Methods: OKF6/TERT2 cells were treated for 90 min (ROS) or 24h and 48 h (intracellular GSH, cell viability) with CQ ± exogenous applied glutathione (GSH). Pre-treatment (30 min) with L-buthionine-sulfoximine (BSO) was used to decrease intracellular GSH. CQ-dependent ROS generation was determined by 2´,7´-dichlorodihydrofluorescein diacetate . Intracellular GSH content and cell viability were assessed by monobromobimane and propidium iodide . Hydrogen peroxide (H2O2) ± GSH was used as positive control showing the GSH protecting effect. Cells incubated in culture medium served as 100% control (c2). All experiments were performed at least three times with six replicates each. Data were statistically analyzed with Bonferroni ANOVA (p < 0.05).
Results: CQ (0.5-2.5 mM) induced a concentration-dependent generation of intracellular ROS in OKF6/TERT2 cells (2.5 mM CQ: 1928% ± 293%, solvent control (c1): 130% ± 7%). Co-incubation with exogenous GSH (5 mM) markedly decreased the CQ-induced ROS generation to 43% ± 9% of c2. However, the combined incubation (CQ/GSH) for 24h (viability: 50% ± 6%) and 48h (43% ± 3%) did not prevent the cytotoxic effect of CQ (2.5 mM CQ: 51% ± 7% (24h), 40% ± 8% (48h)), even with the significant increase of intracellular GSH-concentration to 300-560% in the presence of exogenously supplied GSH (p < 0.05). Furthermore, BSO decreased intracellular GSH to approx. 50% of untreated control cells, but showed also no effect on CQ-reduced viability.
Conclusion: The cytotoxicity of CQ was not attenuated by co-incubation with antioxidant-acting GSH and not enhanced by GSH depletion suggesting more complex mechanisms that are causative for CQ-induced cytotoxicity in OKF6/TERT2 cells.