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Cisplatin (CDDP) is an anti-neoplastic drug extensively used in cases of head and neck cancer. Cisplatin induces numerous untoward side-effects including ototoxicity. In this study, cisplatin ototoxicity in Sprague-Dawley rat animal model has been evaluated and the oto-protection provided by the systemic administration of the antioxidant drug D-methionine has been tested. A total of 12 Sprague-Dawley rats were used: 8 were treated intra-peritoneally with D-methionine (300 mg/kg) and cisplatin (16 mg/kg, slow 30 min-infusion), 4 only with cisplatin. The hearing threshold of the animals was evaluated by electrophysiological procedures as Otoacoustic Emissions and Auditory Brainstem Responses. The effects of protection were evaluated after 72 hours. The data from the Otoacoustic Emissions (in the 4.0–12 kHz band) and Auditory Brainstem Responses recordings suggested that D-methionine can partially protect from Cisplatin ototoxicity.
Cisplatin (CDDP) is a widely used anti-neoplastic drug presenting numerous side-effects such as: nausea and vomiting, neurotoxicity 1 2, nephrotoxicity 3, vestibulotoxicity 3 and ototoxicity 4–6. The latter has been reported in many studies and is mainly characterized by an initial threshold shift at the higher frequencies (i.e., 4.0 and 8.0 kHz) 7. Frequent and continuous CDDP administration can affect the lower frequencies resulting in a progressive hearing loss and difficulties in speech recognition in noisy environments.
The ototoxic effects of cisplatin have been primarily evaluated in various laboratory animals 8. The first ototoxic impact seems to involve the cochlea, leading to anatomical changes on the organ of Corti and of the stria vascularis 9. In such cases, it is possible to observe damage in the outer hair cells starting from the first row of the basal turn, alterations of the supporting cells and Reissner’s membrane 10. These morphological alterations are considered as the direct effect of blocking the transduction channels of the outer hair cells 11 12. The hearing loss caused by ototoxicity is progressive and irreversible and, with nephro-toxicity, is the main limiting factor of the CDDP dosage in current clinical therapeutic strategies.
Traditionally, in experimental animals, the overall alteration of the hearing threshold, due to cisplatin administration, has been studied by means of auditory brainstem responses (ABR) 4 5 7 13 14. These measurements represent the integration (contribution) of individual responses from many neural fibres, therefore minute changes in cochlear micro-mechanics, caused by possibly transitory ototoxic effects, are not revealed. A detailed description of eventual dysfunction in cochlear micro-mechanics caused by cisplatin ototoxicity can be obtained via recordings of the otoacoustic emissions (OAEs) 8 15. These are considered responses of cochlear origin, generated when the auditory periphery is stimulated by a click or a pure tone stimulus and their close relationship with the non-linear micro-mechanics of the outer hair cells has been well established. In this context, use of OAEs can establish not only the presence of an ototoxic effect, but also evidence regarding the progress of ototoxicity as seen from the perspective of the OHCs.
Species differences between Humans and experimental animals exist in the susceptibility of the inner ear. In general, doses inducing an ototoxic effect of cisplatin in experimental animals exceed the doses used in the treatment of patients. It has been demonstrated, in several species, that there is a significant individual variability of hearing loss related to cisplatin treatment 15. Even though several parameters, such as the pharmacokinetic pattern and pre-treatment hearing status, have been taken into consideration 7, no predictive factor for cisplatin-induced hearing loss has been identified.
The fact that a CDDP ototoxic insult results in the loss of outer hair cells due to apoptotic mechanisms has generated great clinical interest for substances: i. which might protect the inner ear from CDDP; and ii. which do not interfere with the activity of the anti-neoplastic agent. Animal studies 1 4 6 16 and clinical observations 1 17 have demonstrated that the family of thiosulphate compounds can protect from platinum ototoxicity including the drug carboplatin (a newer platinum compound). For the latter, Muldoon et al. 18 have shown that administration of sodium thiosulphate, following carboplatin treatment, significantly reduces ototoxicity in guinea pigs, times and doses being consistent with the anti-tumoural activity. In previous investigations 8 15, we evaluated the toxic effects, induced by CDDP, in the Sprague-Dawley rat model, by means of electrophysiological and morphological studies of the cochlea. From these studies, it was concluded that hearing loss related to apoptosis of the outer hair cells can be reliably predicted by otoacoustic emission measurements. In the present study, we evaluated the protective efficacy of a systemic administration of D-methionine using, as measuring technique, otoacoustic emissions (OAEs) verified by auditory brainstem responses (used as gold standard) 19.
A total of 12 male Sprague-Dawley albino rats have been used, mean weight 200 ± 20 g. Animals were divided into two groups: Group 1 (n = 8), and Group 2 (n = 4). Group 1 animals were treated with D-methionine (300 mg/kg), prior to the 30 min slow cisplatin infusion (16 mg/kg). Group 2 animals were considered as controls and received an equal volume of saline solution (instead of D-methionine) and cisplatin.
The experiment was performed as follows:
Group 1 animals:
Group 2 (controls):
Post-treatment auditory function evaluation tests were performed, in both groups, 72 hours after CDDP administration.
The anaesthesia solution contained 1 ml of ketamine (100 mg/ml, Ketavet 100, Intervet, Aprilia, Italy), 1 ml of xylazine (20 mg/ml, Rompun, Bayer Leverrkuen, Germany), and 1 ml of saline. This solution was injected into the rat in 2 phases: in the first phase, a 1 ml/kg was administered by ip injection, and when the animal had reached, or shown signs of muscular relaxation another 0.5 ml/kg dose was administered under the skin (second phase).
CDDP was administered as 16 mg/kg body weight of the rat, at a concentration of 1.0 mg/ml. To simulate a clinical context, cisplatin was administered as a slow 30-min infusion using a Harvard apparatus micro-pump.
Distortion product otoacoustic emission (DPOAE) was recorded in a soundproof box by means of a Starkey 2000 device (Starkey Labs, Eden Praire MN, USA). The DPOAE analysis was set at a 4.0-16.0 kHz band (referring to f2), and 12 points per octave were sampled. The primary tone ratio was fixed at 1.21. The DPOAE responses were evoked by three non-symmetrical DPOAE protocols characterized by L1 > L2. Such protocols are generally considered the best choice to identify cochlear dysfunction 20 21. The protocols used were defined as follows: P1 = low level (L1 = 40 and L2 = 30 dB SPL); P2 = middle level (L1 = 50 and L2 = 40 dB SPL); P3 = high level (L1 = 60 and L2 = 50 dB SPL).
During the DPOAE recordings, the body temperature was maintained at 37 ± 0.5 °C by a temperature control device (Harvard Apparatus, Holliston MA, USA). A typical DPOAE response is shown in Figure Figure11.
To optimize the transient evoked otoacoustic emission (TEOAE) recordings, the anaesthetized animal was placed under a stereotaxic device which held, without any movement, a neonatal ILO probe. This was introduced in the right external acoustic meatus through a small tube (diameter 3 mm) 35 mm in length.
Recordings of the TEOAE were made in a soundproof box by ILO-92 (Otodynamics Ltd, Herts, UK), at the beginning of the experiments (time = 0) and again 72 hours after CDDP administration. The TEOAEs were evoked by a 80 µs click of an intensity 63 ± 2 dB p.e. SPL, according to the standard non-linear ILO protocol 1 8. To eliminate residues related to a stimulus artefact, data have been analysed with a temporal window ranging from 1.5 to 4.5 ms. The TEOAE responses were evaluated in the frequency domain (FFT), by estimating signal to noise ratios (SNR) at 1.5, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 kHz.
During the TEOAE recordings, the animal’s body temperature was maintained at 37 ± 0.5 °C by a temperature control device (Harvard, USA). A typical TEOAE response is shown in Figure Figure22.
The procedure for recording Auditory Brainstem Responses (ABRs) in the Sprague-Dawley rat has been described elsewhere 8 15. Briefly, the ABRs were recorded by means of three platinum-iridium needle electrodes, placed subdermally over the vertex (positive), the mastoid (negative) and the dorsum area (reference/ground) of the animal. The recordings were made in a sound-treated cabin, the walls and ceiling of which were covered by phono-absorbent material. Calibration of the sound field was achieved using a Bruel & Kjaer (Naerum, Denmark) microphone (type 2209), placed 4 cm above the animal’s head and facing the loudspeaker.
ABRs were amplified 20000-fold and filtered from 20 to 5000 Hz. Each recording was the average of 500-1000 individual responses. The ABRs were generated in response to 100 µs alternated clicks and 8, 10, 16, 20, kHz tone pips (1 ms rise-fall time, 10 ms plateau), in the range 100-30 dB SPL. The sound transducer, a Motorola (Schamburg IL, USA) tweeter (flat response ± 1 dB from 4.0 to 35 kHz), was placed 4 cm away from the rat’s ear. At the minimum threshold level, two recordings were acquired. No responses were present below a stimulus level of 30 dB SPL, which was considered the threshold level for our experimental set-up. During all measurements, the animal’s body temperature was maintained at 37 ± 0.5 °C by a rectal probe connected to a Harvard Apparatus homeothermic blanket. Ear plugs were used to occlude the contra-lateral ear in order to avoid a binaural stimulation at high stimulus intensities.
Statistical analyses were performed using Student’s t test (p < 0.05).
Analysis of the DPOAE recordings of the three protocols (L3: 60-50, L2: 50-40, L1: 40-30 dB SPL) suggested that all untreated animals (Group 2) presented a significant reduction of the DPOAE amplitude. The latter affected the entire spectrum of the frequencies considered (4-16 kHz). The most significant differences were observed in the recordings from the L3 protocol (60-50 dB SPL) DPOAE. These data are shown in Figure Figure33 and analysed in Table TableII.
The protected animals from Group 1, presented DPOAE amplitudes which showed no significant differences between the pre- and post-treatment recordings. It should be pointed out, however, that the DPOAE amplitudes from the post-cisplatin DPOAE recordings were characterized by lower values. The TEOAE amplitude and the TEOAE spectra (measured at 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 kHz) were significantly impaired in Group 2 (data not shown), while no significant differences were observed in Group 1, between the pre- and post-recording data (Tab. (Tab.IIII).
The ABR data from the unprotected group presented threshold shifts reaching 45 ± 10 dB SPL at 20 kHz and 25 ± 10 dB SPL at 16 kHz and 30 ± 10 dB SPL both at 10 and 8 kHz (a Table with the threshold shifts is also presented in the Appendix). The ABR responses presented small threshold shifts of 15 ± 10 dB at 20 kHz and less than 10 ± 5 dB SPL at 8.0, 10 and 16 kHz. The differences at 20 kHz reached borderline significance (Tables III– V).
The otoprotective effects of D-methionine are summarized in Figures Figures4–4– 6. Figure Figure44 shows the mean pre-administration TEOAE and DPOAE data from Group 1. Figure Figure55 shows the post-administration TEOAE and DPOAE data. Some structural differences were observed in the TEOAE response but the spectral estimate differences remained non-significant. The DPOAE post-administration responses showed a steeper slope at high frequencies (i.e., > 8.0 kHz) but the post-/pre-differences remained non-significant. Figure Figure66 shows the mean post-administration data from Group 2. A comparison with data in Figures Figures44 and and55 indicates structural differences in the TEOAE responses and signal-to-noise ratio differences in the DPOAE responses (in this case, the responses are very close to the noise floor).
Clinical use of chemoprotectors has always been limited due to the negative interactions with the chemotherapeutic drugs, thus reducing their effectiveness on the neoplastic processes. In this context, it is very important to establish whether D-methionine interferes with the anti-tumoural action of CDDP. Some studies have shown that in vivo the anti-tumoural action does not decrease following pre-administration of D-methionine 8 16 17 19 and similar results have been reported in ovarian cancer in vivo 14. Controversial results have been obtained in an in vitro study on the simultaneous administration of CDDP and D-methionine, demonstrating an effective reduction of the anticancer action of cisplatin 22.
As revealed by Reser et al. 19, sulphurs containing antioxidants have the potential to compensate the side-effects of CDDP, and it has been observed that both isomers of sulphur methionine block the CDDP toxic effects in the ear and in the kidney. The D-methionine-treated animals 19, following CDDP administration, did not present auditory damage, according to the post-treatment ABR recordings, and the data from the electron microscopy scan of the cochlea. Campbell et al. 4 13 evaluated the use of D-methionine in a Wistar rat model. The animals received 16 mg/kg CDDP, and various dosages (75, 150 and 300 mg/kg) of D-methionine, 30 minutes before and 3 days after the treatment. The ABR data suggested that animals which received 300 mg/kg D-methionine presented good to full otoprotection.
The fact that data presented in the literature favour both procedures, suggests that the administration of a chemoprotector should not be given with the same modality as cisplatin administration (i.e., i.v.). Several more recent studies, using laboratory animals, have supported the hypothesis that to reliably treat the inner ear, a chemoprotector should be released locally, administered through the round window 23 24. Although such an approach has beneficial effects on the efficacy of oto-protection and the anti-tumoural efficacy of cisplatin, it, nevertheless, raises the question of the type of technology needed to minimize the traumatic administration of a chemoprotector in the standard audiological setting.
This study tested the hypothesis that the administration of antioxidant agents could prevent CDDP-induced hearing loss. The results obtained suggest that the systemic administration of D-methionine has a potential oto-protective role. Data from the OAE recordings suggest good recovery of the post-treatment responses. Albeit, the post-cisplatin OAE data did not demonstrate 100% recovery. This implies that, in the tested conditions (very high cisplatin dosage), D-methionine can partially protect the outer hair cells. The data from the ABR recordings at 20 kHz also support this hypothesis. It could be argued that the cisplatin dosage tested exceeds many times the dosages used in clinical practice. In this context, it is feasible that D-methionine can protect, completely and more efficiently, the inner ear at lower dosages of cisplatin (i.e., 6-12 mg/kg) but further data are necessary to confirm this hypothesis.
The figures shown in this article were prepared using in-house software (ILO-viewer) for the processing of TEOAEs. The software was developed as a collaborative project between the Polytechnic of Warsaw, Poland (Dr. Grzanka) and Ferrara University (Dr. Stavros Hatzopoulos). This software can be downloaded, free of charge, from the Otoacoustic Emissions Portal (http://www.otoemissions.org).