International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669

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International Journal of Pediatric Otorhinolaryngology

journal homepage: www.elsevier.com/locate/ijporl

Protective effect of trimetazidine on amikacin-induced ototoxicity

in rats
Fadlullah Aksoy a, Remzi Dogan a,*, Orhan Ozturan a, Sabri Baki Eren a, Bayram Veyseller a,
Alev Pektas b, Önder Hüseyinbas c
a
Bezmialem Vakif University, Department of Otorhinolaryngology, Fatih, Istanbul, Turkey
b
Bezmialem Vakif University, Faculty of Health Sciences, Department of Audiology, Fatih, Istanbul, Turkey
c
Bezmialem Vakif University, Research Center, Fatih, Istanbul, Turkey

A R T I C L E I N F O A B S T R A C T

Article history: Objective: Aminoglycoside antibiotics are known to have ototoxic effects and may induce sensorineural
Received 22 September 2013 hearing loss. This study investigated the protective effect of trimetazidine, which has antioxidant and
Received in revised form 20 January 2014 cytoprotective properties, against amikacin ototoxicity.
Accepted 22 January 2014
Methods: Thirty-two male rats were divided into four groups – amikacin, amikacin + trimetazidine,
Available online 5 February 2014
trimetazidine, and control groups. Trimetazidine, 10 mg/kg per day, was given for 14 days by oral gavage.
Amikacin, 600 mg/kg per day, was also given for 14 days, by the intramuscular route. Distortion product
Keywords:
otoacoustic emission (DPOAE) and auditory brainstem response (ABR) tests were applied to the rats for
ABR
Amikacin
hearing assessment. At the termination of the study, the biochemical parameters were calculated to
DPOAE evaluate the oxidative status.
Oxidative status Results: The DPOAE values of the amikacin group were significantly lower on the 7th and 14th days than
Trimetazidine those of the trimetazidine + amikacin group and there was an increase in the ABR thresholds. The ABR
thresholds for the amikacin group on the 7th and 14th days were significantly higher than the levels on
the first day of the study, while there was no significant increase in those values in the
trimetazidine + amikacin group. The total oxidant status (TOS) and oxidant status index (OSI) values
of the amikacin group were significantly higher than those of the trimetazidine + amikacin group. The
total antioxidant status (TAS) values of the amikacin group were lower than those of the
trimetazidine + amikacin group.
Conclusions: The audiologic tests and biochemical parameters investigated in this study both point to
the protective effect of trimetazidine against amikacin-induced ototoxicity.
ß 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction AK is a semi-synthetic AG, produced by the acetylation of

Ototoxicity is a clinical condition, usually brought about by the
detrimental effects of some chemical agent on the auditory and
balance functions of the ear [1]. More than 130 agents are known to
have ototoxic effects, with aminoglycoside (AG) and macrolide
antibiotics, loop diuretics, NSAIDs, antineoplastic and antimalarial
drugs taking the lead [1].
While amikacin (AK), kanamycin and neomycin cause damage
primarily to the cochlea, streptomycin and gentamicin primarily
cause vestibular damage [2]. Cochlear damage may lead to
permanent hearing loss, and vestibular damage may lead to
vertigo, ataxia and/or nystagmus [3].
* Corresponding author at: Bezmialem Vakif University, Medical Faculty,
Department of Otorhinolaryngology, Fatih, Istanbul, Turkey.
Tel.: +90 505 7915844; fax: +90 212 533 2326.
E-mail address: dr.remzidogan@gmail.com (R. Dogan).
kanamycin A. The specific properties of its structure make it
resistant to bacterial enzymes which can inactivate natural AGs
such as gentamicin, kanamycin and tobramycin, thus it has the
broadest spectrum among all AGs. AK is a frequently preferred
antibiotic due to its rapid action, broad spectrum, lower bacterial
resistance, its synergetic effects with beta-lactam antibiotics, and
its lower cost [4]. However, 10–80% of patients who are treated
with AK are reported to suffer from its ototoxic effects. The hearing
loss is typically neurosensorial, nonsyndromic, bilateral, progres-
sive, and is a high-frequency hearing loss [5]. Since the hair cells of
the cochlea cannot regenerate, the hearing loss is irreversible [6].
AK ototoxicity is brought about by the drug’s excitotoxic effects,
caused by impairment of mitochondrial protein synthesis and
overactivation of glutamatergic receptors (N-methyl-D-aspartate),
which increase the formation of free radicals and induce apoptosis
[7,8]. There have been numerous studies of oxidative stress leading
to sensory neural hearing loss [9–11]. Experimental studies have
shown that antioxidant agents may prevent AG ototoxicity [12–16].

0165-5876/$ – see front matter ß 2014 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijporl.2014.01.031
664 F. Aksoy et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669
Trimetazidine (TRM) is a selective mitochondrial 3-ketoacyl
coenzyme A thiolase (3-KAT) inhibitor. Thus, it decreases free
fatty acid (FFA) metabolism and regulates cardiac metabolism. It
controls oxidative stress, preserves mitochondrial respiration in
ischemia–reperfusion injury, and prevents cardiac ischemia
[17,18]. TRM has also been shown to have gastroprotective,
hepatoprotective, anti-inflammatory, antinociceptive and
anti-apoptotic properties [18,19]. Previous studies have shown
that TRM’s cytoprotective effects are brought about by
preventing injury in neurosensorial tissue secondary to
overstimulation of the cochleovestibular system by glutamate,
and by its antioxidant effects [20,21]. In experimental
animal studies, it was reported that the ototoxic effect
induced by gentamicin and neomycin is prevented by TRM
[22,23].
The fields of use of TRM in otorhinolaryngology include the
reduction in vertigo duration and frequency [24], treatment of
Ménière’s disease related cochleovestibular conditions [25],
correction of inner ear ischemia [26], correction of isolated
tinnitus [27], and correction of hypoacusis [28].
Clinical studies report that TRM has excellent tolerability
[21]. Oxidative stress means disruption of the balance between
pro-oxidants and antioxidants in favor of the pro-oxidants [29].
Measuring the amount of different antioxidants individually is
difficult, requires a significant amount of time, increases
laboratory burden, is high cost and is a complicated technique.
Furthermore, there will be complicated interactions among
different antioxidants in the serum, therefore, these measure-
ments may not be sufficiently objective. The recently developed
method of measuring the total antioxidant status (TAS) enables
all antioxidants to be recorded at a very low cost in a short time
as a single parameter by a simple measurement in serum
[30,31]. Similarly, there are no practical methods for the
measurement of individual pro-oxidant molecules, but again,
a single parameter, the total oxidant status (TOS), can be
measured in serum instead [32].
The purpose of this study was to investigate the protective
effect of TRM, which has antioxidant and cytoprotective properties,
against AK ototoxicity.
2. Materials and methods
2.1. Animals
After obtaining permission for experimental studies from the
Local Ethics Committee (2011/39), 32 healthy female Wistar
Albino rats weighing 200–240 g were included in the study. Those
rats which had a negative Preyer response (when clapping, a rapid
movement clearly seen in the bodies of animals indicates a positive
Preyer reflex) were examined, and/or those which had ear
pathologies (cerumen, otitis media with effusion, acute otitis
media, etc.) seen in endoscopic ear examinations were not
included in the study.
The rats were placed in an environment which was illuminated
for 12 h and dark for 12 h, had a temperature of 21  1 8C, with free
access to food and water, and with a background noise level of under
50 dB. The animals were treated in accordance with the Guide for the
Care and Use of Laboratory Animals [33].
2.2. Anesthesia
Before distortion product otoacoustic emissions (DPOAE) and
auditory brainstem responses (ABR) were recorded, all of the rats
were anesthetized intraperitoneally with ketamine hydrochloride
(45 mg/kg) and xylazine (5 mg/kg).
2.3. Experimental design
The 32 rats included in the study were divided into four
groups, with eight rats in each group: Group 1 (AK), Group 2
(AK + TRM), Group 3 (TRM), and Group 4 (control group, no
active treatment (NAT)). TRM was given to Groups 2 and 3 for 14
days by oral gavage, at a dose of 10 mg/kg per day. Rats in
Groups 1 and 2 were administered daily intramuscular injec-
tions of AK for 14 days, at a dose of 600 mg/kg per day. Rats in
Group 4 were given daily intraperitoneal shots of 1 ml saline
solution for 14 days.
At the very beginning of the study, and also on the 7th and 14th
days, DPOAE and ABR tests were performed on all of the rats. On
the 14th day, intracardiac blood samples were drawn from all of
the rats to calculate the biochemical parameters.
2.4. DPOAE measurements
The GSI Audera device was used for DPOAE measurements in
the assessment of the rats’ peripheral hearing system. The smallest
sized rubber tip of the tympanometer probe was used for the tests.
The emissions were measured in General Diagnostic mode, both as
distortion product diagrams (DPgram) and as input/output (I/O)
measurements. Otoacoustic emissions were measured using
stimuli of different frequencies and intensities. The intensity level
of the primary stimuli was adjusted to 65 dB (L1 = L2). Two
different frequencies (f1 and f2) were chosen with a ratio of f2/
f1 = 1:10.
DPgram measurements were taken at 3000, 4008, 5004, 6000,
6996, 8004, 9012, 10008, 11004 and 12000 Hz frequencies.
DPOAEs 3 dB over the 2f1–f2 frequency noise intensity during
measurements were accepted as positive.
2.5. ABR measurements
ABR measurements were carried out in a silent room, with a
Viasys Medelec Synergy instrument, using subcutaneous needle
electrodes (Technomed Europe). Stimuli were delivered in
alternating polarities with ER-3A insert earphones, using 8 kHz
tone-burst stimuli. The filter was set at 30–1500 Hz bandwidth, the
repetition rate was set at 21/s, and the time window was set as
25 ms; 1024 samples were obtained for signal averaging. Stimuli
were delivered at an intensity level of 80 dB nHL, and were reduced
in 20 dB steps until the intensity level approached threshold
values. Nearing the threshold, intensity steps were reduced to
10 dB, and the threshold value was determined. A minimum of two
tracks were generated for each measurement to check for
reproducibility, and for confirmation of the threshold value. The
ABR threshold was defined as the minimum intensity level where
an ABR wave III was observed.
2.6. TAS, TOS, and OSI measurements
Measuring the total antioxidant status (TAS), the combined
activity of all antioxidants, provides an evaluation of the overall
antioxidant status [30]. Total oxidant status (TOS) is an indicator of
the overall oxidant status of the patients [32]. The oxidant status
index (OSI) is the ratio of TOS/TAS, and is a means to calculate
oxidative stress in the body – comparing TAS to TOS is thought to
be a better indicator of the overall oxidative stress. To measure
these parameters, blood samples taken from all rats in all groups
were centrifuged at 3000 rpm for 15 min, the serum was separated
and stored at 80 8C. After all samples had been prepared in this
way, TAS and TOS values were calculated with the relevant kit (Rel
Assay Diagnostics), and OSI values were calculated with the
relevant formula (OSI: TOS/TAS  100).
 F. Aksoy et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669
Fig. 1. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 1 (AK) at different time points.
2.7. Statistical analysis
Statistical analysis was carried out using the Statistical Package
for the Social Sciences version 13.0 software for Windows (SPSS
Inc., Chicago, IL, USA). All quantitative variables were estimated
using measures of central location (i.e. mean and median) and
measures of dispersion (i.e. standard deviation (SD)). Data
normality was checked using the Kolmogorov–Smirnov tests of
normality.
2.7.1. Assessment of DPOAE and ABR results
For the comparison within group, the Repeated ANOVA test was
applied (the difference within group was considered to be
statistically significant if p < 0.05). To determine the days between
which there were differences, the Bonferroni test was adminis-
tered as a post hoc test. Since this was a multiple comparison, the
Bonferroni correction was applied and p < 0.016 was accepted as
statistically significant.
For the comparison between groups, the one-way analysis of
variance (ANOVA) test was applied (the difference between groups
was considered to be statistically significant if p < 0.05). Tukey’s
HSD was administered as a post hoc test to identify between-group
differences. Since this was a multiple comparison, the Bonferroni
correction was applied and p < 0.008 was accepted as statistically
significant.
2.7.2. Assessment of biochemical results
One-way of analysis (ANOVA) was used for comparing the data
between groups. Tukey’s HSD was administered as a post hoc test.
Since this was a multiple comparison, the Bonferroni correction
was applied and p < 0.008 was accepted as statistically significant.
3. Results
3.1. DPOAE
There were statistically significant differences between the
baseline values of DPOAE and the 7th and 14th day values in Group
1 (AK) (n = 8, 16 ears) (p < 0.016) (Fig. 1). There were no
statistically significant differences between the baseline values
of DPOAE and the 7th and 14th day values in Groups 2 (AK + TRM),
665
3 (TRM) and 4 (NAT) (n = 8 and 16 ears in each group) (p > 0.016)
(Figs. 2–4).
Comparisons between groups showed that there were no
statistically significant differences in the baseline values of DPOAE
(p < 0.05), while on the 7th and 14th days of the study, there were
statistically significant differences between Group 1 (AK) and the
other three groups (p < 0.008).
3.2. ABR
A comparison of ABR thresholds of all the groups is included in
Table 1.
In Group 1 (AK), there were statistically significant differences
between the baseline ABR threshold values and those of the 7th
and 14th days, and also between the ABR threshold values of the
7th and 14th days (p < 0.016) (Table 1) (Fig. 5).
When the ABR threshold values of Groups 2 (AK + TRM), 3
(TRM) and 4 (NAT) were compared within each group, no
statistically significant differences were found between the
baseline, 7th day and 14th day values (p > 0.05) (Table 1)
(Fig. 5).
In comparing the ABR threshold values between groups, while
no statistically significant differences were found between groups
for baseline values (p > 0.05), there were statistically significant
differences in those values on days 7 and 15 (p = 0.002 and
p = 0.001, respectively) (Table 1) (Fig. 5).
The ABR threshold values on days 7 and 15 did not show any
statistically significant difference between Groups 2 (AK + TRM), 3
(TRM) and 4 (NAT) (p > 0.05). There were statistically significant
differences between the 7th and 14th day ABR threshold values of
Group 1 (AK) (p < 0.05) (Table 1) (Fig. 5).
3.3. Biochemical parameters
3.3.1. TOS (total oxidant status)
The highest TOS values were measured in Group 1 (AK)
(7.19  0.68 mmol H2O2 equiv./L) (Table 2). The TOS values mea-
sured in Groups 2 (AK + TRM), 3 (TRM) and 4 (NAT) were
significantly lower than those in Group 1 (p < 0.08). TOS values
in Groups 2 (AK + TRM), and 3 (TRM) were lower than those in
Group 4 (NAT), yet the difference was not statistically significant
(p > 0.05) (Table 2).
666 F. Aksoy et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669

Fig. 2. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 2 (AK + TRM) at different time points.

Fig. 3. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 3 (TRM) at different time points.

Fig. 4. Variations in amplitudes of distortion product otoacoustic emissions (DPOAEs) in Group 4 (control group, no active treatment (NAT)) at different time points.
 F. Aksoy et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669
Table 1
Comparison of ABR thresholds within groups and between groups.
3.3.2. TAS (total antioxidant status)
The highest TAS values were encountered in Group 3 (TRM)
(1.91  0.079 mmol Trolox equiv./L), and the lowest TAS values were
found in Group 1 (AK) (1.43  0.14 mmol Trolox equiv./L) (Table 2).
There was a statistically significant difference between TAS values in
Groups 1 and 3 (p < 0.08). TAS values in Groups 2 (AK + TRM) and 4
(NAT) were higher than those in Group 1 (AK) but the difference was
not statistically significant (p > 0.08) (Table 2).
3.3.3. OSI (oxidant status index)
Group 1 (AK) had the highest OSI values (0.047  0.013)
(Table 2). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were
statistically significantly lower than those in Group 1 (AK) (p < 0.08)
(Table 2). OSI values in Groups 2 (AK + TRM) and 3 (TRM) were also
statistically significantly lower than those in the control group (Group
4) (p < 0.08).
Fig. 5. Variations in amplitudes of ABR threshold values in all groups at different time points.
667
4. Discussion
Among other agents, AG antibiotics are the most frequently
used drugs with ototoxic properties [34].
Amikacin is prescribed, especially to children, for febrile
neutropenia [35], prophylaxis, sepsis, meningitis, bacteremia
[36], urinary infections [37], respiratory tract infections [38] and
particularly for microorganisms that are resistant to other
aminoglycoside antibiotics [39]. We aimed to use AK in this study
since it has such a wide degree of effect.
The ear injury caused by AK begins at the base of the cochlea
and progresses toward the apex. The injury can progress even
further and may damage the stria vascularis and the 8th nerve [40].
The specific injury is brought about through free oxygen radicals
generated by AK, and their interaction with phospholipids,
membrane proteins and DNA, which results in irreversible damage
668 F. Aksoy et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669
Table 2
Biochemical parameters (mean  standard deviation).
to the outer hair cells, impairing their function and finally leading
them to apoptose [6].
Several agents have recently been tested to prevent ototoxicity
caused by AK [11,13,41–47]. Even though there has been an
increase in research on this topic in the last 10 years, there are still
no products that have entered general use.
TRM is being used for the treatment of cochleovestibular
disturbances as the drug has cytoprotective properties and is an
inhibitor of oxygen radicals [48].
AK damages the outer hair cells beginning from the basal region
of the cochlea, and as it progresses to the middle and apical regions,
lower frequencies are affected; this may lead to permanent
damage in speech discrimination scores (SDS) [41]. The damage to
outer hair cells can be evaluated by DPOAE testing [24]. In our
study, rats that were given AK had significantly lower DPOAE
values on the 7th and 14th days compared to their baseline values
(p < 0.016) (Fig. 1). This finding is indicative of AK’s deleterious
effect on outer hair cells. Also, the finding that there was a
significant difference in DPOAE values of the TRM + AK group
compared with the AK group on days 7 and 15 (p < 0.008) is
indicative of the protective effect that TRM exerts on AK
ototoxicity.
AK injury to the ear may progress to disturb the 8th nerve. ABR
testing is an objective method for evaluating the patency of the
auditory pathways proximal to the 8th nerve and the 8th nerve
itself [49]. In our study, the 7th and 14th day ABR thresholds were
significantly higher than the baseline values in the AK group
(p < 0.016) (Table 1). This finding demonstrates the toxic effect of
AK on auditory pathways proximal to the cochlea. We have not
encountered any clinical signs related to the vestibulotoxic effect.
We believe that we did not see any vestibulotoxic effects in the
experimental process since AK is primarily cochleotoxic. If we had
continued to administer AK, we would probably have encountered
potentially vestibulotoxic effects at later stages.
The 7th and 14th day ABR values showed a significant
difference among the TRM + AK and AK groups (p < 0.008)
(Fig. 5); this finding is indicative of the protective effect of TRM
against AK ototoxicity. However, a lack of histologic examination,
preferably as a histocochleogram under electron microscopy, is a
shortcoming of this study.
AK-induced free oxygen molecules cause damage to the inner
ear [50]. TRM inhibits the production of free oxygen radicals, thus
decreasing membrane lipid peroxidation levels [51]. Previous
studies have reported a significant correlation between AK-
induced hearing loss and antioxidant enzyme activity [52]. In this
study, we used objective and sensitive biochemical parameters
that measured the total oxidative/antioxidative balance. No other
study in the medical literature has so far evaluated the ototoxic
effects of AK biochemically, investigating the TAS, TOS and OSI
values. The finding that the TOS values for the TRM + AK group
were significantly lower than those of the AK group (p < 0.008)
(Table 2) indicates that TRM decreases the oxidative stress caused
by AK.
TAS values in the TRM group were significantly higher than
those in the AK group (p < 0.008); this finding supports previous
reports that TRM increases antioxidant activity. TAS values in the
TRM + AK group were higher than those in the AK group, but no
statistically significant difference was observed between these two
groups (p > 0.008) (Table 2). These findings show that TRM by
itself does increase antioxidant activity strongly, but since AK
increases oxidative stress, TAS values in the TRM + AK group were
lower than those in the TRM group. Further studies are needed to
see if an increase in TRM dosage may overcome this situation.
OSI values in the AK group were higher than in all the other
groups (0.047  0.005). OSI values in Groups 2 (AK + TRM) and 3
(TRM) were significantly lower than those in Group 4 (control)
(p < 0.008) (Table 2). Various studies have reported that AGs
increased oxidative stress, thus causing a toxic effect [12–15]. Our
study is the first to introduce a correlation between OSI and AK.
AK blocks the calcium-activated potassium channels in the
vestibular system [15]. AK acts like an N-methyl-D-aspartate (NMDA)
agonist which is a subtype of glutamate receptors, and has cytotoxic
effects [15]. TRM inhibits excess accumulation of Na+ and Ca2+ inside
cells and prevents K+ escape, which results in a decrease in
intracellular edema [53]. TRM exerts its cytoprotective effects
through preventing the overstimulation of glutamate, thus prevent-
ing its detrimental effects on neurosensorial tissues [48]. These data
also support the protective effect of TRM against AK ototoxicity.
It has been demonstrated that TRM plays a role in protecting
against the ototoxicity of gentamicin and neomycin as a result of
its antioxidant and cytoprotective effects [22,23]. The study we
conducted demonstrated that TRM was effective against AK
ototoxicity and this finding is in line with the literature. Judging
by these findings, we are of the opinion that TRM may be effective
in the treatment of chemicals, acoustic traumas, and autoimmune
events, which may have an ototoxic effect on the inner ear owing to
its antioxidant and cytoprotective effects.
Currently, there is no drug clinically in use for preventing AK-
induced ototoxicity. Our study experimentally demonstrated that
TRM could prevent AK ototoxicity. Large prospective randomized
studies are needed to confirm the beneficial effects of TRM in this
regard and before implementing TRM in routine clinical usage.
 F. Aksoy et al. / International Journal of Pediatric Otorhinolaryngology 78 (2014) 663–669
5. Conclusion
Numerous agents have been studied previously in the search for
protection against AK ototoxicity but TRM was not one of them.
This study is the first to investigate TRM’s otoprotective effects,
and shows that TRM does exert such an effect. However, additional
electron microscopic studies are needed to determine and confirm
the relevant histopathologic changes taking place. Since AK is a
drug with a high tolerability and minimum side effects, in the light
of the findings of this study, it may be beneficial to add TRM to the
treatment protocols of AK-prescribed patients.
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