Effects of In Vitro Combination of Nitric Oxide Donors and Hypochlorite on Acanthamoeba castellanii Viability

Abstract

Purpose: To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human corneal epithelial cells (HCECs).

Methods: Acanthamoeba castellanii and primary cultured HCECs and keratocytes were treated with sodium hypochlorite (NaOCl), NO donors (sodium nitroprusside [SNP] and sodium nitrite [NaNO₂]), or a combination of hypochlorite and NO donors. The viability of A. castellanii, HCECs, and keratocytes was assessed. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration of NaOCl and NO donors were determined. The activation of mammalian targets of rapamycin (mTOR) and ERK and the expression of nitrite reductase and Nrf2 were assessed in HCECs using Western blot analysis. The cysticidal effects of combined NaOCl and NO donors were also evaluated.

Results: A dose-dependent toxicity was observed in A. castellanii, HCECs, and keratocytes when treated with NaOCl and SNP. The range of tested NaNO₂ concentrations showed no significant toxicity to HCECs; however, dose-dependent toxicity to A. castellanii was observed. The MIC of NaOCl against HCECs and A. castellanii was 8.0 mg/mL. The MIC of NaNO₂ and SNP was 500 mM and 10 mM in both HCECs and A. castellanii, respectively. Weak attenuation of the mTOR and ERK phosphorylation was observed and Nrf2 expression decreased slightly after exposure of HCECs to 2.0 mg/mL NaOCl. For the combination treatment, NaOCl (0.125 mg/mL) was selected based on the safety of HCECs and the toxicity of A. castellanii. A more potent anti-Acanthamoeba effect and HCEC toxicity were observed when NaOCl was combined with SNP rather than NaNO₂.

Conclusions: Combined NaOCl and NO donors had a stronger anti-Acanthamoeba effect compared to either drug alone.

Translational Relevance: This study demonstrates that the combined use of various drugs for the treatment of Acanthamoeba infection can enhance the anti-Acanthamoeba effect while minimizing the toxicity of the individual drug.

Introduction

Acanthamoeba keratitis (AK) is a rare infectious corneal disease that can lead to blindness, with an incidence of 0.15 to 1.4 per million in developed countries.^1–5 Acanthamoeba is the most common amoeba causing infectious keratitis, although other free-living amoebas, such as Harmannella and Vahlkampfia, have also been isolated from patients with contact lens–related keratitis.⁶ 

The major risk factors for AK are contact lens wear and exposure to contaminated water.^5,7 Among various species of Acanthamoeba, Acanthamoeba castellanii accounts for 94.3% of AK.⁷ The most desirable treatment strategy for AK is to selectively eradicate Acanthamoeba without damaging host corneal cells. However, treatment in the real world falls short of this goal: because Acanthamoeba is a protozoa, antibiotics targeting bacteria and fungi have little effect against AK.⁸ The current most common therapeutic regimen against AK includes two types of topical biaguanides: chlorhexidine 0.02% and polyhexamethylene biguanide (PHMB) 0.02%.¹ These two topical agents induce structural and permeability changes, ionic leakage, and cytoplasmic disruption of Acanthamoeba.⁸ However, the corneal toxicity when used under intensive treatment and questionable cysticidal effect of these treatments are major drawbacks.^6,9,10 As is well known, cysts are the major source of recurrent AK.^2,9 Therefore, effective treatment against AK is an important area of research in ophthalmology. 

We previously reported the anti-Acanthamoeba effect of nitric oxide (NO).¹¹ NO is a radical gas and a key player in a common antibacterial mechanism in mammalian hosts.¹² NO synthesized by activated macrophages is a broad-spectrum bactericidal agent that can kill both gram-positive and gram-negative bacteria.^13–15 Exogenous delivery of NO by adding NO donors, sodium nitrite (NaNO₂) and sodium nitroprusside (SNP), in planktonic cultures of A. castellanii demonstrated that killing Acanthamoeba was concentration dependent for both trophozoite and cyst forms.¹¹ However, we also found that the anti-Acanthamoeba effect and human corneal epithelial cell (HCEC) toxicity were proportional: NaNO₂ showed a weak anti-Acanthamoeba effect and less corneal epithelial toxicity, while SNP showed a strong anti-Acanthamoeba effect and severe HCEC toxicity.¹¹ 

Hypochlorite or hypochlorous acid is a potent oxidant with the chemical formula HOCl or ClO⁻. Hypochlorite is produced in neutrophils and plays roles in killing pathogens via oxidative damage.^16,17 Sodium hypochlorite (NaOCl), a donor of hypochlorite, is commonly used as a household bleach. The anti-Acanthamoeba effect of sodium hypochlorite, due to its strong oxidation effect, has been previously reported.^18,19 Exposure of Acanthamoeba to high concentrations (2.5–25 mg/mL) of NaOCl resulted in a dramatic decrease in Acanthamoeba in both trophozoites and cyst forms.¹⁹ Hypochlorite is also used as a disinfectant for skin preparation during surgical procedures.²⁰ The ophthalmic application of hypochlorite for the treatment of blepharitis by reducing the bacterial burden has been proven to be effective.^21,22 However, unlike in the skin and eyelid, the dose-dependent toxicity of hypochlorite on HCECs still remains unclear. 

NO mainly acts through nitrosative stress, and hypochlorite mainly acts through oxidative stress; therefore, there is a possibility of reducing toxicity to HCECs while doubling the antibacterial activity against Acanthamoeba when a combination of these materials is applied. However, there have been few reports about this possibility. In this study, we investigated the combined effects of NO and hypochlorite on A. castellanii viability. Various concentrations of two commonly used NO donors—NaNO₂ and SNP—were combined with sodium hypochlorite and added to the planktonic cultures of A. castellanii. Cell viability was assessed for both trophozoites and cysts. In addition, the dose-dependent toxicity of sodium hypochlorite and NO donors on cultured HCECs and keratocytes was investigated. In doing so, we tried to find the optimal combination concentrations of sodium hypochlorite and NO donors necessary to kill Acanthamoeba effectively while sparing the maximum HCECs. 

Methods

Acanthamoeba Culture

A. castellanii (cat. ATCC-30010) were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA) and grown axenically in a T25 tissue culture flask containing 5 mL ATCC Medium 712 (basal medium containing proteose peptone 2% (w/v), yeast extract 0.1%, and 50 mM CaCl₂, 400 mM MgSO₄, 250 mM Na₂HPO₄, 250 mM KH₂PO₄, 0.1% Na Citrate, 5 mM Fe (NH₄)₂(SO₄)₂, and 2 M glucose as the final concentrations) and incubated at 25°C. When the cultures reached near-peak density, 0.25 mL of culture was transferred to a fresh tube containing 5 mL of fresh medium. 

HCEC Culture

The primary culture of HCECs (cat. PCS-700-010) was purchased from ATCC. The cells were resuspended in corneal epithelial cell basal medium supplemented with a growth kit supplied by ATCC. The cells were plated in 75-cm² tissue flasks and then were maintained at 37°C in a 5% CO₂ and 95% air humidified atmosphere. The culture medium was changed every 3 days, and the cells were passed using 0.05% Trypsin-EDTA (Gibco BRL, Grand island, NY, USA). Cells with a passage number of ≤5 were used in this study. 

Acanthamoeba Viability Assay

Acanthamoeba viability assay was performed using AlamarBlue Cell Viability Reagent (cat. DAL1100; Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. AlamarBlue is a cell viability assay reagent that contains weakly fluorescent blue indicator dye called resazurin. Resazurin is an oxidation reduction indicator that undergoes colorimetric change in response to cellular metabolic reduction. The intensity of fluorescence is proportional to cell viability. Briefly, A. castellanii was cultured at 1.0, 0.5, 0.3, and 0.2 × 10⁴ cells/well for 1, 3, 5, and 7 days, respectively. NaNO₂ and SNP were used as NO donors for the study. NaOCl solution (cat. 33015-1501; Junsei Chemical Co., Ltd., Tokyo, Japan) was tested as an oxidant in this study. NO donors were added to the culture media in a dose-dependent manner (NaNO₂ and SNP: 0, 0.1, 1, 10, 100, and 1000 µM), and NaOCl was added (0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/mL). The stock solution of the NO donors was dissolved in Dulbecco's phosphate-buffered saline (DPBS); thus, the vehicle for NO donors was 0.1% (v/v) of DPBS. The solvent of the NaOCl solution was distilled water; thus, the vehicle for NaOCl was 2.5% (v/v) of distilled water. After appropriate incubation, 10% (v/v) of AlamarBlue solution was added to each culture well, and absorbance was measured at 570 nm. Finally, the absorbance values were normalized to the wavelength values at 600 nm. 

To investigate the cysticidal effect of NaOCl and NO donors, A. castellanii cystic transformation was induced by culturing A. castellanii in Neff's encystment medium (100 mM KCl, 0.4 mM CaCl₂, 8 mM MgSO₄, 1 mM NaHCO₃, and 20 mM Tris-HCl), and the pH was adjusted to 8.9 ± 0.2. The induced cysts were then transferred to an axenical culture medium and exposed to 0.125 mg/mL NaOCl and NO donors (NaNO₂ and SNP: 0, 0.1, 1, 10, and 100 µM) for 1, 3, and 5 days. After incubation, a living cell viability marker, NucGreen Dead 488 ReadyProbes Reagent (SYTOX Green; cat. R37109, Thermo Fisher Scientific), was directly added to the cystic Acanthamoeba (2 drops/mL, 10 minutes at room temperature [RT]) and analyzed for cell death. SYTOX Green can penetrate the compromised cell membranes of dead cells and stain nucleic acid. Images of stained Acanthamoeba cysts were observed and obtained using a confocal live imaging system (Leica Microsystems CMS GmbH, Mannheim, Germany). The ratio of the number of NucGreen-positive cysts/number of total cysts was measured. The cysticidal effect was also demonstrated using AlamarBlue Cell Viability Reagent, which stained living cysts as bright orange and dead cells as dark blue. After induction of encystment, cells were exposed to 0.125 mg/mL NaOCl and NO donors (sodium nitrite and SNP: 10 and 100 µM) for 3 days. After incubation, 20% (v/v) of AlamarBlue solution was added and Acanthamoeba cysts were observed using a confocal live imaging system. 

HCEC Viability and Cytotoxicity

HCEC viability assays were performed using AlamarBlue Cell Viability Reagent according to the manufacturer's protocol. Briefly, HCECs were cultured at 1 × 10⁴ cells/well in a 96-well plate and incubated for 24 hours. Following the adherence of the cells, NO donors were added to the culture media in a dose-dependent manner (NaNO₂ and SNP: 0, 0.1, 1, 10, 100, and 1000 µM), and NaOCl was added (0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/mL). The stock solution of the NO donors was dissolved in DPBS; thus, the vehicle for NO donors was 0.1% (v/v) of DPBS. The solvent of the NaOCl solution was distilled water; thus, the vehicle for NaOCl was 2.5% (v/v) of distilled water. After appropriate incubation, 10 µL AlamarBlue solution was added to each culture well, and absorbance was measured at 570 nm. The absorbance values were finally normalized to the wavelength values at 600 nm. 

The cellular toxicity of each experimental condition was measured by a colorimetric assay using a lactate dehydrogenase (LDH) cytotoxicity detection kit (Takara Bio, Shiga, Japan), and experiments were performed according to the manufacturer's protocol. The LDH assay is a quantitative analysis of LDH secreted from dead cells. HCECs were cultured and incubated at 1 × 10⁴ cells/well in a 96-well plate. After cell attachment, cells were exposed to 0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/mL NaOCl for 24, 48, and 72 hours. After the appropriate incubation period, the activity of LDH released by the cells was measured in supernatants collected after each incubation time, to which was added 10% (v/v) of LDH solution. The wells to which NaOCl was not added and those to which 1% Triton X-100 was added were used as the negative and positive controls, respectively, and absorbance was measured at 490 nm. 

Keratocyte Viability

Keratocyte viability assays were performed using AlamarBlue Cell Viability Reagent according to the manufacturer's protocol. Briefly, keratocytes were cultured at 0.5 × 10⁴ cells/well in a 96-well plate and incubated for 24 hours. Following the adherence of cells, NO donors were added to the culture media in a dose-dependent manner (NaNO₂ and SNP: 0, 0.1, 1, 10, 100, and 1000 µM), and NaOCl was added (0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg/mL). The stock solution of NO donors was dissolved in DPBS, and thus the vehicle for NO donors was 0.1% (v/v) of DPBS. The solvent of NaOCl solution was distilled water, and thus the vehicle for NaOCl was 2.5% (v/v) of distilled water. After appropriate incubation, 10 µL AlamarBlue solution was added to each culture well, and absorbance was measured at 570 nm. The absorbance values were finally normalized to the wavelength values at 600 nm. 

Acanthamoeba and HCEC Minimum Inhibitory Concentration and Fractional Inhibitory Concentration Test

Acanthamoeba and HCEC minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) tests were performed using AlamarBlue Cell Viability Reagent according to the manufacturer's protocol. Briefly, A. castellanii or HCECs were cultured at 1.0 × 10⁴ cells/well for 48 hours, with NaOCl, NO donors (sodium nitrite; NaNO₂ and SNP), or a combination of NaOCl and NO donors. NaOCl was twofold serial diluted in the culture media (0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mg/mL). NaNO₂ was twofold serial diluted and treated in the culture media (1.953125, 3.90625, 7.8125, 15.625, 31.25, 62.5, 125, 250, and 500 mM), and SNP was also twofold serial diluted and treated in the culture media (0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5.0, and 10.0 mM). The stock solution of NaOCl was dissolved in distilled water, and thus the vehicle for NaOCl was 2.5% (v/v) of distilled water. Chlorhexidine (0.05%) and Triton X-100 (0.2%) were used for positive control against A. castellanii and HCECs, respectively. After 48 hours, 10% (v/v) of AlamarBlue solution was added to each culture well, and absorbance was measured at 570 nm. The absorbance values were finally normalized to the wavelength values at 600 nm. The FIC test was performed with the same method using the combination of NaOCl and NO donors. 

Western Blot Analysis

Western blot analysis was performed in accordance with the previously reported method.²³ All NaOCl-treated HCECs were collected and lysed 72 hours after treatment in an ice-cold radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) for 30 minutes. The debris was removed by centrifugation at 16,000 × g for 1 minute. Equal amounts (20 µg) of total cell protein were separated by SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). After blocking with 3% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline (10 mM Tris [pH 8.0], 150 mM NaCl) with 0.1% Tween 20 at RT for 1 hour, the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti–phospho-mTOR (1:1000; cat. 5536; Cell Signaling, Danvers, MA, USA), rabbit anti-mTOR (1:1000; cat. 2983; Cell Signaling), rabbit anti–P-p44/42 MAPK (ERK 1/2) (1:1000; cat. 4370; Cell Signaling), rabbit anti-p44/42 MAPK (ERK 1/2) (1:1000; cat. 4695; Cell Signaling), mouse anti-Nrf2 (1:1000; cat. MAB3925; R&D Systems, Minneapolis, MN, USA), rabbit anti–ferredoxin-nitrite reductase (1:1000; cat. ab243179; Abcam, Cambridge, UK), and mouse anti–β-actin (1:50,000; cat. A5441; Sigma-Aldrich). The membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies at RT for 1 hour. The blots were developed with an enhanced chemiluminescence kit (cat. RPN2232; GE Healthcare, Buckinghamshire, UK) and visualized with a Fujifilm LAS-3000 image reader (Fujifilm, Tokyo, Japan). Densitometric analysis was performed with Multi Gauge V3.0 software (Fujifilm Life Science, Tokyo, Japan). Each experiment was performed in triplicate at a minimum. 

Statistical Analysis

Data are presented as mean ± standard error, and statistical significance was determined by one-way analysis of variance, followed by Dunnett's multiple comparison test. GraphPad Prism Version 9.0 (GraphPad Software, La Jolla, CA, USA) was used for analysis, and P values less than 0.05 were regarded as significant. 

Results

HCEC Viability After Exposure to NaOCl

HCECs’ viability was not affected by NaOCl up to 0.125 mg/mL for 72 hours of culture. However, the dose-dependent toxicity of NaOCl equal to or over 0.25 mg/mL was significant. Exposure to 2.0 mg/mL NaOCl for 24, 48, and 72 hours decreased HCECs’ viability by more than 50% (Fig. 1A). The LDH released from HCECs also showed similar dose-dependent findings (Fig. 1B). However, significant elevations of LDH were observed when HCECs were exposed to NaOCl at equal to or over 0.5 mg/mL. Although the results of the two experiments do not match exactly due to the difference between the LDH and AlamarBlue assay mechanisms, it is clear that the two methods show similar trends. The MIC test for NaOCl in HCECs was 8.0 mg/mL and is shown in Supplementary Figure S1. 

Figure 1.

 

The effect of various concentrations of NaOCl on the viability of primary cultured HCECs. The addition of NaOCl to the culture media showed a dose-dependent toxicity to HCECs. Decreased viability and increased LDH were observed at 72 hours of culture. Control (Ctrl) = culture media without NaOCl. Vehicle = 2.5% (v/v) of distilled water. Triplicates of each treatment group were used in each independent experiment. Values are presented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

The addition of NaOCl to the HCEC cultures showed that cell survival signals, such as phosphorylation of mammalian targets of mammalian targets of rapamycin (mTOR) and ERK, were well maintained even with 1.0 mg/mL of NaOCl exposure (Fig. 2). Weak attenuation of mTOR and ERK phosphorylation was observed when HCECs were exposed to 2.0 mg/mL NaOCl. Nrf2 is a protein that regulates antioxidant mechanisms and protects living organisms from oxidative stress. Nitrite reductase is an intracellular enzyme that converts nitrite to NO and is the major NO producer in mammalian cells under hypoxic conditions. We found that Nrf2 and nitrite reductase expression of HCECs were not affected by the exposure of HCECs to NaOCl in amounts up to 1.0 mg/mL. Although the expression of nitrite reductase was not affected after exposure to 2.0 mg/mL NaOCl, Nrf2 expression slightly decreased at the same concentration. 

Figure 2.

 

Cellular survival signals, such as mTOR and ERK, after 72 hours exposure to NaOCl in primary cultured HCECs were well maintained up to 1.0 mg/mL concentration. Nrf2 and nitrite reductase expressions were also unchanged after exposure to NaOCl. High doses of NaOCl (2.0 mg/mL) induced slight but significant decreases in mTOR and ERK phosphorylation and Nrf2 expression. Triplicates of each treatment group were used in each independent experiment. Values are presented as mean ± SEM from three independent experiments.

Acanthamoeba Viability After Exposure to NaOCl

The viability of A. castellanii showed a dose-dependent decrease when NaOCl was added to the culture media. Even low concentrations of NaOCl, such as 0.031 mg/mL, induced a significant anti-Acanthamoeba effect after 72 hours of culture. The anti-Acanthamoeba effect further increased with prolonged exposure to NaOCl (Fig. 3A). When cultured for 7 days, 2 mg/mL NaOCl decreased the viability of A. castellanii by over 80%. Accordingly, the concentration of NaOCl for the further combination experiment was selected as 0.125 mg/mL because this concentration decreased the viability of A. castellanii by about 30% and 60% at day 5 and day 7 of culture, respectively, while maintaining stable HCEC viability. The effect of NaOCl on the viability of A. castellanii was not believed to be due to the hypertonic effect of the Na ion, because we found that the exposure of A. castellanii to NaCl solutions up to 1 mg/mL showed little cytotoxic effect (Fig. 3B). The MIC test for NaOCl in A. castellanii was 8.0 mg/mL and is shown in Supplementary Figure S1. 

Figure 3.

 

The effect of NaOCl on A. castellanii viability. The addition of NaOCl to the culture media showed dose-dependent toxicity to A. castellanii for up to 7 days. The hypertonic effect of Na ion was measured by exposing A. castellanii to various concentrations of NaCl solution up to 2 mg/mL. The viability was not affected with exposure to NaCl solution up to 1 mg/mL. The culture media were changed every 72 hours. Control (Ctrl) = culture media without NaOCl. Vehicle = 2.5% (v/v) of distilled water. Triplicates of each treatment group were used in each independent experiment. Values are presented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Effect of NO Donors on Acanthamoeba and HCECs

The two NO donors showed different results regarding their toxicity to HCECs and their anti-Acanthamoeba effects. While NaNO₂ showed no toxicity to HCECs even after 72 hours of culture at 1000 µM concentration, SNP toxicity to HCECs was prominent even at concentrations of 1 µM or higher after 48 hours of culture (Figs. 4A, 4B). SNP at concentrations of 100 µM or higher resulted in more than 50% toxicity to HCECs after 48 and 72 hours of culture. Almost all HCECs died after exposure to SNP concentrations of 1000 µM after 72 hours of culture. 

Figure 4.

 

The effect of NaNO₂ and SNP on the viability of HCECs and A. castellanii. While NaNO₂ showed no toxicity to HCECs up to concentrations of 1000 µM, SNP showed significant toxicity to HCECs at concentrations of 1 µM or higher after 48-hour incubation. On the other hand, the anti-Acanthamoeba effect was more prominent with SNP. NaNO₂ showed an anti-Acanthamoeba effect at concentrations of 100 µM or higher after 24-hour incubation and needed both the highest concentration of 1000 µM and 7 days of incubation to reach over 50% of the anti-Acanthamoeba effect; however, SNP showed a significant anti-Acanthamoeba effect even after 24-hour incubation at a concentration of 0.1 µM. SNP showed a potent anti-Acanthamoeba effect of 50% or more when cultured for 5 days at a concentration of 1.0 µM or higher. The culture media were changed every 72 hours. Control (Ctrl) = culture media only. Vehicle = 0.1% (v/v) of DPBS. Triplicates of each treatment group were used in each independent experiment. Values are presented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

The anti-Acanthamoeba effect of NaNO₂ became significant at concentrations of 100 µM or higher after 24 hours of culture (Fig. 4C). However, it needed both the highest concentration of 1000 µM and 7 days of culture to reach the maximum anti-Acanthamoeba effect, which was only 53% cell death. On the other hand, SNP showed a potent anti-Acanthamoeba effect (Fig. 4D). A weak but significant anti-Acanthamoeba effect was observed even after just 24 hours of culture with a low concentration of 0.1 µM. Higher concentrations of SNP showed a more potent anti-Acanthamoeba effect, and culture for 5 days at a concentration of 1.0 µM resulted in more than 50% toxicity. The toxic effect of 1000 µM SNP was over 70% after 3 days of culture. The MIC for NaNO₂ was 500 mM in HCECs and A. castellanii, and the MIC for SNP was 10 mM in HCECs and A. castellanii (Supplementary Fig. S1). 

Keratocyte Viability After Exposure to NaOCl and NO Donors

Keratocyte viability was not affected by NaOCl up to 0.25 mg/mL for 72 hours of culture. However, the dose-dependent toxicity was observed with NaOCl equal to or over 0.5 mg/mL. Exposure to 2.0 mg/mL NaOCl for 72 hours decreased keratocyte viability by 90% (Supplementary Fig. S5). While NaNO₂ showed no toxicity to keratocytes even after 48 hours of culture at 1000 µM concentration, 72 hours of incubation resulted in mild but significant toxicity at concentrations of 100 µM or over. SNP toxicity to keratocytes was prominent at concentrations of 10 µM or higher after 24 hours of culture. High concentration (1000 µM) of SNP decreased keratocytes’ viability by more than 50% (Supplementary Fig. S5). 

Combination Effect of NO Donors and NaOCl

Combining various concentrations of NO donors with NaOCl (0.125 mg/mL) significantly increased the anti-Acanthamoeba effect, as shown in Figure 5. Compared to the monotreatment with NaOCl, the addition of 1 µM NaNO₂ further enhanced the anti-Acanthamoeba effects observed from 1 day of culture. The addition of 100 µM NaNO₂ to 0.125 mg/mL NaOCl resulted in the anti-Acanthamoeba effect almost doubling through day 7. A similar enhancement of the anti-Acanthamoeba effect was observed when NaOCl and SNP were combined. As previously shown, SNP has a stronger anti-Acanthamoeba effect than NaNO₂. When 0.1 µM SNP was combined with 0.125% NaOCl, the amoebicidal effect increased more than twofold from day 1 to day 7. The addition of 10 µM SNP to 0.125 mg/mL NaOCl increased the anti-Acanthamoeba effect more than four times over 7 days. 

Figure 5.

 

The combined effect of NaOCl (0.125 mg/mL) and NO donors. The combination of NO donors with NaOCl (0.125 mg/mL) further increased the anti-Acanthamoeba effect for 7 days. A more potent effect was observed for combined SNP compared to NaNO₂. When HCECs were exposed to the combination of NaOCl (0.125 mg/mL) and NO donors, NaNO₂ combined with NaOCl showed no significant decrease in HCEC viability for 7 days; however, when 10 µM SNP was combined with NaOCl, the viability of HCECs significantly decreased at day 5. At day 7, even the combination of 0.1 µM SNP with NaOCl showed HCEC toxicity. The culture media were changed every 72 hours. Control (Ctrl) = culture media containing NaOCl (0.125 mg/mL). *P < 0.05. Triplicates of each treatment group were used in each independent experiment. Values are presented as mean ± SEM from three independent experiments. **P < 0.01, ***P < 0.001, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. Between-day comparison: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Combining various concentrations of NaNO₂ (0.1–100 µM) with NaOCl (0.125 mg/mL) showed minimal toxicity to HCECs at 7 days. However, when 10 µM SNP was combined with NaOCl (0.125 mg/mL), the viability of HCECs significantly decreased at day 5. On day 7, even the combination of 0.1 µM SNP with NaOCl (0.125 mg/mL) showed significant HCEC toxicity, and few HCECs survived after exposure to both 1.0 and 10 µM SNP combined with NaOCl (0.125 mg/mL). The FIC test for NaOCl and NO donors in A. castellanii is shown in Supplementary Figure S2 and Supplementary Figure S3. In addition, the FIC test for NaOCl and NO donors in HCECs is shown in Supplementary Figure S6 and Supplementary Figure S7. These figures clearly demonstrate the enhanced cytotoxicity by the combination of NaOCl and NO donors. 

Combining NaOCl and NaNO₂ (0.1–100 µM) increased the cysticidal effect on Acanthamoeba. As shown in Figure 6 and Supplementary Figure S4, a dose-dependent booster effect was observed when NaNO₂ was added to NaOCl. The addition of 10 µM NaNO₂ to NaOCl doubled the cysticidal effect on days 1, 3, and 5. Furthermore, the addition of 100 µM NaNO₂ to NaOCl increased the cysticidal effect about six times compared to NaOCl treatment alone. The dose-dependent cysticidal effect of SNP when combined with NaOCl was also observed. The addition of 10 µM SNP to NaOCl resulted in over 50% cysticidal effect (Fig. 7). 

Figure 6.

 

Cysticidal effects of the combination of NaOCl and NO donors were verified. The Acanthamoeba cyst was stained with SYTOX Green for apoptosis detection. Adding 0.125 mg/mL NaOCl to the culture media induced apoptosis and necrosis of the cyst at days 1, 3, and 5 compared to the control. Further addition of NaNO₂ increased the number of dead cells, especially necrosis, at 100 µM NaNO₂. The ratio between the numbers of live cells and dead cells was calculated and presented as graphs. The culture media were changed every 72 hours. Triplicates of each treatment group were used in each independent experiment. Values are presented as mean ± SEM from three independent experiments.

Figure 7.

 

Cysticidal effects of the combination of NaOCl and SNP were verified. The Acanthamoeba cyst was stained with SYTOX Green for apoptosis detection. Adding 0.125 mg/mL NaOCl to the culture media induced apoptosis and necrosis of the cyst at days 1, 3, and 5 compared to the control. Further addition of SNP increased the number of dead cells. The ratio between the numbers of live cells and dead cells was calculated and presented as graphs. The culture media were changed every 72 hours. Triplicates of each treatment group were used in each independent experiment. Values are presented as mean ± SEM from three independent experiments.

Discussion

In this study, we demonstrated that sodium nitrite, SNP, and NaOCl have dose-dependent anti-Acanthamoeba effects on both trophozoite and cyst forms. Although SNP and NaOCl showed potent anti-Acanthamoeba effects compared to sodium nitrite, significant HCEC toxicity was observed at high concentrations and longer incubations. The combination of NaNO₂ with NaOCl might potentiate the anti-Acanthamoeba effect while preserving HCEC viability. However, while the combination of SNP with NaOCl might potentiate the anti-Acanthamoeba effect, a significantly increased HCEC toxicity was observed when the two were used. High concentrations of NaOCl and NO donors also decreased human keratocyte viability in a dose-dependent manner (Supplementary Fig. S5). 

To date, most AK treatment modalities have been shown to be toxic to parasites and host cells. Current medications, such as PHMB and chlorhexidine, can be toxic to the cornea. Although a recent randomized clinical trial in healthy volunteers showed that PHMB eye drops (0.04%, 0.06%, and 0.08%) were tolerable by topical application for 14 days,²⁴ patients with AK usually have compromised corneal epithelial barrier function and may sometimes require months of topical treatment. Therefore, corneal toxicity during AK treatment has to potential to induce severe pain and cause significant corneal scarring to remain, even after complete resolution of the disease. Therefore, it is essential to develop an effective anti-Acanthamoeba regimen to minimize corneal toxicity. Furthermore, Acanthamoeba has a two-phase life cycle: trophozoites and cysts. Because the cyst has a double wall that preserves Acanthamoeba in hostile environments, it is resistant to most amoebicidal drugs, and the remnant cyst is the major source of the recurrence of AK. For effective control of AK, the cysticidal effect of the treatment regimen is mandatory. 

Recently, various drugs have been reported as anti-Acanthamoeba therapeutics. For example, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) induced disassembly of actin cytoskeleton and resulted in increased apoptosis of Acanthamoeba.²⁵ By blocking Acanthamoeba HMG-CoA reductase, the synthesis of ergosterol and 7-dehyrostigmasterol, which are the main sterols of Acanthamoeba’s membrane, is inhibited, and the parasite with a defective cell membrane undergoes apoptosis.²⁶ The combination of statins and azoles also showed a synergistic effect in killing Acanthamoeba.²⁷ Benzalkonium chloride has been reported to be effective in killing Acanthamoeba polyphaga. When exposed to 50 mg/L and 100 mg/L benzalkonium chloride, the viability of Acanthamoeba decreased more than 50% and 90%, respectively.¹⁸ 

The hypochlorite used in this study is frequently used as a disinfectant. Most commercial NaOCl products used as disinfectants have a concentration of 2.5% (w/w; 25 mg/mL). Previous studies have demonstrated the anti-Acanthamoeba effect of hypochlorite.¹⁸ Exposure of A. polyphaga to 5, 10, and 30 mg/mL NaOCl for 15 minutes resulted in decreases of about 50%, 75%, and 85%, respectively, in live Acanthamoeba populations.¹⁸ Exposure to 2.5% NaOCl for 10 minutes resulted in a 4.7 log10-unit reduction of A. castellanii.¹⁹ In the human body, hypochlorite is produced mainly by leukocytes, including neutrophils and macrophages, and plays a vital role in killing various pathogens, including bacteria and protozoa. The mechanism of cell death by hypochlorite is multifactorial.²⁸ The Cl atom of OCl⁻ behaves as Cl⁺ and acts as a strong electrophile attacker to the amide bonds, amino groups, and thiol groups of protein and nucleic acid.²⁸ This reaction is believed to induce injury to the essential proteins, membranes, and DNA of pathogens. For example, hypochlorite damaged the proteins involved in adenosine triphosphate production and depleted metabolic energy in Escherichia coli.²⁹ In addition, stress-caused hypochlorite increases reactive oxygen species generation inside the pathogen, further exacerbating the damage.²⁸ Since these injury mechanisms also apply to mammalian cells, high concentrations of hypochlorite inevitably cause damage to corneal cells. 

The concentration of NaOCl used in our study was 0.125 mg/mL, which is about 200 times the dilution of most commercial NaOCl products used as disinfectants. Previously, Yildiz et al.³⁰ observed trophozoite toxicity of over 90% when 0.109 mg/mL NaOCl was applied to A. castellanii for 90 minutes. However, we found that 0.125 mg/mL NaOCl decreased the viability of A. castellanii by about 30% and 60% at days 5 and 7 of culture, respectively, with no significant toxicity to HCECs. The discrepancy in amoebicidal potency between our study and previous studies may be due to differences in experimental conditions. 

NO is one of our body's most important defensive mechanisms against pathogens. NO can induce DNA damage, protein alteration, and strong oxidative stress in pathogens, causing death of pathogens.³¹ NO is also important amoebic infection, and mice deficient in NO synthase are more vulnerable to amoebic liver abscesses.³² The amoebicidal effect of exogenous NO supply using S-nitrosoglutathione and S-nitroso-N-acetylcysteine was also reported.³³ 

NO production from NaNO₂ requires nitrite reductase. Recently, Park et al.³⁴ verified the expression and function of xanthine oxidoreductase (mammalian nitrate/nitrite reductase) in porcine corneas and sclera. SNP is one of the most widely studied NO donors, and the production of NO from SNP is believed to be dependent on the interaction between SNP and sulfhydryl-containing molecules, such as cysteine and glutathione, in the local microenvironment.³⁵ 

In this study, we tried to find out whether the use of NaOCl and NO donors, which have different mechanisms of action, can enhance antiamoeba effects within the range of corneal epithelium nontoxicity by using them together rather than using them separately, and this hypothesis was successfully proved through experimentation. The combination of 0.125 mg/mL NaOCl with 10 µM sodium nitrite decreased A. castellanii viability by about 60% after 7 days of culture with no significant HCEC toxicity (see FIC data in Supplementary Fig. S6). In addition, about 20% of the cysticidal effect was observed when using this combination. However, it is noteworthy that these combinations also increased HCEC toxicity, especially when SNP and high concentrations of NaOCl were combined (see FIC data in Supplementary Fig. S7). 

We believe that the enhancement of the anti-Acanthamoeba effect caused by using the two chemicals (hypochlorite and NO donor) in combination is probably due to the combined effects of different Acanthamoeba killing mechanisms. As previously described, NO acts primarily through nitrosative stress, and hypochlorite acts primarily through oxidative stress. Even if Acanthamoeba’s defense mechanisms can overcome one stressor and survive, it eventually dies if it cannot overcome an additional stressor. The failure to totally eradicate Acanthamoeba at the dosages used in our study means that some Acanthamoeba can overcome both stresses and survive. However, the use of high concentrations of combined chemicals capable of eradicating all Acanthamoeba is also intolerable to HCECs; hence, its clinical application is impossible. 

Because most of the drugs used to eradicate Acanthamoeba are toxic to human corneal cells, it is necessary to adjust the concentration of the drugs to balance the anti-Acanthamoeba effect and cornea cell toxicity. If our knowledge about the efficacy and safety data of each drug is increased and additional combination effects are verified, we will then be able to select as many drugs as possible to achieve an optimal combination to enhance both the efficacy and safety of the combination regimen. This approach could result in an alternative for effectively controlling Acanthamoeba infection while avoiding the risk of toxicity associated with using one powerful drug. In this sense, our data will have future clinical value. 

Our study has several limitations. We included only A. castellanii in the investigation. Therefore, the application of our results to other species of Acanthamoeba that can also cause corneal infection should be done with caution. The lack of an in vivo experiment is another limitation of this study. As always, there is the possibility that the influence of complex in vivo factors may produce different results. Additionally, NO is a gas with a tendency to release from NO donors early and in bursts, rather than releasing from the NO donors steadily. In our study, the culture medium was changed every 72 hours; therefore, the concentration of NO in the culture medium could not always be kept constant. This should be considered when interpreting the data. Another limitation that cannot be overlooked is that the dose used in this study did not completely eradicate Acanthamoeba. Of course, treatment using a very high dose of NaOCl or NO donor, which is far beyond the range of our current study, is highly likely to reach the eradication level of Acanthamoeba, but as mentioned above, the purpose of our study is to present a new combination treatment method that has the potential for clinical application while minimizing corneal toxicity. In this study, the combination of SNP and NaOCl showed very severe corneal epithelial toxicity despite the anti-Acanthamoeba effect; therefore, clinical application may be difficult. 

In summary, we demonstrated the enhancement of the anti-Acanthamoeba effect achieved by combining hypochlorite and NO donors. The combination anti-Acanthamoeba effect was verified for both the trophozoite and cyst forms. In a situation in which the development of an effective treatment for Acanthamoeba is urgently needed, our study is meaningful in that it suggests a new potential treatment modality that differs from existing ones. 

Acknowledgments

Supported by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant NRF-2021R1A2C1006087) and National Priority Research Center Program Grant funded by the Korean government (NRF-2021R1A6A1A03038865). 

Disclosure: J.-H. Park, None; C.Y. Park, None