The Role of Antioxidants in Protection

from Impulse Noise

D. HENDERSON,a S. L. MCFADDEN, C. C. LIU, N. HIGHT,
AND X. Y. ZHENG
Center for Hearing and Deafness, State University of New York at Buffalo, Hearing
Research Laboratory, 215 Parker Hall, Buffalo, New York 14214, USA

ABSTRACT: The hearing loss from exposure to noise and ototoxic drugs share
a number of audiological and pathological similarities. Recent research has
shown that reactive oxygen species (ROS) may be a common factor in both
noise- and drug-induced hearing loss. This review describes three experiments
that point to ROS as a causative factor in both noise- and drug-induced hearing
loss and antioxidants as a protective agent. In the first experiment, the ears of
chinchillas were treated with R-N6-phenylisopropyladenoisine (R-PIA) and exposed to 150-dB impulse noise. The treated ears developed substantially less
permanent hearing loss (PTS) and hair cell loss than the untreated ears. One
interpretation of this experiment is that R-PIA increases the availability of glutathione (GSH). In the second experiment, the role of GSH was specifically examined. The ears of chinchillas were treated with glutathione monoethylester
(GEE), a pro-GSH drug that has been shown to readily cross cell membranes
and increase GSH levels. The GEE-treated ears had significantly less PTS and
hair-cell loss than the nontreated ear. Previous research has shown that moderate levels of noise exposure can increase a subjects resistance to noise, and also
increase the availability of antioxidant enzymes in the cochlea. In the third experiment, chinchillas were given a series of toughening exposures (i.e., 6 h of
a 0.5-kHz OB noise at 95 dB for 10 days). After the series of toughening exposures, the subjects were treated with carboplatin, a drug that causes massive inner-hair-cell lesions in the chinchilla. The animals receiving the 10-day toughening exposure developed less PTS and hair-cell loss than the control animals.

INTRODUCTION
The pattern of hearing loss and cochlear pathology after exposure to traumatic
noise or ototoxic drugs such as aminoglycosides or cisplatin can be quite similar. In
fact, Hawkins8 has pointed out that it is impossible to identify the causative agent
from knowing either the audiogram or the pattern of hair cell loss. Twenty-five years
later, we are now learning that noise-induced and drug-induced hearing loss could
share a common damaging agent, specifically toxic free-oxygen radicals (FOR) and
other reactive oxygen species (ROS). The direct evidence for free-radical generation
and damage in the cochlea is sparse,2,16,29,30 but free-radical damage is a reasonable
hypothesis that is supported both by in vitro studies of FOR in cochlear cultures treata
To whom correspondence may be addressed. Phone: 716/829-2001; fax: 716/829-2980; email: donaldhe@acsu.buffalo.edu

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ed with cisplatin18,24,25 and by indirect evidence showing the protective effects of antioxidant enzymes.7,11,26,27
High levels of noise are likely to create damaging free radicals by at least two
pathways. First, high levels of noise tax the mitochondrial respiratory process leading
to free-radical generation.14,23 Second, noise exposures lead to cochlea ischemia with
the consequence of the excitotoxic reaction at both the inner hair cell/VIII nerve
synapse22,28 and free radical generation at the surface of stria vascularis.29,30 Yamane
and colleagues exposed guinea pigs to high-level noise and assessed strial blood flow
and the presence of FOR, by using 3,3-diaminobenzidine to react with the O2 radical. When guinea pigs were sacrificed immediately after the exposure, there was a
sharp reduction in cochlear blood flow and a strong presence of O2 at the scala media surface of the stria. By 2 h after the exposure, there was evidence of cochlear
blood flow returning to normal, and most of the free radical activity subsided. The
Yamane study is provocative, but its scope is limited because it only measures O2
when the OH or ONOO radicals are potentially more toxic,4,5 and it focuses on the
medial wall of the stria vascularis and does not examine the presence of free radicals
in the organ of Corti.

ANTIOXIDANT ENZYMES AND SUSCEPTIBILITY

The evidence for free-radical activity in the cochlea following exposure to noise
or ototoxic drugs is limited because of the short, fragile life span of ROS, as well as
the difficult access to the functioning cochlea. The evidence for antioxidant enzymes
as a protective factor in noise-induced hearing loss (NIHL), however, is much
stronger and comes from two lines of research. First are studies of the toughening
phenomenon, whereby the ear is made more resistant to the temporary (TTS) and
permanent (PTS) effects of noise as a consequence of prior exposure to a series of
nontraumatic noise exposures.3 Over a series of 6-h/day exposures for 10 days, the
TTS after one or two days is at its maximum level, and with repeated exposures, the
amount of TTS decreases by as much as 20 to 30 dB. More importantly, after the ear
has completely recovered, it is more resistant to the permanent threshold shifts associated with exposure to even higher levels of noise. Recently, we discovered15 that
noise exposure is associated with significant increases in activity levels of endogenous antioxidant enzymes. Catalase, glutathione reductase, and -glutamyl cysteine
synthtase (GCS) were all increased in concentration in both the organ of Corti and
stria vascularis following a potentially traumatic noise exposure, but the largest increase was found in ears that were previously exposed to the prophylactic series of
toughening exposures. Thus, one possible interpretation of these results is that the increased resistance to noise associated with the toughening phenomenon is a reflection of an increased effectiveness of the endogenous antioxidant system.
The second line of evidence that points to antioxidant activity as a factor in reducing the effects of noise comes from studies of direct pharmacological intervention.27
In our laboratory, Hu et al.13 used R-N6-phenylisopropyladenoisine (R-PIA), an
adenosine receptor agonist, as a prophylactic treatment before exposures to noise.
Briefly, chinchillas were anesthetized and both bullae were opened. A drop of saline
was placed on one round window and R-PIA (60 l of 103-M solution) was applied

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to the other round window. The drug and saline were wicked off the round windows
after 30 min. One hour after the initial dose of R-PIA the chinchillas were exposed to
a 4-kHz octave band noise at 105 dB for 4 h. The R-PIA-treated ears recovered faster
and more completely (FIG. 1A), and had smaller hair cell lesions (FIG. 1B) than control ears. In a second complementary experiment, the same procedures were used, except the experimental treatment was buthionine-sulfoximine (BSO), a drug that
specifically inhibits GCS, the rate-limiting enzyme in the synthesis of GSH.10 There
were no differences between the ears treated with the small dose of BSO and the
saline-treated control ears immediately after the exposure. However, at 4 days postex-

(A)

(B)
FIGURE 1. (A) Evoked potential recovery curves following exposure to 4 kHz OBN. (B) Average (N = 8) OHC (top) and IHC (bottom) lesions.

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posure, differences were apparent in three different measures obtained from separate
groups of animals: TS, GSH levels in the hair cells (assessed by mercury orange
staining of intracellular thiols), and specific activity levels of GCS. As shown in FIGURE 2, the difference in average TS between the ears ranged from approximately 5 dB
to nearly 20 dB. Presumably, TS differences were more pronounced at higher frequencies simply because the concentration of BSO was greater in the basal region of
the cochlea due to diffusion gradients from the round window. Data from histochemical staining of cochleas with mercury orange, and from biochemical analysis of
GCS activity in noise-exposed cochleas supported the physiological findings at 4
days postexposure. FIGURE 3 shows GCS activity levels determined at various times
after noise exposure in BSO- and saline-treated ears. The values shown are averages
determined from ears of three animals at each time point, and are expressed in units
per mg of protein. Separate analyses were performed for organ of Corti and stria vascularis fractions. At 4 days (96 h) postexposure, ears treated with BSO had lower
GCS levels than control ears in both stria and organ of Corti fractions. In the organ of
Corti fraction (FIG. 3, left panel), GCS level in the BSO-treated ears was 43% that in
the control ears. In the stria fraction (FIG. 3, right panel), GCS specific activity in the
BSO-treated ears was 61% that in the control ears. Mercury orange staining confirmed that GSH levels were reduced in BSO-treated ears relative to control ears.
OHCs of BSO-treated ears showed much less intense staining and more cell edema
and distortion of cell bodies than OHCs of control ears.
Despite the clear differences between BSO- and saline-treated ears at 4 days postexposure, there were no significant differences between ears in either PTS or hair cell
loss at 20 days postexposure. The lack of a permanent effect of BSO treatment in this
experiment is most likely due to the small dose of BSO used, and the short amount of
time it was allowed to penetrate into the cochlea. Nevertheless, the results show that

FIGURE 2. Average threshold shifts at 0, 4, and 20 days exposure for BSO and saline-treated
ears.

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FIGURE 3. GCS concentration in stria and organ of Corti following noise exposure in BSO
and control ears.

treatment with a drug that inhibits GSH synthesis alters the pattern of recovery from
a traumatic noise exposure. The significant protection afforded by R-PIA and the altered pattern of recovery with BSO is consistent with the hypothesis that NIHL is
partially related to the action of toxic free radicals. The results raise the possibility of
reducing the degree of NIHL by either increasing the endogenous level of the antioxidant enzymes by certain prophylactic exposures or by direct drug intervention.

PROTECTION FROM IMPULSE/IMPACT NOISE

The positive results of protection from continuous noise raise the issue of whether
ROS toxicity also plays a role in the hearing loss caused by high levels of impulse
noise. Impulse noise, such as gunfire or explosives, creates direct and immediate mechanical damage in the cochlea (FIG. 4). The cochlear pathology can include a disruption of the mechanical support of the pillar cells, ripping apart the tight cell junctions, and, in the extreme, the separation of the organ of Corti from the basilar membrane. The mechanical-induced pathologies are accompanied by a complicated pattern of threshold shift (FIG. 4B). Immediately after the exposure, there is an initial
recovery of hearing sensitivity. However, after 1 to 4 h the recovery process stops and
hearing loss begins to grow again. At 8 to 12 h recovery may begin again and proceeds over several days to a stable hearing loss or full recovery of sensitivity.
The first clue that ROS toxicity can be a factor in the ears response to impulse
noise comes from a toughening experiment by Henselman et al.9 In this experiment,
chinchillas were given a standard prophylactic toughening exposure (95 dB of 0.5kHz octave band noise for 6 h/day for 10 days). Five days after the last exposure,
when the subjects had completely recovered from TTS, they were exposed to 50 pairs
of 150-dB pSPL impulses that had a waveform that mimicked an M16 rifle sound.
Chinchillas who had the toughening exposure developed significantly less TTS immediately after the exposure, and 10 to 25 dB less PTS 30 days later (FIG. 5) and significantly less hair cell loss. The magnitude of protection provided by the toughening
exposure is one of the largest effects seen in our series of toughening experiments.
Since the prophylactic exposures are the same as the exposures in the Jacono et al.

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FIGURE 4. Scanning electron micrograph of chinchilla cochlea following exposure to 155dB impulses. Note cleft between pillar cells and OHC region.

experiment,15 a reasonable hypothesis is that the protection received by the toughened group is related to the increased presence of antioxidant enzymes.
Experiment I: Intervention with R-PIA
The first experiment was designed to test whether the presence of R-PIA renders
the ear more resistant to the mechanical damage associated with impulse noise. A
group of chinchillas (N = 6) were prepared for evoked potential recording by surgically implanting an electrode in the left and right inferior colliculus. Before exposure
to impulse noise, each chinchilla was anesthetized, both bullae were opened, and RPIA (103 M) was placed on the right round window membrane. Saline was placed on
the left round window in three animals, and nothing was done to the left ear of three
animals. Baseline audiograms were measured after the R-PIA was applied but before
the noise exposure. The animals were surgically repaired and allowed to recover for
several hours. When the animals were awake, they were exposed to 50 pairs of 150dB pSPL impulses. Hearing function was monitored after the exposure, and 20 days
later the animals were sacrificed and the cochleas were analyzed. As seen in FIGURE
6, the R-PIA-treated ears had less TTS immediately after the exposure and they recovered more completely. FIGURE 7 shows the R-PIA ear/control ear difference in
PTS at each frequency tested. The R-PIA-treated ears had less PTS at all frequencies.
The differences ranged from approximately 6 dB to 13 dB on average. The protection
against hair cell loss is seen in FIGURE 8. The R-PIA-treated ears have significantly
fewer missing IHCs (FIG. 8A) and OHCs (FIG. 8B).
The protection afforded by the R-PIA treatment may be due to several factors, as
R-PIA has multiple effects. It is an adenosine agonist; it is an inhibitor of glutamate
production; and it is a promoter of blood flow through the stimulation of nitric oxide
(NO) production.19 Each of these effects can influence the degree of hearing loss

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FIGURE 5. Permanent threshold shifts for chinchillas exposed to either 150-dB impulses ()
or 10 days conditioning exposure and 150-dB impulses ().

caused by exposure to noise. For example, the activation of the adenosine receptor
can lead to an increase in GSH and a resultant decrease in FOR.2,6 High levels of
stimulation lead to glutamate excitotoxicity reaction/edema at the postsynaptic membrane of the VIII nerve. R-PIA partially suppresses glutamate synthesis. Finally, high
levels of noise exposure lead to cochlear ischemia.29,30 R-PIA may partially counteract the ischemia by the stimulation of NO that promotes local blood flow.21
Experiment II: Reduction of NIHL with GSH Monoethyl Ester
Each of the three mechanisms described earlier could possibly reduce the effects
of noise. A second experiment was designed to evaluate whether the protective effects were related to increased availability of GSH. Glutathione is synthesized in two
enzyme-catalyzed steps from glutamate, cysteine, and glycine:

-glutamylcysteine synthetase

and

-glutamyl-L-cysteine + ADP + Pi

1. L-glutamate + L-cysteine +ATP

glutathione synthetase
2. L--glutamyl-L-cysteine + glycine + ATP

glutathione + ADP + Pi.

Cellular glutathione levels are regulated by feedback inhibition of -glutamyl cys-

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FIGURE 6. Evoked potential recovery curves following exposure to 150-dB impulses in RPIA and control ears.

FIGURE 7. Permanent threshold differences between R-PIA and control ears.

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(A)

(B)

FIGURE 8. Cochleograms showing IHC (A) and OHC (B) losses for R-PIA treated and control ears.

teine synthtase. GCS normally functions at about 20% of its maximal activity, because it is feedback-inhibited by glutathione. GSH disappears rapidly from plasma
and is not readily transported across the cell membrane.1 Consequently, attempts to
upregulate endogenous levels of GSH directly are often ineffective. A more effective
way to increase GSH levels is to provide a derivative of glutathione that is easily
transported into cells, and the most promising of these is the monoethyl ester of GSH
(GSHEE). GSHEE is rapidly deesterified by intracellular estrase to GSH, and because
it does not involve the enzymes of glutathione it does not require the expenditure of
cellular energy. GSHEE is able to bypass the normal GSH pathway because it does not
involve glutathione synthesis and is not subject to GSH feedback inhibition, so that
intracellular levels can be elevated significantly.20
The chinchillas were assessed audiometrically prior to treatment. The animals
were anesthetized, and the right bulla was surgically opened to provide access to the
round-window membrane. Approximately 60 L of GSHEE was applied to the round
window, and the area was surgically closed approximately 15 min later. The animals
were exposed to 50 pairs of 150-dB pSPL impulse noise simulating gunfire bursts.
FIGURE 9 shows the permanent hearing loss (21 days postexposure) difference between treated and nontreated ears. The amount of PTS seen in the GSHEE-treated ears
was significantly less than the control ears. FIGURE 10 shows the difference in IHC
(FIG. 10A) and OHC (FIG. 10B) losses between the control and the GSHEE-treated
ears. The difference is quite dramatic, with the GSHEE-treated ears exhibiting almost
no damage, while the control ears show marked hair cell damage despite nearly normal thresholds. It must be remembered that the GSHEE experiment is a pilot project,
and based on only three animals, but the results, particularly the hair cell pathology,
are quite dramatic.
The first two experiments convincingly demonstrate that prior treatment with
GSH-promoting drugs (R-PIA and GSHEE) can dramatically reduce the effects of
noise. The results are strong, circumstantial evidence that antioxidant enzymes can
serve as a protective agent against NIHL.

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FIGURE 9. Permanent threshold differences between GSHEE and control ears after exposure
to 150-dB impulses.

Experiment III: Conditioning-Induced Protection from Carboplatin

The third experiment begins to explore the concept of a common etiology for
noise and certain ototoxic drugs. Experiments by Rybak et al.25,26 have shown that
the effects of cisplatin can be partially prevented by prior application of antioxidants
such as diethyldithiocarbamate and 4-methylthiobenzoic acid. Experiments by Van
De Waters group17 have shown that the effects of cisplatin on a cochlear organ culture can be prevented with R-PIA alone or in combination with procysteine drugs.
When reviewing the recent literature on both NIHL and ototoxicity, it is apparent that
antioxidant drugs can have a protective effect. Given that Jacono et al.s experiment15
showed that GHSR, GCS, and catalase are upregulated with a series of conditioning

(A)

(B)

FIGURE 10. Cochleogram showing IHC losses for (A) GSHEE and (B) control ears.

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exposures, it is interesting to see if the prophylactic effects of the conditioning exposure extend to protecting the ear from platinum-based ototoxic drugs.
Six chinchillas were made monaural and a recording electrode was permanently
placed in the inferior colliculus. Two weeks after the initial surgery the subjects
hearing thresholds were estimated using evoked potentials recorded from the awake
animal. The conditioning noise exposure was a series of exposures to 95-dB SPL 0.5
kHz OBN for 6 h/day for 10 days. This is the same conditioning paradigm that was
shown to be effective in reducing PTS and hair cell loss from impulse noise (see reference 9 and FIG. 5). The chinchillas daily hearing loss dropped between the first
and the fifth day of conditioning and then plateaued. After the last conditioning exposure, the animals were rested in quiet for 5 days and then were given carboplatin
(63 mg/kg IP) on two consecutive days.
The magnitude of the hearing loss 20 days after the exposure was minimal. As
shown in FIGURE 11, however, the animals who had the conditioning exposure before
the dose of carboplatin developed substantially less IHC as well as OHC loss, compared to nonexposed chinchillas given the same dose of carboplatin in a previous
study.12 Hofstetters animals12 had a mean IHC loss of 8090% throughout the

(A)

(B)

FIGURE 11. Cochleogram showing IHC (A) and OHC (B) for chinchilla given carboplatin or
a conditioning noise exposure and carboplatin.

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cochlea and OHC losses averaging approximately 20%. In contrast, the six conditioned chinchillas had much less IHC loss in the apex than in the base, and average
OHC losses of less than 10%. It is important to note that the results are preliminary.
However, the initial findings provide support for the notion that a common etiological mechanism underlies damage caused by noise and by carboplatin. The likely
common feature leading to pathology is an increase in the free-radical population in
the cochlea, even though the source of the free radicals may be completely different.
For example, noise exposure is likely to lead to free-radical production by taxing the
mitochondrial system, by ischemia, or by rupturing cell membranes. The effects of
carboplatin are complicated. Studies with a related platinum-based anticancer compound, cisplatin, have shown that cisplatin treatment leads to an increase in superoxide dismutase (SOD), catalase, and malondialdehyde activity in the cochlea, but a
suppression of GSH, GSH-peroxidase, and GSH-reductase activity levels.24 Thus,
the critical factor may be the ratio between FOR/GSH and other antioxidants. The intervention with R-PIA or GSHEE may have a direct effect on this equation by simply
making more GSH available.

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