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Free radical formation in the cochlea plays a key role in the development of noise-induced hearing loss (NIHL). The amount, distribution, and time course of free radical formation have been defined, including a clinically significant formation of both reactive oxygen species and reactive nitrogen species 7-10 days after noise exposure. Reduction in cochlear blood flow as a result of free radical formation has also been described. Here we report that the antioxidant agents vitamins A, C, and E act in synergy with magnesium to effectively prevent noise-induced trauma. Neither the antioxidant agents nor the magnesium reliably reduced NIHL or sensory cell death with the doses we used when these agents were delivered alone. In combination, however, they were highly effective in reducing both hearing loss and cell death even with treatment initiated just 1 h before noise exposure. This study supports roles for both free radical formation and noise-induced vasoconstriction in the onset and progression of NIHL. Identification of this safe and effective antioxidant intervention that attenuates NIHL provides a compelling rationale for human trials in which free radical scavengers are used to eliminate this single major cause of acquired hearing loss.
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Free radical scavengers, vitamins A, C, and E, plus magnesium
reduces noise trauma
Colleen G. Le Prella, Larry F. Hughesb, and Josef M. Millera,c
a Kresge Hearing Research Institute, University of Michigan, Ann Arbor, MI, USA
b Department of Surgery/Otolaryngology, Southern Illinois University Medical School, Springfield, IL, USA
c Center for Hearing and Communication, Karolinska Institutet, Stockholm, Sweden
Abstract
Free radical formation in the cochlea plays a key role in the development of noise-induced hearing
loss (NIHL). The amount, distribution, and time course of free radical formation have been defined,
including a clinically significant formation of both reactive oxygen species and reactive nitrogen
species 7–10 days following noise exposure. Reduction in cochlear blood flow as a result of free
radical formation has also been described. Here we report that the antioxidant agents, vitamins A, C,
and E, act in synergy with magnesium to effectively prevent noise-induced trauma. Neither the
antioxidant agents nor magnesium reliably reduced NIHL or sensory cell death with the doses we
used when these agents were delivered alone. In combination, however, they were highly effective
in reducing both hearing loss and cell death even with treatment initiated just one hour prior to noise
exposure. This study supports roles for both free radical formation and noise-induced
vasoconstriction in the onset and progression of NIHL. Identification of this safe and effective
antioxidant intervention that attenuates NIHL provides a compelling rationale for human trials in
which free radical scavengers are used to eliminate this single major cause of acquired hearing loss.
Keywords
cochlea; free radical; noise; hearing; antioxidant; vasodilation
Mechanical destruction of cells in the organ of Corti was once assumed to be the primary cause
of noise-induced hearing loss (NIHL) [1–8], with perhaps some effect of reduced blood flow
to the inner ear [9–18]. We now know that another key factor is intense metabolic activity that
results in production of excess free radicals [19–23] and lipid peroxidation products [24].
Noise-induced production of reactive oxygen species (ROS) in the cochlea has now been well
characterized, and several recent reviews are available [25–27]. Mitochodrial dysfunction and
ROS production have been implicated in numerous neurodegenerative syndromes and diseases
[28–34, see 35,36]. The use of antioxidant agents holds significant therapeutic promise for
many neurodegenerative processes [32,33,37–42], and there is some suggestion that
Correspondence to: C. G. Le Prell, Kresge Hearing Research Institute, University of Michigan, 1301 East Ann Street, Ann Arbor, MI
48109-0506, USA. Tel: +1-734-763-5104; fax +1-734-764-0014. E-mail address: colleeng@umich.edu.
Disclosure. Dr. Miller is a founding member and Chairman of the Board of Directors at OtoMedicine, Inc., a company that has an option
to license the intellectual property described in this report. Dr. Le Prell is a paid consultant to OtoMedicine, Inc. Drs. Miller and Le Prell
have disclosed these relationships to the Medical School Conflict of Interest Board at the University of Michigan; conflict management
plans including semi-annual review by the Board are in place.
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Author Manuscript
Free Radic Biol Med. Author manuscript; available in PMC 2007 August 20.
Published in final edited form as:
Free Radic Biol Med. 2007 May 1; 42(9): 1454–1463.
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combinations of antioxidants are more effective than single agents [43]. The similarity of free
radical production across multiple neurodegenerative diseases, including NIHL, and the
putative efficacy of antioxidants in reducing neurodegenerative processes, provides a
compelling rationale for study of antioxidants to prevent NIHL.
Consistent with antioxidant protection in neurodegenerative-cell death, many antioxidants are
well known to partially protect sensory cells in the organ of Corti from stress-induced
destruction. Upregulation of the endogenous antioxidant glutathione reduces NIHL and cell
pathology [44,45], whereas the opposite is observed with reduced endogenous antioxidants
[20,22,23,44]. That exogenous antioxidant agents reduce sensory cell death and NIHL has been
well demonstrated in animal studies using a variety of free radical scavengers [24,44–56],
including several studies with dietary antioxidants [50,57–60]. Magnesium supplements also
reduce NIHL [61–69].
Given the potential for synergy of multiple agents in protection from neurodegenerative-cell
death, in this investigation, we evaluated protection from NIHL and noise-induced sensory cell
death with the combination of vitamins A, C, and E, or magnesium, or vitamins A, C, and E,
plus magnesium. Each of these agents has a distinct mechanism of action. The primary
antioxidant action of β-carotene (metabolized to vitamin A in vivo) is to scavenge singlet
oxygen; because singlet oxygen reacts with lipids to form lipid hydroperoxides, the removal
of singlet oxygen prevents lipid peroxidation [for review, see 70]. Vitamin E, present in lipids
in cells [see 71], is a donor antioxidant that reacts with and reduces peroxyl radicals and, thus,
inhibits the propagation cycle of lipid peroxidation [for review, see 70]. Vitamin C detoxifies
free radicals by reducing them [for review, see 72]. Scavenging of oxygen radicals by vitamin
C occurs in the aqueous phase [73,74], which is in contrast to the site of action of vitamin E,
within membranes. Given differences in mechanism and site of action, there are differences in
antioxidant free radical scavenging ability [70,75] as well as synergistic interaction of
hydrophilic and lipophilic antioxidants [73,74,76–78]. Antioxidant scavenging of ROS also
potentially reduces vasoconstriction that occurs with ROS production [46, for review see 27].
One effect of magnesium is reduction of noise-induced vasoconstriction [67,79–81].
Demonstration of an additive efficacy of these agents would provide greater insight into their
mechanisms of action. A treatment that prevents, or more effectively reduces, NIHL would be
of significant clinical benefit to the millions of workers exposed to high levels of noise, military
personnel, and millions of others exposed to high levels of noise during recreational activities.
Methods
Animals
Pigmented male guinea pigs (250–300g; Elm Hill Breeding Labs, Chelmsford, MA) with a
normal Preyer’s reflex were used. Male guinea pigs were selected based on description of sex
differences in ROS detoxification [82], activity of glutathione S-transferase in the cochlea
[83], and susceptibility to NIHL [84]. The experimental protocol was reviewed and approved
by the University Committee for the Care and Use of Animals (UCUCA) at the University of
Michigan; all procedures conform to the National Institutes of Health Guidelines for the Care
and Use of Laboratory Animals.
Experimental groups
Guinea pigs were divided into four groups. All groups received once daily treatments beginning
one-hour prior to noise exposure and continuing once daily at 24-hour intervals until day 5
post-noise, for a total of 6 daily treatments. Control animals (N=9) received saline injections
(1 cc, i.p.). The second group was treated with vitamins A (2.1 mg/kg beta-carotene, p.o.), C
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(71.4 mg/kg L-threoascorbic acid, s.c.), and E (26 mg/kg (±)-6-hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid, “trolox”, s.c.) (“ACE”, N=8). Trolox is a cell-
permeable, water-soluble derivative of vitamin E. The third group was treated with magnesium
sulfate (“Mg”, 2.85 mmol/kg, equivalent to 343 mg/kg, s.c., N=6). The fourth group received
a combination of ACE and Mg (at the same doses as groups 2 and 3, “ACEMg”, N=6). All test
substances were purchased from Sigma-Aldrich (St. Louis, MO) (beta-carotene, #C9750, CAS
7235-40-7; L-threoascorbic acid, #A5960, CAS 50-81-7; Trolox, Fluka Chemika #56510, CAS
53188-07-1; magnesium sulfate, #M7506, CAS 7487-88-9).
Noise exposure
All subjects were exposed to octave-band noise (centered at 4 kHz, 120 dB SPL, 5 hours). This
noise exposure is routinely used in our laboratory [85,86]. As in those investigations animals
were exposed, two at a time in separate cages, in a ventilated sound exposure chamber. The
sound chamber was fitted with speakers (Model 2450H, JBL, Salt Lake City, UT) driven by a
noise generator (ME 60 graphic equalizer, Rane, Mukilteo, WA) and power amplifier
(HCA-1000 high current power amplifier, Parasound Products, San Francisco, CA). Sound
levels were calibrated (Type 2203 precision sound level meter, Type 4134 microphone, Bruel
and Kjar Instruments, Norcross, GA) at multiple locations within the sound chamber to ensure
uniformity of the stimulus, using a fast Fourier transform network analyzer with a linear scale.
The stimulus intensity varied by a maximum of 3 dB across measured sites within the exposure
chamber. During noise exposure, noise levels were monitored using a sound level meter, a pre-
amplifier, and a condenser microphone positioned in the center of the chamber at the level of
the animal’s head.
Auditory brainstem responses
Auditory brainstem response (ABR) thresholds at 4, 8, and 16 kHz were measured for both
right and left ears at two time points. Baseline ABR thresholds were established within 7 days
prior to experimental day 1 (the first day of saline or micronutrient treatment, delivered on the
day of noise exposure 1 hour prior to noise onset). Post-noise thresholds were established on
day 10 post noise exposure. There is some recovery of ABR thresholds initially post-noise;
both ABR threshold shift and loss of outer hair cells (OHCs) stabilize within 10 days of noise
exposure [86].
Prior to ABR tests, animals were anesthetized with xylazine (10 mg/kg i.m.) and ketamine (40
mg/kg i.m.) and placed on a heating pad in one of two sound-isolated chambers (Industrial
Acoustics Company, Bronx, New York, or C-A Tegnér, Bromma, Sweden). The external ear
canals and tympanic membranes were inspected using an operating microscope to assure the
ear canal was free of wax, there was no canal deformity, no inflammation of tympanic
membrane, and no effusion of the middle ear [as in 85], then needle electrodes were placed
subcutaneously below the test ear, at the vertex, and below the contralateral ear.
Acoustic stimuli were 4, 8, and 16 kHz tone bursts (15-ms duration; 1-ms rise-fall; 10/s)
generated using Tucker-Davis Technology (TDT, Alachua, FL) software (SigGen 3.2, or
SigGenRP) and TDT System II/III hardware (DA1, FT6-2, PA5, or RP2.1, PA5, HB7). Signals
were presented to the external auditory meatus in a closed acoustic system through a tube
connected to a transducer (Beyer DT-48, Beyer Dynamic, Farmingdale, NY). Starting levels
were 80–85 dB during baseline tests, and 100–105 dB SPL during post-noise threshold tests.
Sound intensity was initially decreased in 10 to 20 dB steps with 5-dB decrements presented
near threshold. Evoked responses to 1024 tone presentations were amplified (100,000x) and
filtered (300–3,000 Hz) (DB4 BioAmp, or RA4PA/RA4L1 Medusa) then digitized (AD1, or
RA16 Medusa) and averaged (BioSig 3.2, or BioSigRP). Threshold, tested separately for each
ear, was defined as the lowest intensity of stimulation that yielded a repeatable waveform based
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on an identifiable ABR wave III or IV. ABR wave III is the most robust component of the
guinea pig waveform [87,88].
Histological examinations
On day 14, after ABR measurement, the deeply anesthetized animals were decapitated and the
cochleae were immediately removed for immunohistochemical staining with rhodamine
phalloidin and hair cell counts. Upon removal, cochleas were transferred into 4%
paraformaldehyde in 0.1M phosphate-buffered saline (PBS, pH 7.4). Under a dissecting
microscope, the bone nearest the apex and the round and oval windows was opened, followed
by gentle local perfusion from the apex. The tissue was kept in fixative for 12 hours, then the
bony capsule and the lateral wall tissues were removed, and the modiolar core was carefully
removed from the temporal bone. Following permeabilization with Triton X-100 (0.3%, 30
min), the organ of Corti was stained for f-actin using rhodamine phalloidin (1%, 60–120 min)
to outline hair cells and their stereocilia [89]. After washing the tissues with PBS, the organ of
Corti was dissected and surface preparations were mounted on glass slides. The tissues were
observed under fluorescence microscopy, and the number of missing inner hair cells and outer
hair cells were counted from the apex to the base in 0.19 mm segments [as described in 86].
Counting was begun approximately 0.76–1.14 mm from the apex, thus omitting the initial
irregular most-apical part of the cochlear spiral. Percentages of hair cell loss in each 0.19 mm
length of tissue were plotted along the cochlear length.
Statistical analysis
All data values in the text and figures are mean ± S.E.M.; all statistical comparisons were
performed using SPSS. Statistical reliability of group differences in threshold and threshold
shift were via ANOVA; frequency (4, 8, and 16 kHz) and ear (left, right) were treated as within-
subject factors and treatment (saline, ACE, Mg, and ACEMg) was the between-subjects factor.
Adjustment for multiple comparisons was accomplished using the Bonferroni correction. Inner
and outer hair cell loss in different treatment groups were also compared via ANOVA; cochlear
place (0–4.99 mm, 5–9.99 mm, 10–14.99 mm, and 15–20 mm from the apex) and ear (left,
right) were treated as within-subject factors and treatment (saline, ACE, Mg, and ACEMg)
was the between-subjects factor. Specific analyses are described below.
Results
Noise-induced threshold deficits were significantly smaller in animals treated with a
combination of antioxidant agents and magnesium (see Figure 1). ABR threshold shift
measured 10-days post-noise varied with treatment group (F=15.289; df=3,25; p<0.001) as
well as frequency (F=43.844; df=1.854, 46.338; p<0.001); there was no effect of side (right/
left), and there were no significant interactions of frequency x treatment, side x treatment,
frequency x side, or frequency x treatment x side (all p’s > 0.12). Adjusted pair-wise
comparisons revealed that ABR thresholds were reliably lower in the group treated with
vitamins A, C, E and magnesium compared to all other groups (all p’s<0.001); lack of
statistically reliable interactions among factors indicates that protection was equivalent across
all test frequencies.
The significant reduction in noise-induced hearing loss in animals treated with both dietary
antioxidants and magnesium reflects synergistic effects of these agents. ABR thresholds for
this group of animals were lower post-noise, with no systematic group differences in threshold
sensitivity prior to noise exposure. To verify that the group differences in noise-induced
threshold shift were not a consequence of systematic group differences other than assigned
treatment, threshold data used to calculate shift measures were also compared. Baseline ABR
thresholds varied with frequency (F=427.011; df=1.763, 44.071; p<0.001) but were not reliably
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different based on side (right/left) (F=0.217; df=1, 25; p=0.645) and did not vary as a function
of group (F=1.430; df=3, 25; p=0.258). There were no statistically reliable interactions for
frequency x group, side x group, or frequency x side x group (all p’s > 0.15). In contrast, ABR
thresholds measured 10-days post-noise varied with treatment group (F=15.383; df=1,25;
p<0.001) as well as frequency (F=111.660; df=1.380, 34.506; p<0.001), and there were
significant interactions of frequency x treatment (F=2.840; df=4.141, 34.506; p=0.037) and
frequency x treatment x side (F=2.456; df=5.7, 47.499; p=0.40) but no reliable differences
associated with side, or interactions of side x group or frequency x side (all p’s > 0.2). Adjusted
pair-wise comparisons revealed that post-noise ABR thresholds were reliably lower in the
group treated with vitamins A, C, E and magnesium compared to all other groups (all p’s<0.01).
There were no statistically reliable differences between post-noise ABR thresholds in control
animals and animals treated with either the antioxidant agents or magnesium (all p’s > 0.4).
Additional analysis revealed that the threshold differences between animals treated with
vitamins A, C, and magnesium, and the other groups, were statistically significant at all test
frequencies (all p’s 0.011), with no effects of side and no interactions for side x group
comparisons (all p’s >0.13).
Sensory cell death was observed primarily within the basal half of the cochlea, i.e., 10–20 mm
from the apex of the cochlea (see Figure 2), which corresponds to frequencies of approximately
3 kHz and above [90]. Within the basal half of the cochlea, treatment-based differences in outer
hair cell survival exceeded the standard criteria limiting the probability that observed results
are due to chance alone to 5% or less (rows 1–3, p=0.111). However, statistical power for this
between-group comparison was 0.511 and, thus, was not sufficient to detect mean differences
of this magnitude given the observed variability (see error bars, figure 2). To reduce the number
of pair-wise comparisons, and maintain statistical power, we compared outer hair cell loss in
the 10–15 and 15–20 mm from the apex segments of the cochlea in the two groups most crucial
to testing our hypothesis: saline and combination treatment with antioxidants and magnesium
(see Figure 3). Loss of row 1 outer hair cells was location dependant (F=5.577; df=1, 13;
p=0.034) and the reliability of group differences approached the accepted definition of
statistical significance (F=4.134; df=1, 13; p=0.063). Loss of row 2 outer hair cells was more
homogenous across segments (F=3.057; df=1, 13; p=0.104) and the group difference was
statistically reliable (F=4.829; df=1, 13; p=0.047). Outer hair cell loss in row 3 was generally
equivalent to row 2 (location: F=3.364; df=1, 13; p=0.090; treatment: F=4.550; df=1, 13;
p=0.053). Both the mean and the median hair cell loss for all 24 of the 0.19 mm segments of
the cochlea in which hair cells were counted were less for the combined treatment group than
for the saline control group; the probability of this outcome occurring by chance is less than
0.0001. Taken together, the combination treatment appears to preserve not only threshold
sensitivity but also hair cell survival.
Discussion
Synergistic Effects
Treatment with a combination of vitamins A, C, E, and magnesium, initiated 1 hour prior to
noise exposure, produced a compelling reduction in NIHL and cell death. Effects of either the
antioxidant agents (vitamins A, C, and E), or magnesium, were very small and not statistically
reliable. Thus, the combination of agents was clearly more effective than any single category
of agents. High oral doses of some nutrients can have adverse effects in some populations; for
example, high-dose vitamin A is associated with an increased rate of lung cancer in smokers
[91–93]. However, the Institute of Medicine’s Food and Nutrition Board has defined the highest
level of daily nutrient intake that is likely to pose no risks of adverse health effects to almost
all individuals in the healthy population (upper limit). The lack of significant adverse effects
with long-term high-dose intake of several of these micronutrients has now been confirmed in
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several investigations [94,95]. In addition, the US Food and Drug Administration has
established recommended daily allowances that are safe for human use; these are readily
available ‘over-the-counter’.
Mechanism of Action
We know that noise induces free radical formation; there is a nearly 4-fold increase in hydroxyl
(OH) radicals within 1–2 hours of noise exposure [21], and a similarly significant early increase
in superoxide (O2) with reaction products evident at 5 min and 2 hours post noise [19]. There
is also a significant late formation of ROS and RNS, occurring 7–10 days post noise [86]. We
also know that noise induces lipid peroxidation; significant noise-induced lipid peroxidation
and peroxynitrite (ONOO) formation have been described 15–30 min post-noise [25,96], and
lipid peroxidation products increase in level from hour 1 to hour 5 of noise exposure [24]. We
assume that pre-treatment with a variety of scavengers (including vitamins A, C, and E) reduced
the early formation of free radicals that has been well characterized by Ohinata, Ohlemiller,
Yamane, Nicotera, and their colleagues [19,21,24,25,96]. Daily treatment that continued
through day 5 post-noise presumably reduced the late forming radicals revealed in the studies
of Yamashita et al. [86]. Unlike most tissues, where increased metabolism increases blood flow
to provide additional oxygen to stressed cells, reduced blood vessel diameter and red blood
cell velocity [97,98] and decreased blood flow [9,10,16,26,99] are observed in the cochlea
post-noise. This noise-induced vasoconstriction is a direct consequence of noise-induced
formation of 8-isoprostane-F2α, a vasoactive by-product of free radicals [46], and thus,
antioxidant agents that reduce free radical formation may eliminate noise-induced
vasoconstriction. The combination of vitamins A, C, and E, delivered one hour pre-noise
presumably did not adequately preserve inner ear blood flow, as prevention of noise-induced
deficits was achieved only by the combination of vitamins A, C, E, and magnesium.
Together, the agents used here scavenge singlet oxygen (Vitamin A), react with and reduce
peroxyl radicals in cell membranes (Vitamin E), detoxify free radicals by reducing them in the
aqueous phase (Vitamin C), and reduce noise-induced vasoconstriction (magnesium).
Although there are well-characterized differences in the primary mechanism and site of action
of these agents, there is also potential for overlap in their effects. Specifically, antioxidant
scavenging of ROS may reduce the vasoconstriction that occurs with ROS production. In
addition to well known effects of magnesium on blood flow, other biochemical mechanisms
may further contribute to the protective effects of magnesium. For example, magnesium
modulates calcium channel permeability, influx of calcium into cochlear hair cells, and
glutamate release [100,101], each of which may reduce NIHL. Regardless of the specific
mechanism of action of vitamins A, C, and E, and magnesium, a combination of these agents
clearly attenuates NIHL even when treatment is initiated very near the time of noise exposure
(i.e., 1 hour prior to noise).
Individual Effects
Given that vitamins A, C, E, and magnesium, have been shown to attenuate NIHL and hearing
loss from other stressors, it was somewhat surprising that protection was not observed for either
the antioxidant (ACE) or magnesium treatments. Deficits were reduced to some small extent,
but these effects were not statistically reliable. Stable plasma and tissue levels of vitamin C are
obtained (in humans) approximately 3 weeks after beginning dietary treatment [102], and
vitamin E levels similarly stabilize over an initial month-long window [for review, see 103].
Thus, it may be the case that dietary treatments must be provided on a daily basis for some
longer period of time pre-noise for single-agent therapies to be effective. Indeed, the best
protection against NIHL obtained with vitamin C [L--2-pascorbylolyphosphate, delivered as
5,000 mg per kg chow for 35 days, 60] included a 1-month pre-treatment protocol. Treatment
initiated shortly before noise exposure failed to prevent cell death [500 mg/kg ascorbic acid,
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i.p., 48 h, 24 h, and 5 min prior to noise exposure, 104]. In contrast, vitamin E (alpha-tocopherol,
10–50 mg/kg, i.p.) reduced NIHL with treatment initiated 3 days pre-noise [50], and vitamin
A reduced NIHL with treatment initiated two days pre-noise [all-trans retinoic acid, 1 mg/kg
p.o., in sesame oil, 105].
Taken together, there may be differences in the pharmacodynamics of different agents, and
protection may be observed with antioxidants or magnesium alone if treatment is initiated 48
hours (or longer) prior to noise exposure to produce higher serum levels of the agents in the
bloodstream. Agents that are effective shortly post-treatment have greater clinical utility. While
the demonstration of significant protection with vitamins A, C, E and magnesium, delivered
within 1-hour of noise exposure, suggests a compelling new clinical strategy to prevent NIHL,
several of the active agents were injected. Thus, it is possible that the most efficacious treatment
window, assuming oral administration for all agents, will be somewhat longer than 1 hour pre-
noise exposure. Indeed, treatments based on other antioxidant combinations are the most
effective when treatment begins several days prior to noise exposure [85]. Although increasing
total daily doses of single agents might reduce the need for lengthy pre-treatment, clinical
strategies tested in human subjects must not exceed the upper limits established by the Institute
of Medicine’s Food and Nutrition Board.
Efficacy of delayed treatment?
The compelling protection observed when treatment with vitamins A, C, and E, and magnesium
was initiated shortly prior to noise exposure raises the possibility that treatments initiated post-
noise may also be effective. Ohlemiller et al. [21] were among the first to suggest oxidative
stress begins early and becomes substantial over time. Free radical insult that builds over time
would explain hair cell death that continues up to 14 days post-noise [106]. Using 4-
hydroxynonenal and nitryotyrosine, Yamashita et al. [86] confirmed that peak ROS and RNS
production in cells in the organ of Corti occurs 7–10 days post-noise, and that noise-induced
hair cell death is similarly delayed. As a consequence of delayed ROS production, ROS and
RNS scavengers reduce NIHL not only when administered prior to noise, but also when
treatment begins on days 1 or 3 post-noise [85]. Treatment delayed 5 days relative to noise
insult was not effective, suggesting the therapeutic “window of opportunity” for successful
antioxidant-based intervention against NIHL occurs within the first three days post-noise.
Determining the efficacy of vitamins A, C, E, and magnesium when treatment is delayed
relative to the noise insult is a compelling topic for future research.
Alternative Strategies
Multiple groups have pursued preservation of auditory function using glutathione (GSH) -
based therapeutic strategies in recent years. Ebselen is a potent glutathione peroxidase (GPx)
mimic; GPx catalyzes the anti-oxidant actions of GSH [107]. Pretreatment with ebselen reduces
NIHL [108–111]. Given that one of the major determinants of GSH levels is bioavailable
cysteine, and that cysteine can be derived from methionine, other studies have evaluated the
potential for protection by pre-treatment with D-methionine [45]. D-methionine specifically
alters the cochlear oxidative state; superoxide dismutase, catalase, GPx, and glutathione
reductase levels all increase in animals treated with D-Methionine [112].
N-L-Acetylcysteine (L-NAC), which increases intracellular GSH and acts as a ROS scavenger,
has also been used singly [47], and in combination with salicylate [113] to effectively attenuate
NIHL. The utility of comparisons across investigations is compromised by variation in dose
schedule and duration, species, and noise exposure. However, the significant reduction of
NIHL (up to 35 dB at 16 kHz, with treatment initiated 1 hour prior to noise exposure), with
vitamins A, C, E, and magnesium, suggests that this combination will prove to be one of the
most effective therapies when different agents are compared under identical test conditions.
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Relatively greater efficacy could be explained by the combination of antioxidants reducing
both ROS and RNS production, with additional protection as a consequence of magnesium-
mediated reductions in noise-induced vasoconstriction and excitotoxicity.
Identifying treatments to reduce neurodegenerative disease
Mitochondrial dysfunction and ROS production have been implicated in many, if not all,
neurodegenerative processes, and also in association with changes in local blood flow, such as
stroke [28–36]. The use of antioxidant agents to prevent neurodegeneration has been widely
described [32,33,37–42]. As in our investigation, combinations of antioxidants have been
shown to be more effective than single agents in at least some cases [43]. Vasoregulators are
also clearly of potential benefit given observations of reduced cortical blood flow in both
Alzheimer’s and Parkinson’s disease [114–118]. This similarity of free radical production and
reduced blood flow in neurodegenerative processes and NIHL, and the demonstrated efficacy
of antioxidants plus magnesium in reducing sensory cell death and neurodegeneration in the
cochlea, provides a compelling rationale for study of combinations of antioxidants, with and
without magnesium, to ameliorate neurodegeneration from other stresses and disease
processes, at other sites of the nervous system, of more heterogeneous origins, such as
Alzheimer’s disease, Parkinson’s disease, and stroke.
Conclusions
NIHL is a significant clinical issue [for recent review, see 26]. We now know many of the
molecular pathways leading to apoptotic cell death, which are triggered by noise and other
environmentally mediated trauma such as aminoglycoside antibiotics and chemotherapeutics,
as well as aging; and there is increasing evidence for their similarity. Free radical formation
has been implicated in all. Interventions can be directed at preventing initial ROS formation
and maintaining cochlear blood flow (as reported here and by others); alternative therapeutic
interventions and strategies include neurotrophic growth factors [119–122], steroids [123,
124], calcineurin inhibitors [125,126], caspase inhibitors [127–132], and Src protein tyrosine
kinase (Src-PTK) inhibitors [133]. Each of these are at least partially effective in prevention
of hearing loss and hair cell death; none of these strategies have alone been sufficiently
effective. Given the various points of intervention along the cell death pathways, there are
clearly an abundance of potential therapeutic targets. For clinical purposes, the most effective
strategy may include targeting initiating events as well as early molecular processes, thus
maintaining a cell in a relatively ‘normal’ physiological state. Although additional pre-clinical
investigation is essential to the task of defining the most effective combination of agents, given
that safe and effective interventions are now available, we should no longer delay the initiation
of systematic human clinical trials to demonstrate the potential for pharmaceutical treatment
of the inner ear in man.
Acknowledgements
Support for this research was provided by the National Institutes of Health (NIH-NIDCD R01 DC03820 and P30
DC005188), the General Motors Corporation/United Automotive Workers Union, and the Ruth and Lynn Townsend
Professor of Communication Disorders. We thank Susan DeRemer, Chris Ellinger, Alice Mitchell, Diane Prieskorn,
and Hollie Valentine for technical assistance.
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Figure 1.
Noise-induced hearing loss, estimated using auditory brainstem response thresholds prior to
and 10 days post noise (octave-band noise centered at 4 kHz, 120-dB SPL, 5 hours), was
reduced at 4, 8, and 16 kHz by treatment with a combination of vitamins A, C, E and magnesium
(ACEMg), but not by treatment with the antioxidants (ACE) or magnesium (Mg). Asterisks
indicate statistically reliable differences (p’s < 0.001) between ACEMg and all other groups.
Le Prell et al. Page 16
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Figure 2.
Sensory cell death was observed primarily within the basal half of the cochlea, i.e., 10–20 mm
from the apex of the cochlea, and corresponding to frequencies of approximately 3 kHz and
above [90] for both inner hair cells (A, C, E, and G) and outer hair cells (B, D, F, and H). Both
the mean and the median hair cell loss for all 24 of the 0.19 mm segments of the cochlea in
which hair cells were counted were less for the combined treatment group (ACEMg) than for
the saline group; the probability of this outcome occurring by chance is less than 0.0001.
Le Prell et al. Page 17
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Figure 3.
Percent inner (A, B) and outer (C, D) hair cell loss in the 10–15 and 15–20 mm from the apex
segments of the cochlea is illustrated. Treatment with a combination of vitamins and
magnesium reduced outer hair cell loss by approximately 30% in both the 10–15 mm region
(Saline: 42%; ACEMg: 13%) and the 15–20 mm region (Saline: 28%; ACEMg: <1%). This
protection was statistically reliable when group comparisons were limited (to reduce the
number of pair-wise comparisons, and increase statistical power) to the two groups most crucial
to testing our hypothesis: saline and combination treatment with antioxidants and magnesium.
Le Prell et al. Page 18
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... Thus, exogenous regulation and regeneration of glutathione levels [12,13]; potentiation of antioxidant enzymes such as GPX by using enzyme mimics such as ebselen [14][15][16]; or free-radical scavengers such as sulfur-containing amino acids and derivatives including d-methionine, or N-acetyl-cysteine, which are direct antioxidants besides its role in glutathione regeneration, have shown otoprotective potential in NIHL [17,18]. Among free-radical scavengers, antioxidant vitamins, in particular vitamins C and E, also have been shown to protect the auditory receptor from NIHL damage [19][20][21]. ...
... A powerful otoprotective potential of a combination of antioxidant vitamins (A, C, and E) along with Mg 2+ has been proposed [20,22]. It has been suggested that the different antioxidation mechanisms of each vitamin combined with cochlear vasodilation induced by Mg 2+ , along with other effects of this cation mainly related to Ca 2+ antagonism, give rise to a synergistic interaction resulting in efficient otoprotection against NIHL [20]. ...
... A powerful otoprotective potential of a combination of antioxidant vitamins (A, C, and E) along with Mg 2+ has been proposed [20,22]. It has been suggested that the different antioxidation mechanisms of each vitamin combined with cochlear vasodilation induced by Mg 2+ , along with other effects of this cation mainly related to Ca 2+ antagonism, give rise to a synergistic interaction resulting in efficient otoprotection against NIHL [20]. However, otoprotective mechanisms of orally administered ACEMg have not been investigated, and it is even unclear whether increased survival of sensory hair cells may be involved [20,22]. ...
Article
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Noise induces oxidative stress in the cochlea followed by sensory cell death and hearing loss. The proof of principle that injections of antioxidant vitamins and Mg2+ prevent noise-induced hearing loss (NIHL) has been established. However, effectiveness of oral administration remains controversial and otoprotection mechanisms are unclear. Using auditory evoked potentials, quantitative PCR, and immunocytochemistry, we explored effects of oral administration of vitamins A, C, E, and Mg2+ (ACEMg) on auditory function and sensory cell survival following NIHL in rats. Oral ACEMg reduced auditory thresholds shifts after NIHL. Improved auditory function correlated with increased survival of sensory outer hair cells. In parallel, oral ACEMg modulated the expression timeline of antioxidant enzymes in the cochlea after NIHL. There was increased expression of glutathione peroxidase-1 and catalase at 1 and 10 days, respectively. Also, pro-apoptotic caspase-3 and Bax levels were diminished in ACEMg-treated rats, at 10 and 30 days, respectively, following noise overstimulation, whereas, at day 10 after noise exposure, the levels of anti-apoptotic Bcl-2, were significantly increased. Therefore, oral ACEMg improves auditory function by limiting sensory hair cell death in the auditory receptor following NIHL. Regulation of the expression of antioxidant enzymes and apoptosis-related proteins in cochlear structures is involved in such an otoprotective mechanism.
... Vitamin A, which is required for normal development of the inner ear, comprises several compounds, including retinol, retinal, and several pro-vitamin A carotenoids (most notably β-carotene) [55][56][57]. Antioxidant activity has been reported for vitamin A, as well as for many pro-vitamin A carotenoids [58][59][60][61][62]. Vitamin A and carotenoids were shown to be an effective singlet oxygen and peroxyl radical scavenger [58][59][60]. ...
... Vitamin A, which is required for normal development of the inner ear, comprises several compounds, including retinol, retinal, and several pro-vitamin A carotenoids (most notably β-carotene) [55][56][57]. Antioxidant activity has been reported for vitamin A, as well as for many pro-vitamin A carotenoids [58][59][60][61][62]. Vitamin A and carotenoids were shown to be an effective singlet oxygen and peroxyl radical scavenger [58][59][60]. Thus, vitamin A could scavenge the free radicals produced with aging and may prevent cochlear damage caused by high levels of toxic free radicals. ...
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Age-related hearing loss (ARHL) is a major and rapidly growing public health problem thatcauses disability, social isolation, and socioeconomic cost. Nutritional status is known to cause manyaging-related problems, and recent studies have suggested that there are interaction effects betweenARHL and dietary factors. We aimed to investigate the association between ARHL and dietaryassessment using data from the fifth Korean National Health and Nutrition Examination Survey,which is a nationwide cross-sectional survey that included 5201 participants aged≥50 years from2010 to 2012. All participants had normal findings on otoscopic examination and symmetric hearingthresholds of <15 dB between both sides. Nutritional survey data included food consumption andnutrient intake using the 24 h recall method. Data were analyzed using multiple regression modelswith complex sampling adjusted for confounding factors, such as age, sex, educational level, andhistory of diabetes. Higher intake of seeds and nuts, fruits, seaweed, and vitamin A were positivelyassociated with better hearing. Our findings suggest that dietary antioxidants or anti-inflammatoryfood may help reduce ARHL
... Animal studies highlighted the protective role of antioxidants against noise-, virus-, age-or drug-induced hearing loss (Le Prell et al., 2007;Tavanai and Mohammadkhani, 2017;Pecha et al., 2020). In noise-induced hearing loss, one study underlined the mitochondrial generation of reactive oxygens species (ROS) and showed that D-Methionine (D-Met) treatment protected animals from temporary threshold shifts (Cheng et al., 2008). ...
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Hearing loss affects roughly 466 million people worldwide. While the causes of hearing loss are diverse, mechanistically, inflammation and oxidative stress have been identified as major players in hearing loss regardless of pathogenesis. Treatment options remain extremely limited and there is currently no FDA approved drug therapy. Studies indicate that antioxidants such as d -Methionine have shown some protective effects; however, these studies involved systemic or invasive localized delivery methods and highlighted the need for the development of minimally invasive localized therapeutic approaches. Described herein is the development of an antioxidant-conjugated system that shows prophylactic potential against oxidative damage and appears suitable for topical delivery. Specifically, our covalent conjugate of hyaluronan with d -Methionine shows cytocompatibility and protection from oxidative stress in two mouse cochlear cell lines (HEI-OC1 and SV-k1). Mechanistically, the data indicate that the protective effects of the conjugate are due to the hyaluronan-mediated cellular internalization of the antioxidant. Most notably, the conjugate can efficiently permeate through an in vitro round window membrane model without the loss of the attached antioxidant, for subsequent delivery of the therapeutic cargo to the hearing sensory cells. Collectively these findings show that the novel conjugate could be a potential topical preventive agent against hearing loss.
... In other studies where the noise was centered at 4 kHz octave band and 120 dB SPL intensity for 5 h, 4 kHz, 8 kHz, and 16 kHz ABR thresholds on day 1 were observed around 50 dB and 60 dB. 15,16 In the model of Ogurlu et al 17 using thymoquinone in rats, the noise was centered at 4 kHz octave band with 120 dB SPL intensity for 4 hours and the heads of the rats were placed 5-7 cm away from the speakers. On the first day after trauma, ABR measurements revealed 45-50 dB thresholds at frequencies of 2 kHz and 4 kHz. ...
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Background: To investigate the possible protective activity of oleuropein compound on noise-induced hearing loss in rats. Methods: Twenty-eight adult male albino rats were divided into 4 groups. Control normal saline (n=7) group was kept noise-free. Control oleuropein group (n=7) group was kept noise-free and was administered with 50 mg/kg/day oleuropein. The experimental normal saline (n=7) group was subjected to noise. The experimental oleuropein (n=7) group was subjected to noise and was administered with 50 mg/kg/day oleuropein. The experimental groups were subjected to 4 kHz octave noise with a frequency of 120 dB Sound Pressure Level (SPL) for 4 hours. Hearing level measurements were performed with auditory brainstem response and distortion-product otoacoustic emission tests before and after the 1st, 7th, and 10th day of the noise exposure. On the 10th day, rats were sacrificed. The temporal bones of the rats were removed and the cochlea and spiral ganglion cells were evaluated using hematoxylin-eosin staining under light microscopy. Results: Better hearing thresholds were achieved in the experimental oleuropein group compared to the experimental normal saline group at 8 kHz, 12 kHz, 16 kHz, and 32 kHz frequencies (P < .05). Although no statistically significant difference was found between the groups, in the experimental normal saline group, the percentage of damaged spiral ganglion cells was higher than the experimental oleuropein group. Conclusion: Our findings suggest that oleuropein may have a partial protective effect against noise-related hearing loss. However, further research with higher doses is needed to justify this protective effect.
... Supporting this, antioxidant-based therapies should mitigate the hearing damage induced by oxidative stress. Accordingly, several antioxidant and combinations has proven to be effective, mainly at the level of proof principle, for the treatment of NIHL (Yamasoba et al., 1999;Kopke et al., 2000Kopke et al., , 2007Tanaka et al., 2005;Le Prell et al., 2007a, 2011, DIHL (Campbell et al., 2007;Tokgoz et al., 2011;Le Prell et al., 2014) and ARHL (Alvarado et al., 2018;Cai et al., 2020). This confirms the central role that oxidative stress plays in the development and/or progression of many auditory pathologies. ...
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As it is well known, a worldwide improvement in life expectancy has taken place. This has brought an increase in chronic pathologies associated with aging. Cardiovascular, musculoskeletal, psychiatric, and neurodegenerative conditions are common in elderly subjects. As far as neurodegenerative diseases are concerned dementias and particularly, Alzheimer’s disease (AD) occupy a central epidemiological position given their high prevalence and their profound negative impact on the quality of life and life expectancy. The amyloid cascade hypothesis partly explains the immediate cause of AD. However, limited therapeutical success based on this hypothesis suggests more complex remote mechanisms underlying its genesis and development. For instance, the strong association of AD with another irreversible neurodegenerative pathology, without curative treatment and complex etiology such as presbycusis, reaffirms the intricate nature of the etiopathogenesis of AD. Recently, oxidative stress and frailty syndrome have been proposed, independently, as key factors underlying the onset and/or development of AD and presbycusis. Therefore, the present review summarizes recent findings about the etiology of the above-mentioned neurodegenerative diseases, providing a critical view of the possible interplay among oxidative stress, frailty syndrome, AD and presbycusis, that may help to unravel the common mechanisms shared by both pathologies. This knowledge would help to design new possible therapeutic strategies that in turn, will improve the quality of life of these patients.
... Acetylcysteine is a precursor substance of glutathione [77]. Antioxidant effect characterizes dietary supplements like vitamins D, E, A and polyphenols [78]. The main therapeutic approach for synaptopathy is with administration of neurotrophic substances, like neurotrophin-3 [79]. ...
Article
Noise in human societies is unavoidable, but it tends to become a modern epidemic that induces various detrimental effects to several organs and functions in humans. Increased cardiovascular danger, anxiety and sleep disturbance are just few of these effects. It is noteworthy that children, even neonates and their developing organism are especially vulnerable to noise-related health problems. Noise is measured with special noise-meters. These devices express results in decibels by transforming random noise to a continuous sound. This sound is characterized by equivalent acoustic energy to the random noise for a defined time interval. Human auditory apparatus is principally endangered by acute noises but also by chronic noise exposure, in the context of both occupational and recreational activities. Various mechanisms are implicated in the pathogenesis of noise-induced hearing loss that can cause either temporary or permanent damage. Among them, emphasis is given to the impairment by free radicals and inflammatory mediators, to the activation of apoptotic molecular pathways, but also to glutamate excitotoxicity. A hidden hearing loss, synaptopathy, is attributed to the latter. The irreversible nature of hearing loss, as well as the idiosyncratic sensitivity of individuals, imposes the necessity of early diagnosis of auditory impairment by noise. Super high frequency audiograms, otoacoustic emissions and electrophysiological examinations can address diagnosis. Thankfully, there is extensive research on acoustic trauma therapeutic approaches. However, until we succeed in regenerating the sensory organ of hearing, chronic noise-induced hearing loss cannot be treated. Thus, it is fundamental that society protects people from noise, by laws and regulations.
... Long-chain polyunsaturated fatty acids (LC-PUFAs), such as linoleic acid and linolenic acid, are known to improve blood lipid metabolism and exhibit anti-inflammatory effects, which exhibit preventive effects on conditions such as hypertension and diabetes (Abedi and Sahari, 2014). While vitamin E compounds found in cellular lipids block free radical chain reactions, they are nontoxic and serve as powerful inhibitors, reportedly preventing oxidative damage on lipids by reducing peroxyl radicals (Le Prell et al., 2007). EPSP was shown to contain an abundance of antioxidant vitamin E compounds compared with EPS extracted by other processing methods. ...
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This study proposes the processing method that could maximize the functional properties of evening primrose seeds (EPS) and its various nutritional components. EPS can be prepared by different methods, such as being left raw, roasting, steaming, and powdering. Processing of EPS to create EPS powder (EPSP) resulted in higher levels of vitamin E, fatty acids, total phenolic contents, and antioxidant activity, compared with the other processing methods. Also, EPSP maintained lipid peroxidation inhibitory activity for 49 days. In particular, the EPSP ethyl acetate (EtOAc) fraction exhibited high antioxidant, antidiabetic, and angiotensin I-converting enzyme inhibitory activities. The EPSP ethyl acetate fraction showed a high cytoprotective effect against H2O2-induced cell damage in both RAW264.7 and EA.hy926 cells. In addition, the EtOAc fraction showed anti-inflammatory activity by the inhibitory activity of nitric oxide (NO) in RAW264.7 cells, and antihypertensive activity by the activity of NO in EA.hy926 cells. These results suggest that EPSP could be useful as a food ingredient that assists the prevention of various diseases caused by oxidative stress.
... 12 Steroids, nonsteroidal anti-inflammatory drugs, and vitamins have been tested in rat nerve crush injury models to analyze their potential effects on nerve regeneration processes. [13][14][15][16][17][18] Alpha lipoic acid (ALA) is a natural compound that is found in many prokaryotic and eukaryotic cell types. 3 ALA acts as a scavenger of different reactive oxygen species and can further interact with other antioxidants such as vitamin C and E, resulting in their regeneration. ...
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Objective: Investigating regenerative effects of ALA on recovery of SNCI in rats. Design: Randomized, experimental, and sham-controlled study. Sciatic nerves of 28 rats in 4 groups were traumatized for 60 seconds. G1; sham-operated + saline, G2; SNCI + saline, G3; SNCI + ALA 50 mg/kg/day, G4; SNCI + ALA 100 mg/kg/day. SFI values were measured on day 0, 1, 7, 14, 21, and 28. Sciatic nerve stimulation threshold values (STV) were recorded on day 1, 14, and 28. Endpoint histopathologic evaluation was conducted. Results: Mean SFI value of G2 but not G3/G4 on day 7 was significantly lower than day 0 (p=0.035, p=0.447/p=0.800). Mean SFI value of G2 but not G3/G4 increased significantly between day 7 and 14 (p=0.035, p=0.447/p=0.438). Day 14 mean sciatic nerve STV of G3/G4 but not G2 were decreased significantly compared to day 1 (p=0.022/p=0.022, p=0.933). Mean sciatic nerve STV of G3/G4 on day 14 were similar to day 0 (p=0.106/p=0.418). Regeneration in muscle and nerve connective tissues, and nerve structures were observed in G3/G4. Inflammation in muscle and nerve tissues of G4 was suppressed down to similar levels of G1. Myelinated nerve fibers were less degenerated in G3/G4. Conclusion: ALA has the potential to accelerate the process of nerve healing in the context of SNCI in rats.
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Both vitamin A and E support female reproduction and embryonic development. These vitamins have been associated with decreased fertility or failure to end the pregnancy in animals. An observational study was conducted on follicular fluid (FF) samples to determine the concentrations of fat-soluble vitamins of women undergoing in vitro fertilization and its correlation with assisted reproductive technology characteristics and pregnancy outcomes. Moreover, the effects of all-trans-retinoic acid (atRA) and alpha-tocopherol on granulosa cell viability, apoptosis, autophagy and hormonal production were evaluated. No association was identified between fat-soluble vitamin concentrations in FF and infertility aetiology, body mass index or woman’s age. There were differences in follicular antioxidant profiles and ovarian response stimulation. In vitro evaluation of atRA and alpha-tocopherol reveals that, at physiological concentrations, both compounds may affect the viability of granulosa cells. In addition, these compounds are able to protect granulosa cells from oxidative stress, as well as to affect estradiol and progesterone production. Our data suggest that atRA and alpha-tocopherol levels should be well controlled as they may have implications in the function and viability of granulosa cells and highlights retinol as a marker of the oxidative defenses within ovary environment.
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Background: Observational and experimental data suggest that antioxidant and/or zinc supplements may delay progression of age-related macular degeneration (AMD) and vision loss. Objective: To evaluate the effect of high-dose vitamins C and E, beta carotene, and zinc supplements on AMD progression and visual acuity. Design: The Age-Related Eye Disease Study, an 11-center double-masked clinical trial, enrolled participants in an AMD trial if they had extensive small drusen, intermediate drusen, large drusen, noncentral geographic atrophy, or pigment abnormalities in 1 or both eyes, or advanced AMD or vision loss due to AMD in 1 eye. At least 1 eye had best-corrected visual acuity of 20/32 or better. Participants were randomly assigned to receive daily oral tablets containing: (1) antioxidants (vitamin C, 500 mg; vitamin E, 400 IU; and beta carotene, 15 mg); (2) zinc, 80 mg, as zinc oxide and copper, 2 mg, as cupric oxide; (3) antioxidants plus zinc; or (4) placebo. Main outcome measures: (1) Photographic assessment of progression to or treatment for advanced AMD and (2) at least moderate visual acuity loss from baseline (> or =15 letters). Primary analyses used repeated-measures logistic regression with a significance level of.01, unadjusted for covariates. Serum level measurements, medical histories, and mortality rates were used for safety monitoring. Results: Average follow-up of the 3640 enrolled study participants, aged 55-80 years, was 6.3 years, with 2.4% lost to follow-up. Comparison with placebo demonstrated a statistically significant odds reduction for the development of advanced AMD with antioxidants plus zinc (odds ratio [OR], 0.72; 99% confidence interval [CI], 0.52-0.98). The ORs for zinc alone and antioxidants alone are 0.75 (99% CI, 0.55-1.03) and 0.80 (99% CI, 0.59-1.09), respectively. Participants with extensive small drusen, nonextensive intermediate size drusen, or pigment abnormalities had only a 1.3% 5-year probability of progression to advanced AMD. Odds reduction estimates increased when these 1063 participants were excluded (antioxidants plus zinc: OR, 0.66; 99% CI, 0.47-0.91; zinc: OR, 0.71; 99% CI, 0.52-0.99; antioxidants: OR, 0.76; 99% CI, 0.55-1.05). Both zinc and antioxidants plus zinc significantly reduced the odds of developing advanced AMD in this higher-risk group. The only statistically significant reduction in rates of at least moderate visual acuity loss occurred in persons assigned to receive antioxidants plus zinc (OR, 0.73; 99% CI, 0.54-0.99). No statistically significant serious adverse effect was associated with any of the formulations. Conclusions: Persons older than 55 years should have dilated eye examinations to determine their risk of developing advanced AMD. Those with extensive intermediate size drusen, at least 1 large druse, noncentral geographic atrophy in 1 or both eyes, or advanced AMD or vision loss due to AMD in 1 eye, and without contraindications such as smoking, should consider taking a supplement of antioxidants plus zinc such as that used in this study.
Chapter
The two most common causes of hearing loss in adults are (1) acoustic overexposure, resulting in noise-induced hearing loss (NIHL), and (2) age-related cochlear degeneration, resulting in age-related hearing loss (AHL), or presbycusis. The mechanisms underlying NIHL and AHL are not well understood. However, recent investigations implicate a common etiological factor in NIHL and AHL: an imbalance between levels of reactive oxygen species (ROS) produced during cellular metabolism, and the body’s defenses against them. Produced in excess of the body’s ability to remove them, ROS can cause extensive cellular damage. One important ROS is the superoxide radical (·O). Superoxide has many important functions in the body, but it can also be cytotoxic under certain conditions (Halliwell and Gutteridge, 1999). In addition, ·O can be easily converted to other ROS that have greater potential for causing cellular damage, such as peroxynitrite (ONOO-) and the extremely reactive hydroxyl (·OH) free radical molecule. Superoxide dismutase (SOD), an antioxidant enzyme present in virtually every cell of the body, plays a crucial role in regulating ·O levels. An imbalance between ·O generation and SOD availability would be expected to have serious consequences for cellular function and survival. We have been exploring the effects of such an imbalance on AHL and NIHL, using mutant mice with a targeted deletion of Sod1, the gene that codes for one copper/zinc isoform of SOD, Cu/Zn SOD, or SOD1. Our experiments with Sod1 knockout mice (129/CD1- Sod1) have provided us with insights into possible chemical pathways underlying NIHL and AHL. Before describing our experiments with Sod1 knockout mice, it may be useful to provide a broad overview of ROS and cellular defenses against them. What are ROS and how are they generated? What do they do, and when are they harmful? How are ROS levels normally regulated in the body?.
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It has been suggested that increased intake of various antioxidant vitamins reduces the incidence rates of vascular disease, cancer, and other adverse outcomes. METHODS: 20,536 UK adults (aged 40-80) with coronary disease, other occlusive arterial disease, or diabetes were randomly allocated to receive antioxidant vitamin supplementation (600 mg vitamin E, 250 mg vitamin C, and 20 mg beta-carotene daily) or matching placebo. Intention-to-treat comparisons of outcome were conducted between all vitamin-allocated and all placebo-allocated participants. An average of 83% of participants in each treatment group remained compliant during the scheduled 5-year treatment period. Allocation to this vitamin regimen approximately doubled the plasma concentration of alpha-tocopherol, increased that of vitamin C by one-third, and quadrupled that of beta-carotene. Primary outcomes were major coronary events (for overall analyses) and fatal or non-fatal vascular events (for subcategory analyses), with subsidiary assessments of cancer and of other major morbidity. FINDINGS: There were no significant differences in all-cause mortality (1446 [14.1%] vitamin-allocated vs 1389 [13.5%] placebo-allocated), or in deaths due to vascular (878 [8.6%] vs 840 [8.2%]) or non-vascular (568 [5.5%] vs 549 [5.3%]) causes. Nor were there any significant differences in the numbers of participants having non-fatal myocardial infarction or coronary death (1063 [10.4%] vs 1047 [10.2%]), non-fatal or fatal stroke (511 [5.0%] vs 518 [5.0%]), or coronary or non-coronary revascularisation (1058 [10.3%] vs 1086 [10.6%]). For the first occurrence of any of these "major vascular events", there were no material differences either overall (2306 [22.5%] vs 2312 [22.5%]; event rate ratio 1.00 [95% CI 0.94-1.06]) or in any of the various subcategories considered. There were no significant effects on cancer incidence or on hospitalisation for any other non-vascular cause. INTERPRETATION: Among the high-risk individuals that were studied, these antioxidant vitamins appeared to be safe. But, although this regimen increased blood vitamin concentrations substantially, it did not produce any significant reductions in the 5-year mortality from, or incidence of, any type of vascular disease, cancer, or other major outcome.
Article
Background. Epidemiologic evidence indicates that diets high in carotenoid-rich fruits and vegetables, as well as high serum levels of vitamin E (alpha-tocopherol) and beta carotene, are associated with a reduced risk of lung cancer. Methods. We performed a randomized, double-blind, placebo-controlled primary-prevention trial to determine whether daily supplementation with alpha-tocopherol, beta carotene, or both would reduce the incidence of lung cancer and other cancers. A total of 29,133 male smokers 50 to 69 years of age from southwestern Finland were randomly assigned to one of four regimens: alpha-tocopherol (50 mg per day) alone, beta carotene (20 mg per day) alone, both alpha-tocopherol and beta carotene, or placebo. Follow-up continued for five to eight years. Results. Among the 876 new cases of lung cancer diagnosed during the trial, no reduction in incidence was observed among the men who received alpha-tocopherol (change in incidence as compared with those who did not, -2 percent; 95 percent confidence interval, -14 to 12 percent). Unexpectedly, we observed a higher incidence of lung cancer among the men who received beta carotene than among those who did not (change in incidence, 18 percent; 95 percent confidence interval, 3 to 36 percent). We found no evidence of an interaction between alpha-tocopherol and beta carotene with respect to the incidence of lung cancer. Fewer cases of prostate cancer were diagnosed among those who received alpha-tocopherol than among those who did not. Beta carotene had little or no effect on the incidence of cancer other than lung cancer. Alpha- tocopherol had no apparent effect on total mortality, although more deaths from hemorrhagic stroke were observed among the men who received this supplement than among those who did not. Total mortality was 8 percent higher (95 percent confidence interval, 1 to 16 percent) among the participants who received beta carotene than among those who did not, primarily because there were more deaths from lung cancer and ischemic heart disease. Conclusions. We found no reduction in the incidence of lung cancer among male smokers after five to eight years of dietary supplementation with alpha-tocopherol or beta carotene. In fact, this trial raises the possibility that these supplements may actually have harmful as well as beneficial effects.
Article
Background: It has been suggested that increased intake of various antioxidant vitamins reduces the incidence rates of vascular disease, cancer, and other adverse outcomes. Methods: 20,536 UK adults (aged 40-80) with coronary disease, other occlusive arterial disease, or diabetes were randomly allocated to receive antioxidant vitamin supplementation (600 mg vitamin E, 250 mg vitamin C, and 20 mg beta-carotene daily) or matching placebo. Intention-to-treat comparisons of outcome were conducted between all vitamin-allocated and all placebo-allocated participants. An average of 83% of participants in each treatment group remained compliant during the scheduled 5-year treatment period. Allocation to this vitamin regimen approximately doubled the plasma concentration of alpha-tocopherol, increased that of vitamin C by one-third, and quadrupled that of beta-carotene. Primary outcomes were major coronary events (for overall analyses) and fatal or non-fatal vascular events (for subcategory analyses), with subsidiary assessments of cancer and of other major morbidity. Findings: There were no significant differences in all-cause mortality (1446 [14.1%] vitamin-allocated vs 1389 [13.5%] placebo-allocated), or in deaths due to vascular (878 [8.6%] vs 840 [8.2%]) or non-vascular (568 [5.5%] vs 549 [5.3%]) causes. Nor were there any significant differences in the numbers of participants having non-fatal myocardial infarction or coronary death (1063 [10.4%] vs 1047 [10.2%]), non-fatal or fatal stroke (511 [5.0%] vs 518 [5.0%]), or coronary or non-coronary revascularisation (1058 [10.3%] vs 1086 [10.6%]). For the first occurrence of any of these "major vascular events", there were no material differences either overall (2306 [22.5%] vs 2312 [22.5%]; event rate ratio 1.00 [95% CI 0.94-1.06]) or in any of the various subcategories considered. There were no significant effects on cancer incidence or on hospitalisation for any other non-vascular cause. Interpretation: Among the high-risk individuals that were studied, these antioxidant vitamins appeared to be safe. But, although this regimen increased blood vitamin concentrations substantially, it did not produce any significant reductions in the 5-year mortality from, or incidence of, any type of vascular disease, cancer, or other major outcome.
Article
Twelve squirrel monkeys were exposed to pure‐tone stimuli at intensities and durations sufficient to produce, in six, repeated temporary threshold shifts (TTSs) from 15 to 25 dB and, in the remaining six, permanent threshold shifts (PTSs) from 10 to 20 dB. Thresholds were determined by means of an avoidance conditioning procedure similar to that of Clack and Herman (1963), in which observation intervals were signaled by the houselight and the animals required to press a bar if an auditory signal was presented, and to withold response if not. Following final behavioral testing, animals were sacrificed and the effect of the exposures upon hair cells was determined. This was accomplished with surface preparation techniques and phase microscopy. The organ of Corti was examined along its length and the number of missing and visibly damaged hair cells was determined for each of the inner and outer rows from a point 1 mm from the apex to a point 17 mm from the apex. There was no clear evidence of hair cell damage due to the exposure. It is concluded that pure‐tone stimulation sufficient to produce PTSs of 10–20 dB may not produce anatomical injuries to the organ of Corti that are detectable by phase microscopy.
Article
Noise‐induced hearing loss (NIHL) is significantly greater in rats fed a magnesium‐deficient diet than in rats on a magnesium‐rich diet. The hearing loss was found to be negatively correlated with the magnesium concentration of the perilymph. It is suggested that also in man, the magnesium concentration in the perilymph may be of importance in determining susceptibility to NIHL.