Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
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Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage Camila Donato Mahl a,b, Camile Saul Behling a,b, Fernanda S. Hackenhaar a,b, Mélany Natuane de Carvalho e Silva b, Jordana Putti b, Tiago B. Salomon a,b, Sydney Hartz Alves c, Alexandre Fuentefria d, Mara S. Benfato a,b,⁎ a
Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Laboratório de Estresse Oxidativo, Departamento de Biofísica, IB, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Laboratório de Pesquisas Micológicas, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil d Laboratório de Micologia Aplicada, Departamento de Análises, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 10 March 2015 Accepted 22 March 2015 Available online xxxx Keywords: Oxidative stress C. glabrata Fluconazole
a b s t r a c t In this study, we assessed the generation of reactive oxygen species (ROS) induced by subinhibitory concentration of fluconazole in susceptible and resistant Candida glabrata strains at stationary growth phase and measured their oxidative responses parameters: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathioneS-transferase (GST), consumption of hydrogen peroxide, and total glutathione, as well as oxidative damage in lipids, proteins, and DNA. Data showed that fluconazole increased generation of ROS and GPx and SOD enzymatic activity in treated cells; however, these enzymatic activities did not differ between resistant and susceptible strains. Susceptible strains exhibited higher GST activity than resistant, and when susceptible cells were treated with fluconazole, GST activity decreased. Fluconazole treatment cause oxidative damage only in DNA. There are a possible participation of ROS, as organic peroxides and O2•−, in antifungal mechanism of fluconazole, which results in higher GPx and SOD enzymatic activities and oxidative DNA damage in C. glabrata. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Since the early 1980s, fungi have emerged as major causes of human infections, particularly in immunocompromised and hospitalized patients with serious underlying diseases (Pfaller and Diekema, 2007). Of the fungi regarded as human pathogens, Candida spp. have been described as medically important fungi that cause severe mucosal and life-threatening invasive infections (Smeekens et al., 2013). Epidemiological studies reveal C. glabrata as an important emerging pathogen in nosocomial infections (Colombo et al., 2013; Tortorano et al., 2006), and due to the widely use of fluconazole in clinic, infections caused by fluconazole-resistant C. glabrata strains have been rising. Fluconazole is widely employed in part because of its metabolic stability, relatively high water solubility, and good tolerability (Grant and Clissold, 1990). It inhibits the biosynthesis of ergosterol by interfering with the fungal lanosterol demethylase (Silva et al., 2012). Three main mechanisms of fluconazole resistance among Candida species, including C. glabrata, are known, and although those mechanisms are the most well defined, there is evidence that products of genes involved in the oxidative stress response are involved in fluconazole resistance (Rogers
⁎ Corresponding author. Tel.: +55-51-33087603; fax: +55-51-33087003. E-mail address: [email protected] (M.S. Benfato).
and Barker, 2002; Rogers et al., 2006). In recent years, it has been proposed that reactive oxygen species (ROS) participate in the antifungal mechanism of action of azoles (Da Silva et al., 2013; Ferreira et al., 2013; Kobayashi et al., 2002). However, this putative antifungal mechanism is not fully understood, and the generation of reactive species by azoles, particularly fluconazole, still needs to be investigated. C. glabrata was shown to be resistant to ROS and to possess a potent antioxidant system that protects the fungal cell against endogenous and exogenous (Abegg et al., 2010). However, the antioxidant response of C. glabrata against ROS generation by fluconazole remains poorly explored, and the possible oxidative damage caused by fluconazole in this species is unknown. The elucidation of possible associations among antifungal mechanisms of action, antifungal resistance, and oxidative stress responses is important for identification of new targets for new antifungal agents and rational development of antifungal drugs. To better understand the relation between oxidative stress response, ROS generation by fluconazole, and fluconazole resistance in C. glabrata, we determined whether fluconazole induced generation of ROS in C. glabrata and evaluated the antioxidant defenses of fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated or not with fluconazole. We also asked whether fluconazole at subinhibitory concentrations caused oxidative damage to proteins, lipids, and nucleic acids in these strains.
http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
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C.D. Mahl et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
2. Methods
2.3. Measurement of ROS generation
2.1. Strains and culture growth
To measure ROS generation, cells were incubated with 2′,7′dichlorofluorescein diacetate (DCFH-DA) 10 μmol L−1 in 0.9% NaCl for 30 min at 37 °C in the dark. Cells were then transferred to glass slides for analysis of intracellular fluorescence by confocal laser scanning microscopy (CLSM; Olympus FluoView™ 1000; Miami, Florida, USA) at the Electron Microscopy Center of the Federal University of Rio Grande do Sul (CME-UFRGS). Images were processed by Olympus FluoView ver.4.0a Viewer software. The fluorescence intensity of a hundred cells was measured in 2D by ImageJ software. Values are expressed as corrected total cell fluorescence.
Two groups of C. glabrata clinical strains isolated from HIV patients with oropharyngeal candidiasis were assessed. The strains are part of the fungi collection of Mycological Research Laboratory (LAPEMI, Federal University of Santa Maria, Brazil) and were identified using the BioMerieux ID 32C yeast identification system according to manufacturer's recommendations. One group consists of 8 fluconazole-susceptible clinical strains (LAPEMI2010.Cg1, LAPEMI2010.Cg5, LAPEMI2010.Cg7, LAPEMI2010.Cg15, LAPEMI2010.Cg16, and LAPEMI2010.Cg20). The other group was derived in the Mycological Research Laboratory (cited above) from susceptible strains employing the technique of fluconazole resistance induction (Fekete-Forgacs et al., 2000; Mario et al., 2012) and consists of 8 fluconazole-resistant strains (LAPEMI2010.Cg1R, LAPEMI2010.Cg5R, LAPEMI2010.Cg7R, LAPEMI2010.Cg15R, LAPEMI2010.Cg16R, and LAPEMI2010.Cg20R). All of the yeasts were tested by the broth microdilution method using the CLSI M27-A3 and third informational supplement (M27-S3/2008) standardized reference method. No patients received prior treatment with fluconazole before the collection and isolation of fluconazole-susceptible strains. To ensure that the pattern of resistance remained stable in fluconazole-resistant strains, cells were maintained in Sabouraud agar containing 64 μg fluconazole mL−1, and subcultures were always made from this same primary cell culture. To evaluate the relationship between fluconazole and oxidative stress, these 2 groups were grown in the presence (or absence) of fluconazole at a subinhibitory concentration determined from their MIC as shown in Table 1. Growth curves were performed to identify the time required for all strains to reach stationary phase. In brief, from a 24-h culture in Sabouraud agar, an inoculum was added to a 30-mL Erlenmeyer flask containing Sabouraud broth, and the optical density (OD) at 640 nm was adjusted to 0.08 using a spectrophotometer. This subculture was incubated on a shaker at 200 rpm at 32 °C for 48 h. The OD was determined every 30 min during the first 6 h and every 12 h during the following 42 h. The results showed that at 24 h of growth all strains had reached stationary phase (Supplementary Fig. 1). For oxidative stress assays, strains were subcultured on Sabouraud agar for 24 h at 32 °C. They were then inoculated into Sabouraud broth without fluconazole or with fluconazole at subinhibitory concentrations according to their MIC (Table 1) and grown in flasks on an orbital shaker at 32 °C and 200 rpm until stationary phase was reached. Cells were transferred to centrifuge tubes, and cell-free extracts were obtained according to Abegg et al. (2010). Total protein concentration was measured using the Bradford method (Bradford, 1976).
2.2. ATP binding cassette (ABC) efflux pumps The MICs of fluconazole were redetermined in the presence of verapamil, an ABC efflux pump blocker, according to the CLSI M27-A2 protocol (Guinea et al., 2006; Pina-Vaz et al., 2005; Prates et al., 2011).
Table 1 Fluconazole MIC and subinhibitory concentrations of fluconazole-susceptible and fluconazoleresistant C. glabrata. C. glabrata strains
Fluconazole MIC (na)
FS
2 4 128 256
FR
μg μg μg μg
mL−1 (n mL−1 (n mL−1 (n mL−1 (n
= = = =
5) 3) 4) 4)
FS = fluconazole susceptible; FR = fluconazole resistant. a Number of strains.
Subinhibitory concentration of fluconazole for growth 1 2 64 128
μg μg μg μg
mL−1 mL−1 mL−1 mL−1
2.4. Activity of antioxidant enzymes Glutathione peroxidase (GPx) activity was evaluated in cellular extract by measuring the oxidation of NADPH in the presence of reduced glutathione (GSH), glutathione reductase, and tert-butyl hydroperoxide at 340 nm over 3 min (Pinto and Bartley, 1969). GPx is expressed as U mg−1 of protein. Superoxide dismutase (SOD) activity was measured in the cellular extract using a protocol of commercial kit RANSOD (RANSOD SD 125®; Randox Laboratories, Crumlin, UK). SOD activity was measured over 3 minutes and is expressed as U mg−1of protein. Glutathione-S-transferase (GST) activity was assayed in the cellular extract based on the formation of S-(2,4-dinitrophenyl)-glutathione by GST enzymatic activity (Tsuchida, 2000). The absorbance of the compound formed was measured over 2 min on a spectrophotometer at 340 nm. GST activity is expressed as U mg−1 of protein. The consumption of hydrogen peroxide (H2O2) was evaluated in the cellular extract by spectrophotometric assay over 1 min at 240 nm (Aebi, 1984). Activity is expressed as U mg−1 of protein. 2.5. Quantification of glutathione Quantification of total glutathione was measured in the cellular extract on a spectrophotometer at 412 nm as the formation of p-nitrophenol from the reduction of 5,5-dithiobis(2-nitrobenzoic acid). This reduction occurs when NADPH, cofactor for glutathione reductase, is oxidized (Rahman et al., 2006). Glutathione levels were compared with reduced glutathione standard solutions purchased from Sigma-Aldrich (Saint Louis, Missouri, USA) and are expressed as μmol mg−1 of protein. 2.6. Quantification of oxidative damage Quantification of malondialdehyde (MDA) provides an index of oxidative damage in lipids. MDA was determined in the cellular extract by HPLC using a reversed-phase SUPELCOSIL™ LC-18-DB High-performance liquid chromatography (HPLC) column (15 cm × 4.6 mm, 5 μm). A mobile phase flow rate of 0.5 mL min−1 in 30 mmol monobasic potassium phosphate L −1 (pH 3.6) and methanol (9:1, v/v) was employed (Karatepe, 2004). The absorbance of the column effluent was monitored at 254 nm, and the MDA retention time was 5.6 min. MDA levels were determined by comparison with MDA standard solutions purchased from Sigma-Aldrich, and the results are expressed as μmol mg−1 of protein. Oxidative damage in proteins was assessed in the cellular extract on a spectrophotometer according to Levine et al. (1990). Carbonyl content was calculated using a millimolar extinction coefficient of hydrazone of 21,000 M −1 cm −1. Absorbance at 370 nm was measured, and the results are expressed as nmol mg−1 of protein. The indirect method of measuring nitric oxide on a spectrophotometer was performed in the cellular extract by determining the levels of nitrites and nitrates (NO2 and NO3) at 543 nm according to Grisham et al. (1996). NO2 and NO3 levels are expressed as nmol mg−1 of protein. To determine oxidative damage in DNA, we quantified 8-oxo-7,8dihydro-2′-deoxyguanosine (8-oxodG) by High-performance liquid
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
C.D. Mahl et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
chromatography with electrochemical detection (HPLC-ECD) (Dirmeier et al., 2002; ESCODD et al., 2005). The column employed was a reversed-phase LC-18 NUCLEOSIL HPLC column (15 cm × 4.6 mm, 5 μm). A mobile phase flow rate of 0.5 mL min −1 in 50 mmol monobasic potassium phosphate L −1 (pH 5.5) and 4% methanol was employed. A UV detector at 254 nm was used to detect deoxyguanosine, and an electrochemical detector (DECADE II, 20 ηV) was used to detect 8-oxodG. DNA was extracted using lysing enzymes from Trichoderma harzianum (10 mg mL −1 ) purchased from Sigma-Aldrich and a commercial genomic DNA extraction kit (Real Biotech Corporation, Banqiao City, Taipei County, Taiwan). Extracted DNA was quantified by Nanodrop, cleaved with Nuclease P1 from Penicillium citrinum (50 μg 100 μg −1 of DNA) and alkaline phosphatase from bovine intestinal mucosa (1 U 100 μg −1 of DNA), both purchased from Sigma-Aldrich, to release the corresponding nucleosides. 8-oxodG and deoxyguanosine retention time were 5.1 and 11.5 min, respectively. Measurement of 8-oxodG is expressed as the amount of 8-oxodG 10 −6 deoxyguanosine calculated by comparison to standard solutions of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 2′deoxyguanosine hydrate, both purchased from Sigma-Aldrich. Results were normalized against total protein concentration. All assays were independently performed in triplicate. 2.7. Statistical analysis Two-way analysis of variance was performed followed by Bonferroni post hoc test. P ≤ 0.05 was considered statistically significant, and results are expressed as mean ± SE. Statistical analysis was accomplished with the support of the Statistical Nucleus of the Federal University of Rio Grande do Sul. 3. Results 3.1. Evaluation of ABC efflux pumps After incubation with verapamil, a decrease in MIC values of no more than 1 dilution was registered in resistant C. glabrata strain. No alteration was observed on MIC values of fluconazole-susceptible strain (Supplementary Table 1). 3.2. ROS generation in C. glabrata treated with fluconazole The CLSM analysis demonstrated a significant increase (P ≤ 0.05) in fluorescence of fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated with fluconazole compared to untreated strains. There was no significant difference in the generation of intracellular
Fig. 1. ROS generation induced by treatment with fluconazole (Flc) at a subinhibitory concentration in fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains in stationary growth phase. The values reported are mean ± SE. ⁎Statistically significant difference (P ≤ 0.05).
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reactive species between susceptible and resistant cells (Fig. 1 and Supplementary Fig. 2). 3.3. The effect of fluconazole on the antioxidant response of fluconazolesusceptible and fluconazole-resistant C. glabrata The results showed a higher (P ≤ 0.05) enzymatic activity of GPx and SOD in fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated with fluconazole compared to untreated cells. There was no significant difference in GPx and SOD enzymatic activity between susceptible and resistant cells, as shown in Fig. 2A and B. GST enzymatic activity was higher (P ≤ 0.05) in fluconazole-susceptible C. glabrata strains without fluconazole treatment compared to the same strains treated with fluconazole and compared to resistant strains (Fig. 2C). The quantification of total glutathione and the consumption of H2O2 showed no significant differences among the 4 groups (Table 2). 3.4. Treatment with fluconazole and oxidative damage Statistical analysis showed no significant differences among carbonyl, MDA, and NO2/NO3 levels in the studied groups (Table 2). The endogenous levels of 8-oxodG in the DNA of fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated with fluconazole at subinhibitory concentrations were significantly higher (P ≤ 0.05) than these same strains without treatment (Fig. 3). Furthermore, following fluconazole treatment, there was a significantly higher (P ≤ 0.05) level of 8-oxodG in fluconazole-resistant C. glabrata strains than in fluconazole-susceptible strains (Fig. 3). 4. Discussion The antioxidant defenses of C. glabrata against various generators of oxidative stress have been studied (Arana et al., 2010; Petrova et al., 2013; Roetzer et al., 2011). However, antioxidant response and oxidative damage caused by fluconazole in C. glabrata remain poorly explored. Multiple mechanisms of azole resistance have been reported in Candida spp., and overexpression of multidrug efflux transporters CgCDR1, CgCDR2, and CgPDH1 and the transcription factor CgPDR1 are mechanisms of fluconazole resistance most observed in C. glabrata strains (Berila et al., 2009; Parkinson et al., 1995). Our data from analysis of ABC efflux pumps showed that verapamil did not reverse fluconazole resistance in C. glabrata, indicating that resistance in these strains is probably through another mechanism. The results of CLSM with DCFH-DA showed notable induction of ROS generation by fluconazole in C. glabrata under the tested conditions. In a recent study, it was shown that fluconazole also generated ROS in fluconazole-susceptible Candida tropicalis strains (Da Silva et al., 2013) as well as in Candida albicans (Kobayashi et al., 2002). However, studies with Candida krusei (Costa-De-Oliveira et al., 2012) and Cryptococcus gattii (Ferreira et al., 2013) showed no increase in the intracellular amount of ROS when the cells were treated with fluconazole. ROS generation appears to be species dependent, which is comprehensible, since microorganisms differ in their sensitivity and response to oxidative stress (Missall et al., 2004). Moreover, our data showed that fluconazole resistance or susceptibility in C. glabrata did not affect ROS generation induced by fluconazole. This finding contrasts with results in C. tropicalis, in which fluconazole induced generation of reactive species only in susceptible strains (Da Silva et al., 2013). Abegg et al. (2010) showed that these species had different response to oxidative stress generated by H2O2; oxidative damage in lipids and proteins, for example, was significantly higher in C. tropicalis compared to C. glabrata, and the antioxidant defenses were also significantly different. These results demonstrate that oxidative metabolism and possibly fluconazole metabolism vary from one species to another, since the generation of ROS by fluconazole should not be directly through the antifungal molecule but by some other route that is to be studied in the future.
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
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C.D. Mahl et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
Fig. 2. GPx (A), SOD (B), and GST (C) enzymatic activity of fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains treated or not with fluconazole (Flc) at a subinhibitory concentration. The values reported are mean ± SE. ⁎Statistically significant difference (P ≤ 0.05).
Moreover, the experimental conditions that isolates of C. tropicalis and C. glabrata were submitted, as fluconazole concentration, exposure time, and growth phase, should be consider. In view of the evidence of induction of ROS generation by fluconazole in C. glabrata, we evaluated the antioxidant response of fluconazole-susceptible and fluconazole-resistant C. glabrata strains following treatment with fluconazole. Members of the GPx enzymatic family remove H2O2 and organic peroxides by coupling their reduction to H2O with oxidation of reduced glutathione, GSH (Halliwell and Gutteridge, 2007). C. glabrata possesses 1 GPX2 and 2 GPX3 antioxidant genes, with cytoplasmic and mitochondrial localizations (Petrova et al., 2013). The SOD enzymes are highly efficient in catalytic removal of O2•− , reducing it to H2O2 and also oxidizing it to O2 (Halliwell and Gutteridge, 2007). Thus, observing our data, it might be that fluconazole induces generation of organic peroxides and O2•− as part of its antifungal mechanism against C. glabrata, which results in the increased GPx and SOD enzymatic activity as an antioxidant response of the yeast. The mechanisms by which these organic peroxides and O2•− are generated in C. glabrata remain to be elucidated. However, under similar conditions, fluconazole did not increase SOD activity in C. gattii (Ferreira et al., 2013); the authors suggested that this occurs because fluconazole did not induce generation of reactive species in C. gattii. We observed that fluconazole-susceptible C. glabrata strains at stationary phase
presented higher GST enzymatic activity compared to resistant strains, and we also observed that this activity decreased when the susceptible cells were treated with fluconazole at a subinhibitory concentration. It could be proposed that susceptible cells lose part of their GST activity and do not externalize the antifungal, thereby becoming susceptible to it. However, further verification of the role of GST in C. glabrata and its activity against fluconazole is needed. A possible role for the enzymatic activity of catalases, peroxidases, and peroxiredoxins against oxidative stress generated by fluconazole was assessed by H2O2 consumption. Results of H2O2 consumption showed no significant differences among the 4 groups. These results in combination with the total glutathione levels indicated no role of these antioxidants against the oxidative stress generated by fluconazole under the conditions tested. We asked whether fluconazole at a subinhibitory concentration caused oxidative damage in lipids, proteins, and nucleic acids in C. glabrata. MDA is an end-product of lipoperoxidation, and our data showed no significant difference between C. glabrata strains treated or not with fluconazole, indicating that there was no lipid oxidative damage. A recent study showed that fluconazole also did not induce lipoperoxidation in C. gattii after 1 h and 24 h of treatment (Ferreira et al., 2013), which is corroborated by these findings. Our results also showed that fluconazole did not induce oxidative damage in proteins
Table 2 Oxidative stress analysis among fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated or untreated with a subinhibitory concentration of fluconazole (Flc). Assays
MDA (μmol mg−1 protein) Protein carbonylation (nmol mg−1 protein) NO2/NO3a (nmol mg−1 protein) Total glutathione (μmol mg−1 protein) Consumption of H2O2b (U mg−1)
C. glabrata strains FS
FS + Flc
FR
FR + Flc
0.33 (±0.13) 0.07 (±0.008) 20.11 (±2.47) 30.47 (±6.068) 5835 (±750.24)
0.29 (±0.05) 0.08 (±0.02) 17.92 (±2.27) 41.61 (±13.72) 7222 (±1536.54)
0.22 (±0.07) 0.06 (±0.01) 12.76 (±1.83) 50.14 (±9.36) 5843 (±788.07)
0.21 (±0.06) 0.07 (±0.01) 23.48 (±6.04) 46.22 (±16.39) 7269 (±1575.08)
The values are mean ± SE. a Nitrates and nitrites. b Hydrogen peroxide.
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
C.D. Mahl et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
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development of antifungal agents for clinical use, since new antifungal mechanisms are a field to be explored against the broad antifungal resistance seen in epidemiology. Funding This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazilian agency for research support). Transparency declarations None to declare. Acknowledgments Fig. 3. Endogenous levels of 8-oxodG in DNA of fluconazole-susceptible (FS) and fluconazoleresistant (FR) C. glabrata strains treated or not with fluconazole (Flc) at a subinhibitory concentration. The values reported are mean ± SE. ⁎Statistically significant difference (P ≤ 0.05).
under the tested conditions. The high GPx and SOD activity observed in cells treated with fluconazole may protect the cell against lipid and protein oxidation by neutralizing reactive species. NO is a reactive species that can cross membranes; it diffuses readily between and within cells (Halliwell and Gutteridge, 2007). The results of NO2 and NO3 levels showed no significant differences among the groups. The levels of 8-oxodG were also quantified, and the data showed that induction of ROS generation as part of the fluconazole antifungal mechanism of action can cause oxidative DNA damage in C. glabrata. We propose that fluconazole induces generation of O2 •− and organic peroxides in C. glabrata. Although these reactive species do not react at significant rates with DNA bases, they can react with radicals formed after the DNA has been attacked by more aggressive reactive species such as hydroxyl radical (OH ). Furthermore, O2 •− can accelerate OH production by the so-called superoxide-assisted Fenton reaction (Halliwell and Gutteridge, 2007). A recent study showed that fluconazole induced oxidative DNA damage in C. tropicalis after 24-h exposure (Da Silva et al., 2013), which reinforces our findings. To our knowledge, the present study is the first to used HPLC-ECD to evaluate oxidative DNA damage in C. glabrata. Regarding our observation that fluconazole induced more oxidative DNA damage in fluconazole-resistant strains than in fluconazole-susceptible strains, it could be thought that this was a consequence of the fact that the subinhibitory concentration of fluconazole was higher for resistant strains than for susceptible strains (Table 1); thus, resistant strains were treated with a higher concentration of fluconazole. Fluconazole induces generation of ROS only in some yeasts (Da Silva et al., 2013; Kobayashi et al., 2002), and our data demonstrated that fluconazole is able to induced generation of ROS in C. glabrata at stationary growth phase, and this generation, including the generation of organic peroxides and O2 •−, could be part of its antifungal mechanism. The role of the GST enzyme in resistance or susceptibility of C. glabrata to fluconazole should be investigated because this enzyme could be an important key to fluconazole resistance. More studies involving pathogenic C. glabrata and the wide use of the antifungal fluconazole are needed. Yeast cells in exponential or stationary phase respond differently to oxidative stress (Cuellar-Cruz et al., 2008; Jamieson, 1992), and fluconazole could also induce oxidative and nitrosative stress in a time- and dose-dependent manner (Arana et al., 2010) in C. glabrata. The limitations of our study should be noted, which are mainly the use of 1 single fluconazole concentration to each strain and the analysis of oxidative stress response just in stationary growth phase. The antioxidant response is directly related to pathogenicity, so inhibition of this response could attenuate the virulence of pathogenic species such as C. glabrata. Our results help in the understanding about antioxidant metabolism of C. glabrata, and they are important for the rational
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Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
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Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage Camila Donato Mahl a,b, Camile Saul Behling a,b, Fernanda S. Hackenhaar a,b, Mélany Natuane de Carvalho e Silva b, Jordana Putti b, Tiago B. Salomon a,b, Sydney Hartz Alves c, Alexandre Fuentefria d, Mara S. Benfato a,b,⁎ a
Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Laboratório de Estresse Oxidativo, Departamento de Biofísica, IB, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Laboratório de Pesquisas Micológicas, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil d Laboratório de Micologia Aplicada, Departamento de Análises, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 16 December 2014 Received in revised form 10 March 2015 Accepted 22 March 2015 Available online xxxx Keywords: Oxidative stress C. glabrata Fluconazole
a b s t r a c t In this study, we assessed the generation of reactive oxygen species (ROS) induced by subinhibitory concentration of fluconazole in susceptible and resistant Candida glabrata strains at stationary growth phase and measured their oxidative responses parameters: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathioneS-transferase (GST), consumption of hydrogen peroxide, and total glutathione, as well as oxidative damage in lipids, proteins, and DNA. Data showed that fluconazole increased generation of ROS and GPx and SOD enzymatic activity in treated cells; however, these enzymatic activities did not differ between resistant and susceptible strains. Susceptible strains exhibited higher GST activity than resistant, and when susceptible cells were treated with fluconazole, GST activity decreased. Fluconazole treatment cause oxidative damage only in DNA. There are a possible participation of ROS, as organic peroxides and O2•−, in antifungal mechanism of fluconazole, which results in higher GPx and SOD enzymatic activities and oxidative DNA damage in C. glabrata. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Since the early 1980s, fungi have emerged as major causes of human infections, particularly in immunocompromised and hospitalized patients with serious underlying diseases (Pfaller and Diekema, 2007). Of the fungi regarded as human pathogens, Candida spp. have been described as medically important fungi that cause severe mucosal and life-threatening invasive infections (Smeekens et al., 2013). Epidemiological studies reveal C. glabrata as an important emerging pathogen in nosocomial infections (Colombo et al., 2013; Tortorano et al., 2006), and due to the widely use of fluconazole in clinic, infections caused by fluconazole-resistant C. glabrata strains have been rising. Fluconazole is widely employed in part because of its metabolic stability, relatively high water solubility, and good tolerability (Grant and Clissold, 1990). It inhibits the biosynthesis of ergosterol by interfering with the fungal lanosterol demethylase (Silva et al., 2012). Three main mechanisms of fluconazole resistance among Candida species, including C. glabrata, are known, and although those mechanisms are the most well defined, there is evidence that products of genes involved in the oxidative stress response are involved in fluconazole resistance (Rogers
⁎ Corresponding author. Tel.: +55-51-33087603; fax: +55-51-33087003. E-mail address: [email protected] (M.S. Benfato).
and Barker, 2002; Rogers et al., 2006). In recent years, it has been proposed that reactive oxygen species (ROS) participate in the antifungal mechanism of action of azoles (Da Silva et al., 2013; Ferreira et al., 2013; Kobayashi et al., 2002). However, this putative antifungal mechanism is not fully understood, and the generation of reactive species by azoles, particularly fluconazole, still needs to be investigated. C. glabrata was shown to be resistant to ROS and to possess a potent antioxidant system that protects the fungal cell against endogenous and exogenous (Abegg et al., 2010). However, the antioxidant response of C. glabrata against ROS generation by fluconazole remains poorly explored, and the possible oxidative damage caused by fluconazole in this species is unknown. The elucidation of possible associations among antifungal mechanisms of action, antifungal resistance, and oxidative stress responses is important for identification of new targets for new antifungal agents and rational development of antifungal drugs. To better understand the relation between oxidative stress response, ROS generation by fluconazole, and fluconazole resistance in C. glabrata, we determined whether fluconazole induced generation of ROS in C. glabrata and evaluated the antioxidant defenses of fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated or not with fluconazole. We also asked whether fluconazole at subinhibitory concentrations caused oxidative damage to proteins, lipids, and nucleic acids in these strains.
http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
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2. Methods
2.3. Measurement of ROS generation
2.1. Strains and culture growth
To measure ROS generation, cells were incubated with 2′,7′dichlorofluorescein diacetate (DCFH-DA) 10 μmol L−1 in 0.9% NaCl for 30 min at 37 °C in the dark. Cells were then transferred to glass slides for analysis of intracellular fluorescence by confocal laser scanning microscopy (CLSM; Olympus FluoView™ 1000; Miami, Florida, USA) at the Electron Microscopy Center of the Federal University of Rio Grande do Sul (CME-UFRGS). Images were processed by Olympus FluoView ver.4.0a Viewer software. The fluorescence intensity of a hundred cells was measured in 2D by ImageJ software. Values are expressed as corrected total cell fluorescence.
Two groups of C. glabrata clinical strains isolated from HIV patients with oropharyngeal candidiasis were assessed. The strains are part of the fungi collection of Mycological Research Laboratory (LAPEMI, Federal University of Santa Maria, Brazil) and were identified using the BioMerieux ID 32C yeast identification system according to manufacturer's recommendations. One group consists of 8 fluconazole-susceptible clinical strains (LAPEMI2010.Cg1, LAPEMI2010.Cg5, LAPEMI2010.Cg7, LAPEMI2010.Cg15, LAPEMI2010.Cg16, and LAPEMI2010.Cg20). The other group was derived in the Mycological Research Laboratory (cited above) from susceptible strains employing the technique of fluconazole resistance induction (Fekete-Forgacs et al., 2000; Mario et al., 2012) and consists of 8 fluconazole-resistant strains (LAPEMI2010.Cg1R, LAPEMI2010.Cg5R, LAPEMI2010.Cg7R, LAPEMI2010.Cg15R, LAPEMI2010.Cg16R, and LAPEMI2010.Cg20R). All of the yeasts were tested by the broth microdilution method using the CLSI M27-A3 and third informational supplement (M27-S3/2008) standardized reference method. No patients received prior treatment with fluconazole before the collection and isolation of fluconazole-susceptible strains. To ensure that the pattern of resistance remained stable in fluconazole-resistant strains, cells were maintained in Sabouraud agar containing 64 μg fluconazole mL−1, and subcultures were always made from this same primary cell culture. To evaluate the relationship between fluconazole and oxidative stress, these 2 groups were grown in the presence (or absence) of fluconazole at a subinhibitory concentration determined from their MIC as shown in Table 1. Growth curves were performed to identify the time required for all strains to reach stationary phase. In brief, from a 24-h culture in Sabouraud agar, an inoculum was added to a 30-mL Erlenmeyer flask containing Sabouraud broth, and the optical density (OD) at 640 nm was adjusted to 0.08 using a spectrophotometer. This subculture was incubated on a shaker at 200 rpm at 32 °C for 48 h. The OD was determined every 30 min during the first 6 h and every 12 h during the following 42 h. The results showed that at 24 h of growth all strains had reached stationary phase (Supplementary Fig. 1). For oxidative stress assays, strains were subcultured on Sabouraud agar for 24 h at 32 °C. They were then inoculated into Sabouraud broth without fluconazole or with fluconazole at subinhibitory concentrations according to their MIC (Table 1) and grown in flasks on an orbital shaker at 32 °C and 200 rpm until stationary phase was reached. Cells were transferred to centrifuge tubes, and cell-free extracts were obtained according to Abegg et al. (2010). Total protein concentration was measured using the Bradford method (Bradford, 1976).
2.2. ATP binding cassette (ABC) efflux pumps The MICs of fluconazole were redetermined in the presence of verapamil, an ABC efflux pump blocker, according to the CLSI M27-A2 protocol (Guinea et al., 2006; Pina-Vaz et al., 2005; Prates et al., 2011).
Table 1 Fluconazole MIC and subinhibitory concentrations of fluconazole-susceptible and fluconazoleresistant C. glabrata. C. glabrata strains
Fluconazole MIC (na)
FS
2 4 128 256
FR
μg μg μg μg
mL−1 (n mL−1 (n mL−1 (n mL−1 (n
= = = =
5) 3) 4) 4)
FS = fluconazole susceptible; FR = fluconazole resistant. a Number of strains.
Subinhibitory concentration of fluconazole for growth 1 2 64 128
μg μg μg μg
mL−1 mL−1 mL−1 mL−1
2.4. Activity of antioxidant enzymes Glutathione peroxidase (GPx) activity was evaluated in cellular extract by measuring the oxidation of NADPH in the presence of reduced glutathione (GSH), glutathione reductase, and tert-butyl hydroperoxide at 340 nm over 3 min (Pinto and Bartley, 1969). GPx is expressed as U mg−1 of protein. Superoxide dismutase (SOD) activity was measured in the cellular extract using a protocol of commercial kit RANSOD (RANSOD SD 125®; Randox Laboratories, Crumlin, UK). SOD activity was measured over 3 minutes and is expressed as U mg−1of protein. Glutathione-S-transferase (GST) activity was assayed in the cellular extract based on the formation of S-(2,4-dinitrophenyl)-glutathione by GST enzymatic activity (Tsuchida, 2000). The absorbance of the compound formed was measured over 2 min on a spectrophotometer at 340 nm. GST activity is expressed as U mg−1 of protein. The consumption of hydrogen peroxide (H2O2) was evaluated in the cellular extract by spectrophotometric assay over 1 min at 240 nm (Aebi, 1984). Activity is expressed as U mg−1 of protein. 2.5. Quantification of glutathione Quantification of total glutathione was measured in the cellular extract on a spectrophotometer at 412 nm as the formation of p-nitrophenol from the reduction of 5,5-dithiobis(2-nitrobenzoic acid). This reduction occurs when NADPH, cofactor for glutathione reductase, is oxidized (Rahman et al., 2006). Glutathione levels were compared with reduced glutathione standard solutions purchased from Sigma-Aldrich (Saint Louis, Missouri, USA) and are expressed as μmol mg−1 of protein. 2.6. Quantification of oxidative damage Quantification of malondialdehyde (MDA) provides an index of oxidative damage in lipids. MDA was determined in the cellular extract by HPLC using a reversed-phase SUPELCOSIL™ LC-18-DB High-performance liquid chromatography (HPLC) column (15 cm × 4.6 mm, 5 μm). A mobile phase flow rate of 0.5 mL min−1 in 30 mmol monobasic potassium phosphate L −1 (pH 3.6) and methanol (9:1, v/v) was employed (Karatepe, 2004). The absorbance of the column effluent was monitored at 254 nm, and the MDA retention time was 5.6 min. MDA levels were determined by comparison with MDA standard solutions purchased from Sigma-Aldrich, and the results are expressed as μmol mg−1 of protein. Oxidative damage in proteins was assessed in the cellular extract on a spectrophotometer according to Levine et al. (1990). Carbonyl content was calculated using a millimolar extinction coefficient of hydrazone of 21,000 M −1 cm −1. Absorbance at 370 nm was measured, and the results are expressed as nmol mg−1 of protein. The indirect method of measuring nitric oxide on a spectrophotometer was performed in the cellular extract by determining the levels of nitrites and nitrates (NO2 and NO3) at 543 nm according to Grisham et al. (1996). NO2 and NO3 levels are expressed as nmol mg−1 of protein. To determine oxidative damage in DNA, we quantified 8-oxo-7,8dihydro-2′-deoxyguanosine (8-oxodG) by High-performance liquid
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
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chromatography with electrochemical detection (HPLC-ECD) (Dirmeier et al., 2002; ESCODD et al., 2005). The column employed was a reversed-phase LC-18 NUCLEOSIL HPLC column (15 cm × 4.6 mm, 5 μm). A mobile phase flow rate of 0.5 mL min −1 in 50 mmol monobasic potassium phosphate L −1 (pH 5.5) and 4% methanol was employed. A UV detector at 254 nm was used to detect deoxyguanosine, and an electrochemical detector (DECADE II, 20 ηV) was used to detect 8-oxodG. DNA was extracted using lysing enzymes from Trichoderma harzianum (10 mg mL −1 ) purchased from Sigma-Aldrich and a commercial genomic DNA extraction kit (Real Biotech Corporation, Banqiao City, Taipei County, Taiwan). Extracted DNA was quantified by Nanodrop, cleaved with Nuclease P1 from Penicillium citrinum (50 μg 100 μg −1 of DNA) and alkaline phosphatase from bovine intestinal mucosa (1 U 100 μg −1 of DNA), both purchased from Sigma-Aldrich, to release the corresponding nucleosides. 8-oxodG and deoxyguanosine retention time were 5.1 and 11.5 min, respectively. Measurement of 8-oxodG is expressed as the amount of 8-oxodG 10 −6 deoxyguanosine calculated by comparison to standard solutions of 8-oxo-7,8-dihydro-2′-deoxyguanosine and 2′deoxyguanosine hydrate, both purchased from Sigma-Aldrich. Results were normalized against total protein concentration. All assays were independently performed in triplicate. 2.7. Statistical analysis Two-way analysis of variance was performed followed by Bonferroni post hoc test. P ≤ 0.05 was considered statistically significant, and results are expressed as mean ± SE. Statistical analysis was accomplished with the support of the Statistical Nucleus of the Federal University of Rio Grande do Sul. 3. Results 3.1. Evaluation of ABC efflux pumps After incubation with verapamil, a decrease in MIC values of no more than 1 dilution was registered in resistant C. glabrata strain. No alteration was observed on MIC values of fluconazole-susceptible strain (Supplementary Table 1). 3.2. ROS generation in C. glabrata treated with fluconazole The CLSM analysis demonstrated a significant increase (P ≤ 0.05) in fluorescence of fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated with fluconazole compared to untreated strains. There was no significant difference in the generation of intracellular
Fig. 1. ROS generation induced by treatment with fluconazole (Flc) at a subinhibitory concentration in fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains in stationary growth phase. The values reported are mean ± SE. ⁎Statistically significant difference (P ≤ 0.05).
3
reactive species between susceptible and resistant cells (Fig. 1 and Supplementary Fig. 2). 3.3. The effect of fluconazole on the antioxidant response of fluconazolesusceptible and fluconazole-resistant C. glabrata The results showed a higher (P ≤ 0.05) enzymatic activity of GPx and SOD in fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated with fluconazole compared to untreated cells. There was no significant difference in GPx and SOD enzymatic activity between susceptible and resistant cells, as shown in Fig. 2A and B. GST enzymatic activity was higher (P ≤ 0.05) in fluconazole-susceptible C. glabrata strains without fluconazole treatment compared to the same strains treated with fluconazole and compared to resistant strains (Fig. 2C). The quantification of total glutathione and the consumption of H2O2 showed no significant differences among the 4 groups (Table 2). 3.4. Treatment with fluconazole and oxidative damage Statistical analysis showed no significant differences among carbonyl, MDA, and NO2/NO3 levels in the studied groups (Table 2). The endogenous levels of 8-oxodG in the DNA of fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated with fluconazole at subinhibitory concentrations were significantly higher (P ≤ 0.05) than these same strains without treatment (Fig. 3). Furthermore, following fluconazole treatment, there was a significantly higher (P ≤ 0.05) level of 8-oxodG in fluconazole-resistant C. glabrata strains than in fluconazole-susceptible strains (Fig. 3). 4. Discussion The antioxidant defenses of C. glabrata against various generators of oxidative stress have been studied (Arana et al., 2010; Petrova et al., 2013; Roetzer et al., 2011). However, antioxidant response and oxidative damage caused by fluconazole in C. glabrata remain poorly explored. Multiple mechanisms of azole resistance have been reported in Candida spp., and overexpression of multidrug efflux transporters CgCDR1, CgCDR2, and CgPDH1 and the transcription factor CgPDR1 are mechanisms of fluconazole resistance most observed in C. glabrata strains (Berila et al., 2009; Parkinson et al., 1995). Our data from analysis of ABC efflux pumps showed that verapamil did not reverse fluconazole resistance in C. glabrata, indicating that resistance in these strains is probably through another mechanism. The results of CLSM with DCFH-DA showed notable induction of ROS generation by fluconazole in C. glabrata under the tested conditions. In a recent study, it was shown that fluconazole also generated ROS in fluconazole-susceptible Candida tropicalis strains (Da Silva et al., 2013) as well as in Candida albicans (Kobayashi et al., 2002). However, studies with Candida krusei (Costa-De-Oliveira et al., 2012) and Cryptococcus gattii (Ferreira et al., 2013) showed no increase in the intracellular amount of ROS when the cells were treated with fluconazole. ROS generation appears to be species dependent, which is comprehensible, since microorganisms differ in their sensitivity and response to oxidative stress (Missall et al., 2004). Moreover, our data showed that fluconazole resistance or susceptibility in C. glabrata did not affect ROS generation induced by fluconazole. This finding contrasts with results in C. tropicalis, in which fluconazole induced generation of reactive species only in susceptible strains (Da Silva et al., 2013). Abegg et al. (2010) showed that these species had different response to oxidative stress generated by H2O2; oxidative damage in lipids and proteins, for example, was significantly higher in C. tropicalis compared to C. glabrata, and the antioxidant defenses were also significantly different. These results demonstrate that oxidative metabolism and possibly fluconazole metabolism vary from one species to another, since the generation of ROS by fluconazole should not be directly through the antifungal molecule but by some other route that is to be studied in the future.
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
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Fig. 2. GPx (A), SOD (B), and GST (C) enzymatic activity of fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains treated or not with fluconazole (Flc) at a subinhibitory concentration. The values reported are mean ± SE. ⁎Statistically significant difference (P ≤ 0.05).
Moreover, the experimental conditions that isolates of C. tropicalis and C. glabrata were submitted, as fluconazole concentration, exposure time, and growth phase, should be consider. In view of the evidence of induction of ROS generation by fluconazole in C. glabrata, we evaluated the antioxidant response of fluconazole-susceptible and fluconazole-resistant C. glabrata strains following treatment with fluconazole. Members of the GPx enzymatic family remove H2O2 and organic peroxides by coupling their reduction to H2O with oxidation of reduced glutathione, GSH (Halliwell and Gutteridge, 2007). C. glabrata possesses 1 GPX2 and 2 GPX3 antioxidant genes, with cytoplasmic and mitochondrial localizations (Petrova et al., 2013). The SOD enzymes are highly efficient in catalytic removal of O2•− , reducing it to H2O2 and also oxidizing it to O2 (Halliwell and Gutteridge, 2007). Thus, observing our data, it might be that fluconazole induces generation of organic peroxides and O2•− as part of its antifungal mechanism against C. glabrata, which results in the increased GPx and SOD enzymatic activity as an antioxidant response of the yeast. The mechanisms by which these organic peroxides and O2•− are generated in C. glabrata remain to be elucidated. However, under similar conditions, fluconazole did not increase SOD activity in C. gattii (Ferreira et al., 2013); the authors suggested that this occurs because fluconazole did not induce generation of reactive species in C. gattii. We observed that fluconazole-susceptible C. glabrata strains at stationary phase
presented higher GST enzymatic activity compared to resistant strains, and we also observed that this activity decreased when the susceptible cells were treated with fluconazole at a subinhibitory concentration. It could be proposed that susceptible cells lose part of their GST activity and do not externalize the antifungal, thereby becoming susceptible to it. However, further verification of the role of GST in C. glabrata and its activity against fluconazole is needed. A possible role for the enzymatic activity of catalases, peroxidases, and peroxiredoxins against oxidative stress generated by fluconazole was assessed by H2O2 consumption. Results of H2O2 consumption showed no significant differences among the 4 groups. These results in combination with the total glutathione levels indicated no role of these antioxidants against the oxidative stress generated by fluconazole under the conditions tested. We asked whether fluconazole at a subinhibitory concentration caused oxidative damage in lipids, proteins, and nucleic acids in C. glabrata. MDA is an end-product of lipoperoxidation, and our data showed no significant difference between C. glabrata strains treated or not with fluconazole, indicating that there was no lipid oxidative damage. A recent study showed that fluconazole also did not induce lipoperoxidation in C. gattii after 1 h and 24 h of treatment (Ferreira et al., 2013), which is corroborated by these findings. Our results also showed that fluconazole did not induce oxidative damage in proteins
Table 2 Oxidative stress analysis among fluconazole-susceptible and fluconazole-resistant C. glabrata strains treated or untreated with a subinhibitory concentration of fluconazole (Flc). Assays
MDA (μmol mg−1 protein) Protein carbonylation (nmol mg−1 protein) NO2/NO3a (nmol mg−1 protein) Total glutathione (μmol mg−1 protein) Consumption of H2O2b (U mg−1)
C. glabrata strains FS
FS + Flc
FR
FR + Flc
0.33 (±0.13) 0.07 (±0.008) 20.11 (±2.47) 30.47 (±6.068) 5835 (±750.24)
0.29 (±0.05) 0.08 (±0.02) 17.92 (±2.27) 41.61 (±13.72) 7222 (±1536.54)
0.22 (±0.07) 0.06 (±0.01) 12.76 (±1.83) 50.14 (±9.36) 5843 (±788.07)
0.21 (±0.06) 0.07 (±0.01) 23.48 (±6.04) 46.22 (±16.39) 7269 (±1575.08)
The values are mean ± SE. a Nitrates and nitrites. b Hydrogen peroxide.
Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
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development of antifungal agents for clinical use, since new antifungal mechanisms are a field to be explored against the broad antifungal resistance seen in epidemiology. Funding This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazilian agency for research support). Transparency declarations None to declare. Acknowledgments Fig. 3. Endogenous levels of 8-oxodG in DNA of fluconazole-susceptible (FS) and fluconazoleresistant (FR) C. glabrata strains treated or not with fluconazole (Flc) at a subinhibitory concentration. The values reported are mean ± SE. ⁎Statistically significant difference (P ≤ 0.05).
under the tested conditions. The high GPx and SOD activity observed in cells treated with fluconazole may protect the cell against lipid and protein oxidation by neutralizing reactive species. NO is a reactive species that can cross membranes; it diffuses readily between and within cells (Halliwell and Gutteridge, 2007). The results of NO2 and NO3 levels showed no significant differences among the groups. The levels of 8-oxodG were also quantified, and the data showed that induction of ROS generation as part of the fluconazole antifungal mechanism of action can cause oxidative DNA damage in C. glabrata. We propose that fluconazole induces generation of O2 •− and organic peroxides in C. glabrata. Although these reactive species do not react at significant rates with DNA bases, they can react with radicals formed after the DNA has been attacked by more aggressive reactive species such as hydroxyl radical (OH ). Furthermore, O2 •− can accelerate OH production by the so-called superoxide-assisted Fenton reaction (Halliwell and Gutteridge, 2007). A recent study showed that fluconazole induced oxidative DNA damage in C. tropicalis after 24-h exposure (Da Silva et al., 2013), which reinforces our findings. To our knowledge, the present study is the first to used HPLC-ECD to evaluate oxidative DNA damage in C. glabrata. Regarding our observation that fluconazole induced more oxidative DNA damage in fluconazole-resistant strains than in fluconazole-susceptible strains, it could be thought that this was a consequence of the fact that the subinhibitory concentration of fluconazole was higher for resistant strains than for susceptible strains (Table 1); thus, resistant strains were treated with a higher concentration of fluconazole. Fluconazole induces generation of ROS only in some yeasts (Da Silva et al., 2013; Kobayashi et al., 2002), and our data demonstrated that fluconazole is able to induced generation of ROS in C. glabrata at stationary growth phase, and this generation, including the generation of organic peroxides and O2 •−, could be part of its antifungal mechanism. The role of the GST enzyme in resistance or susceptibility of C. glabrata to fluconazole should be investigated because this enzyme could be an important key to fluconazole resistance. More studies involving pathogenic C. glabrata and the wide use of the antifungal fluconazole are needed. Yeast cells in exponential or stationary phase respond differently to oxidative stress (Cuellar-Cruz et al., 2008; Jamieson, 1992), and fluconazole could also induce oxidative and nitrosative stress in a time- and dose-dependent manner (Arana et al., 2010) in C. glabrata. The limitations of our study should be noted, which are mainly the use of 1 single fluconazole concentration to each strain and the analysis of oxidative stress response just in stationary growth phase. The antioxidant response is directly related to pathogenicity, so inhibition of this response could attenuate the virulence of pathogenic species such as C. glabrata. Our results help in the understanding about antioxidant metabolism of C. glabrata, and they are important for the rational
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Please cite this article as: Mahl CD, et al, Induction of ROS generation by fluconazole in Candida glabrata: activation of antioxidant enzymes and oxidative DNA damage, Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.03.019
