The role of glutathione in carboplatin ototoxicity was investigated in the chinchilla. Chinchillas hearing was tested with both distortion product otoacoustic emissions (DPOAE) and evoked potentials recorded from a chronic electrode in the inferior colliculus (IC). All subjects had an osmotic pump fitted to their right ear and it received buthionine sulfoximine (BSO) at a dose of 15 mM delivered at 5 ml per hour for 14 days. A group (N=4) was given a double dose of carboplatin (25 mg/kg i.p. for 2 days). The pump was implanted three days before the carboplatin dose. The BSO treated ears showed a greater loss in both evoked potential and DPOAE measures, as well as substantially fewer missing hair cells. The results implicate reactive oxygen species (ROS) as a common factor in ototoxic reactions because suppression of glutathione antioxidant leads to greater ototoxic reactions.
Keywords: Carboplation ototoxicity, distortion product emissions, glutathione, chinchilla
|How to cite this article:|
Henderson D, Hu B H, McFadden S L, Zheng X Y, Ding D. The role of glutathione in carboplatin ototoxicity in the chinchilla. Noise Health 2000;3:1-10
| Introduction|| |
Ototoxicity: Carboplatin is a chemotherapeutic drug often used as an alternative to cisplatin because its side effects, nephrotoxicity, nausea and hearing loss are typically less severe and less prevalent than treatment with cisplatin. When hearing loss does occur with carboplatin, the loss is very similar to the effects of cisplatin, i.e., the loss starts at the highest frequencies of the audiogram and cochlear lesions begin with outer hair cells (OHC) in the base of the cochlea. However, carboplatin in the chinchilla produces a strange and unique lesion that involves a selective loss of inner hair cells (IHC). In studies of the carboplatin dose/response in the chinchilla, Hofstetter et al. (1997 a and b) showed that up to 70 percent of all IHC could be missing [Figure - 1] without any observable change in either OHC morphology or function as measured by the distortion product otoacoustic emission (DPOAE) [Figure - 2].
The literature on carboplatin is not as well developed but several laboratories (Kopke et al., 1997 and Ryback, 1999) have shown that the ototoxic effects of cisplatin can be avoided or dramatically decreased by the administration of an antioxidant drug. Kopke et al. (1997) in a systematic study of the ototoxicity of cisplatin in cochlear organ cultures found that hair cell damage could be dramatically reduced if the culture was treated with certain antioxidant drugs. Ryback and Somani (1999) using Wistar rats, treated them with cisplatin and 4methylthiobenzoic acid (MTBA), diethyl dithiocarbamate (DDTC), alpha-lipoic acid or ebselen; all the drugs were given I.P. and the antioxidant treated rats developed significantly less threshold shifts as measured by ABR. Biochemically, the cisplatin treated control rats showed a marked depression of glutathione and a decrease in other antioxidant enzymes along with a marked increase in malondialdehyde.
Ryback and Somani (1999) concluded that "these findings suggest that cisplatin ototoxicity is related to lipid peroxidation and . . . protective agents prevent hearing loss and lipid peroxidation by sparing the antioxidant defence system in the cochlea".
Returning to the question of carboplatin, it shows a similar molecular structure to cisplatin; it is less ototoxic but the hearing loss it creates is similar in audiograms and pathology to that of cisplatin so it is reasonable to ask whether there are common pathological processes between carboplatin and cisplatin; i.e., suppression of glutathione (GSH), GSH peroxidase and GSH reductase, and elevation of superoxide dismutase (SOD), catalase and malondialdehyde activity. If depression of glutathione is a factor in carboplatin ototoxicity, then inhibition of glutathione synthesis should lead to additional ototoxic reactions.
L-buthionine-[S,R]-sulfoximine (BSO) is a specific and irreversible inhibitor of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in the synthesis of GSH. BSO has been widely used as a tool to deplete intracellular and extracellular GSH in various cells and organs (Lee et al., 1987; Lee et al., 1992; Luthen et al., 1994; Mistry et al., 1993, Mitchell et al., 1989; Mizui et al., 1992; Morales et al., 1994; Pileblad and Magnusson, 1989; Thanislass et al., 1995), including the cochlea (Hoffman et al., 1988; Lazenby et al., 1988). The purpose of the present study was to determine if BSO enhances carboplatin ototoxicity in the chinchilla cochlea. The severity of carboplatin ototoxicity was assessed physiologically by measuring distortion product otoacoustic emissions (DPOAEs) and evoked potentials from a inferior colliculus (ICPs), and anatomically by the cytocochleograms showing IHC and OHC loss. Since previous studies have shown that carboplatin preferentially damages the IHCs in chinchilla, it was of interest to determine if BSO treatment would increase the amount of damage to the OHCs.
| Materials and Methods|| |
Subjects and surgery for implanting recording electrodes.
Nine adult chinchillas (450-600 g) with normal hearing served as subjects. Tungsten recording electrodes were stereotaxically implanted into the left and right IC and a ground electrode was implanted in the rostral cranium (McFadden et al., 1998). Following surgery, the animals were allowed to recover for at least two weeks prior to testing. The care and use of animals in this study were approved by the State University of New York at Buffalo Institutional Animal Care and Use Committee.
In all subjects, BSO was applied to the right ears and the left ears served as controls. The animals were randomly divided into two groups, carboplatin-treated and drug control. The double-dose carboplatin group (n=4) received BSO in the right ear followed by two doses of carboplatin (25 mg/kg i.p. X 2). The first dose was administered 3 days after the beginning of BSO treatment, and the second dose was administered 4 days after the first. Chinchillas in the drug control group (n=5) only received BSO in the right ear without any carboplatin.
BSO was infused into the right cochlea of each animal using an osmotic pump (2ML4, Alza Corporation) (Brown et al., 1993; Schindler et al., 1995). The pump was filled with 15 mM BSO (Sigma Chemicals) in Hanks balanced salt solution (GIBCO). The pump infusion rate was 5 µl/hr and the infusion period lasted 14 days. Our selection of a 15 mm concentration of BSO was based on previous in vitro studies, most of which used BSO concentrations ranging from 0.05 to 5 mm to deplete cellular GSH. We used a concentration of BSO exceeding the levels used in in vitro studies to allow for intracochlear dilution of the BSO solution by the perilymph. A 14-day pump was used to ensure that GSH levels were reduced over the entire time period that carboplatin was active.
Tone burst stimuli were digitally generated (10 ms duration, 5 ms cosine rise/fall, constant starting phase, 10 stimuli/s) at 0.5, 1, 2, 4, 8 and 16 kHz as described previously (Hu et al., 1997). One hundred samples were averaged at each level. Thresholds were determined by visual inspection of raw waveforms at each frequency. Threshold was defined as the mid-point between the lowest level at which a clear response was seen and the next lower level where no response could be discerned. Mean thresholds were determined by averaging threshold estimates for all animals in a group. I/O functions show mean response amplitudes as a function of input level. The amplitude of the evoked response was measured from the first positive peak to the following negative trough. Thresholds and response amplitudes were measured before and 2, 7 and 14 days after the beginning of BSO treatment.
Input/output (I/O) functions of the cubic (2f 1 -f 2 ) DPOAEs were recorded during the presentation of two primary tones (f 1 and f 2 ), with an f 2 /f 1 ratio of 1.2. The levels of the primaries, L1 and L 2 , were equal. DPOAE I/O functions were recorded in 5 dB steps from 0 to 80 dB SPL. DPOAE I/O functions were collected in random order for f 1 = 1, 2, 4, and 8 kHz.
Fourteen days after BSO treatment, the animals were anesthetized with sodium pentobarbital (50-100 mg/kg) and decapitated. Each bulla was quickly removed and opened to expose the cochlea. The round window and oval window were opened and 0.2M sodium succinate in 0.1M phosphate buffer (pH 7.4) was slowly perfused through the round window; then the cochlea was immersed in the same solution for 1 hr at 37 oC. The cochlea was post-fixed with 10% formaldehyde for 24 hrs. Hair cell counts as reflected in succinia dehydrogenase activity [Figure - 1]for example, were obtained over 0.24 mm intervals along the entire length of the cochlea and the percentage of missing OHCs and IHCs were plotted as a function of percent distance along the length of the cochlea as described previously (Hofstetter et al., 1997 a and b; Hu et al., 1997).
| Results|| |
Effects of Chronic BSO Infusion
Data from the four animals that received BSO (15 mM) for 14 days without carboplatin were used to assess the effects of BSO infusion on cochlear function and morphology. Thresholds and response amplitudes of ICPs were examined before and at various times (2, 7, and 14 days) during the BSO infusion.
[Figure - 3] shows average ICP amplitudes at frequencies from 0.5 to 16 kHz, before and after 2, 7, and 14 days of BSO treatment. Note that 2 days after the beginning of BSO infusion, average ICP response amplitudes were decreased slightly, while at 7 and 14 days after BSO treatment, response amplitudes had returned to pre-treatment levels.
As with ICP I/O functions, DPOAE I/O functions obtained from the drug control subjects showed small amplitude losses 2 days after the beginning of BSO treatment, but normal amplitudes at 7 and 13 days [Figure - 4]. At primary levels of 55 dB SPL, DPOAE amplitudes were decreased by 4.5-9.2 dB 2 days after the beginning of BSO treatment, but were normal by 7 days. OHC and IHC losses were assessed after 14 days of BSO infusion. Light microscopic analysis of SDH-stained cochleas showed no signs of either IHC loss or OHC loss in any of the 8 cochleae from drug control animals.
Changes in ICPs, DPOAEs and HC Losses After a Double Dose of Carboplatin
[Figure - 5] compares the ICP I/O functions of BSO-treated ears and control ears of the four animals in the double-dose carboplatin group. Both control ears and BSO-treated ears showed a decrease in response amplitude after carboplatin treatment. Amplitude losses tended to be slightly greater in BSO-treated ears than in control ears at stimulus levels between 45 and 60 dB SPL.
DPOAE I/O functions were also examined before BSO infusion and 7 days after the second dose of carboplatin application [Figure - 6]. There was no change in amplitude after carboplatin treatment in the control ears. In contrast, BSO treated ears exhibited decreased amplitudes at 2, 4, and 8 kHz following carboplatin treatments. At primary levels of 60 dB SPL, the BSO-treated ears had amplitude shifts of 4-12.3 dB relative to control ears and pre-drug values.
The double dose of carboplatin produced greater IHC loss in both BSO-treated ears than control ears [Figure - 7], upper panel). The magnitude of IHC loss varied across animals. However, all four BSO-treated ears had more IHC loss than their respective control ears. As shown in [Figure - 7], IHC loss in control ears (solid circles) was localised to the middle and high frequency regions of the cochlea, with no losses in the basal 90-100% region or in the apical 0-20% region. In contrast, BSO-treated ears had IHC losses scattered throughout all cochlear turns, with the greatest losses in the 60-80% region of the cochlea. Carboplatin produced an average IHC loss of 18.1% ± 4.62 in control ears and 59.1% ± 2.71 in BSO-pretreated ears. The lower panel of [Figure - 7] compares carboplatin-induced OHC losses in the BSO-treated ears (open circles) and control ears (solid circles). In contrast to the minor OHC losses in control ears, three out of the four BSO-treated ears had much greater OHC losses, and these losses were distributed along all turns of the cochlea.
| Discussion|| |
The results show that infusion of BSO (15 mm) directly into the chinchilla cochlea potentiates the ototoxicity of carboplatin without causing direct damage to IHCs or OHCs. One question that arises from this study is why BSO consistently potentiated carboplatin ototoxicity? The simplest explanation for this is that carboplatin treatment itself reduced cellular GSH, as has been shown to occur following cisplatin treatment (Ravi et al., 1995), and that the double dose of carboplatin, combined with BSO treatment, depleted GSH levels beyond a critical level. The fact that BSO infusion alone did not cause permanent anatomical or functional damage to HCs [Figure - 1] and [Figure - 2] indicates that if GSH depletion is responsible for carboplatin damage, the effect is dose dependent. In future studies, it will be useful to develop techniques for assaying cochlear levels of GSH and other antioxidant enzymes and correlating them with individual susceptibility to address these issues directly.
Since BSO enhanced the sensitivity of both IHCs and OHCs to carboplatin, presumably by inhibiting GSH synthesis, it is reasonable to speculate that chinchilla OHCs are normally protected by higher endogenous levels of GSH than IHCs. To address this hypothesis, we have conducted pilot studies using mercury orange (14 chloro-mercury-phenyl-azo-2-naphthol; Sigma Chemicals) to stain cellular thiol (the most prominent of which is GSH) in the cochlea. Preliminary results show that mercury orange staining is much more intense in the OHCs than in the IHCs. However, we have observed a similar pattern of staining in the guinea pig cochlea, indicating that intracellular distribution of GSH cannot account for the species difference in HC susceptibility. Thus, species differences in hair cell vulnerability may be related to other factors, such as inherent differences in metabolic activity and ROS production, or differences in the ability of other antioxidants to compensate for decreased GSH production. More extensive experiments are required to understand cellular and species differences in susceptibility to carboplatin damage.
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Center for Hearing and Deafness, State University of New York at Buffalo, 3435 Main Street, 215 Parker Hall, Buffalo, New York 14214
Source of Support: None, Conflict of Interest: None
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6], [Figure - 7]