MECHANISMS OF MICROCYSTIN-LR HEPATOTOXICITY

Project: Research project

Description

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.
StatusFinished
Effective start/end date2/1/911/31/96

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $115,531.00

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Hepatocytes
Antibodies
Gold Colloid
Staphylococcal Protein A
Antibody Binding Sites
Rabbits
Cell Fractionation
Actin Cytoskeleton
Cyclic Peptides
Fixatives
Personal Autonomy
Intermediate Filaments
Cyanobacteria
Cricetulus
Transmission Electron Microscopy
Affinity Chromatography
Goats
Liposomes
Microtubules
Cytosol