FIGURE 4.

Effect of antioxidants on AGE-HSA-induced ROI generation and cell death. A, U-937 cells were treated with 100 μg/ml AGE-HSA for different times or 100 pm TNF for 2 h. ROI generation was measured using dihydrorhodamine by FACS and indicated as the percentage detected from mean channel number. U-937 cells were pretreated with N-acetylcysteine (NAC, 10 mm), vitamin C (2 mm), or pyrrolidine dithiocarbamate (PDTC, 100 μm) for 2 h and then stimulated with 100 μg/ml AGE-HSA for 4 h. B, ROI generation was measured using dihydrorhodamine by FACS. Cells were pretreated with N-acetylcysteine (10 mm), vitamin C (2 mm), or pyrrolidine dithiocarbamate (100 μm) for 2 h and then stimulated with 100 μg/ml AGE-HSA for 48 h. C, cell death was determined by MTT assay and indicated in percentage. Error bars in A–C indicate ± S.D. of triplicate samples. D, the amount of cytochrome c was measured from cytoplasmic fraction of AGE-HSA (100 μg/ml)-treated cells for different times, keeping doxorubicin (Dox) (1 μm for 24 h) as positive control for different times by Western blot. E, the amounts of Bad and Bax were determined in the whole cell extracts obtained from AGE-HSA-treated cells for different times by Western blot.