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Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway

Departments of ¹ Radiology and ² Surgery, Cork University Hospital, Wilton, Cork, Ireland

View larger version (39K): [in a new window]   Figure 1. Time course of apoptosis induced by iodinated contrast media (ICM). (a) Representative flow cytometry profiles taken at 12 h culture of annexin v-fluorescein isothiocyanate (V-FITC) and propidium iodide staining of: (A), control; (B) iotrolan 20 mg I ml-1; (C) iohexol 20 mg I ml-1; (D) ioxaglate 20 mg I ml-1; (E), diatrizoate 20 mg I ml-1. The percentage given in the top left quadrant indicate the percentage of annexin V-FITC positive apoptotic neutrophils. (b) Results expressed as mean percentage of apoptotic cells±standard error of the mean. *, p<0.05 and , p<0.01 for ICM treated vs control neutrophils.

View larger version (33K): [in a new window]   Figure 2. Concentration dependence of iodinated contrast media (ICM) induced apoptosis. Results expressed as mean percentage of apoptotic cells±standard error of the mean. *, p<0.05 and , p<0.01 for ICM treated vs control neutrophils; , high necrotic rate.

View larger version (60K): [in a new window]   Figure 3. Cell morphology of control and iodinated contrast media treated neutrophils. Cytospins of (a) control and (b) diatrizoate (20 mg I ml-1) treated neutrophils at 12 h culture. In control neutrophils, normal neutrophil morphology with a multi-lobulated nucleus is seen (arrow). Following diatrizoate treatment, characteristic morphological signs of neutrophil apoptosis, such as cell shrinkage, nuclear pyknosis and chromatin condensation are demonstrated (arrow). Magnification of figures x 1 000.

View larger version (12K): [in a new window]   Figure 4. Effects of physicochemical properties of iodinated contrast media and growth factor dilution on neutrophil apoptosis. (a) Neutrophils were treated with experimental solutions for 12 h and apoptosis quantified by flow cytometry. Control, Rosweli Park Memorial Institute 1640 medium (RPMI)+10% platelet poor plasma; diatrizoate, 20 mg I ml-1; NaCl hypertonic, mannitol, and urea at same hyperosmolality as 20 mg I ml-1 diatrizoate; disodium calcium edetate (EDTA) 0.1 mg ml-1, 0.9% NaCl, RPMI diluted 1:2 with isotonic NaCl solution. Results expressed as mean percentage of apoptotic cells±standard error of the mean; p<0.01 for treated vs control neutrophils. (b) Neutrophils were cultured in RPMI containing 10% platelet poor plasma alone and treated with increasing concentrations (0.1–80 mg I ml-1) of povidone-iodine. Results expressed as mean percentage of necrotic cells±standard error of the mean; n=3–4; p<0.01 for treated vs control neutrophils.

View larger version (15K): [in a new window]   Figure 5 Iodinated contrast media accelerates apoptosis in lipopolysaccharide (LPS) stimulated neutrophils. Neutrophils were cultured in Rosweli Park Memorial Institute 1640 medium containing 10% platelet poor plasma alone and treated with iohexol 40 mg I ml-1, with or without LPS (1000 ng ml-1). Apoptosis was assessed at 1 h, 12 h and 24 h by flow cytometry. Results expressed as mean percentage of apoptotic cells±standard error of the mean; n=4; *p<0.01 for LPS treated vs control neutrophils; p<0.01 for iohexol treated vs control; p<0.01 for LPS+iohexol vs LPS alone. ––, control; ––, iohexol; ––, LPS; ––, LPS+iohexol.

View larger version (15K): [in a new window]   Figure 6. Iohexol promotes mitochondrial depolarization. Neutrophils were cultured in Rosweli Park Memorial Institute 1640 medium containing (a) 10% platelet poor plasma alone and (b) treated with iohexol 40 mg I ml-1. Mitochondrial transmembrane potential was measured using the styryl dye, JC-1, at 4 h, 10 h and 18 h in culture. Iohexol promotes a greater reduction in mitochondrial transmembrane potential compared with the control as assessed by increased formation of monomers (488nm-FL1-H). This effect was evident within 4 h of culture, with progressive loss of mitochondrial transmembrane potential at 10 h and almost complete mitochondrial depolarization at 18 h. Results representative of three experiments.

View larger version (18K): [in a new window]   Figure 7. Induction of apoptosis by iohexol is dependent on activation of the caspase-cascade pathway. Neutrophils were cultured in Rosweli Park Memorial Institute 1640 medium containing 10% platelet poor plasma alone and treated with iohexol 40 mg I ml-1, with or without zVAD-fmk (100 µM). Apoptosis was assessed at 12 h by flow cytometry. Results expressed as mean percentage of apoptotic cells±standard error of the mean; n=3; *p<0.05 for zVAD-fmk vs control neutrophils; p<0.05 for iohexol treated vs control; p<0.05 for zVAD-fmk+iohexol vs iohexol alone.