This journal is c The Royal Society of Chemistry 2011Integr. Biol., 2011, 3,879–886879
Citethis: Integr. Biol.,2011,3,879–886
Intracellular mechanisms of aminoglycoside-induced cytotoxicity
Takatoshi Karasawa* and Peter S. Steyger
Received 8th April 2011, Accepted 4th July 2011
Since introduction into clinical practice over 60 years ago, aminoglycoside antibiotics remain
important drugs in the treatment of bacterial infections, cystic fibrosis and tuberculosis. However,
the ototoxic and nephrotoxic properties of these drugs are still a major clinical problem. Recent
advances in molecular biology and biochemistry have begun to uncover the intracellular actions
of aminoglycosides that lead to cytotoxicity. In this review, we discuss intracellular binding
targets of aminoglycosides, highlighting specific aminoglycoside-binding proteins (HSP73,
calreticulin and CLIMP-63) and their potential for triggering caspases and Bcl-2 signalling
cascades that are involved in aminoglycoside-induced cytotoxicity. We also discuss potential
strategies to reduce aminoglycoside cytotoxicity, which are necessary for greater bactericidal
efficacy during aminoglycoside pharmacotherapy.
Aminoglycoside antibiotics are among the most commonly-used
antibiotics world-wide,1and are highly effective in treating
life-threatening Gram-negative bacterial infections, such as
meningitis and bacterial sepsis in infants.2–4In mammals, amino-
glycosides are both nephrotoxic and ototoxic. Nephrotoxicity
results in increased morbidity during and after treatment, and can
cause acute kidney failure. After systemic delivery, amino-
glycosides are primarily localized in epithelial cells lining the
proximal tubules of the nephron. Distal tubule cells also take up
aminoglycosides, but survive at drug concentrations that kill
proximal tubule cells in vitro.5,6In vivo, aminoglycosides disrupt
distal tubule function by reversibly blocking luminal cation
channels, leading to cation-wasting in urine.7,8Renal cytotoxicity
is reversible due to proximal tubule epithelial cell proliferation.9,10
Aminoglycoside-induced ototoxicity inmammals isfrequently
permanent as these drugs can kill inner ear sensory hair cells
that cannot be spontaneously regenerated following hair cell
death.11,12Within the cochlea, aminoglycosides are prefer-
entially localized in outer hair cells (OHCs) at the base of
the cochlea, and hair cell death initially occurs in basal
OHCs, and extends to inner hair cells (IHCs) and to more
apical cochlear hair cells with increasing total dose.13,14
Aminoglycosides are also localized in stria vascularis and
spiral ligament fibrocytes of the cochlear lateral wall, spiral
ganglion neurons, and in supporting cells within the organ
of Corti.15–18Aminoglycosides also induce morphological
changes in the stria vascularis, decreasing its volume and
altering its structure.19,20
Several aminoglycosides are essential in clinical practice
(Fig. 1). Gentamicin is administered systemically in intensive
care units for prophylaxis in pre-term infants, and topically for
major burn case. Tobramycin is preferentially used for treating
Pseudomonas aeruginosa-induced pneumonia. Amikacin is
most often used for treating severe, hospital-acquired
infections with multidrug resistant Gram-negative bacteria.
Oregon Hearing Research Center, Oregon Health & Science
University, 3181 SW Sam Jackson Park Road, Portland, Oregon
97239, USA. E-mail: firstname.lastname@example.org; Fax: +1 503-494-5656;
Tel: +1 503-494-2373
Insight, innovation, integration
We present a review of aminoglycoside toxicity focusing on
intracellular binding targets and downstream effects. A better
understanding of the molecular mechanisms involved has
approaches such as drug conjugation to agarose beads or
fluorophores, and zebrafish hair cell death assays. Affinity
chromatography or pull-down assays using gentamicin–agarose
conjugates have identified several aminoglycoside-binding
proteins. Zebrafish neuromast hair cells have become a primary
model system to better understand intracellular pathways of cell
death signaling induced by ototoxic drugs. Recent data using
these methods give new insights into how aminoglycosides
induce cytotoxicity. With increasing knowledge of intracellular
drug binding targets and downstream effects, we can now begin
to develop clinical strategies to reduce aminoglycoside-induced
ototoxicity and nephrotoxicity.
Dynamic Article Links
880 Integr. Biol., 2011, 3,879–886This journal is c The Royal Society of Chemistry 2011
Gentamicin and tobramycin are thought to be preferentially
vestibulotoxic, i.e. inducing hair cell death in the balance
(vestibular) end-organs of the saccule, utricle, and the three
ampullae of the semi-circular canals. Amikacin, neomycin
and kanamycin are preferentially cochleotoxic.21Clinically,
gentamicin, tobramycin and amikacin are most frequently
prescribed, while streptomycin remains important for treating
tuberculosis, despite its severe ototoxicity.22
The bactericidal effects of aminoglycosides are largely due
to inhibition and/or mis-translation during protein synthesis.23,24
In mammalian cells, aminoglycoside cytotoxicity occurs via
several mechanisms (Fig. 2), including cytochrome c release
from mitochondria, activation of caspase-9 and caspase-3,
generation of toxic levels of reactive oxygen species (ROS),
activation of c-Jun N-terminal kinases (JNKs), and protein
cleavage by calpains.25–29To exert these intracellular pheno-
mena, aminoglycosides must first enter cells, typically by
endocytosis or cation channel permeation.30–33Endocytosis
transports aminoglycosides to the endoplasmic reticulum (ER)
and lysosomes. In lysosomes, aminoglycosides induce the
release of cathepsins (lysosomal peptidases) and/or lysosomal
rupture, either of which leads to cell death.32,34,35Amino-
glycoside permeation through non-selective cation channels
into the cytosol can induce a wide range of drug–target
Fig. 1Structures of several aminoglycoside antibiotics. Chirality is indicated by the Natta projection method.
Takatoshi Karasawa received
his PhD in Neuroscience in
2002 from Yale University,
USA. Currently, he is Senior
ototoxicity induced by amino-
glycosides and platinum-based
anti-cancer drugs, identifying
drug-binding proteins that are
involved in ototoxicity.
Peter S. Steyger
Peter S. Steyger received his
PhD in Cochlear Anatomy in
1991 from Keele University,
UK. He is Associate Professor
of Otolaryngology—Head &
Health & Science University,
and is also Scientific Director
of the Deafness Research Foun-
dation. The main focus of his
identify and then inhibit the
blood–labyrinth barrier into the
cochlear fluids and hair cells.
This journal is c The Royal Society of Chemistry 2011Integr. Biol., 2011, 3,879–886881
interactions. In this review, we will focus on the intracellular
binding targets of aminoglycosides, and their subsequent effect
on physiological functions and downstream targets, with an
emphasis on ototoxicity.
It has been suggested that aminoglycosides bind to ferric iron
(FeIII) and generate FeII–aminoglycoside complexes in the
cytosol.36,37This redox-active complex catalyzes the formation
of reactive oxygen species (ROS) from molecular oxygen,
using arachidonic acid as an electron donor.37,38This can lead
to excessive cytosolic production of ROS that induces
apoptosis signalling cascades (Fig. 2). Toxic levels of ROS
damage cells by triggering various cell death mechanisms,
including caspase-dependent and independent apoptosis, and
necrosis.39,40Apoptotic signals also induce the release of
mitochondrial ROS into the cytosol, further increasing cyto-
solic ROS levels.41–43Animals that overexpress superoxide
dismutase, a key anti-oxidative enzyme, are more resistant to
aminoglycoside-induced ototoxicity compared to wild-type
animals,44supporting the role of ROS in aminoglycoside-
induced ototoxicity. Although it is not clear how ROS induce
multiple cell death signalling cascades, it is well-established
that ROS activate c-Jun N-terminal kinases (JNK), which in
turn induces apoptosis.45Inhibition of the JNK pathway
promotes acute hair cell survival during treatment with
ototoxic levels of aminoglycosides.28,46,47
located in the cytosolic face of eukaryotic cell membranes.
Phosphoinositides are important second messengers in intra-
cellular signal transduction pathways, and are a source of
arachidonic acid.48Aminoglycoside binding to phosphoino-
sitides induces the release of arachidonic acid, which acts as an
electron donor in FeII–aminoglycoside complex-mediated
ROS formation, as discussed above.37,38
Interactions between the cationic aminoglycosides and
phosphoinositides have long been considered a major compo-
nent of aminoglycoside-induced ototoxicity.49There is a good
correlation between the decline of OHC receptor potential in
response to acoustic stimulation during cochlear perfusion of
aminoglycosides and the binding affinity of aminoglycosides to
phosphoinositides.50A deuterium-NMR study confirmed this
correlation, and proposed that aminoglycosides sequester
drug-bound phosphoinositides,52–54which are cytotoxic and
may be a major cause of ototoxicity.55,56A recent study
postulated that aminoglycosides deplete cytoplasmic levels of
free phosphoinositides that regulate KCNQ4 channel activity,
resulting in the inhibition of potassium efflux necessary for
cochlear sensory function and OHC survival.57,58
Phosphoinositides are ubiquitously expressed in mammalian
cells and regulate a wide variety of ion channels.59,60Because
there is no tissue-specific expression of phosphoinositides, the
cytotoxicity induced by the interactions between aminoglyco-
sides and phosphoinositides is unlikely to be solely responsible
for the selective susceptibility of kidney proximal tubule cells
and inner ear mechanosensory hair cells.
It has been well established that aminoglycosides kill bacteria
by binding to ribosomal 16S rRNA in the 30S subunit of the
ribosome, inhibiting protein synthesis.23,61,62In eukaryotes,
mitochondria are thought to originate from bacteria,63and
mitochondrial ribosomes are highly similar to bacterial
ribosomes compared to mammalian cytosolic ribosomes.64,65
The first analysis of familial aminoglycoside-induced deafness
revealed a nucleotide 1555 A to G substitution (A1555G) in
the mitochondrial 12S rRNA gene.66This A1555G mutation
makes the secondary structure of 12S rRNA more similar to
the corresponding site in 16S rRNA of bacteria.66Indeed,
binding assays using RNA constructs demonstrated that the
A1555G RNA analog binds to aminoglycosides with high
affinity while the wild-type construct does not.67Since the
A1555G mutation also causes non-syndromic hearing loss in
many families, factors other than aminoglycosides must also
contribute to the deafness induced by this mutation.68
Additional mutations (C1494T, T1095C, T961) in the 12S
rRNA also cause drug susceptibility.69–71Biochemical studies
show that these mutations also decrease mitochondrial protein
glycosides can enter cells by permeating cation channels directly into
the cytosol. Binding of aminoglycosides to iron generates ROS, with
arachidonic acid (AA) acting as an electron donor. ROS activates Bax,
which in turn translocates to mitochondrial membranes. (2) Cyto-
chrome c (cyt c) is released from mitochondria through the mito-
chondrial transition pore formed by Bax-dependent mechanisms,
activating caspase-9 and caspase-3, and leading to apoptosis. ROS
are also released from mitochondria, further increasing cytosolic ROS
levels. (3) In caspase-independent mechanisms, EndoG and AIF
released from mitochondria also induce apoptosis. (4) Aminoglyco-
sides can also be endocytosed and are trafficked to the ER and
lysosome by vesicle transport mechanisms. (5) Aminoglycosides can
induce lysosomal rupture, or the release of lysosomal cathepsins, either
of which leads to necrosis. (6) Aminoglycosides within the lumen of
the ER bind to CLIMP-63, inducing oligomerization that can activate
14-3-3 proteins, leading to mitochondrial apoptosis signaling and/or
resulting in JNK activation and c-Jun translocation into nucleus. (7)
The c-Jun transcription factor induces apoptotic gene transcription
and subsequent apoptosis.
Cell death mechanisms induced by aminoglycosides. (1) Amino-
882 Integr. Biol., 2011, 3,879–886 This journal is c The Royal Society of Chemistry 2011
translation efficacy.72Therefore, aminoglycoside binding to
mitochondrial 12S rRNA with these mutations could lead to
high levels of inhibition or mistranslation of protein synthesis
and subsequent cytotoxicity.73
One unexpected consequence of aminoglycoside binding to
RNA has been its ability to readthrough premature termination
codons (PTCs) by binding to the decoding site of 18S rRNA.
Therapeutic approaches to promote readthrough of disease-
causing PTCs have been developed. For example, in patients
with cystic fibrosis (CF) resulting from PTC mutations in the
CF transmembrane conductance regulator (CFTR), amino-
glycoside treatment can lead to expression of full-length
proteins.74,75However, this readthrough efficacy is variable,
depending on genes and mutations,76and more understanding
of the PTC readthrough mechanism is necessary.
Although aminoglycosides bind to at least several proteins,77it
is not clear which proteins, when bound to aminoglycosides,
become dysfunctional, and/or induce cytotoxicity in mammalian
cells. Some aminoglycoside-binding proteins may sequester
aminoglycosides and prevent the noxious intracellular effects
of these drugs.
In kidney, a large glycoprotein called megalin binds to
gentamicin at the apical membrane of proximal tubule cells,
and delivers gentamicin to lysosomes following endocytosis,
suggesting that megalin is involved in renal accumulation of
aminoglycosides.78,79However, a proximal tubule cell line
LLC-PK1 and sensory hair cells in the inner ear do not express
megalin, yet both take up aminoglycosides by endocytosis, and
exhibit aminoglycoside-induced cytotoxicity.30,80,81
Recent studies using gentamicin affinity column chromato-
graphy identified HSP73 in porcine kidney,82and calreticulin
in bovine kidney83as gentamicin-binding proteins (GBPs).
HSP73 is a heat shock protein (HSP) that is constitutively
expressed, and is not induced by cell stress. Constitutively-
expressed HSPs function as molecular chaperones for newly-
synthesized proteins in the ER, and these HSPs assist in
protein folding and assembly, and in transporting proteins
into subcellular organelles.84Although the functional specifi-
city of HSP73 is unknown, it is likely that HSP73 also
functions as a chaperone protein. Since HSP73 is homo-
genously distributed throughout the kidney,85it is unlikely
to contribute to the difference in gentamicin susceptibility
between proximal and distal tubule cells. There has been no
report on HSP73 expression in the inner ear, although the
ubiquitous expression of this protein elsewhere suggests a
similar ubiquitous distribution in the cochlea. Interestingly,
HSP70, another HSP protein, inhibits neomycin-induced hair
cell death in mice.86Unlike HSP73, HSP70 expression is
induced by cellular stress, and protects the cell by stabilizing
Calreticulin is another chaperone protein localized in the
ER.89The ER may play an important role in aminoglycoside-
induced cytotoxicity because endocytosed aminoglycosides are
trafficked to the Golgi body and ER.32,90Calreticulin binds to
glycoproteins and assists in protein folding, quality control,
and degradation.91Calreticulin is expressed in both kidney
proximal and distal tubules.82,142Calreticulin is also expressed
in the cochlea.92We have immunolocalized calreticulin in the
cytoplasm of marginal cells in the stria vascularis and in the
stereociliary bundles of cochlear hair cells.142These locations
are exposed to high levels of aminoglycosides during trans-strial
trafficking into endolymph and hair cell uptake of aminoglyco-
sides (Fig. 3).30,31,93,94The chaperone activities of both HSP73
and calreticulin are inhibited by gentamicin, and this inhibition
could contribute to gentamicin-induced cytotoxicity.82,83
The early GBP studies82,83used CH- or CNBr-activated
Sepharose 4B without a ‘‘spacer’’ molecule between gentamicin
and Sepharose, which could cause steric hindrance between
gentamicin and other GBPs. We employed a neutral 10-atom
spacer between gentamicin and agarose, and identified
another GBP, CLIMP-63, a protein that connects ER to the
expressed in kidney and cochlear cell lines. Many other GBPs
were also observed in kidney cells, including calreticulin, but these
did not show distinct differences in expression or localization
between proximal and distal tubule epithelial cell lines in vitro.
Cell lines derived from the kidney proximal tubule and inner
ear organ of Corti express significant amounts of CLIMP-63
dimers that are resistant to dithiothreitol (DTT) treatment.
Gentamicin treatment increased CLIMP-63 dimerization.
Knock-down of CLIMP-63 with siRNA transfection effectively
reduced CLIMP-63 dimerization while retaining expression of
its electrophysiological environments. The stria vascularis, lining the
spiral ligament on the inside lateral wall of the bony cochlear shell,
contains basal (B) and marginal (M) cells connected together by tight
junctions that form an impermeable paracellular barrier to solutes.
Circulating aminoglycosides within strial capillaries (C) are preferen-
tially transported through the strial blood–labyrinth barrier consisting
of tight junction-coupled endothelial cells, into the intra-strial space
(ISS). From there, aminoglycosides are trafficked through marginal
cells into endolymph, and enter hair cells (HC) across their apical
surfaces by endocytosis and non-selective cation channel permeation.
The electrical potentials of various fluid compartments, separated by
tight junction-coupled endothelial and epithelial cell barrier layers, are
also indicated. Endolymph has a +80 mV, and hair cells have a resting
potential of ?60 to ?75 mV, generating a considerable electrophoretic
driving force across the apical endolymphatic membranes of hair cells.
Schematic diagram of the cochlear duct cytoarchitecture and
This journal is c The Royal Society of Chemistry 2011 Integr. Biol., 2011, 3,879–886883
the CLIMP-63, and cells were more resistant to gentamicin
treatment. Although the nature of these DTT-resistant dimers
of CLIMP-63 is unclear, these dimers could define the tissue
selectivity of aminoglycoside-induced toxicity because they are
not expressed in cells from the kidney distal tubule or other
tissues.77We have identified several 14-3-3 proteins as CLIMP-
63-binding proteins, and that 14-3-3b is also involved in
gentamicin-induced CLIMP-63-dependent cytotoxicity. Since
14-3-3 proteins have been implicated in various cell death and
survival signaling pathways, it is possible that CLIMP-63
association with 14-3-3 proteins induces apoptosis. One possible
pathway leads to JNKs or p38-MAPK-dependent apoptosis.95
One of the major questions in aminoglycoside-induced
cytotoxicity is selective susceptibility of the inner ear hair cells
and kidney proximal tubule cells. The conventional hypothesis
for aminoglycoside susceptibility in these cell types is that
these cells take up higher levels of aminoglycosides compared
to other cells. Since aminoglycoside levels in the cytosol
directly correlate with cytosolic ROS generation through
iron-binding, this could be sufficient to explain the cell type-
specific drug susceptibility. However, it remains to be
explained why these cells retain high intracellular levels of
aminoglycoside, while most cells are able to clear their cytosol
of the drug.96The ability of cells to clear aminoglycosides is
also likely to be important in trafficking the drug across the
tight junction-coupled endothelial and epithelial marginal cells
in the cochlear blood–labyrinth barrier into the intra-strial
space and endolymph, preventing or reducing cytotoxicity in
these cochlear cell types (Fig. 3).
Based on the evidence that we have described of intracel-
lular binding-targets of aminoglycosides, we speculate that
aminoglycoside-binding proteins, like calreticulin, CLIMP-63,
and possibly HSP73, contribute to the tissue selectivity of
aminoglycoside-induced toxicity. Additionally in the cochlea,
the positive endolymphatic potential and low Ca2+level of
endolymph bathing the hair cell apex favor a rapid influx of
aminoglycosides through cation channels, such as mechano-
transduction or TRPV4 channels, concentrating aminoglyco-
sides in hair cells (Fig. 3).31,33
Downstream targets of aminoglycoside-induced
Owing to recent advances in apoptosis research, especially
related to cancer biology, we now have a better understanding
of how aminoglycosides induce cell death signalling after
initial drug–target interactions in the cytoplasm. Aminoglyco-
sides induce phosphorylation of c-Jun in JNK signalling
pathways that trigger hair cell death.28,97Gentamicin treat-
ment of rat cochlear explants also increases the binding
activities of activating protein-1 (AP-1), a heterodimeric
protein consisted of c-Jun and c-Fos family proteins.98
Caspases and Bcl-2 family proteins are essential components
in apoptotic signalling. We will describe how these proteins are
involved in aminoglycoside-induced apoptosis below.
Among a dozen caspase family proteins identified, those that
are activated by tumor necrosis factor receptors (TNFRs),
including caspase-8, do not play a major role in aminoglycoside-
induced ototoxicity.99Activation of caspase-9, on the other
hand, is induced by cytochrome c release from mitochondria
into the cytosol, and has been detected in aminoglycoside-
treated utricles, cochleae and kidney cells in vitro.26,100,101
Caspase-9 activates caspase-3, an ‘‘executioner’’ caspase, which
cleaves anti-apoptotic proteins or inhibits deoxyribonucleases,
to induce cell death.102,103Aminoglycoside-treated hair cells
showed enhanced levels of caspase-3 activity and increased
cell death in vitro and in vivo.99,100,104Direct infusion of the
pan-caspase inhibitor z-VAD-fmk into the vestibule, or systemic
administration of the inhibitor promoted hair cell survival after
In mice, chronic treatment with kanamycin induced hair cell
death by caspase-independent pathway(s).35While cyto-
chrome c release, caspase-9, caspase-3 and JNK activation,
and TUNEL staining were absent, endonuclease G (EndoG)
translocation, calpain activation, and cathepsin D synthesis
and activation were all observed after chronic treatment with
kanamycin.35This raises a question whether in vivo or clinical
aminoglycoside administration induces ototoxicity by caspase-
dependent apoptosis, by other caspase-independent apoptotic,
by necrotic mechanisms, or by a combination of two or more
of these mechanisms. One possible caspase-independent
mechanism involves mitochondrial release of EndoG and
calpain–cathepsin signalling cascades induces necrosis.106–108
Consistent with this, intracellular calcium levels are elevated
by aminoglycoside treatment, and an increase in calcium levels
can activate calpains, which leads to ototoxicity.29,109Since
these mechanisms have not been fully investigated, more
studies using clinically-relevant experimental designs are
Bcl-2 family proteins
The cytoplasmic Bcl-2 family proteins consist of anti-
apoptotic Bcl-2 and Bcl-xLand pro-apoptotic Bax and Bak,
where Bcl-2 and Bcl-xLform heterodimers with Bax and Bak
to inhibit apoptosis and maintain mitochondrial membrane
integrity.110,111When apoptotic signals overcome inhibition/
protection by Bcl-2 and Bcl-xL, Bax translocates from the
cytochrome c that in turn activates caspase-9.26How Bax
triggers release of cytochrome c is not well understood, but it
likely involves the formation of the mitochondrial permeability
There have been numerous reports on Bcl-2 family proteins
in the inner ear, and most early reports discuss their function
in cochlear development. More recent evidence suggests that
Bcl-2 protects against aminoglycoside-induced ototoxicity.115–117
Although little has been reported about the role of pro-
apoptotic proteins, like Bax, in aminoglycoside-induced
ototoxicity, studies on downstream targets suggest that they
are involved. For example, loss of the mitochondrial membrane
potential, an indication of Bax translocation to mitochondria,
has been observed in the auditory hair cells after gentamicin
treatment in vitro.25In addition, cytochrome c release, also
triggered by Bax activity, was detected in sensory hair cells
884 Integr. Biol., 2011, 3,879–886 This journal is c The Royal Society of Chemistry 2011
treated with aminoglycosides.26,97,104It is well-established that
Bax can be up-regulated by the tumor suppressor p53
protein. The involvement of p53 in aminoglycoside-induced
apoptosis is still unclear. However, a recent report suggested
aminoglycoside-induced hair cell death (A. Coffin, personal
communication).118How p53 could be activated by amino-
glycosides needs to be explored further.
Variation in aminoglycoside induction of toxicity
The utilization of zebrafish neuromast hair cells has provided
an important tool to rapidly and reproducibly assess hair cell
dose-response curves for different aminoglycosides. At a given
dose, neomycin induced rapid hair cell death in some but not
all hair cells, while gentamicin induced both acute hair cell
death, and also continuing hair cell death over a longer period
of time for a more complete ablation of neuromast hair
cells.119This effect was also seen with other aminoglycosides
(kanamycin, streptomycin, tobramycin, amikacin) to varying
degrees, implying that these drugs initiate a spectrum of cell
Preclinical studies in aminoglycoside toxicity have also been
confounded by the wide variation in rodent species suscept-
ibility to aminoglycoside toxicity, with mice and rats being
particularly resistant to aminoglycoside toxicity,120compared
to guinea pigs,121,122for example. Furthermore, there is varia-
tion within a single species or strain to different types of
aminoglycosides.120In addition, there is individual variation
within a study to the same dose of aminoglycosides that may
correlate with the efficacy in trafficking aminoglycosides across
the blood–labyrinth barrier.123Nonetheless, with increasing
availability of knockout and transgenic mice, a murine model
for ototoxicity will be extremely advantageous to investigate
specific mechanisms of drug trafficking in drug-induced cyto-
toxicity in the inner ear.120,124
The use of inner ear explants to study ototoxicity has increased
in the last decade, and has both disadvantages and advantages.
Obtaining inner ear sensory epithelial explants is a challenging
procedure, subject to variation in the dissection procedure within
experiments, and sensory cell death prior to ototoxic drug
exposure.125More importantly, explantation removes the unique
three-dimensional electrochemical environment that bathes the
mechano-sensitive hair cells (Fig. 3).126However, inner ear
explants provide high reproducibility in dose and duration of
ototoxic drug exposure to hair cells, and avoid the experimental
variability of animal studies due to species and inter-individual
differences in the blood–labyrinth barrier. In addition, in vitro
micro-Ussing chambers enable electrophysiological investigation
of ion trafficking by the stria vascularis, a major function of the
cochlear blood–labyrinth barrier.127,128
Otoprotection against aminoglycoside toxicity
One way to prevent ototoxicity may be the use of iron chelators,
such as deferoxamine, to reduce ROS formation.120,129Anti-
oxidants also protect against ototoxicity by reducing ROS
levels. Antioxidants that show otoprotective effects in animal
models of aminoglycoside ototoxicity include: lipoic acid,
D-methionine, and salicylate.130–132In clinical studies in
humans, aspirin, an antioxidant that is cheap and widely
available, can ameliorate gentamicin-induced ototoxicity.133
Cellular enzymes with antioxidant properties, such as super-
oxide dismutases and glutathione S-transferase, could also be
used to reduce ROS levels.134–136
Clinical interventions to interfere with aminoglycoside binding
to phosphoinositides, RNA or proteins are far from established,
partly because our understanding of these cytotoxicity mecha-
nisms remains insufficient to devise a pharmacological strategy.
However, in vitro studies and research on animal models are
steadily accumulating evidence that support a few promising
approaches to reduce aminoglycoside toxicity.
The first approach is to decrease cellular uptake of amino-
glycosides in the cochlea or kidney by blocking drug entry into
the cell via modulation of aminoglycoside-permissive ion
channels (or transporters) at the cell membrane.33,137,143This
could be critical at the blood–labyrinth barrier, preventing
aminoglycosides from entering the cochlea and hair cells.18
Another method would be to decrease intracellular accumulation
by increasing cellular clearance of the drug. All cells initially take
up aminoglycosides and most rapidly clear the drug.96However,
how the majorityofcells clear aminoglycosidesfrom theircytosol
remains unknown. Up-regulating the intracellular expression of
aminoglycoside-binding proteins to sequester these drugs and
prevent their cytotoxicity may also be beneficial.
Another approach is to modulate cell death signalling by
targeting specific proteins in cell death signaling, such as JNK
or c-Jun. In order to block the functions of these proteins,
pharmacological inhibitors or RNAi-based approaches may
be useful, and this has been demonstrated in vitro.28,97For
example, L-carnitine, a naturally occurring neuroprotective
agent, prevents expression of harakiri, a proapoptotic factor
implicated in gentamicin-induced ototoxicity.138Alternatively,
increasing expression or efficacy of anti-apoptotic proteins
such as Bcl-2 and Bcl-xLcould promote hair cell survival.
An apoptosis inhibitor protein survivin is a good candidate for
this strategy.139Similar approaches for modulating cell death
signalling could also be used. These intracellular studies can be
accelerated using zebrafish neuromast hair cells as a model
system to identify potential intracellular otoprotectants.140,141
However, it remains to be determined whether hair cell
survival extends beyond the initial assessment period, and that
removal of these inhibitors of cell death signaling pathways
has no adverse effects on hair cell survival.
Regardless of which approach ameliorates aminoglycoside
toxicity, translation from in vitro studies and animal research
to clinical medicine will be complex, and require extensive
verification before clinical application. It will also be critical to
determine how much reduction in eukaryotic toxicity in
sensory receptors and kidney proximal tubules can occur while
maintaining the drug’s inherent bactericidal properties. Any
method that reduces both aminoglycoside ototoxicity and
bactericidal efficacy will be unsuitable for clinical practice.
We thank Dr Hongzhe Li for discussion on the manuscript.
This work was supported by NIH NIDCD R03 DC009501
(TK) and R01 04555 (PSS).
This journal is c The Royal Society of Chemistry 2011 Integr. Biol., 2011, 3,879–886 885
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