RESUMEN
Toxic cyanobacteria have been reported in lakes and reservoirs in several countries. The presence of toxins in drinking water creates a potential risk of toxin transference for water consumers. Besides chemical and physical methods of cyanotoxin removal from water, biodegradation methods would be useful. The aim of the current study was to identify bacterial removal mechanisms of the hepatotoxin microcystin-LR. This was studied by testing the hypothesis of enzymatic degradation of microcystin-LR in the presence of probiotic lactic acid bacterial and bifidobacterial strains and the participation of the proteolytic system of the bacteria in this process. The results suggest that extracellularly located cell-envelope proteinases are involved in the decomposition of microcystin-LR. In particular, a correlation between proteolytic activity and microcystin removal was found and both these parameters were dependent on glucose as an energy source. In addition, EDTA, which was indicated as a main inhibitor of proteinases of the investigated strain, was shown to limit the rate of microcystin removal. The removal of microcystins was shown to be different from the known microcystin-degradation pathway of Sphingomonas. (14)C-labeled microcystin was not found inside the cells and bacterial cell extracts were not able to remove the toxin, which supports the involvement of extracellularly located proteinases. The results confirm the hypothesis of enzymatic degradation of microcystins in the presence of probiotic bacteria.
Asunto(s)
Bifidobacterium/metabolismo , Lactobacillus/metabolismo , Microcistinas/metabolismo , Péptido Hidrolasas/fisiología , Probióticos , Bifidobacterium/enzimología , Biodegradación Ambiental , Extractos Celulares/química , Pared Celular/enzimología , Cromatografía Líquida de Alta Presión , Lactobacillus/enzimología , Toxinas Marinas , Microcistinas/químicaRESUMEN
Cylindrospermopsin (CYN) is a cyanobacterial alkaloid that has been implicated in outbreaks of human morbidity and animal mortality. The principal mode of action for CYN is inhibition of protein and glutathione synthesis, and its toxicity seems to be mediated by cytochrome P-450-generated metabolites. It was also shown that CYN might be responsible for tumor initiation in animals; nevertheless, mechanisms leading to CYN-induced carcinogenesis are scarce and equivocal. The aim of the present study was to investigate the impact of metabolic activation on CYN-induced DNA damage. The effect of different doses of CYN (0.05-2mug/ml) on DNA damage was determined in CHO-K1 cells after 3, 16 and 21h of the treatment. The chromosome aberration assay with and without metabolic activation was applied to evaluate the clastogenic activity of CYN and its metabolite(s). In addition, the occurrence of apoptosis and necrosis was estimated by the annexin method using flow cytometry. The results revealed that CYN is not clastogenic in CHO-K1 cells irrespective of S9 fraction-induced metabolic activation. However, CYN significantly decreases the frequencies of mitotic indices and decreases proliferation irrespective of metabolic activation system. CYN increases the frequency of necrotic cells in a dose- and time-dependent manner, whereas it has a very slight impact on apoptosis. Moreover, the presence of metabolic activation influences a susceptibility to necrotic cell death but not an apoptotic one.
Asunto(s)
Aberraciones Cromosómicas , Uracilo/análogos & derivados , Alcaloides , Animales , Apoptosis/efectos de los fármacos , Toxinas Bacterianas , Biotransformación , Células CHO , Ensayo Cometa , Cricetinae , Cricetulus , Toxinas de Cianobacterias , Índice Mitótico , Necrosis , Uracilo/farmacocinética , Uracilo/toxicidadRESUMEN
Microcystin-LR (MC-LR), a potent inhibitor of PP1 and PP2A protein phosphatases, is related to tumor promotion and initiation. Although the genotoxic properties of this toxin have been extensively investigated with a variety of non-mammalian and mammalian test systems, the existing results are contradictory. Based on our previous results regarding the impact of MC-LR on the processes of DNA repair we decided to examine in greater detail its effect on the capacity of nucleotide excision repair (NER). CHO-K1 cells were pre-treated with increasing doses of MC-LR (1, 10 and 20 microg/ml) and then exposed to UV radiation (25 J/m(2)). Apoptosis was analyzed to exclude the possibility of false positive results in the comet assay. The results suggest that MC-LR targets the nucleotide excision repair mechanisms by interference with the incision/excision phase as well as the rejoining phase of NER and leads to an increased level of UV-induced cytogenetic DNA damage in CHO-K1 cells.
Asunto(s)
Toxinas Bacterianas/toxicidad , Reparación del ADN/efectos de los fármacos , Microcistinas/toxicidad , Animales , Apoptosis , Células CHO , Ensayo Cometa , Cricetinae , Cricetulus , Daño del ADN , Reparación del ADN/efectos de la radiación , Cinética , Toxinas Marinas , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Rayos UltravioletaRESUMEN
The genotoxic activity of microcystin-LR (MC-LR) is a matter of debate. MC-LR is known to be a phosphatase inhibitor and it may be expected that it is involved in the regulation of the activity of DNA-dependent protein kinase (DNA-PK), the key enzyme involved in the repair of radiation-induced DNA damage. We studied the effect of MC-LR on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. A dose of 0.5 microg/ml of MC-LR was chosen because it induced very little early apoptosis which gives no false positive results in the comet assay. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 h and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition the frequencies of chromosomal aberrations were analysed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage and lead to enhanced frequencies of chromosomal aberrations including dicentric chromosomes. The results of a split-dose experiment, where cells were exposed to two 1.5 Gy doses of radiation separated by 3 h with or without MC-LR, confirmed that the toxin increased the frequency of dicentric chromosomes. We also determined the effect of MC-LR and ionizing radiation on the frequency of gamma-H2AX foci. The pre-treatment with MC-LR resulted in reduced numbers of gamma-H2AX foci in irradiated cells. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma MO59J and MO59K cells. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analysed by the comet assay. A strong inhibitory effect was observed in the MO59K but not in the MO59J cells. These results indicate that DNA-PK might be involved in DNA repair inhibition by MC-LR.
Asunto(s)
Daño del ADN , Reparación del ADN , Inhibidores Enzimáticos/efectos adversos , Rayos gamma/efectos adversos , Péptidos Cíclicos/efectos adversos , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Aberraciones Cromosómicas , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/metabolismo , Citometría de Flujo , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/radioterapia , Histonas/metabolismo , Humanos , Cinética , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Toxinas Marinas/efectos adversos , Microcistinas , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Receptores de Neuropéptido Y/antagonistas & inhibidores , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Fase de Descanso del Ciclo Celular/efectos de la radiaciónRESUMEN
Twenty years ago, we detected the interdependence between structure and function of rat liver Golgi complexes that are characteristic for streptozotocin diabetes, which served us in further investigations as a useful indicator of the effectiveness of drugs we were testing. This work presented results obtained in eight groups of rats (four control and four diabetic) that were administered orally either bis(maltolato)oxovanadium(IV) [BMOV] or maltol alone. The activities of the rat liver Golgi marker enzyme, galactosyltransferase [GalT], as well as the morphology of Golgi complexes were studied in situ using an electron microscope; parallel estimations of vanadium concentration and phospholipid percentage were made in Golgi-rich preparations isolated from the liver. Our main findings were normalization in diabetic animals orally treated with 1.8 mmol BMOV in 0.09 mol NaCl solutions over seven days, which demonstrated an accompanying increase in phosphatidic acid (PA) percentage (p < 0.05) compared to controls. In the diabetic groups, Pearson's test showed a positive double correlation between GalT activity, vanadium concentration, and PA percentage in Golgi-rich membrane preparations from the liver. Additionally, a negative correlation was found between vanadium concentration and phosphatidylcholine percentage in the fractions.
Asunto(s)
Diabetes Mellitus Experimental/patología , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Hipoglucemiantes/farmacología , Hígado/efectos de los fármacos , Hígado/patología , Pironas/farmacología , Vanadatos/farmacología , Animales , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/metabolismo , Femenino , Galactosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Hígado/enzimología , Hígado/metabolismo , Microscopía Electrónica , Concentración Osmolar , Fosfolípidos/metabolismo , Ratas , Ratas Wistar , Cloruro de Sodio/farmacología , Vanadio/farmacocinéticaRESUMEN
Oral treatment with maltol or bis(maltolato)oxovanadium(IV) [BMOV] alters the biochemical activity of the rat liver Golgi marker enzyme, i.e., galactosyltransferase (GalT), and the organelle morphology in a relatively short time. Four groups of rats were investigated: control (C), treated with BMOV for 2 days (pVC), treated with BMOV for 7 days (C+V), and treated with maltol alone for 7 days (C+M). All drugs were administered as drinking solutions. These conditions were used, because normalization of galactosyltransferase activity (GalT) and morphology of rat liver Golgi complexes were previously found by us in streptozotocin-induced diabetes. In this paper, we present the influence of BMOV or maltol alone (as a vanadium ligand in BMOV compound) on rat liver Golgi complexes. The lowest statistically significant enzyme activity, in comparison with three other groups of rats (p < 0.01), was found in rats treated with BMOV solution for two days (pVC). Liver Golgi complexes in these rats showed relatively slight changes as compared with controls. The activity of GalT was similar to controls of the C+V and C+M groups. Morphological examinations of the Golgi apparatus in rats treated with vanadium salts revealed a slightly increased secretory activity. In response to various agents used in experiments, the Golgi complexes were generally reduced in size, except for the (C+M) group. Not only cisternae, but also vacuoles and associated vesicles on both sides of stacks were reduced in almost all Golgi structures. Ultrastructural findings were generally in agreement (except for pVC group) with biochemical results (yields of liver Golgi-rich fractions, activity of galactosyltransferase) obtained in the same rats.
Asunto(s)
Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hígado/metabolismo , Hígado/ultraestructura , Pironas/farmacología , Vanadatos/farmacología , Administración Oral , Animales , Femenino , Ratas , Ratas WistarRESUMEN
The relation between bis(maltolato)oxovanadium(IV) (BMOV) influencing the biochemical activity of rat liver Golgi apparatus and the morphology of this organelle was studied in normal and streptozotocin-diabetic rat livers. Ultrastructural examinations revealed marked differences in the morphology of Golgi apparatus in three groups of animals. In the control rats treated only with 0.5% NaCl we did not find any biochemical and morphological changes. Marked changes were found in the rat liver after 1.8 mmol BMOV in 0.5% NaCl (as drinking solution) applied for 7 days, so-called "control" group for vanadium. In this group Golgi apparatus seemed shorter than in the diabetic animals. Finally, the same treatment of rats with previously induced SZ-diabetes, showed relatively small morphological alterations. The ultrastructural observation was compatible with the activity of galactosyltransferase (GalT), the Golgi marker enzyme. In diabetic rats treated with BMOV the activity of this enzyme was almost the same as in controls. Summing up dramatic alterations, previously found in diabetic-untreated rats [22], normalized after orally applied BMOV solution, even after a short time.