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Published online before print December 19, 2006, 10.1148/radiol.2422060245

(Radiology 2006;242:425.)

A more recent version of this article appeared on December 1, 2006
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© RSNA, 2006

Experimental Studies

Cytotoxicity of Iodinated and Gadolinium-based Contrast Agents in Renal Tubular Cells at Angiographic Concentrations: In Vitro Study1

Marc C. Heinrich, MD, Martin K. Kuhlmann, MD, Sonja Kohlbacher, Mario Scheer, Aleksandar Grgic, MD, Martina B. Heckmann, MD and Michael Uder, MD

1 From the Institute of Diagnostic Radiology, University Hospital of Erlangen, Maximiliansplatz 1, 91054 Erlangen, Germany (M.C.H., S.K., M.S., M.B.H., M.U.); and Department of Medicine, Division of Nephrology and Hypertension (M.K.K.), and Department of Radiology (A.G.), Uni-versity Hospital of Saarland, Homburg/Saar, Germany. Received February 8, 2006; revision requested April 7; revision received April 28; final version accepted June 2. Address correspondence to M.C.H. (e-mail: Dr.MarcHeinrich{at}gmx.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To test in vitro whether gadolinium-based contrast agents induce fewer toxic effects on renal tubular cells than does an iodinated contrast medium at concentrations used for angiography.

Materials and Methods: LLC-PK1 cells were incubated with iomeprol, gadopentetate dimeglumine, gadobenate dimeglumine, gadoterate meglumine, gadodiamide, and corresponding mannitol solutions for 24 hours at 37°C in two experimental settings: measurements with equally attenuating solutions and measurements with equimolar solutions. Cytotoxicity was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, trypan blue testing, and an assay to detect apoptosis and necrosis. Data were analyzed with analyses of variance and post hoc tests.

Results: Yielding the same x-ray attenuation, iomeprol-300 and iomeprol-150 at concentrations of 2.34–18.75 mg of iodine per milliliter induced significantly (P < .001) lower inhibition of MTT conversion (74%–102% of undamaged control cells) compared with 15.63–125.00 mmol/L concentrations of the gadolinium-based agents (mean percentages of undamaged control cells: 48%–80%, 50%–87%, 60%–95%, and 56%–92% with gadopentetate dimeglumine, gadobenate dimeglumine, gadoterate meglumine, and gadodiamide, respectively). At equimolar concentrations (62.5 mmol/L), iomeprol-190 induced a mean extent of inhibition of MTT conversion (69% of undamaged control cells) similar to that induced by gadoterate meglumine (71%) and gadodiamide (70%), whereas gadopentetate dimeglumine and gadobenate dimeglumine induced stronger effects (63% and 64%, respectively; P < .001). At trypan blue testing, there were more dead cells after incubation with 125 mmol/L gadopentetate dimeglumine than after incubation with iomeprol-190 (57% vs 19%, P < .001). The 125 mmol/L gadopentetate and gadobenate formulations induced more necrosis and apoptosis than did gadoterate meglumine, gadodiamide, and iomeprol (mean percentage difference between treated and untreated control cells: for necrosis, +124%, +95%, +17%, –6%, and +3%, respectively; for apoptosis, +34%, +35%, +13%, +4%, and +5%, respectively; P < .001).

Conclusion: At angiographic concentrations, gadolinium-based contrast agents do not induce fewer cytotoxic effects on cultured renal tubular cells than does iomeprol.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 
When gadolinium chelates are used for angiography, the image quality achieved is generally inferior to that achieved with iodinated radiographic contrast media (RCM) at approved doses. The available volume of 45 mL of a 0.5 mol/L solution (0.3 mmol per kilogram of body weight, 75 kg body weight) often is not sufficient to perform diagnostic or interventional procedures (1–3). Consequently, administration of much higher gadolinium-based contrast agent doses, with injections of 45–440 mL, has been reported (1–5). Since there have been reports of acute renal failure in patients with chronic renal insufficiency (6–9), even at low gadolinium doses, the use of these media for angiography is still a matter of debate (1,5,10–17).

The pathogenesis of contrast agent–induced nephropathy (CIN) is poorly understood. Renal medullary hypoxia and tubulotoxic effects of RCM, in which mitochondrial dysfunction and generation of adenosine and oxygen radicals may be involved, are thought to contribute to the pathogenesis of CIN (18–22). Despite repeated statements to the contrary, there is little evidence that ischemia is the main mechanism by which RCM induce CIN in humans (20). Most of the evidence is based on the results of animal experiments that have questionable application to humans, and some authors have described an increase in medullary blood flow after RCM administration (20). There is increasing evidence that cytotoxic effects of RCM on renal cells are involved in the pathogenesis of CIN (22). In vitro studies of cultured renal cells are an established method of testing the cytotoxic effects of contrast agents and other nephrotoxic drugs in the absence of confounding variables such as hemodynamic changes (21,23–25).

For clinical practice, it seems appropriate to compare contrast agent concentrations that yield the same in vivo x-ray attenuation. To compare the nephrotoxic potential of different contrast agents, however, it is also necessary to compare them at equimolar concentrations. Thus, the purpose of our study was to test in vitro whether gadolinium-based contrast agents induce fewer toxic effects on renal tubular cells than does an iodinated contrast agent at concentrations used for angiography.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 
Cell Culture
LLC-PK1 cells, a proximal tubular epithelial cell line of porcine origin, were obtained from the American Type Culture Collection (Rockville, Md) and were cultured by using standard techniques (26).

Contrast Media
All contrast agents were used in ready-to-use formulations (Table 1). In addition, we used a water-diluted iomeprol-300 solution (concentration, 190 mg I/mL) to obtain a molar concentration (0.5 mol/L) equal to that of the gadolinium chelates. In a prior study (26), iomeprol was demonstrated to be typical of other low-osmolar nonionic agents.


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  		Table 1. Contrast Media Used in Study

 
Determination of Equally Attenuating Concentrations
We exposed 2-mL syringes filled with 50, 75, 150, 190 and 300 mg I/mL concentrations of iomeprol-300 and 0.5 mol/L concentrations of four gadolinium-based contrast agents (Table 1) in a 20-cm-high water phantom that served as an attenuator to the x-rays of our digital angiographic unit at a range of 70–80 kV. Equally attenuating concentrations were determined in consensus at visual comparison by three radiologists (M.C.H., M.U., M.B.H., with 6, 14, and 5 years experience, respectively, interpreting contrast agent–enhanced images) who were blinded to the contrast agent type and concentration.

Measurement of Cell Viability and Metabolic Activity with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide Assay
All chemicals were purchased from Sigma Chemical (Munich, Germany), unless otherwise noted. Confluent cells were incubated in 96 well plates with either control media (serum-free M199) or various concentrations of the contrast agents diluted in serum-free M199 for 24 hours. The final concentrations were 15.63, 31.25, 62.5, and 125.0 mmol/L of the gadolinium chelates and iomeprol-190 and 2.34, 4.69, 9.38, and 18.75 mg I/mL of iomeprol-300 and iomeprol-150. Cell viability and metabolic activity were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) uptake assay (Appendix A). To compare equally attenuating concentrations, 2.34, 4.69, 9.38, and 18.75 mg I/mL concentrations of iomeprol-300 and iomeprol-150 (dilutions, 1:16 to 1:128) were compared, respectively, with 15.63, 31.25, 62.5, and 125.0 mmol/L concentrations of the gadolinium chelates. To compare the toxic potential of gadolinium-based and iodinated contrast agent molecules, one must compare the contrast agents on a molar basis. To compare equimolar concentrations, 15.63, 31.25, 62.5, and 125.0 mmol/L concentrations of iomeprol-190 were compared with the same concentrations of the gadolinium-based agents.

We tested mannitol solutions that had osmolalities consistent with those of iomeprol-300, iomeprol-190, gadodiamide, gadopentetate dimeglumine, and gadobenate dimeglumine to study the effect of hyperosmolality. At approximately 1900 mOsm/kg H2O, the mannitol solution became saturated. Therefore, the high-osmolar mannitol stock solution could not exactly reach the osmolality of undiluted gadopentetate dimeglumine (1960 mOsm/kg H2O). However, the solubility posed no problem at the final concentrations since the mannitol solutions were used at dilutions corresponding to the dilutions of the contrast agents. Results were obtained from a minimum of 10 separate experiments with 12–15 samples each.

Quantification of Dead Cells with Trypan Blue Test
Cell viability was assessed by using the trypan blue exclusion test after the cells were incubated for 24 hours with 125 mmol/L of control medium, iomeprol-190, or gadopentetate dimeglumine (Appendix B). Results were obtained from a minimum of four independent experiments with two samples each.

Enzyme-linked Immunosorbent Assay for Detection of Necrosis and Apoptosis
Cells were incubated with 125 mmol/L concentrations of the various contrast agents for 24 hours. The Cell Death Detection ELISAPLUS Kit (Roche Diagnostics, Mannheim, Germany) was used to detect necrosis and apoptosis (Appendix C). Results were obtained from three independent experiments with seven or eight samples each. All experiments were performed by three authors (M.C.H., S.K., M.S).

Statistical Analyses
All data are presented as means ± the standard error of the mean. The number of dead cells at trypan blue testing, expressed as a percentage of the total cell number, was determined. MTT and enzyme-linked immunosorbent assay data were reported as percentages of undamaged control cells. MTT assay data were analyzed by using two-factorial analysis of variance (ANOVA), including first-order interactions (two-way ANOVA) modeled for contrast agent type and concentration, followed by the Tukey post hoc test for multiple comparisons. Other test data were analyzed by using one-way ANOVA followed by the Tukey post hoc test. P < .05 indicated statistical significance. For statistical analyses and graphic representations, 2002 Prism 3.03 software (Graph Pad Software, San Diego, Calif) was used. For two-way ANOVA, 2002 SPSS 11.5.1 for Windows (SPSS, Chicago, Ill) was used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 APPENDIX A
 APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 
Comparison of Contrast Media at Equally Attenuating Concentrations
At visual comparison, 75 mg I/mL iomeprol and 0.5 mol/L gadolinium-based contrast agent are essentially equally attenuating in the usual 70–80-kV range (Fig 1). On the basis of this proportion, the concentrations were calculated for comparison of equally attenuating concentrations of the contrast agents. All contrast agents induced concentration-dependent inhibition of MTT conversion (Fig 2). At equally attenuating concentrations, iomeprol-300 and iomeprol-150 induced lower inhibition of MTT conversion compared with the gadolinium-based agents (Table 2, Fig 2).


Figure 1
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  		Figure 1: Comparison of radiopacity of gadolinium-based contrast agents and iomeprol. Syringes filled with dilutions of ready-to-use 50, 75, 150, 190, and 300 mg I/mL concentrations of iomeprol (left) and 0.5 mol/L concentrations of gadodiamide (1), gadoterate meglumine (2), gadobenate dimeglumine (3), and gadopentetate dimeglumine (4), with 20 mL of water attenuation at 70 kV. Image contrast of gadolinium chelates essentially corresponds to that of 75 mg I/mL iomeprol.

 

Figure 2
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  		Figure 2: Comparison of cytotoxic effects on LLC-PK1 cells after 24-hour incubation with iomeprol and different gadolinium-based contrast agents at concentrations yielding equal x-ray attenuation, as assessed with MTT assay. At equally attenuating concentrations, iomeprol-300 and iomeprol-150 induced lower inhibition of MTT conversion than did gadolinium-based agents (P < .001, two-way ANOVA). Data reported as mean percentage of undamaged control cells, ± standard error of the mean, for at least 10 independent experiments.

 

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  		Table 2. Results of MTT Assay, with Comparison of Equimolar and Equally Attenuating Contrast Agent Concentrations

 
Comparison of Contrast Media at Equimolar Concentrations
At two-way ANOVA, equimolar concentrations of gadopentetate dimeglumine and gadobenate dimeglumine induced significantly greater inhibition of MTT conversion compared with iomeprol-190 (Table 2). In contrast, there was no significant MTT conversion difference between iomeprol-190 and either gadoterate meglumine or gadodiamide at equimolar concentrations (Table 2). The two ionic dimeglumine solutions induced greater inhibition of MTT conversion compared with the ionic gadoterate meglumine and the nonionic gadodiamide (Table 2, Fig 3).


Figure 3
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  		Figure 3: Comparison of cytotoxic effects on LLC-PK1 cells after 24-hour incubation with equimolar concentrations of iomeprol-190 and different gadolinium-based contrast agents, as assessed with MTT assay. Ready-to-use gadopentetate and gadobenate formulations induced greater inhibition of MTT conversion than did gadoterate meglumine, gadodiamide, and iomeprol-190 (P < .001, two-way ANOVA). Data reported as mean percentage of undamaged control cells, ± standard error of the mean, for at least 10 independent experiments.

 
Comparison of Contrast Media with Mannitol Solutions
Iomeprol and gadodiamide induced significantly greater inhibition of MTT conversion compared with the corresponding mannitol solutions at all concentrations tested (Fig 4a and 4b). In contrast, only the 31.25 and 62.50 mmol/L concentrations of gadopentetate dimeglumine and gadobenate dimeglumine induced a greater effect compared with the corresponding mannitol solution. At 125 mmol/L, there was no significant difference between any of these contrast agents and the mannitol solution (Fig 4c).


Figure 4
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  		Figure 4a: Comparison of cytotoxic effects on LLC-PK1 cells after 24-hour incubation with (a) iomeprol-190, (b) gadodiamide, and (c) gadobenate and gadopentetate formulations and the corresponding mannitol solutions, as assessed at MTT assay. Contrast agent formulations induced greater inhibition of MTT conversion compared with corresponding mannitol solutions (P < .001, two-way ANOVA). Data reported as mean percentage of undamaged control cells, ± standard error of the mean, for at least 10 independent experiments. * = significant difference between the contrast media and the mannitol solutions.

 

Figure 4
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  		Figure 4b: Comparison of cytotoxic effects on LLC-PK1 cells after 24-hour incubation with (a) iomeprol-190, (b) gadodiamide, and (c) gadobenate and gadopentetate formulations and the corresponding mannitol solutions, as assessed at MTT assay. Contrast agent formulations induced greater inhibition of MTT conversion compared with corresponding mannitol solutions (P < .001, two-way ANOVA). Data reported as mean percentage of undamaged control cells, ± standard error of the mean, for at least 10 independent experiments. * = significant difference between the contrast media and the mannitol solutions.

 

Figure 4
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  		Figure 4c: Comparison of cytotoxic effects on LLC-PK1 cells after 24-hour incubation with (a) iomeprol-190, (b) gadodiamide, and (c) gadobenate and gadopentetate formulations and the corresponding mannitol solutions, as assessed at MTT assay. Contrast agent formulations induced greater inhibition of MTT conversion compared with corresponding mannitol solutions (P < .001, two-way ANOVA). Data reported as mean percentage of undamaged control cells, ± standard error of the mean, for at least 10 independent experiments. * = significant difference between the contrast media and the mannitol solutions.

 
Cell Death and Apoptosis Induced by Equimolar Concentrations of Contrast Medium
Trypan blue test for detection of dead cells.—Incubation with gadopentetate dimeglumine leads to a distinct cell death, as assessed at trypan blue testing. With 125 mmol/L gadopentetate dimeglumine, 57% of cells ± 6 died (P < .001 for comparison with control medium). In contrast, 125 mmol/L iomeprol-190 induced a small increase in the number of dead cells (19% of cells ± 4 vs 12% ± 1 treated with control medium, P > .05). There were significantly more dead cells after incubation with gadopentetate dimeglumine than after incubation with iomeprol-190 (P < .001) (Fig 5).


Figure 5
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  		Figure 5: Comparison of number of dead cells after incubation with control medium, gadopentetate dimeglumine, and iomeprol-190, as assessed with trypan blue test. LLC-PK1 cells were incubated with 125 mmol/L of RCM for 24 hours. Significant differences in induced cell death between gadopentetate dimeglumine and control medium (P < .001) and between gadopentetate dimeglumine and iomeprol-190 (P < .001) were observed. Data reported as mean percentage of total cell number, ± standard error of the mean, for at least four independent experiments. * = significant difference compared with control value. + = significant differences between the two contrast agents.

 
Enzyme-linked immunosorbent assay for detection of necrosis and apoptosis.—At 125 mmol/L, gadopentetate dimeglumine and gadobenate dimeglumine induced significant necrosis and apoptosis (mean percentage difference between treated and untreated control cells: +124% ± 10 and +95% ± 12, respectively, for necrosis [Fig 6a]; +34% ± 1 and +35% ± 3, respectively, for apoptosis [Fig 6b]; P < .001). There was no significant difference in induced necrosis or apoptosis between these two contrast agents (P > .05). Gadoterate meglumine, gadodiamide, and iomeprol-190 induced no significant increases in necrosis (mean percentage difference between treated and untreated control cells: +17% ± 7, –6% ± 8, and +3% ± 9, respectively; P > .05). Incubation with gadoterate meglumine resulted in a small but significant increase in apoptosis (+13% ± 3, P < .01), whereas no significant increase in apoptosis could be detected after incubation with gadodiamide (+4% ± 3, P > .05) or iomeprol-190 (+5% ± 2, P > .05). There were no significant differences in induced necrosis or apoptosis among these three agents (P > .05). At equimolar concentrations, the gadopentetate and gadobenate formulations induced significantly more necrosis and apoptosis compared with the other two gadolinium-based agents and the iodinated RCM (P < .001) (Fig 6).


Figure 6
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  		Figure 6a: (a, b) Comparison of cytotoxic effects on LLC-PK1 cells after 24-hour incubation with 125 mmol/L concentrations of iomeprol-190 and different gadolinium-based contrast agents, as assessed with enzyme-linked immunosorbent assay for detection of (a) necrosis and (b) apoptosis. Ready-to-use gadopentetate and gadobenate formulations induced significant increases in necrosis and apoptosis (P < .001 for comparison with untreated control sample). Gadoterate formulation induced only a small increase in apoptosis (P < .01 for comparison with untreated control sample). Gadopentetate and gadobenate formulations induced significantly greater necrosis and apoptosis than did other two gadolinium-based agents and iodinated RCM (P < .001). Data reported as mean percentage difference in apoptosis and necrosis compared with values for untreated control sample, ± standard error of the mean, for at least three independent experiments. * = significant difference compared with control value. + = significant differences among various contrast agents. ns = no significant difference.

 

Figure 6
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  		Figure 6b: (a, b) Comparison of cytotoxic effects on LLC-PK1 cells after 24-hour incubation with 125 mmol/L concentrations of iomeprol-190 and different gadolinium-based contrast agents, as assessed with enzyme-linked immunosorbent assay for detection of (a) necrosis and (b) apoptosis. Ready-to-use gadopentetate and gadobenate formulations induced significant increases in necrosis and apoptosis (P < .001 for comparison with untreated control sample). Gadoterate formulation induced only a small increase in apoptosis (P < .01 for comparison with untreated control sample). Gadopentetate and gadobenate formulations induced significantly greater necrosis and apoptosis than did other two gadolinium-based agents and iodinated RCM (P < .001). Data reported as mean percentage difference in apoptosis and necrosis compared with values for untreated control sample, ± standard error of the mean, for at least three independent experiments. * = significant difference compared with control value. + = significant differences among various contrast agents. ns = no significant difference.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
APPENDIX A
 APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 
Our study results indicate that gadolinium-based contrast agents do not induce fewer cytotoxic effects on cultured renal proximal tubular cells than do iodinated RCM. In contrast, the gadolinium-based contrast agents induced more pronounced cytotoxicity compared with the iodinated RCM at concentrations yielding the same visual x-ray attenuation.

Our results seem to conflict with clinical observations of good renal tolerance to gadolinium-based contrast agents, even in patients with impaired renal function (27). However, that clinical experience is based on the administration of low doses of contrast agent (≤0.2 to 0.3 mmol/kg). Despite reports of negligible nephrotoxicity at low doses, higher doses of gadolinium-based contrast agents have reportedly caused acute renal failure in patients with underlying chronic renal insufficiency. The prevalences of acute renal failure after administration of 0.27–0.41 mmol/kg of a gadolinium-based agent were reported to be 1.9% at magnetic resonance (MR) angiography and 9.5% at digital subtraction angiography (DSA) in one study, in which 75% of patients had pretest chronic renal insufficiency (8). In a study involving 21 patients with impaired renal function (mean serum creatinine level, 3.2 mg/dL), the use of gadobutrol (0.34–0.90 mmol/kg; mean, 0.57 mmol/kg) for DSA resulted in a significant decrease in glomerular filtration rate, which seemed to exceed even the decrease observed in patients who received iohexol (28). Moreover, several authors have reported acute renal failure after application of low doses of gadolinium-based contrast agent in patients with chronic renal insufficiency (6–9). Similar to our group, Yano et al (29) observed significantly decreased viability of LLC-PK1 cells after the cells were exposed to the gadolinium-based agents.

In our study, the contrast agents with the highest osmolality demonstrated the greatest cytotoxic effects. This finding with iodinated RCM is well documented, so greater toxicity with the high-osmolar gadolinium-based contrast agents can be expected (26,30). Nevertheless, our experiment results suggest that osmolality is not the only reason for the toxicity. At the same concentrations, gadoterate meglumine and gadodiamide induced similar toxicity, even though gadoterate meglumine has a higher osmolality. Furthermore, the gadolinium-based agents, at least at lower concentrations, induced stronger cytotoxic effects than did the corresponding mannitol solutions. Also, clinical observations have indicated that factors other than osmolality play a substantial role in the pathogenesis of CIN in humans (21,31–33).

Other components in commercial formulations of gadolinium chelates may contribute to cytotoxicity. Meglumine has been shown to have only moderate toxic effects on renal epithelial cells and to add to the toxicity of the ionic RCM diatrizoate (34). However, in our study, gadoterate meglumine and gadodiamide induced similar toxic effects, even though the gadoterate solution contains meglumine and gadodiamide does not. Thus, meglumine also may add to the toxicity of gadolinium-based agents; however, as in iodinated RCM, it is not a major contributor. Some gadolinium-based agents contain excess chelate because of the possibility for transmetallation with trace amounts of zinc (35). The gadodiamide solution, which contained by far the greatest excess of chelate (12 mg/mL), was less cytotoxic compared with the gadopentetate dimeglumine (0.4 mg/mL) and gadobenate dimeglumine (no excess chelate) solutions and had toxicity similar to the gadoterate meglumine solution (no excess chelate). In addition, gadopentetate dimeglumine and gadobenate dimeglumine had similar toxicity, even though gadopentetate dimeglumine contains excess chelate and gadobenate dimeglumine does not. Therefore, the excess chelate does not seem to play a major role in the toxicity.

The 2–19 mg I/mL concentrations of iomeprol used in our study correspond to those expected at the proximal tubule in typical clinical conditions (18,36,37). In a 70-kg person, a 100-mL injection of iodinated RCM (300 mg I/mL) would yield a plasma concentration of approximately 2 mg I/mL. The proximal tubule concentration would be markedly higher because the water and solute content of the glomerular filtrate is reabsorbed in this portion of the renal tubule (18). Because of the renal concentration of RCM, urinary RCM concentrations may exceed plasma levels by a factor of 50–100 (38,39). Early clinical investigations revealed mean urinary iodine concentrations of 37–74 mg I/mL after administration of 1.5 mL/kg of RCM in humans (37). The concentrations of the gadolinium chelates had to be matched to these iodine concentrations to enable comparisons at equally attenuating concentrations. A 0.5 mol/L gadolinium-based agent and a 75 mg I/mL iodinated RCM essentially are equally attenuating in the 70–90-kV range commonly used for DSA, according to our and other study results (10).

he gadolinium concentrations used in our experiments are also clinically relevant because the doses of gadolinium-based agents required for satisfactory angiography and interventional procedures are usually larger than the doses approved for MR imaging studies (0.3 mmol/kg) (28). For example, Erley et al (28) administered an average dose of approximately 0.6 mmol/kg for DSA, and the use of even higher doses—up to 3.1 mmol/kg—have been reported by others (1–5). The concentrations used in our study were in the range of concentrations that have been used in previous cell culture studies (18,29,30,40–43).

Since renal insufficiency is the most important risk factor for CIN, the use of gadolinium-based contrast agents has been proposed for these high-risk patients. Although contrast agents are rapidly excreted by means of glomerular filtration in healthy subjects (44), the elimination half-life increases progressively with decreasing glomerular filtration rate (45). Elimination half-lives of 70 and 34 hours for iopamidol and gadodiamide, respectively, have been reported in patients with severely reduced renal function (46,47). In our study, the cytotoxic effects of contrast agents were studied after a 24-hour incubation period, which is shorter than the elimination half-life in patients with renal insufficiency and has been used in several previous studies of renal tubular cells to mimic the conditions in these high-risk patients (18,48,49). Thus, in terms of high urinary contrast agent concentrations and prolonged exposure of the tubular cells to the contrast agents in patients with renal insufficiency, the concentrations and incubation times that we used were in the range of those that may be found in these high-risk patients in clinical settings.

LLC-PK1 cells represent a well-characterized renal epithelial model system for investigation of the tubulotoxic effects of varying substances (21,23,24). RCM cytotoxicity has been investigated in LLC-PK1 cells in previous studies (18,29,40,41,50). A study conducted by Hardiek et al (18) revealed that human proximal tubular cells and LLC-PK1 cells respond to RCM in a similar manner. The validity of comparing different contrast agents in cell culture experiments is supported by in vitro studies, which have revealed greater toxicity with high-osmolar ionic RCM than with nonionic low-osmolar RCM. This greater toxicity is also found in vivo (25,30,40,41).

We assayed the metabolic activity of the cells by using the MTT assay, which is used to measure the activity of mitochondrial dehydrogenases (51). This assay is widely used to evaluate the cytotoxicity of various substances in cell cultures (23,51,52). Previous studies have revealed reduced MTT conversion with RCM in cultured renal tubular cells (18,19,26,53). The reduced MTT conversion could be due to cell death, reduced cell proliferation, and/or reduced mitochondrial activity. In our study, trypan blue testing revealed that use of gadopentetate dimeglumine led to a distinct cell death, whereas iomeprol-190 induced only a small increase in the number of dead cells. Previous studies involving the use of the trypan blue test have revealed a considerable decrease in cell viability induced by high-osmolar iodinated RCM, whereas low-osmolar RCM had no or only minor effects on cell mortality (18,30,40,41).

In our study, gadopentetate dimeglumine and gadobenate dimeglumine induced significant necrosis and apoptosis, whereas gadoterate meglumine caused apoptosis in only a few cells; the other contrast agents caused minimal, nonsignificant necrosis or apoptosis. There are conflicting results regarding RCM-induced apoptosis. In the Hizoh et al (42,43) and Peer et al (49) studies, apoptosis was induced by the high-osmolar agents diatrizoate and ioxithalamate. Peer et al (49) and Yano et al (29,50) observed apoptosis that was induced by some low-osmolar RCM also, whereas Hardiek et al (18) did not observe apoptosis after incubation with the low-osmolar agent iopamidol.

Our study was subject to the limitations of any in vitro experiment, in which the multifactorial pathogenesis of CIN in vivo is not accounted for; therefore, our results cannot be related directly to in vivo nephrotoxicity. Direct cytotoxic effects, however, are thought to be an important part of the nephrotoxic potential of contrast agents (18–21,25,54), and it is believed that this direct cytotoxicity is best studied in vitro because of the absence of confounding variables such as hypoxia and hemodynamic changes that can occur in vivo (25).

In conclusion, our study results demonstrate that, at least in vitro, gadolinium-based contrast agents do not induce fewer cytotoxic effects on renal proximal tubular cells than does an iodinated RCM at the high concentrations used for angiography. Our study data are evidence of the role that direct cytotoxic effects of gadolinium-based agents also have in the pathogenesis of CIN. At equally attenuating concentrations, gadolinium-based agents turned out to be more nephrotoxic than iodinated RCM. Thus, the relatively good renal tolerance to gadolinium-based agents at doses up to 0.3 mmol/kg that has been clinically observed is probably due to the very low dose administered in contrast to the usual doses of iodinated RCM administered.

Practical application: We believe our laboratory investigation may provide a foundation for future clinical studies in which gadolinium-based and iodinated contrast agents at equally attenuating concentrations are compared. Such studies are important, especially given the current off-label use of gadolinium-based agents.


   APPENDIX A
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 
Conversion of MTT, a tetrazolium salt, into formazan depends on the activity of a group of mitochondrial dehydrogenases and thus is an indicator of cell metabolic activity. The MTT assay was performed according to the protocol of Mosmann (51), with modifications. An equal volume of MTT reagent (7.5 mg/mL phosphate-buffered saline) was added to the cell supernatant and incubated for 2 hours at 37°C. The supernatants were removed, 100 µL of lysis buffer (20% sodium dodecyl sulfate, 33.3% dimethylformamide, 2% acetic acid, pH 4.7) per well was added, the plates were shaken at room temperature for 30 minutes, and absorbance was measured at 570 nm, with absorbance at 630 nm as the reference. The measurements were performed automatically by a microplate reader. Incubations with the experimental solutions, MTT, and lysis buffer were performed by one author (S.K.).


   APPENDIX B
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 
Subconfluent LLC-PK1 cell monolayers on six-well microplates were incubated with control medium, iomeprol-190, or gadopentetate dimeglumine at a concentration of 125 mmol/L for 24 hours at 37°C in a humidified atmosphere of 95% air and 5% CO2. Detached cells were collected from the supernatant by means of centrifugation and resuspended in phosphate-buffered saline. Adherent cells in the corresponding wells were washed twice with phosphate-buffered saline, treated with trypsin, and resuspended in phosphate-buffered saline together with the cells collected from the supernatant. The cells were then incubated in a 0.05% solution of trypan blue, and the number of trypan blue–positive nonviable cells, expressed as a percentage of the total number of cells, was determined by using a hemocytometer.


   APPENDIX C
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 APPENDIX C
ADVANCES IN KNOWLEDGE
 References
 
Cells were incubated with the various gadolinium-based contrast agents and iomeprol-190 at a concentration of 125 mmol/L at 37°C in a humidified atmosphere of 95% air and 5% CO2 for 24 hours. To assess cell death due to necrosis and apoptosis, we used the Cell Death Detection ELISAPLUS Kit, which is based on a quantitative sandwich enzyme immunoassay principle that involves the use of mouse monoclonal antibodies directed against DNA and histones. This test allows specific identification of mononucleosomes and oligonucleosomes in the cytoplasmic fraction of cell lysates. After the cells were incubated with the test solutions, the microplates were centrifuged and the cell culture supernatant was removed carefully and stored at 4°C. After incubation of the adherent cells in the microplate wells with lysis buffer, the lysate was centrifuged and the supernatant (ie, cytoplasmic fraction) was carefully removed. The culture supernatants (for analysis of necrosis), lysate of the cells (for analysis of apoptosis), positive control agent (DNA-histone complex), negative control agent (culture supernatant and lysate of untreated cells), and background control agent (incubation buffer) were transferred to a streptavidin-coated microplate. Then, the immunoreagent was added to the wells containing anti–histone-biotin, anti–DNA-peroxidase, and incubation buffer. After incubation in a microplate shaker, the supernatant was removed and the wells were washed with incubation buffer. Then, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt substrate was added to the wells and the absorbance was measured at 405 nm, with absorbance at 492 nm as the reference. The background value was subtracted from the absorbance measurements of the samples, and the absorbance of the samples was recorded as a percentage of undamaged control cells.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
References
 


   ACKNOWLEDGMENTS
 
We thank Mrs Martina Wagner for excellent technical assistance during the experiments.


   FOOTNOTES
 

Abbreviations: ANOVA = analysis of variance • CIN = contrast agent–induced nephropathy • MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide • RCM = radiographic contrast medium

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, M.C.H., S.K., M.U.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, M.C.H., S.K.; experimental studies, S.K., M.S.; statistical analysis, M.C.H., M.S., M.U.; and manuscript editing, M.C.H., M.K.K., S.K., M.B.H., M.U.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 APPENDIX C
 ADVANCES IN KNOWLEDGE
 References
 

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