Relationship between 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbations and cellular glutathione

Exposure of 3T3 cells to micromolar doses of 1-chloro-2,4-dinitrobenzene, a substrate for glutathione-S-transferase, resulted in a rapid depletion of total cellular glutathione accompanied by disassembly of microtubules as visualized by fluorescence microscopy. However, prolonged incubation resulted in cellular recovery from 1-chloro-2,4-dinitrobenzene insult as evidenced by a steady rise in total cellular glutathione accompanied by microtubule reassembly to their normal organization 5 hours after treatment. To evaluate the role of total cellular glutathione in modulating the 1-chloro-2,4-dinitrobenzene-induced cytoskeletal perturbation, we used 1-chloro-2,4-dinitrobenzene and/or buthionine sulfoximine, an effective irreversible inhibitor of glutathione synthesis, to manipulate cellular glutathione levels. Incubation of 3T3 cells with 2.5 microM 1-chloro-2,4-dinitrobenzene and 250 microM buthionine sulfoximine for 5 hours resulted in a complete depletion of total cellular glutathione accompanied by essentially complete loss of microtubules and marked alterations in the density and distribution pattern of microfilaments. Buthionine sulfoximine enhanced markedly the extent and duration of cellular glutathione depletion and the severity of microtubule disruption of 3T3 cells over the level achieved by 1-chloro-2,4-dinitrobenzene treatment alone. Furthermore, buthionine sulfoximine also prevented the restoration of cellular glutathione content and microtubule reassembly that normally were evident 5 hours after 1-chloro-2,4-dinitrobenzene treatment. Exposure of 3T3 cells to 50 microM 2-cyclohexene-1-one, which depletes free glutathione by conjugation, resulted in a complete depletion of total cellular glutathione content without altering the microtubule organization. These results suggest that the total glutathione content may be important for cellular recovery from 1-chloro-2,4-dinitrobenzene-mediated cytoskeletal injuries, and that microtubule disassembly observed in 1-chloro-2,4-dinitrobenzene-treated cells probably results from depletion of cellular glutathione coupled with binding to tubulin and/or other microtubule components.