This application proposes to characterize further the cellular effects of the hepatotoxic peptide from the Cyanobacterium, Microcystis aeruginosa. This toxin (microcystin) has periodically caused morbidity and mortality in both feral and domesticated mammals and poses a threat to food and water sources for humans. Research will compare the effects of crude toxic extracts and one form of purified toxin. To determine the primary target cell of microcystin in liver, cytotoxicity studies will be conducted in vivo and with isolated cell types in vitro Time-course studies of the histological and ultrastructural pathology of lethally intoxicated mice will concentrate on initial hepatic cell alterations in an attempt to elucidate the cell of the liver that is first affected and to characterize the subcellular lesion. Isolated hepatic endothelial cells (HEC) and hepatic parenchymal cells (HPC) will be prepared by liver perfusion with collagenase and subsequent density-gradient centrifugation. Cells will be seeded into multiwell culture plates and HPC will be used 24 hours after attachment and spreading; HEC will be used 24 hours post confluency. Viability and functional state of both cell types will be assessed using biochemical assays, morphological observations and the presence of known antigens. These cells will be exposed to purified toxin in vitro, and the type and extent of cellular alterations will be noted over time from 2.5 minutes to several hours. In order to assess the toxicity of possible HEC and HPC metabolites of microcystin, toxin-containing medium exposed to one cell type will be placed on the other cell type. Co-cultures of HEC and HPC will also be challenged with toxin. In vitro cytotoxicity assays will include: lactate dehydrogenase and arginosuccinic acid lyase release, 51Cr release, [14C]-amino isobutyric acid uptake, and morphological changes using both light and electron microscopy. Age-sensitivity studies will be conducted in mice. Mechanistic investigations will be based on the in vivo and in vitro morphological observations and will include effects of microcystin on cytoskeletal elements (using fluorescently labelled antibodies to nonmuscle actin and myosin and tubulin), protein synthesis (measuring radiolabelled amino acid incorporation) mRNA synthesis (measuring [3H]-uridine incorporation), and glutathione levels. This potent, naturally occurring, biologically active molecule may eventually be a useful research tool and/or a pharmacologic agent as are other biotoxins.
