New cell substrates for in vitro evaluation of microcystin hepato-cytotoxicity

Algal toxins, as Microcystins released in water supplies, may represent a serious health hazard. The behaviour of primary hepatocytes was compared with that of immortalized liver cells, with the intention of providing a new test on Microcystin cellular toxicity. Immortalized liver cells were obtained by transfection with SV40 Large T antigen-bearing plasmids. Primary hepatocytes were used as a reference. Microcystin-LR at 10(-6), 10(-7)and 10(-9)m was added to hepatocytes maintained in suspension or cultured as three-dimensional hepatospheroids for 20 and 90 min at 37 degrees C. Toxic effects were monitored by cytoskeletal disruption ('blebs') using both light and scanning electron microscopy (SEM), lactate dehydrogenase release (LDH) and trypan blue dye exclusion test. Microcystin-LR at all doses induced bleb formation and a loss of microvilli in both primary hepatocytes and immortalized cell suspensions in comparison with controls. A high level of blebbed cells was detected in the absence of increased LDH release. The blebbing phenomenon was readily detectable by light microscopy but its morpho-complexity was unmasked by SEM, with early toxic events being indentified as loss of microvilli prior to bleb formation. Cells of primary hepatospheroids appeared to be less tightly attached to each other and more likely to bleb than immortalized ones. These immortalized cells could limit the use of primary cells and increase the reproducibility of the assay.