RESUMEN
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.
Asunto(s)
Astrocitos/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Monocrotalina/metabolismo , Monocrotalina/toxicidad , Animales , Línea Celular , Supervivencia Celular , Crotalaria/química , Citocromo P-450 CYP1A1/efectos de los fármacos , Glutatión/efectos de los fármacos , Monocrotalina/análogos & derivados , Omeprazol/farmacología , Isoformas de Proteínas/química , RatasRESUMEN
The effects and susceptibility of donkeys to Crotalaria juncea and Crotalaria retusa poisoning were determined at high and low doses. Seeds of C. juncea containing 0.074% of dehydropyrrolizidine alkaloids (DHPAs) (isohemijunceines 0.05%, trichodesmine 0.016%, and junceine 0.008%) were administered to three donkeys at 0.3, 0.6 and 1 g/kg body weight (g/kg) daily for 365 days. No clinical signs were observed and, on liver and lung biopsies, the only lesion was a mild liver megalocytosis in the donkeys ingesting 0.6 and 1 g/kg/day. Two other donkeys that received daily doses of 3 and 5 g seed/kg showed initial respiratory signs 70 and 40 days after the start of the administration, respectively. The donkeys were euthanized following severe respiratory signs and the main lung lesions were proliferation of Clara cells and interstitial fibrosis. Three donkeys ingested seeds of C. retusa containing 5.99% of monocrotaline at daily doses of 0.025, 0.05 and 0.1 g/kg for 365 days. No clinical signs were observed and, on liver and lung biopsies, the only lesion was moderate liver megalocytosis in each of the three donkeys. One donkey that received a single dose of 5 g/kg of C. retusa seeds and another that received 1 g/kg daily for 7 days both showed severe clinical signs and died with diffuse centrilobular liver necrosis. No lung lesions were observed. Another donkey that received a single dose of 2.5 g/kg of C. retusa seeds showed no clinical signs. The hepatic and pneumotoxic effects observed are consistent with an etiology involving DHPAs. Furthermore, the occurrence of lung or liver lesions correlates with the type of DHPAs contained in the seeds. Similarly as has been reported for horses, the data herein suggest that in donkeys some DHPAs are metabolized in the liver causing liver disease, whereas others are metabolized in the lung by Clara cells causing lung disease.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Crotalaria/química , Crotalaria/envenenamiento , Enfermedades Pulmonares/patología , Alcaloides de Pirrolicidina/envenenamiento , Animales , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Crotalaria/clasificación , Equidae , Fibrosis/inducido químicamente , Hígado/efectos de los fármacos , Hígado/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Enfermedades Pulmonares/inducido químicamente , Monocrotalina/análogos & derivados , Monocrotalina/envenenamiento , Intoxicación por Plantas/patología , Intoxicación por Plantas/veterinaria , Semillas/química , Semillas/envenenamientoRESUMEN
Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the Crotalaria species that causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline (DHM). In previous studies using isolated rat liver mitochondria, we observed that DHM, but not MCT, inhibited the activity of respiratory chain complex I and stimulated the mitochondrial permeability transition with the consequent release of cytochrome c. In this study, we evaluated the effects of MCT and DHM on isolated rat hepatocytes. DHM, but not MCT, caused inhibition of the NADH-linked mitochondrial respiration. When hepatocytes of rats pre-treated with dexamethasone were incubated with MCT (5 mM), they showed ALT leakage, impaired ATP production and decreased levels of intracellular reduced glutathione and protein thiols. In addition, MCT caused cellular death by apoptosis. The addition of fructose or dithiotreitol to the isolated rat hepatocyte suspension containing MCT prevented the ATP depletion and/or glutathione or thiol oxidation and decreased the ALT leakage and apoptosis. These results suggest that the toxic effect of MCT on hepatocytes may be caused by metabolite-induced mitochondrial energetic impairment, together with a decrease of cellular glutathione and protein thiols.
Asunto(s)
Apoptosis/efectos de los fármacos , Ditiotreitol/farmacología , Fructosa/farmacología , Hepatocitos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Monocrotalina/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Crotalaria/metabolismo , Glutatión/metabolismo , Hepatocitos/metabolismo , Masculino , Mitocondrias Hepáticas/metabolismo , Monocrotalina/análogos & derivados , Proteínas/metabolismo , Ratas , Ratas Wistar , Compuestos de Sulfhidrilo/metabolismoRESUMEN
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.
Asunto(s)
Crotalaria/toxicidad , Monocrotalina/toxicidad , Mutágenos/toxicidad , Neuroglía/efectos de los fármacos , Neuroglía/patología , Semillas/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Crotalaria/química , Daño del ADN , Relación Dosis-Respuesta a Droga , Humanos , Inmunohistoquímica , Proteínas Asociadas a Microtúbulos/metabolismo , Monocrotalina/análogos & derivados , Monocrotalina/síntesis química , Monocrotalina/aislamiento & purificación , Monocrotalina/metabolismo , Mutágenos/aislamiento & purificación , Mutágenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Semillas/química , Factores de Tiempo , Vimentina/metabolismoRESUMEN
Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM's inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 microM Ca(2+), DHM (50-250 microM) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 microM) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline.
Asunto(s)
Alquilantes/farmacología , Ciclosporina/farmacología , Citocromos c/metabolismo , Inmunosupresores/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Monocrotalina/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Masculino , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Monocrotalina/farmacología , NADP/metabolismo , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Dehydromonocrotaline (DHMC) is the main monocrotaline active cytochrome P450's metabolite, and has already been assessed in the CNS of experimentally intoxicated rats. DHMC effects were here investigated toward rat astroglial primary cultures regarding cytotoxicity, morphological changes and regulation of GFAP expression. Cells, grown in DMEM supplemented medium, were treated with 0.1-500 microM DHMC, during 24- and 72-h. According to MTT and LDH tests, DHMC was toxic to astrocytes after 24-h exposure at 1 microM, and induced membrane damages at 500 microM. Rosenfeld dying showed hypertrophic astrocytes after 72-h exposure to 0.1-1 microM DHMC. GFAP immunocytochemistry and western immunoblot revealed an increase of GFAP labelling and expression, suggesting an astrogliotic reaction to low concentrations of DHMC. At higher concentrations (10-500 microM), astrocytes shrank their bodies and retracted their processes, presenting a more polygonal phenotype and a weaker expression on GFAP labelling Nuclear chromatin staining by Hoechst-33258 dye, revealed condensed and fragmented chromatin in an important proportion (+/-30%) of the astrocytes exposed to 100-500 microM DHMC, suggesting signs of apoptosis. Our results confirm a cytotoxic and dose-dependent effect of DHMC on cultures of rat cortical astrocytes, leading to apoptotic figures. These effects might be related to the neurological damages and clinical signs observed in animals intoxicated by Crotalaria.
Asunto(s)
Alquilantes/toxicidad , Astrocitos/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Monocrotalina/análogos & derivados , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Aumento de la Célula/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Monocrotalina/toxicidad , Ratas , Ratas WistarRESUMEN
Monocrotaline is a pyrrolizidine alkaloid present in plants of the Crotalaria species, which causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline. We evaluated the effects of monocrotaline and its metabolite on respiration, membrane potential and ATP levels in isolated rat liver mitochondria, and on respiratory chain complex I NADH oxidase activity in submitochondrial particles. Dehydromonocrotaline, but not the parent compound, showed a concentration-dependent inhibition of glutamate/malate-supported state 3 respiration (respiratory chain complex I), but did not affect succinate-supported respiration (complex II). Only dehydromonocrotaline dissipated mitochondrial membrane potential, depleted ATP, and inhibited complex I NADH oxidase activity (IC50=62.06 microM) through a non-competitive type of inhibition (K(I)=8.1 microM). Therefore, dehydromonocrotaline is an inhibitor of the activity of respiratory chain complex I NADH oxidase, an action potentially accounting for the well-documented monocrotaline's hepatotoxicity to animals and humans. The mechanism probably involves change of the complex I conformation resulting from modification of cysteine thiol groups by the metabolite.