Dissertação de Mestrado em Biologia Humana e Ambiente apresentada à Faculdade de Ciências da Universidade de Lisboa, 2009Tese orientada por: Professora Doutora Ana Amorim (Professora Auxiliar da Faculdade de Ciências da Universidade de Lisboa); Doutora Elsa Alverca (Investigadora do Laboratório de Biologia e Ecotoxicologia, Departamento de Saúde Ambiental,Instituto Nacional de Saúde Doutor Ricardo Jorge)Microcystin-LR (MCLR) is a natural occurring freshwater cyanotoxin, recognized as
one of the most toxic microcystin variants. It is thought to be responsible for cases of
livestock and human intoxication due to consumption of toxic cyanobacteria-contaminated
water. Although considered a hepatotoxin, MCLR also targets other organs such as the
kidneys and intestines. In spite the cellular mechanisms associated with the toxicity of MCLR
are still unclear, a previous work in a monkey kidney cell line suggested that the endoplasmic
reticulum was an early target of MCLR toxicity and that autophagy was triggered as a cell
defense mechanism at subcytotoxic concentrations of MCLR.
In the present work, cytotoxic, morphological and ultrastructural effects of MCLR were
compared in HepG2 (human liver), Vero (monkey kidney), MDCK (dog kidney) and Caco2
(human intestine) cell lines. MCLR induced a concentration-dependent decrease in cell
viability by the NR assay in all cell lines, with HepG2 and Vero showing the lowest cytotoxic
thresholds of 25 and 50 μM MCLR, respectively. In these cells, MCLR exposure induced
lysosomal damages previously to mitochondrial disruption, reinforcing the role of lysosomes
in MCLR-induced toxicity. Immunolabelling and ultrastructural visualization of
autophagosomes, showed that autophagy was a response transversal to both cell lines,
triggered at subcytotoxic MCLR concentrations, confirming its importance as a defense
mechanism to early damages inflicted by the toxin. The analysis of GRP94, an ER stress
protein, did not undoubtedly demonstrate that MCLR targets the ER. However, together with
the ultrastructural data, suggested that in both HepG2 and Vero cells, the ER has a role in
autophagy induction. Additionally, in HepG2 cells, GRP94 down-regulation with increasing
MCLR concentrations supported the ER role in the triggering of apoptosis. At high toxin
concentrations, ultrastructural alterations consistent with apoptosis were observed for all four
cell lines, proving that this is a general MCLR-induced mechanism