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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:11066 |
| Date | January 2003 |
| Creators | Botha, Nicolette |
| Publisher | University of Port Elizabeth, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | 144 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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