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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES005552-03
Application #
2154211
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1991-02-01
Project End
1996-01-31
Budget Start
1993-02-01
Budget End
1996-01-31
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820