Abstract
Objective
To determine the effects of concurrent treatment with gentamicin and the mitochondria-targeted antioxidant mitoquinone (MitoQ; which may prevent gentamicin ototoxicity) on change in the mitochondrial membrane potential (Δψm), a precursor of apoptosis.
Study Design
Prospective and controlled.
Setting
Academic research laboratory.
Subjects and Methods
LLC-PK1 (Lilly Laboratories Culture–Pig Kidney Type 1) and HEI-OC1 (House Ear Institute Organ of Corti 1) cells—renal and auditory cell lines, respectively—were used in this study. Δψm was assessed by flow cytometry through the MitoProbe JC-1 Kit for Flow Cytometry in untreated LLC-PK1 and HEI-OC1 cells and cells exposed to low- (100µM) or high- (2000µM) dose gentamicin for 24 hours, with and without 0.5µM each of MitoQ or idebenone (IDB; an untargeted ubiquinone).
Results
Δψm was not different in untreated LLC-PK1 cells and cells coincubated with low-dose gentamicin and MitoQ or IDB (P > .05). In HEI-OC1 cells, coincubation with low-dose gentamicin and MitoQ decreased Δψm (P = .002). Coincubation of LLC-PK1 cells with high-dose gentamicin and DMSO, MitoQ, or IDB depolarized Δψm (P < .0001), with MitoQ depolarizing the Δψm to a greater extent than that of IDB (P = .03). In contrast, HEI-OC1 cells demonstrated a hyperpolarized Δψm when coincubated with high-dose gentamicin and DMSO, MitoQ, or IDB (P < .001).
Conclusion
The combination of gentamicin and MitoQ holds the potential to disrupt Δψm. This suggests a heightened need to monitor for toxicity in patients receiving both agents.
Aminoglycoside (AG) antibiotics are a common therapy used to treat aerobic gram-negative bacterial infections, the side effects of which include ototoxicity and nephrotoxicity. 1 AG use can result in cochleotoxicity and vestibulotoxicity, which cause permanent hearing loss in patients.1-3 The ototoxic effects of AGs, such as hearing loss, are irreversible in humans. This poses a problematic and permanent side effect from AG antibiotics despite their efficacy and cost-effectiveness. Thus, development of a protective agent against AG-induced hearing loss will provide a suitable prophylactic therapy against adverse side effects and long-term complications.
AG ototoxicity is thought to be caused by an increased production of reactive oxygen species (ROS) that result in hair cell death.1,2 Consequently, antioxidants or free radical scavengers, such as D-methionine, vitamin E, and aspirin, have been evaluated as potential agents to protect against AG ototoxicity.4-7 Since mitochondria are a major source of ROS, we have been evaluating the utility of mitoquinone (MitoQ), a mitochondria-targeted derivative of the antioxidant ubiquinone,8-10 as a protective agent against AG toxicity.11-13 Oral supplementation with MitoQ attenuated gentamicin-induced hearing loss in guinea pigs. 12 However, we observed a potential toxicity when gentamicin and MitoQ were injected simultaneously in guinea pigs. Since antioxidants are considered generally safe and MitoQ has been shown to be safe in mice, rats, and humans,10,14 this observation was unexpected. As patients can receive an antioxidant and an AG antibiotic simultaneously, the goal of the present study was to determine the effects of concurrent treatment with the AG antibiotic gentamicin and the mitochondria-targeted antioxidant MitoQ on mitochondrial membrane potential (Δψm), a precursor to apoptosis.
Methods
Cell Culture
Lilly Laboratories Culture–Pig Kidney Type 1 (LLC-PK1) and House Ear Institute Organ of Corti 1 (HEI-OC1) renal and auditory epithelial cell lines, respectively, were used for all the experiments in this study. LLC-PK1 cells were purchased from the American Type Culture Collection (Bethesda, Maryland), and HEI-OC1 cells were generously provided by Dr Federico Kalinec (House Ear Institute, Los Angeles, California). HEI-OC1 cells show evidence of apoptosis when exposed to known ototoxic drugs, such as AG antibiotics and cisplatin. 15 The LLC-PK1 cells also demonstrate evidence of apoptosis when exposed to ototoxic drugs such as gentamicin. 16
HEI-OC1 and LLC-PK1 cells were both plated on 100-mm petri dishes containing Dulbecco’s modified Eagle’s medium (Cellgro Mediatech Inc, Manassas, Virginia) with 4.5 g/L of glucose and L-glutamine without sodium pyruvate and 10% fetal bovine serum (Cellgro Mediatech Inc). HEI-OC1 cells were maintained in a humidified incubator at 33°C with 10% CO2, while LLC-PK1 cells were maintained in humidified incubator at 37°C with 5% CO2, respectively. MitoQ and idebenone (IDB), an untargeted ubiquinone, were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, Missouri). The final concentration of DMSO in all treatments was 0.05%, and the DMSO-treated cells were used as solvent control.
Assessment of Δψm
After about 75% confluent, cells were incubated in media containing either 0.05% DMSO (solvent control) or 0.5µM MitoQ and IDB. After 24 hours, growth medium was replaced with either new medium for untreated cells or with medium containing 100µM or 2mM gentamicin (Hospira Inc, Lake Forest, Illinois) for gentamicin-treated cells. The cells were incubated for an additional 24 hours. The cells were then collected for assessment of Δψm.
Δψm was assessed with the MitoProbe JC-1 Assay Kit for Flow Cytometry (Life Technologies, Carlsbad, California) on both untreated and treated HEI-OC1 and LLC-PK1 cells according to the manufacturer’s protocol. Briefly, approximately 2.5 × 105 HEI-OC1 and LLC-PK1 cells were resuspended in 500 µL of warm media and incubated with JC-1 dye (2µM final concentration) at 37°C with 10% and 5% CO2, respectively, for 30 minutes. For the positive control tube, 2 µL of 50mM carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial membrane disrupter, was added simultaneously with JC-1. The cells were pelleted by centrifugation and resuspended in phosphate buffered saline (PBS). Samples were analyzed by BD Accuri C6 Flow Cytometer (BD Biosciences, Ann Arbor, Michigan) via FL1 channel (green fluorescent) and FL2 channel (red fluorescent). Flow cytometry data were collected from 20,000 events per sample. For each cell type, each treatment was run in triplicate, and data were the means of 2 or 3 independent experiments. The potential-dependent accumulation of JC-1 in mitochondria is indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm). Mitochondrial membrane depolarization is indicated by a decrease in the red-to-green fluorescence intensity ratio of JC-1. Data are presented as percentages of the fluorescence intensity ratio of solvent control (DMSO = 100%).
Statistical Analysis
Data were analyzed by 1-way analysis of variance via JMP Pro 11.0 statistical software (SAS Institute, Cary, North Carolina). When analysis of variance was significant (P < .05), a Dunnett post hoc test was used with the DMSO-treated cells as control group. The MitoProbe JC-1 data, with and without 2 mM gentamicin, were also compared within each treatment through a t test for each cell line. Differences were considered significant at P≤ .05.
Results
All data for the MitoProbe JC-1 assay were normalized percentages of the solvent control (DMSO = 100%) and expressed as fluorescence intensity ratios of the red-to-green JC-1 fluorescence. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm); hence, a decrease in red-to-green fluorescence intensity ratio indicates depolarization of the Δψm.
In the renal cell line (LLC-PK1), Δψm was not different in untreated cells and cells coincubated with low-dose gentamicin (100µM) and 0.5µM MitoQ or IDB for 24 hours (P > .05; Figure 1A ). However, concurrent treatment of the LLC-PK1 with high-dose gentamicin (2,000µM) and 0.05% DMSO or 0.5µM MitoQ or IDB resulted in significant depolarization of the mitochondrial membrane when compared to the DMSO solvent control (P < .0001; Figure 1B ). When the Δψm of LLC-PK1 cells, with and without high-dose gentamicin, was compared for each compound, concurrent addition of high-dose gentamicin with DMSO, MitoQ, or IDB significantly depolarized the mitochondrial membrane when compared to each compound alone (P = .003, P = .016, and P = .0003, for DMSO, MitoQ, and IDB, respectively). Furthermore, the depolarization of the Δψm in gentamicin- and MitoQ-treated LLC-PK1 cells was to a greater extent than that of gentamicin- and IDB-treated cells (P = .03).
Fluorescence intensity ratio: a measure of mitochondrial membrane potential (percentage of dimethyl sulfoxide [DMSO] control) of Lilly Laboratories Culture–Pig Kidney Type 1 cells with and without exposure to gentamicin (GM)—(
There were no differences in Δψm in HEI-OC1 cells incubated in 0.05% DMSO or 0.5µM MitoQ or IDB alone. Coincubation with low-dose gentamicin and DMSO or IDB for 24 hours did not alter Δψm. In contrast, coincubation of the HEI-OC1 cells with low-dose gentamicin and 0.5µM MitoQ decreased Δψm (P = .002; Figure 2A ). However, all 3 compounds—DMSO, MitoQ, and IDB—significantly increased Δψm (hyperpolarization) when added to the cells simultaneously with high-dose gentamicin (P = .0005; Figure 2B ). When comparison was made for each compound, with and without the addition of gentamicin, the combination of high-dose gentamicin with DMSO, MitoQ, or IDB significantly increased the Δψm compared to each compound alone without gentamicin (P = .0002, P = .025, and P = .036, for DMSO, MitoQ, and IDB, respectively). The extent at which each compound hyperpolarized the mitochondrial membrane in the presence of high-dose gentamicin was not different (P = .75).
Fluorescence intensity ratio: a measure of mitochondrial membrane potential (percentage of dimethyl sulfoxide [DMSO] control) of House Ear Institute Organ of Corti 1 cells with and without exposure to gentamicin (GM)—(
Discussion
AGs are still commonly used to treat aerobic gram-negative bacterial infections. However, AGs have been shown to be nephrotoxic and ototoxic. 1 As a result, there is a continued interest in potential therapies that would protect against the toxic side effects of AG use. Many studies have reported that antioxidants can attenuate AG ototoxicity.4-7,17 Since the mitochondria are known as major source of ROS production in the cell and since MitoQ—a mitochondria-targeted derivative of the antioxidant ubiquinone—can accumulate several hundred-fold within the mitochondria,8-10 we hypothesized that MitoQ should be a more potent antioxidant when compared to untargeted antioxidants. Our group has been studying whether MitoQ can protect against AG ototoxicity.11-13
Our previous studies indicated that MitoQ holds promise as a means for protecting against AG ototoxicity both in vitro in HEI-OC1 cells and in vivo with oral supplementation of MitoQ to guinea pigs.11-13 In our previous animal study, 12 we observed considerable individual variation in the auditory brain stem response threshold shift data for the MitoQ-supplemented guinea pigs. Since the water intake among guinea pigs was variable, which could result in variable dosing of MitoQ, we tested administering MitoQ to the guinea pigs by intraperitoneal injection. Saline (0.9% sodium chloride) was the vehicle used to deliver MitoQ. However, we found unexpected toxicity with 20 mg/kg of intraperitoneal MitoQ in combination with 130 mg/kg of gentamicin, administered subcutaneously. Two of the 4 guinea pigs that were injected with 20 mg/kg of intraperitoneal MitoQ in combination with 130 mg/kg of gentamicin, administered subcutaneously, died within 24 hours after the injections. The other 2 guinea pigs survived but had to be euthanized after 48 hours due to lethargy, low food consumption, and respiratory distress. The 2 guinea pigs that were euthanized did not show signs of vestibulotoxicity. Necropsy results showed that all 4 guinea pigs had pulmonary congestion and edema, kidney tubular degeneration, and hepatocellular degeneration with hemorrhagic liver. The same MitoQ dose injected intravenously in rats was not toxic. 18 Thus, in consultation with our veterinarians, we tested intraperitoneal injection of 10 mg/kg of MitoQ without gentamicin injection. The guinea pigs receiving a single dose of 10 mg/kg of MitoQ did well. We are currently unaware of the exact cause of the morbidity and mortality associated with concurrent injection of MitoQ and gentamicin. One possibility is the potentiation of gentamicin toxicity by MitoQ administration. Therefore, the present study was conducted to better understand the effects of concurrent treatment with gentamicin (a commonly used AG) and MitoQ. We used an additional cell line, LLC-PK1, to address potential limitations of the HEI-OC1 cells.11,13 Also, different cell lines may respond differently to the same insult. 19 Thus, using 2 cell lines that show evidence of apoptosis when treated with known ototoxic drugs, such as gentamicin, would potentially strengthen our findings.
A distinctive feature of the early stages of programmed cell death (apoptosis) includes disruption of the Δψm and alterations to the oxidation reduction potential of the mitochondria. The changes in the Δψm can be measured by a variety of fluorescent techniques, such as flow cytometry based on cationic dyes that can permeate the mitochondrial membrane (eg, JC-1, DiOC6, and rhodamine 123). The JC-1 dye has been reported to be more specific for mitochondrial versus plasma membrane potential and more consistent in its response to depolarization than the other cationic dyes.20,21 Thus, the MitoProbe JC-1 Assay Kit for Flow Cytometry was used in the present study. A decrease in the red-to-green fluorescence intensity ratio indicates depolarization of the Δψm. Comparative studies based on the ratio of red-to-green JC-1 fluorescence have been extensively used to study effects on Δψm in cells. 21
Our results showed that while LLC-PK1 cells treated with low-dose gentamicin did not exhibit significant mitochondrial membrane depolarization, HEI-OC1 cells coincubated with low-dose gentamicin and MitoQ exhibited significant mitochondrial membrane depolarization. Additionally, the concurrent treatment of LLC-PK1 cells with high-dose gentamicin and DMSO, MitoQ, or IDB significantly decreased Δψm (depolarization), with MitoQ depolarizing Δψm to a greater extent than that of IDB. In contrast, concurrent treatment of HEI-OC1 cells with high-dose gentamicin and DMSO, MitoQ, or IDB significantly increased Δψm (hyperpolarization). A modest depolarization of the Δψm is known to attenuate ROS production. 22 But, in general, mitochondrial membrane depolarization and hyperpolarization can both be precursors to apoptosis.23,24 Therefore, our results suggest that gentamicin can disrupt Δψm in both cell lines, albeit oppositely. That LLC-PK1 and HEI-OC1 cell lines treated with high-dose gentamicin both exhibited significant changes in Δψm (depolarization and hyperpolarization, respectively) but not with low-dose gentamicin indicates that gentamicin toxicity and its ability to disrupt Δψm may also be dependent on concentration. However, single daily dosing of AGs in patients is associated with higher peak tissue concentrations, equivalent efficacy, and lower rates of toxicity. 25
Although our results indicate that concurrent treatment with gentamicin and MitoQ has the potential to disrupt Δψm, findings from a single assay in the present study, based on 2 concentrations of gentamicin and 1 time point (24 hours), are insufficient to arrive at clinically significant conclusions on Δψm variations. Previous literature also discussed that despite the reliability of the JC-1 dye to assess Δψm, the JC-1 aggregate (red fluorescence) can sometime function independent of Δψm, directly affecting the validity of the ratiometric value of JC-1 fluorescence results.21,22 Moreover, the mechanism behind AG ototoxicity is complex, and using cell lines to assess such mechanisms does not allow for a holistic account of cellular interactions that occur in animal models and humans, where levels of Δψm disruption can differ among models. 20 Therefore, additional assays that assess multiple factors of mitochondrial bioenergetics should be performed to accurately interpret Δψm changes. Additional studies—which include assessing several properties of mitochondrial bioenergetics, such as oxygen consumption—are currently underway in our laboratory to further determine the effects of concurrent treatment of gentamicin and MitoQ in LLC-PK1 and HEI-OC1 cells.
In conclusion, although antioxidants are considered generally safe, the combination of gentamicin and the antioxidants IDB or MitoQ holds the potential to disrupt Δψm, with MitoQ disrupting Δψm to a greater extent than that of IDB. This suggests that patients who are receiving both agents simultaneously must take precautions to monitor toxicity. Although earlier studies have shown that MitoQ holds promise as a means for protecting against AG ototoxicity, further investigations—in vitro and in vivo—are needed to evaluate possible side effects of concurrent treatment with MitoQ and AG antibiotics.
Author Contributions
Disclosures
Footnotes
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
This article was presented at the 2014 AAO-HNSF Annual Meeting & OTO EXPO; September 21-24, 2014; Orlando, Florida.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.