Silvia Lima Costa

Biological monitoring of workers occupationally exposed to ethylene oxide

Ethylene oxide (EtO) is an important intermediate industrial chemical which is also used for sterilizing medical products and hospital equipment. In the present study we have evaluated some biological markers, such as chromosomal aberrations, micronuclei and EtO-hemoglobin adducts in the peripheral blood cells, and micronuclei in buccal exfoliated cells of 22 controls and 75 workers employed in an industry in Brazil using EtO as an intermediate. Measurements of EtO in the general area showed that workers were exposed to 2-5 ppm time-weighted average (TWA) for an 8-h working day, during the 3-month sampling. Our results indicate that exposure to EtO resulted in a statistically significant enhancement of chromosomal aberrations (P = 0.01) and of micronuclei in binucleated lymphocytes (P < 0.001). For the frequencies of micronucleated cells in buccal mucosa there was no statistically significant difference between exposed and control groups. The mean values of hemoglobin adduct (HOEtVal) measurements obtained from a selected group of exposed and unexposed donors were statistically different.

A novel multi-target ligand (JM-20) protects mitochondrial integrity, inhibits brain excitatory amino acid release and reduces cerebral ischemia injury in vitro and in vivo

We previously showed that JM-20, a novel 1,5-benzodiazepine fused to a dihydropyridine moiety, possessed an anxiolytic profile similar to diazepam and strong neuroprotective activity in different cell models relevant to cerebral ischemia. Here, we investigated whether JM-20 protects against ischemic neuronal damage in vitro and in vivo. The effects of JM-20 were evaluated on hippocampal slices subjected to oxygen and glucose deprivation (OGD). For in vivo studies, Wistar rats were subjected 90 min of middle cerebral artery occlusion (MCAo) and oral administration of JM-20 at 2, 4 and 8 mg/kg 1 h following reperfusion. Twenty-four hours after cerebral blood flow restoration, neurological deficits were scored, and the infarct volume, histopathological changes in cortex, number of hippocampal and striatal neurons, and glutamate/aspartate concentrations in the cerebrospinal fluid were measured. Susceptibility to brain mitochondrial swelling, membrane potential dissipation, H2O2 generation, cytochrome c release, Ca2+ accumulation, and morphological changes in the organelles were assessed 24 h post-ischemia. In vitro, JM-20 (1 and 10 μM) administered during reperfusion significantly reduced cell death in hippocampal slices subjected to OGD. In vivo, JM-20 treatment (4 and 8 mg/kg) significantly decreased neurological deficit scores, edema formation, total infarct volumes and histological alterations in different brain regions. JM-20 treatment also protected brain mitochondria from ischemic damage, most likely by preventing Ca2+ accumulation in organelles. Moreover, an 8-mg/kg JM-20 dose reduced glutamate and aspartate concentrations in cerebrospinal fluid and the deleterious effects of MCAo even when delivered 8 h after blood flow restoration. These results suggest that in rats, JM-20 is a robust neuroprotective agent against ischemia/reperfusion injury with a wide therapeutic window. Our findings support the further examination of potential clinical JM-20 use to treat acute ischemic stroke.

Effects of the flavonoid casticin from Brazilian Croton betulaster in cerebral cortical progenitors in vitro: Direct and indirect action through astrocytes

Neurodegenerative diseases are a major constraint on the social and economic development of many countries. Evidence has suggested that phytochemicals have an impact on brain pathology; however, both their mechanisms of action and their cell targets are incompletely known. Here, we investigated the effects of the flavonoid casticin, extracted from Croton betulaster, a common plant in the state of Bahia in Brazil, on rat cerebral cortex neurons in vitro. Treatment of neural progenitors with 10 μM casticin increased the neuronal population positive for the neuronal marker β‐tubulin III and the neuronal transcriptional factor Tbr2 by approximately 20%. This event was followed by a 50% decrease in neuronal death. Pools of astrocyte (GFAP and S100β), neural (nestin), and oligodendrocyte (Olig2 and NG2) progenitors were not affected by casticin. Neither neuronal commitment nor proliferation of progenitors was affected by casticin, suggesting a neuroprotective effect of this compound. Culture of neural progenitors on casticin‐treated astrocyte monolayers increased the neuronal population by 40%. This effect was reproduced by conditioned medium derived from casticin‐treated astrocytes, suggesting the involvement of a soluble factor. ELISA assays of the conditioned medium revealed a 20% increase in interleukin‐6 level in response to casticin. In contrast to the direct effect, neuronal death was unaffected, but a 52% decrease in the death of nestin‐positive progenitors was observed. Together our data suggest that casticin influences the neuronal population by two mechanisms: 1) directly, by decreasing neuronal death, and 2) indirectly, via astrocytes, by modulating the pool of neuronal progenitors.

Impact of Plant-Derived Flavonoids on Neurodegenerative Diseases.

Neurodegenerative disorders have a common characteristic that is the involvement of different cell types, typically the reactivity of astrocytes and microglia, characterizing gliosis, which in turn contributes to the neuronal dysfunction and or death. Flavonoids are secondary metabolites of plant origin widely investigated at present and represent one of the most important and diversified among natural products phenolic groups. Several biological activities are attributed to this class of polyphenols, such as antitumor activity, antioxidant, antiviral, and anti-inflammatory, among others, which give significant pharmacological importance. Our group have observed that flavonoids derived from Brazilian plants Dimorphandra mollis Bent., Croton betulaster Müll. Arg., e Poincianella pyramidalis Tul., botanical synonymous Caesalpinia pyramidalis Tul. also elicit a broad spectrum of responses in astrocytes and neurons in culture as activation of astrocytes and microglia, astrocyte associated protection of neuronal progenitor cells, neuronal differentiation and neuritogenesis. It was observed the flavonoids also induced neuronal differentiation of mouse embryonic stem cells and human pluripotent stem cells. Moreover, with the objective of seeking preclinical pharmacological evidence of these molecules, in order to assess its future use in the treatment of neurodegenerative disorders, we have evaluated the effects of flavonoids in preclinical in vitro models of neuroinflammation associated with Parkinson’s disease and glutamate toxicity associated with ischemia. In particular, our efforts have been directed to identify mechanisms involved in the changes in viability, morphology, and glial cell function induced by flavonoids in cultures of glial cells and neuronal cells alone or in interactions and clarify the relation with their neuroprotective and morphogetic effects.

Flavonoids and Astrocytes Crosstalking: Implications for Brain Development and Pathology

Flavonoids are naturally occurring polyphenolic compounds that are present in a variety of fruits, vegetables, cereals, tea, and wine, and are the most abundant antioxidants in the human diet. Evidence suggests that these phytochemicals might have an impact on brain pathology and aging; however, neither their mechanisms of action nor their cell targets are completely known. In the mature mammalian brain, astroglia constitute nearly half of the total cells, providing structural, metabolic, and trophic support for neurons. During the past few years, increasing knowledge of these cells has indicated that astrocytes are pivotal characters in neurodegenerative diseases and brain injury. Most of the physiological benefits of flavonoids are generally thought to be due to their antioxidant and free-radical scavenging effects; however, emerging evidence has supported the hypothesis that their mechanism of action might go beyond these properties. In this review, we focus on astrocytes as targets for flavonoids and their implications in brain development, neuroprotection, and glial tumor formation. Finally, we will briefly discuss the emerging view of astrocytes as essential characters in neurodegenerative diseases, and how a better understanding of the action of flavonoids might open new avenues to develop therapeutic approaches to these pathologies.

Flavonoids inhibit angiogenic cytokine production by human glioma cells

VEGF and TGF‐β1 are cytokines that stimulate tissue invasion and angiogenesis. These factors are considered as molecular targets for the therapy of glioblastoma. Bevacizumab, a recombinant humanized monoclonal antibody developed against VEGF, inhibits endothelial cell proliferation and vessel formation. Flavonoids obtained from Dimorphandra mollis and Croton betulaster have been described as proliferation inhibitors of a human glioblastoma derived cell line. VEGF and TGF‐β1 levels were dosed by ELISA in a GL‐15 cell line treated with bevacizumab and also with the flavonoids rutin, 5‐hydroxy‐7,4′‐dimethoxyflavone, casticin, apigenin and penduletin. Rutin reduced the VEGF and TGF‐β1 levels after 24 h but not after 72 h. The other flavonoids significantly reduced TGF‐β1 production. Bevacizumab reduced only the VEGF levels in the supernatant from GL‐15 cultures. These results suggest that the flavonoids studied, and commonly used in popular medicine, present an interesting subject of study due to their potential effect as angiogenic factor inhibitors. Copyright

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.

Agathisflavone enhances retinoic acid-induced neurogenesis and its receptors α and β in pluripotent stem cells.

Flavonoids have key functions in the regulation of multiple cellular processes; however, their effects have been poorly examined in pluripotent stem cells. Here, we tested the hypothesis that neurogenesis induced by all-trans retinoic acid (RA) is enhanced by agathisflavone (FAB, Caesalpinia pyramidalis Tull). Mouse embryonic stem (mES) cells and induced pluripotent stem (miPS) cells growing as embryoid bodies (EBs) for 4 days were treated with FAB (60 μM) and/or RA (2 μM) for additional 4 days. FAB did not interfere with the EB mitotic rate of mES cells, as evidenced by similar percentages of mitotic figures labeled by phospho-histone H3 in control (3.4% ± 0.4%) and FAB-treated groups (3.5% ± 1.1%). Nevertheless, the biflavonoid reduced cell death in both control and RA-treated EBs from mES cells by almost 2-fold compared with untreated EBs. FAB was unable, by itself, to induce neuronal differentiation in EBs after 4 days of treatment. On the other hand, FAB enhanced neuronal differentiation induced by RA in both EBs of mES and miPS. FAB increased the percentage of nestin-labeled cells by 2.7-fold (mES) and 2.4 (miPS) and β-tubulin III-positive cells by 2-fold (mES) and 2.7 (miPS) in comparison to RA-treated EBs only. FAB increased the expression of RA receptors α and β in mES EBs, suggesting that the availability of RA receptors is limiting RA-induced neurogenesis in pluripotent stem cells. This is the first report to describe that naturally occurring biflavonoids regulate apoptosis and neuronal differentiation in pluripotent stem cells.

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.

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.