A total of 60 Wistar rats were included in the current study. The rats were randomly divided into three groups (20 rats/group) and injected with iodixanol (4 g iodine/kg body weight), iopamidol (4 g iodine/kg body weight) or an equal volume of normal saline via tail vein. The rats were then randomly divided into 4 subgroups according to the time points following injection and were sacrificed at 0.5, 2, 12 or 24 h following injection, respectively. Blood samples, collected from the abdominal aorta for each animal, were treated with heparin to prevent coagulation and centrifuged at 670.8 × g for 10 min at 4°C. Plasma samples were then immediately transferred to −80°C for future tests. All animal experiments were conducted according to the ethical guidelines.

HUVEC cells were cultured in F12K medium containing 10% fetal bovine serum (FBS), 0.1 mg/ml heparin and 0.03–0.05 mg/ml endothelial cell growth supplement. H5V cells (mouse endothelial cells) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. All of these cells were maintained in an atmosphere of 5% CO₂ at 37°C.

Cells were seeded into 96-well plates (5×10⁴/well) and cultured for 24 h before use. Cells were then treated with 10 mg iodine/ml iodixanol or iopamidol for 30 min, 2, 12 or 24 h, respectively. Cell viability was determined by using CCK-8 agents following the manufacturer's manual (Dojindo Molecular Technologies, Inc., Kumamoto, Japan).

Cells were lysed with a protein extraction buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40 and 0.5% sodium deoxycholate], then quantified using a bicinchoninic acid kit (cat no. orb219872; Wuhan Booute Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Proteins (100 µg/well) were then loaded onto 10% SDS-PAGE gels for electrophoresis. Protein was blotted onto nitrocellulose membrane and blocked with 5% skimmed milk at room temperature for 2 h. Then, the membrane was incubated at 4°C for 2 h with antibodies against ABCG1 (cat no. PL0402507; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (cat no. A01020; 1:10,000; Abbkine Scientific Co., Ltd., Wuhan, China). Following washing with TBS containing 0.1% Tween-20 for 4 times, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (cat no. B1254; 1:1,000; Beijing Yonghui Biological Technology Co., Ltd., Beijing, China) at 4°C for 2 h. Specific bands were measured by using SuperSignal West Femto Maximum Sensitivity substrate (Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA). The specific band intensities were quantified with Quantity One software (version, 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Abdominal aortae from each subgroup were washed with PBS and fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin and cut into 4 µm thick sections. The sections were de-paraffinized with xylene and subsequently rehydrated. For routine histological analysis, the sections were stained with hematoxylin and eosin. For immunohistochemistry staining, the sections were treated with 3% H₂O₂ in PBS for 30 min to block the endogenous peroxidase and heat-repaired in 0. 01 M citrate buffer for 15 min. The sections were further blocked by normal goat serum for 30 min at room temperature prior to primary anti-endothelial nitric oxide synthase antibody (cat no. 4231; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) incubation at 4°C overnight. On the next day, the sections were incubated with biotin-conjugated goat anti-rabbit secondary antibody (cat no. sc-2007; 1:1,000; Santa Cruz Biotechnology, Inc.) for 2 h at 37°C followed by incubation in S-A/horseradish peroxidase (Santa Cruz Biotechnology, Inc.) for 15 min at room temperature. Following this, the sections were counterstained with hematoxylin, dehydrated, mounted with neutral gum and observed using Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan).

Plasma ET, von Willebrand factor (vWF), tissue type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI), D-Dimer, fibrinogen (FG), anti-thrombin III (AT-III), plasminogen (PLG) and NOS from cell culture medium were measured by using ELISA kits [Beijing Yonghui Biological Technology Co, Ltd.; Plasma ET (cat no. GD-VN9950), vWF, t-PA, PAI, D-Dimer and FG (cat no. QY-VN3291), AT-III (cat no. E04298), PLG (cat no. E00403) and NOS (cat no. JK-(a)-1609)]. Briefly, samples were applied to a 96-well plate (1×10⁵/well) with pre-coated capture antibodies for 2 h at 37°C. Then, the plates were washed and incubated with biotinylated antibodies (cat no. 500-P39Bt; 1:1,000; PeproTech, Inc., Rocky Hill, NJ, USA) at 37°C for 2 h. The plates were then washed five times and incubated with streptavidin-horseradish peroxidase (Beijing Yonghui Biological Technology Co, Ltd.; cat. no. 554066; 1:1,000) at 37°C for 30 min. The plates were washed again for five times and horseradish peroxidase substrates were added. The reactions were stopped after 30 min. Optical density was measured at 450 nm by using a microplate reader (Thermo Fisher Scientific, Inc.).

All values were presented as means ± standard deviation. Two-tailed Student's t-test was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) to determine the statistical significance of the results between two groups. P<0.05 was considered to indicate a statistically significant difference.

To examine the effect of CMs on endothelial cells in vitro, HUVEC and H5V cells were treated with 10 mg iodine/ml iodixanol or iopamidol for 0.5, 2, 12 or 24 h, and cell viability in each group was evaluated by CCK-8 assay. Iopamidol and iodixanol treatments reduced cell viability at 12 and 24 h in both HUVEC and H5V cells, but iopamidol group had lower cell viability than that in iodixanol group (Fig. 1A and B). These results indicated that damages of endothelial cells treated with iodixanol were less severe than those treated with iopamidol.

To further investigate whether CMs induce endothelial cell injury in vivo, iopamidol, iodixanol or equal volume of normal saline were injected into three groups of rats via tail vein, respectively, and abdominal aorta was dissected and the endothelial structure from dissected tissue was examined at 0.5, 2, 12 and 24 h following injections. The endothelial structure was smooth and intact in both control group and iodixanol group (Fig. 1C). In the iopamidol group, endothelial structure was normal at 0.5, 2 and 12 h following injection, but endothelia were injured and their integrity was disrupted at 24 h following injection (Fig. 1C). These findings indicated that isotonic CM was less toxic than hypotonic CM to endothelial cells in vivo.

To measure the levels of ET, NOS and ABCG1 in CM-treated cells in vitro, ELISA was employed. It was demonstrated that both iopamidol and iodixanol significantly increased the secretion of ET in HUVEC and H5V cells without significant differences between iopamidol and iodixanol (Fig. 2A and B). In addition, both iopamidol and iodixanol increased plasma ET levels at 2 h following injection and no significant difference in plasma ET levels was observed between iopamidol and iodixanol groups (Fig. 2C). In both HUVEC cells and H5V cells, iodixanol dramatically increased NOS levels at 2 h, whereas iopamidol did not. NOS levels were then significantly reduced at 12 and 24 h in HUVEC and H5V cells after treatment with iopamidol or ioxianol (Fig. 3A and B). Similar results were identified in dissected abdominal aorta tissue samples by endothelium-derived (e)NOS immunohistochemistry staining. The data demonstrated that eNOS was upregulated in both iopamidol and iodixanol groups at 2 h following injection, but downregulated at 12 and 24 h after injection compared with the control group (Fig. 3C). Furthermore, both iopamidol and iodixanol inhibited the expression of ABCG1 in HUVEC or H5V cells (Fig. 4). These results together indicated that CMs cause dysfunction of endothelial cells, which may be involved in CM-mediated cytotoxicity.

To compare whether iopamidol and iodixanol have similar effects on thrombin and fibrinolytic system, plasma levels of vWF, t-PA, PAI-1, D-Dimer, FG, AT-III and PLG were measured using ELISA. The data indicated that plasma vWF level was upregulated following iopamidol and iodixanol treatments at 0.5, 2 and 12 h after injection. However, plasma vWF returned to a normal level at 24 h in iodixanol group, but remained high in iopamidol group (Fig. 5A). Plasma t-PA concentration was also significantly increased in rats injected with iopamidol and iodixanol compared to control (Fig. 5B). Compared with the iodixanol group, the iopamidol group showed a lower concentration of plasma t-PA at 0.5 h following injection, but dramatically higher concentration of plasma t-PA at 12 h (Fig. 5B). Plasma PAI-1 levels in iopamidol group were comparable to that of the control, but iodixanol significantly downregulated the expression of PAI-1 at 24 h following injection (Fig. 5C). In addition, iopamidol increased plasma D-Dimer at 2 h after injection, while iodixanol did not significantly alter plasma D-Dimer concentration (Fig. 5D). Both iopamidol and iodixanol increased plasma FG at 2 h after injection compared with the control, but no significant difference was observed between iopamidol and iodixanol (Fig. 5E). Both iopamidol and iodixanol only increased plasma FG levels at 2 h after injection and did not significantly impact plasma FG levels at 0.5, 12 and 24 h following injection compared with control (Fig. 5E). Plasma AT-III levels were elevated at 2, 12 and 24 h after injection, but iopamidol group showed higher levels of AT-III than the iodixanol group (Fig. 5F). Plasma PLG levels were increased in both iopamidol and iodixanol groups, but the peak concentration of plasma PLG in the iopamidol group was at 12 h after injection, which was later than the iodixanol group (2 h after injection) (Fig. 5G). These results suggested that dysfunction of endothelial cells induced by CMs may be involved in thrombin and fibrinolytic system disorder and hence, increasing the risk of thrombus formation.