title: Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells. I. Biochemical and immunocytochemical features.
abstract: Cytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2-methyl-1,4-naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament- and the microtubule-enriched fractions from menadione-treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione-dependent and thiol-sensitive oxidation of actin, leading to the formation of high-molecular-weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione-dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP-induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate-size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation-dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate-size filament distribution in menadione-treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate-size filaments) are actually significant targets in quinone-induced oxidative stress.
authors.affiliation: Dipartimento di Medicina Interna e Terapia Medica, University of Pavia, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Bellomo
authors.forename: F
authors.initials: F
authors.lastname: Mirabelli
authors.forename: M
authors.initials: M
authors.lastname: Vairetti
authors.forename: F
authors.initials: F
authors.lastname: Iosi
authors.forename: W
authors.initials: W
authors.lastname: Malorni
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D000199:Actins / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007382:Intermediate Filaments / Q000187:drug effects / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D012995:Solubility; D013312:Stress, Physiological / Q000473:pathology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013997:Time Factors; D014812:Vitamin K / Q000494:pharmacology*
chemical_list: D000199:Actins; D013438:Sulfhydryl Compounds; D014812:Vitamin K; D006160:Guanosine Triphosphate