Figure 4

Role of glutathione and antioxidant molecules against acute iron toxicity in astrocytes. (A) Reduced glutathione (GSH) was measured in lysate of resting (C) or cytokine-activated (CK) astrocytes (average + SEM of three independent experiments). Statistical significance (*p < 0.05) was calculated using unpaired two-tailed Student’s t-test. BSO was used as a control. (B) GSH was measured in resting (C) or activated astrocytes (CK) in basal condition or after 30 min from iron overload. Columns represent the percent of GSH with respect to the control value of resting astrocytes (average +SEM of eight independent experiments). Statistical significance (*p < 0.05; **p < 0.01) was calculated using one-way ANOVA followed by Bonferroni. (C) A monolayer of resting (BSO) or cytokine-activated (BSO/CK) astrocytes was GSH depleted by BSO, loaded with fura-2 and then challenged by iron. (D) After 90 min from iron overload, cell viability was measured (MTT assay) on astrocytes either in resting condition (C), activated for 24 h with cytokines without (CK) or with (CK/2AAPA) 2AAPA. Each condition was expressed as the percent of viability with respect to the corresponding control without Fe²⁺ challenge. Statistical analysis in this and the following panel was performed using one-way ANOVA followed by Bonferroni (***p < 0.001). (E) After 90 min from acute iron overload, cell viability was measured (MTT assay) on astrocytes in resting condition (C), activated for 24 h with cytokines (CK), or pretreated for 2 h with Mito-TEMPO (MT) or Trolox (*p < 0.05; ***p < 0.001). (F) Astrocytes were plated in culture media containing horse serum from two different companies (BioWhittaker or Invitrogen). Cells were grown for 24 h, then loaded with fura-2 and challenged with iron overload. The traces represent the fluorescence intensity (expressed as RFU) of the astrocytes analyzed by a plate reader.