Assessment of Mitochondrial Membrane Potential in HEI-OC1 and LLC-PK1 Cells Treated with Gentamicin and Mitoquinone
Abstract
Objective: The aim of this study is to investigate the effects of concurrent treatment with gentamicin, an aminoglycoside antibiotic, and the mitochondria-targeted antioxidant mitoquinone (MitoQ). This combination may help prevent gentamicin-induced ototoxicity by assessing changes in mitochondrial membrane potential (Dcm), which is a precursor to apoptosis.
Study Design: This is a prospective and controlled study.
Setting: The research was conducted in an academic laboratory environment.
Subjects and Methods: The study utilized LLC-PK1 (Lilly Laboratories Culture–Pig Kidney Type 1) and HEI-OC1 (House Ear Institute Organ of Corti 1) cell lines, representing renal and auditory cells, respectively. The Dcm was evaluated using flow cytometry with the MitoProbe JC-1 Kit. This assessment was performed on untreated LLC-PK1 and HEI-OC1 cells as well as those exposed to low (100 mM) and high (2000 mM) doses of gentamicin for 24 hours, with or without the addition of 0.5 mM MitoQ or idebenone (IDB), which is an untargeted ubiquinone.
Results: The Dcm levels in untreated LLC-PK1 cells were comparable to those in cells treated with low-dose gentamicin alongside MitoQ or IDB (P > .05). In contrast, HEI-OC1 cells showed a significant decrease in Dcm when treated with low-dose gentamicin and MitoQ (P = .002). High-dose gentamicin coincubated with LLC-PK1 cells and DMSO, MitoQ, or IDB resulted in a notable depolarization of Dcm (P < .0001), with MitoQ causing a greater depolarization effect compared to IDB (P = .03). Conversely, HEI-OC1 cells exhibited a hyperpolarized Dcm when treated with high-dose gentamicin and DMSO, MitoQ, or IDB (P < .001). Conclusion: The findings suggest that the combination of gentamicin and MitoQ has the potential to disrupt Dcm, indicating a need for careful monitoring of toxicity in patients receiving both treatments. Keywords aminoglycoside, gentamicin, ototoxicity, mitoquinone, antioxidants, mitochondrial membrane potential This article was presented at the 2014 AAO-HNSF Annual Meeting & OTO EXPO held from September 21-24, 2014, in Orlando, Florida. The content reflects the authors' views and does not necessarily represent the official views of the National Institutes of Health. Corresponding Author: Carolyn Ojano Dirain, PhD Department of Otolaryngology University of Florida PO Box 100264 1345 Center Drive Gainesville, FL 32610-0264, USA Email: [email protected] Methods Cell Culture The experiments utilized LLC-PK1 and HEI-OC1 cell lines, representing renal and auditory epithelial cells, respectively. LLC-PK1 cells were obtained from the American Type Culture Collection, while HEI-OC1 cells were provided by Dr. Federico Kalinec. Both cell types show signs of apoptosis when exposed to ototoxic drugs, such as gentamicin and AG antibiotics. HEI-OC1 and LLC-PK1 cells were cultured in 100 mm petri dishes using Dulbecco’s modified Eagle’s medium supplemented with glucose, L-glutamine, and fetal bovine serum. HEI-OC1 cells were maintained in a humidified incubator at 33°C with 10% CO2, whereas LLC-PK1 cells were kept at 37°C with 5% CO2. MitoQ and IDB were dissolved in dimethyl sulfoxide (DMSO), with a final concentration of 0.05% DMSO in all treatments serving as the solvent control. Assessment of Dcm Once the cells reached approximately 75% confluence, they were incubated in media containing either 0.05% DMSO (control) or 0.5 mM MitoQ and IDB. After 24 hours, the growth medium was replaced with fresh medium for untreated cells or with medium containing 100 mM or 2 mM gentamicin for gentamicin-treated cells. The cells were incubated for an additional 24 hours before collection for Dcm assessment. Dcm was evaluated using the MitoProbe JC-1 Assay Kit according to the manufacturer’s instructions. Approximately 2.5 x 10^5 HEI-OC1 and LLC-PK1 cells were resuspended in warm media and incubated with JC-1 dye at a final concentration of 2 mM. Positive control included carbonyl cyanide 3-chlorophenylhydrazone, a mitochondrial membrane disruptor. The cells were then pelleted, resuspended in phosphate-buffered saline, and analyzed using a flow cytometer. Statistical Analysis Data were evaluated through one-way analysis of variance (ANOVA) using JMP Pro 11.0 statistical software. When significant differences were detected (P < .05), a Dunnett post hoc test was utilized, with DMSO-treated cells serving as the control group. Additionally, comparisons within each treatment were conducted using a t-test for each specific cell line. Differences were deemed significant at P ≤ .05. Results All data generated from the MitoProbe JC-1 assay were normalized to percentages based on the solvent control, where DMSO was assigned a value of 100%. The results were expressed as fluorescence intensity ratios of the red-to-green JC-1 fluorescence. The JC-1 dye demonstrates potential-dependent accumulation within mitochondria, which is indicated by a shift in fluorescence emission from green (approximately 529 nm) to red (approximately 590 nm). Consequently, a decrease in the red-to-green fluorescence intensity ratio signifies depolarization of the mitochondrial membrane potential (Dcm). In the LLC-PK1 renal cell line, the Dcm did not show significant differences when comparing untreated cells with those exposed to low-dose gentamicin (100 mM) alongside either 0.5 mM MitoQ or idebenone (IDB) for a duration of 24 hours. However, when LLC-PK1 cells were treated concurrently with high-dose gentamicin (2,000 mM) and either DMSO, MitoQ, or IDB, a notable depolarization of the mitochondrial membrane was observed compared to the DMSO solvent control. Furthermore, when the Dcm of LLC-PK1 cells was analyzed in conjunction with high-dose gentamicin, significant depolarization was evident when compared to each compound alone. The extent of depolarization in LLC-PK1 cells treated with gentamicin and MitoQ was greater than that observed with gentamicin and IDB. For the HEI-OC1 cell line, there were no significant differences in Dcm observed in cells incubated with either 0.05% DMSO or 0.5 mM MitoQ or IDB alone. The combination of low-dose gentamicin with DMSO or IDB did not alter the Dcm. In contrast, the combination of low-dose gentamicin and 0.5 mM MitoQ resulted in a significant decrease in Dcm. However, all three compounds—DMSO, MitoQ, and IDB—significantly increased Dcm when added to the cells simultaneously with high-dose gentamicin. When comparing the effects of each compound in the presence of high-dose gentamicin, the degree to which each compound hyperpolarized the mitochondrial membrane did not differ significantly. Discussion Aminoglycoside antibiotics are frequently utilized to treat infections caused by aerobic gram-negative bacteria. Nevertheless, these antibiotics are known to cause nephrotoxicity and ototoxicity. Consequently, there is ongoing interest in exploring potential therapies that could mitigate the toxic side effects associated with the use of aminoglycosides. Numerous studies have indicated that antioxidants may help alleviate aminoglycoside-induced ototoxicity. Given that mitochondria are recognized as significant sources of reactive oxygen species (ROS) production within cells, and considering that MitoQ—a mitochondria-targeted derivative of ubiquinone—can accumulate substantially within the mitochondria, it was hypothesized that MitoQ could serve as a more effective antioxidant compared to untargeted alternatives. Previous investigations have suggested that MitoQ shows promise in protecting against aminoglycoside-induced ototoxicity, both in vitro within HEI-OC1 cells and in vivo through oral supplementation in guinea pigs. Prior studies have documented considerable individual variability in auditory brainstem response threshold shifts among MitoQ-supplemented guinea pigs. Variability in water intake among the guinea pigs was identified as a potential factor influencing the dosing of MitoQ, prompting tests of intraperitoneal administration of MitoQ. Unexpectedly, toxicity was observed with a specific dose of intraperitoneal MitoQ when combined with gentamicin, leading to morbidity and mortality in some subjects. The present study was conducted to gain deeper insights into the effects of concurrent administration of gentamicin and MitoQ. The use of an additional cell line, LLC-PK1, aimed to address potential limitations associated with the HEI-OC1 cells. Different cell lines may exhibit varied responses to similar insults, thus employing two distinct cell lines that demonstrate evidence of apoptosis when exposed to known ototoxic agents such as gentamicin could potentially enhance the robustness of the findings. A key characteristic of the early stages of programmed cell death (apoptosis) includes disruptions to the Dcm and alterations in the oxidation-reduction potential of mitochondria. Changes in the Dcm can be assessed through various fluorescent techniques, including flow cytometry utilizing cationic dyes that permeate the mitochondrial membrane. The JC-1 dye is noted for its specificity in distinguishing between mitochondrial and plasma membrane potentials, and it is more consistent in its response to depolarization compared to other cationic dyes. The results indicated that while LLC-PK1 cells exposed to low-dose gentamicin did not exhibit significant mitochondrial membrane depolarization, HEI-OC1 cells treated with low-dose gentamicin and MitoQ displayed significant depolarization. Concurrent treatment of LLC-PK1 cells with high-dose gentamicin and DMSO, MitoQ, or IDB led to significant depolarization, with MitoQ causing a greater degree of depolarization compared to IDB. Conversely, concurrent treatment of HEI-OC1 cells with high-dose gentamicin and the three compounds resulted in a significant increase in Dcm. These findings suggest that gentamicin can disrupt Dcm in both cell lines, albeit with opposing effects based on concentration. The observation that LLC-PK1 and HEI-OC1 cell lines responded differently to high-dose gentamicin indicates that the toxicity of gentamicin and its impact on Dcm may vary depending on the concentration used. Although single daily dosing of aminoglycosides in clinical settings is associated with higher peak tissue concentrations and lower rates of toxicity, the results of this study indicate that concurrent treatment with gentamicin and MitoQ has the potential to disrupt Dcm. While antioxidants are generally considered safe, the combination of gentamicin with either IDB or MitoQ poses the risk of disrupting Dcm, with MitoQ demonstrating a more pronounced effect than IDB. This underscores the necessity for patients receiving both agents to be closely monitored for potential toxicity. Despite previous studies highlighting the protective potential of MitoQ against aminoglycoside ototoxicity, further investigations—both in vitro and in vivo—are essential to comprehensively evaluate the possible side effects of concurrent treatment with MitoQ and aminoglycoside antibiotics. Author Contributions Maria Raye Anne V. Ng was responsible for the acquisition and interpretation of data, drafting and critically revising the work, providing final approval of the manuscript, and agreeing to manuscript accountability. Patrick J. Antonelli contributed to the analysis and interpretation of data, critically revising the work, giving final approval of the manuscript, and agreeing to manuscript accountability. Jerin Joseph engaged in data acquisition, drafting the manuscript, providing final approval of the manuscript, and agreeing to manuscript accountability. Carolyn Ojano Dirain was involved in the conception and design of the work, acquisition, analysis and interpretation of data, critically revising the work, giving final approval of the manuscript, and agreeing to manuscript accountability. Disclosures Competing interests: Patrick J. Antonelli received grant funding from Medtronic, Auris Medical, and Sound Pharma. He also serves as a consultant to Medtronic, Otonomy, Med-EL, Foresight Biotherapeutics, and Sharklet Technologies. Sponsorships: None. Funding source: Hearing Health Foundation provided funding to Carolyn Ojano Dirain. The Hearing Health Foundation was not involved in the study's design and conduct, data analysis and interpretation, or the writing and approval of the manuscript. The National Institute on Deafness and Other Communication Disorders/National Institutes of Health provided funding (R03DC013659) to Carolyn Ojano Dirain. The National Institute on Deafness and Other Communication Disorders/National Institutes of Health was not involved in the study's design and conduct, data analysis and interpretation, or the writing and approval of the manuscript.