Article Cite This: Chem. Res. Toxicol. 2018, 31, 658−665
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Nickel Oxide (NiO) Nanoparticles Induce Loss of Cell Viability in Yeast Mediated by Oxidative Stress Cat́ ia A. Sousa,†,‡,§ Helena M. V. M. Soares,§ and Eduardo V. Soares*,†,‡ †
Bioengineering Laboratory-CIETI, Chemical Engineering Department, ISEP-School of Engineering of Polytechnic Institute of Porto, Rua Dr. António Bernardino de Almeida, 431, 4249-015 Porto, Portugal ‡ CEB-Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal § REQUIMTE/LAQV, Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal Chem. Res. Toxicol. 2018.31:658-665. Downloaded from pubs.acs.org by TU MUENCHEN on 08/21/18. For personal use only.
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ABSTRACT: The present work aimed to elucidate whether the toxic effects of nickel oxide (NiO) nanoparticles (NPs) on the yeast Saccharomyces cerevisiae were associated with oxidative stress (OS) and what mechanisms may have contributed to this OS. Cells exposed to NiO NPs accumulated superoxide anions and hydrogen peroxide, which were intracellularly generated. Yeast cells coexposed to NiO NPs and antioxidants (L-ascorbic acid and N-tertbutyl-α-phenylnitrone) showed quenching of reactive oxygen species (ROS) and increased resistance to NiO NPs, indicating that the loss of cell viability was associated with ROS accumulation. Mutants lacking mitochondrial DNA (ρ0) displayed reduced levels of ROS and increased resistance to NiO NPs, which suggested the involvement of the mitochondrial respiratory chain in ROS production. Yeast cells exposed to NiO NPs presented decreased levels of reduced glutathione (GSH). Mutants deficient in GSH1 (gsh1Δ) or GSH2 (gsh2Δ) genes displayed increased levels of ROS and increased sensitivity to NiO NPs, which underline the central role of GSH against NiO NPs-induced OS. This work suggests that the increased levels of intracellular ROS (probably due to the perturbation of the electron transfer chain in mitochondria) combined with the depletion of GSH pool constitute important mechanisms of NiO NPs-induced loss of cell viability in the yeast S. cerevisiae.
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INTRODUCTION Nickel oxide (NiO) nanoparticles (NPs) have been the object of a renewed interest due to their multiple applications, such as catalysts, cathode materials for alkaline batteries, diesel−fuel additives, materials for gas or temperature sensors, electronic components, and pigments for ceramics and glass.1 One of the main mechanisms associated with metal oxide NPs toxicity consists of the generation of extracellular or intracellular reactive oxygen species (ROS), leading to the oxidative damage of the cells.2 ROS comprise a broad category of chemical species such as singlet oxygen (1O2), superoxide anion radical (O2•−), hydroxyl radical (HO•), peroxyl radical (ROO•), and hydrogen peroxide (H2O2).3 ROS can originate in the endoplasmic reticulum (through the formation of H2O2 and O2•− during protein folding),4 in peroxisomes (due to the production of H2O2 during fat acids oxidation),5 and in the mitochondria (through generation of O2•− and H2O2 in respiratory chain).6,7 ROS can be found in the cytosol as consequence of the escape of H2O2 from the endoplasmic reticulum and mitochondria.4 Yeast cells are equipped with an elaborate antioxidant defense mechanism to cope with oxidative stress (OS) and © 2018 American Chemical Society
thus to maintain intracellular redox equilibrium. This mechanism includes antioxidant molecules such as reduced glutathione (GSH) and enzymes.8 The yeast S. cerevisiae displays two superoxide dismutases (SOD) enzymes, which catalyze the dismutation of O2•− to H2O2; SOD1 encodes a Cu/ZnSOD, which localizes to the cytosol, mitochondrial intermembrane space and nucleus; SOD2 encodes a MnSOD present in the mitochondrial matrix. S. cerevisiae also displays two catalases (Cat), which reduce H2O2: Cat T, which localizes to the cytosol, and Cat A, which localizes to peroxisomes. In addition, yeast cells also possess peroxidases, which reduce inorganic and organic peroxides into the corresponding alcohols using cysteine thiols. Two classes of peroxidases were found: (a) glutathione peroxidases, which employ GSH such as Gpx3 (located at mitochondrial intermembrane space and peroxisomal matrix) and Grx1 (glutaredoxin, located at cytosol and nucleus); (b) thioredoxin peroxidases (also called peroxiredoxins), which employ Received: February 1, 2018 Published: July 25, 2018 658
DOI: 10.1021/acs.chemrestox.8b00022 Chem. Res. Toxicol. 2018, 31, 658−665
Article
Chemical Research in Toxicology
purchased from EUROSCARF collection (Frankfurt, Germany). The isogenic derivative ρ0 strain (BY4741- ρ0) was obtained as previously described.23 The strains were maintained at 4 °C on YEP agar slants [5 g/L yeast extract (Difco-BD), 5 g/L peptone (Difco-BD), 10 g/L glucose (Merck), and 15 g/L agar (Merck)] and grown in YEP broth at 30 °C on an orbital shaker at 150 rpm. Single-gene deletion mutant strains were maintained on YEP agar with 200 mg/L Geneticin (G418 disulfate salt, Sigma-Aldrich). Cultures in exponential phase of growth were obtained by incubating the cells overnight to an OD600 of ∼1.0, as previously described.22 Treatment of Yeast Cells with NiO NPs. Yeast cells in exponential phase of growth were centrifuged (2.500 × g, 5 min), washed twice, and resuspended in deionized water. Then cells were suspended (1 × 107 cells/mL) in 10 mM MES buffer (SigmaAldrich), pH 6.0, with 20 g/L glucose and incubated with 50 or 100 mg/L NiO NPs, at 30 °C, 150 rpm, for 6 h. Yeast cells were also suspended in MES buffer without NiO NPs (control). The effect of two antioxidants, L-ascorbic acid (AA) or N-tert-butylα-phenylnitrone (PBN) on the quenching of ROS NiO NPs induced was tested. AA efficiently scavenges free radicals and other ROS produced in cell since it reacts rapidly by donating a hydrogen atom to an oxidizing radical.24,25 PBN is a free radical spin trapping agent, which reacts covalently with radicals and form stable adducts.26 For this purpose, yeast cells were preincubated with 10 mM AA (Merck) or 2 mM PBN (Sigma-Aldrich) 30 min before the exposure to NiO NPs. For exposure of yeast cells to NiO NPs under nitrogen atmosphere during 6 h, yeast cells were preincubated in 20 mL of MES buffer in wash bottles under flow of pure N2 (less than 2 mg/L of 02; Linde) at 100 mL/min, 30 min before the exposure to NiO NPs. During cell exposure to NiO NPs, a flow of N2 at 100 mL/min was also maintained. Cell Viability Assay. The toxic impact of NiO NPs on the yeast cells was evaluated by a cell viability assay. Yeast cells were incubated in MES buffer without (control) or with NiO NPs and subsequently plated on YEP agar, as previously described.22 As toxicity end point, the viability was calculated considering the number of colony-forming unit (CFU)/mL at zero time as reference (100%). Determination of Reactive Oxygen Species (ROS). The detection of O2•− was carried out using dihydroethidium (DHE). This compound is a cell permeant probe that can undergo oxidation by superoxide anion radical to form the DNA-binding compound ethidium. DHE allows a specific detection of O2•− since this probe is minimally oxidized by H2O2.27 The H2O2 accumulated in yeast cells was monitored using the probes 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) or dihydrorhodamine 123 (DHR123). H2DCFDA is taken up by yeast cells, being metabolized by intracellular esterases to H2DCF; in the presence of H2O2, H2DCF is oxidized to DCF.28 Other ROS, such as peroxyl radical (ROO•) and peroxynitrite anion (ONOO−), are capable of undergo the oxidation of H2DCF to DCF.29,30 In addition, the probe DHR123, which is a structurally related analogue of H2DCFDA but lacks the diacetate and dichloro substituents of H2DCFDA,29 was also used. DHR123 is a cell permeant compound that is oxidized to rhodamine 123 by oxidants such as, H2O2, HOCl, and ONOO−.29 Because H2DCFDA and DHR123 can be oxidized by different ROS, these probes can be used as general redox sensors in the assessment of cellular oxidative stress.31 All compounds were purchased from SigmaAldrich. Yeast cells were suspended in MES buffer, with 2% (w/v) glucose, at a final concentration of 1 × 107 cells/mL, and incubated at 30 °C for 10 min, in the dark, with 8 μM DHE or with 20 μM H2DCFDA or with 2.88 μM DHR123. Subsequently, cells were treated with NiO NPs (50 or 100 mg/L) or Ni2+ (5.2 to 75 mg/L; from a 1000 mg/L NiCl2 stock solution, Merck) and placed in a 96well flat microplate (Orange Scientific). As control, cells were incubated in the same conditions in the absence of NPs. Fluorescence intensity, as relative fluorescence units (RFU), was measured in a PerkinElmer (Victor3) microplate reader at a fluorescence excitation
thioredoxin such as the cytosolic Tsa1 and the mitochondrial Prx1.5 When the level of ROS overcomes the defense system, the cell redox homeostasis is altered, resulting in OS, which can cause the damage of a wide range of molecules, such as unsaturated lipids via peroxidation, proteins via oxidation, and DNA, leading to reduced cell viability.9 Oxidative stress plays an important role in many human diseases, including diabetes, cancer, and neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease.10 Several studies have reported that NiO NPs induce OS in different cell models. In this context, it has been described that rats exposed to NiO NPs show pulmonary OS.11,12 Exposure to NiO NPs has been shown to induce intracellular accumulation of ROS in multiple human cell lines such as liver (HepG2),13 alveolar basal epithelial (A549),14−16 airway epithelial (HEp-2), and breast cancer (MCF-7) cells.17 Similarly, exposing the aquatic plant Lemna gibba L.18 and the barley Hordeum vulgare L.19 to NiO NPs induce cellular OS. The yeast Saccharomyces cerevisiae has been used as cell model in toxicological evaluations of chemicals. Yeast-based functional genomics and proteomics technologies, which include the use of the yeast deletion strain collection, are important tools for the elucidation of toxicity mechanisms.20 The easy manipulation of mitochondrial respiration, namely, by the loss of mitochondrial DNA,21 makes this simple cell model particularly attractive for investigating the role of mitochondria in ROS generation. A previous study showed that the exposure of the yeast S. cerevisiae to NiO NPs inhibited proliferation capacity, reduced metabolic activity, and enhanced accumulation of ROS.22 The present study aimed to further elucidate the role of ROS generation in the toxic effects of NiO NPs on the yeast S. cerevisiae. Additionally, we sought to determine the mechanism through which NiO NPs generate ROS in yeast cells. This was achieved by investigating the link between ROS production and the loss of cell viability induced by NiO NPs. OS was assessed by measuring both exogenous (acellular, abiotic, cell free) and intracellular ROS generated NPs induced. The different types of ROS produced by exposure to NiO were characterized using fluorescent probes. The involvement of mitochondria as a source of ROS was examined. The role of nonenzymatic (GSH) and enzymatic defenses (using yeast deletion strain collection) in the fight against OS induced by NiO NPs was also investigated.
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EXPERIMENTAL PROCEDURES
Preparation of Nickel Oxide Nanoparticles Stock Suspensions. Nickel oxide (NiO) nanoparticles (NPs) with a particle size
