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| Author(s): |
Paula da Silva Kujbida
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2009-01-09 |
| Examining board members: |
Ernani Pinto;
Yara Cury;
Adriana Lino dos Santos Franco;
Patricia Moriel;
Helenice de Souza Spinosa
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| Advisor: | Ernani Pinto; Elaine Hatanaka |
| Abstract | |
Microcystins (MCs) are a family of heptapeptide toxins produced by some genera of Cyanobacteria. MCs have potent hepatotoxicity and tumor-promoting activity. Leukocyte infiltration in the liver was observed in MC-induced acute intoxication. Although the mechanisms of hepatotoxicity induced by MCs are still unclear, neutrophil infiltration in the liver may play an important role in triggering toxic injury and tumor development. The purpose of this thesis was to investigate the effects of three structurally distinct MCs (MC-LA, MC-YR and MC-LR) in the neutrophil functions: synthesis and expression of adhesion molecules, rolling, adhesion, migration and release of cytokines and ROS. In migration assays of the air pouch, the three MCs similarly induced the migration of leukocytes in vivo in subcutaneous tissue of rats and differentially the secretion of pro-inflammatory cytokines (CINC-2αβ, IL-1-β, TNF--α, VEGF- α and MIP-2) in exudates. Elevated concentrations of CINC-2αβ were found in the inflamed exudates from animals injected with MC-LA, MC-LR or MC-YR, although MIP-2 was only detected in the exudates from animals injected with MC-LR. There were no changes in the secretion of IL-1-β, TNF-α and VEGF--α. Intravital microscopic studies showed that topical application of MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression of L-selectin and -α2-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to fMLP, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. The effects of MC-LA, MC-YR and MC-LR in human neutrophils and mice had the same pattern of response. The analyses of cell viability, DNA fragmentation, mitochondrial membrane depolarization of and release of intracellular ROS were evaluated by the technique of FACS. Extracellular ROS content was measured by lucigenin-amplified chemiluminescence, and cytokines were determined by ELISA. We found that these MCs increased interleukin-8 (IL-8), cytokine-induced neutrophil chemoattractant-2αβ (CINC-2αβ) and extracellular ROS levels in human and rat neutrophils. In conclusion, our results showed that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. This process can significantly contribute to the pathogenesis of hepatic damage due to generation of ROS by neutrophils as well as act on hepatocytes under such conditions and potentially increase injury processes induced by MCs. (AU) | |