Ramon dos Santos El-Bachá

Oxidative Stress in Neurodegenerative Diseases: Mechanisms and Therapeutic Perspectives

The incidence and prevalence of neurodegenerative diseases (ND) increase with life expectancy. This paper reviews the role of oxidative stress (OS) in ND and pharmacological attempts to fight against reactive oxygen species (ROS)-induced neurodegeneration. Several mechanisms involved in ROS generation in neurodegeneration have been proposed. Recent articles about molecular pathways involved in ROS generation were reviewed. The progress in the development of neuroprotective therapies has been hampered because it is difficult to define targets for treatment and determine what should be considered as neuroprotective. Therefore, the attention was focused on researches about pharmacological targets that could protect neurons against OS. Since it is necessary to look for genes as the ultimate controllers of all biological processes, this paper also tried to identify gerontogenes involved in OS and neurodegeneration. Since neurons depend on glial cells to survive, recent articles about the functioning of these cells in aging and ND were also reviewed. Finally, clinical trials testing potential neuroprotective agents were critically reviewed. Although several potential drugs have been screened in in vitro and in vivo models of ND, these results were not translated in benefit of patients, and disappointing results were obtained in the majority of clinical trials.

Astrocytic modulation of blood brain barrier: perspectives on Parkinson’s disease

The blood–brain barrier (BBB) is a tightly regulated interface in the Central Nervous System (CNS) that regulates the exchange of molecules in and out from the brain thus maintaining the CNS homeostasis. It is mainly composed of endothelial cells (ECs), pericytes and astrocytes that create a neurovascular unit (NVU) with the adjacent neurons. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted factors that lead to the adequate association between the cells of the BBB and the formation of strong tight junctions. Under neurological disorders, such as chronic cerebral ischemia, brain trauma, Epilepsy, Alzheimer and Parkinson’s Diseases, a disruption of the BBB takes place, involving a lost in the permeability of the barrier and phenotypical changes in both the ECs and astrocytes. In this aspect, it has been established that the process of reactive gliosis is a common feature of astrocytes during BBB disruption, which has a detrimental effect on the barrier function and a subsequent damage in neuronal survival. In this review we discuss the implications of astrocyte functions in the protection of the BBB, and in the development of Parkinson’s disease (PD) and related disorders. Additionally, we highlight the current and future strategies in astrocyte protection aimed at the development of restorative therapies for the BBB in pathological conditions.

Mitochondrial functions in astrocytes: Neuroprotective implications from oxidative damage by rotenone

Mitochondria are critical for cell survival and normal development, as they provide energy to the cell, buffer intracellular calcium, and regulate apoptosis. They are also major targets of oxidative stress, which causes bioenergetics failure in astrocytes through the activation of different mechanisms and production of oxidative molecules. This review provides an insightful overview of the recent discoveries and strategies for mitochondrial protection in astrocytes. We also discuss the importance of rotenone as an experimental approach for assessing oxidative stress in the brain and delineate some molecular strategies that enhance mitochondrial function in astrocytes as a promising strategy against brain damage.

Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells

Plants of Crotalaria genus (Leguminosae) present large amounts of the pyrrolizidine alkaloid monocrotaline (MCT) and cause intoxication to animals and humans. Therefore, we investigated the MCT-induced cytotoxicity, morphological changes, and oxidative and genotoxic damages to glial cells, using the human glioblastoma cell line GL-15 as a model. The comet test showed that 24h exposure to 1-500microM MCT and 500microM dehydromonocrotaline (DHMC) caused significant increases in cell DNA damage index, which reached 42-64% and 53%, respectively. Cells exposed to 100-500microM MCT also featured a contracted cytoplasm presenting thin cellular processes and vimentin destabilisation. Conversely, exposure of GL-15 cells to low concentrations of MCT (1-10microM) clearly induced megalocytosis. Moreover, MCT also induced down regulation of MAPs, especially at the lower concentrations adopted (1-10microM). Apoptosis was also evidenced in cells treated with 100-500microM MCT, and a later cytotoxicity was only observed after 6 days of exposure to 500microM MCT. The data obtained provide support for heterogenic and multipotential effects of MCT on GL-15 cells, either interfering on cell growth and cytoskeletal protein expression, or inducing DNA damage and apoptosis and suggest that the response of glial cells to this alkaloid might be related to the neurological signs observed after Crotalaria intoxication.

Evaluation of thermal-oxidative stability and antiglioma activity of Zanthoxylum tingoassuiba essential oil entrapped into multi- and unilamellar liposomes

Zanthoxylum tinguassuiba essential oil (ZtEO) contains α-bisabolol, a known antiglioma sesquiterpene, among other potentially active substances. Medical applications of this essential oil require advances in the design of distinctive carriers due to its low water solubility and easy degradation by heat, light, and oxygen. The aim of this work was to evaluate enhancement in oxidative stability and the ability to reduce glioblastoma cell viability of ZtEO loaded into liposomes. Multi- and unilamellar vesicles were prepared to carry ZtEO. By using thermal analysis, it was observed that thermal-oxidative stability of the liposomal ZtEO was enhanced, when compared to its free form. Liposomal ZtEO also presented significant apoptotic-inducing activity for glioma cells. These results show that liposomal systems carrying ZtEO may be a potential alternative for gliobastoma treatment.

Catechol cytotoxicity in vitro: induction of glioblastoma cell death by apoptosis.

The exposure to benzene is a public health problem. Although the most well-known effect of benzene is hematopoietic toxicity, there is little information about the benzene and its metabolites effects on the central nervous system (CNS). This study examined the toxic effects of 1,2-dihydroxybenzene (catechol), a benzene metabolite, to human glioblastoma GL-15 cells. GL-15 cell cultures were used as a model to provide more information about the toxic effects of aromatic compounds to the CNS. Catechol induced time- and concentration-dependent cytotoxic effects. Morphological changes, such as the retraction of the cytoplasm and chromatin clumping, were seen in cells exposed to 200 μM catechol for 48 hours. In cells exposed to 600 μM catechol for 48 hours, 78.0% of them presented condensed nuclei, and the Comet assay showed DNA damage. The percentage of cells labeled with annexin V (apoptotic cells) was greater in the group exposed to catechol (20.7%) than in control cells (0.4%). Exposure to catechol at concentrations greater than 100 μM enhanced Bax levels, and a decrease in Bcl-2 level was observed after the exposure to 600 μM catechol for 48 hours. Furthermore, catechol depleted reduced glutathione. Hence, catechol induced cell death mainly by apoptosis.

Brain rust: Recent discoveries on the role of oxidative stress in neurodegenerative diseases

Abstract Oxidative stress (OS) and damages due to excessive reactive oxygen species (ROS) are common causes of injuries to cells and organisms. The prevalence of neurodegenerative diseases (ND) increases with aging and much of the research involving ROS and OS has emerged from works in this field. This text reviews some recent published articles about the role of OS in ND. Since there are many reviews in this field, the focus was centered in articles published recently. The Scientific Journals Directory supported by the Brazilian Ministry of Education Office for the Coordination of Higher Educational Personnel Improvement (CAPES) was used to search, download, and review articles. The search engine looked for the terms ‘oxidative stress AND neurodegenerative diseases AND nutrition’ in 10 different scientific collections. Biochemical markers for ND lack sensitivity or specificity for diagnosis or for tracking response to therapy today. OS has an intimate connection with ND, albeit low levels of ROS seem to protect the brain. Deleterious changes in mitochondria, OS, calcium, glucocorticoids, inflammation, trace metals, insulin, cell cycle, protein aggregation, and hundreds to thousands of genes occur in ND. The interaction of genes with their environment, may explain ND. Although OS has received much attention over the years, which increased the number of scientific works on antioxidant interventions, no one knows how to stop or delay ND at present. Interventions in vitro, in vivo, and in humans will continue to contribute for a better understanding of these pathologies.

Catechol inhibits FADH2-linked respiration in rat liver mitochondrial fraction.

PURPOSE The aim of this work was to investigate the hypothesis that catechol inhibits FADH -linked basal respiration in mitochondria isolated from rat liver homogenates. Moreover, catechol ability to induce peroxidation of biomolecules in liver nuclear fractions was also studied. METHODS Rat liver homogenates were incubated with 1mM 1,2-dihydroxybenzene (catechol) at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate. Nuclear fractions were incubated in the presence of 1 mM catechol for 17 hours at room temperature and the peroxidation of biomolecules was investigated by the reaction with thiobarbituric acid, which was determined spectrophotometrically at 535 nm. RESULTS Catechol induced a time-dependent partial inhibition of FADH -linked basal mitochondrial respiration, however this substance was unable to induce a direct peroxidation of biomolecules in hepatic nuclear fractions. CONCLUSION Catechol produced an inhibition of basal respiration associated to FADH2 in isolated liver mitochondria that could lead to cytotoxicity, ROS generation and cell death.

Cytotoxicity of catechol towards human glioblastoma cells via superoxide and reactive quinones generation

It is known that the exposure to benzene in the petroleum industry causes lympho-haematopoietic cancer among workers. However, there is little data concerning the toxicity of benzene to the central nervous system. Benzene easily penetrates the brain where it is metabolized to catechol. Since catechol autoxidizes in physiological phosphate buffer, we hypothesized that it could be toxic towards glial cells due to the generation of reactive oxygen species and quinones. In this work we studied the cytotoxic properties of catechol towards human glioblastoma cells. We found that catechol was toxic towards these cells after 72 hours and this toxicity was related to the formation of quinones. Catechol at 230µM killed 50% of cells. The catechol-induced cytotoxicity was prevented by the addition of 100U superoxide dismutase, which also inhibited the formation of quinones. These data suggest that catechol induces cytotoxicity via the extracellular generation of superoxide and quinones.

Effects of IFN-γ, TNF-α, IL-10 and TGF-β on Neospora caninum infection in rat glial cells

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly of the Th1 type. However, infected primary glial cell cultures release IL-10 and IL-6 but not IFN-γ. This suggests a rather protective response of the glia to avoid inflammatory damage of the nervous tissue. In this study, we investigated the effects of pro-inflammatory cytokines in primary mixed cultures of rat astrocytes and microglia infected with N. caninum. The cells were treated with either IFN-γ, TNF-α, anti-IL-10 or anti-TGF-β antibodies and were infected with parasite tachyzoites 24h later. Trypan Blue exclusion and MTT assays were performed to test cell viability. It was observed that cytokines, antibody treatment and in vitro infection did not reveal significant cell death in the various culture conditions. Treatment with 50, 150 and 300 IU/mL of either IFN-γ or TNF-α reduced tachyzoites numbers in cultures by 36.7%, 54.8% and 63.8% for IFN-γ and by 27.6%, 38.4% and 29.7% for TNF-α, respectively. In the absence of IL-10 and TGF-β, tachyzoite numbers were reduced by 52.8% and 41.5%, respectively. While IFN-γ (150 and 300 IU/mL) increased the nitrite levels in uninfected cells, parasite infection seemed to reduce the nitrite levels, and this reduction was more expressive in IFN-γ-infected cells, thereby suggesting an inhibitory effect on its production. However, TNF-α, IL-10 and TGF-β did not affect the nitrite levels. Basal PGE(2) levels also increased by 17% and 25%; 78% and 13% in uninfected and infected cells treated with IFN-γ or anti-TGF-β, respectively. Nevertheless, the antibody neutralization of IL-10 reduced PGE(2) release significantly. These results highlight the possibility of a combined effect between the IFN-γ and parasite evasion strategies and show that the IFN-γ, TNF-α, IL-10 and TGF-β cytokines participate in parasite proliferation control mechanisms.