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Reactive oxygen species are important elements in ototoxic damage to hair cells (HCs), appearing early in the damage process. Higher levels of natural antioxidants are positively correlated with resistance to ototoxins and many studies have shown that exogenous antioxidants can protect HCs from damage. While a very wide variety of antioxidants with different characteristics and intracellular targets exist, most ototoxicity studies have focused upon one or a few well-characterized compounds. Relatively little research has attempted to determine the comparative efficacy of large variety of different antioxidants. This has been in part due to the lack of translation between cell culture and
• A medium-throughput assay based on micro-explants of the organ of Corti was developed to screen mammalian cochlear hair cells for protection from damage by ototoxins.
• Eighty one antioxidants and 3 pro-oxidants were evaluated for hair cell protection from high-dose gentamicin.
• Thirteen antioxidants were significantly protective, while 6 proved to be damaging.
• The use of a common assay permitted an evaluation of the relative capacity of different antioxidants for the protection of hair cells.
Ototoxicity, hearing loss and vestibular disorders are significant side effects of a number of valuable medications. This includes important categories of drugs used to treat life-threatening illnesses, such as aminoglycoside antibiotics and platinum-based anti-neoplastic agents. Hearing loss due to aminoglycosides is estimated to occur in almost 50% of patients (
The most vulnerable elements of the inner ear to ototoxic drugs are the sensory HCs (
This success in animal experiments has led to a limited number of clinical trials that have evaluated the effects of antioxidant treatment on ototoxic or noise-induced HC and hearing loss.
The reasons for the variability in clinical trial results are unclear. However, the degree of experimental control in clinical trials is much less than that in animal studies. Another possibility is that the antioxidant dose actually reaching the cochlea after systemic administration may be non-optimal. Moreover, different antioxidants can exert their effects via several distinct mechanisms and targets. This includes scavenging the radical species that initiate peroxidation, quenching singlet oxygen, chelating metals, breaking free radical chain reactions, reducing the concentration of O2, preventing oxidation of proteins or DNA, and/or stimulating endogenous antioxidant enzymes (
It should also be noted that while many antioxidants have been tested for their ability to protect HCs, there are many other compounds with antioxidant properties. Moreover, few studies have compared HC protection by antioxidants in a standardized model, so that relative effectiveness can be estimated. For the reasons noted above, differences in their potential for HC protection seem likely. Hence, a comparative screen of a large number of antioxidants using a standardized model of HC damage could identify novel antioxidants with protective properties and might also provide evidence regarding differences in protective efficacy.
To address these issues, we screened a commercial library of antioxidants in a single model of aminoglycoside-induced HC damage. Micro-explants from the neonatal murine oC were exposed to a high dose of the ototoxic aminoglycoside gentamicin to elicit oxidative stress. We used a transgenic mouse line in which HCs express GFP under the control of a HC-specific promoter (
Experiments were performed on transgenic animals in which eGFP was selectively expressed in HCs under the control of a
The oC was dissected from the cochleas of postnatal day 3–5
Screening was performed using the Screen-Well Redox Library (BML-2835, Enzo Life Sciences, Farmingdale, NY, United States). The library consists of 81 antioxidant and 3 pro-oxidant compounds. The library represents a variety of classes of compounds, including some that have been shown previously to protect HCs from damage. This includes glutathione (
Each experimental oC micro-explant was pretreated for 24 h with one of the library compounds at concentration of 10, 100, or 1000 μM, performed in triplicate wells. The next day, the media were withdrawn, fresh media containing 200 μM gentamicin plus the pharmacological compound with the appropriate concentration was added, and the micro-explants were cultured for 72 h. Untreated (negative) controls were maintained in media alone and positive controls were treated with 200 μM gentamicin alone. Media for both control groups contained 0.1% DMSO, to match the experimental groups. Compounds were screened in duplicated 96-well plates with seven compounds per plate, plus controls. The size of the testing plates and performance of experiments on different days and with different media or gentamicin batches required replication of both control conditions on each plate, for comparison with the results from the compounds evaluated on that plate.
Green florescent protein-positive HCs were imaged by fluorescence microcopy on each day of treatment, and survival curves were generated for each compound and condition. HC counts, including both inner and outer HCs, were evaluated in ImageJ, and normalized as percentages to the number of HCs present on D1, prior to the start of gentamicin treatment. Any micro-explants that did not attach and flatten in the well by D1 were excluded (usually less than 3% per plate), because HC counts could not be accurately quantified at that time. There were sufficient wells on each plate that three micro-explants per condition could almost always be accommodated even with some unattached samples.
Statistical analysis was performed using GraphPad Prism 6, StatView 5, using the Kruskal–Wallis non-parametric ANOVA to detect treatment effects. Individual condition comparisons were performed using the Mann–Whitney
Redox compound “hits” were identified in the initial round of screening as deviating significantly from the controls. Following this initial identification, repeat plates were prepared in an identical manner for all hits, for a total N of 6 micro-explants. Statistical analysis was then repeated. Hits that demonstrated a repeatable effect in the following round were considered to be confirmed.
Imaging of GFP-positive HCs in control wells typically showed HC survival similar to that illustrated in
Control cultures. Montage of a typical negative control oC micro-explant
Control culture cell counts. All of the positive and negative control oC micro-explants from all plates were counted and the results pooled to illustrate the control survival curves between D1 and D4. Data points represent medians, and error bars present the interquartile range.
Of the 81 antioxidants and 3 pro-oxidants in the redox library, 68 antioxidants and 2 pro-oxidants had no effect on either untreated or gentamicin-treated micro-explants. Two examples of antioxidants with no effect are illustrated in
Normalized D1-4 HC survival curves for two antioxidants (canthatraxin and terbinafine HCl) that had no significant effect on normal or gentamicin-treated micro-explants. HC survival with 1000 μM of the compound alone (open green circles) was not significantly different from an untreated, negative control micro-explant (open black circles). Treatment with the compound at 10 μM (solid red circles), 100 μM (solid blue) or 1000 μM (solid green) plus 200 μM gentamicin was not significantly different from 200 μM gentamicin alone (solid black).
Montages illustrating representative oC micro-explants treated with 1000 μM canthatraxin alone, or 100 μM terbinafine HCl plus 200 μM gentamicin, for 72 h.
Thirteen antioxidants exhibited statistically significant protection of HCs in micro-explants treated with both the compound and gentamicin. The results for these compounds are illustrated in
Montages illustrating representative micro-explants treated with 100 μM seratrodast plus 200 μM gentamicin or 1000 μM hinokitiol plus 200 μM gentamicin, for 72 h.
The pro-oxidant β-lapachone and the antioxidants disuliram, ferulic acid ethylester, gossypol, gentisisc acid and caffeic acid were significantly toxic to HCs in the absence of gentamicin, although none significantly worsened gentamicin-induced HC damage. HC survival curves for the pro-oxidant and two damaging antioxidants are presented in
Normalized D1–4 HC survival curves for 1 pro-oxidant (β lapachone) and 2 representative antioxidants (ferulic acid ethylester and gentisisc acid). When each of these compounds was applied to micro-explants in the absence of concurrent gentamicin, far more HC damage was observed than in untreated (negative controls) micro-explants.
Montages illustrating representative oC micro-explants treated with 1000 μM β-lapachone alone, or 1000 μM ferulic acid ethylester alone, for 72 h, illustrating HC toxicity without gentamicin.
We have developed an assay based on micro-explants of the neonatal mouse oC to screen a variety of antioxidants for their ability to alter aminoglycoside damage to mammalian cochlear HCs
Many assays for the evaluation of compounds on HCs have been developed. The assay presented here, like any preclinical assay, has both advantages and disadvantages. A key advantage of the micro-explant screening assay is that it employs mammalian HCs rather than HCs from different animal classes or mammalian cell lines. This is important since mammalian HCs, and especially cochlear HCs, are quite different from the HCs of different animal classes such as birds or fish. The outer HC, the most vulnerable element in the mammalian cochlea, is not present in other classes of animals. HCs are of course quite different from mammalian cell lines. Thus, it might be argued that an assay based on the mammalian oC gives results more applicable to humans.
Another advantage of the model is the ability to evaluate the effects of a much larger number of compounds than can be achieved with an
Of course, there are also disadvantages to this assay system. Since adult HCs do not survive in culture, the assay is based on neonatal HCs that are not yet functionally mature. They may respond differently to gentamicin or to antioxidants than adult HCs. In addition, the number of compounds that can be tested is limited. Thus, screening very large compound libraries is beyond the capacity of our method. Similarly, including a very large number of conditions, such as a large range of gentamicin dosages, would be difficult when also varying compound concentrations. Finally, this is a screening assay, which as with all screens does not provide definitive data, but rather identifies candidates that warrant further study. These limitations must be considered when interpreting the results of the assay.
A relatively small number of antioxidants among those tested proved to be protective to HCs. The range of protection varied from nearly complete to modest. There was no single category of antioxidant that proved to be superior to others. Several different categories of antioxidants were represented in the protective compounds. Moreover, often some antioxidants of the same class were found to have no effect on HC survival, or even in a few cases to be harmful. Hence, the mechanism of protection is believed to be associated with their antioxidant capacity but is not fully understood.
Seratrodast is a quinone antioxidant, and was one of the most protective compounds identified in the screen. It has not previously been studied as a protective agent for HCs. Seratrodast acts as a free radical scavenger. It is not only an antioxidant, but also a blocker of the thromboxane A2 receptor and is used in the treatment of asthma. While thromboxane A2 has been implicated in vascular disorders of the inner ear (e.g.,
Idebenone, is a quinone antioxidant and a synthetic analog of co-enzyme Q. It is a free radical scavenger that was also highly effective in protecting against high-dose gentamicin-induced HC damage in the assay.
Resveratrol is a naturally occurring stilbene polyphenolic antioxidant that acts as a free radical scavenger. It was very effective in protecting HCs from gentamicin toxicity. A number of studies have shown resveratrol to be protective against various forms of HC damage including cisplatin (
Butylated hydroxyanisole is a phenolic antioxidant and potent free radical scavenger that is often used as a food additive to prevent oxidative damage, especially to lipids. It was a very effective HC protectant against high-dose gentamicin. BHA has not previously been studied as a HC protectant. Reports that BHA is a carcinogen at high levels have since been repudiated, but they led to limits on permissible BHA levels and a decline in BHA use in some foods from the 1990s (
DL-α-lipoic acid is a sulfur-containing antioxidant. It is a free radical scavenger and metal chelator that also enhances intracellular levels of glutathione (
Hinokitiol (β-thujaplicin) is a naturally occurring antioxidant, found in the heartwood of certain plants. It acts as a metal chelator and, also, enhances the activity of superoxide dismutase (
Butylated hydroxytoluene is a phenolic antioxidant and potent free radical scavenger that, like BHA, is a frequent food additive. It was moderately effective in the prevention of HC damage in the assay. It has not previously been studied as a HC protectant.
Dithiotreitol is a thiol-containing, reducing agent that is a free radical scavenger and metal chelator. It was moderately protective in the assay. It has not previously been evaluated for its ability to protect HCs.
MC-186 (MCI-186; edaravone) is a non-phenolic antioxidant. It is a potent free-radical and protein carbonyl scavenger and inhibitor of lipid peroxidation that is used in clinical trials for the treatment of chronic obstructive pulmonary disorder. It was moderately protective against high-dose gentamicin damage to HCs. Several previous studies have shown MC-186 to be protective against various forms of HC damage (e.g.,
Procysteine (
Trolox is a water-soluble, short chain vitamin E analog. It is a potent free radical scavenger. It was modestly protective in the assay. It has previously been shown to slightly inhibit HC damage due to gentamicin
Thiourea is a thiol-containing reducing agent and free radical scavenger. It was modestly protective in the assay. Thiourea is toxic when administered at high systemic dosages. It preferentially inhibits the peroxidase in the thyroid gland and thus inhibits thyroxine production. The reduced synthesis of thyroid hormone causes an increased pituitary secretion of thyreotropic hormone and so hyperplasia of the thyroid which, on continuous stimulation in animals, can lead to tumor formation and, in man, to various thyroid-treated illnesses (
Thymoquinone is a quinone antioxidant. It was minimally effective in our assay, showing protection only after 24 h of gentamicin treatment, and only at the lowest dosage. It is therefore possible that this represents a false positive. However, thymoqunone has previously been shown to protect HCs against damage due to gentamicin (
It should be noted that some of the antioxidants that failed to exhibit protection have been shown in previous studies to protect HCs and hearing. Consequently, their failure to provide protection in our assay was unanticipated and surprising. Nonetheless, there are many potential explanations for these differences. They may be related to the relatively high concentration of gentamicin employed in our assay (200 μM), which presumably provided a very strong oxidative stress response. Differences could also be related to the
The range of protective and toxic responses observed in the assay illustrates the complexity of antioxidative compounds and their underlying mechanisms. Antioxidant effectiveness depends on several important intrinsic factors: permeability, activation energy, rate constants, molecular stability, oxidation–reduction potential, and solubility (
As noted above, antioxidants also vary considerably in their mechanisms. These include scavenging the species that initiate peroxidation, quenching singlet oxygen, metal chelating, interrupting free radical chain reactions, and reducing oxygen concentrations (
VN, AK, and AR designed, performed the experiments and wrote the manuscript. VN, KP, and RJ performed the studies.
AR is a co-founder of, shareholder in and consultant to Otonomy, Inc. which develops slow-release therapeutics for the treatment of ear disease. This relationship has been approved by the Committee on Conflict of Interest at UCSD. The company played no role in this research. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Julie Lightner proofed the manuscript and provided valuable editorial assistance.
The Supplementary Material for this article can be found online at:
butylated hydroxyanisole
butylated hydroxytoluene
dithiotreitol
green florescent protein
hair cells
nuclear factor kappa B
organ of Corti
hydroxyl
reactive oxygen species