Figure 4.

Toxicology and apoptotic effects of pirfenidone. Cells were treated with 0, 0.1, 0.5, or 1.5 mg/ml pirfenidone for 48 h and then used for subsequent experiments. A. Apoptotic rates were assessed using annexin V/propidium iodide (PI) double staining and flow cytometry. The cells shown in the bottom right quadrant are annexin V-fluorescein isothiocyanate (FITC)-positive and PI-negative, indicating an early stage of apoptosis. The cells in the top right quadrant stained positive for annexin V-FITC and PI, indicating late apoptotic/necrotic cells. The total percentage of cell apoptosis is shown in bold. B. Statistical analysis of the total recorded apoptotic cells was performed. The results are shown in the bar graphs. C. A lactate dehydrogenase (LDH) assay was performed to detect the cytotoxicity of pirfenidone. Data represent means ± standard error of the mean (SEM) of the percentage cytotoxicity calculated in cells from three independent experiments. No significant cytotoxic effects were observed for any treatment.