Nanoparticles containing ß-cyclodextrin potentially useful for the treatment of Niemann-Pick C.

ß-Cyclodextrin (ß-CD) is being considered a promising therapy for Niemann-Pick C (NPC) disease because of its ability to mobilise the entrapped cholesterol from lysosomes, however, a major limitation is its inability to cross the blood-brain barrier (BBB) and address the central nervous system (CNS) manifestations of the disease. Considering this, we aimed to design nanoparticles able to cross the BBB and deliver ß-CD into the CNS lysosomes. The physicochemical characteristics of ß-CD-loaded nanoparticles were evaluated by dynamic light scattering, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The in vitro analyses were performed with NPC dermal fibroblasts and the ß-CD-loaded nanoparticles were tracked in vivo. The nanoparticles showed a mean diameter around 120 nm with a disordered bicontinuous inner structure. The nanoparticles did not cause decrease in cell viability, impairment in the antioxidant enzymes activity, damage to biomolecules or release of reactive species in NPC dermal fibroblasts; also, they did not induce genotoxicity or alter the mitochondrial function in healthy fibroblasts. The ß-CD-loaded nanoparticles were taken up by lysosomes reducing the cholesterol accumulated in NPC fibroblasts and reached the CNS of mice more intensely than other organs, demonstrating advantages compared to the free ß-CD. The results demonstrated the potential of the ß-CD-loaded nanoparticles in reducing the brain impairment of NPC.

Exercise during pregnancy decreases doxorubicin-induced cardiotoxic effects on neonatal hearts.

Cancer treatment with Doxorubicin (DOX) is limited due its dose-dependent cardiotoxicity, mainly related to the oxidative stress production. In experimental models of DOX treatment exercise can be used as a beneficial adjuvant therapy. This work aimed to investigate the effects of exercise during pregnancy on DOX-induced cardiotoxicity in cardiomyocytes of progeny, examining the possible intergenerational cardioprotective effects of maternal exercise. For this purpose pregnant rats were divided in control and exercise groups and pre-treated during gestational days. Hearts of newborns were used to obtain a culture of cardiomyocytes to be treated with DOX for analyses of cell viability, apoptosis and necrosis; ROS production; DNA damage; SOD and CAT activities; and Sirt6 protein expression. The results showed that exercise during pregnancy induced an increase in the viability of neonatal cardiomyocytes and a decrease in DOX-induced apoptotic and necrotic death which were correlated to the decrease in ROS production and an increase in antioxidant defenses. Exercise also protected neonatal cardiomyocytes from DOX-induced DNA damage, demonstrating a reduction in the oxidative DNA breaks. Likewise, exercise induced an increase in expression of Sirt6 in neonatal cardiomyocytes. Therefore, these results demonstrate for the first time that exercise performed by mothers protects the neonatal heart against DOX-induced toxicity. Our data demonstrate the intergenerational effect of exercise in cardiomyocytes of progeny, where the modulation of oxidative stress through antioxidant enzymes, and DNA integrity via Sirt6, were induced due to exercise in mothers, increasing the resistance of the neonatal heart against DOX toxicity.

The plasma membrane-enriched fraction proteome response during adaptation to hydrogen peroxide in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, adaptation to hydrogen peroxide (H2O2) decreases plasma membrane permeability to H2O2, changes its lipid composition and reorganizes ergosterol-rich microdomains by a still unknown mechanism. Here we show, by a quantitative analysis of the H2O2-induced adaptation effect on the S. cerevisiae plasma membrane-enriched fraction proteome, using two-dimensional gel electrophoresis, that 44 proteins are differentially expressed. Most of these proteins were regulated at a post-transcriptional level. Fourteen of these proteins contain redox-sensitive cysteine residues and nine proteins are associated with lipid and vesicle traffic. In particular, three proteins found in eisosomes and in the eisosome-associated membrane compartment occupied by Can1p were up-regulated (Pil1p, Rfs1p and Pst2p) during adaptation to H2O2. Survival studies after exposure to lethal H2O2 doses using yeast strains bearing a gene deletion corresponding to proteins associated to lipid and vesicle traffic demonstrated for the first time that down-regulation of Kes1p, Vps4p and Ynl010wp and up-regulation of Atp1 and Atp2 increases resistance to H2O2. Moreover, for the pil1Δ strain, H2O2 at low levels produces a hormetic effect by increasing proliferation. In conclusion, these data further confirms the plasma membrane as an active cellular site during adaptation to H2O2 and shows that proteins involved in lipid and vesicle traffic are important mediators of H2O2 adaptation.

Kinetics of alloxan-induced inhibition on δ-aminolevulinate dehydratase activity in mouse liver homogenates.

This study evaluated the effects of alloxan on the kinetics properties of the δ-aminolevulinate dehydratase (δ-ALA-D) using mouse liver homogenates. δ-ALA-D is an important sulfhydryl enzyme that catalyses the second step in heme biosynthesis and is commonly diminished in experimental and human diabetes. Despite the known effects of alloxan in models of experimental diabetes, there are no data in the literature demonstrating the effects of alloxan on the kinetics properties of the δ-ALA-D. The results showed that alloxan (1.25-20 µM) caused a concentration-dependent inhibition of hepatic δ-ALA-D activity. The inhibition constant (K(i)) for alloxan-induced inhibition on δ-ALA-D was 3.64 µM. The alloxan (5 µM) caused a decrease in V(max) (65.8%) and in K(m) (53.1%), which is suggestive of an uncompetitive inhibition of enzyme. In addition, dithiothreitol (700 and 1,000 µM) completely prevented the δ-ALA-D activity inhibition induced by 10 and 20 µM alloxan. Similar protection was obtained in the presence of 2,000 µM glutathione. Therefore, this work showed that the inhibition of hepatic δ-ALA-D activity can be obtained in vitro at low micromolar levels of alloxan, and can also be prevented by reducing agents. Moreover, these results may help to understand the abnormalities in heme pathway found in models of experimental diabetes in vivo.

Inhibition of δ-aminolevulinate dehydratase is not closely related to the development of hyperglycemia in alloxan-induced diabetic mice.

Alloxan is a compound widely used in models of diabetes mellitus due to its ability for damage insulin-producing ß-cells. The aim of this study was to investigate acute (after 24h) and sub-acute (after seven days) effects of 200mg/kg alloxan administration on mice. Biochemical parameters as liver, kidney, and blood δ-ALA-D activity, total sulfhydryl content of hepatic and renal tissues, and hepatic and renal content of malondialdehyde (MDA) were evaluated. The histopathology of hepatic and renal tissues of alloxan-treated and control animals was carried out. Further, blood glucose levels were determined in an attempt to correlate alloxan-induced hyperglycemia with changes in thiol status. Results showed that mice exhibited a significant inhibition of hepatic and renal δ-ALA-D activity in addition to a significant decrease in total sulfhydryl groups of same tissues in both acute and sub-acute alloxan administrations. Moreover, alloxan-induced inhibition of δ-ALA-D activity was partly suppressed when enzymatic assay was performed in the presence of dithiothreitol, suggesting that inhibitory effect of alloxan on δ-ALA-D activity is, at least partially, related to the oxidation of the enzyme's essential thiol groups. Blood δ-ALA-D activity was significantly inhibited only 24h after alloxan administration; however, at this time, a hyperglycemic status was not observed in animals. In contrast, a significant increase in blood glucose levels was observed seven days after alloxan administration. Despite of alterations in biochemical parameters, histological tissue examination of alloxan-treated mice revealed typical renal and hepatic parenchyma. Therefore, these results showed that acute toxic effects of alloxan are related, at least partially, to depletion of sulfhydryl groups, and do not closely relate to the development of hyperglycemia in mice.

Diphenyl diselenide and diphenyl ditelluride increase the latency for 4-aminopyridine-induced chemical seizure and prevent death in mice.

In this work was investigated the effect of pre-treatment with (PhSe)(2) and (PhTe)(2) on chemical seizure and 4-aminopyridine-induced lethality in mice. Additionally, lipid peroxidation levels of whole brain after treatment with 4-aminopyridine and effect of pre-treatment with (PhSe)(2) and (PhTe)(2) on these levels were investigated. Mice were pre-treated with (PhSe)(2) or (PhTe)(2) (50, 100, or 150 micromol/kg) 30 min before 4-aminopyridine (12 mg/kg) administration. The treatment with 4-aminopyridine caused a significant incidence of seizures (clonic, tonic) and death. Pre-treatment with (PhSe)(2) and (PhTe)(2) significantly increased the latency for clonic and tonic seizures, and prevented 4-aminopyridine-induced death. Significantly, the pre-treatment with (PhSe)(2) or (PhTe)(2) increased the latency for clonic seizures in a dose-dependent manner. Additionally, a significant increase was observed in the brain lipid peroxidation level after treatment with 4-aminopyridine, which was significantly inhibited by pre-treatment with 150 micromol/kg (PhSe)(2) or (PhTe)(2). These results demonstrate that (PhSe)(2) and (PhTe)(2) counteract the harmful effects of 4-aminopyridine. It is possible that this effect results from modulation of the redox state of N-methyl-d-aspartate receptors and/or of Ca(2+) channel activity with subsequent alteration in neurotransmitter release. Importantly, this study provides evidence for anticonvulsant and antioxidant properties of (PhSe)(2) and (PhTe)(2), which indicates a neuroprotective activity of these compounds.

Long-term sucrose and glucose consumption decreases the delta-aminolevulinate dehydratase activity in mice.

OBJECTIVE: This study evaluated the long-term effects of high-glucose (GLU) and high-sucrose (SUC) diets on the development of obesity, abdominal fat deposition, glucose intolerance, oxidative stress and effects on delta-aminolevulinate dehydratase (delta-ALA-D) activity in various organs. In particular, the effect of aging on these parameters was evaluated. METHODS: Mice were assigned to a baseline, control, or experimental group. The control group was provided with tap water and experimental groups with solutions of glucose or sucrose for 30 wk. To verify the effect of aging, young mice (baseline group, 8 wk old) were compared with aged animals (control and experimental groups, 38 wk old). RESULTS: Consumption of GLU or SUC diets caused increases in body weight, abdominal fat index, and fasting plasma glucose levels. A positive correlation was observed between the abdominal fat index and fasting glucose levels. There was a significant increase in levels of thiobarbituric acid-reactive species (TBARS) and a significant decrease in delta-ALA-D activity in various tissues of GLU and SUC feeding mice. Importantly, the dithiothreitol-induced enzymatic reactivation in the GLU and SUC groups was significantly higher than in the control group, and in the aged group it was significantly higher than in the baseline group. After 30 wk, the experimental groups had a decrease in delta-ALA-D activity and an increase in TBARS levels in relation to the baseline group. CONCLUSION: Alterations in the activity of the delta-ALA-D found in this work demonstrate the possible contributions of hyperglycemia and aging for protein oxidation, leading to impairment of its biologic function.

Diphenyl diselenide and 2,3-dimercaptopropanol increase the PTZ-induced chemical seizure and mortality in mice.

The aim of the present study was to evaluate the interaction between a classic GABAergic antagonist -- pentylenetetrazol (PTZ) with an organoselenium compound -- diphenyl diselenide (PhSe)(2) and with the metal chelating agent -- 2,3 dimercaptopropanol (BAL). Mice were pre-treated with 150 micromol/kg (PhSe)(2) or BAL (250, 500 or 1000 micromol/kg) before treatment with PTZ. Pre-treatment with (PhSe)(2) reduced the latency for PTZ-induced seizure at doses of 40 and 60 mg/kg and cause a decrease in the latency for PTZ-induced death at the dose of 60 mg/kg. However, treatment with PTZ at dose of 80 mg/kg was not affected by (PhSe)(2) pre-treatment. Pre-treatment with BAL reduced the latency for PTZ-induced seizure at doses of 40 and 50 mg/kg. In addition, the latency for PTZ-induced death at the dose of 40 mg/kg was decreased significantly by pre-treatment with all doses of BAL. At the dose of 50mg/kg, a significant decrease in the latency for death occurred only in mice pre-treated with 500 and 1000 micromol/kg of BAL. Our results indicate that the PTZ-induced chemical seizures and mortality was enhanced by (PhSe)(2) and BAL. These results indicated that (PhSe)(2) and BAL interact with PTZ possibly by modulating the GABAergic system.

High fat diet increases the incidence of orofacial dyskinesia and oxidative stress in specific brain regions of rats.

Haloperidol-induced orofacial dyskinesia (OD) is a putative animal model of tardive dyskinesia (TD) whose pathophysiology has been related to free radical generation and oxidative stress. Schizophrenic patients have been reported to eat a diet higher in fat than the general population and dietary fat intake can lead to an increase in oxidative stress in animal models. The objective of this study was to determine whether association of ingestion of a high fat diet with prolonged haloperidol treatment could lead to OD and oxidative stress in the rat brain. Haloperidol decanoate administration (38 mg/kg, IM, which is equivalent to 1 mg/kg/day) monthly for a period of 6 months to rats fed previously with a high fat and normo fat diets (6 months) caused a increase in vacuous chewing (VCM) and duration of facial twitching (FT). Haloperidol caused a reduction in body weight gain and the loss of body weight occurred after 4 months of treatment with haloperidol. The effects on body weight were more accentuated in HF diet group. HF diet ingestion was associated with an increase in TBARS levels in cerebellum and cerebral cortex (regardless of haloperidol treatment). A significant dietxhaloperidol treatment interaction in striatum, subcortical parts and the region containing the substantia nigra was observed for TBARS. In fact, haloperidol caused an increase in TBARS levels of these regions only in rats fed with the HF. These results indicate that a high fat diet caused a transitory increase in haloperidol-induced OD in rats and this in part can be related to the haloperidol-induced oxidative stress in brain structures involved with OD.

High sucrose consumption potentiates the sub-acute cadmium effect on Na+/K+ -ATPase but not on delta-aminolevulinate dehydratase in mice.

High sucrose consumption and sub-acute cadmium effects on delta-ALA-D activity, Na+/K+ -ATPase activity, and lipid peroxidation were studied in different tissues of mice. Experimental groups were control, high sucrose (200 g/L), cadmium (5 mg/kg/day, subcutaneously, two consecutive doses in different days in each week, during 4 weeks), and sucrose plus cadmium. There was a significant increase in TBARS levels for spleen and liver in cadmium and sucrose plus cadmium groups. Testicular delta-ALA-D activity of cadmium and sucrose plus cadmium-treated animals was significantly inhibited, whereas the enzyme activity increased in blood (cadmium and sucrose plus cadmium groups) and spleen (sucrose plus cadmium group). Na+/K+ -ATPase activity was significantly decreased in brain and kidney of sucrose plus cadmium-treated animals. Our data indicate that sub-acute cadmium treatment inhibits significantly testicular delta-ALA-D activity, demonstrating the prevalent cadmium effect in vivo on reproductive systems. Furthermore, high sucrose consumption and sub-acute cadmium treatment have interactive effects on cerebral and renal Na+/K+ -ATPase, showing that a short-term intake of high quantity of sucrose can aggravate the toxicity of Cd2+.