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 [1] and a theoretical model with which the rate of aminoglycoside uptake rate can be calculated has been developed [2]. 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 [3]. 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 [4]. It has been demonstrated by Kros et al. that myosin VIIA is required for the normal gating of the MET channel [4]. The myosin VIIA mutation impairs the uptake of gentamicin by hair cells [5]. In other words uptake of aminoglycosides in hair cells which lack resting transducer currents (all MET channels being closed) is limited.

FM1-43

FM1-43 is a styryl pyridinium dye that enters inner ear hair cells through open MET channels [6] and behaves like a permeant blocker [7].Hair cells take up FM1-43 but surrounding (non-transducing) cells show little of nor labelling [6]. Analogously, within the cochlea haircells are damaged by aminoglycosides whereas surrounding (non-transducing) cells remain unharmed [8]. 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 [7]. 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 [7]. Haircells from mice which are homozygous for the myosin VIIA mutation Myo7a6J do not load with FM1-43 [7]. Initially during a dye pulse FM1-43 strongly labels the hairbundles [7]. It is clear from these observations that results obtained from studies with FM1-43 can give insights into aminoglycoside uptake by haircells.

Endocytosis?

It has been reported that aminoglycosides enter haircells via receptor mediated endocytosis [9]. Endocytosis cannot explain why within the cochlea only the haircells degenerate in the presence of aminoglycosides [8] whilst surrounding cells remain unharmed. Also, hair cells which express mutated myosin (important for normal MET gating) do not take up aminoglycosides [5].

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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. [10] 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 [8] 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 [11]. These haircells will not transduce.

Adapted from Figure 1 in [11]

The study by Dulon et al. [10] employs essentially the same method of isolating hair cells, which probably leads to hairbundle damage.

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In another study by Zajic and Schacht [12] isolated outer hair cells appear to have compromised stereocilia (see figures 1-5 in [12]).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) [15].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 [7].Hair cell loss due to AGs starts in the basal coil and progresses apically [16].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 [14]. 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 [17] and OHCs have larger transducer currents than IHCs [18]. 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 coil [19].

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 [20]. 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 [21].The endocochlear potential in mice is only fully developed the 12th day after 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 [7].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 [7]. This could indicate a temperature dependence of the opening of MET channels possibly due to a dependence on myosin ATPase.

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Gentamicin gets taken up quicker by OHCs in the presence of background noise as opposed to in animals maintained under noise-attenuated conditions [19]. This suggests that the MET channels were open during the background noise allowing more AGs in than under noise-attenuated conditions.

The rate of FM1-43 loading into basal coil HCs is much faster than in apical coil HCs [7]. 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 [22]. 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. [1].

Spermine

Spermine is a polyamine which is naturally present in cells [23] 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 [8]. 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 [8].

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 direct surroundings?)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 [24]. These cilia are moved by fluid flow within the lumen of the proximal tubule. Bending these cilia in

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model kidney cells leads to the influx of calcium [25]. The lanthanide gadolinium (Gd3+) is a powerful blocker of the MET current in hair cells [26]. In the presence of Gd3+ bending of the cilium does not lead to influx of calcium [24, 25]. In the presence of Gd3+ and La3+ (another blocker of MET current REF) the uptake of gentamicin in kidney cell lines was decreased [27].

Single channel recordings from renal primary cilia show that single channel conductivity is around 80 pS [28], which is within the range of what is accepted as MET single channel conductance [29]. 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 [30]. This is corroborated by the fact that depolarisation of cultured kidney cells leads to a reduced AG uptake [30], whereas hyperpolarization leads to increased AG uptake [31]. Myosine VIIa is an important protein associated with the MET channel in hair cells [4]. This protein is also present in kidney cell cilia [32].

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 still.

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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 [33]. 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 MET channel.

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 disease [35].

PKD2 localises at the primary cilium of kidney epithelial cells where it is expected to be activated by mechanical stimulation [36].

TRPP2 localizes to both motile and primary cilia and recent evidence strongly implicates it as a mechanosensor in the nonmotile, primary cilia [37]

PKD1 and PKD2 when expressed together in cultured cells form functional ion channels [38]. 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 [39].

Another candidate is the TRPV1 channel. It was shown that TRPV1 regulators mediate the gentamicin uptake by cultured kidney cells [27]. The same study showed that in the presence of Gd3+ or La3+ (inhibitors of mechanoelectrical transduction channel [26] 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 [6]. The uptake of FM1-43 was blocked by ruthenium red [6] which is a specific inhibitor of TRP channels [40] and apparently also a blocker of hair cell transduction [41].

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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 [33], 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 [42] and according to Steyger et al. TRPV1 regulators modulate gentamicin uptake in inner ear hair cells [27].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 [43] which corresponds to the ranking order of ototoxicity found by Kotecha and Richardson [8]: 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 [37]. This is relevant because the MET channel in hair cells is known to interact with PDZ-domain proteins REFS adaptation motor complex

Homozygous mutants (both TRPML3 alleles mutated) do not load with FM1-43 or gentamicin [44]. Although it has been shown that TRPML3 is not the MET channel it still indicates that it could be a TRP channel REFS

Gadolinium (Gd3+) blocks MET in kidney cells and inner ear sensory neurons [26, 27]. Gadolinium is a potent blocker of TRP channels [37].

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.

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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 [16]. A study by Walker et al. showed that gentamicin enhanced production of hydrogen peroxide in a dose-dependent manner in kidney cells [45]. 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 [46] and generation of free radicals in the presence of AGs was demonstrated in explants of the inner ear [47, 48].

This section needs some more refs

Glutathione

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 [19]. Usami et al. showed that GSH levels in both IHCs and OHCs are very low compared to other organ of Corti cells [49]. It has been reported by Sha et al. that glutathione levels in basal OHCs are lower than apical OHCs by [50].

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 information.

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 [51].

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 [52]. According to Feldman et al. N-acteylcysteine protects against

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gentamicin induced ototoxicity. The greatest otoprotective effect was noticed at high frequencies [53], which means that NAC best protects basal OHCs which suggests that ROS levels are higher in basal OHCs than in apical OHCs.

It has been suggested that NAC can help to replenish depleted glutathione content when cells are exposed to elevated oxidative stress [54]. As NAC can cross the blood brain barrier [53] 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 [50]. 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 [50]. 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.

Other antioxidants

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 [55]. 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:

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• Aminoglycosides do not need to undergo a biotransformation to be ototoxic

• Iron chelators also function as radical scavengers.• What is the identity of the iron that aminoglycosides supposedly

complex with?• Is there real, direct evidence of aminoglycosides forming a complex

with iron?• 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 [51]. 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 [51].

Exogenous SOD (superoxide dismutase) protects against AG induced HC degeneration, however exogenous SOD also elicits an immune response [14].Overexpression of superoxide dismutase in inner-ear tissue of transgenic mice prevented kanamycin induced hearing loss [54].

Iron chelators 2,2’-DPD (2,2’-dipyridyl) and Deferoxamine decreased gentamicin induced damage of OHCs in organotypic cultures [56].Iron chelators deferoxamine and DBH reduce gentamicin induced ototoxicity [57] and the efficacy of DBH against gentamicin appears to be dose-dependent [55].

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 [45].

Iron chelators deferoxamine and DHB reduce gentamicin induced damage to the kidney [57]. Another study showed that the iron chelators DFO and DHB reduce gentamicin induced damage to the kidney [55].

N-acteylcysteine can ameliorate gentamicin induced kidney damage [53].

Apparently iron supplementation potentiates gentamicin induced nephrotoxicity in rats [55]. 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 [58].

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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.

Conclusion

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.

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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 [56].

A correlation is found between aminoglycoside ototoxicity and inhibition of protein synthesis of mitochondrial ribosomes [59]. 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 [22]. 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 [56].

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

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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 [60]. 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 unlikely.

Discuss our results with isolated mitochondria in the presence of neo and spec.

There are EM studies showing changes in mitochondrial structure in response to aminoglycoside treatment [61]. It was shown that GTTR co-localizes with mitochondria in HCs [62]. According to Ding et al. gentamicin associates with lysosomes [63]. 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 cells [14].

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 [58]. 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 induced ototoxicity.

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 [64].

According to Servais et al. gentamicin traffics to the intermembrane space of mitochondria [65]. Figure 5E in [66] suggests that gentamicin goes into the intermembrane space, but not into the matrix. Apparently gentamicin

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associates with the outer membrane and the intermembrane space of mitochondria [66].

It has been reported that aminoglycosides lead to reduced mitochondrial respiration in the inner ear and in the kidney [56]. Aminoglycoside poisoning has been shown to selectively inhibit mitochondrial function in kidney [67].

Aminoglycosides have been shown to form free radicals in isolated kidney mitochondria [19]. It was found that gentamicin enhances hydrogen peroxide production in a dose dependent fashion in isolated kidney and liver mitochondria [45].

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 are formed.

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 [68]. Salvi and Toninello report that there are binding sites for polycations on the inner mitochondrial membrane (IMM) [69]. Weinberg et al. suggest that aminoglycosides interact with mitochondria at the IMM [70]. Walker et al. report that gentamicin causes morphological changes in mitochondrial membrane [45]. 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 [71]. Rustenbeck et al. report that aminoglycosides induce a depolarization of the IMM [68].

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 [72]. Work by Dehne et al. showed that CsA provides partial protection against gentamicin toxicity [56]. 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:

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neomycin > gentamicin > streptomycin

This corresponds remarkably well to the ototoxic ranking potential as found by Kotecha and Richardson:

Neomycin > gentamicin > dihydrostreptomycin > amikacin > neamine > spectinomycin [8].

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 several ways:

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 streptomycin.

(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 two sections:

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?

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Sundin et al [66] 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) [72]. 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 [8].

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 [69]. 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 [68]. 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 phosphorylation?

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 [68].

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Weinberg and Humes however found that gentamicin stimulates state 4 respiration and inhibits state 3 and uncoupled respiration in renal cortical mitochondria [74]. 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) [45].

Tiede et al. found that metabolic imaging of the organ of Corti revealed that gentamicin decreases the level of NADH in outer hair cells [22]. 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 [56]. Aminoglycoside poisoning has been shown to selectively inhibit mitochondrial function in kidney [67].

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 ototoxicity

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 [67].A mitochondrial mutation disrupting the penultimate stem in which 1555G resides confers paromomycin resistance in yeast [67]. 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 [76]. A correlation is found between aminoglycoside ototoxicity and inhibition of protein synthesis of mitochondrial ribosomes [59].

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 [77]. 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 [13]. The A1555G mutation on its own can lead to deafness [77]. 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 [63].

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

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described and in which aminoglycoside binding has been documented in bacteria [77].

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 genome [77].

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.

Conclusion

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.

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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 [56] which suggests that aminoglycosides do not induce necrosis. 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 [80].Several studies have shown that exposure to aminoglycosides leads to increased levels of calcium concentration in hair cells [46, 63]. Find more refs.

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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 [82]. 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 [61]. 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 [83]. 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 [84]. 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 [85]. Dehne et al. found that incubation with CsA partially protected against gentamicin induced hair cell damage [56]. 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 mitochondria.

Is there any evidence that aminoglycoside induced nephrotoxicity is also mediated through mitochondria?

Aminoglycoside induced apoptosis in kidney cells

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Gentamicin induces apoptosis in proximal tubule epithelium at low therapeutically relevant doses, whereas at supra-therapeutic doses extensive necrosis is observed [86]. 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 [65].

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 cells [86].

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 [65].

Overexpression of Bcl-2 and co-incubation with cycloheximide prevents gentamicin induced apoptosis in kidney cells (MDKC, not LLC-PK) [86].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 [86].

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

Conclusion

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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References

1. Marcotti, W., S.M. van Netten, and C.J. Kros, The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels. J Physiol, 2005. 567(Pt 2): p. 505-21. Epub 2005 Jun 30.

2. van Netten, S.M. and C.J. Kros, Insights into the pore of the hair cell transducer channel from experiments with permeant blockers, in Mechanosensitive Ion Channels, Pt B. 2007. p. 375-398.

3. Steel, K.P. and C.J. Kros, A genetic approach to understanding auditory function. Nat Genet, 2001. 27(2): p. 143-9.

4. Kros, C.J., et al., Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations. Nat Neurosci, 2002. 5(1): p. 41-7.

5. Richardson, G.P., et al., Myosin VIIA is required for aminoglycoside accumulation in cochlear hair cells. J Neurosci., 1997. 17(24): p. 9506-19.

6. Meyers, J.R., et al., Lighting up the senses: FM1-43 loading of sensory cells through nonselective ion channels. J Neurosci., 2003. 23(10): p. 4054-65.

7. Gale, J.E., et al., FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel. J Neurosci, 2001. 21(18): p. 7013-25.

8. Kotecha, B. and G.P. Richardson, Ototoxicity in vitro: effects of neomycin, gentamicin, dihydrostreptomycin, amikacin, spectinomycin, neamine, spermine and poly-L-lysine. Hear Res, 1994. 73(2): p. 173-84.

9. Hashino, E. and M. Shero, Endocytosis of aminoglycoside antibiotics in sensory hair cells. Brain Res., 1995. 704(1): p. 135-40.

10. Dulon, D., et al., Aminoglycoside antibiotics impair calcium entry but not viability and motility in isolated cochlear outer hair cells. J Neurosci Res., 1989. 24(2): p. 338-46.

11. Williams, S.E., H.P. Zenner, and J. Schacht, Three molecular steps of aminoglycoside ototoxicity demonstrated in outer hair cells. Hear Res., 1987. 30(1): p. 11-8.

12. Zajic, G. and J. Schacht, Comparison of isolated outer hair cells from five mammalian species. Hear Res., 1987. 26(3): p. 249-56.

13. Fischel-Ghodsian, N., R.D. Kopke, and X. Ge, Mitochondrial dysfunction in hearing loss. Mitochondrion, 2004. 4(5-6): p. 675-94. Epub 2004 Nov 6.

14. Ding, D. and R. Salvi, Review of cellular changes in the cochlea due to aminoglycoside antibiotics. The Volta Review, 2005. 105: p. 407-438.

15. Stauffer, E.A. and J.R. Holt, Sensory transduction and adaptation in inner and outer hair cells of the mouse auditory system. J Neurophysiol., 2007. 98(6): p. 3360-9. Epub 2007 Oct 17.

16. Sha, S.H., Physiological and molecular pathology of aminoglycoside ototoxicity. Volta Review, 2005. 105(3): p. 325-334.

23

17. He, D.Z., S. Jia, and P. Dallos, Mechanoelectrical transduction of adult outer hair cells studied in a gerbil hemicochlea. Nature., 2004. 429(6993): p. 766-70.

18. Kros, C.J., A. Rusch, and G.P. Richardson, Mechano-electrical transducer currents in hair cells of the cultured neonatal mouse cochlea. Proc Biol Sci, 1992. 249(1325): p. 185-93.

19. Forge, A. and J. Schacht, Aminoglycoside antibiotics. Audiol Neurootol, 2000. 5(1): p. 3-22.

20. Dai, C.F., et al., Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo. Hear Res., 2006. 213(1-2): p. 64-78. Epub 2006 Feb 8.

21. Kennedy, H.J., et al., Fast adaptation of mechanoelectrical transducer channels in mammalian cochlear hair cells. Nat Neurosci, 2003. 6(8): p. 832-6.

22. Tiede, L., et al., Metabolic imaging of the organ of corti - A window on cochlea bioenergetics. Brain Res, 2009. 6: p. 6.

23. Toninello, A., et al., Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter. J Biol Chem, 1992. 267(26): p. 18393-7.

24. Praetorius, H.A. and K.R. Spring, Bending the MDCK cell primary cilium increases intracellular calcium. J Membr Biol, 2001. 184(1): p. 71-9.

25. Praetorius, H.A., et al., Bending the primary cilium opens Ca2+-sensitive intermediate-conductance K+ channels in MDCK cells. J Membr Biol, 2003. 191(3): p. 193-200.

26. Kimitsuki, T., et al., Gadolinium blocks mechano-electric transducer current in chick cochlear hair cells. Hearing Research, 1996. 101(1-2): p. 75-80.

27. Myrdal, S.E. and P.S. Steyger, TRPV1 regulators mediate gentamicin penetration of cultured kidney cells. Hear Res., 2005. 204(1-2): p. 170-82.

28. Raychowdhury, M.K., et al., Characterization of single channel currents from primary cilia of renal epithelial cells. J Biol Chem, 2005. 280(41): p. 34718-22. Epub 2005 Aug 3.

29. Farris, H.E., et al., Probing the pore of the auditory hair cell mechanotransducer channel in turtle. J Physiol., 2004. 558(Pt 3): p. 769-92. Epub 2004 Jun 4.

30. Myrdal, S.E., K.C. Johnson, and P.S. Steyger, Cytoplasmic and intra-nuclear binding of gentamicin does not require endocytosis. Hear Res., 2005. 204(1-2): p. 156-69.

31. Steyger, P.S., Cellular uptake of aminoglycosides. Volta Review, 2005. 105(3): p. 299-324.

32. Wolfrum, U., et al., Myosin VIIa as a common component of cilia and microvilli. Cell Motil Cytoskeleton., 1998. 40(3): p. 261-71.

33. Corey, D.P., What is the hair cell transduction channel? J Physiol, 2006. 576(Pt 1): p. 23-8. Epub 2006 Aug 10.

34. Ricci, A.J., et al., Mechano-electrical transduction: New insights into old ideas. Journal of Membrane Biology, 2006. 209(2-3): p. 71-88.

35. Hsu, Y.J., J.G. Hoenderop, and R.J. Bindels, TRP channels in kidney disease. Biochim Biophys Acta, 2007. 1772(8): p. 928-36. Epub 2007 Feb 12.

36. Ma, R., et al., PKD2 functions as an epidermal growth factor-activated plasma membrane channel. Molecular and Cellular Biology, 2005. 25(18): p. 8285-8298.

24

37. Pedersen, S.F., G. Owsianik, and B. Nilius, TRP channels: an overview. Cell Calcium., 2005. 38(3-4): p. 233-52.

38. Lin, S.Y. and D.P. Corey, TRP channels in mechanosensation. Curr Opin Neurobiol, 2005. 15(3): p. 350-7.

39. Nauli, S.M., et al., Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nature Genetics, 2003. 33(2): p. 129-137.

40. Christensen, A.P. and D.P. Corey, TRP channels in mechanosensation: direct or indirect activation? Nat Rev Neurosci., 2007. 8(7): p. 510-21.

41. Nagata, K., et al., Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. J Neurosci., 2005. 25(16): p. 4052-61.

42. Zheng, J., et al., Vanilloid receptors in hearing: altered cochlear sensitivity by vanilloids and expression of TRPV1 in the organ of corti. J Neurophysiol., 2003. 90(1): p. 444-55. Epub 2003 Mar 26.

43. Raisinghani, M. and L.S. Premkumar, Block of native and cloned vanilloid receptor 1 (TRPV1) by aminoglycoside antibiotics. Pain., 2005. 113(1-2): p. 123-33.

44. van Aken, A.F., et al., TRPML3 mutations cause impaired mechano-electrical transduction and depolarization by an inward-rectifier cation current in auditory hair cells of varitint-waddler mice. J Physiol., 2008. 586(Pt 22): p. 5403-18. Epub 2008 Sep 18.

45. Walker, P.D. and S.V. Shah, Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria. Am J Physiol., 1987. 253(4 Pt 1): p. C495-9.

46. Hirose, K., et al., Dynamic studies of ototoxicity in mature avian auditory epithelium. Ann N Y Acad Sci., 1999. 884: p. 389-409.

47. Clerici, W.J., et al., Direct detection of ototoxicant-induced reactive oxygen species generation in cochlear explants. Hear Res., 1996. 98(1-2): p. 116-24.

48. Hirose, K., D.M. Hockenbery, and E.W. Rubel, Reactive oxygen species in chick hair cells after gentamicin exposure in vitro. Hearing Research, 1997. 104(1-2): p. 1-14.

49. Usami, S., O.P. Hjelle, and O.P. Ottersen, Differential cellular distribution of glutathione--an endogenous antioxidant--in the guinea pig inner ear. Brain Res, 1996. 743(1-2): p. 337-40.

50. Sha, S.H., et al., Differential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals. Hearing Research, 2001. 155(1-2): p. 1-8.

51. Wu, W.J., et al., Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat. Hear Res., 2001. 158(1-2): p. 165-78.

52. Tylicki, L., B. Rutkowski, and W.H. Horl, Antioxidants: a possible role in kidney protection. Kidney Blood Press Res., 2003. 26(5-6): p. 303-14.

53. Feldman, L., et al., Gentamicin-induced ototoxicity in hemodialysis patients is ameliorated by N-acetylcysteine. Kidney Int., 2007. 72(3): p. 359-63. Epub 2007 Apr 25.

54. Tepel, M., N-Acetylcysteine in the prevention of ototoxicity. Kidney Int., 2007. 72(3): p. 231-2.

55. Song, B.B., D.J. Anderson, and J. Schacht, Protection from gentamicin ototoxicity by iron chelators in guinea pig in vivo. J Pharmacol Exp Ther., 1997. 282(1): p. 369-77.

56. Dehne, N., et al., Involvement of the mitochondrial permeability transition in gentamicin ototoxicity. Hearing Research, 2002. 169(1-2): p. 47-55.

25

57. Song, B.B. and J. Schacht, Variable efficacy of radical scavengers and iron chelators to attenuate gentamicin ototoxicity in guinea pig in vivo. Hearing Research, 1996. 94(1-2): p. 87-93.

58. Hyde, G.E. and E.W. Rubel, Mitochondrial role in hair cell survival after injury. Otolaryngol Head Neck Surg, 1995. 113(5): p. 530-40.

59. Hobbie, S.N., et al., Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity. Proc Natl Acad Sci U S A., 2008. 105(52): p. 20888-93. Epub 2008 Dec 22.

60. Owens, K.N., et al., Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response. J Comp Neurol., 2007. 502(4): p. 522-43.

61. Cunningham, L.L., A.G. Cheng, and E.W. Rubel, Caspase activation in hair cells of the mouse utricle exposed to neomycin. J Neurosci, 2002. 22(19): p. 8532-40.

62. Steyger, P.S., et al., Uptake of gentamicin by bullfrog saccular hair cells in vitro. J Assoc Res Otolaryngol, 2003. 4(4): p. 565-78. Epub 2003 Nov 12.

63. Ding, D., A. Stracher, and R.J. Salvi, Leupeptin protects cochlear and vestibular hair cells from gentamicin ototoxicity. Hear Res., 2002. 164(1-2): p. 115-26.

64. Sandoval, R.M. and B.A. Molitoris, Gentamicin traffics retrograde through the secretory pathway and is released in the cytosol via the endoplasmic reticulum. Am J Physiol Renal Physiol., 2004. 286(4): p. F617-24. Epub 2003 Nov 18.

65. Servais, H., et al., Gentamicin-induced apoptosis in LLC-PK1 cells: involvement of lysosomes and mitochondria. Toxicol Appl Pharmacol., 2005. 206(3): p. 321-33.

66. Sundin, D.P., R. Sandoval, and B.A. Molitoris, Gentamicin inhibits renal protein and phospholipid metabolism in rats: implications involving intracellular trafficking. J Am Soc Nephrol., 2001. 12(1): p. 114-23.

67. Cortopassi, G. and T. Hutchin, A molecular and cellular hypothesis for aminoglycoside-induced deafness. Hear Res., 1994. 78(1): p. 27-30.

68. Rustenbeck, I., et al., Polyamine modulation of mitochondrial calcium transport. II. Inhibition of mitochondrial permeability transition by aliphatic polyamines but not by aminoglucosides. Biochem Pharmacol, 1998. 56(8): p. 987-95.

69. Salvi, M. and A. Toninello, Effects of polyamines on mitochondrial Ca(2+) transport. Biochim Biophys Acta, 2004. 1661(2): p. 113-24.

70. Weinberg, J.M., P.G. Harding, and H.D. Humes, Mechanisms of gentamicin-induced dysfunction of renal cortical mitochondria. II. Effects on mitochondrial monovalent cation transport. Arch Biochem Biophys., 1980. 205(1): p. 232-9.

71. Robert, F. and T.K. Hevor, Abnormal organelles in cultured astrocytes are largely enhanced by streptomycin and intensively by gentamicin. Neuroscience, 2007. 144(1): p. 191-197.

72. Mather, M. and H. Rottenberg, Polycations induce the release of soluble intermembrane mitochondrial proteins. Biochimica Et Biophysica Acta-Bioenergetics, 2001. 1503(3): p. 357-368.

73. Rustenbeck, I., et al., Polyamine modulation of mitochondrial calcium transport. I. Stimulatory and inhibitory effects of aliphatic polyamines,

26

aminoglucosides and other polyamine analogues on mitochondrial calcium uptake. Biochem Pharmacol., 1998. 56(8): p. 977-85.

74. Weinberg, J.M. and H.D. Humes, Mechanisms of gentamicin-induced dysfunction of renal cortical mitochondria. I. Effects on mitochondrial respiration. Arch Biochem Biophys., 1980. 205(1): p. 222-31.

75. Hutchin, T. and G. Cortopassi, Proposed molecular and cellular mechanism for aminoglycoside ototoxicity. Antimicrob Agents Chemother., 1994. 38(11): p. 2517-20.

76. Sinswat, P., et al., Protection from ototoxicity of intraperitoneal gentamicin in guinea pig. Kidney Int., 2000. 58(6): p. 2525-32.

77. Fischel-Ghodsian, N., Hearing loss and mitochondrial DNA mutations: Clinical implications and biological lessons, in Genetics and Auditory Disorders, B.J.B.P. Keats, Arthur N.; Fay, Richard R., Editor. 2002, Springer. p. 228-246.

78. Forge, A. and L. Li, Apoptotic death of hair cells in mammalian vestibular sensory epithelia. Hear Res, 2000. 139(1-2): p. 97-115.

79. Matsui, J.I., J.M. Ogilvie, and M.E. Warchol, Inhibition of caspases prevents ototoxic and ongoing hair cell death. J Neurosci., 2002. 22(4): p. 1218-27.

80. Matsui, J.I., J.E. Gale, and M.E. Warchol, Critical signaling events during the aminoglycoside-induced death of sensory hair cells in vitro. J Neurobiol., 2004. 61(2): p. 250-66.

81. Nakagawa, T. and H. Yamane, Cytochrome c redistribution in apoptosis of guinea pig vestibular hair cells. Brain Res., 1999. 847(2): p. 357-9.

82. Green, D.R. and J.C. Reed, Mitochondria and apoptosis. Science., 1998. 281(5381): p. 1309-12.

83. Cheng, A.G., L.L. Cunningham, and E.W. Rubel, Hair cell death in the avian basilar papilla: characterization of the in vitro model and caspase activation. J Assoc Res Otolaryngol., 2003. 4(1): p. 91-105. Epub 2002 Nov 7.

84. Zhai, D., et al., Characterization of tBid-induced cytochrome c release from mitochondria and liposomes. FEBS Lett., 2000. 472(2-3): p. 293-6.

85. Zhu, S., et al., Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature., 2002. 417(6884): p. 74-8.

86. El Mouedden, M., et al., Gentamicin-induced apoptosis in renal cell lines and embryonic rat fibroblasts. Toxicol Sci., 2000. 56(1): p. 229-39.

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