Comparative study of the cytotoxic effects of microcystin-LR in mammalian cell lines: Vero, HepG2, Caco2 and MDCK

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