Aminoglycosides and Deafness review

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A review on aminoglycosides and deafness

A review on aminoglycosides and deafness

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  • 1. Literature summary • Summary of clinical data • Mechanoelectrical transduction • TRP channels • Reactive Oxygen Species (ROS) • Mitochondria • Apoptosis • Aminoglycosides • Various
  • 2. Summary of clinical data Clinical use of aminoglycosides (AGs) to treat bacterial infections is hampered by nephro- and ototoxicity. The use of AGs as first line antibiotics has decreased due to the discovery of non-ototoxic antibiotics. AGs are undergoing a revival at the moment since organisms are starting to show resistance against most first-line agents, e.g. multidrug resistant tuberculosis, complicated nosocomially-acquired (hospital-acquired infection1 ) urinary tract infections. Because of this the clinical use of aminoglycosides is again on the rise which poses clinicians again with the problems of ototoxicity [1]. The toxic effects of aminoglycosides were realised already in the 1940s [2]. To this day the underlying mechanisms with which AGs cause hair cell induced damage are not clearly understood. For the time being industrialised countries could avoid aminoglycoside induced deafness by using non-ototoxic first-line antibiotics which are more expensive than AGs. But now that the usage of AGs can no longer be avoided the research into aminoglycoside induced ototoxicity needs to be stepped up in order to prevent the imminent rise in numbers of people who will suffer complete destruction of the cells within the inner ear, which will lead to hearing disorders and balance problems, with no chance of regenerating these neurons. Studies using stem cell research aimed at regenerating inner ear cells are only just starting to show limited preliminary results and it is estimated that any possible treatment will not be available for at least two decades [3]. A logical consequence is that there will be a large increase in aminoglycoside induced deafness in the foreseeable future. Although the current first-line antibiotics are proving to be less and less efficient, the investment into research and development of new antibiotics by the pharmaceutical industry is actually declining at the moment. It is feared that this decrease in antimicrobial research comes at a time when there is fear of an emerging epidemic of multidrug-resistant, or possibly even pan-drug resistant bacteria [4]. With the lack of newly developed antibiotics and the currently used first line antibiotics becoming inefficient, using aminoglycosides is the only alternative. Thus far the industrialised countries were able to avoid large scale aminoglycoside induced hearing disorders through the use of non-ototoxic antibiotics. Developing countries never had this luxury and aminoglycosides have been used there for decades because of their cost-efficiency [5]. Studies from southern China claim that 2/3 of all deaf-mutism in that area was due to administration of aminoglycosides to children [6]. South Africa has one of the highest incidences of multidrug-resistant tuberculosis (MDR-TB) in the world and aminoglycosides are routinely used [7]. 1 Hospitals are ideal breeding grounds for super resistant microorganisms. 2
  • 3. Aminoglycosides are an integral part of the currently recommended treatment against MDR-TB by the World Health Organization (WHO, 2005) [2]. As South Africa is a low-resource country it is unavoidable that those individuals suffering from MDR-TB will be treated with AGs and as a consequence thereof develop hearing, balance and kidney disorders. To confound this problem it has been found that there is a relatively high occurrence of the A1555G mutation in the Black population [7]. This mutation occurs in the mitochondrial DNA in the gene coding for 12S ribosomal RNA. Individuals expressing this mutated gene develop severe to complete hearing loss after a single dosage of aminoglycosides [8]. The widespread usage of AGs and the increased susceptibility to AGs induced hearing disorders in South Africa necessitates a better understanding of the mechanisms causing AG induced deafness. The aminoglycosides gentamicin and streptomycin are routinely used in Tanzania to treat septicaemia and tuberculosis, the use of non-ototoxic antibiotics being prohibited due to economical reasons [9]. It has been reported that in Nigeria substantial numbers of children incur hearing loss due to the administration of streptomycin [10]. In Angola it is suspected that the use of ototoxic drugs in the management of meningitis and malaria leads to hearing loss in children [10]. Meningitis is very common in the sub-Saharan African countries (all African countries except for Algeria, Egypt, Libya, Morocco and Tunisia). There is in fact a region dubbed the ‘meningitis belt’ which extends from Gambia to Somalia where meningitis epidemics are common and recur every 5-12 years [10]. Aminoglycoside induced ototoxicity is not routinely monitored in Africa, but given the fact that two thirds of the world’s least developed nations are in sub-Saharan Africa it stands to reason that meningitis has to be managed with the cheapest available drugs. The WHO recommends treatment with gentamicin for neonates infected with meningitis 2 . Both tuberculosis and meningitis are life threatening conditions and the occurrence of these diseases is widespread on the African continent. Even without taking into consideration any other conditions treated with aminoglycosides the incidence of aminoglycoside induced hearing loss on the African continent is therefore undoubtedly substantial. In order to be able to on-site monitor the damaging effects of aminoglycoside administration in low resource countries in Africa, economical and simple diagnostic tools need to be used. At present the best means of testing for aminoglycoside induced hearing disorders are otoaccoustic emission (OAE) tests [11]. Here follows a list of conditions treated with aminoglycosides: Bacterial endocarditis [1]. Pseudomonal infections [1]. (Pseudomonal infections opportunistically infects individuals who are immunocompromised leading to urinary tract infections, sepsis, pneumonia, 2 http://www.who.int/csr/resources/publications/meningitis/WHO_EMC_BAC_98_3_EN/en/ 3
  • 4. pharyngitis. Pseudomonas colonizes lungs of patients suffering from cysticfibrosis and contributes to the progressive pulmonary disease.) Peritonitis [1]. Ménière’s disease [11, 12]. AIDS [6]. Tuberculosis [7]. Leukaemia [11]. Visceral Leishmaniasis [13]. Sepsis induced acute renal failure (ARF) [14] AGs are routinely administered to prevent infection in patients with severe burns or large wounds and also in premature babies [15]. AGs have the ability to suppress premature stop codons in Duchenne muscular dystrophy and cystic fibrosis, thereby allowing translation to continue to the normal termination of the transcript [5]. The use of gentamicin has also been shown to be beneficial for the treatment of Hurler’s syndrome [14] Is this a stop codon suppression? - In countries where aminoglycosides are used commonly, aminoglycoside induced ototoxicity is a major cause of hearing loss. One study showed that all deaf-mutes in a district of shanghai, 21.9% had aminoglycoside induced hearing loss, representing 167 individuals in a population of nearly half a million. The A1555G mutation accounted for at least 30% of these. In the USA the A1555G mutation accounts for about 15% of all aminoglycoside-induced deafness cases. This difference may reflect that AGs in the USA are only used for severe in-hospital infections [16]. Gene therapy might be useful. Attenuation of acute ototoxicity of kanamycin/ethacrynic acid has recently been achieved by overexpressing a neutrophin in the guinea pig cochlea [6]. Get the Wu 2002 paper from Guthrie 2008, it should contain info on TBC in developing countries. - There are some medical conditions caused by mitochondrial mutations which have hearing loss frequently as one of their early clinical signs: Kearns-Sayre syndrome, mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS) and mitochondrial encephalomyopathy with ragged red fibers (MERFF) [16]. - An association between diabetes mellitus, hearing loss and mtDNA mutations has been found [16]. 4
  • 5. Mechanoelectrical transduction Introduction Inner ear sensory neurons and kidney cells are preferentially targeted by aminoglycosides. Both cell types express mechanoelectrical transduction channels which allow for a fast entry of aminoglycosides. In this section a summary is given of all the relevant findings on mechanoelectrical transduction in inner ear sensory neurons and kidney cells. Mechanoelectrical transduction of inner ear hair cells - Aminoglycosides enter the hair cells through the MET channels [17, 18]. Are there AGs which are too big to enter through the MET channels? Could non-reactive chemical groups be attached to existing AGs which will prevent them from entering though the MET channels but will still be effective against bacterial infections? - Aminoglycosides also lead to degeneration of vestibular hair cells [2] Has it been shown that aminoglycosides enter the VHCs through the MET channels? - Aminoglycosides lead to destruction of initially OHCs and then IHCs [8] Why this difference? Does this reflect the fact that IHCs have less stereocilia (48) then OHCs (81) ? [19]. Do IHCs therefore have less MET channels than OHCs? - OHC loss far exceeds ICH loss when the cochlea is exposed to AGs [5]. Can this be explained by the fact that OHCs have more MET channels and hence load up with AGs more quickly? - It has been reported that the destruction of OHCs by cisplatin occurs in a base-to-apex gradient [6]. Does this mean that cisplatin enters the HCs through the METs? Does cisplatin affect non-hair cells in the organ of Corti? What does cisplatin do? What is its structure? According to Guy Richardson Schacht et al. did experiments on isolated OHCs which did not respond to neomycin. These isolated OHCs do not transduce, hence there is no uptake of AGs in these cells. On the basis of 5
  • 6. that result it was postulated that AGs form an interaction with an iron complex which generates cytotoxic agents. [20]. - Apparently streptomycin is almost exclusively vestibulotoxic whereas dihydrostreptomycin is almost exclusively cochleotoxic [2]. Why is this? Because of size differences? streptomycin Is there a difference in structure of the cochleal HC MET channel and the VHC MET channel? - Hair cell loss due to aminoglycosides starts in the basal coil and progresses apically [2]. This is not attributable to a concentration gradient because cochleae in culture (i.e. with a homogenous concentration of AGs) display the same behaviour [5]. Therefore the pattern of sensitivity must be based on some inherent properties of the hair cells in different regions of sensory epithelia. Does this reflect a difference in mitochondria (number, size, etc) between basal and apical HCs? Basal OHCs have larger transducer currents than apical OHCs [21]. OHCs have larger transducer currents than IHCs [22]. So if the HC degeneration is dependent upon the entry of AGs through the METs then the difference in conductivity of the METs in basal and apical OHCs can account for the pattern of sensitivity. However, it has also been reported that there is a regional difference in mitochondrial density within the cochlea. Mitochondrial density apparently is higher in the basal turn of the cochlea and within a HC it is higher in the infra-nuclear region [8]. 6
  • 7. Has this been demonstrated though? Apparently the source of this information is from a 2003 ARO abstract by Kopke et al. If there are indeed more mitos in the basal coil then there are more targets for AGs to bind to. - It has been reported that immature hair cells take up less gentamicin than mature hair cells, also mature cells die faster than immature cells upon exposure to AGs [23]. Could this be due to the fact that transducer currents in adult animals are larger than in immature ones? No, transduction current amplitude stays constant during maturation of the animal, see Figure 4 [24]. Is the establishment of the endocochlear potential involved? Increased driving force for AGs to go into the HCs? Since it is postulated by people who do not believe that AGs enter the HCs through the METs that AGs enter cells via endocytosis, how come non HCs are not affected? Isn’t it a coincidence that the only other cells which are affected by AGs in mammals are kidney cells which, just like HCs, also transduce? -It has been reported that AGs enter HCs via receptor mediated endocytosis [25]. Has the identity of these illustrious receptors been confirmed yet? Also, if AGs enter via receptor mediated endocytosis how come cells with mutated myosins do not accumulate AGs? How would myosin molecules be instrumental in receptor mediated endocytosis? - HCs from homozygous Myo7ash1 accumulate high levels of gentamicin, whereas HCs from homozygous Myo7a6J do not accumulate gentamicin [26]. Are there any differences in mechanoelectrical transduction between these two types? Apparently, the shaker-1 mutants have relatively normal transduction, including a resting transducer current, which is absent in the myo7a6j type. Probably AGs can enter cells in general via endocytosis. However, in cells which display mechanotransduction there is an extra port of entry which allows for flooding of the cell with AGs in a relatively short time. - Hair cells take up FM1-43 but surrounding cells show little or no labelling. [27]. 7
  • 8. This indicates that HCs have uptake mechanisms that other cells are lacking, i.e. MET channels serve as routes of entry for many large polycations. It is reported that non HCs in the cochlea are not affected by AGs [28]. This is probably due to the fact that these cells do not transduce. - FM1-43 labelling is much stronger in the basal coil than in the apical coil [27]. How does that compare with AG loading? - FM1-43 competes with AGs for entry into the HCs. The ototoxicity of neomycin was reduced in the presence of FM1-43 [27]. Can FM1-43 prevent entry of AGs into kidney cells? Temperature dependence of FM1-43 or AG entry into HCs may be due to temperature dependence of the opening of MET channels which may be dependent on myosin ATPase [27]. - TRP channels interact with PDZ-domain containing scaffold proteins [29]. This suggests a role for TRP channels in the process of mechanotransduction since PDZ-domain proteins are known to be important components as part of the adaptation-motor complex. Are there PDZ domains in kidney cells? - According to Dulon et al. [30] isolated OHCs (by means of trituration) are not affected by the presence of 5 mM gentamicin when incubated for 6 hours. When you look at the paper by Kotecha and Richardson it is very clear that in cochleal cultures in the presence of 1 mM gentamicin for 1 hour the OHCs are severely damaged [28]. This suggests that transduction is negatively affected in the isolated OHCs in Dulon’s study. - Gentamicin gets taken up quicker by OHCs in the presence of background noise as opposed to in animals maintained under noise-attenuated conditions [6]. Channels were open because of the noise? 8
  • 9. - According to Gale et al. within the apical coil there is a gradient of FM1-43 loading during development of cochlea in culture. What aspect of development influences the uptake of FM1-43? [27]. - The rate of FM1-43 loading in BC HCs is much faster than in AC HCs [27]. FM1-43 loading is inhibited by incubation with EGTA, which breaks tip- links [27]. Does EGTA prevent AG induced HC damage? Are there other methods that break sever tip-links? Do they prevent AG induced HC damage? Myo7a mutant hair cells do not load with FM1-43, unless stimulated Mechanically [27]. - Apparently type I HCs are generally more sensitive than type II HCs [31]. - When incubated with gentamicin it first labels the stereocilia of hair cells before diffusing through the cell [32]. This suggests a role for the MET channels in AG uptake. - Spermine causes HC damage, whereas other cells in the organ of Corti are not affected [28]. Does this mean that spermine specifically targets HCs entering through the MET? - Meyers [33] suggest that in mature mice FM1-43 loses access to the endolymph. This might explain why mature mice, in vivo, are not affected (that much) by aminoglycosides. - It can be seen that loading of FM1-43 in control HCs is immediate whereas in homozygous Myo7a6j HCs no immediate loading is seen [27]. Is there any loading if you let the culture incubate with FM1-43 (or AGs) for several hours? Myo7a6J does not have a resting IT. - In the vestibular organ AGs affect type I hair cells before type II hair cells [6]. Do type 1 and type 2 hair cells have different MET conductances? - In explants of vestibular organs from mature animals exposed in culture to AGs are preferentially susceptible in the central regions of the epithelia with damage spreading to the periphery with increased time of exposure [6]. Read the Forge and Li paper 9
  • 10. Depolarization of HCs (organotypic cultures) by incubation with high extracellular concentrations of K+ did not block the effects of neomycin [34]. Reducing the driving force for AGs to go into the HCs should attenuate the effects. Was the membrane potential completely dissipated? Even at depolarized values of the membrane potential (-20 mV) a concentration of 50 µM DHS effectively blocks the MET current, which means it can also enter the HCs [17]. In the Richardson and Russell study a concentration of 200 µM was used. This suggests that under depolarized conditions AGs can easily enter the HCs and cause damage. - Immunolabeled gentamicin and GTTR were observed at the apical membranes of HCs particularly in their hairbundles [15]. The Steyger 2003 paper has many good REFS - Steyger claims he would test the hypothesis that GTTR can permeate cation channels [15]. Did they? Clearly neomycin and gentamicin have different blocking potencies with neomycin being the more potent blocker. Does this reflect the ototoxic potential as seen in Kotecha’s study? Dihydrostreptomycin has a binding site at 0.8 relative electrical distance [18]. Neomycin and gentamicin have binding sites at 0.5 and 0.7 relative electrical distance (Kros unpublished). Could it be that neomycin binds the easiest to the MET channel, making it the most potent blocker and also the most potent ototoxic AG? 10
  • 11. Our results with isolated mitochondria suggest that neomycin is very reactive with mitochondria. Still perhaps neomycin also has the fastest uptake rate. - It was shown that gentamicin ototoxicity was prevented by reducing the endolymphatic potential [35]. This suggests that gentamicin uptake is dependent on the presence of a potential difference, i.e. uptake requires a driving force which means that AG uptake is electrophoretic. - According to Takada et al OHCs have a more negative membrane potential than IHCs [35]. This could explain also why OHCs are more sensitive to AG induced ototoxicity than IHCs.  get a recent ref about OHC and IHC membrane potentials. Read Marcotti 2003 and Housley 2006. Song et al report that in gentamicin treated guinea pigs higher frequencies were more affected (higher threshold shift at 18 kHz than at 3 kHz). This was corroborated by the fact that OHCs in the basal coil were more affected than OHCs in the apical coil [36]. 11
  • 12. Table 1 taken from [37] shows the similarity between aminoglycoside uptake by bacteria and mammalian cells which clearly suggests that uptake occurs through ion channels, potentially via TRP channels. It was found that aminoglycoside induced cytoplasmic elevation of Ca2+ in hair cells occurs rapidly (<10 seconds) [37]. This suggests immediate uptake of AGs, i.e. via ion channels, not endocytosis. Read Staecker 1997. Aminoglycosides are present in hair cell cytoplasm 15 minutes following systemic injection, 2 hours before endosomal localization of AGs can first be identified [37]. Again this suggest fast uptake, i.e. via ion channels. Read Dai 2006. It has been reported that perilymphatic perfusion of the scala tympani with aminoglycoside-laden artificial perilymph did not affect hair cell transduction until 24 hours after perfusion commenced. This suggests that aminoglycosides enter apically. - From figure 7A in [15] it can be seen that supporting cells show very little labeling with AGs in comparison to HCs. - It was found that OHCs are most sensitive to gentamicin toxicity followed by IHCs [38]. - Supporting cells (Deiters and Hensen cells) were not affected by gentamicin exposure [38]. - Schacht claims that AGs destroy both sensory and supporting cells in the organ of Corti [39]. Does he show any evidence of the destruction of supporting cells in any of his work? - OHCs exposed to neomycin (1 hour) show blebbing. Whereas OHCs pre- treated with calcium chelators are protected against AGs [27]. The blebbing suggests that these cells go into apoptosis. The exposure to neomycin was 1 hour, during that interval, when did the blebbing start? Also, if there is any endocytosis of AGs then this process takes longer than 1 hour since the OHCs pre-treated with calcium chelators did not show any blebbing. It could of course be the case that AGs were in fact getting into the hair cells through endocytosis. Again it would be useful to know when exactly the blebbing started in the experiment with just neomycin present. Suppose AGs do enter HCs also via endocytosis, 12
  • 13. how slow/fast is this process and how long does it take to reach a steady state AG concentration within the HC? Calcium chelators prevent apoptosis through tip-link breakage? Sha and Schacht [40] found that aminoglycoside induced hearing loss is most severe at high frequencies. When determining threshold shifts in guinea pigs treated with gentamicin they found: 61 ± 2 dB at 18 kHz 41 ± 13 dB at 9 kHz 37 ± 23 dB at 3 kHz This means that basal hair cells are more sensitive to aminoglycosides than apical hair cells. - Aminoglycosides are initially apparent at the apical end of hair cells [6]. - When incubated with gentamicin it first labels the stereocilia of hair cells before diffusing through the cell [32]. - Neomycin causes more morphological damage basally than apically in cochlear cultures [28]. Hirose et al found that basal coil hair cells are more sensitive to gentamicin induced cell death than apical coil hair cells [41]. This indicates that basal coil haircells have a faster AG uptake rate than apical coil HCs. This can explained by the difference in MET channel conductivity. Cochleotoxic effects of AGs is characterized by a hearing loss initially confined to the high frequencies corresponding to hair cell damage in the lower basal turn [6]. Changes in level of acoustic distortion product emissions were detectable before other physiological measures revealed significant effects, indicating an early effect on OHCs. Changes in the response were first detected with tones of high frequency [6]. Age and noise exposure have been claimed to enhance ototoxicity [6]. This suggest that during noise MET channel conductivity is increased and more AGs and other ototoxic agents can enter the HCs. In the rat the ototoxic effect of kanamycin was weak before the onset of cochlear potentials, but strong thereafter [6]. Difference in driving force Postmortem studies on cystic fibrosis patients with signs of ototoxicity confirmed loss of HCs in the lower cochlear turns [6]. 13
  • 14. The GT and GTTR fluorescence in the cell bodies of both IHCs and OHCs were similar in contrast to the greater susceptibility of OHCs to aminoglycosides [42]. This argues against the idea that IHCs are less susceptible to AG ototoxicity due to a lower MET conductivity when compared to OHCs. Read Taylor 2008 who did similar experiments with FM1-43. GM uptake in haircells is begins in the base and spreads to the apex. Uptake is first seen in in OHCs initially in the 3rd row after two days of treatment and then in IHCs after 8 days of treatment [43]. Apparently EA accelerates the influx of GM from the stria vascularis into endolymph followed by entry through the apical surface of the hair cell into the cytoplasm [43]. Avian hair cells accrued GTTR more rapidly at the base of the basilar papilla [23]. Guinea pig OHCs accrued more GTTR fluorescence than IHCs [23]. Mechanoelectrical transduction of kidney cells - Bending of the cilium in model kidney cells leads to influx of calcium [44]. What is the route of entry of the calcium ions ? Also, is the membrane potential measured in this study or any other study where the cilium is mechanically stimulated? - Proximal tubule kidney cells exhibit primary cilia [45]. - It takes a few seconds, after bending of the cilium, before intracellular calcium concentration starts to increase [44]. It is hypothesized by Nauli et al. [46] that bending of the cilium induces minimal calcium influx, which then later leads to release of intracellular calcium, cicr, Calcium induced calcium release. - Gd3+ completely blocks the influx of calcium (induced by movement of the cilium) [44]. Does this imply that mechanoelectrical transduction in these kidney cells is inhibited? - It has been reported that in the presence of Gd3+ and La3+ the uptake of gentamicin in kidney cell line cells was decreased [47]. Gadolinium is known to block MET [48] and it is known to block most TRP channels [29]. 14
  • 15. Does this indicate that gentamicin enter these cells through MET channels? Do these cations block TRP channels? Most TRPC channels are inhibited by lanthanides (Gd3+ , La3+ ) however, TRPC4 is activated by micromolar concentrations of extracellular La3+ . Also, TRPC5 is activated by micromolar concentrations of extracellular La3+ or Gd3+ [29]. - Bending of the cilium of MDCK cells in the presence of Gd3+ does not lead to Ca2+ influx [45]. - Single channel recordings from renal primary cilia show that the single channel conductivity is around 80 pS [41]. This conductivity is within the range to what is accepted for the MET single channel conductance [49]  get more refs Unfortunately the Raychowdhury paper does not show if the channels in the primary cilium are permeable to Ca2+ . - It has been reported that within the kidney gentamicin targets proximal tubule cells strongly. Labelling of distal tubules and glomerulus is negligible [23]. In vitro both proximal and distal tubule cells take up AGs. Are there MET channels in proximal tubule cells? Dai et al. suggest that the lack of labelling in distal tubule cells in vivo could be due to: -GTTR uptake by proximal cells reduces availability to distal cells -Distal tubule cells are able to rapidly clear GTTR from the cytoplasm. -The electrophysiological conditions in the nephron lumen changes between the proximal and distal sectors and may not favour uptake of GTTR by distal tubule cells, i.e. different driving forces for GTTR. - It is reported that gentamicin uptake (by cultured kidney cells used as model cells for HCs) is not dependent on endocytosis [50]. This suggests that gentamicin uptake by kidney cells occurs via a channel. Is there in fact real experimental evidence which shows that AGs can enter HCs through endocytosis? - It has been reported that membrane depolarisation of cultured kidney cells reduced the uptake of AGs [50]. Does this mean that entry of AGs into HCs is electrophoretic? Does this also mean that when the endocochlear potential is established the rate of entry of AGs is increased? Can this explain why in vivo HCs in adult animals seem to degenerate at a higher rate than HCs in immature animals? 15
  • 16. - It has been recorded that TRPP2 localises to both motile and primary cilia. It is also hypothesized that TRPP2 functions as a mechanosensor in the nonmotile, primary cilia [29, 51]. Could it be the case that mechanosensation in kidney cells is dependent on TRPP2? Is there expression of other TRP channels in kidney tubule cells? - It has been found in proximal tubule cells that gentamicin localises to both mitochondria and lysosomes. Initially AGs accumulate in the brush border [52]. Does the brush border contain the kidney epithelial cells containing the mechanoelectrical transducing cilia? - It has been found that TRPP channels in kidney cells (PKD1 and PKD2) flux calcium upon stimulation with fluid flow, suggesting the presence of mechanoelectrical transduction [53]  Lin refers to the 2003 Nauli paper. - It has been found that HEK cells expressing TRPV1 channels readily take up FM1-43 when these channels are opened. Ruthenium red, a TRPV1 antagonist, blocked the uptake of FM1-43 into the HEK cells [33]. It is well known that FM1-43 can enter HCs through the MET. The results obtained with HEK cells suggest that the MET might be a TRP channel. Also, does Ruthenium red block HC mechanotransduction? Try superfusing with ruthenium red to inhibit MET currents. - The presence of Myosine VIIa has been confirmed in kidney cell cilia [54]. Do Myosine VIIa knockout kidney cells show decreased AG uptake? We could isolate kidney cells from shaker-1 mice and test for this. - All of the epithelia of the mammalian kidney (except for the intercalated cells of the collecting duct) express a single primary cilium on their apical (lumenal) surface [45]. So not just proximal tubule cells then? Mechanoelectrical transduction of other cell types - It was found that FM1-43 enters various cells (Merkel cells, neurites innervating Merkel cells, nociceptors and enteric neurons, in various structures (hair follicles, skeletal muscles, cornea), all of these structures have mechanoelectrical transduction properties [33]. What is the effect of aminoglycosides on these structures? 16
  • 17. Does FM1-43 enter native kidney cells? - Apparently ryanodine receptors are blocked by AGs [17]. Do kidney cells express ryanodine receptors? - In chick basilar papillae aminoglycoside induced HC damage spreads from proximal to distal, i.e. it starts with the high frequency range HCs similar to what happens in the mammalian cochlea (Bryant thesis). Do proximal HCs have larger transducer currents than distal HCs? - Apparently, chick embryonic basilar papillae demonstrate a pronounced insensitivity to neomycin (Bryant thesis). Lack of transduction? However, FM1-43 does load at the same ages. Nifedipine (inhibitor of low voltage-gated calcium channels, L-type) inhibits the increase of intracellular calcium induced by gentamicin [55]. This suggests the calcium has an extracellular origin. Does this have implications for the MET channel? Does Nifedipine block the channel? Mechanotransduction in HCs from the bullfrog sacculus is blocked in a way that is Ca2+ competitive, reversible and dependent on transmembrane potential [34]. Get the original REF from Hudspeth 1983 and Kroese 1989. GTTR enters zebrafish neuromast hair cells via apical cation channels [42]. Addition of 10 mM calcium significantly reduces GTTR fluorescence intensity in zebrafish neuromast hair cells [42]. Mariner zebrafish has defective myosin VIIa. In mariner zebrafish GTTR fluorescence in most neuromast hair cells was significantly reduced [42]. Hair cell bodies apparently can survive ototoxic treatment without their hairbundles [42]. Read the Gale 2002 paper to see if aminoglycosides were used. The 2009 Wang and Steyger paper has some interesting references. Huang 2000, Khakh 1999, Virginio 1999 and Karasawa 2008. 17
  • 18. TRP channels TRP channels in haircells - TRP channels interact with PDZ-domain containing scaffold proteins [29]. This suggests a role for TRP channels in the process of mechanotransduction since PDZ-domain proteins are known to be important components as part of the adaptation-motor complex. Are there PDZ-domains in kidney cells? Homozygous mutant varitint mice (both TRPML3 alleles mutated) do not load with FM1-43 or gentamicin. Is there an interaction between TRPML3 channels and TRPV1 receptors? Is the MET channel a supercomplex of various TRP subunits? What about endocytosis of AGs in varitint HCs? - It has been reported that in HCs TRPML3 shares a similar expression profile with TRPA1 [29]. Is there interaction between these two types of subunits? Is the MET channel a heteromer of TRP subunits? Are there specific TRPV1 inhibitors? It is worth trying to measure transduction of hair cells in the presence of these inhibitors. How does capsaicin influence mechanoelectrical transduction? Corey [56] doesn’t think the MET channel is a TRPV channel but he doesn’t comment on TRPV1 at all. It is known that TRPV4 can be found in HCs, what about TRPV1 though? It has been reported that TRPV1 is present in OHCs (guinea pig) [57]. The evidence given in the Zheng paper is mostly indirect. The only immuno assay shown is not all that convincing. - TRPV4 is found in hair cells [56]. 18
  • 19. - TRPA channels are permeable to FM1-43 [53]. Are TRPA channels found in hair cells? - It has been reported that TRPA localises in the stereocilia of hair cells and has characteristics reminiscent of the MET channel [58]. - It was found that -/- TRPA knockout mice were not deaf and had transducer currents which were not different from control mice [56]. Upregulation of other TRP channels? Apparently regulators of TRPV-1 modulate gentamicin uptake in inner ear hair cells [47].  a reference is made to a 2004 ARO abstract by Steyger et al. - It has been found that AGs can inhibit TRPV1 in the order: Neomycin ≅ Streptomycin > Gentamicin [59]. How does this compare with the potency of AGs inducing HC damage? Effects of AGs on cultured OHCs: Neomycin>gentamycin>dihydrostreptomycin>amikacin>neamine>spectinomy cin [28]. It would be very interesting to see the effects of these AGs on kidney cells. Would the toxic potency for kidney cells of these AGs be comparable to their ototoxicity? Maybe Charley can do this experiment? Regulators of TRP channels modulate gentamicin uptake in kidney cells [47] and inner ear hair cells. TRP channels in kidney cells - It has been reported that in the presence of Gd3+ and La3+ the uptake of gentamicin in kidney cell line cells was decreased [47]. Does this indicate that gentamicin enters these cells through MET channels? Do these cations block TRP channels? Most TRPC channels are inhibited by lanthanides (Gd3+ , La3+ ) however, TRPC4 is activated by micromolar concentrations of extracellular La3+ . Also, TRPC5 is activated by micromolar concentrations of extracellular La3+ or Gd3+ [29]. Could it be the case that mechanosensation in kidney cells is dependent on TRPP2? Is there expression of other TRP channels in kidney tubule cells? 19
  • 20. - It has been found that TRPP channels in kidney cells (PKD1 and PKD2) flux calcium upon stimulation with fluid flow, suggesting the presence of mechanoelectrical transduction [46]. Does Praetorius say anything about TRPP channels? - It has been found that HEK cells expressing TRPV1 channels readily take up FM1-43 when these channels are opened. Ruthenium red, a TRPV1 antagonist, blocked the uptake of FM1-43 into the HEK cells [33]. It is well known that FM1-43 can enter HCs through the MET channel. The results obtained with HEK cells suggest that the MET might be a TRP channel. Also, does Ruthenium red block HC mechanotransduction? According to Nagata 2005 [58] Ruthenium Red does block hair cell transduction. Read Nagata 2005 When incubated with FM1-43, if left to incubate for a long time, it does enter the Myo7a6j OHCs, i.e. endocytosis does occur, very slow (unpublished results Kros group). - TRPV4 is expressed in epithelial cells of kidney tubules [60]. Is TRPV4 involved in mechanotransduction in kidney tubule cells? - Hsu et al give a breakdown of TRP channels present in the kidney [51], but TRPV1 is not in the list. Is there evidence that TRPV1 is present in kidney cells? - Apparently, TRPML and TRPP channels are similar, much more so than any of the other TRP channels [61]. Could this indicate that kidney diseases are related to ototoxicity through dysfunction of TRP channels? - TRPV1 regulators mediate gentamicin penetration of cultured kidney cells [47]. If AGs inhibit TRPV1 channels and if TRPV1 regulators mediate the entry of gentamicin into kidney cells could it then be that the MET channel is a TRPV1 channel? Are there specific TRPV1 inhibitors? 20
  • 21. It is worth trying to measure transduction of hair cells in the presence of these inhibitors. How does capsaicin influence mechanoelectrical transduction? Corey [56] doesn’t think the MET channel is a TRPV channel but he doesn’t comment on TRPV1 at all. It is known that TRPV4 can be found in HCs, what about TRPV1 though? It has been reported that TRPV1 is present in OHCs (guinea pig) [57]. The evidence given in the Zheng paper is mostly indirect. The only immuno assay shown is not all that convincing. - In the kidney nephron the following TRP channels have been found: TRPC1, TRPC3, TRPC6, TRPP2, TRPV4, TRPV5, TRPV6 and TRPM6. [51]. - PKD1-like subgroup of the TRPP family is somewhat unusual because it has 11 TM domains. It is considered a TRP channel because it assembles with PKD2-like proteins to form functional complexes [51]. That sounds completely wrong to me. - TRPV4 is highly expressed in kidney cells [44]. - Apparently HEK cells do not express TRPV1 [33]. Why else would you express TRPV1 in this system? Perhaps to boost the number of channels? However, according to Cortright et al. TRPV1 is expressed in kidney cells [62]. TRP channels in other cell types - In oocytes TRPC1 was identified as the mechanosensitive cation channel, which transduces membrane stretch into cation currents [29]. This suggests a role for TRP channels in mechanotransduction. Whereas most TRPC channels are inhibited by lanthanides (which suggests a role for TRPC channels in mechanotransduction) TRPC4 and TRPC5 is activated by micromolar concentrations of extracellular La3+ [29]. - One of the physiological functions of TRPV4 is thought to be mechano sensing (including endothelial cell responses to shear stress) [29]. This again may imply that a TRP channel is involved in mechanotransduction. - TRPN1 is an important mechano-transduction channel in C. elegans, Drosophila and zebra fish, but not detected in mammals [29]. 21
  • 22. - Formation of heteromeric TRP channels has been observed. The only heteromer, so far, which combines TRP subunits from different subfamilies is a combination of PKD2/TRPC1 [63]. Is there any evidence that TRPML3 subunits may combine with TRPA or TRPV subunits? It has been demonstrated that TRPML subunits can form heteromers of all combinations of TRPML1, TRPML2 and TRPML3 [61]. - Both TRPN1 and TRPA1 contain many ankyrin repeats in their N-termini. It has been hypothesized that these ankyrin repeats could function as a gating spring [64]. - Ruthenium red blocks TRP channels and also of mitochondrial calcium influx [64]. Is there a relationship between TRP channels and mitochondria? Do TRPML channels associate with PDZ proteins? Apparently gentamicin binds to PIP2 and PIP2 binding to TRPV-1 inhibits cation currents through the channel. Perhaps gentamicin, by binding PIP2 relieves the block on the TRPV-1 channel [47]. Neomycin appears to induce analgesia of dorsal root ganglions (Zhou 2002 as referenced in [47]). TRP channel modulation? - Apparently systemic treatment with AGs does not affect sensory neurons with TRP channels as much as HCs [17]. Maybe because of the high driving force from the endolymph? How do isolated/cultured sensory neurons respond to AGs? 22
  • 23. Reactive Oxygen Species (ROS) ROS in haircells - Aminoglycosides induce ROS and anti-oxidants can protect against AG induced hearing loss [2]. Does the formation of ROS occur through the mitochondria? Do AGs target mitochondria which then produce ROS and go into apoptosis? Or do the AGs induce ROS formation which then triggers the mitochondria to go into apoptosis? How about culturing cochleae incubated with AGs and determining the mitochondrial ∆Ψ per day (per hour) ? - It appears that basal OHCs are much more sensitive to ROS than apical ones.  REF Why? Because of a higher concentration of mitochondria in basal coil HCs than in apical coil HCs? See the Kopke et al. 2003 ARO abstract. More mitos and therefore more targets for aminoglycosides? - Glutathione in vivo, protects against aminoglycosides induced ototoxicity [6]. Glutathione is a cellular antioxidant, does this mean that the level of glutathione in HCs is too low to effectively deal with aminoglycoside induced ROS formation? 23
  • 24. According to Usami et al. [65] the levels of GSH in IHCs are low and within OHCs very low, see figure. . Figure 2 taken from [65] - It was shown that hair cells have lower concentrations of glutathione than neighbouring cells [65]. But quite a few other cochleal cells have low levels of GSH. How come these cells are not as sensitive to AGs as HCs? - It has been reported that administration of both iron chelators (deferoxamine and 2,3-dihydrobenzoate) and ROS scavengers (mannitol) lends complete protection against gentamicin induced HC damage [36]. Iron chelators alone only lend partial protection. It is also reported that these same iron chelators (DFO and DHB) protect against GM induced nephrotoxicity. Does this indicate that there are multiple sites and mechanisms of action underlying aminoglycoside induced HC damage? Also, the Song 24
  • 25. paper mentions that mannitol may scavenge hydroxyl radicals. I didn’t know mannitol was a ROS scavenger? Song et al further hypothesize that gentamicin forms a complex with an iron complex. Does the fact that iron chelators protect against AG induced ototoxicity show that AGs form a complex with iron? Do iron chelators directly neutralize ROS? What if AGs induce ROS which is subsequently neutralized by iron chelators? According to Schacht iron chelators bind iron, decreasing the available concentration of iron for AGs to react with. Is this really plausible? Which iron atoms are targeted by AGs? Also, isn’t it the case that iron chelators are effective with non auditory cells?  Read the Yun 2003 paper. - It has been reported that OHCs in culture show a base to apex viability gradient, i.e. basal OHCs die sooner than apical OHCs in the absence of AGs [66]. What are the inherent differences between basal and apical OHCs (apart from conductivity) that could account for these differences? - It has been reported that glutathione levels in basal OHCs are lower than apical OHCs by [66] however, the method used for quantifying these levels 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. Perhaps Charley can test for ROS and GSH levels in basal and apical coils? Is the base – apex pattern of viability as seen in OHCs also present in IHCs?  Read it in Forge and Schacht?  no, find different REF Hirose et al found that there is no difference in the levels of gentamicin induced ROS formation between apical coil and basal coil haircells [41]. This suggests that there are no differences in GSH levels between apical and basal coil haircells. - It has been reported that the OHCs in cochleal cultures incubated with radical scavengers (n-acetyl cysteine, p-phenylenediamine, glutathione, mannitol, salicylate) show improved viability. This is in the absence of aminoglycosides! Although the rest of that specific paper seems rather weak, this result appears genuine [66]. 25
  • 26. And other cell types? Do HCs have higher levels of free-radical species than other cells? If so, why? Does ROS formation lead to apoptosis or does apoptosis lead to ROS generation? Or both? - 2,3-Dihydrobenzoate (an antioxidant) protects against kanamycin induced ototoxicity [67].  Isn’t DHB also an iron chelator? - 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 [67]. Can glutathione protect against AG induced HC degeneration in cultures? - 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 [67]. Would homozygous varitint mice benefit from antioxidants? - Exogenous SOD protects against AG induced HC degeneration, however, exogenous SOD also elicits an immune response [5]. - Overexpression of superoxide dismutase in inner-ear tissue of transgenic mice prevented kanamycin induced hearing loss [68] - Somehow Dehne et al. conclude that aminoglycosides induce ROS formation which leads to MPT which induces apoptosis [38]. Although this is an interesting scenario I don’t see how it can be concluded on the basis of the data in their paper. - It has been shown in cultured avian sensory epithelia that exposure to gentamicin induces increased levels of ROS [55]. - According to Feldman et al. N-acteylcysteine protects against gentamicin induced ototoxicity. The greatest otoprotective effect was noticed at high frequencies [69]. This suggests that NAC best protects the basal OHCs. Does this suggest that ROS levels are higher in basal OHCs than in apical OHCs? - Iron chelators 2,2’-DPD (2,2’-dipyridyl) and Deferoxamine decreased gentamicin induced damage of OHCs in organotypic cultures [38]. get Sha et al. 2001 (not available online) 26
  • 27. - Iron chelators deferoxamine and DHB reduce gentamicin induced ototoxicity and damage to the kidney [70]. - The efficacy of DHB against gentamicin appears to be dose-dependent [36]. - Generation of free radicals in the presence of AGs was demonstrated in explants of the inner ear and avian auditory epithelium [41, 71].  more refs? It was found that catalase and glutathione can quench the production of gentamicin induced ROS formation in hair cells [41]. Hirose et al found that treatment with antioxidants (glutathione, catalase) has deleterious effects on hair cells [41]. Hair cells rapidly produce ROS when exposed to gentamicin in a time and dose-dependent fashion [41]. Get the Pierson and Muller 1981, 1982 papers, see Hirose 1997. Hirose et al hypothesize that the rapid onset of ROS production suggests a process other than interference with mitochondrial protein synthesis as the underlying cause of aminoglycoside induced ototoxicity [41]. Read the Conlon 1998 paper Read Gonzalez-Lopez 1999 for aminoglycoside induced ROS formation in haircells How about adding iron to hair cell cultures exposed to AGs to see if that stimulates ototoxicity even further. ROS in kidney cells - It was found that gentamicin enhances hydrogen peroxide production in a dose dependent fashion in isolated kidney and liver mitochondria [72]. - Iron chelators deferoxamine and DBH reduce gentamicin induced ototoxicity and damage to the kidney [70]. Suggesting a common mechanism? - N-acetylcysteine can ameliorate gentamicin induced kidney damage [69]. - Apparently the iron chelators DFO and DHB reduce gentamicin induced damage to the kidney [36]. 27
  • 28. - Apparently iron supplementation potentiates gentamicin induced nephrotoxicity in rats [36]. But that can be explained by the fact that increased free iron levels in itself can induce ROS. Adding AGs would simply exacerbate things. Get Swann and Acosta 1990, see Hirose 1997 Get Shah and Walker 1992 and Yang 1991, see Hirose 1997. ROS miscellaneous - Aminoglycosides have a high affinity for polyphosphoinositides. Phosphoinositides are hypothesized to function as electron donors and contribute to ROS formation. Could it be the case that aminoglycosides induce ROS formation, which subsequently triggers mitochondria to induce apoptosis? That does not explain the short time interval between AG exposure and mitochondrial damage - Aminoglycosides bind strongly to phosphoinositides (PIP2) which are constitutive components of all membranes [23]. Does this relate to mitochondria? - It is hypothesized that aminoglycosides can chelate iron. This complex can then subsequently mediate between oxygen and an electron donor [6]. To which iron atom do the AGs bind? Would they be part of any of the electron transport chain proteins? Also, would incubating with iron chelators prevent hair cell degeneration in cultures? Is there real evidence that AGs can chelate iron? - It has been reported that gentamicin forms complexes with mitochondrial Fe2+ [73]. This could mean the aminoglycosides interact with iron complexes on the outside of the IMM, i.e. without entering the matrix. The Mingeot-Leclerc paper refers to the 1997 Song paper [36] and the 1998 Schacht paper [39]. Which report on iron chelators preventing gentamicin induced toxicity. It also refers to a 1997 Priuska et al. paper (get ref, not available online) where apparently it is reported that AGs associate with mitochondrial iron. Do AGs lead to ROS formation directly? Do AGs generate ROS via the mitochondria? Can ROS stimulate caspase-8 and caspase-9? Do AGs directly stimulate caspase-8 and caspase-9? - Combining KM with ethacrynic acid leads to massive apoptosis [5, 6]. 28
  • 29. Do KM and EA work synergistically? What does EA do ? - EA generates free radicals [5]. - Ethacrynic acid can deplete mitochondrial and cytosolic GSH [71]. EA is a loop diuretic If antioxidants are depleted than an increase in ROS can be expected. - According to Ding [5] AGs generate ROS. How? Du and Yang 1994 and Walker, Barry and Shah 1999 Our results with isolated mitochondria which were incubated with aminoglycosides for 20 min show that AGs interact directly with mitochondria. These mitochondria are purified, i.e. cell free. How could the aminoglycosides within 20 minutes generate ROS which then would have to destroy the mitochondria? We should measure the amount of ROS produced during 20 minute incubation of mitochondria with aminoglycosides. - N-acetylcysteine can cross the BBB [69]. - It is suggested that N-acetylcysteine can help to replenish depleted gluthathione content when cells are exposed to elevated oxidative stress [68]. - ● O- 2 and H2O2 may react with metal ions such as iron and copper, to promote additional radical generation, with the release of the indiscriminately reactive hydroxyl radical (● OH), considered as the final mediator of most free radical induced damage [74]. So ROS generated by AGs can interact with iron and create ● OH - Superoxide dismutase (SOD) catalyzes the dismutation of ● O- 2 and H2O2 which is further degraded by gluthathione peroxidise and catalase [74]. - Statin anti-oxidant activity may be mediated by the decreases of NAD(P)H oxidase expression and in turn by the decrease of ● O- 2 production, the upregulation of catalase activity or by direct scavenging properties [74]. - N-acetylcysteine (NAC), a synthetic precursor of reduced gluthathione (GSH) is a thiol-containing compound which stimulates the intracellular synthesis of GSH, enhances gluthathione-S-transferase activity, and acts solely as a RS scavenger [74]. 29
  • 30. Stressed animals (infections, less than optimal diet) display an enhanced susceptibility to AGs. Dietary supplementation, e.g. glutathione will reduce the toxicity induced by AGs [6]. Read the Lesniak paper on aminoglycoside induced ROS formation. ROS can inactivate proteins, which can lead to the release of ferrous iron which then can serve as a reactant in the Fenton reaction [75]. This means that ROS has to be present first before ferrous iron is released which then can be used to generate more ROS. Does Schacht argue that aminoglycosides directly interact with ferrous iron which then can undergo a Fenton reaction? Where does the ferrous iron come from? The Fenton reaction: H2O2 + Fe2+ = OH- + OH + Fe3+ [75] Hydrogen peroxide is processed by glutathione peroxidase (GPX) to water in a reaction that converts reduced glutathione (GSH) to oxidized glutathione (GSSG): H2O2 + 2GSH = 2H2O + GSSG [75] Two electron transfer chain complexes are responsible for much of the superoxide generated by mitochondria: Complex I and Complex III [75]. Superoxide is especially damaging to 4Fe-4S type of iron sulphur centres [75]. Dichlorofluorescin diacetate (DCFH diacetate) will convert into its fluorescent form, dichlorofluorescein (DCF) only after oxidation by hydrogen peroxide [41]. Doesn’t DCFH detect other ROS species as well? In a cell free system gentamicin was found to stimulate ROS formation, but only in the presence of arachidonic acid. The following ranking order was found: Neomycin > gentamicin ≥ kanamycin > streptomycin An excellent correlation between the ototoxicity of AGs and their binding to polyphosphoinositides lipids has long been recognized. The predominant fatty acid in the 2’-position of these lipids is arachidonic acid [76]. I think AGs bind to polyphosphoinositides (perhaps arachidonic acid) in mitochondrial membranes. This leads to ROS formation. AGs do not react with iron. 30
  • 31. Get Yang 1995 and Huang and Schacht 1990 from Sha 1999 Guthrie refers to 2,3-dihydroxybenzoate (DHB) as an antioxidant [11]. But DHB is also an iron chelators. I think reduction of ROS levels in the presence of DHB is sometimes mistakenly attributed to iron chelation. It has been suggested that triggering of MPT by calcium overloading results from the interaction between calcium and cardiolipin which enhances ROS production [77] Mitochondria preloaded with calcium are more susceptible to MPT activation by AGs [77]. 31
  • 32. Mitochondria Mitochondria in haircells - Somehow Dehne et al. conclude that aminoglycosides induce ROS formation which leads to MPT which induces apoptosis [38]. Although this is an interesting scenario I don’t see how it can be concluded on the basis of the data in their paper. - 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 [6, 78, 79]. How come only the HCs are affected? Do individuals who carry this mutation also show AG induced nephrotoxicity? According to Ding 2002 [80] vestibular hair cells do not show an increased sensitivity to aminoglycosides. Only cochleal hair cells show an increased susceptibility to aminoglycosides. Why do vestibular hair cells in the A1555G individuals not show an increased susceptibility to aminoglycosides? - 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 [78]. - A mitochondrial mutation disrupting the penultimate stem in which 1555G resides confers paromomycin resistance in yeast [78]. - Spermine causes damage to OHCs comparable to DHS and amikacin [28]. Does spermine lead to mitochondrial damage in OHCs which subsequently leads to OHC degeneration? Also, does spermine enter the OHCs through the MET? - According to Mather [77] spermine induces the release of SIMP from mitochondria in vitro. Also, spermine has a high affinity for the anionic phospholipids of the mitochondrial membranes (spermine = neomycin > gentamicin > streptomycin = spermidine). 32
  • 33. How does this ranking order compare to ototoxicity? Effects of AGs on cultured OHCs: Neomycin>gentamycin>dihydrostreptomycin>amikacin>neamine>spectinomy cin. Also, on the basis of SEM data spermine is placed between DHS and amikacin. Poly-L-Lysine is more toxic than any of the antibiotics [28]. - Cloramphenicol can be used to specifically inhibit mitochondrial biogenesis. It binds to the 50S subunit of mitochondrial ribosomes and inhibits transcription of proteins encoded by the mitochondrial genome. It was shown that the presence of chloramphenicol led to an increase in degree of HC loss in chick cochleae exposed to gentamicin [81]. Does chloramphenicol exacerbate the effects of aminoglycosides on cochleal cultures exposed to aminoglycosides? It would be interesting to culture mouse cochleae and incubate them with various inhibitors of mitochondria for several days and patch HCs during this period. To distinguish long term effects from acute effects. - Iron deficiency sensitizes animals to acoustic trauma [81]. Can this be attributed to mitochondrial dysfunction? Does the iron deficiency affect those respiratory complexes which require iron atoms? - The paper of Ding and Salvi [5] mentions specifically that AGs enter the mitochondria. Where is the experimental proof for it? - Figures 3A,C&D and 4B&E and 7A&B in the Ding and Salvi [5] paper show TEM pics of guinea pig OHCs. The mitos look small. The diameter being smaller than 1 micron. Unfortunately it is difficult (if not impossible) to ascertain the age of the animals used as these papers are either in Chinese or never published. - It has been found that AGs target mitochondria [ref], but in [25] Immunogold labeling shows labeling of lysosomes and not to mitochondria. How come mitochondria were not labeled at all? Perhaps first lysosomes are targeted and then with a time delay the mitochondria? - Apparently FM1-43 labels ER and mitochondria [33]. - EM pictures of rat and guinea pig vestibular HCs show clearly that mitochondria are very small, less than a micron along the long axis, look at figure 2 in [82]. 33
  • 34. - Apparently ocsyn and mitochondria co-localise in vestibular HCs [82]. To me the fluorescence images look oversaturated, not sure about the quality of this work. What is ocsyn? According to Vautrin [82] Ocsyn apparently is a syntaxin-interacting protein involved in protein trafficking and is hypothesized to be involved in the formation of canalicular-mitochondrial complexes as previously depicted by Spicer et al  insert reference The Ding paper [5] shows in figure 4B that tritium labeled kanamycin co- localises with mitochondria, the text claims that AG labeling is IN the mitochondria. - IHC mitochondria appear to be small, maximum length, along the long axis being about 1 micron, see figures 1-3 in [83]. - It has been shown, in both IHCs and OHCs, that mitochondria make contact with cisternae. It is assumed that these cisternae have ATPases [83]. I don’t see the relevance at all, but maybe AGs find their way into these cisternae and can target mitochondria more directly? - It has been reported that noise exposure induces Hensen’s bodies. Hensen’s bodies are circular formations composed of ER and encased by mitochondria [84]. Are Hensen’s bodies specific for HCs? (Hensen's bodies - assemblies of vesicles in upper region of cochlear outer hair cells and related to ion transport) - It has been reported that FM1-43 staining distributes homogeneously within the IHCs whereas in the OHCs the staining appears to coincide with Hensen’s bodies [85]. - EM micrographs from OHCs show that they are loaded with mitochondria, See the figures in [86]. It is also very clear that mitochondria in OHCs are not homogeneously distributed. See Figure 2 where mitochondria are lined up along the lateral wall. Do OHCs (and IHCs) have more mitochondria than other cell types? - It has been reported that mitochondria from HCs are not distinguishable from liver mitochondria [87]. That study however compared labeling for dehydrogenase activities in isolated cochleae with labeling of isolated liver mitochondria. I don’t know if you can actually conclude from the results in the Spector paper that HC mitochondria are similar to liver mitochondria. The proper way of comparing 34
  • 35. would be to isolate mitochondria from HCs directly and biochemically characterise them. - It has been reported that dehydrogenase activity is greater in IHCs than in OHCs by Vosteen (get ref) whereas it was found by Koide et al (get ref) that OHCs were either equally or more metabolically active than IHCs. According to Ding 1997 (get ref) SDH activities in IHCs and OHCs are comparable. There is clearly confusion about HC metabolism and also, some of these reports are very old, using outdated techniques by current standards. So HC metabolism should be investigated again. - 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 [88]. (Get the Prezant TR 1993 papers) Is this based on only one patient? - 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 [16]. - A correlation is found between aminoglycoside ototoxicity and inhibition of protein synthesis of mitochondrial ribosomes [89]. - It has been reported that exposure to aminoglycosides leads to ultrastructural changes in hair cell mitochondria [90]. - Metabolic imaging of the organ of Corti revealed that gentamicin decreases the level of NADH in outer hair cells but not in inner hair cells [32]. How? - It has been reported that noise exposure leads to a decrease in SDH staining density in IHCs and OHCs (Canlon 2001)  get ref - It has been reported that aminoglycosides lead to reduced mitochondrial respiration in the inner ear and in the kidney [38].  Get the original paper This suggests that aminoglycosides affect mitochondria. Would respiration in isolated cochleal cells be affected by aminoglycosides? Compare with liver cells? - Heat shocked utricle hair cells (utricles kept at 43 degrees Celsius for 30 min) inhibits neomycin induced hair cell death [91]. 35
  • 36. What is the mechanism? Interfering with apoptosis? - According to [80] gentamicin associates with lysosomes. If you look a figure 1 in Ding 2002 it looks as if actually mitochondria are labelled instead of lysosomes. - There are some medical conditions caused by mitochondrial mutations which have hearing loss frequently as one of their early clinical signs: Kearns-Sayre syndrome, mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS) and mitochondrial encephalomyopathy with ragged red fibers (MERFF) [16]. - An association between diabetes mellitus, hearing loss and mtDNA mutations has been found [16]. I think I read in another paper that the A1555G condition was cochleotoxic but not vestibulotoxic. Is that based on this one particular individual? That would be misrepresenting the facts. Find that reference. How about treating A1555G patients with both AGs and antioxidants? - It is suggested that the hypersensitivity of cochleal cells for mitochondrial mutations is due to an abnormal interaction of cochlea-specific subunits of mitochondrial ribosomes or RNA processing proteins with the mitochondrial defect [16]. I think this is too far-fetched. Probably it is the high concentration levels of aminoglycosides reached in cochleal sensory cells which causes accelerated cell death in individuals with the A1555G mutation who already have labile mitochondria. -Within zebrafish lateral line hair cells ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of neomycin exposure. 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 following neomycin treatment. 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 electrondense with fewer cristae present. Also, mitochondria surrounding the nuclei appear more affected than those located distantly within the HC [31]. If aminoglycosides do not interact with mitochondria directly then the cellular events occurring prior to the mitochondrial response must occur rapidly (<15 minutes). 36
  • 37. - Mitochondria in haircells appear to have a tubular appearance [31]. - The mitochondrial membrane potential in zebrafish lateral line hair cells decreases within 30 minutes in the presence of 50 micromolar neomycin [31]. - There are EM studies showing changes in mitochondrial structure in response to aminoglycoside treatment [90]. Check de Groot 1991 and Bagger-Sjoback 1978) The Owens 2007 paper has a lot of interesting references, see if these are available on WOK. - It was shown that GTTR colocalizes with mitochondria in HCs [15]. - Dehne et al. found that exposure to gentamicin induces a loss of mitochondrial membrane potential in OHCs [38]. Are there more studies where mitochondrial membrane potential was monitored in hair cells? (apart from the Owens 2007 paper) - 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 [81]. - The mitochondrial mutation A3243G is associated with both hearing loss and diabetes mellitus [8]. - The A1555G mutation on its own can lead to deafness [16]. Several polycations (spermine, spermidine and neomycin) induced SIMP release without inducing significant swelling and this release was not inhibited by CsA [77]. Get the Ding 1999 and the McFadden 2004 papers from [43] in which SDH histochemistry experiments are described. Apparently SDH is heavily expressed in OHCs and IHCs but is largely absent from the surrounding support cells in the organ of Corti [43]. Ding et al measured mitochondrial membrane potential in chinchilla cochlear hair cells using Mitotracker M-7514 [43]. Does this dye actually indicate mitochondrial membrane potential? Isn’t mitotracker red the only suitable dye for this? 37
  • 38. Could it be the case that OHC Hensen bodies, which are hypothesized to be in contact with mitochondria, accumulate AGs? In this way AGs would very effectively target mitos in OHCs. Figure 5 in [55] (Hirose 1999) shows swollen mitochondria in haircells in response to aminoglycoside exposure. Mitochondria in kidney cells - It was found that gentamicin enhances hydrogen peroxide production in a dose dependent fashion in isolated kidney and liver mitochondria [72]. - 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 [92]. Apparently the appearance of gentamicin in the cytosol coincides with the decrease of mitochondrial membrane potential. In an older report by Sundin and Sandoval from 2001 they did not see AGs entering the cytosol. Would it be safe to assume that AGs enter cells by both endocytosis and MET channels? Do non transducing HCs (such as Myo 7A and varitints) not load with AGs at all? What if you leave the cultures for 4 to 6 hours? According to Guy Richardson these cells do load with AGs over time. According to Servais et al. gentamicin traffics to the intermembrane space of mitochondria [93]. If so, how does gentamicin cross the OMM? Figure 5E in [94] suggests that gentamicin goes into the intermembrane space, but not into the matrix. - It has been reported that aminoglycosides lead to reduced mitochondrial respiration in the inner ear and in the kidney [38].  Get the original paper This suggests that aminoglycosides affect mitochondria. Would respiration in isolated cochleal cells be affected by aminoglycosides? Compare with liver cells? - Apparently gentamicin associates with the outer membrane and the intermembrane space of mitochondria [94]. Also, they did not observe aminoglycosides entering the cytosol. 38
  • 39. This suggests that aminoglycosides do not enter the mitochondrial matrix. But in that same study they show that Ags do not enter the cytosol. Aminoglycoside poisoning has been shown to selectively inhibit mitochondrial function in kidney [78]. Aminoglycosides have been shown to form free radicals in isolated kidney mitochondria [6]. Read Yang 1995 Schacht thinks that gentamicin in itself is not toxic to isolated hair cells (but what about the Kotecha study then?) and it requires activation to a cytotoxic species [6]. This conclusion is not warranted since the OHCs he used were not transducing and other studies clearly show gentamicin toxicity to hair cells. Mitochondria general - Spermine is reported to induce release of cyt c by disrupting the mitochondrial OMM. Spermine inhibits PTP and does not lead to swelling [77]. - It is reported that aminoglycosides stimulate electrogenic uptake of Ca2+ by mitochondria but do not inhibit PTP [95]. - Aminoglycosides stimulate calcium uptake in mitochondria [77]. - It has been reported that aminoglycosides stimulate calcium uptake in mitochondria [96]. - aminoglycosides lead to decreased velocity of calcium uptake in mitochondria but an increased accumulation of calcium [95]. - polyamines affect calcium transport into mitochondria [97]. Increased calcium uptake? Spermine at 50 μM speeds up the initial uptake of Ca2+ but at 180 μM Ca2+ uptake is inhibited, however overall calcium accumulation increases [97]. Does that mean polyamines interact with the Ca2+ uniporter? Has the effect of aminoglycosides been investigated properly? (aside from the Rustenbeck work?) 39
  • 40. Calcium influx can induce MPT, so perhaps aminoglycosides can induce MPT via calcium influx? - Calcium influx into mitochondria is electrophoretic and dependent on the mitochondrial membrane potential [97]. - Ruthenium red is a polycation and inhibits the Ca2+ uniporter [97] (and it inhibits TRP channels). - The release of SIMP (soluble mitochondrial intermembrane proteins) induced by aminoglycosides is inhibited by CsA to various extents depending on the AG used. CsA highly effective with streptomycin, gentamicin induced SIMP release partially inhibited by CsA and neomycin induced SIMP release only slightly inhibited by CsA [77]. So, aminoglycosides target mitochondria in different ways, is this reflected in the severity of ototoxicity induced by those aminoglycosides? Also, Dehne et al found that CsA provided partial protection against gentamicin toxicity in HCs (decreased cell death) [38], this corroborates Mather’s results on isolated mitochondria. It would be interesting to see if CsA also protects against ototoxicity induced by other aminoglycosides, especially streptomycin and neomycin. -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 [98]. I have no idea what this means and whether the work done in fact is any good, but it does show some effect of AGs on mitos, so it may be relevant. - 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 [95]. - Apparently spermine and aminoglycosides can compete with each other for binding sites on mitochondria [95]. - Apparently there are binding sites for polycations on the IMM [97]. - It has been reported that spermine is transported in the matrix [97]. Can AGs be transported into the matrix of mitochondria through the same transport systems? - it has been reported that aminoglycosides antagonise the effects of natural polyamines like spermine. Spermine inhibits PMT. Perhaps aminoglycosides 40
  • 41. compete with spermine for binding sites. Also it was found using a TPP+ electrode that aminoglycosides induced a depolarization of the IMM [95]. But I thought spermine had a harmful effect on mitochondria. - It has been reported that gentamicin stimulates respiration and reduction of cytochrome c (Sha 1998)  that reference does not say anything about cytochrome c. What is the real source for this? (not Mather) I’m not sure about the methods, but if the findings are true, they suggest that gentamicin might uncouple mitochondria (given the increased vO2). Or because of increased cytochrome c reduction there is more O2 reduction. Cytochrome P450 is found in the IMM Why is this relevant? Relevant to ROS? See Schacht. - It has been reported that gentamicin is metabolised into some cytotoxin in the cytosol of liver cells. Incubation of gentamicin with other fractions, such as the mitochondria do not metabolise gentamicin [99]. It would be interesting to study HCs from a mouse with a mitochondrial mutation Johnson et al. [100] are the first to report on a mouse model in which a mitochondrial DNA mutation affecting a clinical phenotype. - Apparently gentamicin stimulates state 4 respiration and inhibits state 3 and uncoupled respiration in renal cortical mitochondria [101]. This is different from what was reported by Rustenbeck - It is suggested that gentamicin interacts with renal mitochondria at the IMM [102]. - a technique has been established where mitochondria in situ can be imaged using label-free optical imaging [103]. - It has been reported that with age mitochondrial respiration decreases [104]. - 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 [80]. Does this indicate that aminoglycosides do not reach the vestibular hair cells, or does it reflect differences between cochlear and vestibular hair cells? On how many patients is this based? Get the 1993 Prezant paper. 41
  • 42. - 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 bacteria [16]. - 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 [16]. So no dysfunctional oxphos proteins? But maybe oxphos will be dysfunctional because the amount of oxphos protein is too low, or the ratio of expression is not balanced. (There are some nice pictures of mitos in OHCs in the Richardson (1997) paper, see Figure 1.) Aminoglycosides exert their antibacterial effect at the level of the ribosome. It is suggested that due to the similarities between bacterial and mitochondrial ribosomes aminoglycosides target mitochondrial ribosomes [78]. - One of the most common phenotypes of mitochondrial diseases is sensorineural deafness suggesting that mitochondrial function is critical for cochlear function [78]. But this may pertain to the stria vascularis being affected and not a direct effect on haircells. - Cortopassi and Hutchin [79] speculate that mistranslation of mitochondrial mRNA leads to a deficiency of Complex I leading to the generation of ROS. - It has been reported that gentamicin alters mitochondrial respiration (stimulation of state 4 and inhibition of state 3) [72]. - Studies have shown that gentamicin causes morphological changes in mitochondrial membrane. [72] Read the references from Walker 1987 It has been reported that exposure to aminoglycosides leads to upregulation of mitochondrial uncoupling proteins 2 and 3 [37]. Two electron transfer chain complexes are responsible for much of the superoxide generated by mitochondria: Complex I and Complex III [75]. Superoxide is especially damaging to 4Fe-4S type of iron sulphur centres [75]. The inner mitochondrial membrane has a very high content of integral membrane proteins. The anionic phospholipid cardiolipin is known to be 42
  • 43. mostly associated with the integral membrane proteins. This association may protect the IMM against destabilization by AGs [77]. - Gentamicin and polylysine are able to enhance mitochondrial Ca2+ accumulation [97]. Apoptosis Apoptosis in hair cells - OHCs subjected to aminoglycosides display apoptosis. It seems to be the case that first row OHCs are more severely affected than the other rows (see Figure 4 in [6]). Why? Also, if aminoglycosides induce apoptosis, can this be prevented by incubation with CSA? Apparently caspase inhibitors, such as BAF and z-VAD-fmk, can prevent gentamicin induced HC degeneration [105]. - incubation with z-VAD-fmk (general caspase inhibitor) protects hair cells against neomycin induced damage [90]. - Apparently the death receptor pathway does not play a key role during aminoglycoside induced hair cell death [106]. - In contrast (?) to what Cheng reports, Cunningham [90] reports that treatment with neomycin leads to Caspase 9 activity primarily with only slight Caspase 8 activity. Is one of them incorrect? Or does neomycin specifically lead to Caspase 9 activity? - incubation with Caspase 9 inhibitors protects against neomycin induced cell damage. Caspase 8 inhibitors have no protective effect [90]. Also, inhibition of Caspase 9 prevents activation of Caspase 3. Does this show conclusively that aminoglycosides specifically induce apoptosis through the mitochondrial pathway? Does neomycin specifically induce caspase 9? - It has been reported that exposure to streptomycin leads to release of cytochrome c in vestibular guinea pig hair cells, although the experimental evidence given isn’t very strong [107]. Link to the Mahter study? - It has been reported that aminoglycoside HC degeneration is concentration 43
  • 44. dependent, see figure 2 in [108]. Is this apoptosis related? - It has been reported that gentamicin exposure leads to significant activation of caspase-9 which indicates that mitochondria are involved in aminoglycoside induced hair cell degeneration. It was also found that caspase-8 was activated by gentamicin, but anti-body staining did not show any staining for caspase-8 paradoxically enough [108]. This means that aminoglycosides induce HC degeneration via both the mitochondrial pathway and the death receptor pathway. - It has been reported that in Va mutants staining for SDH activity was decreased when compared to control mice [109]. This would imply that the hair cell degeneration seen in varitint waddler mice is to an extent dependent on the mitochondria. As these cells load up with calcium this might trigger apoptosis. Perhaps we should stain varitint HCs for Caspase-9 and determine mitochondrial membrane potential. - It has been reported that blocking of caspase-8 does not prevent neomycin induced activation of caspase-3, whereas caspase-9 inhibitors prevented caspase-3 activation and apoptosis [90]. This means that neomycin induced HC death via the mitochondria. Read Cheng and Cunningham papers again. - It has been reported that minocycline protects against gentamicin induced HC loss by preventing MPTP and the release of cyt c [110]. Based on the work of Mather and Rottenberg this would mean that minocycline would not be effective in protecting against aminoglycosides, such as neomycin as that aminoglycoside does not appear to induced MPTP. Determine if minocycline indeed has been tried with other AGs. Also, Charley should try minocycline with kidney cells that are incubated with neomycin. - OHCs exposed to neomycin (1 hour) show blebbing. Whereas OHCs pre- treated with calcium chelators are protected against AGs [27]. The blebbing suggests that these cells go into apoptosis. The exposure to neomycin was 1 hour, during that interval, when did the blebbing start? Also, if there is any endocytosis of AGs then this process takes longer than 1 hour since the OHCs pre-treated with calcium chelators did not show any blebbing. It could of course be the case that AGs were in fact getting into the hair cells through endocytosis. Again it would be useful to know when exactly the blebbing started in the experiment with just neomycin present. Suppose AGs do enter HCs also via endocytosis, 44
  • 45. how slow/fast is this process and how long does it take to reach a steady state AG concentration within the HC? Calcium chelators prevent apoptosis through tip-link breakage? - Continued exposure to FM1-43 leads to some blebbing of OHCs [27]. Does this tie in with the fact that FM1-43 also targets mitochondria? See [33]. - AG induced influx of calcium is hypothesized to occur through L-type calcium channels [5]. Does Calcium influx induce PMT in mitochondria? Could it be that AGs induce apoptosis via parallel mechanisms? Both calcium influx and ROS formation? - Bax can translocate from the cytoplasm to the mitochondria and promote the formation of pores in the mitochondrial membrane [106]. What is the nature of the interaction of other Bcl-2 members with mitochondria? Do AGs stimulate BAX? - Bcl-2 proteins act upstream of caspase activation [106]. How do AGs (if they do) stimulate Bcl-2 protein activity? Does Caspase-9 induce release of cytochrome c or not? - Activated nuclear p53 can directly translocate to and damage mitochondria [106]. This is a separate apoptosis pathway from the caspase pathway? It has been shown that gentamicin localizes within the nucleus of hair cells [15]. Does gentamicin activate nuclear p53? - Gentamicin localises to the nucleolus because AGs bind to ribosomal RNA [50]. - It has been shown that mitochondria-associated oxidants are involved in pathways regulating cytochrome c translocation and caspase activation [106]. - Phosphorylation of JNK and c-jun have been demonstrated in inner ear hair cells when treated with neomycin and cisplatin [106]. What is the role of JNK and c-jun in apoptosis? 45
  • 46. - It has been reported that incubation with CsA partially protects against gentamicin induced HC damage [38]. - Aminoglycoside induced hair cell death has been characterized as apoptotic, both morphologically and molecularly [93]. The Cunninham 2002 has many interesting references. - Exposure to gentamicin in vitro leads to increased levels of free intracellular calcium in hair cells [55]. Does the calcium increase lead to apoptosis? It is reported that gentamicin does not cause apoptosis via the death receptor pathway. Kanamicin however seems to involve the death receptor pathway [12]. This suggests that different aminoglycosides induce different apoptotic pathways. Read the Selimoglu paper again, it has many interesting references. Read the Bodmer paper and the Lei paper. - According to Ding [5] inhibiting calcium activated proteases prevents AG induced HC loss. How about calcium release by damaged mitochondria (induced by AGs) ? What is the role of calpains in apoptosis? - Exposure to gentamicin leads to apoptosis in HCs in organotypic cultures [38] Aminoglycoside toxicity in vivo leads to apoptosis in both cochlear and vestibular hair cells [38]. - No signs of inflammation which would be expected in necrosis are described in the inner ear after exposure to AGs [38]. Dehne et al found that CsA provided partial protection against gentamicin toxicity in hair cells (decreased numbers of cell death) [38]. Try CsA with other aminoglycosides, both cultures and confocal microscopy. Cisplatin is used for chemotherapy. - It has been reported that cisplatin therapy results in loss of high frequency hearing and deafness. Cisplatin primarily damages OHCs. Also, there apparently is cross talk between the two pro-caspase pathways, i.e. activation of caspase-8 can lead to activation of caspase-9. Cisplatin leads to caspase-8 activity before caspase-9 activity [111]. 46
  • 47. Does this mean that cisplatin induced HC degeneration starts in the basal coil? What is the effect (if any) of cisplatin on IHCs? In the Devarajan paper [111] it is reported that PMT was determined, however, this was done on the basis of measuring mitochondrial membrane potential. So, how does this specifically implicate PMT? - It was found by [112] that upon exposure to neomycin chick utricle HCs went into apoptosis, the mitochondria however appeared to be intact. This was on the basis of morphology, it would be interesting to determine the membrane potential of these mitos. Haircells exposed to neomycin showed blebbing [27]. Haircells exposed to gentamicin show clear characteristics of apoptosis [105]. Read Forge and Li 2000 again Morphological evidence from many vertebrate species suggests that the loss of hair cells in response to treatment with aminoglycoside antibiotics occurs via apoptosis (Jørgensen, 1981, 1991; Forge, 1985; Li et al., 1995; Torchinsky et al., 1999; Forge and Li, 2000; Matsui et al., 2002). Taken from [113]. Print out and read the Matsui 2004 paper in more detail. Figure 4 in [6] shows clear characteristics of apoptosis in cochleal haircells and vestibular haircells. Where did that figure come from? Forge and Schacht 2000 has some good references on aminoglycoside induced apoptosis, get: Forge 1985, Brown 1989, Li 1995, Nakagaw 1998 and Forge and Li 2000. Incubation of cultures with gentamicin in the presence of caspase-specific inhibitors prevents almost all hair cell death that occurs with gentamicin alone [6]. Get Li et al 1997 Co-treatment with neomycin and CEP-1347 results in reduced haircell DNA fragmentation and inhibition of the JNK pathway. Treatment with neomycin alone leads to haircell DNA fragmentation and stimulation of the JNK pathway [11]. Read Pirvola 2000 47
  • 48. Explanted OHCs from guinea pig exposed to gentamicin show that basal OHCs are more vulnerable to disruption in calcium homeostasis by gentamicin than apical OHCs [11]. Chinchilla cochlear hair cells were exposed to gentamicin and EA. Condensation and fragmentation of OHC nuclei, morphological features of apoptosis were first observed 5-6 hours post treatment. Metabolic function reflected by SDH histochemistry and mito staining decreased significantly in the basal turn 4h following GM and EA treatment. These early changes were accompanied by the release of cytochrome c from the mitochondria into the cytosol and intense expression of initiator caspase-9 and effector caspase-3. Also, GM/EA failed to initiate caspase-8 [43]. Activation of caspase-3 was blocked by X-linked inhibitor of apoptosis (XIAP) [43]. Apoptosis in kidney cells -It has been reported that HEK cells expressing mutated TRPML3 (both A419P and A419P/I362T) label positive for annexin V (an indicator for early apoptosis. Also, these same cells load with Ca2+ (Grimm 2007). This suggests a role for mitochondria. Do varitint mutant HCs show morphological signs of apoptosis? - Apparently one of the hallmark features of programmed cell death (PCD) is membrane blebbing [112]. Does exposure to AGs to HCs lead to blebbing? And more specifically, do varitint HCs show blebbing? Richardson [26] shows blebbing as a response to AG exposure. What about varitints though? - It has been reported that lysosomes are the first site of accumulation of AGs in kidney proximal tubules, in vivo as well as in cultured cells [93]. - Depending on concentration of AG present, cells go into apoptosis or necrosis. It was 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 [93]. - 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 [93]. 48
  • 49. Gentamicin induces apoptosis in proximal tubule epithelium at low therapeutically relevant doses, whereas at supra-therapeutic doses extensive necrosis is observed [114]. Apoptosis developed linearly with time and gentamicin concentration in kidney cells [114]. Overexpression of Bcl-2 and co-incubation with cycloheximide prevents gentamicin induced apoptosis in kidney cells (MDKC, not LLC-PK) [114]. 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 [114]. El Mouedden et al. demonstrated that aminoglycosides with lower nephrotoxic potential such as netilmicin, amikacin or isepamicin cause considerable less apoptosis when compared to gentamicin at equitherapeutic doses [114]. 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 [114]. The El Mouedden 2000 paper is very well written and has useful references  READ AGAIN. Apoptosis general Release of cytochrome c induces activation of caspase 9, see figure 1 in [115]. The release of cyto c before or in the absence of a drop in ΔΨ in some cells suggests that different regulatory events control permeability of the inner and outer mitochondrial membranes. A rapid opening and closing of the PT pore at its reversible low conductance state may allow a repeated, respiration- driven reestablishment of ΔΨ REF so that outer membrane disruption and cyto c release can occur before ΔΨ collapse [115]. If apocytochrome c is encoded by the nucleus it has to be transported across the OMM. How does that work? And is this transport process involved in the release of holocytochrome c ? - It has been reported that caspase-8, apart from directly stimulating 49
  • 50. downstream caspases can also induce release of cytochrome c from mitochondria. Apparently caspase-8 interacts with mitochondria indirectly using Bid as an intermediate [116]. Does this mean that mitochondria are always involved in apoptosis? - It has been reported that Bid (and tBid) can induce release of cytochrome c from mitochondria in the presence of CsA, i.e. PTP is not involved in this process [116]. Check references for the caspase-8 cytochrome c interaction. There was a wrong reference there. Read the Green article again. - Minocycline inhibits cytochrome c release, it crosses the BBB and is a non- toxic drug [110]. Would minocycline be able to protect against AG hair cell degeneration in HC cultures? Given the non-toxicity of this drug, it may be a candidate drug to be co-administered with AGs. How does apo-cytochrome c cross the OMM? - Signals such as Ca2+ , nitric oxide and ROS may induce release of proapoptotic proteins from mitochondria independent of proapoptotic Bcl-2 proteins [77]. - MPT allows for a total calcium efflux from mitochondria [97]. Does exposure to AGs lead to increased intra-cellular calcium in hair cells due possibly through efflux from mitos via MPT? - Aliphatic polyamines are able to inhibit MPT with the following efficacy ranking order: spermine > spermidine > putrescine. But gentamicin and polylysine are ineffective in protecting against MPT [97]. The release of SIMP (soluble mitochondrial intermembrane proteins) induced by aminoglycosides is inhibited by CsA to various extents depending on the AG used. CsA highly effective with streptomycin, gentamicin induced SIMP release partially inhibited by CsA and neomycin induced SIMP release only slightly inhibited by CsA. Streptomycin, gentamicin and neomycin induced the release of cytochrome c from isolated mitochondria [77]. 50
  • 51. Aminoglycosides Aminoglycosides in hair cells Aminoglycosides can cause ototoxicity to varying degrees depending on the specific AG. What is the ranking order of aminoglycosides with respect to causing ototoxicity. Is this list the same as for nephrotoxicity? Effects of AGs on cultured OHCs: Neomycin>gentamycin>dihydrostreptomycin>amikacin>neamine>spectinomy cin [28]. Kotecha and Richardson also refer to some other studies where ranking orders were determined, look into those studies. Neomycin is regarded as highly toxic, gentamicin, kanamycin and tobramycin of intermediate toxicity and amikacin and netilmicin somewhat less toxic [6]. This is based on the work done by Kotecha 1994 and Lohdi 1980 - Neomycin causes more morphological damage basally than apically in cochlear cultures [28]. - Apparently adult mice, rats and gerbils show little, if any, drug induced Toxicity under conditions that would produce severe auditory and vestibular deficits in guinea pigs or chinchillas. Apparently, adult mice are not susceptible to aminoglycoside induced ototoxicity. In cochlear explants however there appear to be no differences between adult mouse, gerbil and guinea pig OHCs with respect to their response to AGs [67]. The AGs cannot get into the endolymph? - Apparently OHCs are affected in a particular order, first the first row then the second row then the third row OHCs [6]. Is this seen with aminoglycosides? - It has been reported that streptomycin and gentamicin are primarily vestibulotoxic whereas amikacin, neomycin, dihydrostreptomycin and kanamycin are primarily cochleotoxic.  which ref? Also, it was reported that gentamicin has less ototoxic and vestibulotoxic 51
  • 52. effects in newborns than in older children or adults.  which ref? - Apparently neomycin is more cochleotoxic than vestibulotoxic [31]. - Apparently streptomycin is almost exclusively vestibulotoxic whereas dihydrostreptomycin is almost exclusively cochleotoxic [2]. - The effects of AGs on haircells depends on the type of AG used. Not only is there a ranking order in ototoxicity but there are also qualitative differences in toxicity: neomycin, poly-L-lysine and spermine induce whorls of tightly packed membrane resembling myelin. Only a small amount on membrane whirling is observed with gentamicin. Apical surface changes induced by DHS and amikacin are simply large distensions of the cell filled with cytoplasmic organelles of normal appearance [28]. - It has been shown that neomycin has several (possibly independent) dose dependent effects on hair cells. At low doses (up to 50 μM) causes an irreversible increase in the steady state stiffness of the hairbundle and a reversible inhibition of MET without causing any morphological damage to the cells. At higher concentrations (> 200 μM) there is a rapid formation of numerous blisters on the apical surface of the haircells, these lesions are filled with whorls of membrane resembling myelin. The morphological damage observed in vitro is more severe in basal cells than apical cells [28]. Get the Richardson papers from 1989 and 1991. Also, get the Wang 1984 paper. -Neamine is a neomycin fragment and it has little ototoxicity [28]. What is the structural difference between neomycin and neamine? - Aminoglycosides are the preferred treatment against bacterial infections in developing countries. Studies from southern China claim that 2/3 of all deaf- mutism in that area was due to administration of aminoglycosides to children. Two populations of patients, suffering from AIDS or cystic fibrosis are also facing the prospect of developing ototoxicity due to aminoglycoside treatment [6]. - The glycoprotein megalin has been hypothesized to transport aminoglycosides. In OHCs (so far) the presence of megalin has not been determined [6] Is megalin present in IHCs and OHCs ? According to Mingeot-Leclercq [73] megalin IS present in inner ear epithelia. However in the paper referenced by M-L the word megalin doesn’t even occur, i.e. the M-L paper is inaccurate. However, according to Selimoglu [12] megalin is indeed found within the cochlea. According to Mizuta et al. [117] megalin is indeed found within the cochlea, but not in the hair cells. This excludes the possibility that AG uptake in hair cells occurs via megalin. 52
  • 53. It has been concluded that the uptake mechanism of AGs in kidney cells is fundamentally different from that of HCs because kidney cells express megalin and HCs do not. This way of thinking suggests that there is only one uptake mechanism for AGs possible per cell. My hypothesis is that both kidney cells and hair cells have mechanoelectrical transduction channels which are selective for AGs. This ion channel mediated uptake system exists alongside the megalin mediated uptake system within kidney cells. We know that HCs also show AG endocytosis. According to [80] megalin facilitates entry of aminoglycosides in inner ear epithelial cells. check the references for this. Zajic and Schacht suggest that megalin is present in the apex of hair cells [6]. Get the Ylikoski 1997 paper - Williams et al. [6] report a polyamine-like transport system in haircells. Read the Williams 1987 paper Characteristics of gentamicin uptake in the isolated crista ampullaris of the inner EAR of the guinea pig  not available online - Aminoglycosides are initially apparent at the apical end of hair cells [6]. Associated with hairbundles? Look at de Groot 1990, Hiel 1992 and Hashino 1997 - When incubated with gentamicin it first labels the stereocilia of hair cells before diffusing through the cell [32]. - It has been reported that labelling with gentamicin is restricted to the organ of Corti. Other cochlear tissues such as the stria vascularis, Reissner’s membrane and the spiral ganglion did not demonstrate any labeling [118]. If AGs would enter the cell via endocytosis how come there is only labeling for gentamicin in HCs and not in the other cell types? Is this issue ever explained in any of the papers that advocate endocytosis? Incidentally, de Groot et al. never claim that AGs enter HCs through endocytosis; they only hypothesize that this might be the case. Also, I don’t find the paper by de Groot very convincing, Ding (2005) is not very critical however. - It has been reported that AGs enter HCs via receptor mediated endocytosis on the basis of colocalisation of immunogold labeled KM and ferritin [25]. It has also been reported for cultured kidney cells that AGs enter via endocytosis [119]. Is the presence of ferritin a good measure for determining that? I have the impression that the Hashino 1995 paper isn’t all that good. - It was found that serum from individuals susceptible to AG induced hearing loss can be cytotoxic to isolated OHCs. It is hypothesized that these 53
  • 54. individuals can metabolise AGs into a cytotoxic compound [11]. - It has been reported that aminoglycosides distribute to different regions of the inner ear, on the basis of their molecular structure [1]. How is this possible? It would be very interesting to see the effects of a range of AGs on vestibular cultures and compare them to the results obtained by the Kotecha 1994 study. - It has been reported that concanavalin A prevents uptake of gentamicin in rat cochlear explants [120]. Guy Richardson doesn’t believe this study was very good. - It is reported that megalin is expressed in cochleal sensory cells and in kidney proximal tubule cells [12].  megalin is not present in cochleal sensory cells [117]. Is this true? Check references. Could this be some connecting mechanism which accounts for ototoxicity and nephrotoxicity? - It has been shown that gentamicin localizes within the nucleus of hair cells [15]. - Eardrops containing 0.35% neomycin led to severe ototoxicity in three patients that did not have a mitochondrial predisposition to aminoglycoside induced hair cell damage [12]. - It is reported that hair cells eliminate gentamicin in two phases, a fast phase with a half life of 2 days and a slow phase with a half life of 6 months [80]. Get the 1993 Dulon paper - Leupeptin protects against gentamicin induced hair cell death in cochleal cultures. It is concluded that leupeptin does this by inhibiting calpains (serine- and cysteine-like proteases). Interestingly enough Ding et al. expose hair cell cultures to both gentamicin and ethacrynic acid [80]. Why did Ding et al. use ethacrynic acid? Why not just gentamicin? Because ethacrynic acid is a loop diuretic? - During chronic treatment the presence of gentamicin in OHCs was detected after 2 days and in IHCs after 8 days in vivo in guinea pigs. High frequency hearing loss was not observed until 10-14 days of GM treatment [80]. - Regenerated avian hair cells which are morphologically immature fail to take up kanamycin and are drug resistant until they become functionally mature (Hashino 1997)  get reference 54
  • 55. - In teleost fish, hair cells in the striolar region of the utricle and lagena are especially susceptible to gentamicin ototoxicity (Saidel 1990 and Yan 1991)  get references (look in Ding 2002). - Streptomycin causes a large transient increase in intracellular calcium in isolated OHCs (Li 1995, see Ding 2002)  get reference - In tissue cultures of chick sensory epithelium gentamicin caused a dose- dependent increase in intracellular calcium levels in hair cells [55]. Selective destruction of haircells when treated for 48 hours with neomycin at a concentration of 20 μM can be observed [28]. What about non-hair cells? - Spermine causes HC damage, whereas other cells in the organ of Corti are not affected [28]. Does this mean that spermine specifically targets HCs entering through the MET? - It has been reported that OHCs contain an apical network of so called ‘canalicular reticulum’. Apparently, similar structures have been found in kidney proximal tubule cells [86]. - Hair cells mutant for the inositol lipid phosphatase Ptprq were found to be hypersensitive to aminoglycoside exposure (Jane Bryant thesis). Why? What does Ptprq do? How do increased PIP2 levels lead to increased aminoglycoside sensitivity? More binding of AGs? Apart from a basal to apical coil gradient there is a lateral gradient of OHC degeneration, first OHCs from the first row are affected, then from the 2nd row and then from the 3rd row [6]. - Neomycin leads to degeneration of HCs. Morphological changes are restricted to hair cells and are not observed in supporting cells within the organ of Corti. HCs in apical coil cultures are less sensitive than those in basal coil cultures and at any given point along the cochlea OHCs appear to be more extensively damaged than IHCs. The damage done by neomycin is considerably worse in the absence of Ca2+ /Mg2+ [34]. This suggests that calcium and magnesium compete with neomycin for the MET channel? The effects of neomycin on HCs can be blocked by lowering the temperature to 4 °C [34]. 55
  • 56. This suggests active transport. How does that relate to the activity of the MET? - Blocking the effects of neomycin on organotypic cultures with concanavalin A was not effective [34]. - In hair cells aminoglycosides have long half-lives (5-6 months) and are poorly degraded [15]. Get Dulon 1993 and Imamura 2003 - From figure 7A in [15] it can be seen that supporting cells show very little labeling with AGs in comparison to HCs. - It was found that OHCs are most sensitive to gentamicin toxicity followed by IHCs [38]. - Supporting cells (Deiters and Hensen cells) were not affected by gentamicin exposure [38]. - Gentamicin damages HCs in a dose dependent manner [38]. - Schacht claims that AGs destroy both sensory and supporting cells in the organ of Corti [39]. Does he show any evidence of the destruction of supporting cells in any of his work? - Apparently the cochlear microphonic potential is reduced by gentamicin in two stages: an initial acute phase which is readily reversed by calcium followed by a second phase insensitive to calcium [35]. Aminoglycoside antibiotics also induce a dose dependent increase in intracellular calcium in avian hair cells [55]. Aminoglycosides are found in surviving haircells after 6 months [37] See Dulon 1993 and Imamura 2003 - it has been reported that NMDA antagonist dizocilpine (MK-801) and ifenprodil limit ototoxicity of aminoglycosides. However, it should be taken into consideration that these compounds were dissolved in DMSO and that their maleic or tartaric salts were used [40]. Neutralising ROS? - apparently aminoglycosides can inhibit ornithine decarboxylase [40]. 56
  • 57. What does ornithine decarboxylase do? polyaspartic acid was shown to prevent GM induced ototoxicity in guinea pigs [80]. Read Hulka 1993 Under conditions where neither AGs or diuretics cause a hearing deficit, the combination of AGs and diuretics leads to complete destruction of the organ of Corti and total deafness [6]. Hair cells of the human vestibulum seem to be directly targeted as opposed to their corresponding neurons. For instance, Tsuji et al. (2000) conducted a seminal study on the vestibular histopathologic effects of aminoglycoside ototoxicity. Temporal bones were harvested from patients treated with streptomycin (n = 4), kanamycin (n = 3) and neomycin (n = 3). Audiologic analysis before death showed that both kanamycin and neomycin induced bilateral hearing loss while streptomycin resulted in no hearing loss but induced disequilibrium. Streptomycin induced loss of type I and II hair cells in all vestibular organs. Type I hair cells of all three crista showed the greatest loss. None of the three drugs affected Scarpa’s (vestibular) ganglion. Kanamycin induced little damage to the vestibulum and neomycin did not induce any vestibular damage [11]. So kanamycin and neomycin are ototoxic and streptomycin is vestibulotoxic. The protection of both hair cells and neurons would be a highly attractive strategy to combat aminoglycoside ototoxicity. A few animal studies have espoused such a possibility. For instance, Hester et al. (1998) co-treated guinea pigs with aminoglycosides (neomycin or polymyxin B; 50_l, r.w.) and the _-phenyl-tertbutyl-nitrone (60 mg/kg, i.p.) spin trap. Aminoglycoside treatment alone resulted in high-frequency hearing loss which progressed to the low frequencies as determined by neural CAP and hair cell cochlear microphonics (CM). Co-treatment resulted in significantly less hearing loss [11]. Ylikoski et al. (2002) co-treated Dunkin–Hartley guinea pigs (300–400 g) with gentamicin (120 mg/kg, s.c.) and CEP-1347 (1mg/kg, s.c.). The results showed reduced ABR threshold shifts, reduced cochlear hair cell damage (particularly IHCs) and reduced ampullary crista hair cell damage (particularly type I). These results suggest that the JNK pathway may underlie gentamicin ototoxicity and blocking this pathway may be a treatment option. Additionally,Wu et al. (2001) co-treated CBA/J, C57BL/6 and BALB/c mouse strains and Sprague Dawley rats with kanamycin (400–900mg/kg for 15 days, s.c.) and the antioxidant 2,3-dihydroxybenzoate (300mg/kg, s.c.). Treatment with kanamycin alone induced ABR threshold shifts of 70 dB at 24 kHz and vestibular hypofunction. Hair cell losswas revealed in both cochlear and 57
  • 58. vestibular neurosensory epithelia. Co-treatment reduced ABR threshold shifts, hair cell loss and vestibular hypofunction [11]. What does CEP-1347 do? Aminoglycoside cochleotoxicity leads to permanent severe high-frequency hearing loss. The high-frequency hearing loss develops as soon as 4 h post- treatment and eventually progresses to the low frequencies [11]. Absent from animal experiments on otoprotection are issues of individual susceptibility. Some individuals may develop hearing loss after 1 day of treatment while others may experience no hearing loss even after several months of treatment (Wang et al., 1999). The issue of individual susceptibility has been explored in the seminal work of Wang et al. (1999). These investigators sampled serum from patients who were either resistant or vulnerable to aminoglycoside ototoxicity. The serum was used to treat isolated OHCs from pigmented guinea pigs. The results showed that serum from patients who were vulnerable to aminoglycoside ototoxicity were significantly more cytotoxic to the OHCs than serum from patients who were resistant. The serum was cytotoxic even after one year post-treatment. Additionally, the combination of streptomycin and the toxic serum was more cytotoxic than streptomycin alone. This suggests that the serum from individuals who are susceptible to aminoglycoside induced ototoxicity may metabolize streptomycin to a toxic metabolite [11]. Or these individuals are able to somehow neutralize AGs. The affinity of polyamines to the anionic phospholipids of the mitochondrial membranes (spermine = neomycin > gentamicin > streptomycin = spermidine) correlates roughly with their ability to induce PTP-independent release of SIMP, suggesting that binding of the polycations to the anionic phospholipids of the OMM facilitates the rupture of this membrane [77]. How does this compare to the ototoxic ranking order found by Kotecha and Richardson? Effects of AGs on cultured OHCs: Neomycin>gentamycin>dihydrostreptomycin>amikacin>neamine>spectinomy cin. That compares well. Are there other reports that link affinity of AGs for phosphoinositides to ototoxic ranking order? I believe Schacht mentions it in the 1998 Sha and Schacht paper. Pro-apoptotic Bcl-1 proteins can trigger cytochrome c release [77]. Do AGs stimulate pro-apoptotic Bcl-2 proteins? Ca2+ , nitric oxide and ROS may induce the release of pro-apoptotic proteins from the mitochondria [77]. 58
  • 59. Li, L. and Forge, A., 1995. Cultured explants of the vestibular sensory epithelia from adult guinea pigs and effects of gentamicin: a model for examination of hair cell loss and epithelial repair mechanisms. Audit. Neurosci. 1, pp. 111–125. Where is this ref? Aminoglycosides are toxic to zebrafish neuromast hair cells [42]. - It has been reported that aminoglycoside HC degeneration is concentration dependent, see figure 2 in [108]. Is this apoptosis related? Aminoglycosides in kidney cells - Apparently polyaspartic acid when co-administered with either gentamicin or amikacin protects against early and late signs of aminoglycoside nephrotoxicity [73]. Does polyaspartic acid also prevent AG induced degeneration of HCs in cultures? Polyaspartic acid apparently can protect against gentamicin induced nephrotoxicity by binding to gentamicin, displacing it from negatively charged phospholipids and preventing the inhibition of lysosomal phospholipases. Also, polyaspartic acid was shown to prevent GM induced ototoxicity in guinea pigs [80]. - It has been reported that AGs enter HCs via receptor mediated endocytosis on the basis of colocalisation of immunogold labeled KM and ferritin [25]. It has also been reported for cultured kidney cells that AGs enter via endocytosis [119]. Is the presence of ferritin a good measure for determining that? I have the impression that the Hashino 1995 paper isn’t all that good. - It is reported that megalin is expressed in cochleal sensory cells and in kidney proximal tubule cells [12].  megalin is not present in cochleal sensory cells [117]. Is this true? Check references. Could this be some connecting mechanism which accounts for ototoxicity and nephrotoxicity? - It has been reported that after glomerular filtration a small but sizable proportion (5%) of aminoglycosides is retained in the epithelial cells lining the S1 and S2 segments of the proximal tubules [73]. 59
  • 60. Sundin et al [94] conclude that gentamicin inhibits phospholipid degradation. They also conclude that gentamicin reduces cellular protein synthesis (which makes sense considering that aminoglycosides bind to ribosomal RNA). Can the effects of gentamicin on phospholipid metabolism be related to the results in the Bryant thesis? Are there gentamicin congeners or enantiomers which are bactericidal but not ototoxic? Sandoval et al. [14] have reported on a component isolated from the native gentamicin congener mixture which retains its bactericidal properties but with minimal or no apparent nephrotoxicity. This particular congener (C2) did not induce the formation of ‘myeloid bodies’ which have been classically identified with gentamicin toxicity. They also conclude that variation seen in gentamicin induced nephrotoxicity is due to the naturally occurring variation in the percentage of C2 congener composition of native gentamicin from commercial lot to lot. - It has been reported that OHCs contain an apical network of so called ‘canalicular reticulum’. Apparently, similar structures have been found in kidney proximal tubule cells [86]. Aminoglycosides general - Aminoglycosides can also induce neuromuscular paralysis, block of calcium channels at the neuromuscular synapse [2]. What is the mechanism? Is it related to ototoxicity and nephrotoxicity? TRP channels? AGs affect Ca2+ channels, HCs, the neuromuscular junction, kidney cells and mitochondria. - It is reported that AGs are nonmetabolizable [119]. Is this in fact true? Find out about AG metabolism. Is there a correlation between ototoxicity of various aminoglycosides and their effects on mitochondria? i.e. are mitochondria more severely affected by strong ototoxic AGs (such as neomycin) than weaker ones, such as spectinomycin? Our results from experiments with isolated rat liver mitochondria exposed to various AGs shows there is a clear correlation. Nifedipine (inhibitor of low voltage-gated calcium channels, L-type) inhibits the increase of intracellular calcium induced by gentamicin [55]. 60
  • 61. This suggests the calcium has an extracellular origin. Does this have implications for the MET channel? Does Nifedipine block the channel? Does Nifedipine block TRP channels? - Commercially available gentamicin is a mixture of various congeners. The bactericidal and cellular toxicity vary significantly. Pure bacterial components which do not show ototoxicity are not available yet [68]. Are there gentamicin congeners or enantiomers which are bactericidal but not ototoxic? Sandoval et al. [14] have reported on a component isolated from the native gentamicin congener mixture which retains its bactericidal properties but with minimal or no apparent nephrotoxicity. This particular congener (C2) did not induce the formation of ‘myeloid bodies’ which have been classically identified with gentamicin toxicity. They also conclude that variation seen in gentamicin induced nephrotoxicity is due to the naturally occurring variation in the percentage of C2 congener composition of native gentamicin from commercial lot to lot. They didn’t look for ototoxicity. We should really test gentamicin C2 on haircells. - Modification of Kanamycin A yielded Amikacin and tobramycin was purified from nebramycin , these commercially available AGs are less toxic than the original AGs from which they are derived [14]. How were the AGs changed? Get the original reference - Apparently gentamicin and amikacin can suppress premature stop codon in the IDUA and p53 genes, but tobramycin can’t [14]. This shows that different AGs have different binding properties which may reflect differences in reactivity? Bactericidal activity of AGs occurs by interference with bacterial translation. AGs bind to the 16S ribosomal subunit, altering its conformation at the A-site and disrupting codon-anticodon interaction and thus disrupt the fidelity of the translation process. The eukaryotic mitochondrial ribosome more closely resembles the prokaryotic ribosome and may share its aminoglycoside sensitivity [31]. - Polyphosphoinositides have a high binding affinity for aminoglycosides [6]. Compare with the results from the Bryant thesis. - Streptomycin charge = +3 (maximum charges) neomycin charge = +6 tobramycin charge = +5 61
  • 62. gentamicin charge = +5 At pH = 7 Tobramycin charge = + 3.39 ± 0.04 Neomycin charge = + 4.09 ± 0.24 From [96] Is the ototoxic potential of AGs correlated to their charge? That is, high positive charge of AG  large driving force for AG uptake? Does the pH have an effect on AG induced mito damage? Isolate mitos and incubate at different pH values, e.g. mitos at pH 7, mitos at pH 5, mitos + neo at pH 7 and mitos + neo at pH 5. What is the connection between lysosomes and AGs? Do AGs get taken up by them? - Polyamines such as putrescine, spermine and spermidine appear to be actively transported into mitochondria [97]. - Gentamicin and polylysine are able to enhance mitochondrial Ca2+ accumulation [97]. Gentamicin has three isoforms with molecular weights: (C1) 449.5 (C2) 463.5 (C3) 477.6 [15] Aminoglycoside antibiotics are recovered from the body largely unchanged and it has been assumed that they do not undergo significant metabolism [99]. It is hypothesized by Crann et al that gentamicin when incubated with liver cell cytosol undergoes a chemical transformation making it ototoxic [99]. The reason why Schacht et al think that gentamicin needs to be converted chemically is because they think that isolated hair cells are not affected by gentamicin. The work by Kotecha and others (REFS) clearly shows that gentamicin has a clear effect on hair cells. According to Crann et al OHCs incubated in cytosol were not affected, but OHCs incubated with cytosol and gentamicin were. Maybe there is a synergistic effect of gentamicin and the cytosolic enzymes? Importantly, in the Crann 1992 study the effects of so called biotransformed gentamicin in liver cytosol was not tested on other cell types. So this study does not address the issue why gentamicin preferentially targets hair cells. Also, it is not clear from the study if indeed Crann et al did the following control: 62
  • 63. Incubate the isolated OHCs in just cytosol without gentamicin. Perhaps it is just the presence of enzymes which causes the degeneration of OHCs? How do aminoglycosides get taken up by bacteria? Taken from ‘Measuring and modelling the transducer channel of mammalian hair cells’ grant proposal. Refs: Nakashima T, Teranishi M, Hibi T, Kobayashi M & Umemura M. (2000). Vestibular and Cochlear Toxicity of Aminoglycosides - A review. Acta Oto-Laryngol 120, 904 - 911. Jezowska-Bojczuk M & Lesniak W. (2001). Coordination mode and reactivity of copper(II) complexes with kasugamycin. Journal of Inorganic Biochemistry 85, 99-105. Botto RE & Coxon B. (1983). N-15 Nuclear Magnetic-Resonance Spectroscopy Of Neomycin-B And Related Aminoglycosides. Journal of the American Chemical Society 105, 1021-1028. Ziv G, Bogin E, Shani J & Sulman FG. (1973). Distribution and Blood-to-Milk Transfer of Labeled Antibiotics. Antimicrob Agents Chemother 3, 607-613. Kane RS, Glink PT, Chapman RG, McDonald JC, Jensen PK, Gao H, Pasa-Tolic L, Smith RD & Whitesides GM. (2001). Basicity of the Amino Groups of the Aminoglycoside Amikacin Using Capillary Electrophoresis and Coupled CE-MS-MS Techniques. Anal Chem 73, 4028-4036. 63
  • 64. Clearly neomycin and gentamicin have different blocking potencies with neomycin being the more potent blocker. Does this reflect the ototoxic potential as seen in Kotecha’s study? Some bacteria have developed enzymes which can modify aminoglycosides, conferring resistance [37]. Are there mammalian analogs of these enzymes? Sha and Schacht found that gentamicin stimulated the formation of superoxide and hydrogen peroxide in EBV-transformed lymphoblastoid cells. For some reason no ROS is formed in the absence of PMA, apparently this needs to be present to ‘stimulate the cells’. I don’t understand this aspect. It is not explained in the materials and methods [76]. The generation of ROS stimulated by gentamicin in lymphoblastoid cells is dose-dependent [76]. At a drug concentration of 10 mM the stimulation of ROS in lymphoblastoid cells by gentamicin and kanamycin was higher than by neomycin and streptomycin [76]. (See figure 3 in the article). This does not correspond to the ototoxic ranking order found by Kotecha and Richardson. It does mean that different AGs have different ototoxic potencies. Apparently the iron chelators DFO and DBH attenuated ROS formation in both cell and cell free systems [76]. Aren’t DFO and DBH ROS scavengers as well, next to being iron chelators? Is iron chelation really involved here? Get the Lohdi et al 1980 paper OTOTOXICITY OF AMINOGLYCOSIDES CORRELATED WITH THEIR ACTION ON MONO-MOLECULAR FILMS OF POLYPHOSPHOINOSITIDES  not available online Get Wang 1984 CHARACTERIZATION OF AMINOGLYCOSIDE LIPID INTERACTIONS AND DEVELOPMENT OF A REFINED MODEL FOR OTOTOXICITY TESTING  not available online Gentamicin was discovered in 1963 and is currently the most widely used AG [11]. 64
  • 65. Various - It has been reported that during noise exposure OHCs are more readily damaged than IHCs  REF? It is unlikely that the difference in sensitivity of the HCs for noise is attributable to a difference in conductivity of the METs. So what could be the cause? Is there also a base-to-apex gradient of OHC destruction upon noise exposure? If not this would go against Schacht’s hypothesis. - Apparently regeneration of mammalian vestibular hair cells has been reported [6]. - In avian neuroepithelia haircells regenerate through transdifferentation or by mitosis [3]. Gene therapy is a promising technique when it comes to regeneration of haircells in mammals. Introducing exogenous Atoh 1 leads to differentiation into hair cells [3]. Vestibular hair cells apparently have regenerative potential [11]. 65
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