bromochloroacetic-acid and 2-5-hexanedione

Several neurotoxic compounds cause aggregation of neurofilaments in peripheral axons. This may represent a primary action of these chemicals or a secondary response to other cellular damage. To distinguish between these possibilities, the effects of acrylamide and 2,5-hexanedione (2,5-HD) on vimentin were examined in PtK2 cultured cells. Vimentin intermediate filaments were chosen because they are closely related, in structure, to neurofilaments. Effects on other components of the cytoskeleton (cytokeratin filaments, microtubules, and microfilaments) were also determined. Both acrylamide and 2,5-HD caused aggregation of vimentin filaments in a concentration-dependent fashion; these effects occurred at a lower concentration than alterations in other cytoskeletal filaments. The effects of both acrylamide and 2,5-HD were reversible, except at high concentrations of 2,5-HD. Crosslinking of cytoskeletal proteins was also examined. High-molecular-weight proteins with vimentin-like immunoreactivity were detected on blots from cells exposed to high concentrations of 2,5-HD. No crosslinked protein was detected after acrylamide treatment. These results suggest that both acrylamide and 2,5-HD cause a primary collapse of vimentin intermediate filaments in cultured cells. The initial redistribution of vimentin filaments occurred without apparent crosslinking of cytoskeletal proteins. The aggregation of vimentin filaments in cultured cells and of neurofilaments in vivo may share a common molecular mechanism.

Topics: Acrylamide; Acrylamides; Actin Cytoskeleton; Actins; Animals; Cell Line; Cytoskeleton; Dose-Response Relationship, Drug; Hexanones; Intermediate Filaments; Keratins; Ketones; Microtubules; Neurotoxins; Protein Binding; Tubulin; Vimentin

Using a variety of cell types in culture, we have investigated the relevance of intermolecular crosslinking involving intermediate filament proteins (IF-proteins) to the changes in distribution of intermediate filaments induced by the neurotoxicant, 2,5-hexanedione (2,5HD). Aggregation of vimentin-filaments (vimentin-IF), glial fibrillary acidic protein (GFAP)-IF and neurofilaments was preceded by appearance on immunoblots of a high molecular weight complex (IF-complex) labeled by antibodies against the IF-protein of the cell type: pV-170 from human skin fibroblasts and pV-130 from 3T3 mouse fibroblasts, which were labeled by anti-vimentin and, from cultures of dissociated mouse spinal cord and dorsal root ganglia, pNFH-300 labeled by antibody against the 200 kDa neurofilament protein (NF-200 or NF-H) and a doublet labeled by antiserum to GFAP (pGFAP-145).pV-170 was detected in human skin fibroblasts within one hour of exposure to 2,5HD and the amount of both pV-170 and pNFH-300 was related to the concentration of 2,5HD and the duration of exposure. Intermediate filament-complexes were not detected in PtK1 epithelial cells by labeling of transblots with anti-cytokeratin, nor are keratin-IF aggregated by 2,5HD. Intermediate filament-complexes were not detected in fibroblasts with IF-aggregates secondary to disruption of microtubules by colchicine or in fibroblasts from patients with giant axonal neuropathy.

Topics: Cells, Cultured; Cytoskeleton; Fibroblasts; Glial Fibrillary Acidic Protein; Hexanones; Humans; Intermediate Filament Proteins; Intermediate Filaments; Keratins; Ketones; Skin; Vimentin

2,5-hexanedione (2,5HD) induces focal accumulation of neurofilaments in nerve axons and juxtanuclear aggregation of vimentin-intermediate filaments (vimentin-IF) in cultured human skin fibroblasts. It has been postulated that 2,5HD prevents the cross-filament associations of intermediate filaments (IF) with microtubules which are required for their transport. If this is true, only subclasses of IF which depend on microtubules for their cellular distribution should be affected by 2,5HD-treatment and the aggregates formed should resemble the juxtanuclear coils which form following dissolution of microtubules by colchicine. We have tested this hypothesis in PtK1 cells which contain two separate networks of IF: vimentin-IF which aggregate in the presence of colchicine, and keratin-filaments (keratin-IF) whose distribution is not altered by depolymerization of microtubules. Treatment of confluent monolayers of PtK1 cells with 2,5HD (4 to 6 mM for 14 to 21 days) induced aggregates of vimentin-IF which resembled those induced by colchicine (5 X 10(-6)M for 48 hours), but had no effect on the distribution of keratin-IF.

Topics: Animals; Cell Line; Colchicine; Cytoskeleton; Fluorescent Antibody Technique; Hexanones; Immune Sera; Intermediate Filaments; Keratins; Ketones; Vimentin