RESUMEN
ß-N-methylamino-l-alanine (BMAA) has been shown to inhibit vesicular monoamine transporter 2 (VMAT2), thereby preventing the uptake of monoaminergic neurotransmitters into platelet dense granules and synaptic vesicles. The inhibition is hypothesized to be through direct association of BMAA with hydroxyl groupê·containing amino acid residues in VMAT2. This study evaluated whether BMAA-induced inhibition of VMAT2 could be prevented directly by co-incubation of BMAA with amino acids, and if this protection was specific for BMAA inhibition of VMAT2. l-tyrosine, and to a lesser extent l-serine, was able to prevent BMAA-induced VMAT2 inhibition in a concentration-dependent manner, whereas neither l-threonine nor amino acids without side chain hydroxyl groups could reduce this inhibition. Reserpine-induced VMAT2 inhibition was unaffected by any of the amino acids. These data support the hypothesized interaction between BMAA and hydroxyl groupê·containing amino acids and suggests that this interaction might be leveraged to protect against the toxicity of BMAA.
Asunto(s)
Aminoácidos Diaminos , Aminoácidos , Aminoácidos/farmacología , Proteínas de Transporte Vesicular de Monoaminas , Aminoácidos Diaminos/toxicidad , Tirosina , Neurotoxinas/metabolismoRESUMEN
The first mechanism of toxicity proposed for the cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) was excitotoxicity, and this was supported by numerous in vitro studies in which overactivation of both ionotropic and metabotropic glutamate receptors was reported. However, the excitotoxicity of BMAA is weak in comparison with other known excitotoxins and on par with that of glutamate, implying that to achieve sufficient synaptic concentrations of BMAA to cause classical in vivo excitotoxicity, BMAA must either accumulate in synapses to allow persistent glutamate receptor activation or it must be released in sufficiently high concentrations into synapses to cause the overexcitation. Since it has been shown that BMAA can be readily removed from synapses, release of high concentrations of BMAA into synapses must be shown to confirm its role as an excitotoxin in in vivo systems. This study therefore sought to evaluate the uptake of BMAA into synaptic vesicles and to determine if BMAA affects the uptake of glutamate into synaptic vesicles. There was no evidence to support uptake of BMAA into glutamate-specific synaptic vesicles but there was some indication that BMAA may affect the uptake of glutamate into synaptic vesicles. The uptake of BMAA into synaptic vesicles isolated from areas other than the cerebral cortex should be investigated before definite conclusions can be drawn about the role of BMAA as an excitotoxin.
Asunto(s)
Aminoácidos Diaminos , Toxinas de Cianobacterias , Ácido Glutámico , Vesículas Sinápticas , Neurotoxinas/toxicidad , Aminoácidos Diaminos/toxicidadRESUMEN
The neurotoxic, non-proteinogenic amino acid ß-N-methylamino-l-alanine (BMAA) has been implicated in the development of neurodegenerative diseases; however, the mechanism(s) and mode(s) of toxicity remain unclear. Similarities in the neuropathology and behavioural deficits of neonatal rats exposed to either BMAA or reserpine, a known vesicular monoamine transporter 2 (VMAT2) inhibitor, suggest a similar mode of action. The aims of this study were therefore to determine if BMAA could prevent the uptake of serotonin into dense granules via inhibition of VMAT2, and, if so, the type of inhibition caused by BMAA. Exposing platelet dense granules to BMAA resulted in a concentration-dependent reduction in serotonin uptake. The inhibition of VMAT2 was non-competitive. The findings from this study support previous reports that BMAA-associated neuropathologies in a neonatal rat model may be due to VMAT2 inhibition during critical periods of neurogenesis.
Asunto(s)
Aminoácidos Diaminos , Proteínas de Transporte Vesicular de Monoaminas , Ratas , Animales , Serotonina , Aminoácidos Diaminos/toxicidad , Aminoácidos Diaminos/metabolismo , Aminoácidos/metabolismo , Neurotoxinas/farmacologíaRESUMEN
Historically, reserpine was widely used as an antihypertensive drug. However, severe motor and non-motor symptoms such as dyskinesia and depression led to the discontinuation of reserpine as a first-line treatment for hypertension. Reserpine functions by inhibiting vesicular monoamine transporter 2 (VMAT2), reducing sequestration of monoamines into synaptic vesicles. The consequent reduction in monoamines, most notably dopamine, serotonin and norepinephrine, in the central nervous system, causes well-defined symptoms such as catalepsy, hypoactivity and sedation in animals, and these motor and non-motor symptoms are well defined for reserpine treatment. However, no gross neuropathological changes in response to reserpine treatment have been reported previously in any animal model. In contrast, reducing VMAT2 expression in genetically modified VMAT2 LO mice leads to the production of âº-synuclein-positive aggregates and progressive nigrostriatal neuronal loss. These VMAT2 LO mice have reduced VMAT2 functionality during critical brain developmental stages and this could be the key to producing a reserpine model with matching histopathologies. The aim of this study was therefore to investigate the effect of neonatal reserpine administration on brain histology. We report here that a single dose of 5 mg kg-1 reserpine administered subcutaneously to neonatal rats on postnatal day 3 leads to widespread neuronal loss in various brain regions including the substantia nigra pars compacta, ventral tegmental area, striatum, hippocampus, locus coeruleus, amygdala and cerebral cortex, and the presence of âº-synuclein-positive inclusions in the substantia nigra pars compacta and the dorsal striatum within 30 days of administration.
Asunto(s)
Encéfalo/efectos de los fármacos , Cuerpos de Inclusión/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Reserpina/efectos adversos , alfa-Sinucleína/metabolismo , Animales , Encéfalo/patología , Femenino , Cuerpos de Inclusión/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Embarazo , Ratas , Ratas Sprague-Dawley , Reserpina/administración & dosificaciónRESUMEN
With accumulating evidence that supports the role of ß-N-methylamino-l-alanine (BMAA) in neurodegeneration, it is necessary to elucidate the mechanisms and modes of BMAA toxicity so as to facilitate the search for potential preventative/therapeutic strategies. Daily supplementation with l-serine was suggested as a possible therapy to treat BMAA-induced neurotoxicity, based on the hypothesized mechanism of BMAA misincorporation into proteins for l-serine. As an alternative to misincorporation, it was hypothesized that BMAA toxicity may, in part, be due to its high affinity for associating with hydroxyl group-containing amino acids, and that a dietary excess of the hydroxyl-containing l-serine might offer protection by binding to BMAA and reducing its toxicity. Additionally, l-serine can also reduce the uptake of BMAA into human cells by competitive uptake at ASCT2, and l-phenylalanine, by competitive uptake at LAT1, and l-alanine, by competitive uptake at SNAT2, can also reduce BMAA uptake into human cells. The aim of this study was therefore to determine the protective value of l-serine, l-phenylalanine and l-alanine in reducing the effects of neonatal exposure to BMAA in a Sprague Dawley rat model. Pre-treatment with l-phenylalanine reduced the observed behavioral abnormalities and neuropathologies by 60-70% in most cases. l-serine was also effective in reducing some of the behavioral abnormalities and neuropathologies, most markedly spinal cord neuronal loss. However, the protective effect of l-serine was obfuscated by neuropathies that were observed in l-serine-treated control male rats. l-alanine had no effect in protecting against BMAA-induced neurotoxicity, suggesting that competitive amino acid uptake plays a minor role in protecting against BMAA-induced neurotoxicity.
Asunto(s)
Aminoácidos Diaminos/toxicidad , Aminoácidos/farmacología , Enfermedades del Sistema Nervioso Central/inducido químicamente , Animales , Conducta Animal/efectos de los fármacos , Toxinas de Cianobacterias , Femenino , Aprendizaje por Laberinto/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Sprague-DawleyRESUMEN
The naturally produced, nonprotein amino acid ß- N-methylamino-l-alanine (BMAA) has been proposed as a significant contributor to sporadic neurodegenerative disease development worldwide. However, the existing hypothesized mechanisms of toxicity do not adequately explain the role of BMAA in neurodegenerative disease development. There is evidence for BMAA-induced enzyme inhibition, but the effect of BMAA on human stress response enzymes has received little attention, despite the well-described role of oxidative stress in neurodegenerative disease development. The aim of this study was therefore to investigate the effect of BMAA on human catalase activity and compare it to the known inhibitor 3-amino-1,2,4-triazole. BMAA inhibited human erythrocyte catalase in a cell-free exposure to the same extent as the known inhibitor. Based on enzyme kinetics, the inhibition appears to be noncompetitive, possibly as a result of BMAA binding in the nicotinamide adenine dinucleotide phosphate (NADPH) binding site. BMAA-induced catalase inhibition was also observed in a human cell line culture. We therefore propose that BMAA-induced enzyme inhibition, specifically catalase inhibition, is a mechanism of toxicity that may contribute to the neurotoxicity of BMAA, further supporting the role of BMAA in neurodegenerative disease development.
Asunto(s)
Aminoácidos Diaminos/toxicidad , Catalasa/antagonistas & inhibidores , Catalasa/metabolismo , Línea Celular , Toxinas de Cianobacterias , Humanos , Enfermedades NeurodegenerativasRESUMEN
ß-N-methylamino-l-alanine (BMAA), is commonly found in both a free and proteinassociated form in various organisms exposed to the toxin. The long latency of development of neurodegeneration attributed to BMAA, is hypothesized to be the result of excitotoxicity following slow release of the toxin from protein reservoirs. It was recently suggested that these BMAA-protein associations may reflect misincorporation of BMAA in place of serine, as occurs, for example, when canavanine misincorporates in place of arginine. We therefore compared BMAA and canavanine toxicty in various bacterial species, and misincorporation of these amino acids into proteins in a bacterial protein expression system. None of the bacterial species showed any physiological stress responses to BMAA in contrast to the growth reduction observed when cultures were incubated in media containing canavanine. LC-MS analysis confirmed uptake of BMAA from growth media. However, after immobilized metal affinity chromatography and SDS-PAGE purification of proteins produced in an E scherichia coli expression system, no BMAA was detected by either LC-MS or LC-MS/MS analysis using two derivatization methods, or by orbitrap MS of trypsin digests of the protein. We therefore conclude that BMAA is not misincorporated into proteins in bacteria and that the observed BMAA-protein association in bacteria is superficial.
Asunto(s)
Aminoácidos Diaminos/metabolismo , Aminoácidos Diaminos/toxicidad , Bacterias/química , Canavanina/metabolismo , Canavanina/toxicidad , Neurotoxinas/metabolismo , Neurotoxinas/toxicidad , Proliferación Celular/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Toxinas de CianobacteriasRESUMEN
ß-N-methylamino-l-alanine (BMAA), produced by cyanobacteria, is a neurotoxin implicated in Amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). BMAA concentrations in cyanobacteria are lower than those thought to be necessary to result in neurological damage thus bioaccumulation or biomagnification is required to achieve concentrations able to cause neurodegeneration. Many cyanobacteria produce BMAA and uptake routes into the food web require examination. In this study we investigate the uptake of BMAA by adult phytoplanktivorus Daphnia magna via exposure to dissolved pure BMAA and BMAA containing cyanobacteria, as well as the subsequent oxidative stress response in the daphnia. Free BMAA and protein-associated BMAA were quantified by LC-MS/MS. Dissolved BMAA was taken up and was found as free BMAA in D. magna. No protein-associated BMAA was detected in D. magna after a 24-h exposure period. No BMAA was detectable in D. magna after exposure to BMAA containing cyanobacteria. BMAA inhibited the oxidative stress defence and biotransformation enzymes within 24-h exposure in the tested Daphnia and could therefore impair the oxidant status and the capability of detoxifying other substances in D. magna.
Asunto(s)
Aminoácidos Diaminos/metabolismo , Toxinas Bacterianas/metabolismo , Daphnia/metabolismo , Toxinas Marinas/metabolismo , Microcistinas/metabolismo , Neurotoxinas/metabolismo , Aminoácidos Diaminos/química , Aminoácidos Diaminos/farmacología , Animales , Antioxidantes/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacología , Biotransformación , Cromatografía Liquida , Cianobacterias , Toxinas de Cianobacterias , Daphnia/efectos de los fármacos , Daphnia/enzimología , Monitoreo del Ambiente , Toxinas Marinas/química , Toxinas Marinas/farmacología , Microcistinas/química , Microcistinas/farmacología , Neurotoxinas/química , Neurotoxinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
Microcystins (MCs) are cyclic heptapeptide compounds [where X(2) (position 2) and Z(4) (position 4) are variable l-aminoacids] produced by cyanobacteria and responsible for severe liver damage in animals ingesting acute doses of the toxic compounds. Certain variants of microcystins are more toxic than others, the differences being commonly ascribed to the hydrophobic nature of the variant. Microcystin-LR (MCLR) [X = l-leucine (L); Z = l-arginine (R); R1 = R2 = CH(3)] is the most toxic of all the microcystins investigated to date. This study investigates the similarity of the structures of MCLR and selected MC variants to the liver specific hormone vasopressin. Structures were compiled in HyperChem(®) (professional version 5.1). Initial comparisons of the MCLR and vasopressin indicated comparable volumes, surface areas and masses. Further studies using RMS overlays show that the microcystin derivative MCLR(Dha(7)) is comparably similar to vasopressin in terms of tertiary structure.
RESUMEN
Toxin-producing cyanobacteria pose a world-wide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. The predominant cyanotoxin, microcystin-LR (MCLR), targets the liver and its toxicity depends on the uptake and removal rates in the liver. The role of the glutathione detoxification pathway in protecting the liver from the effects of MCLR was investigated. Mice exposed to a single 75% LD(50) dose of pure MCLR were sacrificed at 8, 16, 24 and 32 h post-exposure (pe). Toxin induced liver damage was observed 8 and 16 h pe as evidenced by raised serum ALT and LDH levels, reduced glycogen levels and liver histology. A significant increase in lipid peroxidation was seen at 16 h pe that decreased after 24 and 32 h pe, the time-points which showed significant increases in GPX activity. An increase in soluble GST activity was noted between 8 and 16 h pe, levels of total GSH increased at 24 h while oxidised glutathione increased throughout the investigation. The increase in activity of both GPX and GST corresponded with increased transcription of these enzymes, as well as the rate-limiting enzyme in GSH synthesis, gamma-glutamyl transferase. In conclusion, this study confirms that an increase in GST activity is critical for the detoxification of MCLR, that this is regulated at the transcriptional level, and that exposure to MCLR induces the de novo synthesis of GSH. Finally, we report the involvement of GPX in the removal of MCLR-induced lipid hydroperoxides.
Asunto(s)
Glutatión/metabolismo , Hígado/efectos de los fármacos , Péptidos Cíclicos/toxicidad , Animales , Femenino , Expresión Génica , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/efectos de los fármacos , Glutatión Transferasa/metabolismo , Glucógeno/metabolismo , Inactivación Metabólica , Peroxidación de Lípido/efectos de los fármacos , Hígado/citología , Hígado/metabolismo , Toxinas Marinas , Ratones , Ratones Endogámicos BALB C , Microcistinas , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos Cíclicos/farmacocinéticaRESUMEN
The increasing presence of toxic cyanobacteria in drinking and recreational water bodies, and their potential to impact on human and animal health is cause for concern. Recent work suggests that apoptosis plays a major role in the toxic effects induced by microcystin-LR (MCLR) in the gastrointestinal tract; however, the biochemical pathway remains elusive. Exposure of CaCo2, a human colon carcinoma cell line, and MCF-7, a cell line deficient in pro-caspase-3, cells to 50 microM MCLR induced similar reductions in cell viability as measured by MTT and LDH leakage. The role of MCLR induced oxidative stress in the initiation of apoptosis was investigated over a 2-hr period, and it was found that there was an increase in the release of H(2)O(2) in the first 30 min of exposure for both cell lines. Both cell lines exhibited a dose-dependent increase in both micro- and millicalpain after 24 h exposure to MCLR suggesting a role for protease activation in MCLR-induced apoptosis in non-hepatic human derived cell lines.
Asunto(s)
Apoptosis , Neoplasias de la Mama/enzimología , Células CACO-2/enzimología , Carcinoma/enzimología , Caspasas/metabolismo , Péptidos Cíclicos/toxicidad , Toxinas Bacterianas/toxicidad , Células CACO-2/efectos de los fármacos , Caspasas/efectos de los fármacos , Cianobacterias , Inhibidores Enzimáticos/toxicidad , Humanos , Peróxido de Hidrógeno/metabolismo , Toxinas Marinas , Microcistinas , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales CultivadasRESUMEN
Microcystin-LR (MCLR) has been associated with the development of gastrointestinal complaints in people ingesting cyanobacterial bloom contaminated water. The symptoms usually present a day or two after exposure raising questions as to the toxic effects of MCLR on the gastrointestinal tract. This study investigated the apoptotic effect of ip administered MCLR over time on the duodenum, jejenum and ileum of mice receiving a single 75% LD(50) dose. The apoptotic index was significantly raised in all sections at 8 h post exposure (pe) and continued to rise for the 16, 24 and 32 h pe groups, while the glycogen levels were normal at 24 h pe. The duodenum exhibited the most significant increase in apoptotic index overall, followed by the jejenum and ileum. Immunohistochemistry indicated the presence of MCLR in the lamina propria suggesting a role for MCLR in the induction of apoptosis in the GIT of mice exposed to a single sublethal dose of MCLR.
Asunto(s)
Toxinas Bacterianas/farmacología , Intestino Delgado/efectos de los fármacos , Péptidos Cíclicos/farmacología , Animales , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/administración & dosificación , Cianobacterias , Duodeno/efectos de los fármacos , Femenino , Íleon/efectos de los fármacos , Inmunohistoquímica , Inyecciones Intraperitoneales , Yeyuno/efectos de los fármacos , Toxinas Marinas , Ratones , Ratones Endogámicos BALB C , Microcistinas , Péptidos Cíclicos/administración & dosificación , Organismos Libres de Patógenos Específicos , Microbiología del AguaRESUMEN
Toxin-producing cyanobacteria pose a worldwide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. Detection of microcystins in water generally relies on specialised equipment and a delay of several days for transport and analysis. Little work has, however, been done on establishing a simple, cost-effective and sensitive plant bioassay for the detection of microcystin-LR (MCLR) in water at the WHO Tolerable Daily Intake guideline level of 1 microg/l. We investigated the effect of a MCLR extract at 1 and 10 microg/l on the growth of Lepidium sativum over 6 days. Exposure to 10 microg/l MCLR resulted in a significant decrease in root and leaf lengths and fresh weights of seedlings when compared to the controls. These results were consistent with seedlings exposed to pure MCLR at 10 microg/l. Seedlings exposed to 1 microg/l MCLR showed a significant decrease in root development from day 2 to day 6. Glutathione S-transferase and glutathione peroxidase activities were also significantly raised in plants from days 5 and 4, respectively, at both toxin levels investigated.