Evaluation of Cisplatin Ototoxicity in a

Rat Animal Model

S. HATZOPOULOS,a M. DI STEFANO,b A. ALBERTIN,a
AND A. MARTINIa,c
a
Department of ENT, Service of Audiology, University of Ferrara, 203 Corso Giovecca,
44100, Ferrara, Italy
b
Department of Biology, Section of General Physiology, University of Ferrara, 44100,
Ferrara, Italy

ABSTRACT: The ototoxic effects of cisplatin were evaluated by otoacoustic

emissions and evoked auditory responses. A transient otoacoustic emissions
protocol indicated no significant ototoxic effects in rats treated intravenously
with 7.5 mg/kg/week for 2-weeks. A chronic 6-week treatment (2.5 mg/kg/week)
monitored by 2F1–F2 distortion product emissions presented significant SNR
alterations in a narrow range of frequencies (5.04–5.66 kHz). An acute treat-
ment of 15 mg/kg, using slow 30-min intraperitoneal infusion, presented the
highest DP and ABR alterations. The SNR at the 2F1–F2 frequencies 6.34, 7.13,
and 7.56 kHz was found significantly decreased, and ABR latency measure-
ments from 8-kHz burst stimuli verified these alterations.

INTRODUCTION

Cisplatin is an antineoplastic agent that is widely used in combination chemother-

apy. It is known to generate a long series of side effects such as nephrotoxicity1–4 and
ototoxicity.5–12 The ototoxic effects of cisplatin include morphological and functional
changes of the organ of Corti, the stria vascularis,9,13 and the outer hair cells
(OHCs).14
Traditionally, the overall alteration of hearing threshold due to cisplatin adminis-
tration has been studied by the use of auditory brainstem responses (ABR).7,10–12
These measurements represent the integration of individual responses from many
neural fibers. Therefore, minute changes of cochlear micromechanics caused by pos-
sible transitory ototoxic effects cannot be revealed. A more detailed description of
cochlear dysfunction caused by ototoxic side effects15,16 can be obtained via record-
ings of otoacoustic emissions (OAE), whose close relationship with the nonlinear mi-
cromechanics of the OHCs has been well established in the last 15 years.17–19 The

c
To whom correspondence may be addressed. Phone: 39-532-236429; fax: 39-532-236887; e-
mail: mma@dns.unife.it

211
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OAE are considered to be responses of cochlear origin generated when the auditory
periphery is stimulated by a click or a pure-tone stimulus. Within this context, it can
be said that the use of OAEs can provide not only a verification of the presence of an
ototoxic effect but evidence regarding the progress of ototoxicity, as seen from the
perspective of the OHCs.
The rat has been extensively used as a clinical model of nephrotoxicity,2,8 but its
utility as an ototoxic animal model is still a subject of dispute. Laurell6 has presented
data supporting the hypothesis that the rat can reach the cisplatin LD50 level
(ⱖ 12.0 mg/kg) without any significant ototoxic effects. Other studies10,12 have pre-
sented data from administration of high doses of cisplatin (ⱖ 15 mg/kg) and have re-
ported low mortality rates, concluding that the rat is a useful model for studying cis-
platin-induced biochemical changes for a posttreatment period of 72 hours. A possi-
ble explanation for these contrasting reports may be related to the variability of the
cisplatin plasma concentration following an intravenous (bolus) or an intraperitoneal
administration, which may cause different levels of ototoxicity, due to different kinet-
ics. Recent evidence20 has indicated that this hypothesis is not valid in the guinea pig
model; therefore, this is an argument requiring further research.
OAE responses from the rat have been reported recently.21,22 They are character-
ized by very small latencies, on the order of 1.0–2.5 ms, and a relatively narrow
bandwidth (2–4 kHz), probably caused by the spectral content of the acoustic stimu-
lus. The OAE recordings have been described as stable and repetitive, similar to
recordings from human subjects. Therefore, OAEs represent an ideal noninvasive
method for studying the perturbation of cochlear micromechanics caused by the dis-
ruption of the antioxidant defense system of the organ of Corti.23
Our hypothesis was that the level of cisplatin ototoxicity is reflected in the me-
chanics of the OHCs, whose proper function can be monitored by means of OAEs.
We expected that the ototoxic effects would be manifested as a significant decrease
of the spectral content of the OAE recordings. The following experiments were con-
ducted in order to indicate the best OAE protocol parameters for the identification of
medium or severe cisplatin ototoxic effects. The ABR measurements were used only
as validators of the possible ototoxic effect present; therefore, compatible OAE/ABR
protocols were used in terms of frequency of stimulation.

MATERIALS AND METHODS

Chemicals
The cisplatin formulation used was Platinex by Bristol Myers (0.5 mg/mL in
normal saline) and it is the same product that is being used in clinical treatments in
Italy. For animal anesthesia, an equal-volume combination of ketamine and xylazine
(Rombun: Bayer, Italy) and saline were used in dosages of 0.1 mL/100 g of body
weight. The anesthetic was administered in two consecutive phases. In phase, one
the animal received an intraperitoneal dose and upon the first signs of muscular re-
laxation (phase two), a second equal-volume dose was administered subcutaneous-
ly.
HATZOPOULOS et al.: CISPLATIN OTOTOXICITY 213

Electrophysiological Studies
Transiently Evoked Otoacoustic Emissions
The transiently evoked otoacoustic emissions (TEOAEs) were evoked by a 80-␮s
click stimulus of 63 ± 2-dB p.e. SPL, following a nonlinear protocol (a stimulus train
composed of four clicks: three positive and one negative with an amplitude 9.5 dB
higher for the negative click than the positive clicks). Each response was on average
1000 stimuli, which were presented at a rate of 74/s. The length of each recording
was 13.5 s. To avoid possible contamination of the TEOAE response by the tail of the
preceding acoustic click, the initial 1.0 ms of each recording was modified by a co-
sine window function (rise time 0.64 ms). The data were collected in a soundproof
room using an ILO-92 apparatus. Signal-to-noise-ratio (SNR) estimates were calcu-
lated at 3.0, 4.6, 6.1, and 7.6 kHz. Estimates at higher TEOAE frequencies are not
feasible with this method. Due to the tonotopic-like structure of the TEOAE re-
sponse, frequencies higher than 7–8 kHz occur in the first ␮s of the recording, but
cannot be measured because they are masked by the stimulus artifact.
The distortion product otoacoustic emissions were recorded in a soundproof room
with Virtual 330 equipment. The data were generated by two stimulus protocols (F1 =
65, F2 = 57 and F1 = 61, F2 = 57-dB SPL).24 The 2F1–F2 DP responses lie in the 4.0 –
8.0-kHz frequency range. Input–output DPOAE curves at 7.0 and 8.0 kHz were com-
puted from 50–75-dB SPL. The ratio of F2–F1 was set to 1.22, and each acceptable
record was the average of 32 responses. The recording of 2F1–F2 DPs at frequencies
higher than 8 kHz in the rat, are a serious technical problem caused by the presence
of constructive interference between the incoming stimulus and standing waves in the
meatus.25,26 This difficulty is enhanced by the fact that most commercial DP probes
(the Virtual included) become severely nonlinear above 12 kHz25 (personal commu-
nication from Dr. Jen Birch, president of Virtual).
In order to record TEOAE/DPOAE responses, the sedated rat was placed in a
stereotaxic device supporting a plastic cone (large diameter: 1.2 mm; small diameter:
3 mm: total length: 22 mm) that was inserted into the rat’s external meatus. An adult
TEOAE ILO/Virtual probe was then inserted inside the cone by multiple layers of
parafilm. In the tested rats the average distance from the end of the probe to the tym-
panic membrane (TM) was on the order of 5 ± 2 mm.

Auditory Brainstem Responses

The auditory brainstem responses (ABR) were recorded by three platinum needle
electrodes placed subdermally over the vertex (positive), the mastoid (negative), and
the dorsum area (reference/ground). The responses were amplified 20,000 times and
filtered from 20 to 5000 Hz. Each recording was on average 250–500 individual re-
sponses.
The data were collected in a soundproof room and were generated in response to
100-␮s condensation clicks and 4 and 8 kHz tone bursts (1 ms rise–fall time, 8 ms
plateau) presented at 90-, 80-, 10-, and 60-dB SPL. The sound transducer (KLH
tweeter, flat response between 2.5 and 14.5 kHz, 10-dB SPL roll-off above 14.5 kHz)
was placed 4 cm away the rat’s ear. During all measurements, the body temperature
of the animal was maintained at a 38±0.5°C by a Harvard homeothermic blanket.
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Experiments

Experiment I
Twenty male Spague-Dawley rats (mean weight 170 ± 10 g) were treated with an
intravenous bolus of 7.5 mg/kg/week of cisplatin for two weeks (final cumulative
dosage of 15 mg/kg). Three measurements of TEOAEs were conducted before and 48
h after the treatments. Due to the short duration of the experiments, the rats were
used as their own controls. The ototoxic effect of cisplatin was expected to produce a
reduction of TEOAE Amplitude reproducibility and of the SNR in the 3.0-, 4.6-,
6.1-, and 7.6-kHz range.

Experiment II
Twenty-four male Sprague-Dawley rats (mean weight 200 ± 30 g) were treated
with a bolus of 2.5 mg/kg/week of cisplatin for six consecutive weeks. Another group
of 6 equal-weight rats was used as a control group (equal-volume saline treatments
were conducted for 6 weeks). IO curves and 2F1–F2 DP responses were measured at
7.0 and 8.0 kHz 24 h prior and post treatments. This experiment was designed to (a)
provide evidence of whether the 2F1–F2 distortion products could detect light ototox-
ic effects caused by a chronic cisplatin regimen within a 24-h monitoring interval,
and (b) determine the minimum cumulative cisplatin dosage causing a detectable
ototoxic effect.

Experiment III
Sixteen male Spague-Dawley rats (mean weight 180 ± 20) were divided into three
groups. Group 2 (4 rats ) was treated with 7.5 mg/kg of cisplatin, Group 3 (8 rats)
was treated with 15.0 mg/kg of cisplatin, and Group 1 (4 rats) was used as a control
for Group 3 (equal-volume saline treatments were conducted). The cisplatin was ad-
ministered by an intraperitoneal slow infusion (30 min) using a micropump from
Harvard Apparatus. ABR responses to compressed clicks and tone bursts at 4.0 and
8.0 kHz and 2F1–F2 DPs from a 65–57 asymmetrical protocol were collected 72 h
before and after treatment. This experiment was designed to provide evidence of
whether the DP parameters could match the threshold alteration provided by the
ABR. In comparison to experiment II, in this experiment the measurements were
conducted with an additional delay of 48 h to match the data presented previously10
for the 15-mg/kg cisplatin dosage.
A flowchart of the cisplatin chronic and acute experiments is presented in FIGURE
1. The label “Not Sign” indicates that for the specific experiment the group differ-
ences were not significant.

Statistical Analyses Employed

The TEOAE and DPOAE data were analyzed with a 2-factor ANOVA and with
modified Wilcoxon pair tests (the distributions of the SNRs proved to be nonnormal).
The criterion for statistical significance for all measures was p ⱕ 0.01.
HATZOPOULOS et al.: CISPLATIN OTOTOXICITY 215

FIGURE 1. Flowchart describing the development of experiments I, II, and III.

RESULTS

Experiment I
Two data sets were formed from the measurements of the first (7.5 mg/kg) second
cisplatin administration (cumulative 15 mg/kg). The TEOAE responses were ana-
lyzed for mean differences between the pre- and postmeasurements from the first and
the second data set in terms of the following parameters: TEOAE correlation, and
TEOAE SNR at 1.5, 3.0, 4.6, 6.1, and 7.6 kHz. The mean difference between pre-
and posttreated rats, in both data sets, was not significant across all tested variables.
A 2-factor analysis of variance indicated that there was no variable that could be con-
sidered significant.
In terms of differences across individual rats, one rat demonstrated significant al-
terations of the SNRs at 3.0, 4.0, 6.1, and 7.6 kHz from the first treatment. This case
is depicted in FIGURE 2. FIGURE 2A shows the pretreatment TEOAE response, while
the bottom (FIG. 2B) graph shows the response after the cisplatin treatment. The cis-
platin caused a significant decrease of the amplitude of the TEOAE response, which
translated into lower SNRs in the 1.5–7.6-kHz range. Three rats from the second data
set also showed altered SNRs at 4.0 and 6.1 kHz. This number (4 rats) indicates that
there were S/N alterations in 20% of the total rat population, but this was too small to
influence the mean data. Within 48 h of the last treatment, all rats were still alive.

Experiment II
The data were analyzed in terms of a pre- and posttreatment (cumulative dose of
15 mg/kg) and across consecutive treatments (2.5 versus 7.5, 2.5 versus 10.0 mg/kg,
etc.). From the two 2F1–F2 DP protocols employed, the 65–57-dB protocol generated
the largest differences across individual rats. An initial exploratory ANOVA of the
DP data across the first (2.5 mg/kg), third (7.5 mg/kg), and fourth (10.0 mg/kg) treat-
ment did not indicate any significant mean differences. For this reason, additional
analyses for between treatment differences were not conducted. An analysis of the IO
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FIGURE 2. TEOAE responses from a rat: (A) pretreatment data, and (B) after 7.5 mg/kg of
cisplatin. The middle panels at the right provide information on the frequency characteristics
(SNR and reproducibility) of the response. The envelope of the response is defined from 1.0 ms
to 2.2 ms in (A) and from 1.0 ms to 1.8 ms in (B). A decrease of TEOAE amplitude corre-
sponds to a decrease of the OHC activity caused by the ototoxic effect of cisplatin.
HATZOPOULOS et al.: CISPLATIN OTOTOXICITY 217

data across all treated rats at the pre- and sixth treatment, with a Wilcoxon statistic,
indicated no significant mean differences at 7.0 and 8.0 kHz, but only an interesting
pattern in the treated rats. FIGURES 3 and 4 show this pattern. The first figure presents
IO data at 8.0 kHz from the control group and the group that received cumulatively
15 mg of cisplatin. In the mean estimates from the treated group, there is a notch at
the stimulus level of 70-dB SPL, which is not observable in the normative data. This
behavior may indicate a subtle change in the DPOAE generation mechanics. FIGURE
4 shows that the notch pattern in the data from the treated rats is observable from the
third (cumulative cisplatin dosage of 7.5 mg/kg) to the sixth treatment. Combining
the spectral amplitude for all the tested frequencies, we see a significant difference
between the control rats and the groups that received a cumulative dose of 7.5–15.0
mg/kg. This difference was strongly correlated with the weight of the animals.
A Wilcoxon statistic comparing the DPOAE data between the pre- and sixth treat-
ment (15 mg/kg) indicated that there were significant mean differences at the fre-
quencies 5.04, 5.66, and 5.99 kHz. The data are shown in FIGURE 5. The frequencies
where the SNRs were attenuated are also associated with artificial notches caused by
the interaction of the incoming stimulus with multicomponent standing waves. We
believe that this hypothesis is not valid for at least two reasons: (1) the data refer to
average S/N estimates from the total number of tested rats; and (2) the decrease of

FIGURE 3. Mean (±1 s.d) input–output curves at 8 kHz from treated vs. nontreated rats. (A)
Cumulative cisplatin treatment of 15 mg/kg. (B) Control group treated with saline.
218 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 4. Surface 3D plot showing the input–output data of treated rats across all eight
measurements in experiment II. The X-axis shows stimulus intensity in dB SPL, the Y-axis
shows the treatment (pretreat, treatment 1, treatment 2, etc.), and the Z-axis shows the S/N val-
ue at 8 kHz (no units). On the Y-axis, the number “0” corresponds to the pretreatment data and
the number “7” to posttreatment data. Arrow indicates the dip at 70-dB SPL, observed from
exp 3 (7.5 mg/kg).

the SNR at these frequencies was not observed in the control and the early treatment
rats (see FIG. 3B).
The analysis of the normative 2F1–F2 SNRs across the pre- and posttreatment pre
data indicated that the SNRs increased in the 6.73–8.0-kHz range. It is plausible that
the increase of 2F1–F2 amplitude, in the 6-week interval may reflect an ongoing mat-
uration mechanism.

Experiment III
The data from the pre- to posttreatment data sets were analyzed across all 2F1–F2
DP frequencies. No significant differences were observed between the normal group
(G1) and the group treated with 7.5 mg/kg of cisplatin (G2).
An analysis of the data from group G3 indicated significant mean differences be-
tween group G1 and the pretest responses of group G3. For the latter, significant
SNR differences were observed at frequencies of 5.66–8.0 kHz (FIG. 6). The data
from the G3 group show a decrease in the mean and an increase in the standard devi-
ation (s.d.) per SNR value in the previously mentioned frequencies. The increase in
the s.d can be interpreted as a lack of homogenous 2F1–F2 responses in the treated
G3 group. FIGURE 7 shows star-plot representations from pre- and posttreatment data
HATZOPOULOS et al.: CISPLATIN OTOTOXICITY 219

FIGURE 5. Histogram of the mean SNRs plus standard error of the treated rats in experiment
II. The data depict responses from pretreatment, treatment with 2.5 mg/kg, treatment with a cu-
mulative dose of 15 mg/kg, and post 15 mg/kg treatment. The asterisk and plus signs indicate
significant differences in the SNR between pre- and 15-mg or posttreatment measurements, re-
spectively.

in the groups G1 (control) and G3. This type of plot provides the opportunity to study
simultaneously the increased alterations of the 2F1–F2 DP responses in terms of how
many frequency components are present and their SNR values. In five rats, partial
(G3B, G3TC) and complete losses (G3D,G3T, G3TD) of the SNR values were ob-
served.
FIGURE 8 shows a pre- to posttreatment histogram of the responses from groups
G2 and G3. For the latter, the larger significant S/N differences were observed at the
frequencies 6.34, 7.13, and 7.56 kHz. Borderline differences were also observed at
5.66 and 8.0 kHz.
The parameters we have considered in the ABR analysis were changes of ampli-
tude and latency of wave III at various stimulus levels (90-, 80-, 70-, 60-dB SPL).
There were no significant differences in these parameters between group G1 and
group G2 (7.5 mg/kg) for both types of stimulation (click and tone burst). The mem-
bers of the 15-mg/kg group demonstrated a significant increase of latency for the 8-
kHz tone burst at all stimulus levels (FIG. 9) and a decrease in the relative amplitude
of wave III at 90-dB SPL. The responses to clicks and the 4-kHz tone burst were not
statistically different from the control group (FIG. 10). All members of the G3 group
FIGURE 6. Mean SNR responses (±1 s.d) of the rats treated with 15 mg/kg in experiment III
across all DPOAE frequencies. (A) Pretreatment data; (B) posttreatment data. In the latter
graph, note increase of the standard deviation and decrease of the mean S/N values from 5.34
kHz.

FIGURE 7. Star-plot graphs of the pre- and posttreatment DPOAE responses from the rats
treated with saline (group G1) and 15 mg/kg (group G3) in experiment III. Bottom right shows
the profile of a response containing all frequency and amplitude characteristics. Deformations
of this pattern implies attenuation of the SNR at various frequencies.
HATZOPOULOS et al.: CISPLATIN OTOTOXICITY 221

FIGURE 8. Histogram of the mean SNR values (+1 s.d.) from (A) rats treated with 15 mg/kg
of cisplatin (group G3), and (B) rats treated with 7.5 mg/kg (group G2) from the pre- and post-
treatment data sets. The star symbol indicates a significant difference between pre- and post-
treatment SNR values. The X-axis shows 2F1–F2 DPOAE frequencies in kHz, and the Y-axis
shows SNRs ratios plus the standard error in dB SPL.

were alive 96 h posttreatment.

DISCUSSION

Experiment I
The data presented indicate that TEOAEs are not useful descriptors of the ototox-
ic status of the organ of Corti. It is quite probable that some signal alterations occur
at higher frequencies (in the initial ms segment of the TEOAE recording); however,
this portion of the response is nulled in order to compensate for stimulus artifacts.
We have considered another possibility, namely that the peak of the ototoxic effect
requires more time to develop than the 48-h interval used. Data from experiment III
indicated that rats treated with a dose of 15 mg/kg survive up to 96 h after treatment.
It is also known that the chronic cisplatin effects are less pronounced than the acute
effects.9,20 This suggests that TEOAE monitoring of ototoxicity may require longer
intratreatment intervals than 48 h.
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FIGURE 9. Histograms of the mean latency values (+1 s.d.) of wave III from 8-kHz tone
burst ABR recordings in experiment III. The X-axis shows stimulus intensity in dB SPL, and
the Y-axis mean latencies in ms. (A) Data from the pretreatment ABR recordings; (B) data
from posttreatment recordings. The mean latency of group G3 at 60-dB SPL is significantly
longer that the corresponding value calculated from the pretreatment recordings.

Experiment II
This experiment was designed to indicate whether the ototoxic effects of a cumu-
lative protocol can be identified by means of 2F1–F2 distortion product otoacoustic
emissions. The data indicate that significant alterations occur in the mean SNR val-
ues across a narrow range of DPOAE frequencies. The hypothesis of this experiment
assumed that the ototoxic effect of cisplatin should result in a reduction in the SNR at
higher frequencies (> 5.0 kHz). However, the data show altered S/N alterations at
lower frequencies, while the SNRs at the higher frequencies remained unchanged.
The data in FIGURE 5 show that the frequencies where the SNRs significantly de-
creased (5.04–5.66 kHz), the corresponding values from the posttreatment measure-
ments show an even higher attenuation. A comparison between the pre- and the 2.5-
mg/kg treatment conditions shows significant differences over a wider range of fre-
quencies (5.04–5.99 kHz). Within this context, we can speculate that (1) the ototoxic
effect of a chronic treatment can be identified at lower frequencies than expected, and
(2) the ototoxic effect develops over a time interval greater than 24 h and involves a
wider range of 2F1–F2 DP frequencies.
The data from the input–output functions also suggest the presence of borderline
ototoxic complications. The SNR dip, shown in FIGURE 4, may represent a mechani-
HATZOPOULOS et al.: CISPLATIN OTOTOXICITY 223

FIGURE 10. Histograms of mean latency values (+1 s.d.) of wave III from click ABR record-
ings in experiment III. The X-axis shows stimulus intensity in dB SPL, and the Y-axis shows
the mean latencies in ms. (A) Data from the pretreatment ABR recordings; (B) data from post-
treatment recordings. No significant increase of mean latency value was observed in either G2
or G3 treated groups.

cal disturbance in local cochlear micromechanics. If this hypothesis is true, then low-
er-level stimuli (i.e., 45–50 dB) should be used in the DPOAE protocol to explore
this possibility further.

Experiment III
The data from the DPOAE and ABR analysis suggest that this experimental
modality (slow infusion) is a very good model for acute cisplatin monitoring. The
low infusion limits the mortality of the treated animals (only one out of eight rats
died) 96 h after treatment.
The higher-frequency DPOAE data and the ABR responses, from the 8-kHz-tone-
burst stimulus, demonstrated good statistical agreement. The 2F1–F2 DP responses
showed attenuated SNRs in the 5.34- to 8.0-kHz frequency interval (in some frequen-
cies the differences were borderline), which does not coincide with the frequency in-
terval defined in experiment II. This observation could suggest that different mecha-
nisms are involved in different levels of ototoxicity.
It is still unclear why the click-evoked ABR was not significantly altered. One hy-
pothesis is that the acoustic clicks do not stimulate the high-frequency regions of the
cochlea. For the majority of ABR transducers the spectrum of the acoustic click has a
224 ANNALS NEW YORK ACADEMY OF SCIENCES

main peak in the midfrequencies (2–4 kHz), and then a gentle roll-off of a relative
10–15 dB/octave.27,28 Within this context, the proposed hypothesis does not seem
valid, because a 90-dB click would produce an 8-kHz signed of 60–70-dB SPL.
Therefore, responses from this frequency region should be still identifiable.

SUMMARY

The present experiments suggest that only acute cisplatin treatments can be moni-
tored efficiently by means of otoacoustic emissions. The data from the TEOAE re-
sponses from our chronic experiment (7.5 mg/kg/week for 2 weeks) showed no
TEOAE alterations. A subsequent chronic treatment study (2.5 mg/kg/week for 6
weeks) monitored by 2F1–F2 DPs showed significant spectral alterations in a very
narrow range of frequencies and an SNR dip in the input–output function at 8 kHz.
These changes could be related to changes in local cochlear mechanics. An acute,
slow-infusion treatment (15-mg/kg dosage) generated the most significant ototoxic
effects manifested as (1) significant increases of ABR latencies (8 kHz), and (2) sig-
nificant decrease in the SNRs ratios at 6.34, 7.13, and 7.56 kHz.
The data from experiments I, II, and III indicate that the rat can be used to study
severe ototoxicity (experiment III), but not light or medium ototoxicity (experiment
II).

ACKNOWLEDGMENTS

We thank Fidia S.p.A. for the technical assistance in this project and particularly
M. Finesso. Our thanks also to M. Rosignoli for assistance with the statistical analy-
sis of the data.

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