RESUMEN
The relationship between Microcystis composition and the production of microcystins and nontoxic peptides in bloom cells, which was regularly collected in Lake Suwa, Japan, in the summer season from 1991 to 1994, was investigated. In order to determine the structures of the nontoxic peptides, we collected large amounts of bloom materials from the same lake on July 23, 1991, and isolated three nontoxic peptides. They were named as aeruginopeptins 917S-A, -B, and -C, and their structures were mainly determined by a mass spectrometry/mass spectrometry (MS/MS) technique as 19-membered cyclic depsipeptides possessing the Ahp (3-amino-6-hydroxy-2-piperidone) moiety. An analysis of the microcystins and aeruginopeptins in the collected blood cells and their Microcystis composition suggested that the M. aeruginosa large cell size produces both microcystins and aeruginopeptins, and the production of both compounds is genetically closely related.
Asunto(s)
Cianobacterias , Eutrofización , Oligopéptidos/análisis , Péptidos Cíclicos/análisis , Tamaño de la Célula , Monitoreo del Ambiente , Espectrometría de Masas , Microcistinas , Oligopéptidos/biosíntesis , Péptidos Cíclicos/biosíntesisRESUMEN
Electrospray ionization mass spectrometry was used to develop a rapid, sensitive, and accurate method for determination and identification of hepatotoxic microcystins, cyanobacterial cyclic heptapeptides. To optimize the electrospray ionization conditions, factors affecting charge state distribution, such as amino acid components of sample, proton affinity of the additives, and additive concentration, were investigated in detail and a method for controlling charge states was developed to provide molecular-related ions for assignment of molecular weight and reasonably abundant precursor ions for MS/MS analysis. A procedure for identification of microcystins consisting of known amino acids was proposed: for microcystins giving abundant [M + 2H]2+ ions, the addition of nitrogen-containing bases to the aqueous sample solution is effective to obtain an increased intensity of [M + H]+ ions, whereas the addition of Lewis acids containing nitrogen can produce increased abundances of [M + 2H]2+ ions for microcystins giving weak [M + 2H]2+ ions. Microcystins possessing no arginine residue always give sodium adduct ions [M + Na]+ as the base peak, and these are difficult to fragment via low energy collision-induced dissociation to yield structurally informative products; the addition of oxalic acid increases [M + H]+ ion abundances, and these fragment readily.
Asunto(s)
Péptidos Cíclicos/análisis , Acetatos/química , Aminoácidos/análisis , Arginina/análisis , Fenómenos Químicos , Enfermedad Hepática Inducida por Sustancias y Drogas , Química Física , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Espectrometría de Masas , Microcistinas , Nitrógeno/análisis , Ácido Oxálico/análisisRESUMEN
Characteristics of electrospray ionization mass spectrometry/collision-induced dissociation (ESIMS/CID) mass spectra of microcystins, cyanobacterial cyclic heptapeptide hepatoxins, were examined. The collision conditions showed remarkable effects on the quality of the CID mass spectra, which were divided into three patterns according to the number of Arg residues. A characteristic cleavage reaction and neutral losses of MeOH, NH3 and guanidine group(s) from the (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4 E,6E-dienoic acid (Adda) and Arg residues were observed in the ESI and ESIMS/CID mass spectra, suggesting the most probable protonation sites in [M + H]+ and [M + 2H]2+ ions of microcystins. Microcystins with no Arg residue showed only [M + H]+ ions with a proton reacting at the methoxyl group in the Adda residue, and the ESIMS/CID/MS data revealed their structures unambiguously. The protonation site in [M + H]+ ions of microcystins with Arg residue(s) was the guanidine group. The [M + 2H]2+ ions of microcystins possessing one Arg residue had one proton on the Arg residue and probably another proton on the Adda residue, while the [M + 2H]2+ ions of microcystins having two Arg residues showed protonation at both Arg residues and the ESIMS/CID/MS data assigned their sequences. Structures of microcystins possessing one Arg residue can be assigned by ESIMS/CID/MS of [M + H]+ ions combined with those of [M + 2H]2+ ions.
Asunto(s)
Espectrometría de Masas/métodos , Péptidos Cíclicos/química , Anabaena/química , Arginina/química , Microcistinas , Estructura Molecular , ProtonesRESUMEN
Microcystin-LR (MCLR) is a potent cyclic heptapeptidic hepatotoxin produced by bloom-forming cyanobacteria. The mutagenicity induced by this compound in cultured human RSa cells was found by determination of ouabain-resistant (OuaR) mutation, with the highest frequency at the concentration of 15 microgram/ml. Moreover, base substitution mutations at K-ras codon 12 in genomic DNA, assessed by polymerase chain reaction (PCR) and differential dot-blot hybridization using digoxigenin-labeled probes, were detected in RSa cells 6 days after exposure to MCLR (7.5-15 microgram/ml).
Asunto(s)
Mutágenos/toxicidad , Péptidos Cíclicos/toxicidad , Células Cultivadas , Resistencia a Medicamentos/genética , Genes ras/efectos de los fármacos , Humanos , Toxinas Marinas , Microcistinas , Pruebas de Mutagenicidad , Ouabaína/farmacologíaRESUMEN
Microcystins, the cyclic heptapeptide toxins produced by cyanobacteria such as Microcystis, show tumor-promoting activity through inhibition of protein phosphatases 1 and 2A. They potentially threaten human health, and are increasing the world-wide interest in the health risk associated with cyanobacterial toxins. In this study, the effect of chlorination on the decomposition of microcystins-LR and -RR was examined. The toxins were easily decomposed by chlorination with sodium hypochlorite, and the decomposition depended on the free chlorine dose. In this operation, many reaction products were formed, one of which was determined to be dihydroxymicrocystin formed through the chloronium ion at the conjugated diene of Adda [3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E), 6(E)-dienoic acid], followed by hydrolysis. Other products may be its stereoisomers and/or regioismers. No noxious products were detected from the chlorination process of microcystin-LR. Although these results suggested that chlorination at an adequate chlorine dose is very effective for the removal of microcystin in raw water, preoxidation of the cell itself with chlorine must be avoided, because it frequently causes toxin release from algae and produce trihalomethanes during water treatment.
Asunto(s)
Toxinas Bacterianas/química , Cloro/química , Cianobacterias/química , Hidrocarburos Clorados/química , Animales , Toxinas Bacterianas/efectos de la radiación , Toxinas Bacterianas/toxicidad , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Hidrocarburos Clorados/efectos de la radiación , Hidrocarburos Clorados/toxicidad , Concentración de Iones de Hidrógeno , Dosificación Letal Mediana , Espectroscopía de Resonancia Magnética , Ratones , Estructura Molecular , Pruebas de Mutagenicidad , Luz Solar , Temperatura , Rayos UltravioletaRESUMEN
We isolated several species of bacteria from the surface water of a Japanese lake. Of the isolated bacteria, Pseudomonas aeruginosa was the only species which could degrade microcystin LR in vitro. Microcystin LR decreased to 4.5% of the spiked microcystin LR quantity in the P. aeruginosa culture, and was metabolized to (2S, 3S, 8S)-3-amino-2, 6, 8-trimethyl-10-phenyldeca-4E, 6E-dienoic acid (DmADDA). It was possible that P. aeruginosa hydrolysed nucleophilically the peptide bond of microcystin LR. We examined if pyochelin, pyocyanin and alkaline protease produced by P. aeruginosa affected the reduction of microcystin LR. In the result, DmADDA was produced from microcystin LR in the presence of 100 microM H2O2 and 100 microM each of pyochelin, pyocyanin or both. However, the production of DmADDA was slight (2 to 12 mole%). After treatment with P. aeruginosa alkaline protease, DmADDA was produced 75 mole% from microcystin LR. Therefore, we concluded that microcystin was degraded mainly by the action of P. aeruginosa alkaline protease.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Toxinas Marinas/metabolismo , Péptidos Cíclicos/metabolismo , Pseudomonas aeruginosa/enzimología , Serina Endopeptidasas/metabolismo , Aminoácidos/análisis , Medios de Cultivo , Japón , MicrocistinasRESUMEN
Accumulation of microcystins mainly produced by cyanobacteria Microcystis was investigated for freshwater mussels and fishes collected from a lake where heavy blooms of Microcystis occurred every year. The identification of microcystins was performed by HPLC equipped with a frit FAB mass spectrometer. Microcystins LR and RR were identified in the mussels Unio douglasiae and Anadonta woodiana, whereas no microcystin was identified by the present method in fishes, such as Cyprinus carpio, Carassius carassius, and Hypomesius transpacificus.
Asunto(s)
Bivalvos/química , Carcinógenos/análisis , Cianobacterias/química , Péptidos Cíclicos/análisis , Animales , Bivalvos/microbiología , Cromatografía Líquida de Alta Presión , Cianobacterias/aislamiento & purificación , Peces/microbiología , Agua Dulce , Microcistinas , Espectrometría de Masa Bombardeada por Átomos Veloces , Microbiología del AguaRESUMEN
An epidemiological survey for the causes of a high incidence of primary liver cancer (PLC) in Haimen city, Jian-Su province and Fusui county, Guangxi province in China, found a close correlation between the incidence of PLC and the drinking of pond and ditch water. With an aim to clarify whether microcystins (MC), a hepatotoxic peptide produced by water bloom algae, contaminate the drinking water in the endemic areas of PLC in China, a highly sensitive enzyme-linked immunosorbent assay with a detection limit of 50 pg/ml, was introduced to monitor the MC. Three trials to survey the drinking water were carried out in 1993-1994. Samples, 1135 in total, were collected from different sources such as: ponds, ditches, rivers, shallow wells and deep wells in Haimen city. The first survey in September 1993 found that three out of 14 ditch water specimens were positive for MC, with a range of 90-460 pg/ml. Several toxic algae such as Oscillatoria agardhii were present in some of the ditches. In the second trial, samples were collected from five ponds/ditches, two rivers, two shallow wells and two deep wells monthly for the whole year of 1994. These data showed that MC was highest in June to September, with a range of 62-296 pg/ml. A third trial on the 989 different water samples collected from the different types of water sources in July 1994 revealed that 17% of the pond/ditch water, 32% of the river water, and 4% of the shallow-well water were positive for MC, with averages of 101, 160 and 68 pg/ml respectively. No MC was detected in deep well water. A similar survey on 26 drinking water samples in Fusui, Guangxi province, demonstrated a high contamination frequency of MC in the water of ponds/ditches and rivers but no MC in shallow and deep wells. These data support a hypothesis that the blue-green algal toxin MC in the drinking water of ponds/ditches and rivers, or both, is one of the risk factors for the high incidence of PLC in China. Based on previous findings on the epidemiology of PLC and the present results from the mass screening of MC in the drinking water, an advisory level of MC in drinking water was proposed to below 0.01 microg/l. The combined effect of a potent hepatocarcinogen AFB1 and an intermittent intake of MC in drinking water in the summer season was discussed as an etiology of PLC.
Asunto(s)
Cianobacterias , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/epidemiología , Péptidos Cíclicos/análisis , Contaminantes del Agua/análisis , Abastecimiento de Agua , Anticuerpos Monoclonales , China/epidemiología , Ensayo de Inmunoadsorción Enzimática , Humanos , Incidencia , Microcistinas , Péptidos Cíclicos/toxicidad , Sensibilidad y Especificidad , Contaminantes del Agua/toxicidadRESUMEN
Microcystins are very potent hepatotoxins and strong liver tumor promoters produced by cyanobacteria, and their occurrence has been reported all over the world. They could threaten human health when toxic Microcystis occurs in water supply reservoirs. In this study, we examined the stability of microcystins during photolysis with UV light. The toxins were easily decomposed by UV light at wavelengths around the absorption maxima of the toxins and the decomposition depended on the intensity of the light. The half-life of microcystin LR by 147 microW/cm2 UV irradiation was 10 min, and the toxin was completely decomposed by 2550 microW/cm2 UV after 10 min. When the toxins were irradiated with weaker UV light, isomerization was also observed by a different mechanism from that during photolysis by sunlight and pigment, and several products including three geometrical isomers of the conjugated diene of Adda were detected. Microcystin RR showed almost the same behavior as that of microcystin LR under the same conditions. Since no noxious products were formed in the present study, a water treatment including UV irradiation is very possible for removing microcystins from raw water.
Asunto(s)
Cianobacterias/efectos de la radiación , Inhibidores Enzimáticos/metabolismo , Péptidos Cíclicos/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Rayos Ultravioleta , Cromatografía Líquida de Alta Presión , Cianobacterias/metabolismo , Inhibidores Enzimáticos/efectos de la radiación , Microcistinas , Pruebas de Mutagenicidad , Péptidos Cíclicos/efectos de la radiación , Péptidos Cíclicos/toxicidad , Fosfoproteínas Fosfatasas/análisis , Fosfoproteínas Fosfatasas/metabolismo , Estándares de Referencia , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/metabolismo , Espectrometría de Masa Bombardeada por Átomos Veloces , Estereoisomerismo , Contaminantes Químicos del Agua/metabolismoRESUMEN
Male rats were intraperitoneally administered a lethal dose of microcystin-LR (a toxin of cyanobacteria). Prior to haemorrhage in the liver, blood coagulation and platelet aggregation activities were measured. The number of cellular components, white blood cells, red blood cells and, especially, platelets, decreased 1-1.5 hr after the injection. Plasma kaolin-activated partial thromboplastin time was prolonged and fibrinogen concentration was reduced, but antithrombin III activity and fibrin degradation product concentration were not significantly changed. Platelet aggregation was not affected for up to 0.5-1.0 hr after administration. Sequential analyses of those parameters and hepatotoxic markers indicate that those hematologic changes as well as the hepatic injury occur suddenly after the massive bleeding. These results suggest that microcystin-LR does not directly act on the haemostatic system to cause a disseminated intravascular coagulation-like state. The decrease in blood coagulation activity and platelet particle concentration may be the results of secondary consumptive effects following the hepatic haemorrhage.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas , Coagulación Intravascular Diseminada/inducido químicamente , Hemorragia/inducido químicamente , Péptidos Cíclicos/toxicidad , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Animales , Toxinas Bacterianas/toxicidad , Cianobacterias , Fibrinógeno/metabolismo , Hígado/efectos de los fármacos , Pruebas de Función Hepática , Masculino , Toxinas Marinas , Microcistinas , Tiempo de Tromboplastina Parcial , Agregación Plaquetaria/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
A clean-up method using high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) was developed to pursue trace amounts of microcystins in lake water. The method consisted of the combined usage of octadecyl silanized (ODS) silica gel and silica gel cartridges. In the first clean-up process, the retention behavior of microcystin RR on ODS silica gel cartridge was carefully observed together with microcystin LR, and 10% water-methanol was chosen as the best solvent system to elute microcystins from the ODS silica gel cartridge. Because many impurities still remained in the desired fraction from the raw water even after the clean-up with ODS silica gel, an additional clean-up process was developed using various cartridges. As a result of extensive experiments, the second clean-up process using silica gel cartridge was established, and the impurities were effectively eliminated. The present method including a tandem cartridge system allowed a precise analysis of microcystins in water samples from three different lakes at a 0.02 ppb level.
Asunto(s)
Agua Dulce/análisis , Péptidos Cíclicos/análisis , Dióxido de Silicio/química , Cromatografía Líquida de Alta Presión , Cianobacterias/metabolismo , Japón , Toxinas Marinas , Espectrometría de Masas , Microcistinas , Péptidos Cíclicos/química , Gel de Sílice , Espectrometría de Masa Bombardeada por Átomos Veloces , Microbiología del Agua , Contaminantes Químicos del Agua/análisisRESUMEN
Microcystin-LR is a unique and potent liver tumor promoter, belonging to the okadaic acid class compounds. Although microcystin-LR is a potent inhibitor of protein phosphatases 1 and 2A, as is okadaic acid, microcystin-LR has liver specificity dominance. Two significant aspects, specific binding and liver specificity of [3H]dihydromicrocystin-LR, a reduced form of microcystin-LR, were studied and compared with those of [3H]okadaic acid. [3H]-Dihydromicrocystin-LR had higher affinity for the receptors in both the particulate and cytosolic fractions of rat liver and various tissues than had [3H]okadaic acid. Intraperitoneal administration of [3H]dihydromicrocystin-LR into mice resulted in the highest uptake into the liver, 71.5 +/- 6.9% of the total administered radioactivity, whereas p.o. administration resulted in less than 1% uptake into the liver, suggesting that the mechanisms of the incorporation of microcystin-LR into the liver by i.p. and p.o. administrations are greatly different. The presence of associated forms of [3H]dihydromicrocystin-LR with macromolecules in the liver indicates a need for further investigation.
Asunto(s)
Hígado/metabolismo , Péptidos Cíclicos/metabolismo , Animales , Toxinas Bacterianas/metabolismo , Carcinógenos , Citosol/metabolismo , Femenino , Masculino , Toxinas Marinas/metabolismo , Ratones , Ratones Endogámicos ICR , Microcistinas , Ratas , Ratas Endogámicas F344 , Distribución TisularRESUMEN
The effects of cylindrospermopsin isolated from a blue-green alga Umezakia natans on mice were examined morphologically and biochemically. The main target of the phycotoxin was the liver. The thymus, kidneys and heart were also affected. There were four consecutive phases of the pathological changes in the liver. The initial phase was that of inhibition of the protein synthesis, the second phase of membrane proliferation followed, and then the third phase of fat droplet accumulation and finally the phase of cell death. Using globin synthesis in the rabbit reticulocytes system, it was clearly demonstrated that cylindrospermopsin is a potent inhibitor of the protein synthesis. Protein in microsomes from the mouse livers treated by cylindrospermopsin decreased in amount more significantly than that of phospholipid in microsomes. Furthermore, the amount of total P450 was extensively diminished in the toxin treated with hepatic microsomes.
Asunto(s)
Cianobacterias/química , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Toxinas Marinas/toxicidad , Uracilo/análogos & derivados , Alcaloides , Animales , Toxinas Bacterianas , Toxinas de Cianobacterias , Cicloheximida/envenenamiento , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Globinas/biosíntesis , Globinas/efectos de los fármacos , Riñón/ultraestructura , Hígado/ultraestructura , Masculino , Ratones , Ratones Endogámicos ICR , Microsomas Hepáticos/efectos de los fármacos , Fosfolípidos/metabolismo , Uracilo/toxicidadRESUMEN
In 1987 a cyanobacterium (blue-green alga) Umezakia natans was isolated from Lake Mikata, Fukui, Japan, as a new member of the family of Stigonemataceae. The crude extract of U. natans showed hepatotoxicity to mice, from which a toxic compound was isolated. The toxin was identical in all respects to a recently reported hepatotoxin, cylindrospermopsin, isolated from an Australian tropical cyanobacterium Cylindrospermopsis raciborskii. Because cylindrospermopsin causes fatty liver and central necroses in mice and is suspected of being an agent causing human hepatoenteritis, its monitoring in drinking water supplies has been required. So a rapid screening method including four steps, extraction, clean-up, separation, and determination, has been proposed for cylindrospermopsin. A combination of a clean-up using HP-20 and C18-cartridge, and HPLC with photodiode array detector made it possible to establish a screening method for the toxin. The established method was applied to five samples and cylindrospermopsin was traced in one of them.
Asunto(s)
Cianobacterias/química , Hígado/efectos de los fármacos , Uracilo/análogos & derivados , Alcaloides , Animales , Toxinas Bacterianas , Cromatografía Líquida de Alta Presión , Toxinas de Cianobacterias , Hígado/patología , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos ICR , Uracilo/química , Uracilo/aislamiento & purificación , Uracilo/toxicidadRESUMEN
Amounts of hepatotoxic microcystin and neurotoxic anatoxin-a were estimated in natural blooms and strains of cyanobacteria from freshwaters in Japan. A simultaneous analysis method of anatoxin-a and microcystin was applied to natural bloom samples, which has been dominated by several species and the strains of cyanobacteria which produced simultaneously both toxins. The natural blooms examined in the present study were mainly composed of Anabaena and Oscillatoria, but most also contained Microcystis and other cyanobacteria. Only one sample was almost unialgal, Anabaena spiroides, collected from Lake Sagami. The toxins in 14 samples collected from nine different natural blooms during 1988-1992 were identified as microcystins-RR, -YR, and -LR; desmethyl-7-microcystin-LR (7-DMLR); and anatoxin-a. Microcystins were the main toxins contained in these natural blooms, with anatoxin-a not being detected or of very little quantity. 7-DMLR was detected in samples only from Lake Kasumigaura. Five strains of Anabaena isolated from waters in Japan produced a small amount of anatoxin-a, but no microcystins. One half of the strains of Microcystis produced microcystins and/or anatoxin-a. This is the first study showing Microcystis producing both anatoxin-a and microcystins.
Asunto(s)
Toxinas Bacterianas/análisis , Cianobacterias/patogenicidad , Toxinas Marinas/análisis , Péptidos Cíclicos/análisis , Microbiología del Agua , Secuencia de Aminoácidos , Toxinas de Cianobacterias , Agua Dulce , Japón , Microcistinas , Datos de Secuencia Molecular , TropanosRESUMEN
In order to separate and identify microcystins, a new analytical method was developed using a frit probe as an interface for fast atom bombardment mass spectral analysis of high performance liquid chromatographic (HPLC) effluents. Two types of HPLC conditions were designed for separation of standard microcystins RR, YR and LR. The HPLC conditions, for example, methanol:0.01% trifluoroacetic acid = 61:39 (containing 0.8% glycerol) as a mobile phase and 0.5 ml/min as a flow rate, provided a base line separation of standard microcystins RR, YR and LR. The HPLC conditions were also effective for separation of the non-toxic geometrical isomers of microcystins RR and LR. The total ion chromatogram of a mixture of standard microcystins showed excellent correlation with the HPLC separation using a u.v. detector. The method was subsequently applied to analysis of microcystins contained in both a culture strain and a field sample, and the procedure from toxin extraction to identification of microcystins was performed within 1 day. The mass chromatogram monitored at m/z 135 that is always observed with abundance in the FAB mass spectra of the purified microcystins, differentiated between microcystins and other types of compounds. This technique allowed the rapid identification of unknown microcystins without standard samples. Additionally, compounds other than microcystins were also found, which would not be seen by u.v. detection at 238 nm.
Asunto(s)
Cianobacterias/química , Péptidos Cíclicos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Toxinas Marinas , Microcistinas , Péptidos Cíclicos/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The decomposition process of toxic blue-green alga (cyanobacteria), Microcystis aeruginosa, under dark and aerobic condition was investigated in relation to the change of the amounts of heptapeptide toxins (microcystins YR and LR) by two experiments: one with Microcystis cells and the other with two purified microcystins. In the experiment with Microcystis cells, an increase of heterotrophic bacteria observed from the beginning of the experiment, was followed by decomposition of the algal cells and the subsequent release of microcystins into the filtrate fraction. The amounts of the toxins initially present in the cells were quantitatively detected in the filtrate fraction on the 35th day. The decomposition of microcystin YR began on the 42nd day. The decomposition rate of the two toxins was different. The decomposition rate of purified microcystins YR and LR, compared in distilled water and culture medium, respectively, indicated clearly that microcystin YR was more labile to decomposition than microcystin LR in the culture medium. At the end of the experiment (45th day) microcystin YR decreased to 58.6%, while 86.2% of microcystin LR remained.
Asunto(s)
Toxinas Bacterianas/análisis , Microcystis/metabolismo , Péptidos Cíclicos/análisis , Toxinas Marinas , Microcistinas , Péptidos Cíclicos/metabolismoRESUMEN
Microcystins, isolated from toxic blue-green algae, are potent inhibitors of protein phosphatases 1 and 2A. Recently, we have reported that microcystin LR has a potent tumor-promoting activity on rat liver initiated with diethylnitrosamine. The structure of microcystins is unique in having an unusual amino acid, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E),6(E)-dienoic acid (Adda), which is thought to be significant for the activity. Geometrical isomers at C-7 in the Adda portion of microcystins, 6(Z)-Adda microcystins LR and RR, have been isolated from cyanobacteria. To estimate their tumor-promoting activities and to understand the importance of the Adda portion for activity, the maternal microcystins LR and RR and their isomers were subjected to examination of their interaction with protein phosphatases 1 and 2A and the release of glutamic pyruvic transaminase from rat liver. 6(Z)-Adda microcystins LR and RR bound to protein phosphatases 1 and 2A, inhibited their activities and released glutamic pyruvic transaminase from rat liver into serum, ten to one hundred times more weakly than the maternal microcystins LR and RR. These results indicated that the conjugated diene with 4(E),6(E) geometry in the Adda portion is important in the interaction with protein phosphatases.
Asunto(s)
Carcinógenos/metabolismo , Péptidos Cíclicos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Alanina Transaminasa/sangre , Animales , Carcinógenos/farmacología , Citosol/enzimología , Éteres Cíclicos/metabolismo , Hígado/enzimología , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/metabolismo , Toxinas Marinas , Ratones , Microcistinas , Ácido Ocadaico , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/efectos de los fármacos , Unión Proteica , Piel/enzimología , Estereoisomerismo , Relación Estructura-Actividad , TritioRESUMEN
Three microcystins, YR, LR and RR and nodularin, all of which are hepatotoxic compounds, inhibited dose-dependently the activity of protein phosphatase 2A in and the specific [3H]okadaic acid binding to a cytosolic fraction of mouse skin, as strongly as okadaic acid. However, microcytins and nodularin did not induce any effects on mouse skin or primary human fibroblasts. Microinjection of microcystin YR into primary human fibroblasts induced morphological changes which were induced by incubation with okadaic acid. Microcystins and nodularin penetrate into the epithelial cells of mouse skin and human fibroblasts with difficulty, which reflects tissue specificity of the compounds.