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Keywords:

  • cisplatin;
  • oxaliplatin;
  • carboplatin;
  • apoptosis;
  • ethacrynic acid;
  • caspase-8;
  • TRADD;
  • copper transporter

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED

Cisplatin, carboplatin, nedaplatin, and oxaliplatin are widely used in contemporary oncology; however, their ototoxic and neurotoxic side effects are quite different as discussed in this review. Cisplatin is considered the most ototoxic, but despite its reputation, the magnitude of hair cell loss that occurs with a single, large drug bolus is limited and confined to the base of the cochlea. For all of these platinum compounds, a major factor limiting damage is drug uptake from stria vascularis into the cochlear fluids. Disrupting the blood–labyrinth barrier with diuretics or noise exposure enhances drug uptake and significantly increases the amount of damage. Combined treatment with ethacrynic acid (a loop diuretic) and cisplatin results in rapid apoptotic hair cell death characterized by upregulation of initiator caspase-8 and membrane death receptor, TRADD, followed by downstream executioners, caspase-3 and caspase-6. Unlike cisplatin, nedaplatin and oxaliplatin are highly neurotoxic when applied to cochlear cultures preferentially damaging auditory nerve fibers at low concentrations and hair cells at high concentrations. Carboplatin, considered far less ototoxic than cisplatin, is paradoxically highly toxic to chinchilla inner hair cells and type I spiral ganglion neurons; however, at high doses it also damages outer hair cells. Hair cell death from cisplatin and carboplatin is characterized in its early stages by upregulation of p53; blocking p53 expression with pifithrin-α prevents hair cell death. Major differences in the toxicity of these four platinum compounds may arise from several different metal transporters that selectively regulate the influx, efflux, and sequestration of these drugs. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED

Platinum-based chemotherapeutic drugs are widely used in contemporary oncology, but their life saving benefits are partially offset by their toxic side effects on the peripheral auditory system which lead to hearing loss and impaired social communication. Cisplatin, first approved for the treatment of testicular and ovarian cancer in the late 1970s, is widely used to treat many solid and disseminated cancers. Maximum dosing with cisplatin is limited by its severe nephrotoxic, neurotoxic, and ototoxic side effects (Aggarwal and Fadool, 1993). Carboplatin, a second-generation platinum derivative, is far less ototoxic and nephrotoxic than cisplatin, and therefore has seen increased use in recent years (Teeling and Carney, 1990; Alberts, 1995). Although the main dose-limiting side effects of carboplatin are myelosuppression and emesis, it has been reported to cause ototoxicity in some patients. Surprisingly, carboplatin is highly toxic to the inner hair cells (IHC) and type I auditory nerve fibers in at least one mammalian species for reasons yet unknown (Wake et al., 1993; Hofstetter et al., 1997; Ding et al., 1999). Nedaplatin (247-S) and oxaliplatin, second and third generation cisplatin analogs, are frequently used to treat ovarian cancer and cancers resistant to cisplatin therapy (Kosugi et al., 2005; Ohashi et al., 2011). The dose-limiting side effect of nedaplatin is myelosuppression (Piccart et al., 2001). Nedaplatin-induced nephrotoxicity and ototoxicity have been reported (Horiuchi et al., 1992; Uehara et al., 2005). Oxaliplatin is considered to be far less nephrotoxic and ototoxic than cisplatin; however, it frequently induces sensory neuropathies (Chollet et al., 1996; O'Dwyer et al., 2000), a condition reminiscent auditory neuropathy (Madden et al., 2002; Berlin et al., 2010).

Over the past 30 years, much effort has been expended to determine the relative ototoxicity of various platinum anticancer drugs, drug-dosing conditions that exacerbate ototoxicity, cochlear structures that are most vulnerable to platinum damage, therapies for reducing ototoxicity, and the molecular signaling pathways that lead to cell death in the inner ear. Although all these drugs share a common platinum backbone, the ototoxic effects of cisplatin, carboplatin, nedaplatin, and oxaliplatin can be quite different. Some differences are undoubtedly related to their unique chemical composition. Individual patient differences also play a role because the same drug dose may be ototoxic in one person, but not others (Peters et al., 2003; Oldenburg et al., 2007). Important species differences have also been uncovered among platinum therapeutics (Taudy et al., 1992; Ding et al., 1999; Sockalingam et al., 2000). Here, we review some of our earlier studies as well as more recent findings illustrating some of the common as well as the unique characteristics of cisplatin, carboplatin, nedaplatin, and oxaliplatin ototoxicity.

CISPLATIN OTOTOXICITY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED

Murine Models of Cisplatin Ototoxicity

While the literature indicates that ototoxicity is a serious side effect in patients undergoing therapy, the dosing regimens used with humans typically involves multiple rounds of treatment each separated by several weeks of recovery (Hannigan et al., 1993; Cavaletti et al., 1997). In contrast, most animals studies utilize a single large bolus or short round of high doses to establish the precise temporal relationship between the onset of the cisplatin treatment and the ensuing hearing loss, cochlear pathology, or biochemical changes (Campbell et al., 1996; Giordano et al., 2006; Garcia-Berrocal et al., 2007; Jamesdaniel et al., 2008). The limitations of using a single large bolus include significant mortality (30%–50%) by 5–7 days post-treatment, damage limited to the base of the cochlea, and hearing loss confined to the high frequencies (Ravi et al., 1995; Campbell et al., 1996; Li et al., 2002). Despite these limitations, the single-dose strategy is widely used in research in an attempt to understand the mechanistic nature of cisplatin ototoxicity.

Because of their enormous genetic diversity, mice have seen increased use in ototoxicity research (Oesterle et al., 2008; Ciarimboli et al., 2010). The CBA mouse, which is widely used in auditory research, is extremely resistant to cisplatin ototoxicity when treated with a single large bolus or multiple low doses administered over 4 consecutive days as described in detail in our recent publication (He et al., 2009). To quantify the degree of cisplatin-induced hair cell damage, cochleograms were prepared showing the percent IHC or outer hair cell (OHC) loss as a function of percent distance from the apex (Ding et al., 2001; Li et al., 2011b). Figure 1A,B compares the mean cochleogram from a group of CBA mice (n = 5) treated with 15 mg/kg (i.p.) of cisplatin (10 days post-treatment) versus the mean cochleogram (Fig. 1B) from a control group (n = 5) (He et al., 2009). In the cisplatin-treated group, mean OHC and IHC losses in the base of the cochlea were ∼60% and 20%, respectively. While a single 15 mg/kg dose of cisplatin induced OHC and IHC lesions in the extreme base of the cochlea, mortality with this treatment was nearly 70% and mice experienced significant weight loss (He et al., 2009). In an effort to reduce mortalities, another group of mice was treated with 4 mg/kg/day (i.p.) of cisplatin for 4 consecutive days (total dose 16 mg/kg). Distributing the total cisplatin dose over 4 consecutive days lowered the mortality rate to 10% and reduced weight loss somewhat; however, the OHC and IHC losses were negligible (Fig. 1C) (He et al., 2009). These findings are consistent with others indicating that it is difficult to induce significant and widespread hair cell loss or hearing loss in mice, gerbils, or chinchillas with a single or several, short doses of cisplatin (Gratton et al., 1990; Ding et al., 1999; Hill et al., 2008; Poirrier et al., 2010).

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Figure 1. Mean cochleograms showing the percent missing inner hair cells (IHC) and outer hair cells (OHC) as a function of percent distance from the apex. (A) Treatment with a single, 15 mg/kg dose of cisplatin, (B) normal controls, (C) treatment with 4 mg/kg/day of cisplatin for 4 consecutive days, (D) treatment with transtympanic injection of cisplatin; concentration 1 mg/mL, (E) treatment with 5 μL of cisplatin (1 mg/mL) on the round window, and (F) treatment with saline on the round window membrane.

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Local Administration of Cisplatin

To reduce the nephrotoxicity and mortality associated with systemic cisplatin treatment and increase ototoxicity, cisplatin was delivered transtympanically to the middle ear of CBA mice at a concentration of 1 mg/mL (He et al., 2009). Transtympanic delivery of cisplatin resulted in total OHC loss in the base of the cochlea that gradually declined to ∼30% OHC in the apex (Fig. 1D). A mean IHC loss of ∼80% was confined to the base of the cochlea. In an effort to increase the amount of hair cell loss and minimize damage to the tympanic membrane and ossicles, the middle ear space was opened surgically and a small amount (5 μL) of cisplatin (1 mg/mL, n = 5) or saline (n = 8) was applied to the round window membrane. Afterward, the middle ear space was closed and the cochlea harvested ∼10 days later (He et al., 2009). Round window administration of cisplatin resulted in 80%–100% OHC loss over most of the cochlea except for the apical 20%. The mean IHC loss decreased from ∼90% in the base of the cochlea to roughly 25% near the apex. Little hair cell loss was seen when saline was applied to the round window membrane (Fig. 1F). Taken together, these studies indicate that cisplatin causes extensive hair cell damage when applied to the round window membrane, but rather limited damage when administered systemically, presumably because the blood–labyrinth barrier inhibits uptake into the inner ear (Neuwelt et al., 1983; Gregg et al., 1992).

Furosemide Disrupts the Stria Vascularis

Furosemide, a loop diuretic that inhibits the Na-K-2Cl symporter, can induce transient hearing loss (Rybak, 1985; Shiozaki et al., 2006). If furosemide is coadministered with other ototraumatic agents such as aminoglycoside antibiotics, it can exacerbate the hearing loss and cochlear pathology (Hirose and Sato, 2010). Furosemide exerts its transient ototoxic effects by suppressing blood flow through the capillaries of the stria vascularis; this causes severe strial edema, enlargement of the extracellular spaces, and a large decrease in the endolymphatic potential (Forge and Brown, 1982). These histopathological changes presumably disrupt the blood–ear barrier (Juhn et al., 1981). To evaluate the effects of furosemide on the stria vascularis of CBA mice, we administered three different doses of the drug (i.p.) and harvested the inner ears between 10 min and 6 hr post-treatment (Li et al., 2011b). In some cases, the stria vascularis was fixed, stained with eosin, and prepared as a flat surface preparation to evaluate the blood vessels in the stria (Ding et al., 2001; Li et al., 2011b). Other cochleae were decalcified, embedded in Epon resin, sectioned (3 μm) parallel to the modiolus, and stained with toluidine blue as described previously (Ding et al., 2001; Li et al., 2011b). Furosemide damaged the stria vascularis in a dose-dependent and time-dependent manner. Both the 200 and 400 mg/kg doses caused significant temporary threshold shift; the 100 mg/kg dose caused less hearing loss that recovered rapidly. As mortality with the 400 mg/kg dose was too high to be used practically, the 200 mg/kg dose was used for subsequent studies. Figure 1A is a typical photomicrograph of a surface preparation of the stria vascularis from a normal CBA mouse stained with Harris' hematoxylin solution. The darkly stained blood vessels coursing through the stria vascularis were packed with red blood cells (arrows). Figure 1B is a photomicrograph of a cross section of the stria vascularis from a normal mouse. Staining of the cytoplasm within the marginal and intermediate cells was moderately intense and homogeneous, whereas staining in the spiral ligament was pale and heterogeneous (Fig. 1B). Treatment with 200 mg/kg of furosemide caused a significant reduction in blood flow through the stria vascularis; this decrease was the most severe 2 hr post-treatment (Fig. 2C); the blood vessels were completely devoid of red blood cells at this time (arrowheads). Cross sections through stria vascularis revealed significant shrinkage of the nuclei of the marginal and intermediate cells. Numerous vacuoles and large clear spaces were present throughout the stria (arrowheads). Prominent extrusions (arrows) were evident on the surface of marginal cells lining the endolymphatic space suggesting that the barrier between stria and endolymph had been disrupted. These results are intriguing given that ototoxic drugs are trafficked through the endothelial cells of the stria vascularis and into the marginal cells where they can enter the endolymph and reach the apical surfaces of the hair cells (Wang and Steyger, 2009).

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Figure 2. (A) Representative photomicrograph of surface preparation of the normal stria vascularis of a CBA mouse stained with eosin. The blood vessels (arrows) coursing through the stria are filled with red blood cells. (B) Photomicrograph of typical Epon-embedded cross section through the stria vascularis of a normal CBA mouse stained with toluidine blue. Note the marginal cells (M) facing the endolymph (EL), intermediate cells (I), and cells of the spiral ligament (SL). (C) Representative photomicrograph of surface preparation of the stria vascularis of a CBA mouse 2 hr after treatment with 200 mg/kg (i.p.) furosemide. Note nearly complete absence of red blood cells within the vessels of the stria vascularis (arrowheads). (D) Photomicrograph of a cross section through the stria vascularis of a CBA mouse taken 2 hr following treatment with 200 mg/kg (i.p.) of furosemide. Note protrusions (black arrows) of marginal cells into the endolymphatic space, large vacant spaces in the intermediate, and marginal cell layers of the stria and spiral ligament (arrowheads).

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Furosemide Enhances Cisplatin Ototoxicity in Mice

To determine if furosemide would enhance the ototoxic effects of cisplatin, CBA mice were treated with 200 mg/kg of furosemide (i.p.) followed 1 hr later with 0.5 or 1 mg/kg of cisplatin; doses of cisplatin far below those known to cause hearing loss or cochlear pathology (Fig. 1A,C). Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were measured 10 days after combined treatment to estimate the degree of auditory impairment (Li et al., 2011b). Combined treatment with 200 mg/kg furosemide and 0.5 mg/kg cisplatin (mean ± standard error of mean (SEM), n = 7) resulted in mild ABR threshold shift relative to baseline thresholds from a control group (mean ± SEM, n = 6) (Fig. 3A). Threshold shifts were ∼20 dB from 4 to 20 kHz and 28 dB at 32 kHz. Threshold shifts from combined treatment with furosemide and 1 mg/kg of cisplatin (mean ± SEM, n = 4) were substantially higher, ranging from 35 to 45 dB.

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Figure 3. (A) Mean (±SEM) baseline ABR thresholds of a control group (n = 6) compared with groups treated with furosemide (200 mg/kg, i.p) followed 1 hr later by 0.5 mg/kg (n = 7) or 1 mg/kg (n = 4) of cisplatin. (B) Mean (±SEM) baseline DPOAE amplitudes and noise floor for a control group versus DPOAE amplitudes from groups treated with furosemide and 0.5 mg/kg (n = 6) or 1 mg/kg (n = 6) of cisplatin. Measurements obtained with f2/f1 = 1.2; L1 and L2 intensity set to 70 and 60 dB SPL, respectively. (C and D) Mean cochleograms of groups treated with 200 mg/kg furosemide and 0.5 mg/kg or 1 mg/kg cisplatin. Representative photomicrographs of surface preparations taken from the middle of the cochlea of a mouse treated with 200 mg/kg furosemide and 0.5 mg/kg cisplatin (E) or 1 mg/kg cisplatin (F). Bracket and arrow point to outer hair cells (OHC) and inner hair cells (IHC). Note absence of OHC after treatment with 1 mg/kg cisplatin (panels C and D reproduced from Li et al., 2011b with permission Springer).

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DPOAE provide a rapid, noninvasive method for assessing the functional integrity of the OHC combined with the +80 mV endolymphatic potential, an electromotive drive potential generated by electrogenic ion pumps in the stria vascularis. Figure 3B shows the DPOAE amplitudes (2f1f2) at f1 frequencies of 4, 8, 12, 16, 20, and 32 kHz. The intensity of L1 was 70 dB sound pressure level (SPL), L2 was 60 dB SPL, and the f2/f1 frequency ratio was 1.2. Baseline DPOAE amplitudes in the control group (mean ± SEM, n = 6) ranged from 12 to 26 dB SPL between 4 and 32 kHz, well above the noise floor (ca. −10 dB SPL). DPOAE amplitudes in the group treated with 200 mg/kg furosemide and 0.5 cisplatin (n = 6) were 12 dB less than in baseline controls at 32 kHz, but not different at lower frequencies. In contrast, DPOAE amplitudes in the group treated with 200 mg/kg furosemide and 1 mg/mg of cisplatin (mean ± SEM, n = 6) were near the noise floor at high frequencies (16–32 kHz) and 8–12 dB below baseline controls at lower frequencies.

The reductions in DPOAE amplitudes and increases in ABR thresholds were reflected in the cochlear lesions. The mean (n = 6) cochleogram for the group treated with 200 mg/kg furosemide and 0.5 mg/kg cisplatin is shown in Fig. 3C; percent distance from the apex (lower abscissa) is related to frequency (upper abscissa) using a mouse frequency-place map (Muller and Smolders, 2005). OHC loss decreased from 75% in the base to ∼10% near the 32 kHz region, a frequency associated with moderate ABR and DPOAE impairment. Figure 3E shows a representative photomicrograph of a cochlear surface preparation stained with Harris' hematoxylin solution; the image was taken from the middle of the cochlea of a mouse that had been treated with 200 mg/kg furosemide and 0.5 mg/kg cisplatin (Ding et al., 2001). All the OHC and IHC were present in the 20 kHz region, consistent with normal DPOAE amplitudes at this frequency. The mean (n = 6) cochleogram obtained from mice treated with 200 mg/kg furosemide and 1 mg/kg cisplatin (Fig. 3D) showed nearly a complete loss of OHC in the basal third of the cochlea, roughly an 80% loss in the middle of the cochlea and a 25% loss near the apex; IHC were largely unaffected. OHC losses map reasonably well to the frequency-dependent loss of DPOAE amplitudes (Fig. 3B). Figure 3F is a representative photomicrograph from the middle of the cochlea of a mouse treated with 200 mg/kg furosemide and 1 mg/kg cisplatin. Nearly all of the OHC were missing, but the IHC remained intact. Together, these results indicate that disrupting the stria vascularis with furosemide greatly enhances the ototoxic effects of a low dose of cisplatin that by itself is not ototoxic.

Noise and Ethacrynic Acid Enhance Cisplatin Ototoxicity in Chinchillas

Because the range of hearing of the chinchilla is similar to humans, chinchillas have been used extensively in research related to ototoxicity and acoustic trauma. Like other rodents, chinchillas are resistant to cisplatin ototoxicity (Gratton et al., 1990). When we treated 10 chinchillas with 2.75 mg/kg (i.p.) for 3 consecutive days, seven animals died from nephrotoxicity after 4–5 days. Of those that survived, none showed evidence of hair cell loss. However, this resistance was overcome by exposure to moderate intensity noise (85 dB SPL, 5 days on, 2 days off, and noise repeated three times) interspersed with two cycles of cisplatin (2.75 mg/kg/day for 2 consecutive days during the two noise-off intervals; total dose 11 mg/kg) as previously described (Gratton et al., 1990). Animals exposed to cisplatin showed little hair cell loss while those exposed to noise developed only mild OHC loss in the apex of the cochlea. However, when chinchillas were treated with both cisplatin and noise, they developed moderate to severe hair cell lesions over a broad region of the cochlea (Gratton et al., 1990). These results are consistent with earlier findings showing that noise greatly enhances aminoglycoside ototoxicity (Ryan and Bone, 1978, 1982) presumably by increasing drug trafficking through the stria vascularis and into the endolymph (Li et al., 2011b).

Ethacrynic acid, another diuretic that inhibits the sodium–potassium–chloride cotransporter, also damages the stria vascularis (Forge, 1981; Syka and Melichar, 1981; Rybak, 1993) and enhances the uptake of gentamicin into the endolymph (Tran Ba Huy et al., 1983). As cisplatin alone failed to induce significant hair cell loss in chinchillas (Gratton et al., 1990), we hypothesized that ethacrynic acid would greatly enhance the ototoxicity of cisplatin. To test this hypothesis, we measured DPOAE before and 4–6 weeks after treating chinchillas with ethacrynic acid (40 mg/kg, i.v.) followed by 0.2, 0.4, or 0.8 mg/kg of cisplatin. DPOAE amplitudes decreased in a frequency-dependent and dose-dependent manner. Treatment with ethacrynic and 0.2 mg/kg of cisplatin caused a drastic decline in DPOAE amplitude at f1 frequencies of 3,000 and 8,000 Hz; however, only a slight decline occurred at 1,000 Hz. In contrast, 0.8 mg/kg cisplatin and ethacrynic acid caused a massive reduction in DPOAE amplitudes at all frequencies. Figure 4C shows the mean (n = 4) cochleogram from the group treated with 40 mg/kg ethacrynic acid and 0.2 mg/kg cisplatin. OHC losses decreased from 100% near the base of the cochlea to ∼10% in the apex (Ding et al., 2007). Unlike OHC, the IHC lesion was relatively uniform, ranging from 30% to 60% loss, along the length of the cochlea. The scanning electron micrograph in Fig. 4D illustrates the condition of the OHC and IHC ∼35% of the distance from the apex. Many OHC had missing or damaged stereocilia (arrows) and some IHC were missing (arrowheads) (Ding et al., 2007).

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Figure 4. Mean (n = 4, ±SD) 2f1f2 DPOAE input/output functions obtained from chinchillas before (Pre) and 4–6 weeks after treatment with 40 mg/kg of ethacrynic acid (EA, i.v.) and (A) 0.2 mg/kg of cisplatin or (B) 0.8 mg/kg of cisplatin (Cis). DPOAE obtained with f2/f1 = 1.2, L1 = L2; intensity varied from 0 to 80 dB SPL; f1 shown above each panel. Noise floor of measurement system was typically between 0 and −10 dB SPL for L1 levels between 40 and 70 dB SPL. (C) Mean (n = 4) cochleogram for chinchillas treated with 40 mg/kg EA and 0.2 mg/kg Cis. Percent IHC and OHC loss plotted as a function of percent distance from the apex of the cochlea. Upper abscissa shows the frequency–place map for chinchillas. (D) Scanning electron micrograph (arrow shows the location of scanning electron micrograph in panel D) from 35% region of chinchilla cochlea (see black arrow in panel C). Arrows identify OHC with missing or damaged stereocilia; arrowheads point to missing or damaged IHC. Pillar cells (PC). Magnification and scale bar shown in lower right (panels C and D reproduced from Ding et al., 2007 with permission Elsevier).

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Cell Death from Cisplatin–Ethacrynic Acid

Hearing loss from cisplatin and ethacrynic acid tends to be rapid and severe. These characteristics may not only be due to the fact that ethacrynic acid enhances the influx of ototoxic drugs into the cochleae, but also because ethacrynic acid increases the production of toxic-free radicals (Clerici et al., 1996) and decreases protective antioxidant enzymes (Lucas et al., 1998). To investigate the underlying mechanisms responsible for rapid hair cell death from cisplatin–ethacrynic acid treatment, we evaluated the histopathological changes in the chinchilla cochleae shortly after cisplatin–ethacrynic acid treatment (Ding et al., 2007). To determine if cochlear hair cells were dying by apoptosis, cochleae were double-labeled with FITC-conjugated phalloidin, which binds to hair cell stereocilia and pillar cells, and propidium iodide (PI), which heavily labels the DNA and RNA in the nuclei of hair cells and supporting cells. In normal controls, the PI-labeled nuclei of OHC, IHC, and support cells were large, round, and uniformly labeled, morphological features of healthy cells (Fig. 5A). The nuclei of OHC were arranged in three orderly rows lateral to the pillar cells, whereas the nuclei of the IHC were arrayed in a single row medial to the pillar cells. Twenty-four hours after combined treatment with cisplatin and ethacrynic acid, the nuclei of most OHC were severely shrunken, anatomical features indicative of cells dying by apoptosis. In contrast, the nuclei of supporting cells retained their normal appearance. To investigate the mechanisms involved in programmed cell death from cisplatin–ethacrynic acid, we labeled cochleae with fluorogenic caspase inhibitors that are activated by specific caspases as previously described (Ding et al., 2007). Caspase-8 and caspase-9 are two important initiator caspases that activate downstream executioner caspase-3 and caspase-6. Caspase-8 is activated by death receptors on the cell membrane, whereas caspase-9 is activated following the release of cytochrome c from mitochondria. Strong caspase-8 labeling was present in OHC 24 hr following cisplatin–ethacrynic acid treatment. Caspase-8 was present in regions where the OHC nuclei were condensed or fragmented (Fig. 5C), but not in support cells or IHC with large, round nuclei. In contrast, caspase-9 labeling was absent 24 hr after cisplatin–ethacrynic acid treatment even in regions where the OHC nuclei were condensed or fragmented (Fig. 5D). Strong labeling of executioner caspase-3 and caspase-6 was also present in the OHC region 24 hr post-treatment. OHC nuclei in these regions were condensed or fragmented, characteristics of apoptotic cells, whereas nuclei of support cells and IHC were large and round features of healthy cells. Tumor necrosis factor associated death domain (TRADD) is a death domain adaptor protein that interacts with tumor necrosis factors receptors that initiates apoptotic cell death through NFκB. TRADD immunolabeling was absent from normal control cochlea (Fig. 5G), but was first evident in OHC 6 hr after cisplatin–ethacrynic acid treatment (Fig. 5H) as previously reported (Ding et al., 2007). Taken together, these results suggest that cisplatin–ethacrynic acid mediated hair cell death is initiated by membrane death receptors, which subsequently activate initiator caspase-8 followed by downstream executioner caspase-3 and caspase-6.

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Figure 5. Representative confocal photomicrographs of surface preparations from the middle turn of the chinchilla cochlea. (A) Control cochlea stained with FITC-conjugated phalloidin (green) and propidium iodide (red). Heads of pillar cells (PC) intensely labeled with phalloidin; note large, round nuclei of three rows of outer hair cells (OHC, bracket) and one row of inner hair cells (IHC; arrow). (B) Cochlear surface preparation 24 hr after treatment with ethacrynic acid (40 mg/kg, i.v.) and cisplatin (0.8 mg/kg, i.p.); specimen labeled with FITC-phalloidin and propidium iodide. Note shrunken OHC nuclei (arrowheads); morphological feature of apoptosis; nuclei of support cells (open circle) and IHC remain large and round. (C) Cochlear surface preparation 24 hr after cisplatin-ethacrynic acid treatment. Specimen labeled with propidium iodide (red) and fluorescently labeled caspase-8 (green). Note intense caspase-8 labeling in OHC region. OHC nuclei are condensed and/or fragmented, morphological features of apoptotic cells. IHC and support cells have large, round nuclei (open circle) characteristic of normal cells. (D) Cochlear surface preparation 24 hr after cisplatin-ethacrynic acid treatment. Specimen labeled with propidium iodide (red) and fluorescently labeled caspase-9 (green). Note absence of caspase-9 labeling in OHC with condensed and/or fragmented nuclei (arrowheads). Nuclei of support cells (open circle) and IHC (arrows) are large and round, characteristic of normal cells. (E) Cochlear surface preparation 24 hr after cisplatin-ethacrynic acid treatment. Specimen labeled with propidium iodide (red) and fluorescently labeled caspase-3 (green). Note intense caspase-3 labeling in OHC region; OHC nuclei are condensed and/or fragmented, features characteristic of apoptotic cells. Nuclei of support cells (open circle) and IHC (arrows) are large and round. (F) Cochlear surface preparation 24 hr after cisplatin-ethacrynic acid treatment. Specimen labeled with propidium iodide (red) and fluorescently labeled caspase-6 (green). Note intense caspase-6 labeling in OHC region; OHC nuclei are condensed and/or fragmented, features characteristic of apoptotic cells. Nuclei of support cells (open circle) and IHC (arrows) are large and round. (G) Cochlear surface preparation from normal control chinchilla. Specimen labeled with propidium iodide (red) and an antibody against TRADD (green). OHC and IHC nuclei are large and round; note absence of TRADD labeling. (H) Cochlear surface preparation 6 hr after cisplatin–ethacrynic acid treatment. Specimen labeled with propidium iodide (red) and an antibody against TRADD (green); TRADD labeling confined to OHC region.

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Cisplatin Ototoxicity In Vitro

Cochlear organotypic cultures are a powerful tool for studying ototoxicity, because drug timing and concentration can be precisely controlled and the effects of the blood–labyrinth barrier can be bypassed (Liu et al., 1998; Cheng et al., 1999; Qi et al., 2008; Ding et al., 2011b; Yu et al., 2011). To investigate the ototoxic effects of cisplatin in vitro, cochlear organ cultures were prepared from postnatal day 3 rats, maintained in culture 1 day, and then treated with increasing doses of cisplatin to establish a complete dose–response curve (Ding et al., 2011b). In normal cochlear cultures, the nerve fibers from the spiral ganglion neurons project out to the three rows of OHC and one row of IHC (Fig. 6A). The hair cells, spiral ganglion neurons, and nerve fibers were extremely sensitive to cisplatin toxicity. When cochlear cultures were treated with as little as 50 μM of cisplatin for 48 hr, virtually all the hair cells, neurons, and nerve fibers were destroyed (Fig. 6B). Unexpectedly, when the dose of cisplatin was increased, hair cell and nerve fiber survival increased (Fig. 6C,D) with nearly complete survival occurring at 400 μM. Figure 6E shows the complete dose–response curve for cisplatin. The number of hairs cells per mm started to decline with 10 μM cisplatin, reached its lowest point around 50 μM, recovered to half maximum at 100 μM and returned to normal at 400 μM or higher. Paradoxically, hair cells were more resistant to high doses of cisplatin than low doses. To determine if other cells manifested a similar resistance, we treated human RB143 retinoblastoma cancer cells with similar doses of cisplatin. Unlike hair cells, RB143 cancer cells showed a systematic decrease in cell number with increasing cisplatin dose (Ding et al., 2011b). These results suggest that hair cells have an intrinsic mechanism that protects them from high doses of cisplatin in the extracellular environment (Ding et al., 2011b).

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Figure 6. Postnatal day 3 rat cochlear organotypic cultures. Hair cells labeled with Alexa Fluor 488-conjugated phalloidin (green) and an antibody against neurofilament 200 kD (red). (A) Normal cochlea cultured for 48 showing three rows of outer hair cells (OHC), one row of inner hair cells (IHC), spiral ganglion neurons (SGN), and nerve fibers (NF) projecting to the hair cells. (B and D) Cochlear cultures treated for 48 hr with 50, 400, or 1,000 μM cisplatin, respectively. Note complete loss of hair cells and massive loss of nerve fibers and spiral ganglion neurons with the 50 μM dose and lack of damage with the 400 and 1,000 μM doses. (E) Dose–response curve showing number of hair cells per mm versus cisplatin dose (panel E reproduced from Ding et al., 2011b with permission Elsevier).

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Cisplatin Uptake

To monitor drug uptake into hair cells, cisplatin was conjugated to Alexa Fluor 488 (Ding et al., 2011b). When cochlear cultures were treated for 48 hr with 50 μM of cisplatin-Alexa Fluor 488, considerable uptake of the labeled cisplatin was observed in OHC and to a lesser extent IHC, consistent with previous in vivo and in vitro results (Cardinaal et al., 2000; Zhang et al., 2003). Uptake of Alexa Fluor 488-cisplatin was associated with considerable damage to the stereocilia and hair cell body indicating that the labeled probe retained its ototoxic potential. Interestingly, the support cells lateral and medial to the hair cells showed relatively little uptake of the fluorescent probe. In contrast to the effects seen with low concentrations, cultures treated for 48 hr with high concentrations of Alexa Fluor 488-cisplatin showed relatively little uptake of the labeled drug. In addition, the condition of the hair cells appeared to be relatively normal (Fig. 7B, 1,000 μM).

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Figure 7. Confocal images of cochlear organotypic cultures from postnatal day 3 rats. (A) Representative photomicrograph of surface preparation treated with 50 μM Alexa Fluor 488 conjugated to cisplatin (green) for 48 hr. Specimen stained with TRITC-conjugated phalloidin (red). Note heavy uptake of Alexa Fluor 488-cisplatin in OHC region and a few IHC. OHC and to a lesser extent IHC were severely damaged by cisplatin. (B) Conditions the same as in panel A except the sample was treated with 1,000 μM of Alexa Fluor 488-cisplatin. Note lack of Alexa Fluor 488-cisplatin labeling in hair cells and relatively normal appearance of OHC and IHC. (C) Normal control culture treated for 30 min with FM1-43 (red) and stained with Alexa Fluor 488 (green)-conjugated phalloidin. Note intense FM1-43 labeling in cytoplasm of OHC and IHC and lack of labeling in support cells. (D) Conditions similar to panel C except that the specimen was treated with 10 μM cisplatin for 48 hr before labeling with FM1-43. Note FM1-43 labeling in IHC cytoplasm and lack of FM1-43 in OHC. Cuticular plate of OHC and IHC brightly labeled with Alexa Fluor 488-phalloidin. (E and F) Cochlear cultures treated for 18 hr with 1,000 μM cisplatin; culture medium replaced with serum-free medium for 18 hr and then labeled with FM1-43. Radial (E) and longitudinal (F) sections taken through the organ of Corti.

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The reduced uptake of labeled drug at high concentrations (Fig. 7B) could be due to stereocilia damage (Fernandez-Cervilla et al., 1993; Kimitsuki et al., 1993). To evaluate the functional status of stereocilia, FM1-43, a small styryl dye that readily enters hair cells through the mechanoelectric transduction apparatus was applied to cochlear cultures (Meyers et al., 2003). FM1-43 was applied to normal cochlear cultures for 30 min, and the cultures fixed, stained with Alexa 488-phalloidin, and evaluated by confocal microscopy. The cytoplasm of OHC and IHC was intensely labeled with FM1-43 (Fig. 7C) except for the nucleus (Ding et al., 2011b). Importantly, the surrounding support cells failed to take up FM1-43. When cultures were treated with 10 μM cisplatin for 48 hr and subsequently labeled with FM1-43 and Alexa 488-phalloidin, there was a significant reduction of red fluorescent label in the three rows of OHC, but strong labeling in the IHC (Fig. 7D). Incubating cochlear cultures with 50 μM or more of cisplatin resulted in complete loss of FM1-43 labeling in both IHC and OHC (data not shown). As the stereocilia and hair cell body appeared relatively normal following high concentrations of cisplatin (1,000 μM, see Fig. 7B), we evaluated the ability of cisplatin-treated hair cells to take up FM1-43 after washing out the cisplatin and culturing the explants for 18 hr in serum-free medium. When FM1-43 was applied to these cochlear cultures, robust FM1-43 labeling was observed in the hair cell body (Fig. 7E,F) (Ding et al., 2011b). These results suggest that the hair cells were still able to take up FM1-43 after a heavy dose of cisplatin.

Copper Transporters Flux Platinum

Recent studies indicate that influx, sequestration, and efflux of cisplatin are mediated in large part by copper transporters expressed in most cells including those of the inner ear (More et al., 2010; Ding et al., 2011b). The influx of cisplatin is largely mediated by Ctr1 located on the plasma membrane (Howell et al., 2010). Ctr1 is rapidly downregulated by extracellular copper and cisplatin thereby providing cells with a negative feedback system that reduces the uptake of these metals (Holzer et al., 2004). The sequestration and efflux of copper and cisplatin is mediated by ATP7A and ATP7B. Interestingly, increasing the expression of ATP7A and ATP7B makes tumor cells resistant to cisplatin killing (Samimi et al., 2004; Mangala et al., 2009). To determine if these copper transporters were expressed in the inner ear, immunolabeling studies were carried out on postnatal day 3 rat organ cultures using antibodies against Ctr1, ATP7A and ATP7B as described in a recent publication (Ding et al., 2011b). Figure 8A–C shows a series of confocal photomicrographs taken through slices of cochlear surface preparations that cut through the OHC. The samples were double-labeled with an antibody against Ctr1, ATP7A or ATP7B, and phalloidin conjugated to either TRITC or Alexa Fluor 488 to label F-actin that is expressed in pillar cells and hair cells. Ctr1 was expressed in discrete puncta in the cytoplasm and plasma membrane of OHC (Fig. 8A). ATP7A was heavily expressed in pillar cells and a few puncta were also present in OHC (Fig. 8B). In contrast, strong ATP7B labeling was present in OHC (Fig. 8C) (Ding et al., 2011b).

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Figure 8. Confocal images of surface preparations from postnatal day 3 rat organ cultures immunolabeled with: (A) Antibody against Ctr1 (green) and TRITC-conjugated phalloidin. Ctr1 expressed in cytoplasm and along the plasma membrane of outer hair cells (OHC). (B) Antibody against ATP7A (green) and Alexa 488-conjugated phalloidin (red). ATP7A heavily expressed in pillar cells (PC); low expression in OHC. (C) Antibody against ATP7B (green) and TRITC-conjugated phalloidin (red). ATP7B expressed in OHC. (D) Western blots for GAPDH, Ctr1, ATP7A, and ATP7B performed on cochleae (n = 6 per condition) cultured for 24 hr. Results shown for normal control cultures and cultures treated with 10 μM cisplatin (Cis), 10 μM cisplatin and 50 μM CuSO4, and 50 μM CuSO4 alone. Expression of GAPDH stable across conditions. Ctr1 expression greatly reduced in cochlear cultures treated with 50 μM CuSO4 and10 μM cisplatin and 50 μM CuSO4. Expression of ATP7A reduced in cochlear cultures with 50 μM CuSO4 or 50 μM CuS04 and 10 μM cisplatin. Expression of ATP7B increased in cochlear cultures treated with 10 μM cisplatin; further increase in ATP7B expression in cultures treated with 10 μM cisplatin and 50 μM CuSO4 or 50 μM CuSO4. (E) Average (n = 6, SEM) number of hair cells per mm length of the organ of Corti in normal cochlear cultures and cultures treated with 10 μM cisplatin and 0–100 μM CuSO4 (abscissa). Cisplatin (10 μM) reduced hair cell counts by roughly half. Adding 10 μM or more of CuSO4 increased hair cell counts to nearly normal levels (panel E reproduced from Ding et al., 2011b with permission Elsevier).

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Copper Sulfate Modulates Transporter Expression

As extracellular copper is likely to influence the levels of these three transporters, the expression of Ctr1, ATP7A, and ATP7B, were evaluated in normal control cultures and cultures treated with 10 μM of cisplatin, 50 mM CuSO4, or cultures treated with the 10 μM cisplatin and 50 CuSO4. Tissues were harvested from six cochlear cultures per condition and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Ctr1, ATP7A, and ATP7B expression levels evaluated by western blots using published methods and antibodies against each of the proteins (Ding et al., 2011b; Jamesdaniel et al., 2011). Expression of the housekeeping protein, GAPDH, remained stable across conditions (Fig. 8D). Expression of Ctr1 and ATP7A showed little change after 24 hr of cisplatin treatment, but was strongly downregulated after treatment with CuSO4 alone or CuSO4 and cisplatin. These results suggest that extracellular copper decreases the influx of copper and platinum through Ctr1. In contrast, the ATP7B export pump was moderately upregulated by cisplatin alone and strongly upregulated by CuSO4 alone or cisplatin and CuSO4. These results suggest that cisplatin and extracellular copper strongly upregulates the ATP7B efflux pump thereby protecting cells against the buildup of cisplatin and copper.

To determine if CuSO4 would protect hair cells from cisplatin toxicity, cochlear cultures were treated with 10 μM cisplatin alone or in combination with different concentrations of CuSO4. CuSO4 alone had no adverse effects on hair cells at concentration of 200 μM or less, whereas 500 μM was toxic to hair cells as recently reported (Ding et al., 2011b). Treatment of cochlear cultures with 10 μM cisplatin alone caused significant hair cell loss (Fig. 5E); however, cisplatin toxicity was almost completely abrogated by 10–100 μM of CuSO4. These results are consistent with other recent results (More et al., 2010). Under combined treatment, CuSO4 presumably protects cells by competitively inhibiting the uptake of cisplatin, by increasing the expression of the ATP7B export pump (Fig. 8D) or a combination of both mechanisms. However, the protective effects of CuSO4 were limited to low doses of cisplatin. When cisplatin was increased to 50 μM, CuSO4 failed to provide any protection for reasons that remain unclear (Ding et al., 2011b).

CARBOPLATIN

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED

Carboplatin Ototoxicity in Chinchillas

Previous studies have suggested that carboplatin is far less ototoxic than cisplatin (Saito et al., 1989; Teeling and Carney, 1990; Taudy et al., 1992; Alberts, 1995; Ding et al., 1999). However, in chinchillas carboplatin is highly toxic to IHC and type I auditory nerve fibers (Wake et al., 1993; Takeno et al., 1994; Hofstetter et al., 1997; Ding et al., 1999). High doses of carboplatin can also damage OHC, but this only occurs after virtually all the IHC have been destroyed. The IHC lesions produced by carboplatin are also peculiar. Low and moderate doses (e.g., 50 mg/kg, i.p.) of carboplatin destroy every second, third, or fourth IHC along the length of the cochlea (cf. Fig. 9A,B); this tends to thin out the IHC population and results in a lesion that is roughly the same magnitude along the entire length of the cochlea (Trautwein et al., 1996; Hofstetter et al., 1997; Wang et al., 1997). When the carboplatin dose is increased to 100 mg/kg, nearly all of the IHC are destroyed whereas most of the OHC are intact (Fig. 9C). Chinchillas treated with higher doses or multiple doses of carboplatin begin to develop OHC lesions (Hofstetter et al., 1997). Unlike IHC lesions, carboplatin-induced OHC lesions tend to decrease from the base towards the apex much like what is seen with cisplatin (Fig. 1D,E).

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Figure 9. Photomicrographs of cochlear surface preparations from chinchillas. Specimens in panel A–C labeled with succinate dehydrogenase. Representative surface preparations from: (A) Normal control, (B) Chinchilla treated with 50 mg/kg (i.p.) carboplatin, and sacrificed ∼4 weeks post-treatment. Note selective loss of every second, third, or fourth IHC resulting in a patchy IHC lesion. (C) Chinchilla treated with 100 mg/kg carboplatin (i.p.) and sacrificed ∼4 weeks post-treatment. Note nearly complete loss of IHC will full retention of OHC. (D) Surface preparation from chinchilla treated with 50 mg/kg (i.p.) carboplatin and sacrificed 2 days post-treatment. Specimen immunolabeled with an antibody against phospho-p53 serine 15 and biotinylated secondary antibody; label visualized using avidin-biotinylated horseradish peroxidase and diaminobenzidine. Note p53 immunolabeling in IHC region (arrowheads).

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Carboplatin and P53

Cisplatin and carboplatin cause oxidative stress, downregulate antioxidant enzymes, induce DNA damage and upregulate p53, a tumor suppressor protein that plays an important role in programmed cell death (Husain et al., 2001b; Boulikas and Vougiouka, 2003; Zhang et al., 2003; Park et al., 2006). Recent studies indicate that p53 plays an important role in the early stages of cisplatin induced hair cell death. Phospho-p53 serine was largely absent from normal cochlear cultures, but was strongly upregulated as early as 6 hr following treatment with 10 μM cisplatin and continued to be highly expressed out to 48 hr post-treatment (Zhang et al., 2003). Pifithrin-α, a small molecule that inhibits p53, suppressed the expression of p53, caspase-1, and caspase-3 and protected hair cells from cisplatin damage (Zhang et al., 2003). In a cochlear cell line, cisplatin upregulated p53 as early as 3 hr post-treatment; this was followed by upregulation of Bax, cytochrome-c, caspase-8, caspase-9, and apoptotic cell death (Devarajan et al., 2002). To determine if p53 also contributes to the unique carboplatin-induced IHC lesions, we treated chinchillas with 50 mg/kg (i.p.) of carboplatin and harvested their cochleae 2 days post-treatment, a time during which IHC loss is underway (Wang et al., 2003). Cochleae were immunolabeled with an antibody against phospho-p53 serine 15, and the location of p53 was visualized using a biotinylated secondary antibody, avidin-biotinylated horseradish peroxidase and diaminobenzidine following published methods (Zhang et al., 2003; Ding et al., 2008). Many dark, circular clumps of phospho-p53 serine 15 immunolabeling were present in IHC (Fig. 9D, arrowheads), consistent with the selective IHC lesions induced by this dose of carboplatin (Fig. 9B). In contrast, p53 immunolabeling was largely absent in OHC.

OXALIPLATIN

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED

Oxaliplatin and Spiral Ganglion

Oxaliplatin, a third generation platinum derivative, is widely used in the treatment of colorectal and ovarian cancer (Haller, 2000; Piccart et al., 2001). Oxaliplatin is often associated with transient peripheral neuropathy and persistent neurosensory loss and sensory ataxia (Haller, 2000; Pasetto et al., 2006); however, it seldom causes ototoxicity. Pharmacokinetic studies indicate that the cochlear uptake of oxaliplatin is considerably less than for cisplatin, which may explain its low ototoxic potential (Geoerger et al., 2008; Hellberg et al., 2009). This differences in cochlear uptake may be related to the various drug transporters such as organic cation transporter1 (Oct1) and 2 (Oct2) (Zhang et al., 2006) and the multidrug and toxic extrusion (MATE) pump (Yokoo et al., 2007). Despite its cochlear uptake, oxaliplatin has on occasion been found to cause significant hearing loss (Malhotra et al., 2010). This led us to examine its toxic effects on hair cells and spiral ganglion neurons in cochlear organotypic cultures. Postnatal day 3 rat organ cultures were treated with different doses of oxaliplatin for 48 hr to characterize the pattern of damage. Cochlear cultures treated with 10 μM of oxaliplatin show extensive damage to the nerve fibers projecting to the hair cells; the nerve fibers were thinner, fractured, and pixilated (cf. Fig. 10A,B). Despite extensive nerve fiber damage, the hair cells appeared nearly normal. These results indicate that oxaliplatin is considerably more toxic to spiral ganglion nerve fibers than hair cells, consistent with previous studies showing that oxaliplatin is neurotoxic and causes peripheral neuropathy (Haller, 2000; Pasetto et al., 2006). However, when the dose of oxaliplatin was increased to 50 or 100 μM, hair cells and stereocilia damage eventually appeared along with nerve fiber degeneration (Fig. 10C). Paradoxically, increasing the dose of oxaliplatin to 500 or 1,000 μM (Fig. 10E,F) resulted in less hair cell and nerve fiber damage, similar to what occurred with high doses of cisplatin (Ding et al., 2011b).

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Figure 10. Representative confocal photomicrographs of postnatal day 3 rat cochlear organotypic cultures maintained for 48 hr under control conditions and with different doses of oxaliplatin indicated in panels BF. Hair cells labeled with FITC-conjugated phalloidin (green). Nerve fibers (NF) projecting out to the hair cells labeled with a monoclonal antibody against neurofilament 200 kD and TRITC-labeled secondary antibody (red). (A) Representative control culture showing the stereocilia bundles on the three rows of OHC and single row of IHC. Note thick fascicles of nerve fibers (NF) projecting out to the hair cells. (B) 10 μM oxaliplatin caused extensive damage to NF, but little damage to OHC and IHC. (C and D) 50–100 μM oxaliplatin caused massive loss of NF and significant damage to IHC and OHC. (E and F) Condition of OHC, IHC, and NF improved as the dose of oxaliplatin increased from 500 to 1,000 μM.

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NEDAPLATIN

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED

Nedaplatin and Spiral Ganglion

Nedaplatin, a second generation platinum drug, is used to treat many of the same types of cancers as cisplatin. Nedaplatin is considered less nephrotoxic and neurotoxic than cisplatin (Ito et al., 1999; Uehara et al., 2005), but has been reported to cause myelosuppression and ototoxicity (Horiuchi et al., 1992; Adachi et al., 2001). To evaluate the ototoxic properties of nedaplatin, cochlear organotypic cultures from postnatal day 3 rats were treated with different doses of nedaplatin for 48 hr. Treatment with 10 μM nedaplatin causes extensive nerve fibers damage (cf. Fig. 11A,B); nerve fiber damage remained severe as the dose escalated to 1,000 μM. While most hair cells were still present after treatment with 10 μM nedaplatin, the stereocilia bundles were damaged or absent. Hair cell damage became more severe as the dose of nedaplatin increased to 100 μM, but then reversed course and decreased as the dose escalated to 1,000 μM (Fig. 11C–E). This trend is similar to that seen with oxaliplatin (Fig. 10).

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Figure 11. Representative confocal photomicrographs of postnatal day 3 rat cochlear organotypic cultures maintained for 48 hr under control conditions or with different doses of nedaplatin indicated in panels BF. Hair cells labeled with FITC-conjugated phalloidin (green). Nerve fibers (NF) projecting out to the hair cells labeled with a monoclonal antibody against neurofilament 200 kD and TRITC-labeled secondary antibody (red). (A) Representative control culture showing stereocilia bundles on the three rows of OHC and single row of IHC. Note thick fascicles of nerve fibers (NF) projecting out to the hair cells. (B) Nedaplatin (10 μM) caused extensive damage to NF and moderate damage to hair cells. (C and D) Nedaplatin (50–100 μM) caused extensive damage to NF and significant damage to hair cells. (E and F) Hair cell damage with 500 and 1,000 μM nedaplatin less severe than with lower doses. Note severe damage to NF with 500 and 1,000 μM nedaplatin.

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SYNOPSIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED

Cisplatin, carboplatin, oxaliplatin, and nedaplatin continue to be widely used in oncology. Among this group, cisplatin is considered the most ototoxic although the others have been reported to cause mild or severe hearing loss in some patients. In nearly all human clinical studies, platinum drugs are administered in multiple treatment cycles separated by several weeks. With few exceptions (Gratton et al., 1990; Minami et al., 2004), this contrasts markedly with the majority of animal studies that usually use a single bolus. From a scientific standpoint, the single-dosing regimens are advantageous for investigating the immediate cause and effect relationships and for exploring the time course of ototoxic phenomenon at the histological, biochemical, or physiological levels. However, single-dosing may not adequately reflect the biochemical and histological processes that occur in humans undergoing multiple treatments because the latter likely elicits stress responses and cellular repair processes between treatment (Lautermann et al., 1997; Husain et al., 2001a; Coling et al., 2007).

While it is possible to induce cochlea lesions in rodents using a single dose of cisplatin, the hair cell lesions are of limited magnitude and largely confined to the basal, high-frequency region of the cochlea (Fig. 1A). In our experience, a single large bolus of cisplatin sufficient to induce a cochlear lesion often leads to severe nephrotoxicity, weight loss, or mortality. Because of these side effects, many studies seldom continue for more than a week. A major factor limiting the degree of ototoxicity is uptake of the drug from blood into the inner ear. One method of bypassing the blood–labyrinth barrier and producing a large cochlear lesion is to apply cisplatin or other drugs directly to the cochlea (e.g., round window membrane or cochlear infusions) (Husmann et al., 1998; Li et al., 2004; Bauer et al., 2008; He et al., 2009); however, the magnitude of the lesions can vary considerably between animals and between ears (Reyes et al., 2001; Zhou et al., 2009).

A second approach to rapidly generating large cisplatin-induced lesions is to use loop inhibiting diuretics to temporarily open the blood–labyrinth barrier (Komune and Snow, 1981; Minowa, 1984; Ding et al., 2007; Li et al., 2011b). In our experience, maximal hair cell damage occurs if cisplatin is given 1 hr after administering the diuretic, a time when the lateral wall of the cochlea is severely disrupted (Figs. 2 and 3). When cisplatin is coadministered with ethacrynic acid, remarkably low doses of the drug (<1 mg/kg) can induce massive and widespread hair cell loss and hearing impairment (Fig. 4). The rapid induction of a broad lesion has proven useful in identifying some of the signaling pathways involved in acute ototoxicity (Fig. 5) (Ding et al., 2007). However, one drawback to this approach is that diuretics themselves can induce oxidative stress and deplete antioxidant enzymes thereby confounding the interpretation of data.

A third approach to inducing large lesions is to combine administration of cisplatin with noise (Gratton et al., 1990). As noise can induce oxidative stress and alter antioxidant enzyme levels (Jacono et al., 1998; Ohlemiller et al., 1999; Henderson et al., 2006; Vlajkovic et al., 2009), it may create a state of cochlear vulnerability. Noise exposure can also damage the stria vascularis potentially disrupting the blood–labyrinth barrier (Shi and Nuttall, 2003), thereby increasing the flux of ototoxic drugs through the stria vascularis and into the hair cells (Li et al., 2011a). Surprisingly, noise exposures 2 months before aminoglycoside treatment were still capable of potentiating ototoxicity (Ryan and Bone, 1978).

As a rule, it is extremely difficult to produce large cochlear lesions in rodents with a single dose of any of the platinum drugs. A major exception to this are the massive hair cell lesions produced in chinchillas with one or two doses of carboplatin, which is considered less ototoxic than cisplatin (Hofstetter et al., 1997). No other mammalian species exhibits such extreme susceptibility to carboplatin induced IHC loss. As carboplatin causes significant hair cell loss in chinchillas, the drug must be readily trafficked through the stria into the cochlear fluids. Ctr1 is known to transport carboplatin as well as cisplatin (Larson et al., 2009; Yonezawa and Inui, 2011). As is difficult to damage chinchilla hair cells with cisplatin (Gratton et al., 1990), Ctr1 is unlikely to play a major role in carboplatin ototoxicity. The same reasoning applies to Ctr2 (Blair et al., 2009). Other transporters such as OCT1-2 and MATE1-2 do not transport carboplatin and therefore are unlikely to be involved (Yokoo et al., 2007; Yonezawa and Inui, 2011). While an explanation for the high degree of carboplatin ototoxicity in chinchillas remains elusive, both carboplatin and cisplatin upregulate p53 in chinchilla and mice, suggesting they share a common mechanism once these drugs enter hair cells (Fig. 9) (Zhang et al., 2003).

While performing cisplatin dose–response studies in vitro, we discovered that cisplatin doses of 100 μM or more were far less destructive to hair cells and nerve fibers than lower doses (Fig. 6) (Ding et al., 2011b). While these initial results were greeted with great skepticism, we found similar reversals of the dose–response hair cell killing functions for nedaplatin and oxaliplatin (Figs. 10 and 11). As 100 μM concentrations are well within the solubility range of cisplatin and as retinoblastoma cell numbers decreased systematically as cisplatin concentrations increased, we were forced to conclude that the hair cell results were valid. As Ctr1 transport cisplatin into cells and ATP7A and ATP7B are involved in exporting cisplatin, we evaluated their expression in the presence of 10 μM cisplatin and CuSO4, a competitive inhibitor of cisplatin. CuSO4, which decreased the expression of Ctr1 and increased the expression of ATP7, protected hair cells from cisplatin toxicity presumably by decreasing the influx of cisplatin and increasing its efflux; effects that would lower its intracellular concentration and ototoxic effects (Fig. 8) (Ding et al., 2011b). On the other hand, cisplatin alone, at a dose that caused extensive hair cell damage, increased the expression of the ATP7B export pump by a moderate amount, but had little effect on the Ctr1 import pump. These results and others suggest that the distribution of copper transporters in the cochlea may play an important role in cisplatin ototoxicity (More et al., 2010; Yonezawa and Inui, 2011). Oct2 is involved in the transport of cisplatin and oxaliplatin, whereas MATE1 and 2 transport oxaliplatin (Ciarimboli et al., 2010; Yonezawa and Inui, 2011). The distribution and relative abundance of these transporters in the cochlea contribute likely to the unique ototoxicity profile of each compound.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CISPLATIN OTOTOXICITY
  5. CARBOPLATIN
  6. OXALIPLATIN
  7. NEDAPLATIN
  8. SYNOPSIS
  9. LITERATURE CITED
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