Discussion aminoglycoside project
• Discussion on MET 2
• Discussion on TRP channels 7
• Discussion on ROS 9
• Discussion on Mitochondria 13
• Discussion on Apoptosis 20
Discussion on MET
It has been shown by Marcotti et al. that aminoglycosides enter inner ear
haircells through mechanoelectrical transduction (MET) channels  and a
theoretical model with which the rate of aminoglycoside uptake rate can be
calculated has been developed . Apart from the electrophysiology data by
Marcotti et al. there are many studies that corroborate the finding that
aminoglycosides enter inner ear hair cells via the MET channel.
A small proportion of the MET channels in normal hair cells is open in the
absence of any stimuli . This resting current is absent in hair cells from
mice suffering from various forms of hereditary deafness, e.g. mice
expressing a mutated form of myosin VIIA show both vestibular and cochlear
dysfunction and lack resting transduction currents . It has been
demonstrated by Kros et al. that myosin VIIA is required for the normal gating
of the MET channel . The myosin VIIA mutation impairs the uptake of
gentamicin by hair cells . In other words uptake of aminoglycosides in hair
cells which lack resting transducer currents (all MET channels being closed) is
FM1-43 is a styryl pyridinium dye that enters inner ear hair cells through open
MET channels  and behaves like a permeant blocker .
Hair cells take up FM1-43 but surrounding (non-transducing) cells show little
of nor labelling . Analogously, within the cochlea haircells are damaged by
aminoglycosides whereas surrounding (non-transducing) cells remain
unharmed . It was found that FM1-43 competes with aminoglycosides for
entry into the haircells and the ototoxicity of neomycin is reduced in the
presence of FM1-43 . This means that aminoglycosides and FM1-43 both
target the MET channel. When haircells are treated with EGTA the tiplinks are
broken and the MET channels close. In the presence of EGTA the loading
with FM1-43 by haircells is inhibited . Haircells from mice which are
homozygous for the myosin VIIA mutation Myo7a6J
do not load with FM1-43
. Initially during a dye pulse FM1-43 strongly labels the hairbundles . It is
clear from these observations that results obtained from studies with FM1-43
can give insights into aminoglycoside uptake by haircells.
It has been reported that aminoglycosides enter haircells via receptor
mediated endocytosis . Endocytosis cannot explain why within the cochlea
only the haircells degenerate in the presence of aminoglycosides  whilst
surrounding cells remain unharmed. Also, hair cells which express mutated
myosin (important for normal MET gating) do not take up aminoglycosides .
Undoubtedly aminoglycosides will enter the haircells via endocytosis to a
small extent, but these studies show that aminoglycosides predominantly
enter the haircells via the MET channel.
What about hair cells that do not take up AGs?
There are some studies where no aminoglycoside uptake into haircells was
observed. Dulon et al.  showed that triturated haircells are not affected by
the presence of 5 mM gentamicin for 6 hours. Trituration is a very harsh
isolation method for hair cells and in all likelihood a large proportion of the
hairbundles were destroyed during this procedure.
The hairbundle is the location of the MET channels, triturating the haircells
probably destroyed the stereocilia and these haircells were not transducing.
These observations are in stark contrast with the study done by Kotecha and
Richardson  where an organotypic culture of the cochlea was used. The
haircells in this preparation are transducing and exposure with 1 mM of
gentamicin for 1 hour completely destroyed the haircells.
In another study by Williams et al. it can be clearly seen that isolating OHCs
can lead to the loss of stereocilia. See figure 1 in . These haircells will not
Adapted from Figure 1 in 
The study by Dulon et al.  employs essentially the same method of
isolating hair cells, which probably leads to hairbundle damage.
In another study by Zajic and Schacht  isolated outer hair cells appear to
have compromised stereocilia (see figures 1-5 in ).
The recent observation that aminoglycosides enter hair cells through the MET
channel can explain the apparent discrepancies between the Kotecha and
Richardson study and the results obtained in the Schacht laboratory.
AG uptake via MET channels can explain a lot of observations
Exposure to aminoglycosides leads to a phased degeneration of haircells, first
the OHCs then the IHCs [13, 14]. This can be explained by the fact that an
outer hair cells has more stereocilia (~81) than an inner hair cell (~48) .
This means that an OHC has more MET channels than an IHC and therefore
more pathways for AG uptake. If OHCs load up quicker with aminoglycosides
than IHCs it can be expected that OHCs degenerate quicker.
Are there any studies which show that aminoglycoside uptake in OHCs
is faster than IHCs? FM1-43 uptake in OHCs is quicker than IHCs .
Hair cell loss due to AGs starts in the basal coil and progresses apically .
This is not due to a concentration gradient of AGs within the cochlea because
this same pattern is observed in organotypic cultures that are incubated with a
uniform concentration of AGs . This means that this pattern of sensitivity is
based on inherent properties of the hair cells. Basal OHCs have larger
transducer currents than apical OHCs  and OHCs have larger transducer
currents than IHCs . The uptake rate of AGs will be higher in the basal coil
than in the apical coil due to the differences in conductance.
Clinically the effects of aminoglycosides are characterized by hearing loss
initially at high frequencies which corresponds to hair cell damage in the basal
In vivo studies have shown that immature hair cells take up less gentamicin
than mature hair cells. Also, mature cells die faster than immature cells upon
exposure to AGs . One possible explanation could be a difference in MET
current amplitude between immature and mature hair cells. However,
transduction current amplitude stays constant during maturation .
The endocochlear potential in mice is only fully developed the 12th
birth REF. In immature animals the driving force for positively charged
molecules like AGs is lower than in mature animals, this could explain the
differential sensitivity between immature and mature hair cells in vivo.
FM1-43 labeling is much stronger in the basal coil than in the apical coil .
As described above, FM1-43 enters the hair cells via the MET channel. The
differences in labeling can be explained by the fact that basal coil hair cells
have larger transducer currents than apical coil hair cells.
The uptake of FM1-43 is temperature dependent, i.e. at 4 °C the uptake rate
is reduced . This could indicate a temperature dependence of the opening
of MET channels possibly due to a dependence on myosin ATPase.
Gentamicin gets taken up quicker by OHCs in the presence of background
noise as opposed to in animals maintained under noise-attenuated
conditions . This suggests that the MET channels were open during the
background noise allowing more AGs in than under noise-attenuated
The rate of FM1-43 loading into basal coil HCs is much faster than in apical
coil HCs . This again can be explained by the differences in MET current
amplitudes with basal coil HCs displaying larger MET conductances than
apical coil conductances.
When incubated with gentamicin it first labels the stereocilia of hair cells
before diffusing through the cell . This suggests that the location where
the AGs get taken up by the haircells is situated within the stereocilia. This
corroborates the hypothesis that AGs enter via the MET channel as
demonstrated by Marcotti et al. .
Spermine is a polyamine which is naturally present in cells  and displays
effects on hair cells very similar to those of AGs and FM1-43.
Spermine causes haircell damage without affecting any of the surrounding
cells . This indicates that hair cells have a very specific uptake mechanisms
for polyamines which suggests involvement of the MET channel.
Spermine causes damage to OHCs comparable to DHS and amikacin .
It is important to take spermine into account because it directly targets
mitochondria. I will discuss in the discussion section on Mitochondria.
Aminoglycosides preferentially target inner ear hair neurons and proximal
tubule kidney cells. Although undoubtedly some slow uptake of
aminoglycosides through endocytosis will take place, it is striking that other
cell types in the direct surroundings of inner ear hair cells are not affected by
the presence of aminoglycosides. (What about non-kidney cell types in the
This suggests that inner ear hair cells and kidney proximal tubule cells share
some common uptake system for aminoglycosides. As shown above, there is
convincing evidence that AGs enter the hair cells via the MET channel. What
evidence is there that AGs enter the kidney cells via MET channels as well?
In order to address this question we will first investigate whether or not kidney
proximal tubule cells display mechano-electrical transduction.
Do kidney cells display mechanoelectrical transduction?
Proximal tubule kidney cells exhibit primary cilia . These cilia are moved
by fluid flow within the lumen of the proximal tubule. Bending these cilia in
model kidney cells leads to the influx of calcium . The lanthanide
) is a powerful blocker of the MET current in hair cells . In
the presence of Gd3+
bending of the cilium does not lead to influx of calcium
[24, 25]. In the presence of Gd3+
(another blocker of MET current
REF) the uptake of gentamicin in kidney cell lines was decreased .
Single channel recordings from renal primary cilia show that single channel
conductivity is around 80 pS , which is within the range of what is
accepted as MET single channel conductance . It has been reported that
gentamicin uptake in cultured kidney cells is not dependent on endocytosis
which suggests that gentamicin uptake by kidney cells occurs via a channel
. This is corroborated by the fact that depolarisation of cultured kidney
cells leads to a reduced AG uptake , whereas hyperpolarization leads to
increased AG uptake . Myosine VIIa is an important protein associated
with the MET channel in hair cells . This protein is also present in kidney
cell cilia .
Clearly kidney cells display some remarkable similarities with inner ear
sensory neurons. This may explain why aminoglycosides preferentially target
these two cell type.
The role of megalin in hair cells and kidney cells needs to be discussed
Discussion TRP channels
Both inner ear sensory neurons and kidney proximal tubule cells are
mechanotransducing. The identity of the MET channel in inner ear hair cells
has not yet been identified [33, 34]. Several candidates have been proposed,
namely the Epithelial Sodium Channel (ENaC), the Acid-Sensing Ion Channel
(ASIC), amiloride sensitive sodium channels and Transient Receptor Potential
(TRP) channels . Due to the limitations of the material (~30,000 haircells
per cochlea REF) progress in identifying the MET channel in hair cells has
been painstakingly slow. Kidney proximal tubule cells share several very
interesting similarities with inner ear sensory neurons due to the fact that
these cells are also mechanotransducing and are preferentially targeted by
AGs. On the basis of these similarities I would like to postulate the following
hypothesis: The MET channels in inner ear sensory neurons and kidney
proximal tubule cells are similar and possibly of the same channel type.
Kidney cells are readily available, at higher quantities than hair cells and
therefore could serve as a good model system to investigate the identity of the
Evidence that the kidney mechanosensor is a TRP channel
TRPP1 and TRPP2 (alternatively PKD1 and PKD2 or polycystin-1 and
polycystin-2) are receptors which when mutated can cause polycystic kidney
PKD2 localises at the primary cilium of kidney epithelial cells where it is
expected to be activated by mechanical stimulation .
TRPP2 localizes to both motile and primary cilia and recent evidence strongly
implicates it as a mechanosensor in the nonmotile, primary cilia 
PKD1 and PKD2 when expressed together in cultured cells form functional ion
channels . Nauli et al. found that both PKD1 and PKD2 are expressed on
the cilia of embryonic kidney cells and both proteins need to be functional for
normal mechanotransduction .
Another candidate is the TRPV1 channel. It was shown that TRPV1 regulators
mediate the gentamicin uptake by cultured kidney cells . The same study
showed that in the presence of Gd3+
(inhibitors of mechanoelectrical
transduction channel  REF for La3+
) the uptake of gentamicin in these
kidney cells was decreased.
A study in which TRPV1 channels were expressed in HEK cells showed that
FM1-43 is readily taken up in the presence of capsaicin . The uptake of
FM1-43 was blocked by ruthenium red  which is a specific inhibitor of TRP
channels  and apparently also a blocker of hair cell transduction .
Since FM1-43 behaves in a way similar as aminoglycosides, this suggests
that the mechanoelectrical transduction channel could be a TRPV1 channel.
Corey rules out TRPV4 as a candidate for the hair cell MET channel , but
he does not discuss TRPV1. On the basis of the results described above it is
worthwhile further exploring TRPV1 as a possible candidate for the MET
channel in both inner ear sensory neurons and kidney proximal tubule cells.
Is the MET channel in hair cells a TRP channel?
There is evidence which shows that the MET channel in kidney cells could be
a TRPV1 channel. Is TRPV1 expressed in hair cells? According to Zheng et
al. TRPV1 is present in guinea pig OHCs  and according to Steyger et al.
TRPV1 regulators modulate gentamicin uptake in inner ear hair cells .
In other words, TRPV1 regulators modulate gentamicin uptake in both inner
ear hair cells and kidney cells. It has also been found that AGs can inhibit
TRPV1 in the order: Neomycin ≅ Streptomycin > Gentamicin  which
corresponds to the ranking order of ototoxicity found by Kotecha and
Richardson : Neomycin > gentamicin > dihydrostreptomycin > amikacin >
Neamine > spectinomycin.
Other lines of evidence indicate that the hair cell MET channel is a TRP
channel. It has been found that TRP channels interact with PDZ-domain
containing scaffold proteins . This is relevant because the MET channel in
hair cells is known to interact with PDZ-domain proteins REFS adaptation
Homozygous mutants (both TRPML3 alleles mutated) do not load with FM1-
43 or gentamicin . Although it has been shown that TRPML3 is not the
MET channel it still indicates that it could be a TRP channel REFS
) blocks MET in kidney cells and inner ear sensory neurons
[26, 27]. Gadolinium is a potent blocker of TRP channels .
The findings described above show that aminoglycosides interact with TRP
channels and that there is a strong relationship between MET and TRP
channels in both inner ear sensory neurons and kidney cells.
Discussion on ROS
Reactive oxygen species (ROS) play an instrumental role in aminoglycoside
induced oto- and nephrotoxicity, e.g. aminoglycosides can generate ROS and
anti-oxidants can protect against AG induced hearing loss . A study by
Walker et al. showed that gentamicin enhanced production of hydrogen
peroxide in a dose-dependent manner in kidney cells . In the next sections
experimental evidence for aminoglycoside induced ROS formation in inner ear
hair cells and kidney cells will be discussed.
Aminoglycoside induced ROS formation in inner ear hair cells
ROS generation in inner ear hair cells
It has been shown in cultured avian sensory epithelia that exposure to
gentamicin induces increased levels of ROS  and generation of free
radicals in the presence of AGs was demonstrated in explants of the inner ear
This section needs some more refs
Glutathione (GSH) is a cellular oxidant which can neutralise ROS in cells.
REF It has been found that glutathione in vivo, protects against
aminoglycosides induced ototoxicity . Usami et al. showed that GSH
levels in both IHCs and OHCs are very low compared to other organ of Corti
cells . It has been reported by Sha et al. that glutathione levels in basal
OHCs are lower than apical OHCs by .
However, the method used for quantifying these levels by Sha et al. is
unclear. Also, based on the numbers there don’t appear to be any
differences. It is reported that there is a significant difference, however, it is
not mentioned which statistical test is applied (probably a T-test) and more
seriously, it is not specified whether variation is expressed as SD or SEM.
When testing for statistical significance we found that with using SD there is a
significant difference but with SEM there isn’t. Rather cheeky to omit this
It has been reported that the end of the ‘sensitive period’ for AGs in the
young rat coincides with the maturation of glutathione-S-transferases,
enzymes which use glutathione as a substrate in drug detoxification .
N-acetylcysteine (NAC), a synthetic precursor of reduced glutathione (GSH) is
a thiol-containing compound which stimulates the intracellular synthesis of
GSH, enhances glutathione-S-transferase activity, and acts solely as a ROS
scavenger . According to Feldman et al. N-acteylcysteine protects against
gentamicin induced ototoxicity. The greatest otoprotective effect was noticed
at high frequencies , which means that NAC best protects basal OHCs
which suggests that ROS levels are higher in basal OHCs than in apical
It has been suggested that NAC can help to replenish depleted glutathione
content when cells are exposed to elevated oxidative stress . As NAC can
cross the blood brain barrier  this compound potentially could be used to
attenuate aminoglycoside induced ROS formation.
It has been reported that OHCs in culture show a base to apex viability
gradient, i.e. also in the absence of AGs basal OHCs die sooner than apical
OHCs . This suggests that apart from differences in MET conductivity
there may be other inherent differences between basal and apical haircells.
These observations indicate clearly, be it indirectly, that aminoglycosides
induce ROS formation in hair cells. Glutathione neutralises ROS and
increasing the concentration of glutathione in cells (by injection of GSH in the
bloodstream or by stimulating the intracellular synthesis of GSH) prevents
aminoglycoside induced ototoxicity. Inherent low levels of GSH in hair cells
could be a contributing factor making these cells especially vulnerable to AGs.
Differences in GSH concentration between basal and apical haircells could
also help explain the observed aminoglycoside induced damage gradient .
We find however that the data provided in the study by Sha et al. do not
support this claim and it would be good to repeat this study.
Nevertheless, it is clear that increasing the levels of GSH in hair cells can
prevent aminoglycoside induced ototoxicity.
It has been reported that administration of both iron chelators (deferoxamine,
2,3-dihydrobenzoate) and ROS scavengers (mannitol) lends complete
protection against gentamicin induced HC damage . Iron chelators alone
only lend partial protection.
Schacht et al distinguish between iron chelators and antioxidants. Whereas
iron chelators are know to have radical scavenging properties themselves,
e.g. neutralising hydroxyl ions (REFS).
Schacht focuses completely on the iron chelation aspect. According to his
theories aminoglycosides are not inherently damaging to OHCs but need to
undergo a biotransformation before becoming ototoxic. There are plenty
reports which completely refute this notion but let’s stick with his idea for now.
In order to ‘biotransform’ the aminoglycoside needs to form a complex with
iron. Once this aminoglycoside-iron complex is formed it will start generating
ROS. Adding iron chelators to haircells protects haircells against
aminoglycosides by chelating iron, which is then no longer available to
complex with aminoglycosides and hence there will be no production of ROS.
There are several flaws in this hypothesis:
• Aminoglycosides do not need to undergo a biotransformation to be
• Iron chelators also function as radical scavengers.
• What is the identity of the iron that aminoglycosides supposedly
• Is there real, direct evidence of aminoglycosides forming a complex
• It is generally accepted that aminoglycosides are unreactive.
In conclusion, I think that iron chelators protect against aminoglycoside
induced ototoxicity due to the fact that they scavenge ROS. I don’t think iron
chelation has any part, nor do aminoglycosides form iron complexes.
It was found that 2,3-Dihydrobenzoate (DHB) protects against kanamycin
induced ototoxicity . It has been reported that the order of resistance to
kanamycin correlates with the pigmentation of mouse strain used. It is
hypothesized that this is due to enhanced antioxidant capability of melanin-
containing cochlea .
Exogenous SOD (superoxide dismutase) protects against AG induced HC
degeneration, however exogenous SOD also elicits an immune response .
Overexpression of superoxide dismutase in inner-ear tissue of transgenic
mice prevented kanamycin induced hearing loss .
Iron chelators 2,2’-DPD (2,2’-dipyridyl) and Deferoxamine decreased
gentamicin induced damage of OHCs in organotypic cultures .
Iron chelators deferoxamine and DBH reduce gentamicin induced ototoxicity
 and the efficacy of DBH against gentamicin appears to be dose-
Aminoglycoside induced ROS formation in kidney cells
It was found that gentamicin enhances hydrogen peroxide production in a
dose dependent fashion in isolated kidney and liver mitochondria .
Iron chelators deferoxamine and DHB reduce gentamicin induced damage to
the kidney . Another study showed that the iron chelators DFO and DHB
reduce gentamicin induced damage to the kidney .
N-acteylcysteine can ameliorate gentamicin induced kidney damage .
Apparently iron supplementation potentiates gentamicin induced
nephrotoxicity in rats . Schacht would argue that this results in the
formation of more ‘aminoglycoside-iron complexes’ but it probably just results
in ROS formation by gentamicin on top of the ROS formation induced directly
by the iron supplementation. Also, it was found that Iron deficiency sensitizes
animals to acoustic trauma .
These results show that the response to aminoglycosides and antioxidants by
kidney cells is similar to what is seen in hair cells.
This section needs more kidney specific references on ROS.
Clearly aminoglycosides induce ROS formation in both hair and kidney cells.
Antioxidants protect against aminoglycoside induced damage and there
efficacy as therapeutic agents needs to be further tested.
But neutralising ROS only protects against the aminoglycoside induced
pathological changes in cell physiology. It would be better to prevent the
formation of ROS full stop. Understanding how aminoglycosides induce the
formation of ROS is essential and this question will be addressed in the next
section on mitochondria.
Discussion on mitochondria
Exposure to aminoglycosides induces the formation of ROS and leads to
apoptosis in both inner ear hair cells and kidney cells. This suggests an active
role for mitochondria in aminoglycoside induced toxicity. Compromising
mitochondria results in the generation of ROS and the release of apoptotic
factors (discussed in the section on apoptosis). There are numerous studies
which show that mitochondrial function rapidly becomes affected in cells
exposed to aminoglycosides.
The effects of aminoglycosides on mitochondria in haircells
In a study by Dehne et al. it was found that exposure to gentamicin induces a
loss of mitochondrial membrane potential in OHCs .
A correlation is found between aminoglycoside ototoxicity and inhibition of
protein synthesis of mitochondrial ribosomes . This will be discussed in
more detail further on. Metabolic imaging of the organ of Corti revealed that
gentamicin decreases the level of NADH in outer hair cells but not inner hair
cells . This suggests that aminoglycosides interfere with mitochondrial
metabolism and that the effects on aminoglycosides on inner and outer hair
cells are different. Tiede et al. exposed their preparation for one hour which is
definitely more than enough time for OHCs to load up with aminoglycosides
but not for IHCs probably due to differences in MET conductivity.
It has been reported that aminoglycosides lead to reduced mitochondrial
respiration in the inner ear and in the kidney .
A study by Owens et al. shows an impressive amount of evidence that
demonstrates a clear role for mitochondria in aminoglycoside induced
ototoxicity. Within zebra fish lateral line hair cells ultrastructural analysis
revealed structural alteration among hair cells within 15 minutes of neomycin
exposure. This in itself suggests fast uptake of aminoglycosides, i.e. it
suggests uptake through MET channels as opposed to endocytosis. Animals
exposed to low, 25 micromolar neomycin exhibited hair cells with swollen
mitochondria, but little other damage. Quantification of the types of alterations
observed indicated that mitochondrial defects appear earlier and more
predominantly than other structural alterations. In vivo monitoring
demonstrated that mitochondrial potential decreased within 30 minutes in the
presence of 50 µM neomycin. These results indicate that perturbation of the
mitochondria is an early event in aminoglycoside-induced damage. The most
prevalent effect observed is mitochondrial swelling, mitos within HCs exposed
to neomycin are qualitatively less electron dense with fewer cristae present.
Also, mitochondria surrounding the nuclei appear more affected than those
located distantly within the HC . If aminoglycosides do not interact with
mitochondria directly then the cellular events occurring prior to the
mitochondrial response must occur rapidly (< 15 minutes) which seems highly
Discuss our results with isolated mitochondria in the presence of neo
There are EM studies showing changes in mitochondrial structure in
response to aminoglycoside treatment . It was shown that GTTR co-
localizes with mitochondria in HCs . According to Ding et al. gentamicin
associates with lysosomes . But if you look at their figure 1 it looks as if
actually mitochondria are labelled instead of lysosomes.Results by Ding et al.
show that tritium labeled kanamycin co-localizes with mitochondria in hair
These results show that exposure to aminoglycosides leads to the dissipation
of the mitochondrial membrane potential in hair cells. Aminoglycosides co-
localize with mitochondria, mitochondrial structure changes in response to
aminoglycoside exposure. NADH levels drop and respiration decreases.
This suggests that aminoglycosides target mitochondria, compromising their
structure and function.
Congenital thyroid dysfunctions are associated with hearing loss. Upon
treatment with thyroid extract patients showed improvement of hearing,
it has been shown that thyroid hormone controls mitochondrial function by
regulating the production of nuclear- and mitochondrial encoded
mitochondrial proteins . Although not directly related to aminoglycoside
induced toxicity this study does show that proper mitochondrial function is
critical for hair cells. Perhaps thyroid extract can alleviate aminoglycoside
The effects of aminoglycosides on mitochondria in kidney cells
It has been reported in cultured kidney cells that the presence of gentamicin
leads to a significant reduction of mitochondrial membrane potential (after 4
and 8 hours of exposure). In the same study it is shown that AGs can be
trafficked via retrograde transport through both the Golgi complex and ER to
subsequently be released into the cytosol and interact with other organelles,
such as mitochondria. Apparently the appearance of gentamicin in the cytosol
coincides with the decrease of mitochondrial membrane potential .
According to Servais et al. gentamicin traffics to the intermembrane space of
mitochondria . Figure 5E in  suggests that gentamicin goes into the
intermembrane space, but not into the matrix. Apparently gentamicin
associates with the outer membrane and the intermembrane space of
It has been reported that aminoglycosides lead to reduced mitochondrial
respiration in the inner ear and in the kidney . Aminoglycoside poisoning
has been shown to selectively inhibit mitochondrial function in kidney .
Aminoglycosides have been shown to form free radicals in isolated kidney
mitochondria . It was found that gentamicin enhances hydrogen peroxide
production in a dose dependent fashion in isolated kidney and liver
These results are similar to those presented in the previous section on hair
cell mitochondria. When kidney cells are exposed to aminoglycosides it can
be seen that they co-localize with mitochondria, that the mitochondrial
membrane potential is dissipated, that respiration decreases and that ROS
The results from studies done on hair cells and kidney cells show that
aminoglycosides target the mitochondria. In the next section we will look at
the interaction between aminoglycosides and mitochondria.
Interaction of aminoglycosides with mitochondria
In the previous section it was discussed that within both hair cells and kidney
cells aminoglycosides co-localize with mitochondria [14, 62, 63, 65, 66]. Do
aminoglycosides interact directly with isolated mitochondria? Rustenbeck et
al. report that spermine and aminoglycosides can compete with each other for
binding sites on mitochondria . Salvi and Toninello report that there are
binding sites for polycations on the inner mitochondrial membrane (IMM) .
Weinberg et al. suggest that aminoglycosides interact with mitochondria at the
IMM . Walker et al. report that gentamicin causes morphological changes
in mitochondrial membrane . It has been reported that astrocytes in
culture develop mitochondria with strange shapes. The same study showed
that in the presence of AGs the occurrence of ‘strange’ mitochondria
increased . Rustenbeck et al. report that aminoglycosides induce a
depolarization of the IMM .
Work done by Mather and Rottenberg showed that aminoglycoside induced
release of soluble mitochondrial intermembrane proteins (SIMP) is inhibited
by cyclosporin A (CsA) to various extents depending on which AG used.
CsA is an inhibitor of the mitochondrial permeability transition (MPT), an event
that precedes apoptosis (insert REF). MPT can be triggered by destruction of
the mitochondrial outer membrane. CsA is highly effective with streptomycin
whereas gentamicin induced SIMP release is inhibited partially and neomycin
induced SIMP release is inhibited only slightly by CsA . Work by Dehne et
al. showed that CsA provides partial protection against gentamicin toxicity
. These results show that aminoglycosides have different direct effects on
mitochondria dependent on the AG used. From this it can be concluded that
the aminoglycosides can be ranked according to their mitotoxic potential:
neomycin > gentamicin > streptomycin
This corresponds remarkably well to the ototoxic ranking potential as found by
Kotecha and Richardson:
Neomycin > gentamicin > dihydrostreptomycin > amikacin > neamine >
The fact that CsA prevents release of SIMP strongly suggests that
aminoglycosides damage the mitochondrial outer membrane inducing MPT.
The fact that CsA is highly effective with streptomycin, partially effective with
gentamicin and almost not effective with neomycin could be explained in
Streptomycin, gentamicin and neomycin induced SIMP release occurs via
different reaction mechanisms.
Streptomycin, gentamicin and neomycin induce SIMP release via the same
reaction mechanism but neomycin is far more reactive than gentamicin and
(Corne and Charley, can you think of other alternative interpretations?)
How do aminoglycosides interact with mitochondria?
The release of SIMP can be induced by damaging the outer mitochondrial
membrane (OMM). Do aminoglycosides destroy this membrane directly? One
event which can trigger apoptosis is the influx of calcium into mitochondria.
This causes the inner compartment (the matrix) to swell. The IMM has a large
surface area confined within the intermembrane space. If water is drawn into
the matrix it can expand to a sphere. The OMM however cannot further
expand, i.e. when matrix volume starts to increase the OMM is disrupted.
Do aminoglycosides destroy the OMM indirectly by inducing calcium influx?
There is evidence for both mechanisms, which will be discussed in the next
Do aminoglycosides damage the OMM directly?
It has been reported that aminoglycosides bind strongly to phosphoinositides
(e.g. PIP2) which are constitutive components of all membranes [19, 20].
Jane Bryant found that hair cells from mice with a mutation for inositol lipid
phosphatase (Ptprq) were hypersensitive to aminoglycoside exposure (Jane
Bryant thesis). This suggest that increased levels of PIP2 makes the hair cells
more sensitive to aminoglycoside induced damage. With increased levels of
PIP2 there will be more binding sites for aminoglycosides?
Sundin et al  report that gentamicin inhibits phospholipid degradation,
which suggests that aminoglycosides can interact with membranes directly.
(Do Sundin et al explore how gentamicin inhibits the degradation?)
(Inhibition of phospholipases?)
Mather and Rottenberg investigated the interaction between aminoglycosides
and the anionic phospholipids phosphatidyl-inositol (PI) and cardiolipin (CL)
. The major anionic phospholipid in the OMM is PI whereas in the IMM the
mayor anionic phospholipid is CL. For both PI and CL the following ranking
order in affinity was found:
neomycin > gentamicin > streptomycin
The binding affinity for mitochondrial membranes reflects the propensity with
which these aminoglycosides are able to induce SIMP release. It also reflects
the ototoxic potential of these aminoglycosides when compared to the results
by Kotecha and Richardson .
These results strongly suggest that after aminoglycosides enter the cell they
interact directly with mitochondrial membranes inducing apoptosis.
The differences between toxic efficacy of aminoglycosides can be explained
by the differences in their binding affinities for mitochondrial membranes.
Do aminoglycosides stimulate calcium uptake?
It has been reported that polyamines affect calcium transport into
mitochondria . Rustenbeck et al. found that aminoglycosides stimulate
electrogenic uptake of Ca2+
by mitochondria [68, 73]. They also found that
aminoglycosides lead to decreased velocity of calcium uptake in mitochondria
but an increased accumulation of calcium . These studies looked
specifically at calcium uptake and not at mitochondrial damage or ensuing
apoptosis. Nevertheless, aminoglycosides appear to induce increased
calcium accumulation in mitochondria which can be a trigger for apoptosis.
Find more publications on aminoglycoside induced calcium uptake
Do aminoglycosides induce mitochondrial ROS formation?
Both hair and kidney cells, when exposed to aminoglycosides show
generation of ROS. Mitochondria naturally generate small amounts of ROS
during the process of oxidative phosphorylation. These levels are so low that
natural anti-oxidants such as glutathione can neutralise them. When the
electron transfer chain in mitochondria becomes compromised high levels of
ROS are generated which cause intracellular damage and can induce
apoptosis. Do aminoglycosides interfere with the process of oxidative
It was found by Rustenbeck et al. that oxygen consumption rate under state 4
conditions in liver mitochondria was inhibited to 49.1 ± 4.7 % of control the
rate (9.2 ± 0.4 nmol O2 min-1
* mg of protein-1
) by 250 µM gentamicin .
Weinberg and Humes however found that gentamicin stimulates state 4
respiration and inhibits state 3 and uncoupled respiration in renal cortical
mitochondria . Their findings are corroborated by the work of Walker et al.
who report that gentamicin alters mitochondrial respiration (stimulation of
state 4 and inhibition of state 3) .
Tiede et al. found that metabolic imaging of the organ of Corti revealed that
gentamicin decreases the level of NADH in outer hair cells . Which
suggests dysfunction of the electron transport chain.
It has been reported that aminoglycosides lead to reduced mitochondrial
respiration in the inner ear and in the kidney . Aminoglycoside poisoning
has been shown to selectively inhibit mitochondrial function in kidney .
These results indicate that aminoglycosides interfere with the process of
oxidative phosphorylation which can induce ROS formation. This suggests
that the aminoglycosides induced formation of ROS in both hair and kidney
cells is mitochondrial in nature.
The role of mitochondrial DNA mutations in aminoglycoside induced
Carriers of the 1555 mutation of mitochondrial ribosomal RNA are more
susceptible to AG induced hearing loss. This mutation is present in all
mitochondria of a person affected [19, 67, 75].
The 1555 mutation is inferred to introduce an extra base pair at the
penultimate stem of the mitochondrial 12S rRNA which may create more
space in the are of the ribosome for aminoglycoside binding .
A mitochondrial mutation disrupting the penultimate stem in which 1555G
resides confers paromomycin resistance in yeast . Individuals with the
1555 mutation in mitochondrial ribosomal RNA are extremely sensitive to
aminoglycosides. A single injection may induce deafness. Interestingly
enough the vestibular system in these people seems not to be affected . A
correlation is found between aminoglycoside ototoxicity and inhibition of
protein synthesis of mitochondrial ribosomes .
It has been found that in one individual with a strong familial history of
aminoglycoside induced hearing loss and the A1555G mutation detailed
vestibular examination revealed severe hearing loss but completely normal
vestibular function . Could it be the case that there is only one
documented case of this?
The mitochondrial mutation A3243G is associated with both hearing loss and
diabetes mellitus . The A1555G mutation on its own can lead to deafness
. Individuals with the mitochondrial A1555G mutation show susceptibility
to aminoglycoside induced cell death only in their cochleal hair cells and not in
their vestibular hair cells .
It has been found that the A1555G mutation lies exactly in the region of the
gene for which resistance mutations in yeast and tetrahymena have been
described and in which aminoglycoside binding has been documented in
All mtDNA mutations associated with non-syndromic hearing loss involve
ribosomal or transfer RNA, i.e. none of the known mtDNA mutations cause a
structural change in any of the 13 proteins encoded by the mitochondrial
Mitochondria are very similar to bacteria. Mitochondrial and bacterial
ribosomes are structurally more similar than mammalian ribosomes.
Aminoglycosides have a higher affinity for bacterial and mitochondrial
ribosomes than for mammalian ribosomes. Aminoglycosides will interfere with
mitochondrial protein synthesis. But this cannot account for the rapidity with
which aminoglycosides destroy hair cells. Individuals with the A1555G
mutation can go deaf spontaneously, in the absence of aminoglycosides. This
suggests that hair cells are highly dependent on mitochondria. A condition
that weakens the mitochondria will make the individual susceptible to
ototoxicity. Exposure to aminoglycosides will lead to rapid destruction of
already vulnerable mitochondria.
In other words, aminoglycoside induced ototoxicity is not due to interference
with mitochondrial protein synthesis.
There is an abundance of evidence showing that aminoglycosides target the
mitochondria within both hair and kidney cells. Aminoglycosides co-localize
with mitochondria. Aminoglycosides induce loss of mitochondrial membrane
potential, release of apoptotic factors, generation of ROS and interference
with oxidative phosphorylation.
Compromising mitochondrial structure and function induces apoptosis. A
process which will be discussed in the next section.
Discussion on apoptosis
It is generally accepted that cell death occurs via either necrosis or apoptosis.
Necrosis is cell death due to factors external to the cell, e.g. infection, toxins
or trauma. Necrosis is detrimental and elicits an immune response. Apoptosis
on the other hand is the process of programmed cell death which does not
elicit an immune response. Apoptosis is characterized by morphological
changes to the cell (blebbing, cell shrinkage, nuclear fragmentation, chromatin
condensation en chromosomal DNA fragmentation). Apoptosis is caused by
triggering one or both apoptotic pathways: the death-receptor pathway
(extrinsic) and/or the mitochondrial pathway (intrinsic). The death-receptor
pathway originates with the activation of caspase-8 which activates caspase-3
which is the key mediator of apoptosis in mammalian cells. The mitochondrial
pathway activates caspase-9 which then activates caspase-3.
Mitochondria induced apoptosis is characterized by the following features:
Decrease of mitochondrial membrane potential, triggering of the mitochondrial
permeability transition, release of pro-apoptotic factors (e.g. cytochrome c).
There are several causes that can lead to mitochondria induced apoptosis:
increased levels of mitochondrial calcium, interaction of ROS or
aminoglycosides with mitochondria or combinations of these.
(Write a more detailed introduction on apoptosis)
Aminoglycosides at therapeutic levels can induce apoptosis. In the next
section aminoglycoside induced apoptosis in hair cells will be discussed.
Aminoglycoside induced apoptosis in hair cells
Various studies show that exposure to aminoglycosides lead to morphological
changes characteristic of apoptosis in hair cells [7, 8, 19, 56, 61, 78-80]. No
signs of inflammation are described in the inner ear after exposure to
aminoglycosides  which suggests that aminoglycosides do not induce
In the previous section it was discussed that mitochondria play an important
role in aminoglycoside induced ototoxicity. Is there any evidence showing that
aminoglycoside induced apoptosis in hair cells is mitochondrial in nature?
One of the hallmarks of mitochondrial induced apoptosis is the release of
apoptogenic factors by mitochondria. It has been reported that exposure to
aminoglycosides leads to the release of cytochrome c vestibular hair cells [80,
81]. What about other factors like apaf1 and adenylate kinase?
Find more references. It has been reported that elevation of intra-cellular
calcium levels is implicated in the release of cytochrome c .
Several studies have shown that exposure to aminoglycosides leads to
increased levels of calcium concentration in hair cells [46, 63]. Find more
Another hallmark of mitochondrial induced apoptosis is the dissipation of the
membrane potential across the mitochondrial inner membrane. Several
studies have demonstrated that exposure to aminoglycosides leads to the
dissipation of mitochondrial membrane potential in hair cells [56, 60].
Find more studies on hair cell mitochondrial membrane potential.
Another marker for the mitochondrial apoptosis pathway is the activation of
caspase-9. Release of cytochrome c leads to activation of caspase-9 .
Several studies showed that the general caspase inhibitors such as BAF and
z-VAD.fmk prevent aminoglycoside induced hair cell degeneration [61, 78]
which demonstrates that aminoglycoside exposure leads to apoptosis.
Cunningham et al. report that treatment with neomycin leads to caspase-9
activity primarily, with only slight caspase-8 (the extrinsic pathway) activity
. Their study showed that incubation with caspase-9 inhibitors protect
against neomycin induced cell damage whereas caspase-8 inhibitors had no
effect. Also, inhibition of caspase-9 prevented activation of caspase-3. Cheng
et al. found that gentamicin exposure leads to significant activation of
caspase-9. It was also found that caspase-8 was activated by gentamicin but
anti-body staining did not show any staining for caspase-8. They conclude
that the extrinsic pathway does not play a key-role during aminoglycoside
induced ototoxicity . Zhai et al. reported that caspase-8, apart from directly
stimulating downstream caspases can also induce release of cytochrome c
from mitochondria. Apparently caspase-8 interacts with mitochondria indirectly
using Bid as an intermediate . This suggests that both caspase-8 and
caspase-9 interact with mitochondria, i.e. aminoglycoside exposure will
always lead to mitochondria induced apoptosis. Investigate further. Loss of
mitochondrial membrane potential is another characteristic of the intrinsic
pathway. It has been shown that exposure to aminoglycosides leads to
dissipation of mitochondrial membrane potentials in hair cells [56, 60].
Mitochondrial permeability transition (MPT) is an event occurring in
mitochondria which precedes dissipation of mitochondrial membrane potential
and the release of cytochrome c. Compounds like Cyclosporin A (CsA) and
minocycline prevent MPT and the release of cytochrome c. Zhu et al. report
that minocycline protects against gentamicin induced hair cell loss by
preventing MPT and cytochrome c release . Dehne et al. found that
incubation with CsA partially protected against gentamicin induced hair cell
damage . Find more references
From the evidence presented in the section on mitochondria it is clear that
aminoglycosides target the mitochondria in hair cells. The evidence outlined in
this section clearly shows that aminoglycoside induced apoptosis in hair cells
occurs through the intrinsic pathway, which further corroborates the
hypothesis that aminoglycoside induced ototoxicity is mediated via the
Is there any evidence that aminoglycoside induced nephrotoxicity is also
mediated through mitochondria?
Aminoglycoside induced apoptosis in kidney cells
Gentamicin induces apoptosis in proximal tubule epithelium at low
therapeutically relevant doses, whereas at supra-therapeutic doses extensive
necrosis is observed . This was corroborated by Servais et al. who found
that cultured kidney cells went into apoptosis when incubated with gentamicin
at concentrations up to 3 mM, whereas at higher concentrations the cells
started showing signs of necrosis .
Incubation with gentamicin shows typical apoptosis characteristics in kidney
cells: shrunk cells displaying segregation of chromatin into discrete clumps
abutting the nuclear membrane, whereas cytoplasmic organelles most often
kept a normal appearance. Apoptotic bodies consisting of membrane-bound
entities, containing intact organelles together with condensed chromatin were
observed. The occurrence of a typical apoptotic process in gentamicin treated
cells was further characterised by demonstration of fragmented DNA.
Apoptosis developed linearly with time and gentamicin concentration in kidney
In LLC-PK1 cells (kidney cell line) incubation with gentamicin leads to the
following chain of events: after 2h gentamicin appears to be released from
lysosomes, after 10h loss of mitochondrial membrane potential, after 12h
release of cytochrome c and activation of caspase-9, after 16 to 24h later
caspase-3 activity and appearance of fragmented nuclei .
Overexpression of Bcl-2 and co-incubation with cycloheximide prevents
gentamicin induced apoptosis in kidney cells (MDKC, not LLC-PK) .
Apoptosis was associated with increased activity of caspases. Bcl-2
transfectants showed no increase in caspase activities and Z-VAD.fmk
afforded full protection against gentamicin induced apoptosis .
These results clearly show that aminoglycoside induced apoptosis in kidney
cells is similar to that in hair cells. Clearly aminoglycoside induced
nephrotoxicity is also mediated through the mitochondria as is the case in
aminoglycoside induced ototoxicity.
Get more references on kidney apoptosis studies
Aminoglycosides target the mitochondria in both kidney and hair cells.
Aminoglycoside induced apoptosis is mediated by the mitochondria in both
kidney and hair cells. Possible therapeutic treatments aimed at preventing
apoptosis via the intrinsic (mitochondrial) pathway should be considered.
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