Учебники / Auditory Trauma, Protection, and Repair Fay 2008

232 L.P. Rybak, A.E. Talaska, and J. Schacht

(Prim et al. 2001), others have found none in animals (Myers et al. 1993); even high doses of cisplatin did not induce significant morphological damage on the vestibular neuroepithelium in the guinea pig (Schuknecht et al. 1993; Sergi et al. 2003).

10.2 Aminoglycosides

Vestibular pathology is a major side effect of aminoglycoside antibiotics. It is again the hair cells that are affected in the vestibular organs, and the initial damage occurs in the apex of the cristae and the striolar regions of the maculi (Caussé et al. 1949; Lindemann 1969). From there, hair cell loss progresses toward the periphery of the vestibular receptor organ with type I hair cells affected earlier than the type II hair cells (Wersäll et al. 1969). The otoconial membrane and otolith structures may also be affected.

Like their cochlear counterparts, afferent nerve endings and ganglion cells will eventually also deteriorate (Li et al. 1995) and may be protected by nerve growth factors (Altschuler, Chapter 10). Regeneration of vestibular hair cells has been observed in mammalian species (Forge et al. 1993, 1998) and even the cochlea may have a latent capacity to regenerate hair cells (see Raphael and Heller, Chapter 11).

11. Pharmacokinetics

11.1 Cisplatin

Following systemic injections, more than ninety percent of cisplatin is bound to serum protein, and this cisplatin–protein complex is biologically inactive (Gormley et al. 1979). About 25% of administered cisplatin is eliminated from the body during the first 24 hours, with renal clearance for more than 90%. This clearance follows triphasic pattern, with an initial plasma half-life (t1/2) of 20–30 min, a second phase t1/2 of 60 min, and a terminal t1/2 of more than 24 h (Himmelstein et al. 1981). Cisplatin preferentially collects in the liver, kidneys, and large and small intestines, with little penetration of the central nervous system (Vermorken et al. 1984).

Aiding the antitumor activity of cisplatin is the fact that a large amount of cisplatin may remain in brain tumors after intra-arterial administration. Up to a 10-fold greater amount of radioactivity was detected in brain tumor tissue compared with normal brain using scintigraphic imaging following radiolabeled cisplatin intraarterial injection. After intravenous injection, however, the differential localization of label in tumors was seldom greater than twice that of normal brain (Shani et al. 1989).

The cellular uptake of cisplatin was initially thought to occur by passive diffusion (see review by Wang and Lippard 2005) but more recent studies have established a direct link between the cellular regulation of copper and platinum concentrations. Cisplatin resistance in tumor cells has been associated

with mutations or deletions in copper transporter genes controlling drug uptake (Ctr 1) and drug efflux (ATP7B and ABCC2). These observations are supported by the fact that both copper and cisplatin can prevent the uptake of each other (Ishida et al. 2002). Consistent with its targets in the cochlea, platinated DNA has been localized to the nuclei of OHCs, marginal cells of the stria vascularis, and the cells in the spiral ligament (van Ruijven et al. 2005b). No mention was made regarding uptake into vestibular structures.

11.2 Aminoglycosides

Aminoglycosides exhibit negligible binding to serum proteins. After systemic application, they reach peak plasma levels by 30–90 min and their plasma half-life ranges from 2 to 6 h. The drugs are excreted essentially unaltered. Aminoglycoside antibiotics enter the inner ear via the bloodstream within minutes after an injection and may reach a plateau as early as after 0.5–3 h (Tran Ba Huy et al. 1986). Drug concentrations in the inner ear typically remain at onetenth of peak serum levels (Henley and Schacht 1988) but are less efficiently cleared from the inner ear than from serum. Clearance is biphasic and the halflife of the second phase may exceed 30 days (Tran Ba Huy et al. 1986), and even 11 months after cessation of treatment gentamicin could be found in hair cells (Dulon et al. 1993). This difference in half-lives in serum and cochlear tissues incorrectly gave rise to the idea of an “accumulation” of aminoglycosides in the inner ear and was held responsible for the organ specific toxicity of these drugs. However, the concentrations reached in the inner ear by different aminoglycosides correlate neither with the magnitude of their ototoxic potential (Ohtsuki et al. 1982) nor with their preferential vestibular or cochlear toxicity (Dulon et al. 1986).

The precise mechanisms of aminoglycoside uptake into hair cells remain enigmatic. Even localization studies in the inner ear are contradictory and differences have been reported whether immunocytochemistry or radioactive or fluorescently tagged drugs were employed. While a preferential uptake into hair cells can be seen in some studies, others find a more widespread distribution in the cochlea (Imamura and Adams 2003; see also the review by Steyger 2005).

Potential transport mechanisms include a polyamine-like transport consistent with the polyamine-like nature of the drugs (Williams et al. 1987) and a vesicular transport at the base of hair cells (Lim 1986). The possibility of endocytotic uptake at the apex of the hair cell was suggested by early observations that lysosomes appear in the subcuticular portion of hair cells soon after systemic treatment of guinea pigs with kanamycin (Darrouzet and Guilhaume 1974). An uptake mechanism at the apical region of hair cells is also supported by the apparent involvement of myosin VII-A (Richardson et al. 1997). The myosin VII mutation affects the turnover of the apical plasma membrane of hair cells but not their basolateral membrane and explants from the inner ear of mutant mice do not take up aminoglycosides. Another candidate transporter, the glycoprotein megalin, has been suggested by evidence from the proximal tubules of the kidney

proteins, lipids, and metabolic intermediates. Today most of these are considered secondary events and not causally related to the mechanisms that trigger aminoglycoside toxicity. One of the earliest adverse reactions of aminoglycoside antibiotics was an antagonism of these drugs with calcium, originally discovered as a neuromuscular blocking action (Vital-Brazil 1957). The underlying mechanism was later confirmed as a block of calcium channels, and aminoglycoside antibiotics were widely used as experimental tools to elucidate calcium channel function (Corrado et al. 1989). For example, aminoglycosides may block N-type and P/Q-type channels in neurons (Pichler et al. 1996) as well as prevent calciumentry into hair cells (Dulon et al. 1989). They also block transduction channels at the tips of stereocilia (Kroese et al. 1989). This action does not directly lead to hair cell death (Kossl et al. 1990), and such acute actions may not be causally related to the chronic ototoxicity of the drugs.

Aminoglycoside–calcium interactions are yet another example of the complexity of the actions of these drugs. On the one hand, aminoglycoside antibiotics are confirmed calcium channel blockers; on the other hand, gentamicin was able to increase intracellular calcium in explants of the chick sensory epithelium (Hirose et al. 1999). Aminoglycoside antibiotics frequently exert apparently contradictory actions, often doseor tissue-dependently, for example stimulating or inhibiting free radical formation (Priuska and Schacht 1995) or stimulation or inhibiting lipid metabolism (McDonald and Mamrack 1995). Aminoglycoside antibiotics are also agonists at calcium sensing receptors that respond to these drugs by a mobilization of intracellular calcium (Ward et al. 2005). Such an activation of calcium-sensing receptors and elevation of intracellular calcium has been suggested to contribute to the renal toxicity of aminoglycoside antibiotics by disrupting homeostasis and initiating of calcium-dependent cell death pathways.

13. Oxidative Stress

The overproduction of reactive oxygen species (ROS; free radicals) and the resulting redox imbalance in the cell now appears to be a common mechanism by which many forms of stress cause damage to the inner ear, including age, noise, and ototoxic drugs. Mitochondrial respiration and oxidative enzymatic processes produce reactive oxygen species in all cells under normal conditions. While some ROS are simply byproducts of metabolism (such as mitochondrial “leakage” of superoxide radicals during respiration), others are essential metabolites or physiological mediators and second messengers (such as nitric oxide). Detrimental consequences can arise from an overproduction of ROS whereby, for example, an increased level of superoxide radicals can lead to the formation of hydrogen peroxide. Hydrogen peroxide can be catalyzed in a Fenton-type reaction by iron to form the hydroxyl radical, which is highly reactive and can cause peroxidation products including the highly toxic aldehyde, 4-hydroxynonenal.

236 L.P. Rybak, A.E. Talaska, and J. Schacht

Another source of radicals, reactive nitrogen species, can arise from the activation or induction of the enzyme nitric oxide synthase. The different forms of this enzyme serve a variety of physiological processes in normal tissue physiology and the product of the enzymatic reaction, nitric oxide, is an important second messenger molecule. When produced in excess, nitric oxide is not only potentially damaging as a free radical, but it can also combine with superoxide to produce the highly reactive and destructive peroxynitrite, which can react with proteins to form nitrotyrosine.

Balancing the adverse potential of ROS and maintaining redox homeostasis is the cellular antioxidant system. Perturbations of this physiological balance may result from a direct interaction of ROS with antioxidants such as glutathione (scavenging) or, at the level of antioxidant enzyme activity by (1) direct binding of a toxic drug to essential sulfhydryl groups within the enzymes; (2) depletion of copper and selenium, which are essential for superoxide dismutase and glutathione peroxidase activities; (3) increased ROS and organic peroxides which inactivate antioxidant enzymes; and (4) depletion of glutathione and the cofactor NADPH, which are required for detoxifying glutathione peroxidase and glutathione reductase activities.

The increased production of ROS and the resulting depletion of antioxidant capacity can initiate cell death pathways through the activation of redox-sensitive transcription factors or through calcium influx within cochlear cells, leading to pathological changes resulting in apoptosis. Chapter 3 by Wangemann gives a detailed account of homeostasis and homeostatic perturbations.

13.1 Cisplatin

Several lines of evidence indicate the formation of reactive oxygen species and a resulting redox imbalance in cisplatin-treated tissues. Cisplatin generates reactive oxygen species in cochlear tissue explants (Clerici et al. 1996; Kopke et al. 1997) including superoxide anion (Dehne et al. 2001) which may also be involved in nephrotoxicity (McGinness et al. 1978). The origin of the cochlear superoxide is speculative but an isoform of NADPH oxidase, NOX 3, could be a major source of its generation and constitute part of the pathway leading to cisplatin-mediated hair cell damage (Mukherjea et al. 2006). NOX 3 was upregulated following systemic cisplatin administration in the rat cochlea and after in vitro cisplatin application to hair cell lines derived from the immortomouse. Another source for superoxide anion may be xanthine oxidase although it has not been directly demonstrated that its activity increases in the cochlea after cisplatin exposure. Rather, this hypothesis is based on the partial protection against cisplatin afforded by allopurinol, an inhibitor of this enzyme (Lynch et al. 2005a, b). Nevertheless, in agreement with the emergence of reactive oxygen species, 4-hydroxynonenal has been detected immunohistochemically in the cochlea after cisplatin treatment (Lee et al. 2004a) as has malondialdehyde, an indicator of lipid peroxidation (Rybak et al. 2000).

In addition to superoxide, reactive nitrogen species, such as nitric oxide, may play a role in cisplatin ototoxicity, at least in the lateral wall of the cochlea and in the spiral ganglion cells. Increased nitric oxide levels have been found in cochlear extracts of rat cochleae after treatment with cisplatin (Kelly et al. 2003) and in the stria vascularis the onset of apoptosis was correlated with increased immunolabeling of 4-hydroxynonenal, nitrotyrosine, and iNOS (Lee et al. 2004a).

As a consequence of the increased formation of reactive oxygen and nitrogen species, cochlear tissues are depleted of glutathione and antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) (Ravi et al. 1995; Rybak et al. 2000). The resulting redox imbalance will then trigger pathways of cell death.

13.2 Aminoglycosides

Oxidative stress is also part of the toxic action of aminoglycosides although the details of ROS formation differ. In retrospect, experimental studies in the 1950s and 1960s can be interpreted as indicating the participation of ROS in aminoglycoside ototoxicity. Substances like 2,3-dimercaptopropanol were found to protect against the side effects of streptomycin but positive results were also met with failures laying such attempts to rest (see Federspil 1979 for a review of the early literature). The speculations were revived by the ability of a radical scavenger to protect from the auditory side effects of kanamycin (Pierson and Moller 1981) only to be challenged by a lack of protection by another radical scavenger (Bock et al. 1983). It took another decade to establish that antioxidants could limit ototoxicity and that free radicals were generated in tissues exposed to aminoglycosides (Lautermann et al. 1995; Clerici et al. 1996; Hirose et al. 1997). Clear evidence now exists for the potential of aminoglycoside antibiotics to catalyze ROS formation both nonenzymatically and by stimulating enzymatic reactions.

A step toward the understanding one of the mechanisms of ROS formation was the observation that gentamicin was able to accelerate iron-mediated formation of free radicals (Priuska and Schacht 1995). Since iron-catalyzed oxidations can be greatly accelerated by chelators, it was postulated that gentamicin and iron may form redox-active complexes. These complexes reduce molecular oxygen to superoxide radicals at the expense of electrons provided by polyunsaturated fatty acid with arachidonic acid being a particularly suitable coreactant (Sha and Schacht 1999a). The availability of free arachidonic acid is low in an intracellular environment where most is esterified to phospholipids. Polyphosphoinositides generally contain arachidonate as one of their fatty acid esters, and it had long been known that aminoglycosides strongly bind to phosphatidyl inositol 4,5- bisphosphate (Schacht 1979), suggesting the possibility that the lipid itself could provide reactive electrons through its arachidonic acid content. Redox-active ternary complexes between Fe2+/3+, gentamicin, and arachidonic acid, capable of producing superoxide radicals, indeed exist (Lesniak et al. 2005).

238 L.P. Rybak, A.E. Talaska, and J. Schacht

Enzymatic mechanisms may be an additional source of ROS in aminoglycoside toxicity. One of the initial reactions of aminoglycoside antibiotics in vivo is an activation of redox-dependent molecular signaling pathways linked to RhoGTPases (Jiang et al. 2006b). The activity of Rac-1, a member of the family of Rho-GTPases, is enhanced by aminoglycosides, thereby leading to activation of the NADPH oxidase complex which enzymatically promotes the formation of the superoxide radicals. Such an action would be akin to the stimulation of the NOX 3 isoform of NADPH oxidase by cisplatin.

Although acute exposure of tissues to aminoglycosides in vitro or by local application may generate nitric oxide, for example in vestibular structures (Takumida et al. 2000), evidence for such a reaction is notably absent from the chronically exposed mouse cochlea (Jiang et al. 2005). This resembles the situation with cisplatin, which also has the capability of enhancing NO formation in the stria vascularis and spiral ganglion cells (Liu et al. 2006) but apparently does not do so in hair cells.

The question whether the formation of ROS causally relates to cell death or simply represents an epiphenomenon is best answered by the ability of iron chelators and antioxidants to protect against aminoglycoside ototoxicity (see below and Green, Altschuler, and Miller, Chapter 10).

14. Biochemical Basis of Genetic Susceptibility

Considering the enhanced aminoglycoside ototoxicity due to the mitochondrial mutations, it is interesting to notice that these mutations increase the structural similarity of the mitochondrial RNA in this region to the bacterial ribosomal RNA (Prezant et al. 1993), which is a target site of the antimicrobial actions of the drugs. Binding experiments have proven that aminoglycoside binding to the mitochondrial 12S ribosomal RNA is enhanced (Hamasaki and Rando 1997), potentially resulting in altered protein synthesis in the mitochondria. However, it has not yet been established whether protein synthesis is indeed affected in the cochlea in vivo in response to aminoglycosides. Furthermore, the vestibular system is not involved in the enhanced response to aminoglycosides (Tono et al. 2001), leading to more unresolved questions on how the mutation interacts with proposed mechanisms of toxicity.

15. Pathways of Cell Death

On oxidative insult, a plethora of molecular pathways can be activated or attenuated, leading to cell death or survival. These pathways of cell death and survival are a complex network of interfacing signaling systems involving the activation of a variety of transcription factors and proteases, and the participation of intracellular organelles such as mitochondria or lysosomes (Leist and Jäättelä 2001). Of particular interest in the context of drug-induced hearing loss are caspases, a

family of proteases that are inactive in the basal state and promote apoptotic cell death upon activation. Also frequently investigated is the Bcl-2 family of intracellular proteins consisting of both pro-apoptotic and anti-apoptotic members. The anti-apoptotic members include Bcl-2 and Bcl-XL while the pro-apoptotic proteins include two classes, the Bax and Bak subfamily and the BH-3 proteins such as Bid, Bad, Bim/Bod, and PUMA (Huang and Strasser 2000). Apoptosis is controlled within the cell by a balance between proand anti-apoptotic Bcl-2 family proteins (Cheng et al. 2005). When apoptosis is triggered, Bax can translocate from the cytoplasm to the mitochondria and increase the permeability of the mitochondrial membrane. This can lead to loss of mitochondrial membrane potential, generation of ROS and leakage of cytochrome c into the cytoplasm (Cheng et al. 2005). The p53 tumor suppressor gene, induced by DNA damage, is another important mediator of cell death (Oren 1999). When the DNA repair mechanisms fail, p53 can be activated and upregulate Bax. Alternatively, p53 can translocate to the mitochondria and damage them directly leading to loss of membrane potential and induction of apoptosis (Cheng et al. 2005). Also of relevance, the c-jun NH2-terminal kinases (JNKs) are a group of the mitogenactivated protein (MAP) kinases generally involved in apoptotic events. JNKs are activated following a variety of cell insults, such as irradiation, excitotoxic damage and inflammatory cytokines. Chapter 10 by Green, Altschuler, and Miller provides a comprehensive account of cell death pathways.

15.1 Cisplatin

Morphological and histochemical evidence points to apoptosis as the major form of cisplatin-induced cell death in the inner ear. Apoptotic markers such as terminal transferase dUTP nick end labeling (TUNEL)-staining label primarily the OHCs in the organ of Corti, the stria vascularis, spiral ligament and the spiral ganglion cells (Alam et al. 2000; Watanabe et al. 2003; Liang et al. 2005). Several pathways, individually or on concert, may contribute to this pattern of demise. Caspases appear pivotal, and members of the intrinsic apoptosis caspase cascade, the Bcl-2 family proteins and caspase-9, are activated after cisplatin treatment but c-Jun N-terminal kinases may play a lesser role.

15.1.1 Caspases

General inhibitors of caspases prevent hair cell death after cisplatin exposure (Liu et al. 1998) and, specifically, cochlear hair cells were preserved from cell death and hearing loss was prevented in guinea pigs treated with cisplatin by concomitant perilymphatic perfusion of inhibitors of caspase-3 and caspase-9 (Wang et al. 2004). Likewise, in cochlear and utricular organotypic cultures explanted from postnatal day 3–4 rats cisplatin caused a dose-dependent loss of hair cells and increased immunolabeling for caspase-1 and caspase-3.

Caspase-3 (as well as other caspases) can be activated by caspase-8 which is closely linked to the death domain-containing receptors in the cell membrane

240 L.P. Rybak, A.E. Talaska, and J. Schacht

(Cheng et al. 2005). Although an in vitro study of immortalized mouse hair cells (HEI-OC1 cells) reported an early but transient increase in caspase-8 activity after application of cisplatin (Deravajan et al. 2002), immunohistochemical studies revealed no significant caspase-8 activation in guinea pigs after systemic cisplatin administration, and a caspase-8 inhibitor was unable to protect guinea pig OHCs from apoptosis (Wang et al. 2004). Therefore, caspase-3 might be activated by caspase-9, an upstream caspase which, in turn, is activated by cytochrome c released from permeabilized mitochondria.

Cell death mechanisms in the stria vascularis appear to be similar to those in hair cells. Caspase-3 and caspase-activated deoxyribonuclease were detected by immunohistochemistry in the stria vascularis and spiral ligament (Watanabe et al. 2003). Local application of cisplatin in adult mouse resulted in apoptotic cell death of marginal cells 3 days after treatment and immunostaining was positive for caspases-3 and -9, but not for caspase-8. The finding that cytochrome c was redistributed in affected marginal cells suggested a caspasedependent, mitochondrion-mediated pathway in marginal cells.

15.1.2 Bcl-2 Family Proteins

The expression of Bcl-2 is reduced and that of Bax is increased in auditory hair cells from gerbils treated with cisplatin (Alam et al. 2000). Bax was also translocated from the cytosol to the mitochondria in OHCs (Wang et al. 2004). Consistent with the activation of mitochondrial death pathways was the release of cytochrome c into the cytosol of OHCs and supporting cells in the cochleae of guinea pigs treated with cisplatin (Wang et al. 2004). Similar observations have been made in vitro in hair cell lines treated with cisplatin (Deravajan et al. 2002).

15.1.3 Other Mechanisms

Exposure of mouse hair cell lines and organotypic cultures of the organ of Corti to cisplatin in vitro increased p53 expression (Deravajan et al. 2002; Zhang et al. 2003). Conversely, deletion of the p53 gene prevents cisplatininduced caspase-3 activation, cytochrome c translocation, and hair cell death (Cheng et al. 2005).

Cisplatin can also induce apoptosis in the fibrocytes of the lateral wall by activation of potassium channels, leading to potassium efflux, reducing intracellular ionic and osmotic strength, which in turn can trigger apoptosis by activating pro-apoptotic enzymes, such as caspases and pro-apoptotic nucleases. These cellular losses can then affect ion transport and endocochlear potential generation in the stria vascularis, changes that in turn may affect other cells within the cochlea (Liang et al. 2005).

Studies using JNK inhibitors suggest that the JNK pathway is not involved in the hair cell death induced by cisplatin, but rather that it may play a role in the repair of DNA and in the maintenance of cisplatin-damaged hair cells (Wang et al. 2004).