TY - JOUR
AU - Santos, Daniel, Assis
AB - Abstract Cryptococcosis is a life-threatening fungal infection, and its current treatment is toxic and subject to resistance. Drug repurposing represents an interesting approach to find drugs to reduce the toxicity of antifungals. In this study, we evaluated the combination of N-acetylcysteine (NAC) with amphotericin B (AMB) for the treatment of cryptococcosis. We examined the effects of NAC on fungal morphophysiology and on the macrophage fungicidal activity 3 and 24 hours post inoculation. The therapeutic effects of NAC combination with AMB were investigated in a murine model with daily treatments regimens. NAC alone reduced the oxidative burst generated by AMB in yeast cells, but did not inhibit fungal growth. The combination NAC + AMB decreased capsule size, zeta potential, superoxide dismutase activity and lipid peroxidation. In macrophage assays, NAC + AMB did not influence the phagocytosis, but induced fungal killing with different levels of oxidative bursts when compared to AMB alone: there was an increased reactive oxygen species (ROS) after 3 hours and reduced levels after 24 hours. By contrast, ROS remained elevated when AMB was tested alone, demonstrating that NAC reduced AMB oxidative effects without influencing its antifungal activity. Uninfected mice treated with NAC + AMB had lower concentrations of serum creatinine and glutamate-pyruvate transaminase in comparison to AMB. The combination of NAC + AMB was far better than AMB alone in increasing survival and reducing morbidity in murine-induced cryptococcosis, leading to reduced fungal burden in lungs and brain and also lower concentrations of pro-inflammatory cytokines in the lungs. In conclusion, NAC + AMB may represent an alternative adjuvant for the treatment of cryptococcosis. Cryptococcosis, anticryptococcal therapy, adjuvant, N-acetylcysteine, Amphotericin B Introduction Cryptococcosis is a severe invasive mycosis primarily caused by Cryptococcus neoformans and Cryptococcus gattii and is associated with high morbidity and mortality. Both C. neoformans and C. gattii cause pneumonia and meningoencephalitis, the most severe form of the disease, in immunocompromised and immunocompetent patients.1 Worldwide, these pathogens cause more than 200000 cases of cryptococcal meningitis per year among people with human immunodeficiency virus (HIV)/AIDS, resulting in more than 181000 deaths annually.2 The drug of choice for the treatment of cryptococcosis is amphotericin B (AMB), which may or may not be used with other drugs, such as fluconazole (FCZ) and 5-flucytosine (5FC).3 The success of this treatment might be compromised due to the toxicity of these agents, particularly AMB, and the development of fungal resistance to FCZ, leading to high mortality rates. This has increased the interest in developing alternative treatments of this systemic mycosis.4 In this sense, drug repurposing, where existing drugs are screened for alternative activities, is becoming an attractive approach in antimicrobial discovery programs, and various compound libraries are now commercially available. As these drugs have already undergone extensive optimization and passed regulatory hurdles, this can fast-track their progress to market for new uses.5 N-acetylcysteine (NAC) has been used in clinical practice for several decades for a number of therapeutic purposes, such as a mucolytic agent in lung disease and protect against toxicity induced by some drugs.6–8 In addition, it has been used in psychiatric and neurological disorders, including autism, Alzheimer's disease, bipolar disorder, depression, and drug dependency, such as marijuana and cocaine.9–11 Recently, a range of other applications have been described for NAC in: antibacterial activity,12 antidiabetes,13 the treatment of contrast-induced nephropathy, noise-induced hearing loss,14,15 and pneumococcal meningitis.16 In a model of bacterial meningitis, Christen et al. demonstrated that NAC treatment was able to reduce cortical lipid peroxidation induced by parenchyma oxidative stress.17 Antifungal activity has not previously been described for NAC; however, Xu et al. found that in combination with the antifungal amphotericin B, it reduced oxidative stress and lung injury associated with invasive pulmonary aspergillosis in neutropenic mice.18 Therefore, considering all the previously described applications of NAC, we assessed if this drug would be a promising adjuvant therapy for cryptococcosis. Methods Ethics The protocol for animal studies was approved by the Comissão de Ética no Uso de Animais (CEUA) of the Universidade Federal de Minas Gerais, Minas Gerais, Brazil (protocol 366/2013). Female C57/BL6 mice (16–18 g) aged 6- to 8-weeks (six mice per cage) were housed in clean bedding with food and water ad libitum, in a controlled environment with a 12 hour light/dark cycle. Microorganisms Twelve C. gattii strains were used, all of which were maintained on Sabouraud dextrose agar (SDA) at 4°C prior to the tests (Table 1). Table 1. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) values of N-acetylcysteine (NAC), amphotericin B (AMB), and fluconazole (FCZ) alone and in combination against Cryptococcus gattii strains. . . Combination . Strains . MIC values (µg/ml) . FICI values* . Cryptococcus gattii . NAC . AMB . FCZ . NAC + FCZ . NAC + AMB . ATCC 24 065 >512 0.3 4.0 1.29 1.19 ATCC 32 608 >512 0.5 16.0 1.29 1.19 135 L/03 >512 0.4 8.0 1.37 1.29 L28/02 >512 0.6 11.3 1.19 1.23 23/10 993 >512 0.5 4.0 1.37 1.19 196 L/03 >512 0.5 16.0 1.16 1.11 1913/ER >512 0.5 8.0 1.29 1.10 547 OTTI >512 0.4 16.0 1.29 1.11 L27/01 >512 0.3 16.0 1.19 1.19 LMM 818 >512 0.5 16.0 1.19 1.06 L24/01 >512 0.4 8.0 1.23 1.19 29/10 893 >512 0.3 16.0 1.29 1.19 . . Combination . Strains . MIC values (µg/ml) . FICI values* . Cryptococcus gattii . NAC . AMB . FCZ . NAC + FCZ . NAC + AMB . ATCC 24 065 >512 0.3 4.0 1.29 1.19 ATCC 32 608 >512 0.5 16.0 1.29 1.19 135 L/03 >512 0.4 8.0 1.37 1.29 L28/02 >512 0.6 11.3 1.19 1.23 23/10 993 >512 0.5 4.0 1.37 1.19 196 L/03 >512 0.5 16.0 1.16 1.11 1913/ER >512 0.5 8.0 1.29 1.10 547 OTTI >512 0.4 16.0 1.29 1.11 L27/01 >512 0.3 16.0 1.19 1.19 LMM 818 >512 0.5 16.0 1.19 1.06 L24/01 >512 0.4 8.0 1.23 1.19 29/10 893 >512 0.3 16.0 1.29 1.19 * Synergism FICI < 0.5, no interaction 0.5 < FICI < 4, and antagonism FICI > 4. Open in new tab Table 1. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) values of N-acetylcysteine (NAC), amphotericin B (AMB), and fluconazole (FCZ) alone and in combination against Cryptococcus gattii strains. . . Combination . Strains . MIC values (µg/ml) . FICI values* . Cryptococcus gattii . NAC . AMB . FCZ . NAC + FCZ . NAC + AMB . ATCC 24 065 >512 0.3 4.0 1.29 1.19 ATCC 32 608 >512 0.5 16.0 1.29 1.19 135 L/03 >512 0.4 8.0 1.37 1.29 L28/02 >512 0.6 11.3 1.19 1.23 23/10 993 >512 0.5 4.0 1.37 1.19 196 L/03 >512 0.5 16.0 1.16 1.11 1913/ER >512 0.5 8.0 1.29 1.10 547 OTTI >512 0.4 16.0 1.29 1.11 L27/01 >512 0.3 16.0 1.19 1.19 LMM 818 >512 0.5 16.0 1.19 1.06 L24/01 >512 0.4 8.0 1.23 1.19 29/10 893 >512 0.3 16.0 1.29 1.19 . . Combination . Strains . MIC values (µg/ml) . FICI values* . Cryptococcus gattii . NAC . AMB . FCZ . NAC + FCZ . NAC + AMB . ATCC 24 065 >512 0.3 4.0 1.29 1.19 ATCC 32 608 >512 0.5 16.0 1.29 1.19 135 L/03 >512 0.4 8.0 1.37 1.29 L28/02 >512 0.6 11.3 1.19 1.23 23/10 993 >512 0.5 4.0 1.37 1.19 196 L/03 >512 0.5 16.0 1.16 1.11 1913/ER >512 0.5 8.0 1.29 1.10 547 OTTI >512 0.4 16.0 1.29 1.11 L27/01 >512 0.3 16.0 1.19 1.19 LMM 818 >512 0.5 16.0 1.19 1.06 L24/01 >512 0.4 8.0 1.23 1.19 29/10 893 >512 0.3 16.0 1.29 1.19 * Synergism FICI < 0.5, no interaction 0.5 < FICI < 4, and antagonism FICI > 4. Open in new tab Susceptibility test and drug combination assay The antifungal activity of NAC was investigated using the microdilution broth assay, following the Clinical and Laboratory Standards Institute (CLSI) protocol M27-A3.19 The antifungals FCZ and AMB were also tested. Inocula were prepared from cultures of C. gattii strains grown for 48 hours on SDA at 37°C. The suspensions were adjusted with sterile saline to a transmittance of 75–77% at 530 nm, for a cell density of 1–5 × 106 colony forming units (cfu)/ml, with a further 1000-fold dilution in Roswell Park Memorial Institute (RPMI)-1640 medium. In sum, 100 μl culture was added to each well of a microdilution plate to obtain a final inoculum concentration of 0.5–2.5 × 103 cfu/ml with the addition of the antifungal compounds.20 The microplates containing inocula and drugs were incubated at 37°C, and results were read visually after 72 hours. Minimum inhibitory concentrations (MIC) were defined as the lowest drug concentrations at which no visible growth was observed for AMB and NAC, and prominent decrease (∼50%) in visible growth for FCZ. The combination of NAC + AMB and NAC + FCZ was evaluated against C. gattii strains by use of the checkerboard assay. Fractional inhibitory concentration index (FICI) was calculated by the quotient of the MIC of the combination of the drugs and their MIC values alone. The interaction between these drugs was classified as synergism if FICI was <0.5, no interaction if 0.5 < FICI < 4, and antagonism for FICI > 4.21 Morphometric and zeta potential analysis To investigate influence of NAC and its combination with AMB on C. gattii (Cg), we evaluated the capsule production and the magnitude of negative cell charge. The highest tested concentration of NAC (512 µg/ml), a sub-MIC concentration (0.25 µg/ml) of AMB, and the combination NAC + AMB (512 µg/ml + 0.25 µg/ml) were used to evaluate capsule size of strain L27/01 on minimal medium. The same strain cultivated without drugs was used as a control. After 48 hours at 37°C, the cells were visualized in a suspension in India ink with an Axioplan optical microscope (Carl Zeiss, Göttingen, Germany) and photographed. The capsule and diameter of at least 50 cells were measured using Image J 1.40 g software (http://rsb.info.nih.gov/ij/; National Institutes of Health [NIH] Bethesda, MD, USA).22 The zeta potentials of suspensions of yeast cells were measured with a Zeta sizer Nano ZS90 zeta potential analyzer (Malvern Panalytical, Malvern, United Kingdom) as previously described.23 Lipid peroxidation assay and superoxide dismutase (SOD) activity In order to understand the effect of oxidative stress on Cg membrane damage and the ability of Cg to neutralizing reactive oxygen species, we evaluated lipid peroxidation on Cg cells and superoxide dismutase (SOD) activity. The products of lipid peroxidation were measured as thiobarbituric acid-reactive substances (TBARS).24,25 The L27/01 strain was grown in SDA for 48 hours at 37°C, and then 10.0 mg of the fungal cell mass was transferred to polypropylene tubes containing the drugs at the same concentrations described above. To analyze lipid peroxidation, the cells were frozen and homogenized in 1000 µl ice cold 1.1% phosphoric acid. Four hundred µL of the homogenate was mixed with 400 µL 1% thiobarbituric acid (Sigma-Aldrich, St. Louis, MI, USA) prepared in 50 mM NaOH containing 0.1 mM butylated hydroxytoluene, and 200 µL 7% phosphoric acid; all the solutions were kept on ice during the manipulations. Subsequently, samples (at pH 1.5) were heated for 60 minutes at 98°C, and 1500 µl butanol was added. They were then mixed vigorously using a vortex and centrifuged for 5 minutes at 2000 g. The organic layer was transferred and its absorbance at 532 nm was measured. TBARS values were calculated using the extinction coefficient 156 mM−1cm−1. SOD activity was measured in a cell-free extract of C. gattii L27/01 by the inhibition of pyrogallol autoxidation.24,25 In the test samples, 100 µl cell-free extract was added to pyrogallol and inhibition of autoxidation was monitored every 30 seconds for 3 minutes at a wavelength of 420 nm. The unit of SOD was considered as pyrogallol autoxidation per 200 µl, calculated as follows: Unit of SOD/ml of sample = (A–B)/A × 50 × 100) × df, where A is the difference in absorbance per minute in the control, B the difference in absorbance per minute in the test samples, and df is the dilution factor equal to 0.6. Results were expressed in units/mg protein and represented the means of three independent experiments.24,25 Phagocytosis, reactive oxygen species (ROS) production by macrophages, and intracellular proliferation Fungicidal activity of macrophages induced by NAC was investigated by using bone marrow derived macrophages (BMDM). These cells were isolated from the femur and tibia of mice and were differentiated into bone marrow derived macrophages (BMDM) for seven days in culture medium supplemented with 30% L929 supernatant.26 BMDM at 2 × 105/ml was plated into 24-well plates to determine phagocytosis and intracellular killing and into 96-well plates for the detection of ROS. Prior to infection, the cells were treated for 3 hours with the same concentrations of the drugs described above. The treatments were then removed and the cells were infected with C. gattii L27/01 (0.4 × 105 cells/ml). The phagocytic index was determined by counting the internalized yeasts in at least 100 macrophages at 3 and 24 hours post-inoculation.27 To determine the intracellular killing by BMDM, the supernatant of cell cultures was removed, and non-internalized and adherent yeast cells were removed from the wells by washing three times with 200 μl phosphate-buffered saline (PBS). Next, macrophages were lysed with 200 μl sterile distilled water for 30 minutes at 37°C, and then 200 μl was collected and plated on SDA for cfu determination at 3 and 24 hours post-inoculation with C. gattii.28,29 ROS quantification and fluorescence was measured with 2,7-dichlorofluorescein diacetate (DCFH-DA; Invitrogen, Life Technologies, Carlsbad, CA, USA) and a Synergy 2 SL Luminescence Microplate Reader (Biotek, Winooski, VT, USA).24 Effects of NAC on the concentrations of creatinine and glutamate-pyruvate transaminase (GPT) of mice Nephrotoxicity induced by AMB was assessed by determining the serum creatinine and GPT concentrations. Noninfected (NI) mice (n = 6 per group) were treated daily during 15 days with NAC (100 mg/kg/day) or AMB (2.0 mg/kg/day) or NAC + AMB (100 and 2.0 mg/kg/day, respectively). The serum of the animals was collected for evaluation of creatinine and GPT concentrations according to the manufacturer (Bioclin, Belo Horizonte, Brazil). Intratracheal infection, survival curve, behavioral analysis, fungal burden, histopathological analysis, and cytokines quantification Murine model of pulmonary cryptococcosis were used to investigate the therapeutic effect of NAC combination with AMB. Before infection, mice were anesthetized by intraperitoneal (i.p.) injection with 80 mg/kg ketamine and 10 mg/kg xylazine and then inoculated by intratracheally (i.t.) injection with 30 μl C. gattii L27/01 suspension (1 × 104 cfu/animal) or PBS (uninfected, NI). Mice were divided into different groups, and the treatments begun 24 hours after fungal inoculation, administered once a day by i.p. injection. The behavior and survival of mice were monitored daily until they succumbed. The concentrations of NAC 50 and 100 mg/kg/day were tested initially in the survival assay, and 100 mg/kg/day was then selected for further experiments.30,31 The experimental groups were: (a) 100 mg/kg/day NAC, (b) 0.5 mg/kg/day AMB, (c) 100 mg/kg/day NAC + 0.5 mg/kg/day AMB, (d) untreated (NT), and (e) uninfected (NI).32 During the survival curves, we also studied mice behavior using the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) protocol, which provided standardized protocols for behavioral and functional assessment of neurological disorders in animal models. The protocol was used to evaluate five functional categories: the sensorial reflex, neuropsychiatric state, motor behavior, autonomous function, and muscle tone and strength. The mice were examined daily, and the score for each functional category was calculated as a total of the evaluated parameters25,33 using EpiData 3.1 software. In addition, other groups of mice were infected and treated as described for the determination of fungal burden and analysis of histopathology. Animals were euthanized 15 or 60 days after infection. The lungs and brain of the animals were aseptically removed, weighed, homogenized, diluted in PBS, plated on SDA, and incubated for 48 hours at 37°C. The results were then expressed as CFU per gram of tissue. Bronchoalveolar lavage was performed and cfu/ml was determined for the resulting fluid.25 Lungs were also stained with hematoxylin-eosin (HE) for evaluation of histopathological alterations. In addition, scores of yeasts quantity were determined as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe). The determination of the concentrations of interferon (IFN)-γ, interleukin (IL)-17, and chemokine ligand (CXCL)-1 in the lungs was performed by enzyme-linked immunosorbent assay (ELISA). Statistical analysis Statistical analyses were performed using GraphPad Prism, version 5.00, for Windows (GraphPad Software, San Diego, CA, USA), with P < .05 considered to indicate significance. Survival curves were plotted using Kaplan–Meier analysis, and results were analyzed using the log rank test. SHIRPA data were analyzed using analysis of variance (ANOVA) and Tukey test. The results of capsule size, cfu, phagocytosis assay, intracellular proliferation rate (IPR), ROS, creatinine, GPT, and cytokines were analyzed by ANOVA. For zeta potential, SOD and TBARS detection we used ANOVA followed by Dunn multiple comparison test. Results MIC and drug combination assay NAC was not able to inhibit the growth of C. gattii strains at the concentrations tested. The combinations between NAC + AMB, and NAC + FCZ were considered indifferent (Table 1). Morphometric alterations, zeta potential analysis, lipid peroxidation, and SOD activity Treatment with NAC or NAC + AMB reduced the capsule volume of C. gattii L27/01 cells (P < .01 and P < .001, respectively; Fig. 1A). The zeta potential of C. gattii L27/01 cells treated with AMB and with NAC + AMB was reduced (Fig. 1B). Only treatment with NAC was able to reduce SOD activity (P < .05; Fig. 1C). On the other hand, lipid peroxidation was higher when cells were treated with AMB and with NAC + AMB (P < .05; Fig. 1D). Figure 1. Open in new tabDownload slide Morphophysiological changes in Cryptococcus gattii treated with N-acetylcysteine (NAC) and Amphotericin B (AMB). Capsule volume (A), Zeta potential (B), Superoxide Dismutase (SOD) (C), and Lipid peroxidation (TBARS production) (D). TBARS, thiobarbituric acid-reactive substances. *, P < .05; **, P < .01; ***, P < .001. Figure 1. Open in new tabDownload slide Morphophysiological changes in Cryptococcus gattii treated with N-acetylcysteine (NAC) and Amphotericin B (AMB). Capsule volume (A), Zeta potential (B), Superoxide Dismutase (SOD) (C), and Lipid peroxidation (TBARS production) (D). TBARS, thiobarbituric acid-reactive substances. *, P < .05; **, P < .01; ***, P < .001. Phagocytosis, intracellular proliferation and ROS production by macrophages Phagocytosis was reduced in all treated groups at 3 hours (P < .05; Fig. 2A) and in the groups AMB and NAC + AMB after 24 hours (P < .05; Fig. 2B). The levels of ROS were increased in the NAC + AMB group after 3 hours (P < .05; Fig. 2C). After 24 hours, the levels of ROS were increased for the group treated with AMB (P < .05) but were reduced for the NAC + AMB and NAC groups (P < .05) (Fig. 2D). The number of viable C. gattii L27/01 cells recovered from macrophages had decreased after 3 h of treatment with AMB and with NAC + AMB (P < 0.05; Fig. 2E), and at 24 h, no cells were detected for these treatments (Fig. 2F). Figure 2. Open in new tabDownload slide Effects of the combination of N-acetylcysteine (NAC) and Amphotericin B (AMB) on phagocytosis and reactive oxygen species (ROS) production by macrophages inoculated with C. gattii. Phagocytic rate after 3 h (A) and 24 h (B), ROS production after 3 h (C) and 24 h (D), and fungal killing inside macrophages after 3 h (E) and 24 h (F) of treatment with different groups. NT, untreated; NI, non-inoculated; C.g., Cryptococcus gattii; *, P < .05; **, P < .01; AU, arbitrary units of fluorescence; cfu/ml, colony forming units per ml; ND, undetected. Figure 2. Open in new tabDownload slide Effects of the combination of N-acetylcysteine (NAC) and Amphotericin B (AMB) on phagocytosis and reactive oxygen species (ROS) production by macrophages inoculated with C. gattii. Phagocytic rate after 3 h (A) and 24 h (B), ROS production after 3 h (C) and 24 h (D), and fungal killing inside macrophages after 3 h (E) and 24 h (F) of treatment with different groups. NT, untreated; NI, non-inoculated; C.g., Cryptococcus gattii; *, P < .05; **, P < .01; AU, arbitrary units of fluorescence; cfu/ml, colony forming units per ml; ND, undetected. Creatinine and GPT concentrations NAC reduced the concentrations of serum creatinine and GPT, since these markers were increased when AMB were used alone in comparison to the combination NAC + AMB (P < .05; Fig. 3). Figure 3. Open in new tabDownload slide Concentrations of serum creatinine (mg/dL) (A) and glutamate-pyruvate transaminase – GPT (U/L) (B) in mice treated daily with N-acetylcysteine (NAC), amphotericin B (AMB) or with the combination NAC + AMB. NT, untreated group. Figure 3. Open in new tabDownload slide Concentrations of serum creatinine (mg/dL) (A) and glutamate-pyruvate transaminase – GPT (U/L) (B) in mice treated daily with N-acetylcysteine (NAC), amphotericin B (AMB) or with the combination NAC + AMB. NT, untreated group. Survival curve, behavioral analysis, fungal burden, histopathology and cytokines The NAC concentration of 50 mg/kg/day did not improve survival of mice infected with C. gattii (data not shown). The NAC dose of 100 mg/kg was selected for evaluation in combination with the antifungals FCZ and AMB. The median survival time for animals without treatment (NT) was 19 days, which was the same for those treated with 100 mg/kg NAC (Fig. 4A). In comparison to the NT group, mice treated with FCZ and NAC + FCZ had their average survival extended for 27 (P < .0001) and 32 (P < .0001) days, respectively (Fig. 4A). When mice were treated with 0.5 mg/kg AMB, their median survival was 26 days (P < .0001), while no deaths were recorded until 60 days post-infection in the group treated with NAC + AMB (P < .0001), at which point the experiment was stopped (Fig. 4B). The increased survival of this group was accompanied by reduced manifestations of cryptococcosis, corroborated by the SHIRPA protocol (Fig. 4C–H). In this test, the NI group showed a better performance, since no symptoms were detected; therefore, the closer the scores of different groups were to the NI group, the better their performance. The neuropsychiatric state (P < .05; Fig. 4E), motor behavior (P < .0001; Fig. 4F), autonomous function (P < .05; Fig. 4G), and muscle tone (P < .05; Fig. 4H) of the NAC + AMB group showed better scores (closer to the NI group) in comparison to AMB monotherapy. In addition, the group treated with NAC + AMB demonstrated a pronounced reduction in fungal burden in bronchoalveolar lavage fluid (P < .0001; Fig. 5A), lungs (P < .0001; Fig. 5B) and brain (P < .0001; Fig. 5C) after 15 days, with no colonies being detected after 60 days (Fig. 5A–C), at which point only this group was in the protocol. The results of fungal burden reduction were further confirmed with the histopathological analysis, where treatment with NAC, AMB and the combination NAC + AMB reduced the number of yeast cells in lung tissue (Fig. 6A–G). A small number of yeasts was found in the lungs by histopathology in the group NAC + AMB 60 d.p.i, but no colonies were recovered in the fungal burden determination. Furthermore, the concentrations of the cytokines IFN-γ, IL-17 and the chemokine CXCL-1 were reduced in the lungs of mice treated with NAC + AMB after 60 d.p.i. (P < .05; Fig. 6H–J). Figure 4. Open in new tabDownload slide Survival and behaviour of mice infected with C. gattii L27/01 treated with N-acetylcysteine (NAC) associated with amphotericin B (AMB). Survival curves of mice inoculated intratracheally with C. gattii without treatment (NT), and treated with 100 mg/kg NAC, 10 mg/kg FCZ and 100 mg/kg NAC + 10 mg/kg FCZ (A); and also treated with 0.5 mg/kg AMB and in association with 100 mg/kg NAC (B). ***P < .0001 when compared to the NT group; ###P < .0001 when compared to the AMB group. Behavioural profile evaluation (SHIRPA protocol) of animals infected with C. gattii and treated with NAC and AMB alone and together (C-H): body weight (C), sensorial reflex (D), neuropsychiatric state (E), motor behaviour (F), autonomous function (G), and muscle tone and strength (H). *P < .05; **P < .001; ***P < .0001.The connected lines indicate differences between AMB alone and AMB + NAC. Figure 4. Open in new tabDownload slide Survival and behaviour of mice infected with C. gattii L27/01 treated with N-acetylcysteine (NAC) associated with amphotericin B (AMB). Survival curves of mice inoculated intratracheally with C. gattii without treatment (NT), and treated with 100 mg/kg NAC, 10 mg/kg FCZ and 100 mg/kg NAC + 10 mg/kg FCZ (A); and also treated with 0.5 mg/kg AMB and in association with 100 mg/kg NAC (B). ***P < .0001 when compared to the NT group; ###P < .0001 when compared to the AMB group. Behavioural profile evaluation (SHIRPA protocol) of animals infected with C. gattii and treated with NAC and AMB alone and together (C-H): body weight (C), sensorial reflex (D), neuropsychiatric state (E), motor behaviour (F), autonomous function (G), and muscle tone and strength (H). *P < .05; **P < .001; ***P < .0001.The connected lines indicate differences between AMB alone and AMB + NAC. Figure 5. Open in new tabDownload slide Fungal burden recovered from lungs and brain of mice treated with N-acetylcysteine (NAC) in combination with amphotericin B (AMB). Mice were inoculated with C. gattii and treated daily with 0.5 mg/kg AMB, 100 mg/kg NAC, and 100 mg/kg NAC + 0.5 mg/kg AMB. Colony forming units (CFU) per unit of tissue recovered from the bronchoalveolar lavage (A), lungs (B) and brain (C) at 15 and 60 days post-infection. NT, control, untreated; ND, undetected; dpi, days post-infection. ***, P < .0001 and ###, P < .0001 when compared with NT and AMB groups, respectively. Figure 5. Open in new tabDownload slide Fungal burden recovered from lungs and brain of mice treated with N-acetylcysteine (NAC) in combination with amphotericin B (AMB). Mice were inoculated with C. gattii and treated daily with 0.5 mg/kg AMB, 100 mg/kg NAC, and 100 mg/kg NAC + 0.5 mg/kg AMB. Colony forming units (CFU) per unit of tissue recovered from the bronchoalveolar lavage (A), lungs (B) and brain (C) at 15 and 60 days post-infection. NT, control, untreated; ND, undetected; dpi, days post-infection. ***, P < .0001 and ###, P < .0001 when compared with NT and AMB groups, respectively. Figure 6. Open in new tabDownload slide Score of yeasts of C. gattii in the lungs of mice (A). Representative images of lungs from uninfected mice (NI; B), untreated mice (NT; C), and treated for 15 days with N-acetylcysteine (NAC; D), amphotericin B (AMB; E), and with NAC + AMB for 15 days (F) and 60 days (G) post-infection (dpi). The arrows indicate the presence of yeast cells in the tissue. Levels of IFN-γ (H), IL-17 (I) and CXCL-1 (J) in the lungs. *P < .05. This Figure is reproduced in color in the online version of Medical Mycology. Figure 6. Open in new tabDownload slide Score of yeasts of C. gattii in the lungs of mice (A). Representative images of lungs from uninfected mice (NI; B), untreated mice (NT; C), and treated for 15 days with N-acetylcysteine (NAC; D), amphotericin B (AMB; E), and with NAC + AMB for 15 days (F) and 60 days (G) post-infection (dpi). The arrows indicate the presence of yeast cells in the tissue. Levels of IFN-γ (H), IL-17 (I) and CXCL-1 (J) in the lungs. *P < .05. This Figure is reproduced in color in the online version of Medical Mycology. Discussion NAC, which is an acetylated cysteine compound, has aroused scientific interest for decades due to its important medical applications. It has long been used therapeutically for the treatment of acetaminophen overdose, acting as a precursor for the substrate (L-cysteine) in the synthesis of hepatic glutathione (GSH) depleted through drug conjugation. Other therapeutic uses of NAC have also emerged, including the alleviation of clinical symptoms of cystic fibrosis through cysteine-mediated disruption of disulfide cross-bridges in the glycoprotein matrix in mucus.34 This is the first study to evaluate the anticryptococcal activity of NAC alone and in combination with commercial antifungal agents. Although NAC did not inhibit the growth of the strains in vitro, it was able to cause morphometric changes to the cells of C. gattii. The capsule of Cryptococcus species is primarily composed of two polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), in addition to a smaller proportion of mannoproteins (MP).35 The presence of glucuronic acid residues in cryptococcal polysaccharide imparts a negative charge to the capsule, which is believed to contribute to protection against phagocytosis.36 AMB and NAC made the cells less electronegative, as reflected by a reduction in the zeta potential of the capsule of yeasts. This was consistent with Nosanchuk et al., who treated C. neoformans cells with AMB and fluconazole.23 As an antioxidant, only treatment with NAC alone reduced SOD activity. However, AMB and NAC + AMB led to increased SOD activity, probably due to oxidative stress, which also leads to higher lipid peroxidation. We previously demonstrated high lipid peroxidation of C. gattii cells by amphotericin B.24 The absence of an in vitro pharmacodynamic interaction between NAC and AMB was also found when we tested the ability of NAC + AMB to kill the fungus inside macrophages. AMB and NAC + AMB were efficient in stimulating BMDM, since no yeast cells were recovered from macrophages after 24 hours. However, it was possible to verify the difference between these treatments based on ROS production, since cells treated with AMB alone produced more ROS. By contrast, NAC + AMB treatment was able to provide recovery from the oxidative burst after fungal killing inside macrophages, resulting in ROS levels similar to the NI group. From these results, it might be concluded that AMB was important for fungal killing, while NAC was important in helping macrophages recover from oxidative stresses caused by the fungus and by AMB. The combination NAC + FCZ did not improve on FCZ effects in C. gattii infected mice. By contrast, NAC + AMB extended their survival for more than 60 days, probably due to the reduced toxicity of the treatment, as attested by the lower concentrations of creatinine and GPT. We believe that the preserved functions of kidneys and liver were also important for the reduction of fungal burden in the bronchoalveolar lavage fluid, lungs and brain. Xu and coworkers showed that the combination of 1 mg/kg AMB and 150 mg/kg NAC alleviated oxidative stress and lung injury associated with invasive pulmonary aspergillosis in neutropenic mice.17 Furthermore, besides increased survival, mice also presented with low morbidity, as shown in behavior assay. Although AMB has been used as a gold standard in treating life-threatening systemic mycoses, its use has been shown to be accompanied by dose-limited toxicities, most importantly, infusion-related reactions and nephrotoxicity.37,38 NAC is a safe and well-tolerated drug with no clinically significant side effects.39 Interestingly, Odabasi et al. demonstrated that NAC was able to reduce renal tubular apoptosis induced by AMB,40 and this effect may have contributed to the positive effects of the combination of the two drugs on survival and morbidity. In addition, the reduced concentrations of cytokines reinforce the beneficial effects of NAC in reducing inflammatory response after the reduction of fungal burden. In conclusion, NAC could be a promising adjuvant to AMB in anticryptococcal therapy. The data in this study might encourage clinicians to perform clinical trials with NAC + AMB. Acknowledgments This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES; the Ministry of Healthy and the National Council for Scientific and Technological Development—MS/CNPq (grant numbers 403006/2016-3, 440010/2018-7, and 302670/2017-3 to DAS). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Cogliati M . Global Molecular Epidemiology of Cryptococcus neoformans and Cryptococcus gattii: an atlas of the molecular Types . Scientifica (Cairo) . 2013 ; 2013 : 675213 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 2. Rajasingham R , Smith RM , Park BJ et al. . Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis . Lancet Infect Dis . 2017 ; 17 : 873 – 881 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Perfect JR , Dismukes WE , Dromer F et al. . 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Reduction of amphotericin B-induced renal tubular apoptosis by N-acetylcysteine . Antimicrob Agents Chemother . 2009 ; 53 : 3100 – 3102 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - N-acetylcysteine reduces amphotericin B deoxycholate nephrotoxicity and improves the outcome of murine cryptococcosis
JF - Medical Mycology
DO - 10.1093/mmy/myz129
DA - 2020-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/n-acetylcysteine-reduces-amphotericin-b-deoxycholate-nephrotoxicity-a66it9ZSsE
DP - DeepDyve
ER -