Neurotoxicologyand Teratology, Vol. 19,No. 2, pp.

12Y-140,1997
Copyright 0 1997Elsevier Science Inc.
Printed in the USA. All rights reserved
0892.0362197 $17.00+ .OO
ELSEVIER PIISOS92-0362(96)00214-O

Toluene-Induced Hearing Loss:

A Mid-Frequency Location of the
Cochlear Lesions

P. CAMPO, R. LATAYE, B. COSSEC AND V. PLACID1

Institut National de Recherche et de Stcuritt? (I.N.R.S.), Avenue de Bourgogne, B.P.27, 54501 Vandoeuvre, France

Received 25 April 1995; Accepted 25 September 1996

CAMPO, P., R. LATAYE, B. COSSEC AND V. PLACIDI. Toluene-induced hearing loss: A mid-frequency location of
the cochlear lesions. NEUROTOXICOL TERATOL 19(Z) 129-140, 1997.-inhaled toluene (from loo0 to 2000 ppm, 6 h/day,
5 days/week, 4 weeks) is an ototoxic solvent that severely damaged the cochlea in adult Long-Evans rats. Auditory function
was tested by recording near field potentials from the inferior colliculus. Surprisingly, the electrophysiologic results did not
reflect all the cochlear damage observed by histology. Loss of outer hair cells of the organ of Corti occurred in all toluene-
treated rats in middle and mid-apical turns, whereas the basal turn of the cochlea was fairly well preserved. The third row of
outer hair cells was more injured than the second row, which itself was more injured than the first row. The locations of the
cochlear lesions are reported in the present study with regard to the toluene dose. o 1997 Elsevier Science Inc.

Toluene Ototoxicity Mid-frequency Rat

TOLUENE is a widely used organic solvent, heavily employed lesion in their early studies without really testing frequencies
in many industries (paints, adhesives, glues, coatings and de- higher than 20 kHz. In fact, the pathology data of Pryor et al.
greasing/cleaning agents, plastics, textiles, printing inks, agri- (32) do not point to a classical high-frequency lesion because
cultural, and pharmaceutical products) (1,19,23,24). Chronic there was very little damage to the hook region in toluene-ex-
exposure to toluene, particularly in circumstances of abuse. posed rats, whereas there was extensive damage in the middle
can cause severe, often permanent, damage to the central ner- turn. Given these disparate findings, the purpose of our re-
vous system (14,21,45) resulting in neuropsychological impair- search was to clarify the frequency location of toluene-induced
ments (8,27) and even alterations to hearing function (3,26. hearing loss with the aid of both electrophysiological and his-
37,38). Several authors have studied the effects of toluene on tological techniques.
auditory function (6,X,22,30,32); however, few attempts have To study the effects of toluene on auditory structure and
been made to relate the histological data to electrophysiology. function, auditory evoked potentials were recorded from the
To the best of our knowledge, only two complete cytococh- inferior colliculus, and a detailed histological analysis (optical
leograms have been presented (16,41) and they contain par- and scanning electron microscopy) of the cochlea was carried
tially disparate findings. One area of disagreement between out with adult pigmented rats. Results were obtained with in-
these two works centered on the location of the toluene-in- haled doses of toluene ranging from 1000 to 2000 ppm, 6 h/
duced cochlear lesions. Sullivan et al. (41) reported that le- day, 5 days/weeks, 4 weeks.
sions occurred in a low- to mid-frequency region, whereas
Johnson and Canlon (15) found lesions positioned in the mid- METHOD
to high-frequency area of the organ of Corti. Obviously, the
Animals
experimental conditions (exposure dose and duration, age of
animals, species and intoxication route: inhalation vs. gavage) Male Long-Evans rats (45&500 g) were purchased from a
may have contributed to the discrepancies between the results. local breeder (Janvier Laboratories). These animals were
Additionally, Pryor et al. (31,32) mentioned a high-frequency approximately 7 months of age at the beginning of the experi-

Requests for reprints should be addressed to P. Campo, Institut National de Recherche et de SCcuritC (I.N.R.S.), Avenue de Bourgogne,
B.P.27.54501 Vandoeuvre, France. Fax: 83-50-21-85; E-mail: campo@inrs.fr

129
130 CAMP0 ET AL.

ment, and between 10 and 11 months of age at the end of the stereotaxic reference landmarks. The incisor bar (-5 mm)
experiment. The rats were housed in individual polycarbonate and the interaural line were positioned in a horizontal plane.
cages (350 X 180 X 184 mm) with steam-cleaned pinewood The stereotaxic coordinates were obtained from Paxinos
bedding I month before the start of the experiment. Tempera- and Watsons atlas. Using the atlas, the accurate coordinates
ture in the animal quarters was 22 +- 1C and the humidity for the central nucleus of the right inferior colliculus (IC) were
ranged from SO% to 55%. The cages were lit by fluorescent ~9.3 mm from the bregma point, 1.5 mm from the vertical
lights from 0700 to 1900 h. plane passing through bregma and lambda. and 4.2 mm from
While conducting the research described in this article, the the horizontal plane passing through bregma and lambda. A
investigators adhered to the G&e for Care and Use of Labo- tungsten microelectrode covered with Teflon (except at the
ratory Animals, as promulgated by the French Conseil dEtat tip) was surgically implanted to record the inferior colliculus
through the D&ret No. 87-848 published at the French Journal potentials (ICP). and a ground microelectrode (4 mm) was
Officiel on October 20th. 1987. placed on the dura mater in the nasal region [adapted from
Henderson et al. (IX)]. The electrodes were then fastened to a
transistor socket and fixed with dental cement to the skull.
E.upurinzental Procedure
The animals were allowed to recover for 4 weeks before any
Animal preparation. Half an hour prior to the surgical an- testing.
esthesia, atropine sulfate (0.05 mg/kg) and levomepromazine Atrdionzetry. Figure I is a schematic representation of the
(20 mglkg) were given to animals to minimize stress and respi- equipment used for measuring the audiometric thresholds.
ratory distress. Deep anesthesia was induced with an IP injec- Testing was conducted in an audiometric room 1 month after
tion of ketamine (150 mgikg). Rectal temperature was main- surgery. Awake rats were placed in a restraining device that
tained at 38C with a heating pad. Otoscopic examination was ensures constant distance and orientation relative to the
carried out to verify that the outer ears and tympanic mem- speaker. The animals quickly adapted to the holder and usu-
branes were free from obstruction, infection, or other abnor- ally did not struggle. The sound stimuli were generated by
malities. The animals were then placed in a stereotaxic device Medelec, ISD45 equipment and transduced by a speaker
(model 900 cat: 743200 from Phymep Cie. France) with spe- (JBL, 240.5) positioned 15 cm from the left pinna. The acous-
cial ear bars to prevent damage to the tympanic membrane. tic stimuli (two cycles for the rise/fall ramp. four cycles for the
The head holder left the external meatus free for acoustic plateau; therefore. the duration of the click stimulus is related
stimulation. A midline incision (1.5 cm) was made on the to the frequency) were filtered clicks presented at a rate of 20
scalp to expose the bregma and lambda. which were used as per second. The level used for starting the audiometric test

Audiometric room RAClA

MEDELEC MEDELEC
AAGMKIII DA V62 CARD RTl815F

\
Holder

Measuring
Amplifier - Amplifier

TRADELEC 1% BK 2636

Filter Filter
l/3 octave l/3 octave
Computer
BK 1617 BK 1677

T
t A
Acoustic Stimulation VICTOR
Stimulator Control V386S
I I I I

RACIA ISD45 MEDELEC SC6

FIG. 1. Block diagram of apparatus used for measuring and storing auditory evoked responsc~
TOLUENE-INDUCED HEARING LOSS 131

was 40 dB SPL. Audiometric testing was always performed in 4. 1250 ppm, 6 h/day. 5 days/week, 4 weeks, Eight toluene-
the following order: 2. 4, 6, 8, 10. 12, 16, 20. 24, and 32 kHz. exposed (1265 2 24 ppm) and control animals were used.
An intensity calibration was carried out weekly with a 0.5in. 5. 1000 ppm, 6 h/day. 5 days/week. 4 weeks. Eight toluene-
microphone (B&K. 4133) with a measuring amplifier (B&K. exposed (1014 2 23 ppm) and control animals were used.
2636). The sound level was calculated from the peak value of
the electric signal picked up at the speaker. For each fre-
quency, the calibration was carried out by measuring the The animals were anesthetized with ketamine (3.50 mgikg,
sound pressure level (RMS, re: 20 FPa) emitted by the IM) 7-8 weeks after the end of the toluene exposure. To
speaker when it was driven by a continuous pure tone of a 100 avoid the swelling of outer hair cells in the hook and in the
mV peak level. The sound field was calibrated by positioning apical region due to the air exposure after removal of the co-
the microphone at a point normally occupied by the center of chleae. intracardiac fixation of the tissue was followed by in-
the animals head. tralabyrinthine perfusion. For both perfusions, the fixative
The ICPs were amplified and filtered (Medelec, liquid was cold 2.5% glutaraldehyde in a trihydrate solution
AA6MKIIl: gain: 101. 32 Hz-08 kHz) and then fed into a sig- of sodium cacodylate (0.1 M at pH = 7.4). The next day. the
nal averager (MEDELEC DAV62: n = 256). A -trough-to- cochleae were postfixed with 0~0, (t%) in 0.1 M cacodylate
peak amplitude of 20 p,V was considered as the threshold in buffer for 1 h and finally washed in buffer. Following a partial
our experiment. The analysis window lasted 30 ms. The direct dissection. the cochleae were dehydrated from 30% to 70%
and averaged signals were displayed on an oscilloscope ethanol (EtOH). Next, the cochleae were drilled to obtain a
(Gould, 1604). and stored on a personal computer (PC,
V386S). Figure 2 shows a typical series of ICP response wave-
forms elicited at 8 kHz in a nonexposed rat.
Audiometric thresholds were determined once prior to ex-
posure (Tl). A second record of auditory thresholds (72) was
obtained 24-32 h postexposure. The last threshold (T3) mea-
FREQUENCY : 8 kHz
surement was carried out 6 weeks after exposure. Two thresh-
old shifts were calculated for each animal: TSl = T2 -Tl and
Ts2 = T3 -Tl to measure the change in the hearing function
after the end of the exposure. + 50 dB
Tolurne exposure. Animals were exposed to toluene (Merck
Schuchart. 995%) vapors (1000, 1250, 1500. 1750. or 2000
ppm. 6 h/day. 5 days/week, 4 weeks), in inhalation chambers
(200 I) designed to sustain dynamic and adjustable airflow
(IO-20 mYh). The animals were exposed to toluene from 0900
to 1500 h. Neither food nor water was given to animals during
the exposure period. The chambers were maintained at a neg-
+40dB
ative pressure of no more than 3 mm HzO. Input air was fii-
tered (opacimetric method 99%) and conditioned to a tem-
perature of 22-24C and relative humiditv of 50-55%.
Toluene was vaporized by bubbling an addiiional air tlow
through a flask containing the test compound. The ambient + 30 dB
noise level within the exposure chambers were measured with
an integrating sound level meter (B&K. 2218) used with a
slow time constant. It was equipped with a one-third octave
filter (B&K. 1616) and a 0.5in. microphone (B&K. 4133).
Ambient noise levels in one-third octave band from SO Hz to
40 kHz did not exceed 52 dB SPL (Fig. 3), and the overall (lin-
+20dB
ear) level was 66 dB SPL. The roluene concentration in the
exposure chambers was continuously monitored using a gas-
liquid chromatograph. Five different concentrations were
tested in the prcscnt siudy:

2000 ppm. h h/day. 5 days/week. 4 weeks. Three separate +lOdB

chambers were used simultaneously during this toluene ex-
posure. Eight animals were housed in each inhalation
chamber (n = 3 >: 8). Three other inhalation chambers
without toluene were used for the control group (11 = 3 X 7).
Thus, 24 animals wcrc used in the 2000 ppm toluene-exposed THRESHOLD
group and 21 animals in the control group. The concentra-
tion measured dul-ing the exposure was 2006 ? 89 ppm
(mean and SD).
1750 ppm. 6 hlday. 5 dayslweek. 4 weeks. Eight toluene-
exposed (1741 t 46 ppm) and eight control animals were FIG. 2. Example of brain stem (inferior colliculus) auditory-evoked
used. potentials elicited at 8 kHz in nonexposed rats. Negative polarity is
1500 ppm. 6 h/day. 5 days/week. 4 weeks. Eight toluene- downward. Vertical bar in the basal trace indicates 20 )J-V trough-to-
exposed (1498 ? 42 ppm) and control animals were used. peak amplitude.
132 CAMP0 ET AL.

80

FIG. 3. Noise spectrum (one-third octave bands) in the inhalation chambers. The overall SPL was 66 dB SPL.

thin layer of bone. The bony shell was then removed from on microscope slides. The organ of Corti (including hook) was
apex to base to isolate the entire organ of Corti. dissected away in three turns and mounted in glycerin as a
Light microscopy. The cochleae were dissected in 70% surface preparation for counting hair cells. The dissection
EtOH at room temperature. The trimmed tissue was mounted method was adapted from Bohne (4). Cytocochleograms were

- Mean thresholds obtained width 85 adult Long-Evans rats

t Mean behavioral thresholds obtained width 4 Long-Evans

hooded rats (Heffner et al.)

35

30

25
A...
..
20 ~..,
..
15 ...
...

10 :- ... .
. .
5 . . * -------.-----------..
. . .
. ., _.... ___.:-.
0 1 b
2 4 8 8 10 12 16 20 24 32
-5

1FREQUENCY (kHz) 1

FIG. 4. Preexposure mean auditory thresholds [brain stem (inferior colliculus)-evoked potentials] in the
adult Long-Evans rat. The squares represent the mean thresholds obtained with 85 adult Long-Evans
rats. The triangles represent the mean behavioral thresholds obtained with four Long-Evans hooded rats
[according to Heffner et al. (12)]. The bars represent the 95% confidence intervals.
TOLUENE-INDUCED HEARING LOSS 133

constructed from the surface preparation. The frequency- were averaged across each group of animals to compare
place map established by Mtiller (25) was used to superim- groups. This histological procedure was adapted from a
pose the frequency coordinates on the length coordinates of method described in Lataye and Campo (20).
the organ of Corti. Cells were counted as present if either the Scanning Electron Microscopy (SEM). The organ of Corti
stereocilia, the cuticular plate, or the cell nucleus could be vi- samples were dehydrated in ascending concentrations of EtOH
sualized. No attempt was made to assess the degree of possi- up to 100%. Following the dehydration, the samples were im-
ble cellular damage to surviving cells. A cochleogram showing mersed in hexamethyldisilazane (HMDS, Merck 804324) and
the percent hair cell loss as a function of distance from the placed in a vacuum drying chamber overnight. The dried speci-
base of the cochlea was plotted for each animal. The results mens were mounted on aluminium stubs using epoxy and

A A
30 --
TSI
25 -- - 2000 ppm
-. - 1750 ppm
+- 1500ppm
***
- 1250 ppm
20 --
-+- 1000 ppm
--o-- CTRL

15 --

10 --

5 --

0 --

-51 :
2 4 6 a 10 12 16 20 24 32

1FREQUENCY (kHz) 1

B A
30 --
TS2
26 -- - 2000 fswm
-* - 1750 bpm
- - 1500 ppm
-+- 1260 ppm
-e- 1000 ppm
--Q-- CTRL

-51 :
2 4 a a 12 b
IO 16 20 24 32

) FREQUENCY (kHz) 1

FIG. 5. (A) TS1 vs. frequencies obtained with toluene-treated animals. Confidence
intervals are left off for the sake of clarity. (B) TS2 vs. frequencies obtained with toluene-
treated animals. Confidence intervals are left off for the sake of clarity. 2000 ppm exposure
(4 n = 24) 1750ppm exposure (0, n = 8), 1500 ppm exposure (A, n = 8) 1250 ppm
exposure (X, n = 8) 1000 ppm exposure (0, n = 8). Control animals (0, n = 29). *The
difference between treated and control animals is significant at 95% at the mentioned
frequency. * A *The difference is significant at 99%.
134 CAMP0 ET AL.

OHC3
HAIR CELL LOSS /
IN PERCENT

0HC3
HAIR CELL LOSS /
7% 19%
IN PERCENT

HAIR CELL LOSS

IN PERCENT

0 3 6 9 (mm)
I ,I,, I I I l,llIIIIII
50 40 30 20 15 10 5 1 (kHz)
TOLUENE-INDUCED HEARING LOSS 135

75% 78%
HAIR CELL LOSS
IN PERCENT

50 - ,,/ // ,j. ,,I,

0 /I 1 iI 1 1 I 11
/
I
0 3 6 9 (mm)

50 40 30 20 15 10 5 1 (kHz1

OHC3 90 % 86 %
HAIR CELL LOSS
IN PERCENT

OHCZ

OHC3, 86 %
78 %
HAIR CELL LOSS
IN PERCENT

0 3 6 9 (mm)
I I I I I
II,, I I L II I, I / I, / / I
50 40 30 20 15 10 5 1 CkHz)
136 CAMP0 ET AL.

painted with conductive graphite. The samples were sputter

coated with gold to a depth of approximately 25 nm. The tis-
sues were viewed on a JEOL JSM 840A scanning electron mi-
croscope at an accelerating voltage of 15-20 kV.
Sernithin sections. The cochleae were dehydrated in a graded
series of EtOH, placed in propylene oxide, and finally infil-
trated with resin (Epon/Araldite). When the resin was poly-
merized, semithin sections (1 pm) were cut from the block
and stained with cresyl violet.

Statistical Analysis
Due to the size of several samples (n = 8 for 1750, 1500,
1250, and 1000 ppm), the test for normality (40) was not al-
lowed. Therefore, parametric tests were deemed not suitable
in the experimental context. Two main nonparametric tests
were used in the present investigation. The Mann-Whitney
test was used on unpaired samples for comparisons between
the control and the toluene-exposed group (control vs. TSl and
control vs. TS2) at each frequency. Friedmans test was used on FIG. 7. Scanning electron micrograph (G X 1200) of the 16 kHz
paired samples for comparison between TSl and TS2 within region of the organ of Corti from a control rat.
subject. Alpha levels of 0.05 (significant at 95%) and 0.01 (sig-
nificant at 99%) were used for the significance of the tests.

RESULTS as a function of the toluene concentrations tested. Each cyto-

Electrophysiological Data
The data in Fig. 4 show the ICP thresholds from the controls
and the toluene-exposed animals (n = 85) before exposure.
The greatest sensitivity (10 dB) appears at 10 kHz and increases
at higher and lower frequencies. The pattern of the curve
(squares) is quite similar to that obtained with Long-Evans
rats using a behavioral technique [triangles, data from Heffner et
al. (12)].
Figure SA and B shows, respectively, the TSI and TS2 values
obtained in the present experiment. Only three toluene con-
centrations (1500, 1750, and 2000 ppm) produce significant
auditory threshold shifts. With 2000 ppm. the peak of the TS2
amplitude shift appears at 16 kHz (23 dB), but a narrow plateau
(13.5-14.4 dB) can be observed around 8-12 kHz. With 1750
ppm, the peak of the TS2 amplitude appears also at 16 kHz
(14 dB), but there is no plateau @ = 0.01). At 1500 ppm. the
amplitude shift is relatively weak (4 dB) and restricted to 20
kHz (TSl: p = 0.057 ; TS2: p = 0.01).
Regardless of the dose of toluene considered, no significant
TS2 can be observed at 32 kHz. indicating no high-frequency
hearing loss. Similarly. no effects on TSl nor TS2 were found at
frequencies below 6 kHz. which indicates that the low-frequency
regions were also spared. Globally, there is no significant dif-
fcrcnce between IS1 and TS2 b (Z > 0.19) = 0.841.
Lighr rnicroscop~. The series of cytocochleograms shown
in Fig. hA-F illustrates the percentage of missing hair cells
and the progressive losses that occur along the organ of Corti
cochleogram is an average obtained with five animals.
From a general point of view, the most significant losses
appear at the level of the third row of outer hair cells
(OHC3). The second row (OHC2) is less damaged than the
third (OHC3), but more than the first (OHCl). The inner hair
cells (IHC) seem to be relatively well preserved. Two peaks of
OHC appear as a function of the toluene concentrations. The
first one is positioned at approximately 4 kHz, and the second
one within the frequency range 16-22 kHz. The maximum
percentage of missing hair cells for these two peaks are indi-
cated in Fig. 6B-F. Notice there is more damage in the 4-kHz
region compared to the 20-kHz region for all the toluene con-
centrations. Furthermore, the OHC losses obtained at 1750
and 2000 ppm are equivalent; there is no obvious difference
between these two cochleograms (Fig. 6E, F).
Finally, the average cytocochleogram for the control ani-
mals (Fig. 6A) is flat whatever the row of hair cells. Small
amounts of hair cell loss (less than 1% for any turn) can be
observed in control preparations.
Scanning electron microgruphy. Figure 7 (photo 3046) is a
scanning electron micrograph (G X 1200) of the organ of
Corti from a control rat. Three rows of OHC, and one row of
IHC are arranged in a regular fashion. This micrograph is
from the second turn and corresponds to the 16-kHz region of
the cochlea. Figure 8 (photo 3104) is a SEM micrograph (G X
1300) of the organ of Corti from a toluene (2000 ppm)-treated
rat. It can be observed here that the third row of OHCs in this
area (approximately 20 kHz) has been completely destroyed.
Phalangeal scars (PS) have replaced missing hair cells in the
first and second row. The IHC do not seem to be injured. Fig-
ure 9 (photo 3106) is a SEM micrograph (G X 1600) of the or-

FIG. 6. Average cochleogram obtained from Long-Evans rats. Abscissa-upper trace: length (mm) of the
entire spiral course of the organ of Corti from the hook; lower trace: frequency map according to Miillcr
(25). Ordinate: hair cell loss in percent. IHC: inner hair cells; OHCl: first row of outer hair cells; OHC2:
second row; OHC3: third row. (A) Control rats (n = 5); (B) rats exposed to toluene (1000 ppm, 6 h/day, 5
days/week, 4 weeks) (n = 5); (C) rats exposed at 12.50 ppm (n = 5); (D) 1500 ppm (n = 5); (E) 1750 ppm (n =
8); (F) 2000 ppm (rz = 8).
TOLUENE-INDUCED HEARING LOSS 137

FIG. 8. Scanning electron micrograph (G X 1300) of the 20 kHr FIG. 10. Low power view (G X 950) of the organ of Corti from a
region of the organ of Corti from a toluene (2000 ppm)-treated rat. toluene (1250 ppm)-treated rat. Arrowhead: mid-apical turn.
PS, phalangeal scars.
Figure 12A shows the intraganglionic bundle from a con-
trol rat. At the top right of the picture, the IHC is observable.
The myelin sheaths are stained by the 0S04 at the center of
gan of Corti from a toluene (2000 ppm)-treated rat. In this the picture. Figure 12B shows the intraganglionic bundle from
area, the third row of OHC has disappeared except two cells
(star in the figure) but the IHC are still present. The micro-
graph is from the mid-apical turn and corresponds to the 3-kHz
region. Figure 10 (photo 3194) is a low power (G X 950) view
of the organ of Corti from a toluene (1250 ppm)-treated rat.
From this picture, the trauma in the mid-apical turn (arrow
head) can be observed with regard to the apex of the cochlea.
Semithin sections. Figure 11A shows the organ of Corti
from a control rat. Some of the important structures such as
the outer and inner hair cells, the pillar cells. the Deiters
cells, the tectorial, and the basilar membrane are easily recog-
nizable. Figure 1 iB shows the organ of Corti from a toluene
(2000 ppm)-treated rat (upper turn). Neither OHC nor Deit-
ers cells are present. The nucleus of an OHC is still visible.
The IHC is preserved.

FIG. 11. (A) Light microscopy (G X 400) of an organ of Corti from

a control rat. OHC. outer hair cells: IHC, inner hair cell; P, pillar
cells; D. Deiters cells; T, tectorial membrane: HP, habenula
FIG. 9. Scanning electron micrograph (G X 1600) of the 3 kHz perforata: IB. intraganglionic bundles. BM, basilar membrane. (B)
region of the organ of Corti from a toluene (2000 ppm)-treated rat. j ,I Light microscopy (G X 400) of an organ of Corti from a toluene
Surviving cells. (2000 ppm)-treated rat (mid-apical turn).

FIG. 12. (A) Light microscopy (G x 400) of an intraganglionic
bundle from a control rat. (B) Light microscopy (G x 400) of an
intraganglionic bundle from a tolucne-treated rat (2000 ppm).
a toluene (2000 ppm)-treated rat (upper turn) showing the
lack of neural fibers. Neural fibers of the intraganglionic bun-
dle, probably efferent fibers, are missing.
DISCTJSSION
The pattern of the auditory threshold curve in nonexposed
animals (Fig. 4) is quite similar to that obtained with Long-
Evans rats using a behavioral technique (12). The shift between
the two curves is most likely due to the use of two techniques
(electrophysiological vs. behavioral) having different sensitivi-
ties. Due to the temporal integration, comments on the differ-
ence in absolute thresholds are risky because the signal duration
is shorter in ICP (54 ms) than in behavioral technique (400 ms).
As far as the cochlear trauma is concerned, the electro-
physiological data show that a concentration of at least 1500
ppm is necessary in our experimental conditions to obtain signifi-
cant auditory threshold shifts (TSI or TS2). The concentra-
tions of 1250 and 1000 ppm did not cause any detectable
threshold shifts. Given the histological data, the lack of
threshold shift with the two lowest concentrations of toluene
(1000 and 1250 ppm, 6 h/day, 5 days/week for 4 weeks) might
be due to the lack of sensitivity of the audiometric measures.
For concentrations higher than 1250 ppm, the results demon-
strate that toluene produces a significant hearing deficit in the
mid-frequency range (i.e., 8-24 kHz) of adult pigmented rats
CAMP0 ET AL.
(Fig. 5A, B). The effects of toluene reported here are consistent
with previous reports that positioned the toluene-induced
trauma at 8 and 16 kHz (6,15,32). The present results also
demonstrate a lack of observed effect at 32 kHz, clearly indi-
cating the lack of injury in the high-frequencvregion, as has been
previously reported (6). Furthermore, the mid-frequency effect
appears to be different from that generated by classical ototox-
icants. which first injure the high-frequency region (9,10,37,38).
NO statistically significant difference was obtained between
TSl and TS2 (Fig. SC). Therefore, no deterioration in hearing
over time has been established in our experimental conditions.
The histological data show that toluene has a toxic effect
on the cochlea. Both the hair cell loss and the lack of intragangli-
onic fibers observed in Fig. 12A and B confirms the strong
possibility of a primary cochlear injury as suspected by Johnson
and Canlon (16). More precisely, as was the case in the
present study, they demonstrate that outer hair cells are much
more sensitive to toluene than the inner hair cells (Figs. 6, 8,
11B). The damage to the outer hair cells increases from the
first to the third row. Surprisingly, the loss peak occurring at
4-kHz region (mid-low frequency) seems to be larger than the
peak at 16-20 kHz (mid-frequency). regardless of the row ob-
scrvcd (Fig. 6). The origin of this orderliness is still obscure,
and the results of the present study do not allow us to under-
stand the process of degeneration of the outer hair cells in
adult Long-Evans rats. Along the organ of Corti, toluene in-
duces a loss of outer hair cells in the middle and mid-apical
turns of the cochlea. More precisely. toluene-induced losses
concern mid-frequency (18-20 kHz) and mid- to low-fre-
quency (4 kHz) regions in adult Long-Evans rats. It should be
noted that in this discussion, the frequency range of 18-20
kHr is considered as mid-frequency. whereas other authors
considered it as high frequency (31.32). If most authors are in
agreement with the mid-frequency hearing losses (6.15). only
Sullivan et al. (41) have also mentioned mid- to low-frequency
losses in toluene-treated rats.
At first glance, the comparison between the electrophysio-
logical and histological data, or in other terms, the correlation
between function and structure. seems quite inconsistent at
the level of the mid- to low-frequency range. because large
hair cell losses have been obtained with only weak threshold
shifts below 4 kHI: (Fig. SA). These results could appear contra-
dictory, but there are observations that could explain these data.
It is important to know that the maximum density of affer-
ent neurons (around 3000 units) is positioned half-way be-
tween the base and apex of the basilar membrane, whereas
only 800 neural units come from the apical turn of the cochlea
in the rat (5,7,33,34). In addition to this anatomical observa-
tion, the anatomy and the physiology of the inferior colliculus
are particular in the rat. Indeed, within the inferior colliculus,
the volume of neural tissue that exhibits evoked neural activ-
ity is three to four times higher for high frequencies than it is
for low frequencies. In fact, the maximum level of neural ac-
tivity is reached at 32 kHz (17,36). Furthermore, some authors
think that the band-like responses of the inferior colliculus are
poorly induced by pure-tone stimuli < 5 or > SO kHz (28). In
the present article. audiometric data were obtained using ICP.
Therefore. the opportunity to record evoked neural activity
coming from the middle turn (mid-frequency area) is much
higher than that from the apical turn (low-frequency area) of
the cochlea.
Furthermore. a measure of the cochlear frequency sensitivity
can be obtained at low intensity (near the normal auditory,
threshold in noncxposed animals) from a restricted number ot
cochlear neurons tuned to the stimulus frequency. In the
TOLUENEPINDUCED HEARING LOSS 139

present study, the auditory thresholds obtained with nonex- low-frequency signals, so that the loss of apical hair cells has
posed animals are already higher than those obtained with a less effect on hearing than the loss of an equivalent number of
behavioral technique: around 20 dB at 2 kHz and 15 dB at 4 basal cells. Surprisingly, Prosen et al. (29) found that a heavy
kHz (Fig. 4). After cochlear trauma (e.g., the result of a tolu- loss of outer hair cells (up to 50%) in the apical 30% of the
ene exposure), the measurement of the frequency sensitivity cochlea does not cause hearing loss at any frequency. In the
requires a higher intensity (common situation after trauma) present study, that could partly explain why no threshold
than was previously the case to obtain the same value of shifts were obtained at 1000 and 1250 ppm.
threshold. However. in this last case the threshold may reflect Another possible explanation of the weak correlation be-
the neural activity of a larger number of fibers by the recruit- tween histological and electrophysiological data at 4 kHz could
ment of higher frequency fibers. Given the distribution of the be alterations in the mechanical behavior of the basilar mem-
afferent fibers along the organ of Corti (33-35). it is plausible brane after hair cell loss. Given the lack of outer hair cells in
that thresholds collected after trauma and obtained for low- the mid- and low-frequency regions (Fig. 6). the mechanical
frequency stimulus predominantly reflect the global activity behavior of the basilar membrane, and particularly the active
of a population of nerve fibers, rather than the restricted area phenomenon (42) in the mid and mid-apical turn of the co-
(apical turn) of the organ of Corti, which the experimenters chlea, should be dramatically modified. Indeed, it is well
hoped to test. In fact, the higher intensity may have overcome known that animals having extensive damage to the OHC
the auditory dysfunction produced by toluene. show large differences in the resonance frequency of the basilar
A third possible interpretation of these results might be membrane (l&42,46). Consequently, a modification of the
that the inferior colliculus is capable of enhancing the evoked basilar membrane tuning could explain the results reported in
response amplitudes when the auditory periphery is damaged the literature, even with a behavioral approach. Finally, it is
and more specifically in the low frequency (39). Indeed, previ- not unlikely that both assumptions could: at least partly, be
ous investigations have demonstrated enhancement of single the origin of the lack of hearing deficit observed in the mid- to
unit and gross potential activity in the inferior colliculus fol- low-frequency range with the electrophysiological technique.
lowing acoustic trauma (44). There is a plasticity of response Whatever the answer, the audiometric thresholds obtained after
properties of inferior colliculus neurons (43). It is possible toluene-induced hearing loss should be interpreted with cau-
that the inferior colliculus may enhance the evoked potential tion in terms of frequency localization.
amplitude of the low-frequency tones following a toluene-in-
duced hearing loss. CONCLUSIONS
The aforementioned hypotheses could explain the poor
correlations between hearing loss and hair cell loss. Regard- The results obtained in adult pigmented (Long-Evans)
less of the mechanism(s) involved, it is clear that the evoked rats show a cochlear toxicity induced by inhaled toluene. The
potentials from the central auditory pathway do not simply trauma is localized in the middle (1620 kHz) and the mid-apical
mirror changes that occur in the cochlea. It would be an error (4-S kHz) turn of the cochlea. Within the organ of Corti, the
to make conclusions concerning cochlear damage only with losses progress from the first to the third outer hair cell row.
brain stem (inferior colliculus) auditory-evoked potentials. Given the anatomical features of the rats organ of Corti, the
Obviously, this experimental bias could still be even more present electrophysiological data might be more a reflection
problematic in using auditory-evoked potentials recorded of the global. rather than restricted neural activity. The use of
from the vertex of the animal. One possibility for testing such BAER for estimating toluene-induced hearing loss needs
an assumption could be the use of a masking procedure, as de- caution in the interpretation of the audiometric data in rats.
tailed by Harrison et al. (11). However, such a procedure
might not be enough to detect the neural activity from the ACKNOWLEDGElEtiTS
low-frequency region because Prosen et al. (29) and more re-
The authors wish to thank Dr. K. Crofton for helpful discussions
cently Altschuler et al. (2). used it unsuccessfully with guinea and critiques of the manuscript and Dr. D. Henderson, R. Salvi, S.
pigs. According to these authors, fundamental differences exist McFadden. and R. Burkard from The Center for Hearing and Deaf-
in the processing of auditory signals between the apical and ness at SUNY Buffalo for their review. They also wish to thank the
basal turn of the cochlea (2,29). For instance. there is a redun- laboratory of biochemistry, P. Bonnet (INRSITIE), and S. Vessikre
dancy of encoding mechanisms in the mammalian cochlea for (INRSIESS) for their technical assistance during the experiments.

REFERENCES
I. Abbate, C.: Giorgianni. C.; Munao, F.; Brecciaroli, R.: Neurotox- 6. Crofton, K.: Lassiter, T.; Rebert, C.: Solvent-induced ototoxicity
icity induced by exposure to toluene. Int. Arch. Occup. Environ. in rats: An atypical selective mid-frequency hearing deficit. Hear.
Health 64:38Y-392: lYY3. Res. 80:25-30; 1994.
2. Altschuler, R.; Raphael. Y.: Prosen. C.: Dolan, D.; Moody. D.: Dannhof. B.: Bruns, V.: The innervation of the organ of Corti in
Acoustic stimulation and overstimulation in the cochlea: A com- the rat. Hear. Res. 66:X-22; lYY3.
parison between basal and apical turns of the cochlea. In: Dancer. Deschamps. D.: Gamier, R.: Lille, F.: Tran Dinh. Y.: Evoked poten-
A.,: Henderson, D.: Salvi. R.: Hamernik. R.. eds. Noise-induced tials and cerebral blood flow in solvent induced psycho-organic syn-
hearing loss. St. Louis: Mosby Year Book; 1992:6@72. drome. Br. J. Ind. Med. 50~325-330: 1993.
7 BergstrGm, B.; Nystriim. B.: Development
_. of hearing loss during Forge, A.; Richardson, G.: Freeze fracture analysis of apical mem-
long-term exposure to occupational noise. Stand. Audiol. 15:227- branes in cochlear cultures: Differences between basal and apical-
234: 1986. coil outer hair cells and effects of neomycin. J. Neurocytol. 22:854-
4. Bohne, B. A.: Location of small cochlear lesions by phase contrast X67: 1993.
microscopy prior to thin sectioning. Laryngoscope 82:1-16: 1972. 10. Govaerts. P.; Claes. J.; Van de Heyning, P.: Jorens. P.: Minireviewi
.5 Burda, H.: Ballast. L.: Bruns, V.: Cochlea in old world mice and Aminoglycoside-induced ototoxicity. Toxicol. Lett. 52:227-251:
rats (Muridae). J. Morphol. lY8:269-285; 1988. l9YO.
140 CAMP0 ET AL.

Il. Harrison, R.; Aran, J. M.: Erre, J.: AP tuning curves from normal 29. Prosen, C. A.: Moody, D.; Stebbins. W.; Smith, D.; Sommers. M.;
and pathologic human and guinea pig cochlea. J. Acoust. Sot. Brown, N: Altschuler, R.: Hawkings. R.: Apical hair cells and
Am. 6911374-1385; 1981. hearing. Hear. Res. 44:179-194; 1990.
12. Heffner, H.: Heffner. R.; Contos, C.; Ott. T.: Audiogram of the 30. Pryor, Ci.; Dickinson, J.; Howd, A.; Rebert, C.: Neurobehavioral
hooded Norway rat. Hear. Res. 73:244-247: 1994. effects of subchronic exposure of weanling rats to toiuene or hexane.
13. Henderson. D.; Hamernik, R.: Woodford, C.: Sitler. R.: Salvi, R.: Neurobehav. Toxicol. Teratol. S:47-52; 1983.
Evoked-response audibility curve of the chinchilla. J. Acoust. 31. Pryor, G.: Dickinson, J.; Howd, A.; Rebert, C.: Transient cogni-
Sot. Am. 54:1099-l 101: 1973. tive deficits and high-frequency hearing loss in weanling rats
14. Hormes. J. T.: Filley. C. M.; Rosenberg. N. L.: Neurologic exposed to toluene. Neurobehav. Toxicol. Tcratol. 5:53-57: 1983.
sequelae of chronic solvent vapor abuse. Neurology X6:698-702; 32. Pryor, G.: Dickinson, J.; Feeney, E.; Rebert, C.: Hearing loss in
1986. rats first exposed toluene as weanlings or as young adults. Neu-
15. Johnson, A.: Canlon, B.: Progressive hair cell loss induced by tol- robehav. Toxicol. Teratol. 6:ll l-l 19: 1984.
uene exposure. Stand. Audiol. Suppl. 39:1-9; 1993. 33. Roth, B.: Bruns. V.: Postnatal development of the rat organ of
16. Johnson, A.; Canlon, B.: Toluene exposure affects the functional Corti. I. Anat. Embryo]. 185:559-569; 1992.
activity of the outer hair cells. Hear. Res. 72:189-196; 1994. 34. Roth, B.; Bruns, V.: Postnatal development of the rat organ of
17. Keithley, E.; Lo, .I.; Ryan, A.: 2.Desoxyglucose uptake patterns Corti. II Anat. Embryo]. 185:571-581; 1992.
in response to pure tone stimuli in the aged rat inferior colliculus. 35. Roth, B.: Bruns, V.: Late developmental changes of the innerva-
Hear. Res. 80:79-85; 1994. tion densities of the myelined fibers and the outer hair cell effer-
18. Kellv, J. B.; Khanna, S.: Ultrastructural damage in cochleas used for ent fibres in the rat cochlea. Anat. Embryo]. 187:565-571; 1993.
studies of basilar membrane mechanics. Hear. Res. 14:59-78; 1984. 36. Ryan, A.: Furlow, Z.; Woolf, N.; Keithley. E.: The spatial repre-
19. Kirk-Othmer, R.: Encyclopedia of chemical technology (3rd ed.). sentation of frequency in the rat dorsal cochlear and inferior col-
Economic Aspects 23:257-271; 1983. liculus. Hear. Res. 36:181-190; 1988.
20. Lataye, R.; Campo, P.: Applicability of the Leq as a damage-risk 37. Rybak, L.: Ototoxic mechanisms. In: Altschuler, R., ed. Neurobi-
criterion. An animal experiment. J. Acoust. Sot. Am. 99(3):1621- ology of hearing. New York: Raven Press; 1986:441454.
1632; 1996. 38. Rybak, L.: Hearing: The effects of chemicals. Otolaryngol. Head.
21. Lazar, R.; Ho, S.; Melen, 0.; Daghestani, A.: Multifocal central Surg. 106:677-681; 1992.
nervous system damage caused by toluene abuse. Neurology 39. Salvi, R.; Saunders, S.; Gratton, M.; Arehole, S.; Powers, N.:
33:1337-40; 1983. Enhanced evoked response amplitudes in the inferior colliculus of
22. Mattsson, J.; Gorzinski, S.; Albee, R.; Zimmer, A.: Evoked poten- the chinchilla following acoustic trauma. Hear. Res. 50:245-258;
tial changes from 13 weeks of simulated toluene abuse in rats. 1990.
Pharmacol. Biochem. Behav. 36:683-689; 1991. 40. Shapiro, S.; Wilk, M.: A comparative study of various tests for
23. Morata, T.; Dunn, D.; Sieber, W.: Occupational exposure to noise normality. J. Acoust. Sot. Am. 63:1343-72; 1968.
and ototoxic organic solvents. Arch. Environ. Health 49:359-365; 41. Sullivan, M.; Rarey, K.: Conolly, R.: Ototoxicity of toluene in
1994. rats. Neurotoxicol. Teratol. 10525-530; 1989.
24. Morata, T.; Nylen, P.; Johnson, A.; Dunn, D.: Auditory and vesti- 42. Ulfendahl, M.; Khanna, S.; Lofstrand, P.: Changes in the mechan-
bular functions after single or combined exposure to toluene: A ical tuning characteristics of the hearing organ following acoustic
review. Arch. Toxicol. 69:431443; 1995. overstimulation. Eur. J. Neurosci. 5:713-723; 1993.
25. Mtiller, M.: Frequency representation in the rat cochlea. Hear. 43. Wang, J.; Salvi, R.; Powers, N.: Plasticity of response properties
Res. 511247-254; 1991. of inferior colliculus neurons following acute cochlear damage. J.
26. Odkvist, L.; Bergholtz, L.; Ahlfeldt, H.; Andersson, B.: Oto- Neurophys. 75(1):1-13; 1996.
neurological and audiological findings in workers exposed to 44. Willott, J.: Lu, S. M.: Noise-induced hearing loss can alter neural
industrial solvents. Acta Otolaryngol. Suppl. 386:249-251; 1982. activity and increase excitability in the CNS. Science 216:1331-
27. Orbaek, P.; Nise, G.: Neurasthenic complaints and Psychometric 1332; 1982.
function of toluene-exposed rotogravure printers. Am. J. Ind. 45. Yamanouchi. N.; Okada, S.; Kodama, K.; Hirai, S.; Sekine, H.:
Med. 16:67-77; 1989. White matter changes caused by chronic solvent abuse. Am. J.
28. Pierson, M.; Snyder-Keller, A.: Development of frequency- selective Neuroradiol. 16:1643-1649; 1995.
domains in inferior colliculus of normal and neonatally noise- 46. Zenner, H.; Ernst, A.: Sound preprocessing by ac and dc move-
exposed rats. Brain Res. 6365567: 1994. ments of cochlear outer hair cells. Prog. Brain Res. 97:21-30; 1993.