Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin
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F Mirabelli1, A Salis, V Marinoni
1Dipartimento di Medicina Interna e Terapia Medica, University of Pavia, Italy.
Abstract:
Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) or the thiol oxidant, diamide (azodicarboxylic acid bis(dimethylamide)), resulted in the appearance of numerous plasma membrane protrusions (blebs) preceding cell death. Analysis of the Triton X-100-insoluble fraction (cytoskeleton) extracted from treated cells revealed a dose- and time-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols. These changes were associated with the disappearance of actin and formation of large-molecular-weight aggregates, when the cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis under nonreducing conditions. However, if the cytoskeletal proteins were treated with the thiol reductants, dithiothreitol or beta-mercaptoethanol, no changes in the relative abundance of actin or formation of large-molecular-weight aggregates were detected in the cytoskeletal preparations from treated cells. Moreover, addition of dithiothreitol to menadione- or diamide-treated hepatocytes protected the cells from both the appearance of surface blebs and the occurrence of alterations in cytoskeletal protein composition. Our findings show that oxidative stress induced by the metabolism of menadione in isolated hepatocytes causes cytoskeletal abnormalities, of which protein thiol oxidation seems to be intimately related to the appearance of surface blebs.
Insights
Oxidative stress from menadione in rat hepatocytes causes cytoskeletal damage, leading to cell blebbing. Protecting protein thiols prevents these harmful effects, highlighting their role in cell survival.
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- Menadione and diamide induce oxidative stress in cells.
- Oxidative stress can disrupt cellular structures and functions.
Purpose of the Study:
- To investigate the effects of menadione and diamide on rat hepatocytes.
- To understand the role of cytoskeletal protein thiol oxidation in cell damage.
Main Methods:
- Incubation of isolated rat hepatocytes with menadione or diamide.
- Analysis of cytoskeletal protein composition and thiol content.
- Use of thiol reductants (dithiothreitol, beta-mercaptoethanol) for protection studies.
Main Results:
- Menadione and diamide induced plasma membrane blebbing and cell death.
- Increased cytoskeletal protein and loss of protein thiols were observed.
- Actin disappearance and aggregate formation occurred under nonreducing conditions, but were prevented by thiol reductants.
Conclusions:
- Oxidative stress in hepatocytes causes cytoskeletal abnormalities.
- Protein thiol oxidation is closely linked to the development of surface blebs.
- Thiol protection mitigates menadione-induced cell damage.