cyanoginosin-lr and Necrosis

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 μg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

Topics: Alkaloids; Animals; Chemical and Drug Induced Liver Injury; Comet Assay; Cyanobacteria Toxins; Fatty Liver; Female; Gastric Mucosa; Hepatocytes; Male; Marine Toxins; Microcystins; Micronuclei, Chromosome-Defective; Micronucleus Tests; Necrosis; Rats, Wistar

Microcystins (MCYST) are the most frequently reported cyanotoxins in human poisoning incidents. Despite the well-described mechanism of acute and lethal injury, the sublethal effects of this toxin require further investigation. The aim of this study was to contribute to the knowledge of the variant MCYST-LR effects at sublethal doses by investigating biochemical changes and tissue damage in a murine model. For this purpose, mice were intraperitoneally injected with 45 μg of MCYST-LR/kg body weight. Their organs were collected at 2, 8, 24, 48 or 96 h after injection. Control animals received saline solution. We detected oxidative imbalance in the liver, particularly at 8 h after exposure. Furthermore, biomarkers of liver injury were detected in high concentration in the serum of the exposed animals. Stereological analyses of the liver indicated two different phases in the intoxication process: an initial phase characterized by an increase in steatosis was followed by a second, later phase characterized by increased inflammation and hepatocyte binucleation. Formation of areas of necrosis and increased blood vessel diameter were observed throughout the experimental period. The number of hepatocytes per area unit also decreased. However, these parameters recovered over the period of exposure. MCYST accumulated in liver and was detectable until the end of the monitoring period. These results confirm the necessity for further studies of processes involved in sublethal exposure to MCYST.

Topics: Animals; Hepatocytes; Liver; Male; Marine Toxins; Mice; Microcystins; Necrosis; Oxidation-Reduction; Toxicity Tests

Blooms of cyanobacteria producing very toxic secondary metabolites (especially microcystins) are potent environmental stressors, hazardous not only to aquatic animals but also to public health. The purpose of this study was to investigate the effects of an extract containing microcystins on immune cells isolated from the common carp (Cyprinus carpio L.). In the present study it has been found that the extract induced apoptosis and inhibited in vitro lymphocyte proliferation. In addition, the results indicated the possible role of oxidative stress in this cytotoxicity and apoptosis. The in vivo investigations showed that the extract containing microcystins had greater suppressive effects on the essential functions of immune cells (intracellular reactive oxygen species production and lymphocyte proliferation) than the pure toxin alone. Moreover, immersion of fish in the toxic extract caused changes in the mRNA levels of various pro- and anti-inflammatory cytokines in carp leukocytes, while after exposure to the pure toxin, only IL1-β expression was markedly up-regulated. The observed modulatory effects on immune cells could have important implications for the health of planktivorous fish, which feed more frequently on toxic cyanobacteria.

Topics: Animals; Apoptosis; Carps; Cell Proliferation; Cyanobacteria; Cytokines; Fish Proteins; Gene Expression Regulation; Lymphocytes; Marine Toxins; Microcystins; Necrosis; Phagocytes; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Respiratory Burst; RNA, Messenger

This study evaluated the effects of [D-Leu(1)]Microcystin-LR variants, by the exposure of Hypophthalmichthys molitrix to Microcystis aeruginosa NPLJ4. Fish was placed in aquariums and exposed to 10(5) cells mL(-1). For 15 days, 05 individuals were removed every 05 days, and tissue samples of liver, skeletal muscle and intestinal tract were collected for histopathologic analyses. Following exposure, those surviving were placed in clean water for 15 days to evaluate their recovery. A control without toxins was maintained in the same conditions and exhibited normal histology and no tissue damage. In exposed fish, samples were characterized by serious damages that similarly affected the different organs, such as dissociation of cells, necrosis and haemorrhage. Samples showed signs of recovery but severe damages were still observed. The results should be valuable to analyze the potency of microcystin toxicity and to help in the diagnosis of fish deaths.

Topics: Animals; Chemical and Drug Induced Liver Injury; Chromatography, High Pressure Liquid; Cyprinidae; Digestive System; Hemorrhage; Intestinal Diseases; Liver; Marine Toxins; Mass Spectrometry; Microcystins; Muscle, Skeletal; Muscular Diseases; Necrosis; Survival

Microcystins (MCs) are potent hepatotoxins acting by the inhibition of protein phosphatase 1 and 2A, and may promote liver tumors. Moreover, studies also suggest they are nephrotoxic. The aim of the present study was to assess possible in vitro effects of microcystin-LR (which contains the amino acids leucine and arginine, the most widely studied and distributed variant of all microcystins) on the selected immune functions of the cells isolated from the head kidney of carp. In the experiments, pure microcystin-LR (MC-LR), was used at concentrations of 0.01, 0.1, 0.5, and 1 microg/ml RPMI-1640 medium. Leucocytes (lymphocytes and phagocytes) were isolated by centrifugation on a density gradient. Lymphocyte proliferation, intracellular production of reactive oxygen species by phagocytes, and the presence of apoptotic and/or necrotic cells were assessed. The respiratory burst activity of phagocytic cells was increased at the lowest toxin concentration used in the study, but it was decreased at higher concentrations. Using a sensitive luminescent immunoassay, MC-LR was observed to have no influence on the T-cell proliferation but decreased the proliferation of B lymphocytes. Moreover, it was noted that MC-LR induced necrosis to a higher degree than apoptosis in fish leucocytes. The results of the present study suggest the modulatory potency of microcystin-LR on fish leucocytes.

Topics: Animals; Apoptosis; Carps; Leukocytes; Lymphocyte Activation; Marine Toxins; Microcystins; Necrosis; Reactive Oxygen Species; Respiratory Burst; Water Microbiology

Microcystins, a family of cyclic heptapeptides produced by the cyanobacteria, Microcystis aeruginosa, have documented hepatotoxic and tumor promoting activities. The purpose of this study was to evaluate the toxicity of inhaled microcystin LR (microcystin). Male BALB/c mice were exposed by nose-only inhalation to 260-265 microg microcystin/m(3) for 7 days. The low-, mid- and high-dose groups were exposed for 0.5, 1, and 2h, respectively. Control animals were sham exposed to aerosolized vehicle. Treatment-related microscopic lesions were observed only in the nasal cavity of the mid- and high-dose groups. These lesions consisted of minimal to moderate multifocal degeneration and necrosis of the respiratory epithelium, with variable neutrophilic inflammation and minimal to marked degeneration, necrosis, and atrophy of the olfactory epithelium. The no-adverse-effect dose for the nasal lesions was approximately 3 microg/kg body weight, or 20 ng/cm(2) of nasal epithelium. In serum, only two protein peaks, occurring at m/zs of 11,688 and 11,829 Da, exhibited decreases in intensity that were microcystin dose-dependent. While these proteins have not been positively identified, they may be useful in the future as biomarkers of microcystin exposure in humans.

Topics: Administration, Inhalation; Analysis of Variance; Animals; Blood Proteins; Dose-Response Relationship, Drug; Histological Techniques; Male; Marine Toxins; Mice; Mice, Inbred BALB C; Microcystins; Necrosis; No-Observed-Adverse-Effect Level; Olfactory Mucosa; Peptides, Cyclic; Respiratory Mucosa; Time Factors

Microcystin-LR is a cyanobacterial heptapeptide that presents acute and chronic hazards to animal and human health. We investigated the influence of this toxin on human and chicken immune system modulation in vitro. Peripheral blood lymphocytes were treated with microcystin-LR at environmentally relevant doses of 1, 10 and 25 microg/ml for 12, 24, 48, 72 h (for proliferation assay cells were treated for 72 h). T-cell and B-cell proliferation as well as apoptosis and necrosis were determined in human and chicken samples. IL-2 and IL-6 production by human lymphocytes also was measured. In addition, uptake kinetics of microcystin-LR into human and chicken peripheral blood lymphocytes were calculated by Liquid Chromatography (LS) /Mass Spectrometry (MS) analysis. At the highest dose microcystin-LR decreased T-cell proliferation and all doses of microcystin-LR inhibited B-cell proliferation. The frequency of apoptotic and necrotic cells increased in a dose and time-dependent manner. Human lymphocytes responded to stimulation with microcystin-LR by increased production of IL-6 and decreased production of IL-2. Human lymphocytes were able to uptake from 0.014 to 1.663 microg/ml and chicken lymphocytes from 0.035 to 1.733 microg/ml of the microcystin-LR added to the cultures, depending on the treatment time and dose. In conclusion, microcystin-LR acted as an immunomodulator in cytokine production and down-regulated lymphocyte functions by induction of apoptosis and necrosis. However, further studies dealing with the influence of microcystin-LR on expression cytokine genes and transcription factors are necessary to confirm these hypotheses.

Topics: Animals; Apoptosis; B-Lymphocytes; Cell Division; Chromatography, Liquid; Female; Humans; In Vitro Techniques; Interleukin-2; Interleukin-6; Lymphocytes; Marine Toxins; Mass Spectrometry; Microcystins; Necrosis; Peptides, Cyclic; Phosphatidylserines; T-Lymphocytes

Acute intoxication with MC-LR induces cytoskeletal alterations, apoptosis and necrosis of hepatocytes resulting in intrahepatic hemorrhage. Preliminary results have shown that chronic treatment of rats with intraperitoneal injections of sublethal doses of microcystins MC-LR and MC-YR could induce not only liver, but also kidney injuries. We aimed to investigate whether the induction of the cytoskeletal changes, apoptosis and necrosis could be the mechanisms involved in the injury of kidney cells in the chronic model of microcystin intoxication. Experimental rats were receiving intraperitoneal injections of MC-LR (10 microg/kg) or MC-YR (10 microg/kg) every second day for 8 months, while control rats were receiving only the vehicle. The histopathological investigation revealed collapsed glomeruli with thickened basement membranes and dilated tubuli filled with eosinophilic casts. Rhodamine-phalloidin labeling showed cytoplasmic aggregation and accumulation of fibrilar actin filaments within the epithelial tubular cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed increased number of TUNEL-positive cells in the kidney cortex and medulla. The pathological changes induced by MC-LR appeared more severe than those induced by MC-YR. The results support the view that at the cellular level, the mechanisms that underly the chronic nephrotoxicity are similar to the mechanisms of the acute hepatotoxicity of microcystins.

Topics: Analysis of Variance; Animals; Apoptosis; Bacterial Toxins; Cytoskeletal Proteins; Cytoskeleton; Enzyme Inhibitors; Kidney; Male; Marine Toxins; Microcystins; Necrosis; Peptides, Cyclic; Rats; Rats, Wistar; Renal Circulation; Toxicity Tests, Chronic

Microcystin-LR (MC-LR) is the most commonly encountered of the toxic cyclic peptide hepatotoxins occurring in China. It is a model compound for toxicological studies. In this study, the toxicity of MC-LR (50 and 500 micrograms/L) on isolated carp hepatocytes was determined and the ultrastructural alterations of cells induced by MC-LR were observed. The alterations noted when hepatocytes were exposed to 50 micrograms/L MC-LR were blebbing of cell membrane, shrinking and deformation of nuclei, vesiculation and transformation into concentric membrane whorls of RER, and swelling and rearrangement of the cytoskeleton. However, when the cells were exposed to 500 micrograms/L MC-LR, broken cell membranes, nuclei and cytoskeleton could be observed. These ultrastructural changes paralleled the pathological events, which lead to apoptosis or necrosis of hepatocytes. These results suggest that disruption of the cytoskeletal structures could account for the blebbing of cell membrane and apoptosis induced by MC-LR.

Topics: Animals; Apoptosis; Carps; Cell Membrane; Cell Nucleus; China; Cytoskeleton; Endoplasmic Reticulum; Hepatocytes; Marine Toxins; Microcystins; Microscopy, Electron; Mitochondria; Necrosis; Peptides, Cyclic

Aged mice (32 weeks) were orally administered microcystin-LR at 500 micrograms/kg, and injuries of the liver were estimated by microscopy 2 hr after treatment. Sixty-two per cent of aged mice proved to be sensitive to microcystin-LR, whereas such changes in the liver were not found in young mice (5 weeks). Uptake of the toxin into the liver was confirmed by high-performance liquid chromatography and frit-fast atom bombardment liquid chromatograph/mass spectrometry after clean-up with an immunoaffinity column. To verify the difference in sensitivity to microcystin-LR between aged and young mice, non-treated mice were examined, and among them aged mice were confirmed to have a rough surface of the stomach and small intestinal mucosa. These results suggested that the hepatotoxicity by oral administration of microcystin-LR is deeply related to aging, and particularly to conditions in the small intestine such as the permeability of capillaries in the villi.

Topics: Administration, Oral; Aging; Alanine Transaminase; Animals; Aspartate Aminotransferases; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Liver; Marine Toxins; Mice; Microcystins; Necrosis; Peptides, Cyclic