Monocrotaline induces acutely cerebrovascular lesions, astrogliosis and neuronal degeneration associated with behavior changes in rats: A model of vascular damage in perspective.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites playing an important role as phytotoxins in the plant defense mechanisms and can be present as contaminant in the food of humans and animals. The PA monocrotaline (MCT), one of the major plant derived toxin that affect humans and animals, is present in a high concentration in Crotalaria spp. (Leguminosae) seeds and can induce toxicity after consumption, characterized mainly by hepatotoxicity and pneumotoxicity. However, the effects of the ingestion of MCT in the central nervous system (CNS) are still poorly elucidated. Here we investigated the effects of MCT oral acute administration on the behavior and CNS toxicity in rats. Male adult Wistar were treated with MCT (109 mg/Kg, oral gavage) and three days later the Elevated Pluz Maze test demonstrated that MCT induced an anxiolytic-like effect, without changes in novelty habituation and in operational and spatial memory profiles. Histopathology revealed that the brain of MCT-intoxicated animals presented hyperemic vascular structures in the hippocampus, parahippocampal cortex and neocortex, mild perivascular edema in the neocortex, hemorrhagic focal area in the brain stem, hemorrhage and edema in the thalamus. MCT also induced neurotoxicity in the cortex and hippocampus, as revealed by Fluoro Jade-B and Cresyl Violet staining, as well astrocyte reactivity, revealed by immunocytochemistry for glial fibrillary acidic protein. Additionally, it was demonstrated by RT-qPCR that MCT induced up-regulation on mRNA expression of neuroinflammatory mediator, especially IL1ß and CCL2 in the hippocampus and cortex, and down-regulation on mRNA expression of neurotrophins HGDF and BDNF in the cortex. Together, these results demonstrate that the ingestion of MCT induces cerebrovascular lesions and toxicity to neurons that are associated to astroglial cell response and neuroinflammation in the cortex and hippocampus of rats, highlighting CNS damages after acute intoxication, also putting in perspective it uses as a model for cerebrovascular damage.

Agathisflavone as a Single Therapy or in Association With Mesenchymal Stem Cells Improves Tissue Repair in a Spinal Cord Injury Model in Rats.

Agathisflavone is a flavonoid with anti-neuroinflammatory and myelinogenic properties, being also capable to induce neurogenesis. This study evaluated the therapeutic effects of agathisflavone-both as a pharmacological therapy administered in vivo and as an in vitro pre-treatment aiming to enhance rat mesenchymal stem cells (r)MSCs properties-in a rat model of acute spinal cord injury (SCI). Adult male Wistar rats (n = 6/group) underwent acute SCI with an F-2 Fogarty catheter and after 4 h were treated daily with agathisflavone (10 mg/kg ip, for 7 days), or administered with a single i.v. dose of 1 × 106 rMSCs either unstimulated cells (control) or pretreated with agathisflavone (1 µM, every 2 days, for 21 days in vitro). Control rats (n = 6/group) were treated with a single dose methylprednisolone (MP, 60 mg/kg ip). BBB scale was used to evaluate the motor functions of the animals; after 7 days of treatment, the SCI area was analyzed after H&E staining, and RT-qPCR was performed to analyze the expression of neurotrophins and arginase. Treatment with agathisflavone alone or with of 21-day agathisflavone-treated rMSCs was able to protect the injured spinal cord tissue, being associated with increased expression of NGF, GDNF and arginase, and reduced macrophage infiltrate. In addition, treatment of animals with agathisflavone alone was able to protect injured spinal cord tissue and to increase expression of neurotrophins, modulating the inflammatory response. These results support a pro-regenerative effect of agathisflavone that holds developmental potential for clinical applications in the future.

Reverted effect of mesenchymal stem cells in glioblastoma treated with agathisflavone and its selective antitumoral effect on cell viability, migration, and differentiation via STAT3.

Glioblastoma is the most lethal tumor of the central nervous system, presenting a very poor prognostic, with a survival around 16 months. The interaction of mesenchymal stem cells and tumor cells has been studied, showing a bias in their role favoring or going against aggressiveness. Natural products such as flavonoids have showed their anticancer properties and the synergic potential with the activation of microenvironment cells to inhibit tumor progression. Agathisflavone is a flavonoid studied in neurodegenerative diseases and cancer. The present study investigated the effect of flavonoid in the viability of heterogeneous glioblastoma (GBM) cells considering a coculture or conditioned medium of mesenchymal stem cells (MSCs) effect, as well as the dose-dependent effect of this flavonoid in tumor migration and differentiation via STAT3. Agathisflavone (3-10 µM) induced dose-dependent toxicity to GL-15 and U373 human GBM cells, since 24 h after treatments. It was not toxic to human MSC but modified the pattern of interaction with GBM cells. Agathisflavone also inhibited migration and increased differentiation of human GBM cells, associated with the reduction on the expression of STAT3. These results demonstrate that the flavonoid agathisflavone had a direct anti-glioma effect. However, could be observed its effect in MSCs response that may have an impact in controlling GBM growth and aggressiveness, an important factor to consider for new therapies.

Involvement of astrocytic CYP1A1 isoform in the metabolism and toxicity of the alkaloid pyrrolizidine monocrotaline.

Monocrotaline (MCT) and its pyrrole derivative, dehydromonocrotaline (DHMC), interact with molecular targets in cells of the central nervous system. DHMC presents higher toxicity than MCT indicating that its metabolism of MCT is a critical step of this alkaloid toxicity. This study sought to elucidate the metabolism and the toxicity of MCT in C6 astrocyte cell line and primary cultures of rat astrocytes by investigating metabolic enzymatic mechanisms of the cytochrome P450 (CYP) system and conjugation with glutathione. Treatment with omeprazole (OMP) (20 µM), a non-specific inducer of CYP450 induced approximately 10-fold increase in CYP1A1 activity after 2 h of treatment. Similarly, the 7-Ethoxyresorufin-O-deethylase (EROD) activity was induced by treatment with MCT (100-500 µM), indicating that the P450 CYP1A1 isoform was active and involved in the metabolism of MCT. Analysis of conjugation with glutathione showed a significant depletion of GSH after MCT (500 µM) treatment, and this was partially reversed by pretreatment with a P450 inhibitor (cimetidine 100 µM). These results suggest that not only the alkaloid MCT but, also its metabolite may deplete GSH. Rosenfeld staining showed intense vacuolization after MCT treatment, which was partially inhibited in the presence of a P450 activator. MTT test showed that association of MCT with OMP induced a reduction in cell viability in C6 and primary astrocytic cells. These results demonstrate that MCT is metabolized by astrocytic CYP1A1 to generate metabolites that can deplete GSH. Moreover, changes in the activity of the P450 enzymes interfere with the cytotoxic effects induced by the alkaloid.

Juliprosopine and juliprosine from prosopis juliflora leaves induce mitochondrial damage and cytoplasmic vacuolation on cocultured glial cells and neurons.

Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 µg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 µg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 µg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in ß-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.

The Role of Astrocytes in Metabolism and Neurotoxicity of the Pyrrolizidine Alkaloid Monocrotaline, the Main Toxin of Crotalaria retusa.

The metabolic interactions and signaling between neurons and glial cells are necessary for the development and maintenance of brain functions and structures and for neuroprotection, which includes protection from chemical attack. Astrocytes are essential for cerebral detoxification and present an efficient and specific cytochrome P450 enzymatic system. Whilst Crotalaria (Fabaceae, Leguminosae) plants are used in popular medicine, they are considered toxic and can cause damage to livestock and human health problems. Studies in animals have shown cases of poisoning by plants from the genus Crotalaria, which induced damage to the central nervous system. This finding has been attributed to the toxic effects of the pyrrolizidine alkaloid (PA) monocrotaline (MCT). The involvement of P450 enzymatic systems in MCT hepatic and pulmonary metabolism and toxicity has been elucidated, but little is known about the pathways implicated in the bioactivation of these systems and the direct contribution of these systems to brain toxicity. This review will present the main toxicological aspects of the Crotalaria genus that are established in the literature and recent findings describing the mechanisms involved in the neurotoxic effects of MCT, which was extracted from Crotalaria retusa, and its interaction with neurons in isolated astrocytes.

Assessment of neurotoxicity of monocrotaline, an alkaloid extracted from Crotalaria retusa in astrocyte/neuron co-culture system.

Studies have shown cases of poisoning with plants from the genus Crotalaria (Leguminosae) mainly in animals. They induce damages in the central nervous system (CNS), which has been attributed to toxic effects of the pyrrolizidine alkaloid (PA) monocrotaline (MCT). Previously we demonstrated that both MCT and dehydromonocrotaline (DHMC), its main active metabolite, induce changes in the levels and patterns of expression of the main protein from astrocyte cytoskeleton, glial fibrillary acidic protein (GFAP). In this study we investigated the effect of MCT on rat cortical astrocyte/neuron primary co-cultures. Primary cultures were exposed to 10 or 100 µM MCT. The MTT test and the measurement of LDH activity on the culture medium revealed that after 24h exposure MCT was not cytotoxic to neuron/astrocyte cells. However, the cell viability after 72 h treatment decreased in 10-20%, and the LDH levels in the culture medium increased at a rate of 12% and 23%, in cultures exposed to 10 or 100 µM MCT. Rosenfeld staining showed vacuolization and increase in cell body in astrocytes after MCT exposure. Immunocytochemistry and Western blot analyses revealed changes on pattern of GFAP and ßIII-tubulin expression and steady state levels after MCT treatment, with a dose and time dependent intense down regulation and depolarization of neuronal ßIII-tubulin. Moreover, treatment with 100 µM MCT for 12h induced GSH depletion, which was not seen when cytochrome P450 enzyme system was inhibited indicating that it is involved in MCT induced cytotoxicity in CNS cells.