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Exposure of Macrophages to Low-Dose Gadolinium-Based Contrast Medium: Impact on Oxidative Stress and Cytokines Production

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The toxicity of gadolinium-based contrast agents (GBCAs) has drawn a lot of attention. Nephrogenic systemic fibrosis (NSF), a lethal disease related to the use of GBCAs, is still not understood. Recently, gadolinium retention is found in brain tissues after repeated use of GBCAs in magnetic resonance imaging (MRI). However, most of the works investigating the toxicity of GBCAs are focusing on its high-concentration (0.5–10 mM) part, which is not reflective of the physiological conditions in human beings. Macrophages play a regulatory role in immune responses and are responsible for the fibrosis process. Their role in gadolinium retention and the pathogenesis of NSF, however, has seldom been investigated. This study aimed to evaluate the immune response generated by macrophages (RAW 264.7) exposing to low levels of GBCAs. The incubation concentration of GBCAs, including Omniscan®, Primovist®, Magnevist®, and Gadovist®, is proportional to the level of gadolinium uptake when detected via inductively coupled plasma mass spectrometry (ICP-MS) and imaged by MRI, whereas Primovist® treatment groups have highest gadolinium uptake among all of the tested concentrations. Low-concentration (2.5 μ mol/L) Gd chloride or GBCAs exposure promoted the reactive production of oxygen species (ROS), nitrate/nitrite, prostaglandin E2 (PGE2), and suppressed the potential of mitochondrial membrane. There was higher ROS, nitrate/nitrite, and PGE2 production in the Primovist®, Omniscan®, and Magnevist® groups compared to the Gadovist® group. In face of lipopolysaccharide (LPS) stimulation, Primovist®, Omniscan®, and Magnevist® groups exhibited elevated nitrite/nitrate and suppressed IL-1 β secretion and IL-6 and IL-10 secretion. Moreover, upon LPS stimulation, there is decreased TNF- α secretion 4 hours after Primovist® or Omiscan® exposure but the TNF- α secretion increased at 24 hours. Our data suggest that there is upregulated inflammation even in the presence of low levels of GBCAs, even similar to the physiological condition in murine macrophage. Further investigation of GBCAs on the human macrophage or in vivo animal study may clarify the role of macrophage on the pathogenesis of NSF and other GBCAs-related disease.
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Research Article
Exposure of Macrophages to Low-Dose Gadolinium-Based
Contrast Medium: Impact on Oxidative Stress and
Cytokines Production
Te-I Weng,
1
,
2
,
3
Huang Jen Chen,
4
Chen-Wen Lu,
5
Yu-Chin Ho,
2
Jia-Lun Wu,
3
Shing-Hwa Liu,
4
and Jong-Kai Hsiao
5
,
6
1
Forensic and Clinical Toxicology Center, National Taiwan University College of Medicine
and National Taiwan University Hospital, Taipei, Taiwan
2
Department of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
3
Department of Emergency Medicine, National Taiwan University Hospital, Taipei, Taiwan
4
Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan
5
Department of Medical Imaging, Taipei Tzu Chi Hospital, Buddhist Tzu-Chi Medical Foundation,
New Taipei City, Taiwan
6
School of Medicine, Tzu Chi University, Hualien, Taiwan
Correspondence should be addressed to Jong-Kai Hsiao; jongkai@tzuchi.com.tw
Received 5 June 2018; Revised 25 August 2018; Accepted 17 October 2018; Published 2 December 2018
Academic Editor: Changning Wang
Copyright ©2018 Te-I Weng et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
e toxicity of gadolinium-based contrast agents (GBCAs) has drawn a lot of attention. Nephrogenic systemic fibrosis (NSF), a lethal
disease related to the use of GBCAs, is still not understood. Recently, gadolinium retention is found in brain tissues after repeated use
of GBCAs in magnetic resonance imaging (MRI). However, most of the works investigating the toxicity of GBCAs are focusing on its
high-concentration (0.510 mM) part, which is not reflective of the physiological conditions in human beings. Macrophages play a
regulatory role in immune responses and are responsible for the fibrosis process. eir role in gadolinium retention and the
pathogenesis of NSF, however, has seldom been investigated. is study aimed to evaluate the immune response generated by
macrophages (RAW 264.7) exposing to low levels of GBCAs. e incubation concentration of GBCAs, including Omniscan®,
Primovist®, Magnevist®, and Gadovist®, is proportional to the level of gadolinium uptake when detected via inductively coupled
plasma mass spectrometry (ICP-MS) and imaged by MRI, whereas Primovist®treatment groups have highest gadolinium uptake
among all of the tested concentrations. Low-concentration (2.5 μmol/L) Gd chloride or GBCAs exposure promoted the reactive
production of oxygen species (ROS), nitrate/nitrite, prostaglandin E2 (PGE2), and suppressed the potential of mitochondrial
membrane. ere was higher ROS, nitrate/nitrite, and PGE2 production in the Primovist®, Omniscan®, and Magnevist®groups
compared to the Gadovist®group. In face of lipopolysaccharide (LPS) stimulation, Primovist®, Omniscan®, and Magnevist®groups
exhibited elevated nitrite/nitrate and suppressed IL-1βsecretion and IL-6 and IL-10 secretion. Moreover, upon LPS stimulation, there
is decreased TNF-αsecretion 4 hours after Primovist®or Omiscan®exposure but the TNF-αsecretion increased at 24 hours. Our
data suggest that there is upregulated inflammation even in the presence of low levels of GBCAs, even similar to the physiological
condition in murine macrophage. Further investigation of GBCAs on the human macrophage or in vivo animal study may clarify the
role of macrophage on the pathogenesis of NSF and other GBCAs-related disease.
1. Introduction
Gadolinium-based contrast agents (GBCAs) have been used
clinically in magnetic resonance imaging (MRI) to detect
malignancy. It is also used to verify vascular abnormality and
tissue perfusion defects in stroke and myocardial infarction
[1]. However, safety concerns were recently brought up that
nephrogenic systemic fibrosis may be caused by repeated use
of GBCAs [2]. Several studies on the pathogenesis of
GBCAs-related NSF have proposed that impaired clearance
Hindawi
Contrast Media & Molecular Imaging
Volume 2018, Article ID 3535769, 10 pages
https://doi.org/10.1155/2018/3535769
of gadolinium by kidneys could lead to tissue accumulation
of dissociated gadolinium (Gd) [3]. Recently, gadolinium
deposition in brain tissues has been observed in patients and
animals with normal renal function when receiving repeated
MRI along with GBCAs administration [4–6]. However, the
cause of gadolinium retention in normal tissues and its
potential hazards, as well as its role in the process of NSF,
remain unknown.
Studies have demonstrated a variety of adverse effects
associated with GBCAs administration. Histopathological
and molecular evidences showed obvious damage in the
spleen, lungs, and renal tissues [7]. GBCAs were found to
induce higher cytotoxicity in a confluent proximal tubular
epithelial cell line when compared with iodinated contrast
agents [8]. Moreover, ionic gadolinium dissociated from
gadolinium chloride can cause in vitro neurotoxicity [9].
Because of the widespread use of chelated gadolinium in the
clinical field, the toxicity of chelated and ionic forms of
gadolinium calls for more thorough investigation.
Macrophages play an important role in the regulation
of immune and inflammatory responses. When activated,
macrophages secrete a variety of cytokines, including TNF-
α, interleukin (IL)-1 beta, IL-6, IL-10, nitric oxide (NO),
and eicosanoids such as prostaglandin E2 (PGE2) [10, 11].
ese substances contribute to tissue damage mediated by
activated macrophages. Moreover, it has been demon-
strated that activated macrophages can contribute to early
events in various fibrotic processes [12]. ese results
suggest that macrophages could play a pivotal role in NSF
pathogenesis.
e cytotoxicity of GBCAs has been studied in macro-
phages [13, 14]. Most studies, however, focused on the re-
sponses produced by high concentrations (0.510 mM) of
GBCAs, which may result in the production of IL-1βor iNOS
[13, 14]. Human studies have revealed maximum plasma
gadolinium concentrations of 65.7 μg/mL. e plasma
clearance of GBCAs is 1.12.15 ml/min/kg. Moreover, the
elimination half-life of GBCAs is 1.2 h (1.01.8 h) for persons
with normal renal function, which indicates that very low
concentrations and trace amounts of GBCAs can also interact
with macrophages [15]. Previous studies focusing on changes
due to high gadolinium concentrations (0.510 mM) may not
be directly applicable to the pathogenesis of NSF or gadoli-
nium deposition in the brain [13, 14]. As a result, the present
study tends to examine the oxidative and immune effects of
GBCAs at the concentration level lower than the human
serum level when administered intravenously.
2. Material and Methods
2.1.Chemicals. Gadolinium (as GdCl3·xH2O 99% purity) was
obtained from Sigma-Aldrich. Primovist®(gadoxetic acid;
Gd-EOB-DTPP; 500 mM/mL; human dose, 25 μmol/kg),
Magnevist®(gadopentetic acid; 500 mM/mL; human dose,
100300 μmol/kg), and Gadovist®(gadobutrol; 1000 mM/mL;
human dose, 100300 μmol/kg) were purchased from Bayer
Schering Pharma or Bayer HealthCare Pharmaceuticals.
Omniscan®(gadodiamide; 500 mM/mL; human dose,
100 μmol/kg) was purchased from GE Healthcare Inc.
2.2. Cell Cultures. RAW 264.7, a murine macrophage cell
line, was purchased from Culture Collection and Research
Centre, Hsin-Chu, Taiwan. e cells were cultured in
DMEM medium (Gibco, Grand Island, NY, USA) supple-
mented with 2 mM glutamine, antibiotics (100 U/mL pen-
icillin A and 100 U/mL streptomycin), and 5% heat-
inactivated fetal bovine serum (Gibco). Also the cells were
maintained in a 37°C humidified incubator containing 5%
CO2. e cells were passaged when they reached 70%–80%
confluence.
2.3. Treatment of RAW 264.7 with GBCAs. Equal number (8
×10
5
) of RAW 264.7 was plated in 60 mm plastic culture
dishes. e cells were exposed to different concentrations
of Gd chloride or GBCAs (Primovist®, Omniscan®,
Magnevist®, or Gadovist®) at concentrations of 0.25 μM
to 2.5 μM.
2.4. Cell Viability Assay. Cell viability was evaluated by
MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) assay. e Gd chloride- and GBCAs-treated cells
were grown in triplicate in 24-well plates for 24 h. Later, MTT
was added to the medium at a final concentration of
0.5 mg/mL, and the cells were incubated for one hour at 37°C
in 5% CO
2
. After incubation, when the dark-blue formazan
dye generated by the living cells became proportional to the
number of live cells, the absorbance was measured at 570 nm
using a microplate reader. MTT data were shown as the
percentage of the average values of the control cells.
2.5. Cell Morphology. e RAW 264.7 cells were seeded on
10 cm plates with fresh medium and exposed to 2.5 μM
GBCAs for 24 h. At the end of the treatment, the cells
were washed twice with PBS and visualized using an
inverted microscope (BX51, Olympus, Japan) with 200×
magnification.
2.6. MRI. MRI was performed using a clinical 3.0 T MR
System (Signa Excite; GE Healthcare Bio-Science, Pis-
cataway, NJ, USA) as described previously [16]. Briefly,
the cell samples were centrifuged and placed in a water
tank, which was placed in an 8-channel head coil. Two-
dimensional T1-weighted fast spin-echo pulse sequences
were used (TR/TE 550/13 ms). e slice thickness was
1.0 mm, with a 0.5 mm gap, and the field view was 14 cm ×
10 cm with a matrix size of 288 ×192. e scan time
was 4 min and 5s with a repetition of 2. ese images
were further analyzed at a workstation provided by GE
Healthcare (Advantage workstation 4.2) with the free
Image J software. We measured the signal intensity of each
cell pallets to obtain quantitative data, which is also an
indirect method to determine the gadolinium deposition
in the cells. e gross phenomenon of cells via MRI in-
directly proved the interactions between macrophages
and GBCAs [16].
2Contrast Media & Molecular Imaging
2.7. Reactive Oxygen Species (ROS) Measurements. e
production of ROS under oxidative stress was measured
using the OxiSelectIntracellular ROS Assay Kit (Cell
Biolabs, San Diego, CA, USA). Cells were cultured in 96-well
plates after the treatment of 2.5 μM GBCAs for 4 or 24 h then
loaded with 1 mM of the cell-permeable fluorogenic probe
2’,7’-dichlorodihydrofluorescin diacetate (DCF-DA) for 1 h.
In brief, the DCF-DA was finally oxidized to high fluorescent
2’,7’-dichlorodihydrofluorescin by intracellular ROS. e
fluorescence intensity was measured using a fluorescence
plate reader (480 nm/530 nm) [17].
2.8. Mitochondrial Membrane Potential Measurements.
Alterations in the mitochondrial membrane potential were
analyzed using the tetramethylrhodamine ethyl ester
(TMRE) mitochondrial membrane potential assay (Cayman
Chemical, Ann Arbor, MI). e methods were modified
from those described in a previous study [18]. Cells were
cultured in 96-well plates after the treatment of 2.5 μM
GBCAs for 4 or 24 h. Treated cells were incubated with
5–500 nM TMRE in a serum-free medium at 37°C for
30 min. Active mitochondria absorb positively charged
TMRE due to its negative charge. Depolarized or inactivated
macrophages have low membrane potentials and fail to
absorb TMRE. We used a fluorescence plate reader with
excitation at 530 nm and emission at 580 nm to analyze the
accumulation of TMRE. Changes in fluorescence were
calculated following the manufacturer’s instructions.
2.9. Measurements of Cytokines, Nitrite/Nitrate, and Prosta-
glandin E2 (PGE2) Levels. Enzyme-linked immunosorbent
assay kits were used to measure the levels of interleukin (IL)-
1β, IL-6 (R&D Systems), tumor necrosis factor (TNF)-α
(Assaypro), IL-10 (LEGEND MAX, BioLegend), and PGE2
(Cayman Chemical, Ann Arbor, MI) in supernatants from
macrophages exposed to the Gd
3+
compounds for 4 or 24 h.
Concentrations of nitrite were determined by a
nitrate/nitrite colorimetric assay kit (R&D Systems). In LPS
stimulation experiments, cells were stimulated with LPS for
4 h or 24 h following overnight Gd chloride or GBCAs
incubation.
2.10. Statistics. Data are presented as means ±standard
error (SEM). Statistical analysis was performed using one-
way analysis of variance followed by the Dunnett test for
each paired experiment. p<0.05 was considered as statis-
tically significant.
3. Results
3.1. Effects of Different Kinds of GBCAs in Mouse’s Macro-
phage RAW Cells. We serially diluted Primovist®,
Omniscan®, Magnevist®, and Gadovist®with PBS solution.
Because different dissociation would result in different pH
values, we tested the pH value of PBS diluted GBCAs. It is
found that all of PBS diluted GBCAs kept neutral pH values
(Figure 1(a)). Figures 1(b) and 1(c) show the viabilities of the
cells of murine macrophage (RAW 264.7 cell line) stimu-
lated by Gd chloride, Primovist®, Omniscan®, Magnevist®,
and Gadovist®for 24 hours. No cell viability changes were
observed with different kinds of GBCAs and Gd chloride
(concentrations 0–2.5 μM). No conformational changes in
the cytology of RAW 264.7 were noted after 24 h of exposure
to GBCAs. In the control and GBCAs stimulated cells in our
experiment, the cell morphology generally showed a round
form (Figure 1(d)). After 24 h incubation with 0–2.5 μM
GBCAs, the macrophages increased uptake of gadolinium as
detected by inductively coupled plasma mass spectrometry
(ICP-MS; Figure 2(a)). e incubation concentration of the
GBCAs had a strong influence on the level of gadolinium
uptake; the maximum gadolinium uptake was 2020.0 ±
47.6 ppb/10
6
cells in the 2.5 μM Primovist®treatment group,
which was significant when compared to the control group.
e presence of cellular GBCA uptake was also confirmed
using cellular MRI, which demonstrated hyperintense dots
at the bottom of the test tube. All of the cells, except those in
the phosphate-buffered saline (PBS) treatment group,
exhibited uptake of GBCAs regardless of the type of GBCAs
used (Figure 2(b)).
3.2. ROS Production. We observed increased levels of ROS
when the cells were exposed to either Gd chloride or GBCAs
for 4 h. is effect was more significant in the 24 h exposure
groups (Figures 3(a) and 3(b)). ere was an almost two-fold
increase in ROS levels in the Gd chloride and GBCAs
treatment groups.
3.3. Decrease in Mitochondrial Membrane Potential.
Treatment with Gd chloride or GBCAs resulted in a dramatic
drop of mitochondrial membrane potential in macrophages
at 4 and 24 h after induction (Figures 3(c) and 3(d)). Both Gd
chloride and GBCAs demonstrated similar levels of mito-
chondrial membrane potential depression in macrophages.
However, no statistically significant differences were observed
between the 4 and 24 h treatment groups.
3.4. Effects on IL-6, IL-10, PGE2, Nitrite/Nitrate Production.
We investigated the inflammatory effects of Gd
3+
and
GBCAs on macrophages (Figure 4). e administration of
Gd
3+
or GBCAs did not result in increasing the secretions
of TNF-αand IL-1βin the 4 and 24 h groups (Figures 4(a)
and 4(e)). Interestingly, macrophages treated with
Magnevist®demonstrated a two-fold increase in IL-6
levels at 24 h (Figure 4(d)). Moreover, Omniscan®,
Magnevist®, and Gadovist®inhibited IL-10 secretion in
the same group (Figure 4(c)). Primovist®, Omniscan®, and
Magnevist®increased nitrate/nitrite production at 4 and
24 h, and this effect was more significant in the 24 h group,
except in the case of Magnevist®(Figure 4(b)). Further-
more, both Gd chloride and Gadovist®increased
nitrate/nitrite production at 24 h; the increases were more
noticeable in the Primovist®, and Omniscan®groups than
in the Gd chloride group. Primovist®, Omniscan®,
Magnevist®, and Gadovist®increased PGE2 production
Contrast Media & Molecular Imaging 3
at 24 h (Figure 4(f )). e production of PGE2 in the
Primovist®, Omniscan®, and Magnevist®groups were
higher than that of the Gd chloride group at 24 h.
3.5. Effects on Cytokines Production after the Exposure of LPS.
Followed by overnight exposure of Gd chloride and GBCAs,
macrophages were stimulated with 100 ng/ml or 1 μg LPS for
either 4 or 24 h. en cytokine production was measured
(Figure 5). In most cytokines secretion including TNF-α
(4 h), nitrite/nitrate (4 h), and IL-10 (4 h), IL-1β(24 h), PGE2
(24 h) with a combination of Gd
3+
and LPS is similar to the
LPS treatment group. ere is a decreasing secretion of
TNF-α(24 h) and IL-6 (24 h) in Gd3
+
, in conjunction with
the LPS group, when compared to the LPS stimulation group
(Figures 5(a) and 5(d)). Primovist®, Omniscan®, and
Magnevist®treatment resulted in a decreased secretion of
TNF-αwhen compared to the LPS stimulation group at 4 h
after LPS exposure (Figure 5(a)). Gd chloride, Magnevist®,
and Gadovist®treatment resulted in a decreased TNF-α
production when compared to the LPS stimulation group as
well as the combination of LPS plus Gd chloride at 24 h.
7.35
7.30
7.25
pH value
7.20
7.15
Medium only
PBS only
Control
Primovist
Omniscan
Magnevist
Gadovist
0.25
0.83
2.5
0.25
(µM)
0.83
2.5
2.5
2.5
0.25
0.25
0.83
0.83
(a)
Cell viability
(%, folds of control)
150
100
50
0
Control
Primovist
Omniscan
Magnevist
Gadovist
(µM)
Control
0.25
0.83
2.5
0.25
0.83
2.5
2.5
2.5
0.25
0.25
0.83
0.83
(b)
Cell viability
(%, folds of control)
150
100
50
00.250 0.83 2.5
GdCl3 (µM)
(c)
Control
Primovist Omniscan
Magnevist Gadovist
2.5 µM
(d)
Figure 1: Cell viability of different types of gadolinium-based contrast agents (GBCAs) and gadolinium chloride on RAW 264.7 cells.
Macrophages (RAW 264.7) were incubated in the absence or presence of 0.25, 0.83, and 2.5 µM/mL of various GBCAs or Gd chloride for
24 h. e pH values of PBS diluted GBCAs were checked (a) (n3). e effect of GBCAs or Gd chloride on cell viability was determined by
MTTassays immediately after incubation (b, c) (n3). Representative morphology recorded with a microscope after treatment with GBCAs
for 24 h (d).
4Contrast Media & Molecular Imaging
However, TNF-αsecretion at 24 h post-LPS exposure was
significantly increased in the presence of Primovist®and
Omniscan®. e production of nitrate/nitrite and IL-1βwas
significantly increased in Primovist®, Omniscan®, and
Magnevist®at 4 h or 24 h post-LPS exposure. e secretion
of IL-10 or IL-6 decreased with the presence of Primovist®,
Omniscan®, and Magnevist®at 4 h or 24 h post-LPS ex-
posure (Figures 5(c), and 5(d)). e productions of PGE2 in
Gadolinium 106 cells (pbb)
3
(103)
2
1
00.250 0.83 2.5
(µM)
Gadolinium content
Primovist
Omniscan
Magnevist
Gadovist
(a)
GdCl3
1× PBS
Control
0.25 µM
2.5 µM
1× PBS
Control
0.25 µM
2.5 µM
Primovist
Omniscan
Magnevist
Gadovsit
(b)
Figure 2: MRI and gadolinium content of macrophages treated with various gadolinium chloride and GBCAs. ICP-MS analysis showed that
the RAW cells generally contained gadolinium after treatment with the various Gd chloride and GBCAs (a) (n3). Coronal view and T1-
weighted scanning protocols were performed (b). e cells were centrifuged to the bottom of the test tube and imaged as dark signals. e
signal intensities of the cells revealed a dose-dependent increase on treatment with higher concentrations of Gd chloride and GBCAs for
24 h.
(a)
RFU (folds of control)
15
3
2
1
0
30
Control GdCl3Positive
control
Omniscan
Primovist Magnevist Gadovist
(b)
Control GdCl3
150
100
50
0
RFU (% of control)
#
OmniscanPrimovist Magnevist Gadovist
(c)
150
100
50
0
RFU (% of control)
Control GdCl3Primovist Magnevist GadovistOmniscan
(d)
Figure 3: e effects of the different GBCAs and gadolinium chloride-induced reactive oxidative species and mitochondrial membrane
potential in cultured RAW 264.7. e production of ROS was measured after treatment with 2.5 μM Gd chloride or GBCAs for 4 h (a) and
24 h (b). Similarly, mitochondrial membrane potential was measured after 4h (c) and 24 h (d) of treatment. p<0.05 when compared with
the control, #p<0.05 when compared with Gd chloride. Positive control, H
2
O
2
(2000 μM) for 30 min.
Contrast Media & Molecular Imaging 5
the Omniscan®, Magnevist®, and Gadovist®groups were
lower than both the LPS group and the combination of Gd
chloride and LPS group, but the combination of Primovist®-
LPS exposure increased at 24 h (Figure 5(f )).
4. Discussion
Our novel findings demonstrate that exposing to low con-
centration (2.5 μM) of GBCAs can alter the immune
function of macrophage regardless of the presence of LPS
exposure. Despite that there is no obvious GBCA-mediated
cytological changes, we observed different gadolinium
concentrations in macrophages measured by ICP-MS.
However, the actual subcellular compartment in which
gadolinium accumulates needs further investigation. e
accumulation of gadolinium was most pronounced in the
Primovist®-treated group. It is known that organic anion
transporting polypeptide (OATP) is responsible for trans-
ferring Primovist®into the cytoplasm, and cancer cell
lined with overexpressed OATP has higher intracellular
Primovist®deposition [16, 19]. e expression of OATP in
the macrophage is not fully determined, but it is an evident
route for certain kinds of GBCAs to go into the intracellular
space. Future studies to investigate OATP expression levels
in macrophages and their potential effects following the
Primovist®exposure are clinically relevant.
4.1. Exposure to GBCAs Increases Oxidative Stress. In our
present study, Gd chloride and GBCAs stimulated the pro-
duction of ROS and suppressed the potential of the mito-
chondrial membrane. ese effects were observed at a clinical
practice concentration. Both the increase of ROS and the
decrease of mitochondrial membrane potential have been
reported in some studies related to environmental hazard
toxicity, particularly those induced by heavy metals [20, 21].
Mitochondria have been suggested to be both the source and
target of ROS [22]. Abnormal accumulation of ROS in cells
can trigger downstream events of apoptosis and cytokine
release [23, 24]. Low levels of GBCAs exposure increased
(pg/ml)
25
20
10
15
5
0
Control GdCl3
Mouse TNF-α
Primovist Magnevist GadovistOmniscan
(a)
(µM)
2.5
1.5
2.0
1.0
0.5
0.0
Control GdCl3
Nitrate/nitrite
#
#
#
#
#
4
24 hours
Primovist Magnevist GadovistOmniscan
(b)
(pg/ml)
60
50
30
40
20
10
0
Mouse IL-10
Control GdCl3
#
OmniscanPrimovist Magnevist Gadovist
(c)
(pg/ml)
50
30
40
20
10
0
Mouse IL-6
Control GdCl3
#
Primovist Magnevist GadovistOmniscan
(d)
(pg/ml)
1.0
0.6
0.8
0.4
0.2
0.0
Mouse IL-1β
Control GdCl3Primovist Magnevist GadovistOmniscan
(e)
(pg/ml)
500
300
400
200
100
PGE2
Control GdCl3
#
#
#
Primovist Magnevist GadovistOmniscan
(f)
Figure 4: Effect of gadolinium or GBCAs on the production of TNF-α, nitrate/nitrite, IL-10, IL-1β, IL-6, and PGE2 by the RAW 264.7 cells.
e levels of TNF-α(a) were measured after treatment with 2.5 μM Gd chloride or GBCAs (2.5 μM) for 4 h and those of nitrite/nitrate (b) for
4 h and 24 h IL-10 (c) for 4 h, IL-6 (d) for 24 h, IL-1β(e) for 24 h, and PGE2 (f ) for 24 h were measured. p<0.05 when compared with
control. #p<0.05 when compared with Gd chloride (n5).
6Contrast Media & Molecular Imaging
oxidative stress in murine macrophages in the current study.
is finding might have some impacts in several pathological
conditions such as NSF because previous studies showed that
the increase of ROS is related to this disease [25].
4.2. GBCA Exposure Induces Nitric Oxide and Prostaglandin
E2 Production. Our study showed increased PGE2 secretion
in all macrophages after 24 h exposure to GBCAs.
Nitrate/nitrite levels originating from the murine macro-
phages were also elevated in the Primovist®, Omniscan®,
and Magnevist®groups after 4 and 24 h of exposure. Both
nitrate/nitrite and PGE2 are considered as inflammatory
and immunomodulatory mediators in the mammalian
physiology [26]. ey also play a major role in chemical
carcinogenesis [27]. Several oxidative stressors can induce
the expression of iNOS and COX-2, which synthesize NO
and PGE2, respectively [28]. Some studies have indicated
that iNOS and COX-2 expression pathways are induced in
vivo by models involving both inflammatory and oxidative
stress conditions [29]. Further in vivo studies to verify the
effects of GBCAs at low concentrations are needed.
4.3. Cytokines Secretion after the Combination of GBCAs and
LPS Exposure. We observed differences in cytokine profiles
5
(104 pg/ml)
TNF-α
#
#
#
#
#
#
#
4
2
3
1
0
Medium only
Medium + LPS
GdCl3 + LPS
Primovist+ LPS
Omniscan+ LPS
Magnevist+ LPS
Gadovist+ LPS
4
24 hours
(a)
(μM)
No (nitrate/nitrite)
#
#
12
8
10
6
4
GdCl3 + LPS
LPS only
Control
Primovist+ LPS
Omniscan+ LPS
Magnevist+ LPS
Gadovist+ LPS
(b)
IL-10
#
#
#
(pg/ml)
800
600
200
400
0
Primovist+ LPS
Omniscan+ LPS
Magnevist+ LPS
Gadovist+ LPS
GdCl3 + LPS
LPS only
Control
(c)
(103pg/ml)
IL-6
#
#
#
20
10
15
5
0
GdCl3 + LPS
LPS only
Control
Primovist+ LPS
Omniscan+ LPS
Magnevist+ LPS
Gadovist+ LPS
(d)
IL-1β
#
#
#
#
(pg/ml)
200
150
50
100
0
Medium only
Medium + LPS
GdCl3 + LPS
Primovist+ LPS
Omniscan+ LPS
Magnevist+ LPS
Gadovist+ LPS
(e)
(103 pg/ml)
#
#
#
PGE2
12
8
10
6
4
2
0
GdCl3 + LPS
LPS only
Control
Primovist+ LPS
Omniscan+ LPS
Magnevist+ LPS
Gadovist+ LPS
(f)
Figure 5: Effect of gadolinium or GBCAs on the production of TNF-α, nitrate/nitrite, IL-10, IL-1β, IL-6, and PGE2 by the RAW 264.7 cells
after the stimulation of LPS. RAW 264.7 cells were treated 24 h with 2.5 μM Gd chloride or GBCAs. e following cultures were stimulated
for 4 or 24 h with 100 ng/ml or 1 μM LPS. e levels of TNF-α(a) were measured after treatment for 4 h and 24 h, and those of nitrite/nitrate
(b) for 4 h, IL-10 (c) for 4 h, IL-6 (d) for 24 h, IL-1β(e) for 24 h, and PGE2 (f ) for 24 h were measured. p<0.05 when compared with LPS
only. #p<0.05 when compared with Gd chloride and LPS exposure (n3).
Contrast Media & Molecular Imaging 7
in response to GBCAs and LPS exposure compared to LPS
only. Primovist®, Omniscan®, and Magnevist®upregulated
the expression of nitrate/nitrite at 4 h, IL-1βat 24 h and
TNF-αat 24 h in LPS-stimulated macrophages. ey also
inhibited the expressions of anti-inflammatory molecules of
IL-10 at 4 h and inflammatory cytokines of IL-6 at 24 h.
TNF-αis an early stage cytokine after the LPS stimulation
[30]. However, the levels of TNF-αwere still high even after
LPS exposure for 24 h. is scenario should be studied in the
future. Most cytokines secretion in our studies such as TNF-
α, nitrite/nitrate, IL-10, and IL-1βafter the stimulation of Gd
chloride, Gadovist®, and LPS exposure was similar to the
LPS exposure except IL-6 and PGE2. e combination of
Primovist®, Omniscan®, and Magnevist®and LPS exposure
impaired the immune responses. e impaired levels of
cytokines with this Gadovist®were the least significant when
compared to the other three GBCAs in this study.
4.4. e Relationship between the Chemical Structure of
GBCAs and Inflammatory Changes in the Macrophage.
e current study indicated that Gadovist®was the least
toxic GBCA. Although the maximum and minimum
gadolinium cellular uptake concentrations were observed
with Primovist®and Omniscan®, respectively, the uptake
was not correlated with the toxicity of the GBCAs. is may
be attributed to several reasons. Macrocyclic GBCAs, such as
Gadovist®or Dotaram, exhibited lower dissociation con-
stants, and the molecular structure of macrocyclic GBCAs is
more stable than the linear GBCAs [31]. Among the four
GBCAs investigated in the current study, Gadovist®was a
macrocyclic GBCAs, whereas the remaining three were
linear agents. Macrocyclic GBCAs form ring-shaped
structures with Gd
3+
surrounded by an organic chelating
portion, making it harder for gadolinium to dissociate from
this encircled chelating environment. e higher the dis-
sociation constant is, the freer gadolinium can be released
into the circulation and tissues [32]. e gadolinium released
from the chelating complex induces the activation of various
profibrotic molecular pathways in one or more of the cell
types existed in fibrotic NSF lesions, such as macrophages,
fibroblasts, and fibrocytes [33]. A previous study reported
that according to the chelate model of gadolinium, the entire
Gd
3+
chelating complex, not just the transmetallated
gadolinium, was involved in the pathophysiology of NSF
[34]. In the present study, the macrophages released some
cytokines that may be related to NSF even at very low GBCA
concentrations. We also compared the cellular responses
toward free and chelated gadolinium. However, when Gd
chloride is added in DMEM which contains phosphate, the
formation of insoluble Gd phosphate is unavoidable. We
cannot identify the true toxicity of gadolinium because the
proper amount of gadolinium uptake by the macrophages is
unpredictable. e toxicity of gadolinium is likely to be
underestimated in our study. Although we noticed the
neutral pH of the PBS diluted solution after 24 h incubated
with macrophage in all contrast medium groups, the con-
ditions do not mimic the intracellular and intralysosomal
pH that likely exists in vivo. e concern of the existence of
dechelation of gadolinium after encountering acidic solution
may come from macrophage metabolism. Further studies
are required to differentiate between the toxic levels of
GBCAs originating from dissociated gadolinium and those
from chelated Gd
3+
complexes.
Macrocyclic Gadovist®elicited lower immune re-
sponses from macrophages with marginal ROS elevation and
potential mitochondrial membrane suppression. No nitrate/
nitrite stimulation was observed after 4 h exposure to
Gadovist®. Moreover, the levels of cytokines under LPS
exposure were similar to the combination of the LPS group
and were the least significant when compared with the other
three GBCAs in this study. ese findings may be attributed
to the macrocyclic chemical nature of this GBCA, which
makes it less influential on the macrophage functions.
Previous studies have reported that ROS and cytokines
were induced after incubation with high concentrations
(0.5–10 mM) of GBCAs in monocytes [13, 14]. e human
study has revealed maximum plasma gadolinium concen-
trations of 65.7 μg/mL [15]. We assume that the gadolinium
concentrations releasing from gadolinium retention tissue
are far less than 65.7 μg/mL. In the current study, we focused
on a lower concentration (2.5 μM) of GBCAs to simulate the
real clinical conditions. Interestingly, our results showed
that toxicities existed despite of the low concentration of
GBCAs used.
Although a variety of cytokines were released from
macrophages cultured with different kinds of GBCAs, we
acknowledge that the increase in ROS, IL-6, nitrite/nitrate,
and PGE2 levels and the decrease in mitochondrial mem-
brane potential induced by GBCAs in the current study are
not analyzed in the clinical practice. We think macrophage
morphology is one of the critical issues in the research of
cytokine release. When stimulated, the cells become stellate-
like that is a good indicator for determination of strong
irritation. In the control and GBCAs stimulated cells in our
experiment, the cell morphology generally showed no
conformational changes. Our results showed no strong ir-
ritation from the GBCAs that is compatible with our cy-
tokine analysis. With the increasing reports on the
deposition of gadolinium in brain, bone, and renal tissues of
patients with normal renal function exposed to GBCAs
during MRI examinations [35], the potential toxic effects of
low level GBCAs in various tissues must be investigated
thoroughly. e accumulated GBCAs in the human body
may stimulate macrophage and alter the immune reaction of
macrophages after LPS stimulation. Our data showed that
low levels of GBCAs could induce a potent activation of the
macrophages and suggested a possible mechanism that may
be related to the potential toxicity of GBCAs.
To the best of our knowledge, this is the first study that
focuses on the effects of low concentrations (2.5 μM) of
GBCAs on macrophage responses. GBCAs exerted a variety
of impacts on the macrophages even at low concentrations,
indicating that it is capable of inducing several patho-
physiological events that might be related to NSF or the
accumulation of gadolinium in different tissues. Further in
vitro and in vivo studies to evaluate the effects of low levels of
GBCAs on immune cell response should be conducted.
8Contrast Media & Molecular Imaging
In conclusion, similar to physiological conditions, ex-
posing to low levels of GCBAs can also alter the macrophage
function and elicited a variety of immune responses in
murine macrophages in our present study. Further in-
vestigation of GBCAs on the human macrophage or in vivo
animal study may clarify the role of macrophage on the
pathogenesis of NSF and other GBCAs-related disease.
Data Availability
e data used to support the findings of this study are in-
cluded within the article.
Conflicts of Interest
e authors declare no conflicts of interest.
Acknowledgments
is study was supported by a research grant from National
Taiwan University Hospital (106-S3527) and the Ministry of
Science and Technology, Taiwan (103-2314-B-303-001-MY3,
MOST-106-2314-B-303-007). We thank the staff of the
Eighth Core Lab, Department of Medical Research, National
Taiwan University Hospital, for technical support during the
study.
Supplementary Materials
Supplement 1. e effects of the very low doses of GBCAs
and gadolinium chloride-induced reactive oxidative species
and mitochondrial membrane potential in cultured RAW
264.7 were observed. e production of ROS was measured
after treatment with 0.25 and 0.83 μM Gd chloride or GBCAs
for 4 h (A: 0.25 μM, B: 0.83 μM) and 24 h (C: 0.25 μM, D:
0.83 μM). Similarly, mitochondrial membrane potential
was measured after 4 h (E: 0.25 μM, F: 0.83 μM) and 24 h (G:
0.25 μM, H: 0.83 μM) of treatment. H2O2 (2000 μM) for
30 min (n3). Supplement 2. e effect of gadolinium or
GBCAs on the production of nitrate/nitrite (24 h), IL-1β
(4 h), and IL-6 (4 h) by the RAW 264.7 cells after the
stimulation of LPS was observed. RAW 264.7 cells were
treated 24 h with 2.5 μM Gd chloride or GBCAs. e fol-
lowing cultures were stimulated for 4 or 24 h with 100 ng/ml
or 1 μM LPS. e levels of nitrate/nitrite (A) were measured
after treatment for 24 h, and IL-1β(B) and IL-6 (C) for 4 h
for 24 h were measured. p<0.05 when compared with LPS
only. #p<0.05 when compared with Gd chloride and LPS
exposure (n3). (Supplementary Materials)
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10 Contrast Media & Molecular Imaging
... Macrophages are important in immune and inflammatory responses and are involved in the early events in various fibrotic processes [67]. Moreover, several studies suggest that these could play a pivotal role in NSF [68]. They are also used as cell models in a series of in vitro studies for the evaluation of the potential cytotoxic effects of GBCA [69,70]. ...
... Following these issues, a recent study led by Weng et al. [68] aimed to evaluate the immune response generated by RAW 264.7 cells by exposing them for 24 h to low doses (0.25-2.5 µM) of different types of GBCA (Primovist ® , Omniscan ® , Magnevist ® , Gadovist ® ) and GdCl 3 [68]. During the experiment, none of the GBCA induced negative effects on the growth of RAW 264.7 cells. ...
... Following these issues, a recent study led by Weng et al. [68] aimed to evaluate the immune response generated by RAW 264.7 cells by exposing them for 24 h to low doses (0.25-2.5 µM) of different types of GBCA (Primovist ® , Omniscan ® , Magnevist ® , Gadovist ® ) and GdCl 3 [68]. During the experiment, none of the GBCA induced negative effects on the growth of RAW 264.7 cells. ...
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Although neutrophil elastase (NE) may play a role in lung fibrosis and liver fibrosis, NE involvement in the development of nephrogenic systemic fibrosis has been unclear. We investigated the involvement of NE in the development of nephrogenic systemic fibrosis-like skin lesions post-injections of linear gadolinium-based contrast agents in renal failure mouse models. Renal failure mouse models were randomly divided into three groups: control group (saline), gadodiamide group, and gadopentetate group. Each solution was intravenously administered three times per week for three weeks. The mice were observed daily for skin lesions. Quantification of skin lesions, infiltrating inflammatory cells, and profibrotic cytokines in the affected skin was performed by immunostaining and reverse-transcription polymerase chain reaction (RT-PCR). Blood samples were collected from the facial vein to quantify NE enzymatic activity. The ¹⁵⁸ Gd concentrations in each sample were quantified using inductively coupled plasma mass spectrometry (ICP-MS). In the gadodiamide group, the mRNA expression of fibrotic markers was increased in the skin lesions compared to the control group. In the gadopentetate group, only collagen 1α and TGF-β mRNA expression were higher than in the control group. The expression of CD3+, CD68+, NE cells and the NE activity in the blood serum were significantly higher in the gadodiamide and gadopentetate groups compared to the control group. Gadolinium concentration in the skin of the gadodiamide group was significantly higher than the gadopentetate group, while almost no traces of gadolinium were found in the control group. Although gadopentetate and gadodiamide affected the fibrotic markers in the skin differently, NE may be involved in the development of fibrosis linked to the GBCAs injections in renal failure mouse models.
... GdCl3 is toxic, whereas Gadovist (gadobutrol) is a neutral and macrocyclic molecule with inert properties, resulting in superior thermodynamic and kinetic stability. 41,42 Intriguingly, both molecules showed similar effects regarding leukocyte stiffening; however, the effect was less pronounced than for Magnevist and Dotarem. ...
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... Finally, although GBCA were originally developed to distribute extracellularly after IV administration, it has been speculated that reactive cellular responses may occur [46]. Inhibition of phagocytosis and macrophage apoptosis upon Gd exposure have been reported [47,48]. The threshold we applied on the fluorescence signal detected for the P distribution at 0.5μm resolution analyzed by SR-μXRF corroborates a cellular response since high P distribution marks cell membrane, ATP, or nucleic acids. ...
... Furthermore, during CCl 4 administration, treatment with GdCl 3 and glycine stopped the effect of CCl 4 enhancing liver fibrosis, and there was no -smooth muscle actin staining with GdCl 3 and glycine treatment. Also, when comparing the LPS stimulation group to the Gd 3+ group, there is a decrease in TNF-(24 h) and IL-6 (24 h) secretion in Gd 3+ after long exposure as 24 hours [27]. Taken collectively, Gdch3 had a protective effect against CCl 4 induced liver injury in male albino rats through suppressing expression of TGFb1 and attenuating the effect of CCl4 induced liver fibrosis. ...
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Reporter proteins have broad applications in visualizing molecular events at the cellular, tissue and whole-body levels. Transmembrane transporters recognizing specific molecular domains are of particular interest because they enable the migration of signal-source molecules from the extracellular space to the cytoplasm for subsequent application in multimodality imaging. Organic anion-transporting polypeptides (OATPs) have demonstrated their MRI reporter efficacy. We further expanded their use as a dual-modality reporter in MRI and noninvasive in vivo imaging system (IVIS). We overexpressed OATP1B3 in the HT-1080 sarcoma cell line. Both Gd-EOB-DTPA, an MRI contrast agent, and indocyanine green (ICG), a near-infrared fluorescent dye that provides better deep-tissue detection because of its long wavelength, could be delivered to the intracellular space and imaged in a tumor-bearing nude mouse model. Our in vivo dual-imaging reporter system achieved high sensitivity in MRI and observation periods lasting as long as 96 h in IVIS. Because of the superior temporal and spatial resolutions and the clinical availability of both ICG and Gd-EOB-DTPA, this dual-imaging OATP1B3 system will find biomedical use in tumor biology, stem cell trafficking, and tissue engineering.-Wu, M.-R., Liu, H.-M., Lu, C.-W., Shen, W.-H., Lin, I.-J., Liao, L.-W., Huang, Y.-Y., Shieh, M.-J., Hsiao, J.-K. Organic anion-transporting polypeptide 1B3 as a dual reporter gene for fluorescence and magnetic resonance imaging.
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Objectives: Retrospective studies in patients with primary brain tumors or other central nervous system pathologies as well as postmortem studies have suggested that gadolinium (Gd) deposition occurs in the dentate nucleus (DN) and globus pallidus (GP) after multiple administrations of primarily linear Gd-based contrast agents (GBCAs). However, this deposition has not been associated with any adverse effects or histopathological alterations. The aim of this preclinical study was to systematically examine differences between linear and macrocyclic GBCAs in their potential to induce changes in brain and skin histology including Gd distribution in high spatial resolution. Materials and methods: Fifty male Wistar-Han rats were randomly allocated into control (saline, n = 10 rats) and 4 GBCA groups (linear GBCAs: gadodiamide and gadopentetate dimeglumine, macrocyclic GBCAs: gadobutrol and gadoteridol; n = 10 rats per group). The animals received 20 daily intravenous injections at a dose of 2.5 mmol Gd/kg body weight. Eight weeks after the last GBCA administration, the animals were killed, and the brain and skin samples were histopathologically assessed (hematoxylin and eosin; cresyl violet [Nissl]) and by immunohistochemistry. The Gd concentration in the skin, bone, brain, and skeletal muscle samples were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS, n = 4). The spatial Gd distribution in the brain and skin samples was analyzed in cryosections using laser ablation coupled with ICP-MS (LA-ICP-MS, n = 3). For the ultra-high resolution of Gd distribution, brain sections of rats injected with gadodiamide or saline (n = 1) were assessed by scanning electron microscopy coupled to energy dispersive x-ray spectroscopy and transmission electron microscopy, respectively. Results: No histological changes were observed in the brain. In contrast, 4 of 10 animals in the gadodiamide group but none of the animals in other groups showed macroscopic and histological nephrogenic systemic fibrosis-like skin lesions. The Gd concentrations observed in the skin/brain samples (in nanomole Gd per gram of tissue) for each agent were as follows: gadodiamide: 1472 ± 115/11.1 ± 5.1, gadopentetate dimeglumine: 80.8 ± 6.2/13.1 ± 7.3, gadobutrol: 1.1 ± 0.5/0.7 ± 0.4, and gadoteridol: 1.7 ± 0.8/0.5 ± 0.2. The average detected residual Gd concentration in the brain was approximately 15-fold higher for linear than for macrocyclic GBCAs. The highest amounts of Gd found in brain corresponded to less than 0.0002% of the injected dose per gram of tissue. Using LA-ICP-MS, high Gd concentrations in the deep cerebellar nuclei and in the granular layer of the cerebellar cortex were detected only for linear gadodiamide and gadopentetate dimeglumine but not for gadoteridol or gadobutrol. The energy dispersive x-ray spectroscopy analysis revealed Gd-containing spots in the skin of animals administered gadodiamide and gadopentetate dimeglumine. Transmission electron microscopy revealed several Gd-containing spots in the region of the dentate nuclei in the brain of 1 animal injected with gadodiamide. Conclusions: After repeated high dosing, nephrogenic systemic fibrosis-like macroscopic and histopathological lesions of the skin were observed only in some of the gadodiamide-treated animals. No histopathological findings were detected in the rodent brain. The administration of linear GBCAs was associated with significantly higher Gd concentrations in the brain and skin compared with macrocyclic GBCA administration. The results of LA-ICP-MS demonstrated local accumulation of Gd within the deep cerebellar nuclei and the granular layer only after the administration of linear agents. In summary, the detected low Gd concentrations in the skin and brain were well correlated with the higher kinetic stability of macrocyclic GBCA.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
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Age-related macular degeneration (AMD) is a devastating neurodegenerative disease and a major cause of blindness in the developed world. Owing to its complexity and the lack of an adequate human model that recapitulates key aspects of the disease, the molecular mechanisms of AMD pathogenesis remain poorly understood. Here we show that cultured human retinal pigment epithelium (RPE) from AMD donors (AMD RPE) are functionally impaired and exhibit distinct phenotypes compared with RPE cultured from normal donors (normal RPE). Accumulation of lipid droplets and glycogen granules, disintegration of mitochondria, and an increase in autophagosomes were observed in AMD RPE cultures. Compared with normal RPE, AMD RPE exhibit increased susceptibility to oxidative stress, produce higher levels of reactive oxygen species (ROS) under stress conditions, and showed reduced mitochondrial activity. Measurement of the ratio of LC3-II/ LC3-I, revealed impaired autophagy in AMD RPE as compared with normal RPE. Autophagic flux was also reduced in AMD RPE as compared with normal RPE, as shown by inability of AMD RPE to downregulate p62 levels during starvation. Impaired autophagic pathways were further shown by analyzing late autophagic vesicles; immunostaining with lysosome-associated membrane protein 1 (LAMP-1) antibody revealed enlarged and annular LAMP-1-positive organelles in AMD RPE as opposed to smaller discrete puncta observed in normal RPE. Our study provides insights into AMD cellular and molecular mechanisms, proposes dysfunctional autophagy as an underlying mechanism contributing to the pathophysiology of the disease, and opens up new avenues for development of novel treatment strategies.
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Gadolinium chelates are widely used as contrast media for magnetic resonance imaging. The approved gadolinium-based contrast agents (GBCAs) have historically been considered safe and well tolerated when used at recommended dosing levels. However, for nearly a decade, an association between GBCA administration and the development of nephrogenic systemic fibrosis (NSF) has been recognized in patients with severe renal impairment. This has led to modifications in clinical practices aimed at reducing the potential and incidence of NSF development. Newer reports have emerged regarding the accumulation of gadolinium in various tissues of patients who do not have renal impairment, including bone, brain, and kidneys. Despite the observations of gadolinium accumulation in tissues regardless of renal function, very limited clinical data regarding the potential for and mechanisms of toxicity is available. This significant gap in knowledge warrants retrospective cohort study efforts, as well as prospective studies that involve gadolinium ion (Gd(3+)) testing in patients exposed to GBCA. This review examines the potential biochemical and molecular basis of gadolinium toxicity, possible clinical significance of gadolinium tissue retention and accumulation, and methods that can limit gadolinium body burden.
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Magnetic resonance imaging (MRI) contrast agents with high relaxivity are highly desirable because they can significantly increase the accuracy of diagnosis. However, they can be potentially toxic to the patients. In this study, using a mouse model, we investigate the toxic effects and subsequent tissue damage induced by three T1 MR contrast agents: gadopentetate dimeglumine injection (GDI), a clinically used gadolinium (Gd)-based contrast agent (GBCAs), and oxide nanoparticle (NP)-based contrast agents, extremely small-sized iron oxide NPs (ESIONs) and manganese oxide (MnO) NPs. Biodistribution, hematological and histopathological changes, inflammation, and the endoplasmic reticulum (ER) stress responses are evaluated for 24 h after intravenous injection. These thorough assessments of the toxic and stress responses of these agents provide a panoramic description of safety concerns and underlying mechanisms of the toxicity of contrast agents in the body. We demonstrate that ESIONs exhibit fewer adverse effects than the MnO NPs and the clinically used GDI GBCAs, providing useful information on future applications of ESIONs as potentially safe MRI contrast agents.
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Emerging evidence has linked MRI signal changes in deep nuclei of the brain with repeated administration of gadolinium-based contrast agents. Gadolinium deposits have been confirmed in brain tissue, most notably in the dentate nuclei and globus pallidus. Although some linear contrast agents appear to cause greater MRI signal changes than some macrocyclic agents, deposition of gadolinium has also been observed with macrocyclic agents. However, the extent of gadolinium deposition varies between agents. Furthermore, the clinical significance of the retained gadolinium in the brain, if any, remains unknown. No data are available in human beings or animals to show adverse clinical effects due to the gadolinium deposition in the brain. On behalf of the International Society for Magnetic Resonance in Medicine, we present recommendations for the clinical and research use of gadolinium-based contrast agents. These recommendations might evolve as new evidence becomes available.
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Streptozotocin (STZ) is a glucosamine-nitrosourea compound used for experimental simulation of sporadic Alzheimer’s disease at intracerebroventricular administration in vivo. The studies of STZ influence on neurons of central nervous system performed on the primary cultures are practically absent. We have shown the application of STZ (1–5 mM) in primary culture for 48 h induced strong dose-dependent death in cultured cerebellar granule neurons. This toxic effect was decreased by pyruvate, insulin partially. Using the indicator Fluo-4 AM for measurements of intracellular calcium ions and tetramethylrhodamine ethyl ester (TMRE) for detection of changes of mitochondrial membrane potential in live cells we have shown that 5 h-exposure to STZ induced intensive increase of Fluo-4 and decrease TMRE fluorescence in neurons. STZ exposure caused considerable ultrastructural alterations in granule neurons: chromatin clumping, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. Probably, STZ significantly impaired glucose metabolism and mitochondrial function that, in turn, resulted in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.
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The established class of intravenous contrast media for magnetic resonance imaging is the gadolinium chelates, more generally referred to as the gadolinium-based contrast agents (GBCAs). These can be differentiated on the basis of stability in vivo, with safety and tolerability of the GBCAs dependent upon chemical and biologic inertness. This review discusses first the background in terms of development of these agents and safety discussions therein, and second their relative stability based both on in vitro studies and clinical observations before and including the advent of nephrogenic systemic fibrosis. This sets the stage for the subsequent focus of the review, the current knowledge regarding accumulation of gadolinium in the brain and specifically the dentate nucleus after intravenous administration of the GBCAs and differentiation among agents on this basis. The information available to date, from the initial conception of these agents in 1981 to the latest reports concerning safety, demonstrates a significant difference between the macrocyclic and linear chelates. The review concludes with a discussion of the predictable future, which includes, importantly, a reassessment of the use of the linear GBCAs or a subset thereof.