cells
ArticleDabigatran Etexilate Induces Cytotoxicity in Rat Gastric
Epithelial Cell Line via Mitochondrial Reactive Oxygen
Species Production
Hiromi Kurokawa 1, *, Atsushi Taninaka 2 , Hidemi Shigekawa 2 and Hirofumi Matsui 1
12*

Citation: Kurokawa, H.; Taninaka,
A.; Shigekawa, H.; Matsui, H.
Dabigatran Etexilate Induces
Cytotoxicity in Rat Gastric Epithelial
Cell Line via Mitochondrial Reactive
Oxygen Species Production. Cells

Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan; hmatsui@md.tsukuba.ac.jp
Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan;
jun_t@bk.tsukuba.ac.jp (A.T.); hidemi@bk.tsukuba.ac.jp (H.S.)
Correspondence: hkurokawa.tt@md.tsukuba.ac.jp; Tel.: +81-29-853-3466

Abstract: Dabigatran is a novel oral anticoagulant that directly inhibits free and fibrin-bound thrombins and exerts rapid and predictable anticoagulant effects. While the use of this reagent has been
associated with an increased risk of gastrointestinal bleeding, the reason why dabigatran use increases
gastrointestinal bleeding risk remains unknown. We investigated the cytotoxicity of dabigatran etexilate and tartaric acid, the two primary components of dabigatran. The cytotoxicity of dabigatran
etexilate and tartaric acid was measured in a cell viability assay. Intracellular mitochondrial reactive
oxygen species (mitROS) production and lipid peroxidation were measured using fluorescence dyes.
Cell membrane viscosity was measured using atomic force microscopy. The potential of ascorbic
acid as an inhibitor of dabigatran cytotoxicity was also evaluated. The cytotoxicity of dabigatran
etexilate was higher than that of tartaric acid. Dabigatran etexilate induced mitROS production andlipid peroxidation and altered the cell membrane viscosity. Ascorbic acid inhibited the cytotoxicity
and mitROS production induced by dabigatran etexilate. Therefore, we attributed the cytotoxicity ofdabigatran to dabigatran etexilate, and proposed that the cytotoxic effects of dabigatran etexilate are
mediated via mitROS production. Additionally, we demonstrated that dabigatran cytotoxicity can beprevented via antioxidant treatment.

2021, 10, 2508. https://doi.org/
10.3390/cells10102508

Keywords: dabigatran etexilate; mitochondrial reactive oxygen species; lipid peroxidation; atomic
force microscopy; antioxidant

Academic Editors: Li Xiao and
Takeo Tsutsui
Received: 10 August 2021
Accepted: 13 September 2021
Published: 22 September 2021

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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Dabigatran and warfarin are anticoagulant drugs used to prevent stroke in patientswith atrial fibrillation [1]. Warfarin is a vitamin K antagonist that inhibits the activation ofclotting factors II, VII, IX, and X [2]. Vitamin K antagonists exhibit multiple interactions withfood and drugs; therefore, patients who are prescribed these drugs need to be monitored
frequently. Dabigatran is a novel oral anticoagulant that was approved by the FDA in2010 [3]. Dabigatran directly inhibits free and fibrin-bound thrombin and exhibits rapid
and predictable anticoagulant effects [4,5]. Dabigatran prevents embolic events in patientswith non-hemorrhagic stroke and atrial fibrillation and can be used in a safe and effective
manner without the need for monitoring and dose adjustment, in contrast to warfarin,
which requires continuous monitoring [6]. Moreover, dabigatran is well-tolerated, exhibits
predictable pharmacokinetics, and induces effective anticoagulant effects. Therefore, theuse of dabigatran is increasing worldwide.
Dabigatran capsules are composed of cores of tartaric acid coated with dabigatran
etexilate, which is a prodrug of dabigatran. As a prodrug, dabigatran etexilate does notdirectly inhibit thrombin. After oral administration, the low pH in the stomach facilitates
the conversion of dabigatran etexilate into dabigatran, the active form that acts as a direct
thrombin inhibitor [7]. Moreover, the presence of tartaric acid promotes the acidity of theCells 2021, 10, 2508. https://doi.org/10.3390/cells10102508

https://www.mdpi.com/journal/cells

Cells 2021, 10, 2508

2 of 13environment, which further assists in the conversion of dabigatran etexilate to dabigatran [8]. The widespread use of dabigatran has led to increased reports of gastrointestinal(GI) bleeding risk associated with its use [9]. Additionally, the risk of GI bleeding associatedwith dabigatran has been found to be higher than that associated with warfarin [3,10].
Several researchers have reported that tartaric acid plays an important role in the inductionof GI bleeding [5,11]. It has been postulated that tartaric acid adheres to the esophagus anddamages the esophageal mucosa, which sheds after peristalsis [12]. However, the precise
reason why dabigatran use increases the risk of GI bleeding remains unknown.
To investigate why dabigatran use is associated with an increased risk of bleeding,
we investigated the role of reactive oxygen species (ROS), especially mitochondrial ROS(mitROS). In a previous report, we investigated the relationship between certain drugs and
mitROS. We found that indometacine and bisphosphonate induce cytotoxicity via mitROS
production [13,14]. However, it has been found that antioxidants can prevent mitROS
cytotoxicity [15,16]. MitROS induce lipid peroxidation and DNA damage [17] and act
as mediators of intracellular signaling cascades that can trigger mitochondria-associateddamaging events, such as apoptosis via regulation of the Bcl-2/Bax balance [18]. Therefore,
any agents, such as antioxidants, that reduce mitROS production can help prevent cellular
injury and inhibit dabigatran etexilate-associated cytotoxicity.
In this study, we investigated the cytotoxicity of dabigatran etexilate and tartaric acid,the two main components of dabigatran, using a rat gastric epithelial cell line (RGM-1). We
hypothesized that dabigatran etexilate or tartaric acid induces mitROS production, thereby
causing cytotoxicity in normal cells and inducing GI bleeding when dabigatran is used.
As indometacine and bisphosphonate induce GI bleeding and cytotoxicity via mitROS
production, we focused on the mitROS. We also investigated whether antioxidants canattenuate the cytotoxicity of dabigatran etexilate and/or tartaric acid.
2. Materials and Methods
2.1. Cell Culture
Cells from the rat gastric epithelial cell line RGM-1 were purchased from the Riken
Cell Bank (Ibaraki, Japan) and cultured in DMEM/F12 with L-glutamine (Life Technologies
Japan Ltd., Tokyo, Japan). The culture medium was supplemented with 10% inactivatedfetal bovine serum (GE Healthcare Life Sciences, Amersham Place, UK) and 1% penicillin/streptomycin (Wako Pure Chem. Ind. Ltd., Osaka, Japan). The cells were cultured in
5% CO2 at 37 ◦ C.
2.2. Cell Viability Assay
The cell viability was evaluated using a Cell Counting Kit-8 (Dojindo, Tokyo, Japan)
according to manufacturer’s protocol. Cell Counting Kit-8 is a kit for measuring thenumber of cells in cell proliferation or chemical sensitivity tests. WST-8 (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt),
which is a reagent, is reduced by intracellular dehydrogenase to produce water-soluble
formazan, and the absorbance of this formazan at 450 nm can be directly measured toeasily determine the number of viable cells. Cells were seeded on 96-well plates at adensity of 2.5 × 103 cells/well and incubated overnight. The supernatant was aspirated
and replaced with a medium containing 1 or 5 µM dabigatran etexilate (Chemscene, LLC,
Monmouth Junction, NJ, USA) or tartaric acid (Wako). The cells were then incubated at
37 ◦ C for 24 h. After incubation, the cells were incubated with 10% Cell Counting Kit-8
reagent. The absorbance was measured at 450 nm using a Synergy H1 microplate reader(BioTek Instruments Inc., Winooski, VT, USA). The cells were treated with ascorbic acid todetermine its effect. Cells were seeded on 96-well plates at a density of 2.5 × 103 cells/welland incubated overnight. The supernatant was aspirated and replaced with a medium
containing 0, 50, or 100 µM dabigatran etexilate with or without 500 µM ascorbic acid
(Wako). The cells were incubated at 37 ◦ C for 24 h, and cell viability was measured using
the Cell Counting Kit-8.

Cells 2021, 10, 2508

3 of 132.3. Intracellular mitROS Measurement
MitoSOX (Thermo Fisher Scientific K.K., Kanagawa, Japan) was used to detect mitROS.
Cells were seeded on 96-well plates at a density of 1 × 104 cells/well and incubatedovernight. The supernatant was aspirated and replaced with a medium containing 0,
10, 15, 20, or 25 µM dabigatran etexilate. The cells were then incubated at 37 ◦ C for 6 h.
After aspirating the supernatant, the cells were incubated with 5 µM MitoSOX for 10 min.
After incubation, the medium was replaced with MSF buffer, containing 5.4 mM KCl,
136.9 mM NaCl, 8.3 mM glucose, 0.44 mM KH2 PO4 , 0.33 Na2 HPO4 , 10.1 mM HEPES,
1 mM MgCl2 .6H2 O, and 1 mM CaCl2 .2H2 O. Fluorescence intensity was measured using afluorescence microscope (Olympus, IX83). MitoSOX excitation was induced at 535–555 nm,
and the emission spectra were recorded using a 570–625 nm filter. To determine the effectof ascorbic acid, the cells were seeded on 24-well plates at a density of 1 × 105 cells/welland incubated overnight. The supernatant was aspirated and the cells were incubated in a
medium containing 25 µM dabigatran etexilate with or without 500 µM ascorbic acid. We
confirmed that this ascorbic acid concentration did not cause cell injury in RGM1 (data notshown). The cells were incubated for 6 h, the supernatant was aspirated, and MSF was added.
The fluorescence intensity of MitoSOX was measured using fluorescence microscopy.
2.4. Evaluation of Lipid Peroxidation
Lipid peroxidation was measured using diphenyl-1-pyrenylphosphine (DPPP) (Dojindo). Cells were seeded on a 6-well plate at a density of 5 × 104 cells/well and incubatedfor 2 days. The supernatant was aspirated, and the cells were incubated in a medium
containing 10 µM DPPP for 10 min. After incubation, the supernatant was aspirated, andthe cells were incubated in a medium containing 0, 25, 50, and 100 µM dabigatran etexilate.
The cells were incubated for 1 h, the supernatant was aspirated, and FluoroBrite DMEM
(Thermo) was added. The fluorescence intensity of DPPP was measured using fluorescencemicroscopy. DPPP fluorescence excitation was induced at 340–390 nm, and the emission
spectra were recorded at 420 nm.
2.5. Measurement of Cell Membrane Viscosity using Atomic Force Microscopy
The cultured cells were observed using AC mode atomic force microscopy (AFM),
and the viscoelastic loss tangent (tan δ) was estimated from the relationship between themeasured amplitude image and the phase image [19]. The value of tan δ was estimatedwith following equation.
tan δ =

ωVωfree Vfree − sin ϕ

Q VV
free



2
1 − ωω2free− cos ϕHere, w, Q, V, and j are the cantilever drive frequency, quality factor, the cantileveramplitude, and the phase, respectively. wfree and Vfree are the free reference values of thedrive frequency and the amplitude when the cantilever is separated from the sample by aheight of ∆z.
The viscoelastic loss tangent tan δ of a material is a dimensionless parameter thatmeasures the ratio of energy dissipated to energy stored in one cycle of a periodic deformation [20]; it is the parameter indicating the viscoelasticity of the cell. The cells were seeded
in a 60 mm dish. After incubation, the cells were stimulated with 25 µM dabigatran for 6 h.
After exposure, the cell membrane viscosity was measured using AFM in the loss tangent
mode [19,20].
2.6. Measurement of The Mitochondrial Membrane Potential using JC-1
The mitochondrial membrane potential was measured using JC-1 (5,5’,6,6’-tetrachloro1,1’,3,3’-tetraethyl benzimidazolyl carbocyanine iodide/chloride) [21]. Cells were cultured
in a 4-well chamber at 2.5 × 104 cells/well and incubated for 5 days. The supernatant was2.6. Measurement of The Mitochondrial Membrane Potential using JC-1
Cells 2021, 10, 2508

The mitochondrial membrane potential was measured using JC-1 (5,5’,6,6’-tetra4 of 13
chloro-1,1’,3,3’-tetraethyl benzimidazolyl carbocyanine iodide/chloride) [21]. Cells were
cultured in a 4-well chamber at 2.5 × 104 cells/well and incubated for 5 days. The supernatant was aspirated and the cells were incubated in a medium with or without 25 μM
dabigatran
etexilatefor 6wereh. Afterincubation,
the supernatant
aspiratedthe cells
aspirated andthe cells
incubatedin a medium
with or was
without
25 µManddabigatranwereincubatedinamediumcontaining
2
μM
JC-1.
The
cells
were
then
incubated30
etexilate for 6 h. After incubation, the supernatant was aspirated and the cellsfor
were
min.
The supernatant
wasaspirated2andcells
twiceincubatedwith PBS,for
following
incubatedin a medium
containingµM theJC-1.
Thewerecellsrinsed
were then
30 min.
which
MSF was added.
JC-1 redandfluorescence
excitation
at 535–555
nm,
andThe supernatant
was aspirated
the cells wererinsed was
twiceinducedwith PBS,
followingwhich
theemission
spectrawere
at 570–625
nm. JC-1
fluorescence
excitation
MSF
was added.
JC-1
redrecorded
fluorescence
excitation
was green
inducedat 535–555
nm, andwas
theinducedat
460–480nm,
andtheemission
spectrawererecordedat495–540nm.
emission spectra were recorded at 570–625 nm. JC-1 green fluorescence excitation was
induced at 460–480 nm, and the emission spectra were recorded at 495–540 nm.
2.7. Statistical Analysis
2.7. Statistical
Analysis was performed independently at least three times. Data are exEach experiment
Eachleast threetimes. Data
are expressed
asexperiment
the means was± SDperformed
and were independently
assessed by anatanalysis
of variance.
Individualpressed
as
the
means
±
SD
and
were
assessedbyananalysisofvariance.
Individual
groups were compared by Tukey’s post hoc or Student’s t-test with p < 0.05 consideredgroups werecompared by Tukey’s post hoc or Student’s t-test with p < 0.05 considered
statisticallysignificant.
statistically significant.
3. Results
3. Results
3.1. Dabigatran Etexilate induces Cytotoxicity in Normal Gastric Cells
3.1. Dabigatran Etexilate induces Cytotoxicity in Normal Gastric Cells
The
The cytotoxicities
cytotoxicities ofof bothboth dabigatrandabigatran etexilate
etexilate andand tartaric
tartaric acidacid were
were analyzed
analyzed using
using
the
WST
assay.
Cells
were
stimulatedwithdifferent
concentrationsofdabigatran
etexilatethe WST assay. Cells were stimulated with different concentrations of dabigatran etexilate
andacid forfor 2424 h.
h.The
Thecytotoxicity
cytotoxicityofofdabigatrandabigatranetexilate
etexilateincreasedsignificantly
and tartaric
tartaric acidincreasedsignificantlyininadose-dependent
manner(Figure1A),
while
tartaric
acid
did
not
inducecytotoxicity
a dose-dependent manner (Figure 1A), while tartaric acid did not induce cytotoxicity
at
at
concentrations
less
than
(Figurecytotoxicity
of dabigatran
etexilateconcentrations
less
than
100100µMμM(Figure1B).1B).
TheThecytotoxicity
of dabigatran
etexilatewaswashigherof tartaricof dabigatranhigherthanthanthat thatof tartaricacid.acid.
The TheIC50IC50of dabigatranwas was26.3 26.3µM. μM.

Figure 1. The
The cytotoxicity
cytotoxicity effecteffect ofof (A) dabigatran etexilate and (B) tartaric acid. RGM1 cells were
Figureexposedseveralconcentrationsof dabigatran
etexilateor tartaric
acid
exposedto
toculture
culturemedium
mediumcontaining
containing
severalconcentrationsof dabigatran
etexilateor tartaric
for
h, 24
then
a cellaviability
assayassay
was performed.
Data areas means± SD (n= 6).(n**=p6).
<
acid24forh, thencell viability
was performed.
Dataexpressed
are expressed
as means± SD
0.01.
** p < 0.01.

3.2. Dabigatran
Dabigatran Increases
Increases mitROS
mitROS Production
Production
3.2.
Intracellular mitROS
using
MitoSOX.
Cells
werewereincubated
Intracellular
mitROS production
productionwas
wasmeasured
measuredusing
MitoSOX.
Cells
incuinamediumwithorwithout
dabigatran
etexilate.
The
cellviabilityin25µM
dabigabated in a medium with or without dabigatran etexilate. The cell viability in 25 μM
tran etexilate
was decreased
down down
to 50%tofor
24 forh incubation(Figure(Figure1A). In1A).
thisInstudy,
dabigatran
etexilate
was decreased50%
24 h incubation
thisto evaluate
the mechanismsof cellofinjury
by dabigatran
etexilate,
we weselectedthethe0 tostudy,
to evaluate
the mechanisms
cell injury
by dabigatran
etexilate,selected
0
25 25
µMμM
concentration
andand6 h 6incubation
time.
TheThefluorescenceintensityof MitoSOX
wastoconcentrationh incubationtime.
fluorescenceintensityof MitoSOX
measured.
TheThefluorescencemicroscopic
imagesshowedthatthat
thethefluorescenceintensitywasmeasured.
fluorescencemicroscopic
imagesshowedfluorescenceintenof MitoSOX increased in a dose-dependent manner (Figure 2A). In particular, the intracellular fluorescence intensity of MitoSOX increased significantly upon treatment with
20 and 25 µM dabigatran etexilate compared to that obtained upon treatment with 0 µM
dabigatran etexilate (Figure 2B).

Cells 2021, 10, 2508

sity of MitoSOX increased in a dose-dependent manner (Figure 2A). In particular, the intracellular fluorescence intensity of MitoSOX increased significantly upon treatment withof 13
20 and 25 μM dabigatran etexilate compared to that obtained upon treatment with 0 5μM
dabigatran etexilate (Figure 2B).

Figure2.2. The
of of
MitoSOX.
(A)(A)
Fluorescent
microscopy
utilized
to assesscellularcellular
uptake uptakeof MitoSOX.
Figure
Thefluorescence
fluorescenceintensity
intensity
MitoSOX.
Fluorescent
microscopy
utilized
to assessof Mi(B)Data
areexpressed
as
means
±
SD
(n
=20).
The
samplesize
isthenumber
of
cells
in
the
analyzed
images.
Ex= 535–555
toSOX. (B) Data are expressed as means ± SD (n =20). The sample size is the number of cells in the analyzed
images.
nm and Em = 570–625 nm. **p < 0.01.
Ex = 535–555 nm and Em = 570–625 nm. ** p < 0.01.

3.3. Dabigatran Etexilate induces Lipid Peroxidation
3.3. Dabigatran Etexilate induces Lipid Peroxidation
Since ROS induce lipid peroxidation, we measured the lipid peroxidation of cells usSince ROS induce lipid peroxidation, we measured the lipid peroxidation of cells using
ing DPPP. The cells were incubated in a medium with or without dabigatran etexilate.
DPPP. The cells were incubated in a medium with or without dabigatran etexilate. After
After incubation, the fluorescence intensity of DPPP increased in a dose-dependent manincubation, the fluorescence intensity of DPPP increased in a dose-dependent manner, as
ner, as evident in the fluorescence microscopic images (Figure 3A). The intracellular fluoevident in the fluorescence microscopic images (Figure 3A). The intracellular fluorescence
rescence intensity of DPPP increased significantly upon treatment with 50 and 100 μM
intensity of DPPP increased significantly upon treatment with 50 and 100 µM dabigadabigatran etexilate compared to that obtained upon treatment with 0 μM dabigatrantran etexilate compared to that obtained upon treatment with 0 µM dabigatran etexilate
etexilate (Figure 3B).
(Figure 3B).

Cells2021,
10,2508
x FOR PEER REVIEW
Cells
2021,
10,

6 6
ofof1313

Figureintensity
ofof
DPPP.
(A)(A)
Fluorescent
microscopy
utilized
to assesscellularuptakeof DPPP.
(B) Data
Figure3.3.The
Thefluorescence
fluorescenceintensity
DPPP.
Fluorescent
microscopy
utilized
to assesscellularuptakeof DPPP.
(B)
areexpressed
as means± SD (n= 10).
size istheisnumberof cells
in theimages.
Ex =Ex
340–390
nm nm
and
Data
are expressed
as means± SD(n =The10).sample
The sample
size
the numberof cells
inanalyzed
the analyzed
images.
= 340–390
and
Em
=
420
nm.
**p<0.01.
Em = 420 nm. ** p < 0.01.

3.4. Dabigatran
Dabigatran Etexilate
Etexilate Alters
Alters Cell
3.4.
Cell Membrane
Membrane Viscosity
Viscosity
Cellmembraneviscositywasmeasured
Cell membrane viscosity was measured using
using AFM.
AFM.Figure
Figure4A,B
4A,Bshow
showthethephasephasecontrast
imagesobtainedusing
anopticalmicroscope.
AFM
wasperformed
toobserve
contrast images obtained using an optical microscope. AFM was performed to observethe
theareasurroundedsurrounded byby thethe dasheddashed line
line inarea
in Figure
Figure 4A,B.
4A,B. Figure
Figure 4C–J
4C–J show
showthetheAFM
AFMimages
imagesofof
the topography,
topography, amplitude,
amplitude, phase,
phase, andand lossloss tangent
tangent forfor dabigatrandabigatran etexilate
etexilate ((-)
the
−)and
and(+)(+)
groups,respectively.
The
AFM
amplitude
imagesindicatetheelasticity
component,andgroups, respectively. The AFM amplitude images indicate the elasticity component, andthethe AFM
phase
imagesindicate
the viscosity
component.
The tangent
loss tangent
estimated
AFM
phase
imagesindicate
the viscosity
component.
The loss
was wasestimated
from
from
the
amplitudeandphasevalue,
which
togetherindicatetheviscoelasticity
compo-ofthe amplitude and phase value, which together indicate the viscoelasticity component
nent
of Comparing
the cell. Comparing
Figurewith images
4F and in
Figure
4G4E,G
withare4H,
the stripe
the
cell.
Figure 4E–H,
the4Estripe
Figureclearerthan images
those inin
Figure4E,G
areclearer
than
those
in
Figure
4F,H.
The
loss
tangent
valuesofthe stripe
Figure 4F,H. The loss tangent values of the stripe structure in Figure 4H,J were greaterthanstructureincellFigure4H,J weregreater than
thoseof the elasticity.
cell membrane,
indicating
greaterthoseof themembrane,indicating
greatercytosolic
Therefore,
the dabigatrancytosolic(−elasticity.
Therefore,
dabigatran(-)structure
cells, whichmore clearly
showedetexilate) cells, whichmore theclearly
showedetexilate
the stripe(as observed
using
AFM),
the
stripestructure(asobserved
using
AFM),
were
more
elastic.
Thevaluesoftaninwere more elastic. The values of tan δ in Figure 4J are lower than those in Figure 4I.δThe
Figure
4J
are
lower
than
those
in
Figure4I.
The
mostfrequent
valuesofthelosstangent
most frequent values of the loss tangent in Figure 4G,H were estimated to be 1.1 and 0.25,
in Figure 4G,H
were4K,L).
estimated
1.1parameterand 0.25, respectively(Figure4K,L). Tan
is therespectively(FigureTan δtoisbe
theindicating theviscoelasticityofδthecell.
parameter
indicating
theviscoelasticity
of
the
cell.
Dabigatran
etexilatetreatment
Dabigatran etexilate treatment decreased the elasticity and increased the viscosity ofdethecreasedelasticity
viscosity ofetexilate-treatedthe cell. The valueof thetangent
cell.
Thethevalueof theandlossincreased
tangent thein dabigatran
cells
waslosslower
thanin dabigatran
etexilate-treatedcells was that
lower
than
that in non-treatedcells. Thispromoted
suggeststhatin non-treatedcells. This suggests
thedabigatran
etexilate treatment
thatviscosity.
the dabigatran etexilate treatment promoted cell viscosity.
cell

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7 of 13
7 of 13

Figure 4.4. The
The cellular
cellular membrane
membrane viscosity.
viscosity. Cells
Cells were
were treatedtreated without
without (upper)(upper) andand withwith (lower)(lower)Figuredabigatran
etexilate.
(A,B)
Phase
contrast
images;(C,D)
AFM
topography
images;(E,F)
AFM
amdabigatran etexilate. (A,B) Phase contrast images; (C,D) AFM topography images; (E,F) AFM
plitudeimages;
(G,H)
AFM
phase
images;(I,J)
loss
tangent
images;(K,L)
histogramsofthevaluesamplitude images; (G,H) AFM phase images; (I,J) loss tangent images; (K,L) histograms of the valuesof the loss tangent. The dashed lines in (A,B) indicate the AFM measurement region.
of the loss tangent. The dashed lines in (A,B) indicate the AFM measurement region.

3.5. Ascorbic Acid Suppresses the Cytotoxicity of Dabigatran Etexilate via t
mitROS Production
Cells 2021, 10, 2508

8 of 13

The cells were incubated with ascorbic acid to suppress the cytoto
etexilate. Compared to untreated cells, the ascorbic acid-treated cells re
3.5.
Acid Suppresses
Cytotoxicity
Dabigatran
Etexilate viainduced
the Inhibitionof dabigatra
ureAscorbic
5). Ascorbic
acidthecaninhibitofROS
generation
bymitROS Productionuredusing MitoSOX. Dabigatran etexilate induced intracellular ROS
The cells were incubated with ascorbic acid to suppress the cytotoxicity of dabigatran
Cells 2021, 10, x FOR PEER REVIEW
8 of 13was subsequently
inhibited
to treatment
withviableascorbic
etexilate.
Compared to untreatedcells, inthe response
ascorbic acid-treatedcells remained(Figure5). Ascorbic
acid can inhibit ROS
generation inducedby dabigatran
etexilate, using
Moreover,the mitochondrial
membranepotential
was observed
as measured using MitoSOX. Dabigatran etexilate induced intracellular ROS production,
3.5. Ascorbic Acid
Suppresses the Cytotoxicityof Dabigatran
Etexilate viafluorescence
the Inhibition ofrescence
microscope.
The
ratio
red/greenin the dwhich
was subsequently
inhibitedin response
tooftreatmentwith ascorbic acid (Figure 6A,B).
mitROS Production
Moreover,themitochondrial
membranepotential
using JC-1 undera fluorestreatedcells
cells
was
lower
than
thatwasinobserved
thetheuntreatedcells.
Moreove
wereTheincubatedascorbicfluorescenceacid to suppress
cytotoxicityetexilate-treatedof dabigatrancenceThe
microscope.
ratio ofwith
red/greenin thedabigatran
etexilate. Compared
untreated
the ascorbic
acid-treatedcellsacidremained
viable(Fig- than th
dabigatran
treatedwithascorbic
wasgreatercells
was lower etexilate
thantothat
incells
the cells,
untreated
cells.
Moreover,
this
ratio
in
the
dabigatranure 5). Ascorbic acid can inhibit ROS generation induced by dabigatran etexilate, as measetexilate
cells treatedwithsuggesting
ascorbic acid was
greaterthan that in etexilate-induced
the cells without ascorbicdecrea
outascorbic
acid,
that
dabigatran
ured using MitoSOX. Dabigatran etexilate induced intracellular ROS production, whichacid, suggesting that dabigatran etexilate-induced decrease in the mitochondrial membrane
drial
membranepotential
wasto suppressedby ascorbic
acid(Figure 7A
was subsequently
inhibitedin response
treatment with ascorbic
acid (Figure
6A,B).
potential was suppressed by ascorbic acid (Figure 7A,B).
Moreover, the mitochondrial membrane potential was observed using JC-1 under a fluorescence microscope. The ratio of red/green fluorescence in the dabigatran etexilatetreated cells was lower than that in the untreated cells. Moreover, this ratio in the
dabigatran etexilate cells treated with ascorbic acid was greater than that in the cells without ascorbic acid, suggesting that dabigatran etexilate-induced decrease in the mitochondrial membrane potential was suppressed by ascorbic acid (Figure 7A,B).

Figure5.
5. Theof ofdabigatran
etexilate.
Ascorbic
acid prevented
the dabigatranetexFigure
Thecytotoxicity
cytotoxicityeffecteffectdabigatran
etexilate.
Ascorbic
acid prevented
the dabigatranFigure5. Thecytotoxicity
ofmeans
dabigatran
Ascorbic
acid prevente
ilate-induced
cell cell
injury.
DataData
are expressedas± SD ±(n SD= 6).etexilate.
0.001.
etexilate-induced
injury.
areeffect
expressed
as means(n***= p6).< ***p<0.001.

ilate-induced cell injury. Data are expressed as means ± SD (n = 6). *** p < 0.00

Figure 6. The fluorescence intensity of MitoSOX. The cells were incubated in a medium containing 25 µM dabigatranFigure
6. The fluorescenceofincubated
MitoSOX. The
were
incubated inwas
a mediumcontaining
etexilate with or without 500µM ascorbic
acid. The intensity
cells werefor 6cells
h, thesupernatant
aspirated,
and25 μM dabigatran etexilate with or without 500 μM ascorbic acid. The cells were incubated for 6 h,
MitoSOX was added. The fluorescence intensity of MitoSOX was measured using fluorescence microscopy. (A) Fluorescent
the supernatant was aspirated, and MitoSOX was added. The fluorescence intensity of MitoSOX
microscopy utilized to assess wascellularuptakeusingof MitoSOX.
(B) Data
are expressed
as means microscopy± SD (n = 20).
The sampleismeasured
fluorescencemicroscopy.
(A) Fluorescentutilized
to assesssize
cellularthe number of cells in the analyzed
images.
Ex=
535–555nm
andEm=
570–625nm.
***p<0.001.
uptake of MitoSOX. (B) Data are expressed as means ± SD (n = 20). The sample size is the numberof cells in the analyzed images. Ex = 535–555 nm and Em = 570–625 nm. *** p < 0.001.

Figure 6. The fluorescence intensity of MitoSOX. The cells were incubated in
25 μM dabigatran etexilate with or without 500 μM ascorbic acid. The cells w

Cells 10,
2021,
10, 2508
Cells 2021,
x FOR
PEER REVIEW

9 of 139 of 13

Figure7. Thefluorescenceintensityof JC-1.
The cellscontaining
were incubatedin a medium
containing 25
Figure 7. The fluorescenceintensityof JC-1.
The cells wereincubatedin a medium
25 µM dabigatran
etexilateμM dabigatranwith or without
500 μM wasascorbic
acidandfor the6 h.cells
Afterincubation,with or without 500 µM ascorbic
acid for 6etexilate
h. After incubation,
the supernatant
aspiratedwere
incubated the superwas aspirated
and theintensity
cells werein a medium
μM JC-1. The fluoresin a medium containingnatant
2 µM JC-1.
The fluorescenceof incubatedJC-1 was measured
usingcontaining
fluorescence2 microscopy.
cenceintensityof JC-1
wasuptakemeasuredusing
fluorescencemicroscopy.
(A)(A) Fluorescent microscopy
utilized
to assesscellularof JC-1.
(B) Data
are expressed
as means ±SDFluorescent(n = 20). Themicroscopy
utilizedtoassesscellularuptake
of
JC-1.
(B)
Data
are
expressed
as
means
±
SD
(n = 20). The sample
sample size is the number of cells in the analyzed images. * p < 0.05, ** p < 0.01.
size is the number of cells in the analyzed images. *p < 0.05, **p < 0.01.

4. Discussion

4. Discussion
In this study, we investigated the roles of dabigatran etexilate and tartaric acid inorder
to elucidate
theinvestigatedmechanism underlying
dabigatrancytotoxicity.
We foundthat
In thisstudy, wethe roles ofdabigatran
etexilate andtartaric
acid indabigatran
etexilateinducedgreatercytotoxicity
innormalmucosalcells
thantartaricacid
order to elucidate the mechanism underlying dabigatran cytotoxicity. We found that
via inductionof mitROS
production.
alsomucosal
induced lipid
dabigatran
etexilateinducedgreater Dabigatrancytotoxicityetexilate
in normal
cellsperoxidation
than tartaric acidand altered the cellular viscoelasticity. The cytotoxicity, mitROS production, and decrease
via induction of mitROS production. Dabigatran etexilate also induced lipid peroxidationin the mitochondrial membrane potential with dabigatran etexilate were inhibited upon
and altered the cellular viscoelasticity. The cytotoxicity, mitROS production, and decreasecombined treatment with an antioxidant.
in the mitochondrial
membranedabigatran
etexilatewere inhibited
Dabigatran is anoral direct potential
thrombin with
inhibitor.
Idarucizumab
reverses
the effects upon
combinedtreatmentwithanantioxidant.
of dabigatran by binding to it with an affinity that is 350 times higher than that ofDabigatranis Dabigatranan oral direct
inhibitor. Idarucizumab
reverses
effects ofthrombin[22,23].
is anthrombin
effective anticoagulant
with a favorable
bleedingthe
profileinpatients
without
regularmonitoring.
However,GI
bleeding
isreportedasaside
effectindabigatran by binding to it with an affinity that is 350 times higher than that of thrombin
patients
treated with
dabigatran
Therefore,with
it is important
to identify
theprofile
mecha-in pa[22,23].
Dabigatran
is aneffective[24,25].
anticoagulanta favorable
bleeding
nism
underlying
dabigatran-induced
bleeding.
Dabigatran
etexilateandtartaricacid
tients without regular monitoring. However, GI bleeding is reported as a side are
effect in
the primary components of dabigatran. Dabigatran etexilate is absorbed at a low pH [26];patients
treated with dabigatran [24,25]. Therefore, it is important to identify the mechathus, a tartaric acid core is provided in dabigatran capsules [8]. Tartaric acid can create annism underlying dabigatran-induced bleeding. Dabigatran etexilate and tartaric acid are
acidic environment as it has pKa values of 2.98 and 4.34 [27]. Hence, it has been suggested
thethatprimary
componentsof dabigatran.
Dabigatran
etexilateis absorbed
at a low
pH [26];
dabigatrancytotoxicity
may be inducedby the tartaric
acidcore contained
withinthus,
tartaricwhichacid coreis provided
in dabigatrancapsules[8]. toTartaric
acid
can create antheacapsule,maintainsthe low-pH
environment
required
activate
dabigatranacidicenvironment
as
it
has
pKa
values
of
2.98
and
4.34
[27].
Hence,
ithas
beensuggested
etexilate. In this study, we compared the cytotoxicities of dabigatran etexilate and tartaricacid.
Tartaric acid
did not induce
damageat concentrations
10 mM within(data thethatdabigatrancytotoxicity
may cellular
be inducedby thetartaric acid lesscorethancontained
not shown).
cell viability
decreasedby 40% after treatment
with
100 µMdabigatrandabigatran etexicapsule,whichThe
maintainsthe low-pH
environment
required toactivate
etexilatethethein cytotoxicities
vitro results may
necessarily
indicateandthetartaric
in vivo acid.
late.
In this(Figure
study, 1).
weHowever,comparedof notdabigatran
etexilateresults.
At
least
in
this
study,
thecytotoxicity
ofdabigatran
etexilatewashigherthan
Tartaric acid did not induce cellular damage at concentrations less than 10 mM that(data notof tartaric acid. It is thought that dabigatran cytotoxicity may be induced by unconverted
shown). The cell viability decreased by 40% after treatment with 100 μM dabigatran etexdabigatran etexilate. To our knowledge, this is the first study to investigate this hypothesis.

ilate (Figure 1). However, the in vitro results may not necessarily indicate the in vivo results. At least in this study, the cytotoxicity of dabigatran etexilate was higher than that oftartaric acid. It is thought that dabigatran cytotoxicity may be induced by unconverted
dabigatran etexilate. To our knowledge, this is the first study to investigate this hypothe-

Cells 2021, 10, 2508

10 of 13

We evaluated the relationship between dabigatran and mitROS as a cytotoxic mechanism. We have previously reported the relationship between gastric mucosal injury
and clinical drugs. Nonsteroidal anti-inflammatory drugs (NSAIDs) cause GI complications such as ulcers and erosions. This is most likely due to cyclooxygenase inhibition,
prostaglandin deficiency, and neutrophil activation and infiltration, among other factors [28]. NSAIDs have also been reported to induce mitochondrial injury by reducing
the mitochondrial transmembrane potential (MTP) [29]. We reported that indomethacin
induces both MTP reduction and cellular apoptosis and that rebamipide exerts a protective
effect on mitochondrial membrane stability [30]. We further reported that bisphosphonate,
which is used for the treatment of postmenopausal osteoporosis, also induces injury via
mitROS production, which can be prevented by antioxidants [14,15]. Based on these results,
we concluded that the cytotoxicity of dabigatran etexilate may be strongly influenced by
mitROS. Indeed, the intracellular fluorescence intensities of MitoSOX and DPPP wereincreased upon treatment with dabigatran etexilate (Figures 2 and 3). Therefore, dabigatran
etexilate induced mitROS production and membrane lipid peroxidation.
Membrane lipids are composed of one polar head and two hydrophobic hydrocarbon
tails. Each tail is composed of saturated and poly-unsaturated fatty acyl chains. Lipid
peroxidation is induced by a ROS-mediated attack on lipids containing carbon–carbon
double bonds [31]. The fluidity of a cell membrane depends on its composition [32]. The
length and saturation of fatty acid tails significantly affect the cell membrane fluidity.
We have previously reported that promoting lipid peroxidation of cellular membranes
alters the fluidity [33]. To evaluate the cell membrane fluidity, we measured the cellular
membrane viscoelasticity using AFM. For a fluid mosaic model, the lateral movements anddistributions of membrane components are restrained by such factors as the membrane
domains, cytoskeletal interactions, extracellular matrix interactions, membrane protein
interactions, and lipid–lipid interactions [34]. These membrane components determine
the physical properties of the cells, such as viscoelasticity. Therefore, by measuring theviscoelasticity of cells, a correlation with membrane fluidity can be obtained. However,
the methods for measuring the membrane fluidity without affecting the properties of thecell membrane are limited. Atomic force microscopy (AFM) has shown many successes inmeasuring the shape and mechanical properties of cells without affecting the cell membrane, such as the state of cytoskeletons, the elastic cytoplasm, and the viscoelasticity ofcells [19,35–38]. In this study, the viscoelastic modulus of cells was obtained using theloss-tangent method, which is one of the AFM measurement methods. The viscoelastic loss
tangent tan δ of a material is a dimensionless parameter that measures the ratio of energy
dissipated to energy stored in one cycle of a periodic deformation, and it is the parameterfor the viscoelasticity of a cell.
In this study, we focused on the relationship between mitROS production and cellular
membrane viscoelasticity using AFM. Dabigatran etexilate can enhance mtROS production(Figure 2). An increase in mitROS production accelerates the changes in cell membrane
fluidity and makes the stripe structure more elastic than the cell membrane, as shown in
Figure 4C,D. The stripe structure is composed of actin filaments. As shown in Figure 4D,
the number of actin filaments increased after dabigatran treatment. Since actin filaments are
one of the factors that determine the elastic moduli of cells [39], their reduction reduces theelastic moduli. The values of tan δ decreased after dabigatran treatment, indicating that theviscosity of cells increased with dabigatran treatment. In particular, dabigatran treatment
reduced the elastic modulus and increased the viscosity to change the viscoelasticity of
the cells. Moreover, dabigatran etexilate is involved in cellular lipid peroxidation. Lipid
peroxidation induces structural transitions in membranes and affects their orientationand fluidity [40]. Dabigatran etexilate altered the cellular viscoelasticity, actin formation,and lipid peroxidation. The lipid peroxidation in cellular membrane is promoted by ROS
production. In this study, dabigatran etexilate increased the mitROS production. Taken
together, dabigatran etexilate altered the properties of the cell membrane. Dabigatran etexi-

Cells 2021, 10, 2508

11 of 13late also enhanced the mitROS production; thus, we considered that the alteration of theproperties of the cell membrane may have been induced by increasing mitROS production.
We demonstrated that ascorbic acid could attenuate the cytotoxicity of dabigatran etexilate. Ascorbic acid is a typical antioxidant and it was found to prevent cellular injury and
mitROS production induced by dabigatran etexilate (Figures 5 and 6). The mitochondrial
membrane potential was evaluated using JC-1, which is a cationic dye that accumulates
in energized mitochondria. In cells with low MTP, JC-1 remains in a monomeric form
that emits green fluorescence. In healthy cells with high MTP concentrations, JC-1 forms
complexes known as J-aggregates with intense red fluorescence. A higher ratio of red togreen fluorescence indicates greater mitochondrial membrane polarization [41]. Ascorbic
acid can prevent the decrease in the mitochondrial membrane potential by dabigatran
etexilate (Figure 7). It has been reported that the decrease in the mitochondrial membrane potential is the phenomena associated with apoptosis [42,43]. Ascorbic acid not
only prevents mitochondrial fission but also decreases oxidative stress and apoptosis [44].
Therefore, increased mitROS production can accelerate cytotoxicity caused by dabigatran
etexilate, especially in its contribution to apoptosis signaling. Ascorbic acid inhibits mitROS
production and thereby prevents dabigatran etexilate cytotoxicity.
5. Conclusions
Dabigatran etexilate exhibited greater cytotoxicity than tartaric acid by inducing
mitROS production. Therefore, the lowering of mitROS production by antioxidants, such as
ascorbic acid, can help attenuate the cytotoxicity of dabigatran etexilate. We confirmed thatdabigatran etexilate was responsible for dabigatran-mediated injury and concluded thatdabigatran cytotoxicity can be inhibited by treatment with the antioxidant ascorbic acid.
Author Contributions: H.K., A.T., H.S. and H.M. designed the experiments. H.K., A.T. and H.M.
wrote the manuscript. H.K., A.T. and H.S. performed and analyzed the experiments. All authors
have read and agreed to the published version of the manuscript.
Funding: This study was partially supported by JSPS Grant-in-Aid for Scientific Research JP17H06088,
JP19H02591 and JP19K16854 and CREST (JPMJCR1875).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest associated with this manuscript.

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