This study reports the findings on the effect of Microcystin-LR (MCLR) on the gastrointestinal tract cells of mice and on two different cell lines, Caco2 and MCF-7. The cyanobacterium Microcystis aeruginosa produces the potent toxin, MCLR. This toxin has been implicated in a number of cases of ill-health. It was decided to investigate whether microcystin-LR induced apoptosis in the gastrointestinal tract of mice and also which possible mechanisms were involved in the induction in vitro. Balb/c mice were given a 75% LD50 intraperitoneal dose of pure microcystin -LR and sacrificed at 8, 16, 24 and 32 hours post-exposure. The small intestinal sections were stained with haematoxylin and eosin and examined for apoptotic cells. There was a time-dependent increase in the number of apoptotic cells with most in the duodenum and the jejunum. No change in glycogen content was evident at 24 hours post exposure when PAS-stained sections were examined. To determine that microcystin was the agent responsible for the changes, fluoroscein isothiocyanate (FITC) immunostaining for the toxin was done on the sections. Apoptosis in vitro was investigated in Caco2, a cell line that behaves like normal enterocytes when the cells are differentiated at confluency, and MCF-7, a breast cancer cell line deficient in pro-caspase-3, cells by 3-[dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays and by staining with DAPI and Rhodamine 123. MCLR exposure induced apoptosis, as seen in decreased cell viability and increased leakage of LDH, as well as mitochondrial damage shown by Rhodamine staining. The MCF-7 cells, deficient in pro-caspase-3, and Caco2 cells did not show cleavage of poly(ADP)ribose polymerase (PARP) after exposure to 50μM MCLR after 72 hours exposure. Both micro- and milli-calpain activity was however significantly increased in both cell lines exposed to the toxin. There was a significant increase in H2O2, one of the key reactive oxygen species, production during the first 30 minutes that the cells were exposed to 50 mM MCLR.

Blooms of cyanobacteria have increased in occurrence in the past three decades and have been reported to cause severe problems for animals and humans, leading to death in extreme instances. The majority of poisonings that have taken place have been attributed to a hepatotoxin produced by the species Microcystis aeruginosa, namely microcystin. The appearance of a cyanobacterial bloom does not give any indication as to its toxicity and therefore, it is imperative that simple, yet sensitive, bioassays are developed to overcome this problem. This study was undertaken to evaluate the effects of microcystin-LR on the liver of Xenopus laevis both in vitro and in vivo. This animal provides an opportunity to study the long-term hepatotoxic effects of the toxin compared to in vitro studies performed with mice and rats. The use of the liver slice model system as a potential bioassay to study the effects of microcystin-LR on Xenopus laevis liver was evaluated. Liver slices were cultured in RPMI- 1640 culture medium for periods ranging from 30 hours to 10 days and the liver slices were exposed to toxin concentrations ranging from 1nM to 500nM. The use of frog liver slices to study the longer-term effects of low-dose exposure to microcystin-LR was evaluated by observing the ultrastructural changes within hepatocytes using transmission electron microscopy, the release of the enzymes alanine aminotransferase and lactate dehydrogenase into the surrounding culture medium, as well as using a 3-[4,5-dimethylthiazol-2yl]-2,5- diphenyl tetrazolium bromide assay to determine the viability of the liver slices in culture. The amount of lipid peroxidation in the liver slices after exposure to microcystin-LR was assessed using the Thiobarbituric Acid Test. Results showed the frog liver slice culture system to be an inadequate method to evaluate the hepatotoxic effects of microcystin-LR. An in vivo assessment of the effects of microcystin-LR on Xenopus laevis was carried out using a total of 9 frogs (3 groups of 3 frogs). Frogs received a single intraperitoneal dose of 120mg/kg of microcystin-LR and were sacrificed at 8 and 24 hours post exposure. Microcystin-LR caused no significant change in serum lactate dehydrogenase levels, hepatosomatic index (liver weight as a percentage body weight), glutathione peroxidase activity, glycogen or lipid peroxidation. There was, however, an increase in glutathione sii transferase activity in the liver. The presence of the toxin in the liver was confirmed by immunohistochemistry. This study suggests that Xenopus laevis has, in some way, adapted to detoxifying aquatic toxins in the environment.

This study reports the findings on the effect of nitrates and phosphates on the biomass and toxin production of various strains of the unicellular non-nitrogen fixing cyanobacterium, Microcystis aeruginosa. The occurrence of blooms of Microcystis aeruginosa and microcystin in freshwater impoundments across the globe has been on the increase lately due to increased levels of eutrophication, resulting in human and animal deaths and illness, as well as drinking and recreational water foulment. A range of environmental factors have been shown to effect growth and microcystin production. Existing literature however is somewhat contradictory as to the effects of these physical and chemical factors on toxin production. Therefore Microcystis aeruginosa strains were cultured under batch and continuous conditions to determine the effect of nitrate and phosphate concentrations and ratios on biomass and toxin production. Cultures were analysed with regards to internal nutrient stores, biomass production, nutrient depletion, photosynthetic efficiency and microcystin production. Results showed that microcystin production correlated to growth rate, photosynthetic efficiency and internal nitrogen stores and that an optimal N:P ratio was associated with microcystin levels, growth rate and photosynthetic efficiency. Results therefore led to the conclusion that the nitrogen, carbon, and phosphate balance within the cell is closely associated with microcystin production. Whether or not microcystin is produced to maintain this balance or produced as a function of this balance remains to be determined.