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. Author manuscript; available in PMC: 2014 Nov 21.
Published in final edited form as: J Otol. 2013;8(1):63–71. doi: 10.1016/s1672-2930(13)50009-2

Ototoxic Model of Oxaliplatin and Protection from Nicotinamide Adenine Dinucleotide

Ding Dalian 1,2,3,4, Jiang Haiyan 1, Fu Yong 5, Li Yongqi 6, Richard Salvi 1, Shinichi Someya 7,✉, Masaru Tanokura 4,✉
PMCID: PMC4240522  NIHMSID: NIHMS594882  PMID: 25419212

Abstract

Oxaliplatin, an anticancer drug commonly used to treat colorectal cancer and other tumors, has a number of serious side effects, most notably neuropathy and ototoxicity. To gain insights into its ototoxic profile, oxaliplatin was applied to rat cochlear organ cultures. Consistent with it neurotoxic propensity, oxaliplatin selectively damaged nerve fibers at a very low dose 1 μM. In contrast, the dose required to damage hair cells and spiral ganglion neurons was 50 fold higher (50 μM). Oxailiplatin-induced cochlear lesions initially increased with dose, but unexpectedly decreased at very high doses. This non-linear dose response could be related to depressed oxaliplatin uptake via active transport mechanisms. Previous studies have demonstrated that axonal degeneration involves biologically active processes which can be greatly attenuated by nicotinamide adenine dinucleotide (NAD+). To determine if NAD+ would protect spiral ganglion axons and the hair cells from oxaliplatin damage, cochlear cultures were treated with oxaliplatin alone at doses of 10 μM or 50 μM respectively as controls or combined with 20 mM NAD+. Treatment with 10 μM oxaliplatin for 48 hours resulted in minor damage to auditory nerve fibers, but spared cochlear hair cells. However, when cochlear cultures were treated with 10 μM oxaliplatin plus 20 mM NAD+, most auditory nerve fibers were intact. 50 μM oxaliplatin destroyed most of spiral ganglion neurons and cochlear hair cells with apoptotic characteristics of cell fragmentations. However, 50 μM oxaliplatin plus 20 mM NAD+ treatment greatly reduced neuronal degenerations and hair cell missing. The results suggested that NAD+ provides significant protection against oxaliplatin-induced neurotoxicity and ototoxicity, which may be due to its actions of antioxidant, antiapoptosis, and energy supply.

Keywords: oxaliplatin, apoptosis, copper transporter, nicotinamide adenine dinucleotide

Introduction

Platinum-based antineoplastic drugs are widely used to treat cancer. The member of platinum-based agents includes cisplatin, carboplatin, oxaliplatin, satraplatin, nadaplatin, triplatin, etc[1]. The reason to summarize above drugs as platinum-based agents is because they are derived from the element platinum. The anti-tumor mechanism of platinum-based chemotherapeutic agents works by binding to DNA and forming cross links between the strands, and then inhibits DNA synthesis and eventually destroys the tumor cells [2]. Although platinum compounds are highly effective anti-tumor agents, their clinical usage is limited by a number of serious side effects, such as nephrotoxicity, neurotoxicity, hepatotoxicity, myelosuppression, neutropenia, gastroenteropathy, ototoxicity, etc[3-25]. However, besides those common side-effects, the major side effects of platinum compounds are different from each other. For example, cisplatin has severe nephrotoxic, neurotoxic, and ototoxic side effects[9, 12, 21, 23, 24, 26]. Carboplatin is far less ototoxic and nephrotoxic than cisplatin[5, 27, 28]. The major side effect of nedaplatin is myelosuppression[29]. In contrast, oxaliplatin is considered to be far less nephrotoxic and ototoxic than cisplatin; however, it frequently induces sensory neuropathies which is a condition reminiscent auditory neuropathy[30-33]. As the third-generation of platinum-based antineoplastic agent, oxaliplatin is extremely neurotoxic suggesting that it might preferentially damage spiral ganglion neurons, and might be a potential interesting model for study in axonal degenerations [16, 23].

Nicotinamide adenine dinucleotide, abbreviated NAD+, is a coenzyme with function of electron transfer actions. However, it is also helpful in many cellular processes. The most notable protective function of NAD+ is to delay the onset and extension of axonal degenerations by various neurodegenerative injuries, including traumatic injury, ischemia damage, autoimmune encephalomyelitis, p53-induced neuron apoptosis, radiation-induced immunosuppression, etc. [16, 23, 34-45].

Since axonal destruction was a remarkable feature in oxaliplatin-induced auditory nerve degenerations[16, 23], while NAD+ has been proved to protect against axonal degeneration caused by various neurotoxic agents, we hypothesized that NAD+ might also protect spiral ganglion axons and cochlear hair cells from oxaliplatin injury. To test this hypothesis, the cochlear organotypic cultures were treated with oxaliplatin or combined with NAD+ for evaluation of protective effects of NAD+.

Materials and Methods

Animal procedures

Sprague-Dawley rat pups on postnatal day 3 were purchased from Charles River Laboratories (Wilmington, MA) for this study. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of University at Buffalo, and conform to the guidelines issued by the National Institutes of Health.

Cochlear organotypic cultures

The preparations of cochlear organotypic cultures have been described in detail in our earlier publications[17, 46-48]. Briefly, rat pups were decapitated. The cochleae were carefully removed and placed in Hank's Balanced Salt Solution (1X GIBCO, 14175, Invitrogen, Carlsbad, CA). The cochlear basilar membrane containing the organ of Corti and spiral ganglion neurons was micro-dissected and transfered onto a collagen gel matrix. A droplet of 15 μl rat tail collagen (Type 1, BD Biosciences, 4236 Bedford, MA, 10× basal medium eagle, BME, Sigma B9638, 2% sodium carbonate, 9:1:1 ratio) was freshly made and placed in the center of a 35 mm diameter culture dish (Falcon 1008, Becton Dickinson) and allowed to gel at room temperature for about 30 minutes. Afterward, 1.3 ml of serum-free medium consisting of 2 g bovine serum albumin (BSA, Sigma A-4919), 2 ml Serum-Free Supplement (Sigma I-1884), 4.8 ml of 20% glucose (Sigma G-2020), 0.4 ml penicillin G (Sigma P-3414), 2 ml of 200 mM glutamine (Sigma G-6392), and 190.8 ml of 1X BME (Sigma B-1522) were added to the culture dish. The cochlear explants were placed as a flat preparation on the surface of the collagen gel, and the surface of cochlear explants was exactly even with the culture medium. Cochlear explants were cultured overnight for recovery (Forma Scientific 3029, 37oC in 5% CO2). On day 2, the culture medium was removed and replaced with fresh medium.

Treatment to cochlear explants

After overnight conditioning, cochlear explants were treated with oxaliplatin with various doses ranging from 10 μM to 5000 μM for 48 h for detection of ototoxic dose-response of oxaliplatin. According to our previous experiments dealing with NAD+ for otoprotections, the concentration of NAD+ at 20 mM was efficient for protection to axon degenerations in cochlear organotypic culture system[17, 44, 45]. To test if NAD+ can also protect the auditory neurons and cochlear hair cells from oxaliplatin-induced damage, 20 mM NAD+ was selected for protection against 10 μM or 50 μM oxaliplatin treament. In addition, control cochlear explants were cultured with standard serum-free medium, and run concurrently with the experimental samples.

Histology

At the end of experiment, the cochlear explants were fixed with 10% phosphate buffered formalin for 1 h. After fixation, specimens were rinsed in 0.01 M PBS, incubated overnight (4 oC) in solution containing 20 μl of mouse anti-neurofilament 200 kD antibody (Sigma N0142, clone N52), 20 μl Triton X-100 (10%), 6 μl normal goat serum, 154 μl of 0.01 M PBS. Specimens were then rinsed with 0.01 M PBS, immersed in a solution containing 2 μl of secondary anti-mouse IgG TRITC (Sigma T5393), 12 μl normal goat serum, 40 μl Triton X-100 (10%) and 345 μl of 0.01 M PBS, rinsed again with 0.01 M PBS for labeling of spiral ganglion neurons and auditory nerve fibers. Afterwards, specimens were double stained with Alexa Fluor 488 phalloidin (Invitrogen A12379) for 30 minutes to label the stereocilia and cuticular plate of hair cells. Specimens were rinsed in 0.01M PBS, and mounted on glass slides in glycerin, coverslipped and examined using a confocal microscope (Zeiss LSM-510) with appropriate filters for TRITC (absorption: 544 nm, emission: 572 nm) , and for Alexa Fluor 488 (excitation 495 nm, emission 519 nm). Images were stored on a PC computer and evaluated with software (Zeiss LSM Image Examiner, Adobe Photoshop).

To quantify the neurotoxic activity of oxaliplatin and protective efficiency of NAD+ against oxaliplatin neurotoxicity, the numbers of morphological intact auditory nerve fibers projecting to the cochlear hair cells were counted for the control group, oxaliplatin treated groups, and oxaliplatin (10 μM and 50 μM) plus NAD+ (20μM) treated groups in a determinate square region under a fluorescent microscope on magnification of 630 tines with a diameter of 143 μm. Counts were obtained from 6 cochlear explants (n=6) from each experimental condition. Three separate counts at each location produce an average number to reflect the density of auditory nerve fibers in this location for this cochlea. The statistical analysis was using a one-way ANOVA followed by Newman-Keuls post hoc analyses (GraphPad Prism 5 software) [44, 47].

Results

Ototoxic dose-response of oxaliplatin

48 h after culture, cochlear hair cells and auditory nerve fibers present normal in controls which was cultured with standard culture medium (Fig. 1A). Cochlear hair cells were also intact with the low doses of oxaliplatin treatment (1 μM and 10 μM) (Fig. 1B, 1C). However, the number of auditory nerve fibers began to decrease in comparision with controls (Fig. 1b, 1C, and Fig. 3). When the concentration of oxaliplatin was increased to 50 μM, most hair cells and auditory nerve fibers were destroyed (Fig. 1D). 100 μM oxaliplatin resulted complete destruction in both cochlear hair cells and auditory nerve fibers (Fig. 1E). However, some survival hair cells with weak phalloidin signals and fragmented auditory nerve fibers were seen 48 h after 500 μM oxaliplatin treatment (Fig. 1F). Interestingly, the number of survival cochlear hair cells shown a tendency to increase 48 h after 1000 μM or 5000 μM oxaliplatin treatment, even though the labeling of phalloidin on stereocilia was wearing off, but the profile of cuticular plate in some hair cells were still visible (Fig. 1G, 1H). This may reflect that high dose of oxaliplatin can damage the stereocilia bundles, but remaining the body of hair cells. Unexpectedly, many auditory nerve fibers present 48 h after the highest concentration of oxaliplatin treatment (Fig. 1H).

Figure 1.

Figure 1

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Photomicrographs show cochlear organotypic cultures 48 h after oxaliplatin treatment. Oxaliplatin concentration is shown in each panel. Cochlear hair cells were intact in control (A), 1μM oxaliplatin (B) and 10μM oxaliplatin treated cochlear explants (C). However, the density of auditory nerve fibers began to decrease 48 h after 1μM or 10 μM oxaliplatin treatment (B, C). 50 μM and 100 μM oxaliplatin treatment destroyed most cochlear hair cells and auditory nerve fibers (D, E). Some survival hair cells were seen when the concentration of oxaliplatin increased to 500 μM or higher, but the labeling of stereocilia by phalloidin was very weak (F, G, H). The auditory nerve fibers appear again 48 h after 5000 μM oxaliplatin treatment (H)

Figure 3.

Figure 3

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Comparison of the mean number of auditory nerve fibers for each treatment condition reviled that moderate concentration of oxaliplatin (50 μM, and 100μM) resulted in most heavy damage to auditory nerve fibers. *Significantly different from control (P < 0.05).

A similar unusual concentration-damage response was also seen in the soma of spiral ganglion neurons that neuronal damage increased at moderate doses (50 μM, 100 μM, 500 μM, and 1000 μM) (Fig. 2A-2G), but remained intact at highest concentration of 5000 μM (Fig. 2H).

Figure 2.

Figure 2

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Photomicrographs show spiral ganglion neurons 48 h after oxaliplatin treatment. Oxaliplatin concentration is shown in each panel. Spiral ganglion neurons present normal in control (A), 1μM oxaliplatin (B) and 10μM oxaliplatin treated cochlear explants (C). Spiral ganglion neurons were condensed or fragmentated by oxaliplatin treatmnent with doses of 50 μM, 100 μM, 500 μM, or 1000 μM (D, ED, F, G). In contrast, the body of spiral ganglion neurons was quite close to the normal value 48 h after 5000 μM oxaliplatin treatment, although the size of soma seems smaller than control (H).

The quantifications of auditory nerve fibers were as shown in Figure 3. Clearly, oxaliplatin can damage auditory nerve fibers as low as 1 μM, and the most toxic effects were in the moderate concentrations of 50 μM and 100 μM. However, the damage to auditory nerve fibers was significantly reduced when the concentration of oxaliplatin reached the highest dose, 5000 μM.

Protective effects of NAD+ against oxaliplatin

10 μM oxaliplatin treatment for 48 h resulted in a minimum damage to auditory nerve fibers in comparision with control cochlea (Fig. 4A, 4B). When the cochlear explants were treated with 10 μM oxaliplatin plus 20 mM NAD+ for 48 h, the auditory nerve fibers present normal (Fig. 4C). Treatment of 50μM mefloquine for 48 h resulted in a large loss of cochlear hair cells and auditory nerve fibers (Fig. 4D). Additional NAD + at concentration of 20mM rescued many cochlear hair cells and auditory nerve fibers (Fig. 4E). The quantitative data shows about 20% decrease of auditory nerve fibers in 10 μM oxaliplatin treated cochlear explants, while additional 20 mM NAD+ protected auditory nerve fibers completely (Fig. 4F). 50 μM oxaliplatin destroyed about 80% auditory nerve fibers, however, the survival auditory nerve fibers were increased to 80% by additional NAD + treatment (Fig. 4F).

Figure 4.

Figure 4

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Cochlear hair cells (green fluorescence) and auditory nerve fibers with spiral ganglion neurons (red fluorescence) present normal 48 h after culture with standard culture medium (A). 10 μM oxaliplatin treatment for 48 h did only minimal damage to auditory nerve fibers (B). Cochlear hair cells and auditory nerve fibers were intact 48 h after 10 μM oxaliplatin plus 20 mM NAD+ treatment (C). 50 μM oxaliplatin destroyed most cochlear hair cells and auditory nerve fibers (D). However, additional 20 mM NAD+ rescued many cochlear hair cells and auditory nerve fibers (E). Comparison of the mean number of auditory nerve fibers for each treatment condition reviled that additional 20 mM NAD + treatment significantly rescued the auditory nerve fibers (F). *Significantly different from control (P < 0.05). **Significantly different from oxaliplatin treatment alone (P < 0.05).

As shown in figure 4B, 10 μM oxaliplatin caused minimum damage to auditory nerve fibers, but it did not damage the soma of spiral ganglion neurons (Fig. 5A). This suggested that oxaliplatin-induced damage to auditory peripheral neurons begins with auditory nerve fibers. Therefore, again the morphology of spiral ganglion neurons treated with 10 μM oxaliplatin plus 20 mM NAD+ without any noticeable injuries (Fig. 5B). However, when the concentration of oxaliplatin increased to 50 μM, most spiral ganglion neurons were characteristic of apoptosis, cell condensation or fragmentation (Fig. 5C). In contrast, additional 20 mM NAD+ treatment rescued most spiral ganglion neurons from apoptosis (Fig. 5D).

Figure 5.

Figure 5

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Spiral ganglion neurons were intact in both 10 μM oxaliplatin or 10 μM oxaliplatin plus 20 mM NAD+ treated cochlear explants (A, B). 50 μM oxaliplatin treatment for 48 h caused death of spiral ganglion neurons with characteristics of apoptosis, cell shrinkage and fragmentation (C). Additional 20 mM NAD + treatment greatly reduced the apoptosis characteristics.

Discussion

As a member of platinum-based chemotherapeutics, oxaliplatin has similar antineoplastic effects and toxic mechanisms with other platinum reagents, such as cisplatin. However, its neurotoxic effects have attracted wide attention[29-32, 49-53]. In current study, degeneration to axons and cochlear hair cells were observed after oxaliplatin treatment in cochlear culture system. The results have established that the auditory nerve axons are more vulnerable to oxaliplatin than the hair cells. Because the dose required to damage hair cells and soma of spiral ganglion neurons was 50 folds higher than the dosage required to destroy auditory axons. This is consistent with previous reports that oxaliplatin is in preference to target the nervous system[16, 23, 50, 52, 54].

As a general rule in toxicity, the more toxic chemicals the more severe damage to the tissue. However, we found that oxaliplatin-induced degeneration of auditory axons and sensory hair cells was greater following exposure to moderate doses of oxaliplatin (50 μM, and 100 μM). Unexpectedly, the highest dose of oxaliplatin (5000 μM) did not cause severe damage to auditory axons. Similarly, cochlear hair cells were completely destroyed with moderate dose treatment (100 μM), whereas the survival hair cells were increased by higher dose of oxaliplatin treatment (1000 μM). This unique phenomenon is very similar to our previous findings in other platinum based chemotherapeutics, such as cisplatin, carboplatin, and nedaplatin[11, 12, 17-21, 23-25]. Evidence suggested that cells can detect their surrounding environment, and adjust the particular channels for chemical import and / or export according to intracellular dynamic balance[12, 16, 17, 20, 21, 23-25, 55-60]. It has been proved that extracellular platinum agents do not damage cells. To exert their toxic effects, platinum agents must enter the cell first. For example, cisplatin can be activated once it enters the cytoplasm when the chloride atoms on cisplatin are displaced by water molecules. The aquatic cisplatin becomes potent electrophile and then react with nucleic acid to target DNA. In addition, previous studies have demonstrated that when cisplatin is transported into the cell, it can bind with glutathione, and then becomes a cisplatin-glutathione complex to exert its toxic effects[61]. Therefore, the entry of platinum agents is the first important step responsible for its following intracellular toxic effects. The platinum import is via copper transporter importer, Ctrl, and platinum export is controlled by copper transporter exporters, ATP7A and ATP7B[11, 12, 17, 19-21, 23, 24, 59, 60, 62-68]. In normal circumstances, copper / platinum homeostasis is regulated stringently by copper transporters. If the extracellular concentration of copper or platinum was low, it may be easier to be introduced into the cell by Ctrl. But if the concentration of copper or platinum was extremely high in extracellular environment, the cell may modulate its copper transporters by increasing export and decreasing import to accomplish the purpose of self-protection[11, 12, 16-21, 23-25, 59, 60]. This magic response to toxic effects of platinum agents was investigated in vitro experimental models, because the concentration of platinum agents can be simply adjusted to a high level in culture conditions that can arouse cell's alert[11, 12, 16-21, 23, 25, 60]. However, we have demonstrated that local application of copper sulfate via round window membrane can also efficiently stimulate copper transporters for the reaction of self-protection in vivo[24, 59]. Therefore, regulating copper transporters may be a promising approach to keep cells away from platinum drug-induced injury.

The most prominent ototoxic mechanism of platinum-basedreagentsareinvolvedincellapoptosis[8-10,12,14,17,21,23,24, 69-75]. According to extensive literatures, cell apoptosis becomesapparentinnuclearshrinkagewhichisalwaysassociated with positive TUNEL labeling and/ or caspase activations[8,9,12,17,21-24,48,76-80]. Consequently, nuclear shrinkage, condensationandfragmentationhavebeenidentified as characteristic of apoptosis. In current study, oxaliplatin-induced death of spiral ganglion neurons was characterized by apoptosis with evident cell fragmentations (Fig. 3 and Fig. 5). This is in conformity with previous literatures.

Another toxic factor, reactive oxygen species or free radicals also play an important role in platinum-induced toxicities[12, 81-89]. Besides the direct damage to cells from excessive reactive oxygen species or free radicals, most importantly, the oxidative stress can result in cell apoptosis. For example, immoderate reactive oxygen species can induce protein oxidation, disrupt protein synthesis, alter cytoskeletal components, attack DNA, disrupt ionic balance, interfere with cellular signaling, break calcium homeostasis, damage DNA repair, and interfere with transcription processes etc, which triggers the apoptotic signals to start the programmed cell death[90-93].

NAD+ and its relative NAD phosphate (NADP+) play essential role in many biochemical reactions, especially redox reactions in which oxidoreductase enzymes transfer hydrogen and electrons from one reaction to another in the respiratory chain in the cell. However, a growing evidences support the fact that NAD metabolism regulates many important biological effects including life span. NAD + , through poly-ADP-ribosyl polymerase (PARP), mono-ADP-ribosyltransferase (ARTs) and recently characterized sirtuin enzymes, exerts potential biological effects. NAD+ is also involved in adding or removing chemicals from proteins as a substrate of enzymes in a manner of posttranslational modifications. In addition, the antioxidant effect of NAD+ or NAD+ dependent enzymes could be also as a protective function against cell injury[35, 44, 45]. Many studies have demonstrated that a decrease in axonal NAD+ is common in degenerating axons triggered by various mechanical and chemical insults[38-40]. Lacking of NAD+ may also be involved in oxaliplatin-induced axon degenerations in the cochlea. NAD-dependent deacetylase activity of Sirt1 could regulate death/survival decision against p53 apoptotic pathway in mammalian cells[34, 94-96]. As a crucial substrate of Sir2 (silent information regulator 2), NAD+ has been implicated in influencing for diverse biological roles including gene silencing, DNA damage repair, cell cycle regulation, and life span extension by anti-apoptosis[34, 94-100]. Deacetylase stress was recognized as a key molecule for cellular metabolism in organisms. NAD + serves as cofactor with dehydrogenases for both aerobic and anaerobic ATP generation. In addition, NAD+ acts as an important substrate for protein of PARP [poly (ADP-rebose) polymerase], which is a protein involved in a number of cellular processes. Especially, PARP is in immediate cellular response to apoptosis, involving mainly DNA repair and against programmed cell death. Therefore, one of the most important functions of PARP is to detect and repair the single strand DNA breaks against caspase cleavage in apoptosis, which requires NAD+ as a the essential substrate[101-103]. Considering evident fact of decrease of NAD+ always associate with axon degeneration in various experimental models[38-40], when the level of NAD+ was decreased in the cell, it may trigger multiple apoptotic signaling pathways to start the cell apoptosis. In contrast, increase of NAD+ can prevent axonal NAD + decline efficiently that can protect axons from degeneration[38-41]. In current study, the survival rate of auditory nerve fibers was significantly increased when the cochlear explants were treated with NAD+ which efficiently protected auditory axons from oxaliplatin neurotoxicity. These findings are consistent with previous studies showing that axonal degenerations can be prevented or protected by NAD+ in various experimental models[38, 39, 41]. Surprisingly, we found that NAD+ can also prevent cochlear hair cell damage from oxaliplatin in current study. As particular sensorineural epithelium, cochlear hair cells are directly connected to auditory afferent synaptic junctions, which are characteristic of neurons, such as secreting neurotransmitters that might be injured by oxaliplatin similarly to its neurotoxic effects with decrease of NAD+. The decreased ATP level was always being found in parallel with NAD+ reduction in degenerating cells[39]. Therefore, NAD+ depletion may also impair energy production that triggering the onset of cell apoptosis. However, the exogenously providing NAD can effectively delay the decrease of ATP levels in degenerating cells[39]. Importantly, additional NAD + treatment can activate Sirt1 and Sirt2 which are positive regulators for anti-apoptosis. Therefore, the protective effects of NAD+ to auditory axons and cochlear hair cells against oxaliplatin may be also benefited from mobilizing of intrinsic anti-apoptotic reactions. Taking all factors into consideration, the efficient protective effects of NAD+ against oxaliplatin-induced cochlear degeneration may benefit from antioxidant effects, anti-apoptotic effects, and energy supply from additional NAD+ treatment.

Contributor Information

Shinichi Someya, Email: someya@ufl.edu.

Masaru Tanokura, Email: amtanok@mail.ecc.u-toky.ac.jp.

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