- Email: [email protected]
ROS-independent toxicity of Fe3O4 nanoparticles to yeast cells: Involvement of mitochondrial dysfunction
Accepted Manuscript ROS-independent toxicity of Fe3O4 nanoparticles to yeast cells: Involvement of mitochondrial dysfunction Qi Peng, Da Huo, Hongyue Li, Bing Zhang, Yang Li, Anping Liang, Hui Wang, Qilin Yu, Mingchun Li PII:
S0009-2797(17)30292-2
DOI:
10.1016/j.cbi.2018.03.012
Reference:
CBI 8258
To appear in:
Chemico-Biological Interactions
Received Date: 14 March 2017 Revised Date:
27 February 2018
Accepted Date: 19 March 2018
Please cite this article as: Q. Peng, D. Huo, H. Li, B. Zhang, Y. Li, A. Liang, H. Wang, Q. Yu, M. Li, ROSindependent toxicity of Fe3O4 nanoparticles to yeast cells: Involvement of mitochondrial dysfunction, Chemico-Biological Interactions (2018), doi: 10.1016/j.cbi.2018.03.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT ROS-independent toxicity of Fe3O4 nanoparticles to yeast cells: involvement of
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mitochondrial dysfunction
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Qi Peng,a Da Huo,a Hongyue Li,a Bing Zhang,a Yang Li,a Anping Liang,a Hui Wang,b Qilin Yua,*,
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Mingchun Lia,*
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a
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of Microbiology, College of Life Science, Nankai University, Tianjin 300071, PR China
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b
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Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department
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Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin, 300091, China
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Corresponding authors:
Qilin Yu, Department of Microbiology, College of Life Science, Nankai University, Tianjin
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300071, P. R. China. E-mail address: [email protected].
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Mingchun Li, Department of Microbiology, College of Life Science, Nankai University, Tianjin
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300071, P. R. China. E-mail address: [email protected].
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ACCEPTED MANUSCRIPT Abstract
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Fe3O4 nanoparticles, one kind of magnetic nanomaterials (NMs), are widely used in drug delivery,
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biological imaging, sensors, catalysts and pollution management. However, its toxicity to
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biological systems and related toxicity mechanisms remain to be explored. In this study, we
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investigate the effect of as-synthesized Fe3O4 nanoparticles on growth of Saccharomyces
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cerevisiae, an important model fungus. Growth inhibition assays showed that Fe3O4 nanoparticles
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remarkably inhibited yeast growth. Interestingly, this inhibitory effect was not attributed to the
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well-known plasma membrane damage, cell wall damage and ROS accumulation. Further
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investigations revealed that the nanoparticles strongly impaired mitochondrial functions, resulting
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in abnormal mitochondrial morphology, decreased mitochondrial membrane potential (MMP) and
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attenuated ATP production. Most importantly, the respiratory chain complex
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respiratory chain complexes and ATP synthases, was found to be the main target of the
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nanoparticles. This study uncovers a novel ROS-independent toxicity mechanism of Fe3O4
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nanoparticles to eukaryotic cells.
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Keywords: Fe3O4 nanoparticle; mitochondrion; respiratory chain complex; nanotoxicity;
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Saccharomyces cerevisiae
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1. Introduction With the development of nanotechnology, more and more nanomaterials (NMs) are
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incorporated into the organisms and the ecosystems [1,2]. Fe3O4 nanoparticles are one kind of
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important magnetic NMs [3]. Owing to their excellent property that their action could be easily
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manipulated under the external magnetic field, Fe3O4 nanoparticles were widely used in drug
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delivery, magnetic resonance imaging, sensors, catalysts and pollution management [4-8]. Since
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these NMs frequently contact the organisms, it is urgent to elucidate their potential biological
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effects.
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Several studies have evaluated the potential toxicity of Fe3O4 nanoparticles to mammalian cells
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[9-12]. The nanoparticles may enter into the cells by the endocytosis pathway and release iron
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cations in the lysosomes, resulting in ROS production and oxidative stress-related apoptosis [9,10].
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Also, the nanoparticles may disorganize actin fiber and tubulin network, causing inhibition of cell
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migration and invasion of the human umbilical vein endothelial cells [11]. Furthermore, these
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nanoparticles may enter into the brain, inducing ROS production and mitochondrial dysfunction
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[12]. However, the mechanism by which they disrupt the mitochondrial functions remains
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unknown. Moreover, up to now, little is known about their effect on other organisms, especially on
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the fungal organisms that play important roles in decomposition and recycling of organic materials
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[13].
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In this study, we assessed the effect of as-synthesized Fe3O4 nanoparticles on growth of yeast,
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and found that this kind of NMs remarkably inhibited yeast growth at the concentrations ≥ 200
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mg/L. Interestingly, their inhibitory effect was not attributed to common plasma membrane
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damage-related cell death, cell wall damage and ROS accumulation. Further investigations
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revealed that these nanoparticles targeted to the mitochondrial respiratory chain complex
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leading to dysfunction of the mitochondria and consequent attenuation of ATP production. This
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study uncovers a mitochondrial respiratory chain-related toxicity mechanism of Fe3O4
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nanoparticles in eukaryotic cells.
2. Materials and methods
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2.1. Preparation and characterization of Fe3O4 nanoparticles
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The Fe3O4 nanoparticles used in this study were prepared by the co-precipitation method [14].
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Briefly, 100 mL of 0.3 M FeCl2 (dissolved in distilled water) were mixed with 100 mL of 0.6 M
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FeCl3 solution (dissolved in distilled water). 100 mL of 0.3 M NaOH were then slowly added into
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the mixture, obtaining the black solution. The solution was heated and the Fe3O4 nanoparticles
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were precipitated. After washed with distilled water to pH 7.0, the nanoparticles were dried at the
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room temperature for further use.
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The morphology of the obtained nanoparticles was characterized by a transmission electron
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microscope (TEM, Tecnai G2 F-20, FEI, USA). The crystal structure and composition of the
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nanoparticles were characterized by X-ray diffraction (XRD, D/max-2500, Japan).
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2.2. Strains and growth conditions
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The S. cerevisiae strain InvSc1 (Invitrogen, USA) were used for assessing biological effects of
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Fe3O4 nanoparticles. To investigate their possible effect on the cell wall integrity (CWI) pathway,
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the strain Sc+p2052 containing the LacZ gene under the control of the promoter of the CWI gene
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FKS2 was used [15]. Normally, the yeast cells were cultivated overnight in liquid YPD medium
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with shaking at 30
and were suspended in fresh YPD medium for further testing.
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2.3. Growth inhibition assays To assess the effect of Fe3O4 nanoparticles on yeast growth, overnight cultured yeast cells were
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suspended in YPD medium containing Fe3O4 nanoparticles with the following concentrations, 0,
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100, 200, 400, 800 and 1600 ppm. The initial yeast number in the suspensions was 1×106 cells/mL.
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The suspensions were then cultured with shaking for 12 h at 30
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and used for cell number counting using the haemocytometers. The percent of growth was
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calculated as the cell number of each treatment group divided by that of the control × 100.
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. The cultures were then sampled
To investigate the effect of the ROS scavenger N-acetylcysteine (NAC) on the inhibition effect
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of Fe3O4 nanoparticles, the yeast cells were cultured in YPD medium containing 800 mg/L Fe3O4,
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5 mM NAC or 800 mg/L Fe3O4 plus 5 mM NAC. The suspensions were then cultured for 12 h as
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described above, sampled and used for cell counting.
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To investigate the effect of Fe3O4 nanoparticles on cell death, the yeast cells were treated by
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Fe3O4 nanoparticles for 12 h as described above, harvested and suspended in PBS. The cells were
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then stained by propidium iodide (PI, prepared in distilled water, final concentration 10 µg/mL,
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Sigma, USA) for 5 min. The stained cells were washed twice with PBS and observed by
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fluorescence microscopy (BX-41, Olympus, Japan) using the RFP filtering set. The percentage of
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PI-positive (dead) cells was calculated as the number of PI-positive cells divided by the total cell
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number × 100. At least 20 observed fields were determined.
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2.5. Cell cycle assays The yeast cells were treated by Fe3O4 nanoparticles for 12 h as described above and harvested. Cell cycle progression was then evaluated by the cell cycle assay kit (Beyotime Biotech., China).
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2.6. ROS assays
To determine the number of ROS-accumulated cells, the treated cells were washed and
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suspended in PBS buffer. 500 µL of cell suspensions were stained with 2 µL of
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2’,7’-dichlorofluorescein diacetate (DCFH-DA, 10 000 ppm, dissolved in PBS) for 30 min at 30℃.
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The stained cells were harvested, washed and re-suspended in PBS buffer, and were examined by
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flow cytometry (FACS, Calibur, BD, USA). The percent of ROS-accumulated cells was calculated
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as the number of DCFH-DA-positive cells divided by that of total observed cells × 100. At least
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30 fields were observed.
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2.7. Cell wall staining and chitin measurements
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To detect the yeast cell wall chitin, the cells were treated with Fe3O4 nanoparticles for 12 h as
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described above. The cells were then washed, re-suspended in PBS buffer, and stained with
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Calcofluor White (CFW, final concentration of 100 mg/L, Sigma, USA) for 1 min, followed by
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examination by fluorescence microscopy (BX-41, Olympus, Japan) with the blue filter set. In
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order to test chitin contents in the cell wall, the CFW-stained cells were washed with PBS for three
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times and added into 96-well microplates. The fluorescence density (FLU) of the cells (excitation
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wave 325 nm, emission wave 435 nm) was determined by a fluorescence microplate reader
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(Enspire, Perkinelmer, USA). The cells were also counted with haemocytometers. The relative
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fluorescence intensity was calculated as FLU divided by the total number of tested cells.
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β-Galactosidase assays were performed according to our previous method [16]. In short, yeast
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cells that contain the CWI reporting plasmid p2052 [15] were treated with Fe3O4 nanoparticles for
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12 h as described above, harvested and suspended in 1 mL working Z buffer (60 mM Na2HPO4,
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40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 0.027% (v/v) β-mercaptoethanol, pH 7.0). 150 uL
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of suspensions were permeabilized with 20 µL chloroform and 50 µL SDS (0.1%, m/v) at 30
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5 min, mixed with 500 µL O-nitrophenyl-β-D-galactopyranoside (ONPG, 5000 ppm, BBI, USA),
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and incubated at 30
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(1 M) were added to cease the reactions. The suspensions were centrifuged at 12,000 rpm for 10
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min, and the optical density of the supernatants at 420 nm (OD420) was determined. Miller units of
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β-galactosidase activity were calculated as (OD420 × 1000) / (cell number × 10-6 × T).
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2.9. Real-time PCR (RT-PCR)
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To investigate the effect of Fe3O4 nanoparticles on expression of the cell wall synthesis-related
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genes (i.e., CHS1, CHS2, CHS3 and FKS2), the yeast cells were treated with Fe3O4 nanoparticles
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for 12 h as described above, harvested and used for RNA extraction. The obtained total RNA was
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then used for reverse transcriptional synthesis of cDNA with Oligo(dT)-primed RT reagent Kit
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(Promega, USA). RT-PCR analysis for expression of CHS1, CHS2, CHS3 and FKS2 was
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performed using the RealMasterMix (SYBR Green) Kit (TransGen, China). Their transcription
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levels were normalized against the expression level of ACT1. Each sample was analyzed in three
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independent experiments. The results were expressed as fold change compared with the untreated
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cells
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To determine intracellular ATP contents, the yeast cells were cultured in YPD medium
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containing different concentrations of Fe3O4 nanoparticles for 12 h as described above. The cells
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were then harvested, suspended in PBS buffer and broken by vortexing with glass beads. The
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lysates were centrifuged, and the supernatants were used to detect ATP contents using an ATP
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assay kit (Beyotime, China) based on the ATP-dependent luciferase [17].
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2.11. Mitochondrial membrane potential (MMP) assays
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To determine the effect of Fe3O4 nanoparticles on MMP, the treated yeast cells were harvested and suspended in PBS buffer, and then stained by JC-1 (final concentration 2 µg/mL, Sigma, USA)
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at 30 ℃ for 40 min. Fluorescence intensity of the JC-1 monomer (green) and the aggregate (red)
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were detected using a flow cytometer (Calibur, BD, USA), and the percent of cells with decreased
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red fluorescence intensity (decreased MMP) was recorded [18].
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2.12. TEM observation of yeast cells
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The treated yeast cells were fixed for 12 h with 2% (v/v) glutaraldehyde, post-fixed for 2 h
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with 1% osmium tetroxide solution, dried, and observed by a transmission electron microscope
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(Tecnai G2 F-20, FEI, USA).
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2.13. Respiratory chain enzymatic assays The activities of respiratory chain enzymes were tested according to Medja’s and Spinazzi’s
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methods [19, 20]. The yeast cells were first treated by Snailase (Dingguo, China), obtaining the
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yeast protoplasts. The protoplasts were then homogenized for 50-80 strokes with a Dounce
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homogenizer to break the cells. Cell lysates were then centrifuged at 1,000 g for 5 min to remove
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the nuclei and intact cells. The supernatant was then centrifuged at 10,000 g for 15 min, and the
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pellet was washed twice with the mitochondrial buffer (210 mM surcrose, 70 mM mannitol, 1 mM
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EDTA, 1 mM EGTA), obtaining the yeast mitochondria. The mitochondria were then mixed with
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different concentrations of Fe3O4 nanoparticles in the same buffer, and used to detect the activities
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of respiratory chain complex Ⅰ, Ⅱ, Ⅲ and Ⅳ and ATP synthases using the respiratory chain
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enzymatic assay kits and ATP synthase assay kit (Jinping Bio, China).
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2.14. Statistical analysis
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Each experiment was performed with three replicates, and the values represent the means ±
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standard deviation of three experiments. Significant differences between the treatments were
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determined using one-way ANOVA (P < 0.05). All statistical analyses were performed by
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Statistical Packages for the Social Sciences (SPSS, Version 20).
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3. Results and discussion
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3.1. Characteristics of the synthesized Fe3O4 nanoparticles
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Transmission electron microscope (TEM) images and XRD data of the synthesizedFe3O4
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nanoparticles are shown in Fig. 1. The samples displayed nanoparticle structure with cube-like
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morphology and with the dimension of 10-20 nm (Fig. 1a). XRD analysis revealed that the
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obtained nanoparticles were indexed to the pure Fe3O4 structures (Fig. 1b) [21].
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In order to investigate the potential toxicity of Fe3O4 nanoparticles on yeast cells, the growth
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inhibition test was first performed. As shown in Fig. 2a, there was a remarkable inhibitory effect
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of the nanoparticles on the yeast growth when the concentration reached up to 200 mg/L, and the
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effect became much more obvious as the concentrations increased. The IC50 of the nanoparticles to
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yeast cell growth was 1,353 ± 78 mg/L.
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Cell death assays were then performed to explore whether the inhibitory effect of the
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nanoparticles to yeast growth was attributed to direct plasma membrane (PM) damage and
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consequent cell death caused by these nanoparticles. Interestingly, propidium iodide (PI) staining
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showed that although the percent of PI-positive cells had a tendency of increase when the
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nanoparticles concentrations increased, the highest percent was less than 1%, even that the
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concentrations reached to 1,600 mg/L (Fig. 2b). This indicated that cell death was not the main
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reason of Fe3O4 nanoparticle-related growth inhibition. Some other mechanisms must be included
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for this effect.
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Cell cycle assay was further detected by FACS. With the increase of the Fe3O4 nanoparticle
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concentrations from 0 to 1600 mg/L, the percent of S-phase cells decreased from 63.01% to 52.21%
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(Fig. 2c). This indicated that Fe3O4 nanoparticles partially led to cell cycle arrest, which may be
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involved in growth inhibition of the nanoparticles to yeast cells.
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3.3. ROS accumulated but did not contributed to growth inhibition under treatment of Fe3O4
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nanoparticles Previous studies have shown that cell toxicity of nanoparticles often involved ROS
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accumulation and ROS-dependent growth inhibition [22, 23]. To understand whether ROS
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accumulation is the mechanisms resulting in the observed growth inhibition, we detected
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intracellular ROS levels of the yeast cells after treatment of Fe3O4 nanoparticles. DCFH-DA
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staining showed that the nanoparticles caused remarkable increase of ROS-accumulated cells
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when the concentration reached up to 200 mg/L. The percent of ROS-accumulated cells increased
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from 3.52% to 11.56% when the concentration of Fe3O4 nanoparticle increased from 200 mg/L to
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1600 mg/L (Fig. 3a). This indicated that high-level nanoparticles led to remarkable ROS
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accumulation in the yeast cells.
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To examine whether ROS accumulation is involved in growth inhibition, the ROS scavenger
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N-acetylcysteine (NAC) was used to remove accumulated ROS. However, the addition of NAC
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could not rescue the growth of Fe3O4 nanoparticle-treated cells at all of the tested concentrations
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(Fig. 3b), suggesting that removal of ROS could not eliminate the inhibitory effect of Fe3O4
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nanoparticles. These results manifested that, although Fe3O4 nanoparticles caused ROS
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accumulation, this accumulation did not contribute to growth inhibition.
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3.4. Cell wall damage was not involved in Fe3O4 nanoparticle-related growth inhibition
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The cell wall is essential for the living of fungal cells, and has been proved to be associated
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with nanotoxicity in these cells [24, 25]. Since direct cell death was not responsible for the growth
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one of the important cell wall components essential for cell shape and morphogenesis in yeast
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cells, and the increase of its contents indicates that the cells suffer from cell wall damage [26].
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Interestingly, the cells showed decreased rather than increased chitin contents under the treatment
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of Fe3O4 nanoparticles, indicating that chitin synthesis was partially inhibited by this treatment
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(Fig. 4a). The expression of chitin synthase-encoding genes (i.e., CHS1, CHS2 and CHS3) was
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further examined by RT-PCR. As shown in Fig. 4b, treatment of the nanoparticles led to
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significant decrease of the expression of these genes. Together, these results indicated that chitin
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synthesis was inhibited by Fe3O4 nanoparticles.
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The cell wall integrity (CWI) pathway is an important signal pathway in yeast cells responding
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to cell wall stress. This pathway is activated by the cell wall stress, followed by up-regulation of
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CWI genes, including the β-1,3-glucan synthase-encoding gene FKS2 [34].We further examined
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whether the CWI pathway was activated under the treatment of Fe3O4 nanoparticles. Similarly,
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RT-PCR analysis showed that FKS2 had decreased expression levels than the control at 100-400
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mg/L (Fig. 4b). Moreover, the yeast Sc+p2052 cells, which express β-galactosidase (LacZ) under
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the control of the FKS2 promoter, showed decreased rather than increased β-galactosidase activity
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with 100-400 mg/L nanoparticles (Fig. 4c). Therefore, the CWI pathway was not activated by the
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treatment of Fe3O4 nanoparticles. Together, both chitin assays and β-galactosidase assays revealed
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that the Fe3O4 nanoparticles did not cause obvious cell wall damage. Hence, cell wall damage also
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did not contribute to the inhibition effect of the nanoparticles. An interesting observation is that
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expression of FKS2 was up-regulated under the treatment of 800-1600 mg/L Fe3O4 nanoparticles
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than that under 100-400 mg/L (Fig. 4b, 4c). A possible exploration is that expression of this gene
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mg/L) down-regulate its expression, while high-level nanoparticles (800-1600 mg/L) rescue its
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normal expression. The up-regulation of FKS2 under treatment of high-level nanoparticles as
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compared to under treatment of low-level nanoparticles may be involved in maintenance of CWI.
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3.5. Fe3O4 nanoparticles caused remarkable decrease of intracellular ATP contents
Intracellular ATP is an indicator of cell energy supplying, which is essential for cell growth. We
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then quantified the intracellular ATP contents based on the ATP-dependent luciferase. As shown in
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Fig. 5, Fe3O4 nanoparticles at the concentrations ≥ 200 mg/L caused significant decrease of
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intracellular ATP contents. When the nanoparticle concentration reached up to 800 mg/L, the ATP
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contents decreased from 0.42 mg/g proteins to 0.25 mg/g proteins. This is consistent with the
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growth inhibition tendency of the nanoparticles. The result implied that the decrease of ATP
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contents may be associated with growth inhibition. More specifically mechanisms need to be
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clarified to explain this phenomenon. In addition, the decrease of ATP contents might contribute to
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the nanoparticle-caused attenuation of chitin synthesis as demonstrated in Fig. 4b.
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3.6. Fe3O4 nanoparticles impaired the mitochondrial functions
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ATP production is accompanied by the mitochondrial actions, including electron transport
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mediated by mitochondrial respiratory chain complexes, and proton gradient-dependent ATP
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production catalyzed by mitochondrial ATP synthases. Since Fe3O4 nanoparticles caused a
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decrease of intracellular ATP contents, we hypothesized that the nanoparticles may affect
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mitochondrial functions that are required for ATP production. As expected, TEM observation
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by red arrows), resulting in mitochondrial damage (indicated by white arrows) (Fig. 6a).
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Mitotracker Red staining further showed that the untreated cells had cable-like mitochondrial
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morphology, whereas the treated cells had fragmented mitochondria (as indicated by the white
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arrows, Fig. 6b), suggesting that the nanoparticles resulted in mitochondrial fragmentation in the
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yeast cells. Moreover, MMP assays revealed that the nanoparticles caused remarkable decrease of
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MMP, even that the concentration of the nanoparticles was merely 100 mg/L. When their
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concentrations reached up to 400 mg/L, > 80% cells showed decreased MMP (Fig. 6c). Hence, the
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nanoparticles severely affect mitochondrial morphology and functions.
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We then assessed the effect of Fe3O4 nanoparticles on both the mitochondrial respiratory chain
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complexes and ATP synthases. Interestingly, the activity of the respiratory chain complex I, II and
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III and ATP synthases was not affected (data not shown), while that of the respiratory chain
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complex
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decrease of the activity of complex
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activity (Fig. 6d). Obviously, Fe3O4 nanoparticles had a severe impact on mitochondrial functions
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by specifically inhibiting the activity of complex
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inhibit this complex, leading to dysfunction of the mitochondria and decrease of ATP production,
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followed by growth inhibition. During the application of Fe3O4 nanoparticles, their potential
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toxicity to the mitochondria should not be ignored.
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was severely impaired by the nanoparticles. 100 mg/L nanoparticles led to 40%
. Thus, the nanoparticles might specifically
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4. Conclusion We here reveal that the Fe3O4 nanoparticles, one kind of important magnetic NMs, have an
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inhibitory effect on yeast growth. This inhibition is not attributed to the well-known PM
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damage-related cell death, ROS accumulation and cell wall damage, but is attributed to their
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interaction with the mitochondria, leading to disruption of the mitochondrial respiratory chain
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complex
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uncovers the potential toxicity of Fe3O4 nanoparticles to the mitochondria during their interaction
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with the eukaryotic cells.
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, decreased MMP and consequent attenuation of ATP production (Fig. 7). This study
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302 Acknowledgements
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We thank Professor David E. Levin (Boston University) for friendly providing the plasmid p2052.
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This project was supported by the National Natural Science Foundation of China (31670146,
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31400132,
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15JCQNJC15300),
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(201610055103) and Biological Top-notch Talent Training Project of Nankai University.
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Figure legends
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Fig. 1. Characterization of the synthesized Fe3O4 nanoparticles. (a) Transmission electron
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microscopy (TEM). (b) XRD patterns.
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Fig. 2. Effect of Fe3O4 nanoparticles on the growth of yeast cells (a), cell death (b) and cell cycle
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(c). (a) Yeast cells were treated with Fe3O4 nanoparticles at indicated concentrations for 12 h and
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counted. (b) The treated cells were stained with PI and observed by fluorescence microscopy with
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a RFP filter. The values represent the means ± standard deviation (n = 3). (c) Cell cycle analysis of
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treated cells. The percent of S-phase cells are indicated. * indicates significant difference between
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the treatment group and the control (P < 0.05).
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Fig. 3. Effect of Fe3O4 nanoparticles on ROS accumulation (a) and yeast growth under the
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treatment of the ROS scavenger NAC (b). (a) The treated yeast cells were stained with DCFH-DA
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and the fluorescence intensity of cells were detected by flow cytometry (FACS). The percent of
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ROS-accumulated (DCFH-DA-positive) cells were recorded. (b) The cells were cultured in YPD
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medium containing Fe3O4 NPs alone with the indicated concentrations (-NAC) or Fe3O4 NPs with
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the indicated concentrations plus 5 mM NAC (+NAC). The blue and red bars indicate the
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treatments of Fe3O4 alone (-NAC) and the treatments of Fe3O4 plus NAC (+NAC), respectively.
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The percent of growth was calculated as the cell number of experimental group divided by the
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number of the control group (0). The values represent the means ± standard deviation (n = 3).
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There is no significant difference in the percent of growth between the –NAC group and the
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+NAC group at each Fe3O4 concentration (P < 0.05).
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Fig. 4. Effect of Fe3O4 nanoparticles on cell wall chitin contents (a) and expression of the cell wall
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synthesis-related genes (b, c). (a) Chitin contents of the yeast cells. The yeast cells were treated
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with 200 mg/L or 400 mg/L Fe3O4 nanoparticles for 12 h, stained with CFW and the fluorescence
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intensity was detected. (b) Expression levels of the cell wall synthesis-related genes CHS1, CHS2,
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CHS3 and FKS2 after 12 h of treatment of Fe3O4 nanoparticles at the indicated concentrations. (c)
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Yeast cells that contain the CWI reporting plasmid p2052 in which the β-galactosidase-encoding
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gene LacZ was under the control of the FKS2 promoter were treated with Fe3O4 nanoparticles as
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described above, and then the β-galactosidase activity was determined. The values represent the
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means ± standard deviation (n = 3). * indicates significant difference between the treatment group
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and the control (P < 0.05).
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Fig. 5. Fe3O4 nanoparticles caused significant decrease of intracellular ATP contents. The yeast
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cells were cultured in YPD medium containing different concentrations of Fe3O4 nanoparticles for
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12 h. The cells were then harvested and broken by vortexing. The supernatants were used to
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determine ATP contents by using an ATP assay kit. The error bars indicate the standard deviations
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(n = 3). * indicates significant difference between the treatment group and the control (P < 0.05).
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Fig. 6. Fe3O4 NPs have a severe impact on mitochondrial morphology and function. (a) TEM
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images of the control and Fe3O4 NP-treated cells. The white arrows indicate the damaged
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mitochondria. The red arrows indicate the mitochondrion-localized Fe3O4 NPs. Mit,
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mitochondrion; Nuc, nucleus; CW, cell wall. (b) Observation of mitochondrial morphology. The
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yeast cells were treated by Fe3O4 nanoparticles with indicated concentrations for 12 h, stained
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with Mitotracker Red, and observed by fluorescence microscopy. The white arrows indicate the
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fragmented mitochondria. Scale bar = 5 µm. (c) MMP assays. The treated cells were stained with
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decreased red fluorescence were recorded. (d) Activity of the respiratory chain complex Ⅳ. The
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yeast mitochondria were incubated with Fe3O4 nanoparticles, and used for respiratory chain
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complex Ⅳ assay. The error bars indicate the standard deviations (n = 3). * indicates significant
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difference between the treatment group and the control (P < 0.05).
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Fig. 7. A model illustrating the mechanism of Fe3O4 nanoparticle-caused growth inhibition. NPs,
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nanoparticles; MMP, mitochondrial membrane potential.
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ACCEPTED MANUSCRIPT Highlights The synthesized Fe3O4 nanoparticles inhibit S. cerevisiae growth independent of cell death. The inhibitory effect of Fe3O4 nanoparticles is not attributed to ROS accumulation.
is the main target of Fe3O4 nanoparticles.
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The respiratory chain complex
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S0009-2797(17)30292-2
DOI:
10.1016/j.cbi.2018.03.012
Reference:
CBI 8258
To appear in:
Chemico-Biological Interactions
Received Date: 14 March 2017 Revised Date:
27 February 2018
Accepted Date: 19 March 2018
Please cite this article as: Q. Peng, D. Huo, H. Li, B. Zhang, Y. Li, A. Liang, H. Wang, Q. Yu, M. Li, ROSindependent toxicity of Fe3O4 nanoparticles to yeast cells: Involvement of mitochondrial dysfunction, Chemico-Biological Interactions (2018), doi: 10.1016/j.cbi.2018.03.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT ROS-independent toxicity of Fe3O4 nanoparticles to yeast cells: involvement of
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mitochondrial dysfunction
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Qi Peng,a Da Huo,a Hongyue Li,a Bing Zhang,a Yang Li,a Anping Liang,a Hui Wang,b Qilin Yua,*,
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Mingchun Lia,*
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a
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of Microbiology, College of Life Science, Nankai University, Tianjin 300071, PR China
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b
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Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department
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Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin, 300091, China
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Corresponding authors:
Qilin Yu, Department of Microbiology, College of Life Science, Nankai University, Tianjin
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300071, P. R. China. E-mail address: [email protected].
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Mingchun Li, Department of Microbiology, College of Life Science, Nankai University, Tianjin
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300071, P. R. China. E-mail address: [email protected].
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ACCEPTED MANUSCRIPT Abstract
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Fe3O4 nanoparticles, one kind of magnetic nanomaterials (NMs), are widely used in drug delivery,
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biological imaging, sensors, catalysts and pollution management. However, its toxicity to
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biological systems and related toxicity mechanisms remain to be explored. In this study, we
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investigate the effect of as-synthesized Fe3O4 nanoparticles on growth of Saccharomyces
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cerevisiae, an important model fungus. Growth inhibition assays showed that Fe3O4 nanoparticles
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remarkably inhibited yeast growth. Interestingly, this inhibitory effect was not attributed to the
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well-known plasma membrane damage, cell wall damage and ROS accumulation. Further
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investigations revealed that the nanoparticles strongly impaired mitochondrial functions, resulting
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in abnormal mitochondrial morphology, decreased mitochondrial membrane potential (MMP) and
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attenuated ATP production. Most importantly, the respiratory chain complex
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respiratory chain complexes and ATP synthases, was found to be the main target of the
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nanoparticles. This study uncovers a novel ROS-independent toxicity mechanism of Fe3O4
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nanoparticles to eukaryotic cells.
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Keywords: Fe3O4 nanoparticle; mitochondrion; respiratory chain complex; nanotoxicity;
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Saccharomyces cerevisiae
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1. Introduction With the development of nanotechnology, more and more nanomaterials (NMs) are
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incorporated into the organisms and the ecosystems [1,2]. Fe3O4 nanoparticles are one kind of
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important magnetic NMs [3]. Owing to their excellent property that their action could be easily
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manipulated under the external magnetic field, Fe3O4 nanoparticles were widely used in drug
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delivery, magnetic resonance imaging, sensors, catalysts and pollution management [4-8]. Since
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these NMs frequently contact the organisms, it is urgent to elucidate their potential biological
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effects.
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Several studies have evaluated the potential toxicity of Fe3O4 nanoparticles to mammalian cells
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[9-12]. The nanoparticles may enter into the cells by the endocytosis pathway and release iron
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cations in the lysosomes, resulting in ROS production and oxidative stress-related apoptosis [9,10].
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Also, the nanoparticles may disorganize actin fiber and tubulin network, causing inhibition of cell
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migration and invasion of the human umbilical vein endothelial cells [11]. Furthermore, these
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nanoparticles may enter into the brain, inducing ROS production and mitochondrial dysfunction
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[12]. However, the mechanism by which they disrupt the mitochondrial functions remains
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unknown. Moreover, up to now, little is known about their effect on other organisms, especially on
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the fungal organisms that play important roles in decomposition and recycling of organic materials
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[13].
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In this study, we assessed the effect of as-synthesized Fe3O4 nanoparticles on growth of yeast,
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and found that this kind of NMs remarkably inhibited yeast growth at the concentrations ≥ 200
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mg/L. Interestingly, their inhibitory effect was not attributed to common plasma membrane
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damage-related cell death, cell wall damage and ROS accumulation. Further investigations
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revealed that these nanoparticles targeted to the mitochondrial respiratory chain complex
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leading to dysfunction of the mitochondria and consequent attenuation of ATP production. This
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study uncovers a mitochondrial respiratory chain-related toxicity mechanism of Fe3O4
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nanoparticles in eukaryotic cells.
2. Materials and methods
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2.1. Preparation and characterization of Fe3O4 nanoparticles
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The Fe3O4 nanoparticles used in this study were prepared by the co-precipitation method [14].
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Briefly, 100 mL of 0.3 M FeCl2 (dissolved in distilled water) were mixed with 100 mL of 0.6 M
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FeCl3 solution (dissolved in distilled water). 100 mL of 0.3 M NaOH were then slowly added into
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the mixture, obtaining the black solution. The solution was heated and the Fe3O4 nanoparticles
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were precipitated. After washed with distilled water to pH 7.0, the nanoparticles were dried at the
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room temperature for further use.
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The morphology of the obtained nanoparticles was characterized by a transmission electron
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microscope (TEM, Tecnai G2 F-20, FEI, USA). The crystal structure and composition of the
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nanoparticles were characterized by X-ray diffraction (XRD, D/max-2500, Japan).
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2.2. Strains and growth conditions
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The S. cerevisiae strain InvSc1 (Invitrogen, USA) were used for assessing biological effects of
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Fe3O4 nanoparticles. To investigate their possible effect on the cell wall integrity (CWI) pathway,
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the strain Sc+p2052 containing the LacZ gene under the control of the promoter of the CWI gene
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FKS2 was used [15]. Normally, the yeast cells were cultivated overnight in liquid YPD medium
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with shaking at 30
and were suspended in fresh YPD medium for further testing.
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2.3. Growth inhibition assays To assess the effect of Fe3O4 nanoparticles on yeast growth, overnight cultured yeast cells were
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suspended in YPD medium containing Fe3O4 nanoparticles with the following concentrations, 0,
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100, 200, 400, 800 and 1600 ppm. The initial yeast number in the suspensions was 1×106 cells/mL.
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The suspensions were then cultured with shaking for 12 h at 30
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and used for cell number counting using the haemocytometers. The percent of growth was
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calculated as the cell number of each treatment group divided by that of the control × 100.
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. The cultures were then sampled
To investigate the effect of the ROS scavenger N-acetylcysteine (NAC) on the inhibition effect
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of Fe3O4 nanoparticles, the yeast cells were cultured in YPD medium containing 800 mg/L Fe3O4,
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5 mM NAC or 800 mg/L Fe3O4 plus 5 mM NAC. The suspensions were then cultured for 12 h as
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described above, sampled and used for cell counting.
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Fe3O4 nanoparticles for 12 h as described above, harvested and suspended in PBS. The cells were
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then stained by propidium iodide (PI, prepared in distilled water, final concentration 10 µg/mL,
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Sigma, USA) for 5 min. The stained cells were washed twice with PBS and observed by
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fluorescence microscopy (BX-41, Olympus, Japan) using the RFP filtering set. The percentage of
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PI-positive (dead) cells was calculated as the number of PI-positive cells divided by the total cell
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number × 100. At least 20 observed fields were determined.
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2.5. Cell cycle assays The yeast cells were treated by Fe3O4 nanoparticles for 12 h as described above and harvested. Cell cycle progression was then evaluated by the cell cycle assay kit (Beyotime Biotech., China).
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2.6. ROS assays
To determine the number of ROS-accumulated cells, the treated cells were washed and
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suspended in PBS buffer. 500 µL of cell suspensions were stained with 2 µL of
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2’,7’-dichlorofluorescein diacetate (DCFH-DA, 10 000 ppm, dissolved in PBS) for 30 min at 30℃.
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The stained cells were harvested, washed and re-suspended in PBS buffer, and were examined by
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flow cytometry (FACS, Calibur, BD, USA). The percent of ROS-accumulated cells was calculated
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as the number of DCFH-DA-positive cells divided by that of total observed cells × 100. At least
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30 fields were observed.
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2.7. Cell wall staining and chitin measurements
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To detect the yeast cell wall chitin, the cells were treated with Fe3O4 nanoparticles for 12 h as
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described above. The cells were then washed, re-suspended in PBS buffer, and stained with
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Calcofluor White (CFW, final concentration of 100 mg/L, Sigma, USA) for 1 min, followed by
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examination by fluorescence microscopy (BX-41, Olympus, Japan) with the blue filter set. In
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order to test chitin contents in the cell wall, the CFW-stained cells were washed with PBS for three
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times and added into 96-well microplates. The fluorescence density (FLU) of the cells (excitation
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wave 325 nm, emission wave 435 nm) was determined by a fluorescence microplate reader
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(Enspire, Perkinelmer, USA). The cells were also counted with haemocytometers. The relative
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fluorescence intensity was calculated as FLU divided by the total number of tested cells.
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β-Galactosidase assays were performed according to our previous method [16]. In short, yeast
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cells that contain the CWI reporting plasmid p2052 [15] were treated with Fe3O4 nanoparticles for
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12 h as described above, harvested and suspended in 1 mL working Z buffer (60 mM Na2HPO4,
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40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 0.027% (v/v) β-mercaptoethanol, pH 7.0). 150 uL
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of suspensions were permeabilized with 20 µL chloroform and 50 µL SDS (0.1%, m/v) at 30
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5 min, mixed with 500 µL O-nitrophenyl-β-D-galactopyranoside (ONPG, 5000 ppm, BBI, USA),
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and incubated at 30
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(1 M) were added to cease the reactions. The suspensions were centrifuged at 12,000 rpm for 10
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min, and the optical density of the supernatants at 420 nm (OD420) was determined. Miller units of
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β-galactosidase activity were calculated as (OD420 × 1000) / (cell number × 10-6 × T).
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2.9. Real-time PCR (RT-PCR)
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To investigate the effect of Fe3O4 nanoparticles on expression of the cell wall synthesis-related
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genes (i.e., CHS1, CHS2, CHS3 and FKS2), the yeast cells were treated with Fe3O4 nanoparticles
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for 12 h as described above, harvested and used for RNA extraction. The obtained total RNA was
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then used for reverse transcriptional synthesis of cDNA with Oligo(dT)-primed RT reagent Kit
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(Promega, USA). RT-PCR analysis for expression of CHS1, CHS2, CHS3 and FKS2 was
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performed using the RealMasterMix (SYBR Green) Kit (TransGen, China). Their transcription
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levels were normalized against the expression level of ACT1. Each sample was analyzed in three
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independent experiments. The results were expressed as fold change compared with the untreated
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cells
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To determine intracellular ATP contents, the yeast cells were cultured in YPD medium
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containing different concentrations of Fe3O4 nanoparticles for 12 h as described above. The cells
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were then harvested, suspended in PBS buffer and broken by vortexing with glass beads. The
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lysates were centrifuged, and the supernatants were used to detect ATP contents using an ATP
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assay kit (Beyotime, China) based on the ATP-dependent luciferase [17].
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2.11. Mitochondrial membrane potential (MMP) assays
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To determine the effect of Fe3O4 nanoparticles on MMP, the treated yeast cells were harvested and suspended in PBS buffer, and then stained by JC-1 (final concentration 2 µg/mL, Sigma, USA)
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at 30 ℃ for 40 min. Fluorescence intensity of the JC-1 monomer (green) and the aggregate (red)
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were detected using a flow cytometer (Calibur, BD, USA), and the percent of cells with decreased
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red fluorescence intensity (decreased MMP) was recorded [18].
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The treated yeast cells were fixed for 12 h with 2% (v/v) glutaraldehyde, post-fixed for 2 h
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with 1% osmium tetroxide solution, dried, and observed by a transmission electron microscope
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(Tecnai G2 F-20, FEI, USA).
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2.13. Respiratory chain enzymatic assays The activities of respiratory chain enzymes were tested according to Medja’s and Spinazzi’s
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methods [19, 20]. The yeast cells were first treated by Snailase (Dingguo, China), obtaining the
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yeast protoplasts. The protoplasts were then homogenized for 50-80 strokes with a Dounce
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homogenizer to break the cells. Cell lysates were then centrifuged at 1,000 g for 5 min to remove
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the nuclei and intact cells. The supernatant was then centrifuged at 10,000 g for 15 min, and the
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pellet was washed twice with the mitochondrial buffer (210 mM surcrose, 70 mM mannitol, 1 mM
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EDTA, 1 mM EGTA), obtaining the yeast mitochondria. The mitochondria were then mixed with
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different concentrations of Fe3O4 nanoparticles in the same buffer, and used to detect the activities
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of respiratory chain complex Ⅰ, Ⅱ, Ⅲ and Ⅳ and ATP synthases using the respiratory chain
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enzymatic assay kits and ATP synthase assay kit (Jinping Bio, China).
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2.14. Statistical analysis
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Each experiment was performed with three replicates, and the values represent the means ±
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standard deviation of three experiments. Significant differences between the treatments were
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determined using one-way ANOVA (P < 0.05). All statistical analyses were performed by
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Statistical Packages for the Social Sciences (SPSS, Version 20).
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3. Results and discussion
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3.1. Characteristics of the synthesized Fe3O4 nanoparticles
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Transmission electron microscope (TEM) images and XRD data of the synthesizedFe3O4
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nanoparticles are shown in Fig. 1. The samples displayed nanoparticle structure with cube-like
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morphology and with the dimension of 10-20 nm (Fig. 1a). XRD analysis revealed that the
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obtained nanoparticles were indexed to the pure Fe3O4 structures (Fig. 1b) [21].
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In order to investigate the potential toxicity of Fe3O4 nanoparticles on yeast cells, the growth
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inhibition test was first performed. As shown in Fig. 2a, there was a remarkable inhibitory effect
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of the nanoparticles on the yeast growth when the concentration reached up to 200 mg/L, and the
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effect became much more obvious as the concentrations increased. The IC50 of the nanoparticles to
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yeast cell growth was 1,353 ± 78 mg/L.
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Cell death assays were then performed to explore whether the inhibitory effect of the
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nanoparticles to yeast growth was attributed to direct plasma membrane (PM) damage and
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consequent cell death caused by these nanoparticles. Interestingly, propidium iodide (PI) staining
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showed that although the percent of PI-positive cells had a tendency of increase when the
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nanoparticles concentrations increased, the highest percent was less than 1%, even that the
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concentrations reached to 1,600 mg/L (Fig. 2b). This indicated that cell death was not the main
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reason of Fe3O4 nanoparticle-related growth inhibition. Some other mechanisms must be included
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for this effect.
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Cell cycle assay was further detected by FACS. With the increase of the Fe3O4 nanoparticle
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concentrations from 0 to 1600 mg/L, the percent of S-phase cells decreased from 63.01% to 52.21%
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(Fig. 2c). This indicated that Fe3O4 nanoparticles partially led to cell cycle arrest, which may be
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involved in growth inhibition of the nanoparticles to yeast cells.
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3.3. ROS accumulated but did not contributed to growth inhibition under treatment of Fe3O4
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nanoparticles Previous studies have shown that cell toxicity of nanoparticles often involved ROS
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accumulation and ROS-dependent growth inhibition [22, 23]. To understand whether ROS
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accumulation is the mechanisms resulting in the observed growth inhibition, we detected
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intracellular ROS levels of the yeast cells after treatment of Fe3O4 nanoparticles. DCFH-DA
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staining showed that the nanoparticles caused remarkable increase of ROS-accumulated cells
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when the concentration reached up to 200 mg/L. The percent of ROS-accumulated cells increased
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from 3.52% to 11.56% when the concentration of Fe3O4 nanoparticle increased from 200 mg/L to
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1600 mg/L (Fig. 3a). This indicated that high-level nanoparticles led to remarkable ROS
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accumulation in the yeast cells.
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To examine whether ROS accumulation is involved in growth inhibition, the ROS scavenger
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N-acetylcysteine (NAC) was used to remove accumulated ROS. However, the addition of NAC
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could not rescue the growth of Fe3O4 nanoparticle-treated cells at all of the tested concentrations
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(Fig. 3b), suggesting that removal of ROS could not eliminate the inhibitory effect of Fe3O4
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nanoparticles. These results manifested that, although Fe3O4 nanoparticles caused ROS
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accumulation, this accumulation did not contribute to growth inhibition.
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3.4. Cell wall damage was not involved in Fe3O4 nanoparticle-related growth inhibition
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The cell wall is essential for the living of fungal cells, and has been proved to be associated
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with nanotoxicity in these cells [24, 25]. Since direct cell death was not responsible for the growth
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one of the important cell wall components essential for cell shape and morphogenesis in yeast
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cells, and the increase of its contents indicates that the cells suffer from cell wall damage [26].
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Interestingly, the cells showed decreased rather than increased chitin contents under the treatment
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of Fe3O4 nanoparticles, indicating that chitin synthesis was partially inhibited by this treatment
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(Fig. 4a). The expression of chitin synthase-encoding genes (i.e., CHS1, CHS2 and CHS3) was
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further examined by RT-PCR. As shown in Fig. 4b, treatment of the nanoparticles led to
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significant decrease of the expression of these genes. Together, these results indicated that chitin
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synthesis was inhibited by Fe3O4 nanoparticles.
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The cell wall integrity (CWI) pathway is an important signal pathway in yeast cells responding
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to cell wall stress. This pathway is activated by the cell wall stress, followed by up-regulation of
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CWI genes, including the β-1,3-glucan synthase-encoding gene FKS2 [34].We further examined
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whether the CWI pathway was activated under the treatment of Fe3O4 nanoparticles. Similarly,
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RT-PCR analysis showed that FKS2 had decreased expression levels than the control at 100-400
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mg/L (Fig. 4b). Moreover, the yeast Sc+p2052 cells, which express β-galactosidase (LacZ) under
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the control of the FKS2 promoter, showed decreased rather than increased β-galactosidase activity
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with 100-400 mg/L nanoparticles (Fig. 4c). Therefore, the CWI pathway was not activated by the
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treatment of Fe3O4 nanoparticles. Together, both chitin assays and β-galactosidase assays revealed
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that the Fe3O4 nanoparticles did not cause obvious cell wall damage. Hence, cell wall damage also
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did not contribute to the inhibition effect of the nanoparticles. An interesting observation is that
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expression of FKS2 was up-regulated under the treatment of 800-1600 mg/L Fe3O4 nanoparticles
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than that under 100-400 mg/L (Fig. 4b, 4c). A possible exploration is that expression of this gene
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ACCEPTED MANUSCRIPT is regulated by Fe3O4 nanoparticles in a dose-dependent manner: low-level nanoparticles (100-400
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mg/L) down-regulate its expression, while high-level nanoparticles (800-1600 mg/L) rescue its
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normal expression. The up-regulation of FKS2 under treatment of high-level nanoparticles as
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compared to under treatment of low-level nanoparticles may be involved in maintenance of CWI.
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3.5. Fe3O4 nanoparticles caused remarkable decrease of intracellular ATP contents
Intracellular ATP is an indicator of cell energy supplying, which is essential for cell growth. We
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then quantified the intracellular ATP contents based on the ATP-dependent luciferase. As shown in
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Fig. 5, Fe3O4 nanoparticles at the concentrations ≥ 200 mg/L caused significant decrease of
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intracellular ATP contents. When the nanoparticle concentration reached up to 800 mg/L, the ATP
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contents decreased from 0.42 mg/g proteins to 0.25 mg/g proteins. This is consistent with the
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growth inhibition tendency of the nanoparticles. The result implied that the decrease of ATP
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contents may be associated with growth inhibition. More specifically mechanisms need to be
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clarified to explain this phenomenon. In addition, the decrease of ATP contents might contribute to
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the nanoparticle-caused attenuation of chitin synthesis as demonstrated in Fig. 4b.
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3.6. Fe3O4 nanoparticles impaired the mitochondrial functions
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ATP production is accompanied by the mitochondrial actions, including electron transport
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mediated by mitochondrial respiratory chain complexes, and proton gradient-dependent ATP
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production catalyzed by mitochondrial ATP synthases. Since Fe3O4 nanoparticles caused a
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decrease of intracellular ATP contents, we hypothesized that the nanoparticles may affect
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mitochondrial functions that are required for ATP production. As expected, TEM observation
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by red arrows), resulting in mitochondrial damage (indicated by white arrows) (Fig. 6a).
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Mitotracker Red staining further showed that the untreated cells had cable-like mitochondrial
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morphology, whereas the treated cells had fragmented mitochondria (as indicated by the white
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arrows, Fig. 6b), suggesting that the nanoparticles resulted in mitochondrial fragmentation in the
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yeast cells. Moreover, MMP assays revealed that the nanoparticles caused remarkable decrease of
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MMP, even that the concentration of the nanoparticles was merely 100 mg/L. When their
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concentrations reached up to 400 mg/L, > 80% cells showed decreased MMP (Fig. 6c). Hence, the
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nanoparticles severely affect mitochondrial morphology and functions.
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We then assessed the effect of Fe3O4 nanoparticles on both the mitochondrial respiratory chain
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complexes and ATP synthases. Interestingly, the activity of the respiratory chain complex I, II and
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III and ATP synthases was not affected (data not shown), while that of the respiratory chain
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complex
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decrease of the activity of complex
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activity (Fig. 6d). Obviously, Fe3O4 nanoparticles had a severe impact on mitochondrial functions
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by specifically inhibiting the activity of complex
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inhibit this complex, leading to dysfunction of the mitochondria and decrease of ATP production,
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followed by growth inhibition. During the application of Fe3O4 nanoparticles, their potential
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toxicity to the mitochondria should not be ignored.
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was severely impaired by the nanoparticles. 100 mg/L nanoparticles led to 40%
. Thus, the nanoparticles might specifically
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4. Conclusion We here reveal that the Fe3O4 nanoparticles, one kind of important magnetic NMs, have an
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inhibitory effect on yeast growth. This inhibition is not attributed to the well-known PM
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damage-related cell death, ROS accumulation and cell wall damage, but is attributed to their
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interaction with the mitochondria, leading to disruption of the mitochondrial respiratory chain
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complex
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uncovers the potential toxicity of Fe3O4 nanoparticles to the mitochondria during their interaction
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with the eukaryotic cells.
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, decreased MMP and consequent attenuation of ATP production (Fig. 7). This study
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302 Acknowledgements
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We thank Professor David E. Levin (Boston University) for friendly providing the plasmid p2052.
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This project was supported by the National Natural Science Foundation of China (31670146,
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31400132,
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15JCQNJC15300),
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(201610055103) and Biological Top-notch Talent Training Project of Nankai University.
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Figure legends
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Fig. 1. Characterization of the synthesized Fe3O4 nanoparticles. (a) Transmission electron
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microscopy (TEM). (b) XRD patterns.
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Fig. 2. Effect of Fe3O4 nanoparticles on the growth of yeast cells (a), cell death (b) and cell cycle
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(c). (a) Yeast cells were treated with Fe3O4 nanoparticles at indicated concentrations for 12 h and
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counted. (b) The treated cells were stained with PI and observed by fluorescence microscopy with
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a RFP filter. The values represent the means ± standard deviation (n = 3). (c) Cell cycle analysis of
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treated cells. The percent of S-phase cells are indicated. * indicates significant difference between
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the treatment group and the control (P < 0.05).
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Fig. 3. Effect of Fe3O4 nanoparticles on ROS accumulation (a) and yeast growth under the
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treatment of the ROS scavenger NAC (b). (a) The treated yeast cells were stained with DCFH-DA
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and the fluorescence intensity of cells were detected by flow cytometry (FACS). The percent of
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ROS-accumulated (DCFH-DA-positive) cells were recorded. (b) The cells were cultured in YPD
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medium containing Fe3O4 NPs alone with the indicated concentrations (-NAC) or Fe3O4 NPs with
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the indicated concentrations plus 5 mM NAC (+NAC). The blue and red bars indicate the
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treatments of Fe3O4 alone (-NAC) and the treatments of Fe3O4 plus NAC (+NAC), respectively.
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The percent of growth was calculated as the cell number of experimental group divided by the
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number of the control group (0). The values represent the means ± standard deviation (n = 3).
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There is no significant difference in the percent of growth between the –NAC group and the
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+NAC group at each Fe3O4 concentration (P < 0.05).
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Fig. 4. Effect of Fe3O4 nanoparticles on cell wall chitin contents (a) and expression of the cell wall
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synthesis-related genes (b, c). (a) Chitin contents of the yeast cells. The yeast cells were treated
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with 200 mg/L or 400 mg/L Fe3O4 nanoparticles for 12 h, stained with CFW and the fluorescence
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intensity was detected. (b) Expression levels of the cell wall synthesis-related genes CHS1, CHS2,
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CHS3 and FKS2 after 12 h of treatment of Fe3O4 nanoparticles at the indicated concentrations. (c)
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Yeast cells that contain the CWI reporting plasmid p2052 in which the β-galactosidase-encoding
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gene LacZ was under the control of the FKS2 promoter were treated with Fe3O4 nanoparticles as
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described above, and then the β-galactosidase activity was determined. The values represent the
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means ± standard deviation (n = 3). * indicates significant difference between the treatment group
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and the control (P < 0.05).
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Fig. 5. Fe3O4 nanoparticles caused significant decrease of intracellular ATP contents. The yeast
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cells were cultured in YPD medium containing different concentrations of Fe3O4 nanoparticles for
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12 h. The cells were then harvested and broken by vortexing. The supernatants were used to
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determine ATP contents by using an ATP assay kit. The error bars indicate the standard deviations
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(n = 3). * indicates significant difference between the treatment group and the control (P < 0.05).
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Fig. 6. Fe3O4 NPs have a severe impact on mitochondrial morphology and function. (a) TEM
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images of the control and Fe3O4 NP-treated cells. The white arrows indicate the damaged
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mitochondria. The red arrows indicate the mitochondrion-localized Fe3O4 NPs. Mit,
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mitochondrion; Nuc, nucleus; CW, cell wall. (b) Observation of mitochondrial morphology. The
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yeast cells were treated by Fe3O4 nanoparticles with indicated concentrations for 12 h, stained
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with Mitotracker Red, and observed by fluorescence microscopy. The white arrows indicate the
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fragmented mitochondria. Scale bar = 5 µm. (c) MMP assays. The treated cells were stained with
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decreased red fluorescence were recorded. (d) Activity of the respiratory chain complex Ⅳ. The
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yeast mitochondria were incubated with Fe3O4 nanoparticles, and used for respiratory chain
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complex Ⅳ assay. The error bars indicate the standard deviations (n = 3). * indicates significant
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difference between the treatment group and the control (P < 0.05).
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Fig. 7. A model illustrating the mechanism of Fe3O4 nanoparticle-caused growth inhibition. NPs,
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nanoparticles; MMP, mitochondrial membrane potential.
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ACCEPTED MANUSCRIPT Highlights The synthesized Fe3O4 nanoparticles inhibit S. cerevisiae growth independent of cell death. The inhibitory effect of Fe3O4 nanoparticles is not attributed to ROS accumulation.
is the main target of Fe3O4 nanoparticles.
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The respiratory chain complex
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Fe3O4 nanoparticles strongly impair mitochondrial functions.