title: Assessment of neurotoxicity of monocrotaline, an alkaloid extracted from Crotalaria retusa in astrocyte/neuron co-culture system.
abstract: 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 βIII-tubulin expression and steady state levels after MCT treatment, with a dose and time dependent intense down regulation and depolarization of neuronal βIII-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.
authors.affiliation: Laboratório de Neuroquímica e Biologia Celular, Departamento de Biofunção/Bioquímica, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Av. Reitor Miguel Calmon s/n, Salvador, BA, 40.110-902, Brazil.
authors.forename: Bruno P S
authors.initials: BP
authors.lastname: Pitanga
authors.forename: Victor Diógenes A
authors.initials: VD
authors.lastname: Silva
authors.forename: Cleide S
authors.initials: CS
authors.lastname: Souza
authors.forename: Huiara A
authors.initials: HA
authors.lastname: Junqueira
authors.forename: Breno O N
authors.initials: BO
authors.lastname: Fragomeni
authors.forename: Ravena P
authors.initials: RP
authors.lastname: Nascimento
authors.forename: Ana Rita
authors.initials: AR
authors.lastname: Silva
authors.forename: Maria de Fátima D
authors.initials: Mde F
authors.lastname: Costa
authors.forename: Ramon S
authors.initials: RS
authors.lastname: El-Bachá
authors.forename: Silvia L
authors.initials: SL
authors.lastname: Costa
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D054022:Cerebrum / Q000187:drug effects* / Q000196:embryology / Q000378:metabolism / Q000473:pathology; D018920:Coculture Techniques; D029864:Crotalaria / Q000737:chemistry; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007150:Immunohistochemistry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016686:Monocrotaline / Q000302:isolation & purification / Q000633:toxicity*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; C488416:Tubb3 protein, rat; D014404:Tubulin; D016686:Monocrotaline; D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase; D005978:Glutathione