These studies emphasize experimentation in vitro to characterize the primary site of action of the cyclic peptide hepatotoxin, microcystin-LR (MCLR), a toxin commonly produced by the blue-green alga Microcystis aeruginosa. Studies utilizing exposure to tritiated dihydro-MCLR via osmotic lysis of pinosomes and/or liposome fusion will investigate whether intracellularly exposed cells, including hepatocytes lacking bile acid carriers, L929 fibroblasts, and Chinese hamster ovary cells, respond to tritiated dihydro-microcystin in the same manner as hepatocytes. Cell fractionation studies using highly labelled 3H-dihydro-MCLR will be employed to assess the entry and sites of accumulation of toxin in hepatocytes over time. It is anticipated that, at some time points, a major fraction of the toxin will be free in the cytosol, whereas at others, the toxin will be present in proteins which interact with cytoskeletal fractions. Rabbit anti-MCLR antibody will be purified by affinity chromatography, incubated with toxin-exposed hepatocytes, and labelled with a goat anti- rabbit fluorescent antibody to localize sites of toxin binding (i.e., initially the plasma membrane). Staphylococcus aureus protein A avidin biotin complex (or Streptococcus protein G) will be bound to colloidal gold to detect the antibody binding sites for further examination via light and scanning electron microscopy (SEM). Several fixative solutions and mechanical techniques (as needed) will be explored to ensure entry of the anti-MCLR antibody without disrupting intracellular antigen-antibody interactions. In the same hepatocytes in which actin filaments are visualized with rhodamine-labelled phalloidin, we will use SEM to visualize the toxin. The toxin anti-MCLR antibody complex will be bound with the protein A (or G) colloidal gold tag. Finally, freeze-substitution techniques will also be employed with transmission electron microscopy to construct a more accurate representation of temporal changes in actin filaments, microtubules, and intermediate filaments in affected hepatocytes.
| Wickstrom, M L; Khan, S A; Haschek, W M et al. (1995) Alterations in microtubules, intermediate filaments, and microfilaments induced by microcystin-LR in cultured cells. Toxicol Pathol 23:326-37 |
