Chem.-Biol. Interactions, 80 (1991) 217- 236 217 Elsevier Scientific Publishers Ireland Ltd. CYTOSKELETON AS A TARGET IN MENADIONE-INDUCED OXIDATIVE STRESS IN CULTURED MAMMALIAN CELLS: ALTERATIONS UNDERLYING SURFACE BLEB FORMATION W. MALORNI, F. IOSI, F. MIRABELLIa and G. BELLOMO a Department of Ultrastructures, Istituto Superiore di Sanitd, 00161 Rome and aDepartment of Inter- hal Medicine, Clinica Medica I, University of Pavia, 27100 Pavia (Italy) (Received April llth, 1991) (Revision received July 22nd, 1991) (Accepted August 8th, 1991) SUMMARY Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-l,4-naphtoquinone (menadione) which is known to induce bleb forma- tion and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and elec- tron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione- induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramem- brane particles on their narrow base were detected using freeze-fracture tech- niques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin in- termediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the C~onden~ to: Dr. Walter Malorni, Dept. Ultrastructures, Istituto Superiore di Sanita', Viale Regina Elena 299, 00161, Rome, Italy. Abbreviations: DTT, dithiothreitol; IMP, intramembrane particles; Menadione, 2-methyl-l,4-naphto- quinone; PAP, peroxidase/anti-peroxidase; PBS, phosphate-buffered saline. 0009-2797/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.