MCT/SO was not associated with necrosis (P < .005), whereas OO/SO had higher frequencies of ulcer and necrosis (P < .005). SO was associated with increased expression of interferon-gamma (P = .005) and OO/SO with increased interleukin (IL)-6 and decreased tumor necrosis factor-alpha expression (P < .05). MCT/SO appeared to decrease IL-1 (P < .05) and increase IL-4 (P < .001) expression. Conclusions: Parenteral SO with high concentration of omega-6 fatty acids was not associated with greater tissue damage in experimental colitis. SO partial replacement with MCT/SO decreased the frequency of histological necrosis and favorably modulated cytokine expression in the colon; however, replacement with OO/SO had unfavorable effects. (JPEN J Parenter Enteral Nutr. 2012; 36: 442-448)

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-alpha in the induction of endothelin system genes

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice. CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100 mg/kg) once a day, starting from the day when arthritis was clinically detectable. CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student's t test. Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1 beta, TNF alpha and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNF alpha upregulated ET system genes. These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNF alpha.

Effect of Byrsonima crassa and Phenolic Constituents on Helicobacter pylori-Induced Neutrophils Oxidative Burst

Byrsonima crassa Niedenzu (Malpighiaceae) is used in Brazilian folk medicine for the treatment of diseases related mainly to gastric ulcers. In a previous study, our group described the gastric protective effect of the methanolic extract from the leaves of B. crassa. The present study was carried out to investigate the effects of methanolic extract and its phenolic compounds on the respiratory burst of neutrophils stimulated by H. pylori using a luminol-based chemiluminescence assay as well as their anti-H. pylori activity. The suppressive activity on oxidative burst of H. pylori-stimulated neutrophils was in the order of methyl gallate > (+)-catechin > methanol extract > quercetin 3-O-alpha-L-arabinopyranoside > quercetin 3-O-beta-D-galactopyranoside > amentoflavone. Methyl gallate, compound that induced the highest suppressive activity with IC50 value of 3.4 mu g/mL, did not show anti-H. pylori activity. B. crassa could be considered as a potential source of natural antioxidant in gastric ulcers by attenuating the effects on the damage to gastric mucosa caused by neutrophil generated reactive oxygen species, even when H. pylori displays its evasion mechanisms.

Sugarwin: A Sugarcane Insect-Induced Gene with Antipathogenic Activity

In sugarcane fields, colonization of the stalk by opportunistic fungi usually occurs after the caterpillar Diatraea saccharalis attacks the sugarcane plant. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN), sugarcane wound-inducible proteins SUGARWIN1 and SUGARWIN2, have been identified in sugarcane by an in silico analysis. Antifungal properties have been described for a number of BARWIN homologues. We report that a SUGARWIN:green fluorescent protein fusion protein is located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of sugarwin transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment. The accumulation of transcripts is late induced and is restricted to the site of the wound. Although the transcripts of sugarwin genes were strongly increased following insect attack, the protein itself did not show any effect on insect development; rather, it altered fungal morphology, leading to the apoptosis of the germlings. These results suggest that, in the course of evolution, sugarwin-encoding genes were recruited by sugarcane due to their antipathogenic activity. We rationalize that sugarcane is able to induce sugarwin gene expression in response to D. saccharalis feeding as a concerted plant response to the anticipated invasion by the fungi that typically penetrate the plant stalk after insect damage.

Protective effect of bixin on cisplatin-induced genotoxicity in PC12 cells

Bixin is the main carotenoid found in annatto seeds (Bixa orellana L.) and is responsible for their reddish-orange color. The antioxidant properties of this compound are associated with its ability to scavenge free radicals, which may reduce damage and protect tissues against toxicity caused by anticancer drugs such as cisplatin. In this study, the genotoxicity and antigenotoxicity of bixin on cisplatin-induced toxicity in PC12 cells was assessed. Cytotoxicity was evaluated using the mu assay, mutagenicity, genotoxicity, and protective effect of bixin were evaluated using the micronucleus test and comet assay. PC12 cells were treated with bixin (0.05, 0.08, and 0.10 mu g/mL), cisplatin (0.1 mu g/mL) or a combination of both bixin and cisplatin. Bixin was neither cytotoxic nor genotoxic compared to the controls. In the combined treatment bixin significantly reduced the percentage of DNA in tail and the frequency of micronuclei induced by cisplatin. This result suggests that bixin can function as a protective agent, reducing cisplatin-induced DNA damage in PC12 cells, and it is possible that this protection could also extend to neuronal cells. Further studies are being conducted to better understand the mechanisms involved in the activity of this protective agent prior to using it therapeutically. (C) 2011 Elsevier Ltd. All rights reserved.

Involvement of nitric oxide on the pathogenesis of irinotecan-induced intestinal mucositis: role of cytokines on inducible nitric oxide synthase activation

Purpose Intestinal mucositis and the closely associated diarrhea are common costly side effects of irinotecan. Cytokine modulators, such as thalidomide and pentoxifylline, are found capable of attenuating intestinal mucositis progression. Nitric oxide (NO) seems to be a key mediator of the antineoplastic drug toxicity. The aim of this study was to investigate the role of NO on the pathogenesis of intestinal mucositis, as well as the participation of cytokines upon inducible nitric oxide synthase (iNOS) expression in irinotecan-induced intestinal mucositis. Methods iNOS-knockout (iNOS(-/-)) and C57BL/6 (WT, wild type) animals (n = 5-6) were given either saline or irinotecan (60 mg/kg i.p for 4 days), with or without pretreatment with aminoguanidine (50 mg/kg s.c.), thalidomide (60 mg/kg s.c), infliximab (5 mg/kg i.v.), or pentoxifylline (1.7 mg/kg s.c). On day 5, diarrhea was assessed, and following euthanasia, proximal intestinal samples were obtained for myeloperoxidase (MPO) and iNOS activity, morphometric analysis, western blot and immunohistochemistry to iNOS, cytokine dosage, and for in vitro evaluation of gut contractility. Results Irinotecan induced severe diarrhea and intestinal smooth muscle over-contractility, accompanied with histopathological changes. Additionally, increased MPO and iNOS activity and iNOS immunoexpression were found in WT animals treated with irinotecan. The rise in MPO, smooth muscle over-contractility, and diarrhea were abrogated in aminoguanidine-treated and iNOS(-/-) mice. Moreover, through western blot, we verified that infliximab and pentoxifylline significantly inhibited irinotecan-induced iNOS expression. In addition, cytokine concentration was found only partially decreased in irinotecan-treated iNOS(-/-) mice when compared with wild-type animals that were given irinotecan. Conclusions This study suggests a role of nitric oxide in the pathogenesis of irinotecan-induced intestinal mucositis and also provides evidence for the participation of cytokines on iNOS induction.

Phase-disorder-induced firing activity in excitable neuronal networks with attractive and repulsive coupling

It has been revealed that the network of excitable neurons via attractive coupling can generate spikes under stimuli of subthreshold signals with disordered phases. In this paper, we explore the firing activity induced by phase disorder in excitable neuronal networks consisting of both attractive and repulsive coupling. By increasing the fraction of repulsive coupling, we find that, in the weak coupling strength case, the firing threshold of phase disorder is increased and the system response to subthreshold signals is decreased, indicating that the effect of inducing neuron firing by phase disorder is weakened with repulsive coupling. Interestingly, in the large coupling strength case, we see an opposite situation, where the coupled neurons show a rather large response to the subthreshold signals even with small phase disorder. The latter case implies that the effect of phase disorder is enhanced by repulsive coupling. A system of two-coupled excitable neurons is used to explain the role of repulsive coupling on phase-disorder-induced firing activity.

Mikania glomerata Sprengel (Asteraceae) influences the mutagenicity induced by doxorubicin without altering liver lipid peroxidation or antioxidant levels

As shown in numerous studies, natural compounds may exert adverse effects, mainly when associated with some drugs. The hydroalcoholic extract of Mikania glomerata is the pharmaceutical form present in commercially available syrup used for the treatment of respiratory diseases in popular Brazilian medicine. The objective of the present investigation was (1) to evaluate the preventive effects of standardized hydroalcoholic extract of M. glomerata (MEx) against antitumoral drug doxorubicin (DXR)-induced micronucleated polychromatic erythrocytes (MNPCE) in a subchronic assay in mice, and (2) to determine the liver content of malondialdehyde (MDA) and the antioxidants glutathione (GSH) and vitamin E (VE). Male Swiss mice were treated for 30 d with MEx added to drinking water, combined or not with DXR (90 mg/kg body weight) injected intraperitoneally (ip) 24 h before analysis. The results demonstrated that MEx produced no genotoxic damage, but significantly increased the frequency of MNPCE induced by DXR, indicating a drug-drug interaction. This rise was not accompanied by lipid peroxidation or antioxidants level reduction, as measured by MDA, GSH, and VE. Despite the presence of coumarin (a known antioxidant), MEx may exert adverse effects probably in association with mutagenic compounds, although this effect on DNA damage did not involve oxidative stress.

Low-level laser therapy (LLLT) in human progressive-intensity running: effects on exercise performance, skeletal muscle status, and oxidative stress

The aim of this work was to evaluate the effects of low-level laser therapy (LLLT) on exercise performance, oxidative stress, and muscle status in humans. A randomized double-blind placebo-controlled crossover trial was performed with 22 untrained male volunteers. LLLT (810 nm, 200 mW, 30 J in each site, 30 s of irradiation in each site) using a multi-diode cluster (with five spots - 6 J from each spot) at 12 sites of each lower limb (six in quadriceps, four in hamstrings, and two in gastrocnemius) was performed 5 min before a standardized progressive-intensity running protocol on a motor-drive treadmill until exhaustion. We analyzed exercise performance (VO(2 max), time to exhaustion, aerobic threshold and anaerobic threshold), levels of oxidative damage to lipids and proteins, the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and the markers of muscle damage creatine kinase (CK) and lactate dehydrogenase (LDH). Compared to placebo, active LLLT significantly increased exercise performance (VO(2 max) p = 0.01; time to exhaustion, p = 0.04) without changing the aerobic and anaerobic thresholds. LLLT also decreased post-exercise lipid (p = 0.0001) and protein (p = 0.0230) damages, as well as the activities of SOD (p = 0.0034), CK (p = 0.0001) and LDH (p = 0.0001) enzymes. LLLT application was not able to modulate CAT activity. The use of LLLT before progressive-intensity running exercise increases exercise performance, decreases exercise-induced oxidative stress and muscle damage, suggesting that the modulation of the redox system by LLLT could be related to the delay in skeletal muscle fatigue observed after the use of LLLT.

Toll-like receptor 2/MyD88 signaling mediates zymosan-induced joint hypernociception in mice: Participation of TNF-alpha, IL-1 beta and CXCL1/KC

Arthritic pain is a serious health problem that affects a large number of patients. Toll-like receptors (TLRs) activation within the joints has been implicated in pathophysiology of arthritis. However, their role in the genesis of arthritic pain needs to be demonstrated. In the present study, it was addressed the participation of TLR2 and TLR4 and their adaptor molecule MyD88 in the genesis of joint hypernociception (a decrease in the nociceptive threshold) during zymosan-induced arthritis. Zymosan injected in the tibio-tarsal joint induced mechanical hypernociception in C57BL/6 wild type mice that was reduced in TLR2 and MyD88 null mice. On the other hand, zymosan-induced hypernociception was similar in C3H/HePas and C3H/Hej mice (TLR4 mutant mice). Zymosan-induced joint hypernociception was also reduced in TNFR1 null mice and in mice treated with IL-1 receptor antagonist or with an antagonist of CXCR1/2. Moreover, the joint production of TNF-alpha, IL-1 beta and CXCL1/KC by zymosan was dependent on TLR2/MyD88 signaling. Investigating the mechanisms by which TNF-alpha, IL-1 beta and CXCL1/KC mediate joint hypernociception, joint administration of these cytokines produced mechanical hypernociception, and they act in an interdependent manner. In last instance, their hypernociceptive effects were dependent on the production of hypernociceptive mediators, prostaglandins and sympathetic amines. These results indicate that in zymosan-induced experimental arthritis, TLR2/MyD88 is involved in the cascade of events of joint hypernociception through a mechanism dependent on cytokines and chemokines production. Thus, TLR2/MyD88 signaling might be a target for the development of novel drugs to control pain in arthritis. (C) 2011 Elsevier B.V. All rights reserved.

Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen

Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen (102), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with 102, they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT. (c) 2012 Elsevier Inc. All rights reserved.