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MgO nanoparticles cytotoxicity caused primarily by GSH depletion in human lung epithelial cells
Journal of Trace Elements in Medicine and Biology 50 (2018) 283–290
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Toxicology
MgO nanoparticles cytotoxicity caused primarily by GSH depletion in human lung epithelial cells
T
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Mohd Javed Akhtara, , Maqusood Ahameda, Hisham A. Alhadlaqb,a, Salman A. Alrokayanc a
King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia c Research Chair for Biomedical Applications of Nanomaterials, Biochemistry Department, College of Science, Building 5, PO Box 2455, King Saud University, Riyadh, Saudi Arabia b
A R T I C LE I N FO
A B S T R A C T
Keywords: MgO NPs Cytotoxicity ROS-independent Autophagosome Caspase-3 activity
Bio-response of magnesium oxide nanoparticles (MgO NPs) is emerging, obviously, with a conflicting flavor. This study evaluates the underlying mechanism of bio-responses of MgO NPs in human lung epithelial (A549) cell. TEM size of NPs was 40–50 nm and cuboidal in shape. EDS data showed no detectable impurity. Zeta potential of MgO NPs suggested a fair dispersion in complete culture media and in PBS. MgO NPs induced a concentration dependent cytotoxicity when measured by MTT and NRU. MgO NPs induced cytotoxicity strongly correlated with intracellular depletion of antioxidant GSH. MgO NPs did not induce concentration dependent ROS. All live treatment conditions caused autophagy, a survival mechanism when deprived of nutrients and antioxidant. At highest cytotoxic concentration of MgO NPs, there was significant elevation in MMP and caspase-3 activity. GSH depletion mediated autophagy failure lead to MgO NPs induced death at higher concentrations that might have potentiated by induced ROS. This study suggested a mechanism of cytotoxicity caused by MgO NPs that was primarily dependent on GSH depletion, and ROS induction played secondary role in toxicity. Significantly higher toxicity observed for MgO NPs in comparison to Mg salt clearly indicated the involvement of nanoparticulate form in toxicity.
1. Introduction Magnesium oxide (MgO) nanoparticle (NP; particle having at least one dimension under ≤100 nm), is emerging because of its wide anticipated exploration in many fields [1–3]. Exploration MgO NPs in health sciences, particularly their bactericidal activity, have started relatively recently [4–7]. A series of toxicity studies of micro- and nanoMgO particles performed in wistar rats indicated these particles to cause genotoxicity and alter significantly redox homeostasis [8,9]. According to other investigators, however, the genotoxic effect of MgO NPs was not significant in Hep G2 cells compared with control experiments [10]. Another report indicated MgO NPs to be reasonably safe in HeLa cells and concluded that MgO NPs could be utilized in various medical applications after conducting more careful assessments [11]. Potential mechanism of bio-response of MgO NPs was evaluated in human lung epithelial (A549) cells which is an established cell line for nanotoxicity assessment [12,13]. Concentration dependent cell viability and membrane damaging potential was measured in cells treated
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with NPs. DCFH-DA was used to probe general ROS and a specific sensor of H2O2 (Sigma, MAK164) was used to determine H2O2 inducing potential of NPs. Intracellular glutathione (GSH) was measured in A549 cells due to MgO NPs under different GSH modulating conditions by treating cells with NAC (N-acetyl cysteine, a precursor of GSH) and BSO (buthionine-[S, R]-sulfoximine, an inhibitor of GSH synthesis pathway). H2O2 was used as exogenous oxidant whenever necessary. The role of autophagy is increasingly recognized as a cell survival strategy and failure of which culminates in the death of cells by different mode of cell death [14–18]. In this study, autophagosome formation, an early event in autophagy, was determined using a kit (Sigma, MAK-138) in cells at lethal and sub-lethal concentrations of MgO NPs in order to understand the potential role of autophagy activation on cell survival and death. Physicochemical properties of MgO NPs were characterized by field emission transmission electron microscopy (FETEM) and energy dispersive spectrum (EDS) analysis respectively. Dynamic light scattering (DLS) and zeta potential of NPs in distilled water, phosphate buffer saline, serum free culture medium and complete culture media were measured to see the potential differences caused by serum and
Corresponding author at: King Abdullah Institute for Nanotechnology (KAIN) King Saud University, P.O. Box 2454, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (M.J. Akhtar).
https://doi.org/10.1016/j.jtemb.2018.07.016 Received 5 June 2018; Received in revised form 17 July 2018; Accepted 20 July 2018 0946-672X/ © 2018 Elsevier GmbH. All rights reserved.
Journal of Trace Elements in Medicine and Biology 50 (2018) 283–290
M.J. Akhtar et al.
2. Materials and methods
viable cells were solubilized and absorbance taken at 570 nm using a plate reader (Biotek Synergy HT). Cell viability of treated and control cells is given as cell viability in percentage of control.
2.1. Chemicals and reagents
2.8. Neutral red uptake assay
Fetal bovine serum, penicillin–streptomycin were purchased from Invitrogen Co. (Carlsbad, CA, USA). DMEM F-12, Neutral red dye, MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], Bradford reagent, GSH, H2O2, o-phthalaldehyde (OPT), TBA, NADPH, pyruvic acid, NAC, BSO, H2O2 measuring kit were obtained from Sigma–Aldrich (Sigma–Aldrich, MO, USA). Ultrapure deionized-water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals used were of reagent grade.
The neutral red uptake (NRU) assay is based on the method described by Repetto et al. [22]. A549 cells per well were seeded in 96 well plates in 100 μL of culture medium as for MTT. Before 3 h treatment period to complete, 50 μL of NR dye (0.6 mg/ml in PBS, filtered with 0.22 μm filter) was added to each 96 well and incubated for the remaining 3 h. After NR dye was extracted in an elution solution, it was centrifuged and transferred to new plate. Absorbance was taken at 540 nm using a plate reader (Biotek Synergy HT) and data is given as cell viability in percentage of control.
media components.
2.2. Magnesium oxide NPs 2.9. Lactate dehydrogenase assay NPs of MgO was commercially obtained from Sigma. As per the information provided by supplier, TEM size of MgO was below 50 nm. Color of nanopowder was white.
The activity of cytoplasmic LDH released into the culture media was determined with the method described by Welder et al. [23]. A 100 μL sample from the centrifuged culture media was collected after the cells were treated for 24 h. The LDH activity was assayed by determining the rate of NADH oxidation at 340 nm for 3 min at 30 s interval at 25 ℃ using a spectrophotometer (Thermo-Spectronic Genesys, USA). The amount of LDH released is represented as LDH activity (IU/L) in culture media.
2.3. Transmission electron microscopy of MgO NPs Size of MgO NPs was determined by field emission transmission electron microscopy (FETEM) (JEM-2100 F, JEOL Inc,) using an accelerated voltage of 200 kV [19]. Normal and high resolution (HR) TEM images was also taken to observe the crystallinity of NPs as it happens in HR-TEM texture of typical crystals. Suspension of ultra-sonicated MgO NPs was placed onto a carbon-coated copper grid, air dried and observed with FETEM. Purity of MgO NPs was determined by energy dispersive spectrum (EDS) analysis.
2.10. Determination of lipid peroxidation LPO was assessed by the thiobarbituric acid reactive substances (TBARS) assay, which detects mainly malondialdehyde (MDA), a product of the peroxidation of polyunsaturated fatty acids and related esters. MDA was measured by slight modification of the method of Ohkawa et al. [24]. A 200 μL aliquot of cell suspension was subsequently mixed with 800 μL of LPO assay cocktail containing 0.4% (w/v) thiobarbituric acid, 0.5% (w/v) SDS, 5% (v/v) acetic acid, pH 3.5 and, incubated for 60 min at 95 ℃. The absorbance of the supernatants was read at 532 nm (Thermo-Spectronic Genesys, USA). Results were calculated as nmol TBARS/mg of cellular protein using 1.56 × 105 M−1 as molar extinction of MDA-TBA.
2.4. Agglomeration and zeta-potential of MgO NPs Agglomeration behavior and zeta potential of MgO NPs in water, phosphate buffer saline (PBS) and complete cell culture medium was determined by dynamic light scattering (DLS) system (Nano-ZetaSizerHT, Malvern Instruments, Malvern, UK) as described by Murdock et al. [20]. 2.5. Dissolution measurement of MgO NPs in aqueous solutions
2.11. Measurement of total reactive oxygen species by DCFH-DA and H2O2 specific sensor
MgO NPs were suspended in culture media and phosphate buffer saline (PBS) and left for 1 week at 37 ℃ with a gentle shake for 1 h per day using a shaker. A concentration of 1 mg/mL of MgO NPs was taken in each solution. After the incubation period, tubes were centrifuged at 1000x g for 10 min and supernatant was carefully collected for the estimation of free ions by atomic absorption spectroscopy (AAS) at 285.2 nm that was already calibrated with four standard concentrations of Mg2+ using MgCl2.
The generation of intracellular ROS was measured using 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) probe [25]. Cells were seeded and treated with NPs and salt as for MTT but in black plates with clear bottom. When the treatment period was over the medium was aspirated off and 100 μL of DCFH-DA working solution was incubated for 45 min. Then each well was washed twice with cold PBS and DCF fluorescent intensity was measured at emission from 528 nm band pass of plate reader (Biotek Synergy HT). A specific H2O2 sensor was employed for the detection of intracellular H2O2 according manufacturer’s protocol (Sigma, MAK164). A standard of H2O2 was run and measured similarly using H2O2 sensor.
2.6. Cell culture Human lung carcinoma (A549) cells (ATCC, USA) were maintained in DMEM-F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, at 37 ℃ in a humidified incubator with 5% CO2. The cells were passaged for every 3–4 days before reaching confluence level.
2.12. Measurement of mitochondrial membrane potential by JC-10 Mitochondrial membrane potential in cells treated with NPs and salt was determined by kit obtained from Sigma (#MAK159). According to manufacturer protocol, in healthy cells with high mitochondrial membrane potential, JC-10 spontaneously forms complexes known as J-aggregates with intense red fluorescence the ex/em intensity of which can be quantified at 560/595 nm. On the other hand, in apoptotic or unhealthy cells with low membrane potential, JC-10 remains in the monomeric form showing only green fluorescence the ex/em intensity
2.7. MTT assay MTT assay was carried out according to the protocol described by Mosmann [21] with minor modifications. Briefly, around 20,000 A549 cells per well were seeded in 96 well plates in 100 μL of culture medium. The next day, NPs at various concentration, suspended in equal volume of fresh media were exposed. Blue formazan formed in 284
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Fig. 1. Size confirmation of MgO NPs by TEM microscopy at the resolution of 20 nm (A) and 10 nm (B). TEM calculated MgO NPs to range from 40 to 50 nm. Shape of MgO NPs looked cuboidal. Regular crystal plane observed in high resolution (HR)-TEM image (B) is indicative of crystal structure of MgO NPs. Elemental dispersive spectrum analysis (C) detected no element other than Mg and O2.
cycles of freeze–thaw and centrifuged at 10,000 × g for 10 min at 4 ℃. For intracellular GSH, supernatant was precipitated in 1% perchloric acid and centrifuged at 10,000 × g for 5 min at 4 ℃. A 20 μL from the protein precipitated sample was mixed with 160 μl of 0.1 M phosphate–5 mM EDTA buffer, pH 8.3 and 20 μL o-phthalaldehyde (OPT, 1 mg/mL in methanol) in a black 96 well plate. After 2.5 h of incubation at room temperature in the dark, fluorescence was measured at an emission wavelength of 460 nm (Biotek Synergy HT) along with similarly prepared standards of GSH. Results are expressed as GSH nmol/mg protein.
of which can be quantified at 485/535 nm. Cells were seeded at appropriate densities in 96 well black plate. When treatment period was over, media was aspirated off from each well and 5 μM JC-10 in PBS was added for 45 min. Respective fluorescence ratio of the two fluorescent intensities obtained by plate reader (Biotek Synergy HT) was used as an indicator of cell health. 2.13. Caspase-3 activity Activity of caspase-3 enzyme was determined using a standard fluorometric microplate assay. In brief, 5 × 104 A549 cells/well were seeded in T25 flasks and exposed to NPs at the concentration of 50, 100, 200 and 400 μg/mL for 24 h. After the exposure was complete, cells were harvested in ice cold phosphate buffer saline and prepared cell lysate. Further, a reaction mixture containing 30 μL of cell lysate, 20 μL of Ac-DEVDAFC (caspase-3 substrate), and 150 μL of protease reaction buffer (50 mM Hepes, 1 mM EDTA, and 1 mM DTT) (pH 7.2) was incubated for 15 min in 96 well plates. The fluorescence of the reaction mixture was measured at 5 min intervals for 15 min at excitation/ emission wavelengths of 430/535 nm using a microplate reader (Biotek Synergy HT). 7-Amido-4- trifluoromethylcoumarin (AFC) standard ranging from 5 μM to 15 μM was prepared, and its fluorescence was recorded for calculation of caspase-3 activity in pmol AFC released/ minute/mg protein.
2.15. Detection of autophagosome formation Briefly, around 20,000 A549 cells per well were seeded in 96 well plates in a 100 μL of culture medium. The next day, NPs and salt at various concentration, suspended in equal volume of fresh media, were added to A549 cells. The autophagy assay kit (Sigma, MAK-138) provides a simple and easy detection of autophagosome using an autophagosome detection fluorescent probe. Fluorescence was measured at 520 nm emission using a plate reader (Biotek Synergy HT). Autophagosome formation data is given as percent of control as in [27].
2.16. Estimation of protein
2.14. Determination of intracellular glutathione
The total protein concentration was measured at 595 nm (ThermoSpectronic Genesys, USA) by Bradford method [28] using a ready-touse Bradford reagent with bovine serum albumin as protein standard.
The cellular content of GSH was quantified by fluorometric measurement [26]. After exposure, cell lysate was prepared in distilled water containing 0.1% deoxycholic acid plus 0.1% sucrose by four 285
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3.3. MgO NPs induced membrane damage poorly correlated with lipid peroxidation and ROS induction
Table 1 MgO NP’s properties in powder form and its behavior in relevant aqueous media.
MgO NPs did not cause significant loss of cell membrane integrity in cells at any of MgO NPs concentration. Amount of lactate dehydrogenase (LDH) release from cells into extracellular culture media confirmed the integrity in cell membrane up to 100 μg/mL of MgO NPs (Fig. 3A). Membrane damage is often considered as the result of peroxidation reactions in lipid bilayer constituent of polyunsaturated fatty acids due to ROS. MgO NPs did not induced significant lipid peroxidation (LPO) up to 200 μg/mL of MgO NPs (Fig. 3B). Significant LPO occurred only at 400 μg/ml. Like LPO, MgO NPs did not cause ROS generation in correlation of cytotoxicity. The ability of MgO NPs in general oxidant production was assessed by measuring DCF fluorescence as a reporter of total ROS generation. A concentration of 50, 100, and 200 μg/mL of MgO NPs exposed to 24 h did not cause a significant induction in ROS except at 400 μg/mL of NPs (Fig. 3C). Similarly, a sensor specific to H2O2 measured H2O2 production in cells treated with MgO NPs, and H2O2 as a positive control. Intracellular level of H2O2 in cells treated with MgO NPs for 24 h was not significantly different from that of control cells except at 400 μg/mL of MgO NPs (Fig. 3D). Cells that were treated with 100 μM H2O2 for 1 h before measurement were found to contain 14.9 ± 1.40 μM H2O2. In control cells, concentration of H2O2 was calculated to be 5.3 ± 0.5 μM from the standards of H2O2 measured similarly using H2O2 sensor. Measurements and calculations by several methods in cells have determined the intracellular concentration of H2O2 in the range of low-nanomolar to low-micromolar [29,30]. In essence, MgO NPs did not generate significant ROS at MgO NPs concentrations (i.e. 100 and 200 μg/mL of MgO NPs) that caused significant toxicity in cells.
MgO NPs Physico-chemical properties TEM size Color HR-TEM TEM shape EDS
40–50 nm White Crystallite texture Mostly cubic Elemental impurities not detected
Agglomeration and zeta potential in aqueous media Water Hydrodynamic size 300–350 nm Zeta potential −26.0 ± 4.1 mV PBS Hydrodynamic size Zeta potential
400–470 nm −16.0 ± 2.5 mV
Serum free culture media Hydrodynamic size Zeta potential
380–4300 nm −20.0 ± 3.0 mV
Complete culture media Hydrodynamic size Zeta potential
190–280 nm −34.0 ± 2.2 mV
NPs dissolution Complete culture media Serum free culture media PBS Water
27.2 16.5 15.2 17.5
± ± ± ±
7.4% 4.8% 5.5% 5.4%
2.17. Statistics Data were expressed as the mean ± SD from three independent experiments using four replicates in plate reader (Biotek Synergy HT) and three replicates in cuvette reader (Thermo-Spectronic Genesys, USA). One-way ANOVA and Dunnett’s Multiple Comparison Test was applied using Graph Pad prism (Version 6.0) software for significance testing, using a p value ≤ 0.05 as an indicator for statistical significance
3.4. MgO NPs mediated cellular GSH depletion correlated well with cytotoxicity MgO NPs caused GSH depletion in A549 cells in concentration-dependent that started from 100 μg/ml of MgO NPs and onwards (Fig. 4A). It is notable that non-cytotoxic concentration (i.e. 50 μg/mL) of MgO NPs significantly increased cellular GSH. In the presence of NAC, the significant GSH depletion in A549 cells caused by MgO NPs exposure was restored to that of control level (4B). NAC significantly restored GSH in A549 cells treated with MgO NPs. BSO, an inhibitor of GSH synthesizing enzyme, aggravated further GSH depletion in MgO NPs treated cells (Fig. 4B). Cell viability data correlated well in accordance of GSH level in cells treated under various co-culture conditions (Fig. 4C).
3. Results 3.1. MgO NP characterization Shape of MgO NPs was found to be cuboidal in shape as shown in Fig. 1. Size of MgO NPs was found to 40–50 nm as determined by TEM. Texture in HR TEM image confirms the crystalline nature of MgO NPs (Fig. 1B). EDS spectrum of MgO NPs is given in Fig. 1C. EDS result shows that there are no other elemental impurities present in MgO NPs other than Mg and O2. DLS data shows MgO NPs to be agglomerated potentially due to magnetic property of this NPs. Table 1 summarizes the physico-chemical characterization data of MgO NPs.
3.5. MgO NPs induced autophagy and apoptotic markers at high concentration
3.2. MgO NPs induced cytotoxicity in a concentration dependent manner Mitochondrial outer membrane permeabilization is an early step found in cells undergoing apoptosis. As given in Fig. 5A, MgO NPs induced this early event only at 400 μg/mL of MgO NPs. Recall, excess ROS induction has been suggested to play an important role in the mechanism of toxicity of a number of compounds by causing oxidation of proteins, lipid and DNA. In the present study, however, MgO NPs induced caspase-3 activation (Fig. 3B) at concentration that significantly induced ROS (and LPO). Autophagy is a conserved cell survival mechanism that allows a starving cell, or a cell that is deprived of growth factors, to survive [31]. Major inducing factor of autophagy in cells is the depletion nutrients and antioxidants such as ATP and GSH [32]. In this study, autophagy initiation was ensued at all GSH depleting concentrations of MgO NPs (Fig. 5C).
A549 cells were treated from 50, 100, 200 and 400 μg/mL of MgO NPs after which linearity in cell viability was lost abruptly. A concentration of 50 μg/mL of NPs did not cause significant reduction in cell viability when measured by MTT and NRU. As the concentrations of MgO NPs increased to 100, 200 and 400 μg/mL the cell viability significantly decreased (Fig. 2A). For example, at 400 μg/ml the cell viability decreased to 68% (MTT) and 69% (NRU). The cell viability for positive control was 50%. In summary, cell viability was found to be 101, 88, 76 and 68% respectively at 50, 100, 200 and 400 μg/mL of MgO NPs concentration when determined by MTT. Similar was the cell viability trend observed for NRU. Cell morphological studies (Fig. 2B), a direct evidence, also confirmed the cell viability findings determined by biochemical methods of MTT and NRU. 286
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Fig. 2. Cell viability due to MgO NPs (A) measured by MTT and NRU due to 24 h treatment of MgO NPs in cells. Concentration of NP is taken in μg/mL. Photomicrograph (×20) of control and treated cells (B) correlates well with the cell viability observed by biochemical methods. Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05).
Fig. 3. Membrane integrity, thought to be modulated by oxidative stress, was assessed by measuring LDH release (A), TBARS formation (B), total ROS induction (C) and cellular H2O2 production (D) due to 24 h treatment of MgO NPs at indicated concentration. It should be noted that significant LDH release, TBARS formation, cellular ROS and H2O2 induction occurred only at 400 μg/mL of MgO NPs. Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05).
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Fig. 4. Antioxidant GSH content in cells. Intracellular GSH level was significantly depleted in cell treated with MgO NPs in a concentration dependent manner except at 50 μg/mL of MgO NPs (A). Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05). α, β, and γ denotes a significant GSH changes with MgO NPs (200 μg/mL) when co-treated separately with NAC (2 mM), H2O2 (at IC50 = 680 μM) and BSO (200 μM) respectively when compared with GSH changes caused by NAC, H2O2, and BSO alone (without MgO NPs) (B). A concentration of MgO at 200 μg/mL was chosen because this concentration caused a mild cytotoxicity and GSH depletion. Photomicrograph (×20) of MgO treated cells in conjunction with NAC, H2O2, and BSO correlates well with the cell viability observed by biochemical methods (C).
4. Discussion
spherical NPs tend to dissolute at higher rate than NPs that are large in size and non-spherical in shape [35]. Ge et al. found that once the concentration of MgO NPs was higher than 500 μg/mL, the relative growth rate was lower in treated human umbilical vein endothelial cells than the control cells [37]. Among NPs of ZnO, TiO2 NPs and MgO, NPs of MgO were found to be the least cytotoxic in human astrocytes-like astrocytoma (U87) cells [38]. Toxic potentials of MgO NPs were investigated on liver (HepG2), kidney (NRK-52E), intestine (Caco-2), and lung (A549) cell lines and it was observed that MgO NPs induced cell death was not due to apoptosis [39]. Metal oxide NPs' antibacterial effect might be due to solely by dissoluted ions or by a combined effect of NPs and dissoluted ions or by only NPs [40]. Similarly, bio-response of metal oxide NPs to
Magnesium (Mg2+) is the second most abundant intracellular cation after potassium [33]. Using scanning electron microscopy, transmission electron microscopy, and X-ray diffraction, it was found that MgO NPs less than 10 nm dissoluted within few minutes while MgO NPs within a size range of 10–1000 nm dissolved with a significantly smaller dissolution rate in water [34]. For the cubic isomers, increasing the size of the MgO cluster in any dimension improved the nanostructure stability [35]. In this study, significantly, higher dissolution of MgO NPs occurred both in culture media and PBS as compared with other metal oxide NPs reported previously [36]. Dissolution and, thus, stability of MgO NPs is strongly dependent on its size and shape; low size and
Fig. 5. Effect of MgO NPs on apoptosis and autophagy, the two interrelated mode of cell death. Significant alteration of MMP (A) and increase in caspase activity (B) was found at only high (400 μg/mL) concentration of MgO NPs. Autophagy initiation, however was found to occur at all concentrations of MgO NPs except for 50 μg/ mL (C). Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05). 288
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the first report that find MgO NPs induced toxicity happened to be ROS independent but strongly GSH dependent in A549 cells.
mammalian cells is broad and vague [41,42]. Cell viability was found to decrease with increasing concentration of NPs. ROS is considered as being the main underlying chemical process in nanotoxicology, leading to secondary processes such as inflammations that can ultimately cause cell damage and even cell death. However, in this study sign of MgO NPs mediated ROS induction was not observed in correlation of MgO NPs induced cytotoxicity. Moreover, toxicity caused by Fe3O4 NPs in yeast cells was also ROS-independent [43]. Dihydroethidium (DHE), a specific probe to measure superoxide anion, suggested that 5 mM exogenous Mg2+ (corresponding to 601.83 μg/mL using MgSO4) had no significant effect on intracellular ROS induction in rat cardiomyocytes and decreased LDH release during re-oxygenation of hypoxic cardiomyocytes [44]. In this study, no significant reduction in cell viability was found when cells were treated with 100–800 μg/mL of MgCl2 for 24 h (data not shown). In a recent study, however, alteration in mitochondrial Mg2+ homeostasis lead to dysfunction of mitochondria with disrupted ATP production [45]. Now we are not sure about MgO NPs uptake inside cells, but it is safe to conclude that dissoluted Mg2+ might have primed cells towards cyto-protection [46,47]. Since toxicity was found significantly higher for NPs of Mg when compared to same amount of Mg salt, it is clear that toxicity is attributable to NP form of Mg. Majority of particulate toxicants exhibit a common phenomenon of ROS dependency in the core of toxicity mechanism [48,49]. However, Leung et al found that significant toxicity due to NPs of ZnO and TiO2 to Escherichia coli cells does not necessarily correlate with up-regulation of ROS and ROS-related proteins [50]. A novel ROS-independent toxicity mechanism of Fe3O4 nanoparticles to eukaryotic cells is recently reported [43]. Therefore, it is not surprising that relatively low toxic MgO NPs cause cell death independently of ROS. In the present study MgO NPs started GSH depletion at concentrations that were not cytotoxic and ROS inductive. When treated with BSO, cells were found to be strongly exhausted with GSH but there occurred no cytotoxicity. When cells were simultaneously treated with non-toxic concentration of MgO NPs and BSO, intracellular GSH depletion was apparently observed that was translated into significant cytotoxicity. GSH depletion caused by NPs and BSO was significantly robust than GSH depletion caused by combined action of NPs and H2O2. GSH precursor antioxidant NAC completely abolished the GSH depletion as well cytotoxicity caused by toxic concentration of MgO NPs. Our study concludes the involvement a mechanism of toxicity caused by MgO NPs that is primarily dependent on cellular antioxidant depletion that might be ROS independent particularly at MgO NPs concentration that cause cell viability around 75% or above. Interestingly supporting this mechanism of toxicity in bacteria, MgO NPs exhibited robust toxicity towards Escherichia coli bacterial cells in the absence of ROS [51]. Moreover, Proteomics data also clearly demonstrated the absence of oxidative stress and indicated that the primary mechanism of cell death might be due to cell membrane damage that occurred in the absence of lipid peroxidation [51]. Recently, biodegradable Mg-based alloy (Mg-Nd-Zn-Zr) extract induced necrosis and complete damage of cell function at high-concentration whereas middle-concentration extract induced cell apoptosis and partially impaired cell function in THP-1 cells and THP-1 macrophages [52]. There is ample evidence that nanomaterials can cause surviving autophagic failures and lysosomal dysfunctions resulting in toxicological consequences [53]. Autophagosome accumulation has been found to be associated with the exposure of NPs of Tantalum (Ta) in osteoblasts [54], CuO in A549 cells [55], and CNTs in murine peritoneal macrophages [56]. Further, failure of autophagic survival can activate apoptotic-dependent [57] or apoptotic-independent cell death [58]. In this study, GSH depletion mediated autophagy failure lead to MgO NPs induced death at higher concentrations that might have potentiated by induced ROS. This study also suggest a ROS-independent cytotoxicity by MgO NPs particularly at low concentrations. There are few evidences of ROS-independent toxicity caused by MgO NPs in prokaryotic cells [50,51] and other NPs in eukaryotic cells [43]. This is
5. Conclusion In conclusion, bio-response of MgO NPs seem to be dependent of many factors. Findings of this study suggest that MgO NPs mediated toxicity is ROS-independent at low concentrations and ROS-dependent at high concentration. Significantly higher toxicity observed for MgO NPs in comparison to Mg salt clearly indicated the involvement of nanoparticulate form in toxicity. Conflict of interest Authors do not report conflict of interest. Acknowledgement The authors are thankful to King Abdullah Institute for Nanotechnology, Deanship of Scientific Research, King Saud University for their financial support. References [1] S. Utamapanya, K.J. Klabunde, J.R. Schlup, Nanoscale metal oxide particles/clusters as chemical reagents. Synthesis and properties of ultrahigh surface area magnesium hydroxide and magnesium oxide, Chem. Mater. 3 (1991) 175–181. [2] B.Q. Xu, J.H. Wei, H.Y. Wang, K.Q. Sun, Q.M. Zhu, Nano-MgO: novel preparation and application as support of Ni catalyst for CO2 reforming of methane, Catal. Today 68 (2001) 217–225. [3] Y. Li, Y. Bando, T. Sato, Preparation of network-like MgO nanobelts on Si substrate, Chem. Phys. Lett. 359 (2002) 141–145. [4] S. Makhluf, R. Dror, Y. Nitzan, Y. Abramovich, R. Jelinek, A. Gedanken, Microwaveassisted synthesis of nanocrystalline mgo and its use as a bacteriocide, Adv. Funct. Mater. 15 (2005) 1708–1715. [5] W. Ding, Opportunities and challenges for the biodegradable magnesium alloys as next-generation biomaterials, Regen. Biomater. 3 (2016) 79–86. [6] G. Moussavi, M. Mahmoudi, Removal of azo and anthraquinone reactive dyes from industrial wastewaters using MgO nanoparticles, J. Hazard. Mater. 168 (2009) 806–812. [7] M.R. Bindhu, M. Umadevi, M.K. Micheal, M.V. Mariadhas ValanArasu, N.A. AlDhabi, Structural, morphological and optical properties of MgO nanoparticles for antibacterial applications, Mater Lett. 166 (2016) 19–22. [8] B. Mangalampalli, N. Dumala, P. Grover, Acute oral toxicity study of magnesium oxide nanoparticles and microparticles in female albino Wistar rats, Regul. Toxicol. Pharmacol. 90 (2017) 170–184. [9] B. Mangalampalli, N. Dumala, R. Perumalla Venkata, P. Grover, Genotoxicity, biochemical, and biodistribution studies of magnesium oxide nano and microparticles in albino wistar rats after 28-day repeated oral exposure, Environ. Toxicol. 33 (2018) 396–410. [10] R.S. Kumaran, Y.K. Choi, V. Singh, H.J. Song, K.G. Song, K.J. Kim, H.J. Kim, In vitro cytotoxic evaluation of MgO nanoparticles and their effect on the expression of ROS genes, Int. J. Mol. Sci. 16 (2015) 7551–7564. [11] M.W. Akram, M. Fakhar-E-Alam, M. Atif, A.R. Butt, A. Asghar, Y. Jamil, K.S. Alimgeer, Z.M. Wang, In vitro evaluation of the toxic effects of MgO nanostructure in HeLa cell line, Sci. Rep. 8 (1) (2018) 4576. [12] M.J. Akhtar, M. Ahamed, S. Kumar, H. Siddiqui, G. Patil, M. Ashquin, I. Ahmad, Nanotoxicity of pure silica mediated through oxidant generation rather than glutathione depletion in human lung epithelial cells, Toxicology 276 (2010) 95–102. [13] M.J. Akhtar, M. Ahamed, S. Kumar, M.M. Khan, J. Ahmad, S.A. Alrokayan, Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species, Int. J. Nanomed. 7 (2012) 845–857. [14] J.J. Lum, R.J. DeBernardinis, C.B. Thompson, Autophagy in metazoans: cell survival in the land of plenty, Nat. Rev. Mol. Cell Biol. 6 (2005) 439–448. [15] L. Galluzzi, J.M. Vicencio, O. Kepp, E. Tasdemir, M.C. Maiuri, G. Kroemer, To die or not to die: that is the autophagic question, Curr. Mol. Med. 8 (2008) 78–91. [16] P. Codogno, A.J. Meijer, Autophagy and signaling: their role in cell survival and cell death, Cell Death Differ. 12 (2005) 1509–1518. [17] L. Liu, J.Z. Liao, X.X. He, P.Y. Li, The role of autophagy in hepatocellular carcinoma: friend or foe, Oncotarget 8 (2017) 57707–57722. [18] W. Zheng, M. Wei, S. Li, W. Le, Nanomaterial-modulated autophagy: underlying mechanisms and functional consequences, Nanomedicine (Lond.) 11 (2016) 1417–1430. [19] M. Ahamed, M.J. Akhtar, M. Raja, I. Ahmad, M.K. Siddiqui, M.S. AlSalhi, S.A. Alrokayan, ZnO nanorod-induced apoptosis in human alveolar adenocarcinoma cells via p53, survivin and bax/bcl-2 pathways: role of oxidative stress, Nanomedicine 7 (2011) 904–913. [20] R.C. Murdock, L. Braydich-Stolle, A.M. Schrand, J.J. Schlager, S.M. Hussain,
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Toxicology
MgO nanoparticles cytotoxicity caused primarily by GSH depletion in human lung epithelial cells
T
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Mohd Javed Akhtara, , Maqusood Ahameda, Hisham A. Alhadlaqb,a, Salman A. Alrokayanc a
King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia c Research Chair for Biomedical Applications of Nanomaterials, Biochemistry Department, College of Science, Building 5, PO Box 2455, King Saud University, Riyadh, Saudi Arabia b
A R T I C LE I N FO
A B S T R A C T
Keywords: MgO NPs Cytotoxicity ROS-independent Autophagosome Caspase-3 activity
Bio-response of magnesium oxide nanoparticles (MgO NPs) is emerging, obviously, with a conflicting flavor. This study evaluates the underlying mechanism of bio-responses of MgO NPs in human lung epithelial (A549) cell. TEM size of NPs was 40–50 nm and cuboidal in shape. EDS data showed no detectable impurity. Zeta potential of MgO NPs suggested a fair dispersion in complete culture media and in PBS. MgO NPs induced a concentration dependent cytotoxicity when measured by MTT and NRU. MgO NPs induced cytotoxicity strongly correlated with intracellular depletion of antioxidant GSH. MgO NPs did not induce concentration dependent ROS. All live treatment conditions caused autophagy, a survival mechanism when deprived of nutrients and antioxidant. At highest cytotoxic concentration of MgO NPs, there was significant elevation in MMP and caspase-3 activity. GSH depletion mediated autophagy failure lead to MgO NPs induced death at higher concentrations that might have potentiated by induced ROS. This study suggested a mechanism of cytotoxicity caused by MgO NPs that was primarily dependent on GSH depletion, and ROS induction played secondary role in toxicity. Significantly higher toxicity observed for MgO NPs in comparison to Mg salt clearly indicated the involvement of nanoparticulate form in toxicity.
1. Introduction Magnesium oxide (MgO) nanoparticle (NP; particle having at least one dimension under ≤100 nm), is emerging because of its wide anticipated exploration in many fields [1–3]. Exploration MgO NPs in health sciences, particularly their bactericidal activity, have started relatively recently [4–7]. A series of toxicity studies of micro- and nanoMgO particles performed in wistar rats indicated these particles to cause genotoxicity and alter significantly redox homeostasis [8,9]. According to other investigators, however, the genotoxic effect of MgO NPs was not significant in Hep G2 cells compared with control experiments [10]. Another report indicated MgO NPs to be reasonably safe in HeLa cells and concluded that MgO NPs could be utilized in various medical applications after conducting more careful assessments [11]. Potential mechanism of bio-response of MgO NPs was evaluated in human lung epithelial (A549) cells which is an established cell line for nanotoxicity assessment [12,13]. Concentration dependent cell viability and membrane damaging potential was measured in cells treated
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with NPs. DCFH-DA was used to probe general ROS and a specific sensor of H2O2 (Sigma, MAK164) was used to determine H2O2 inducing potential of NPs. Intracellular glutathione (GSH) was measured in A549 cells due to MgO NPs under different GSH modulating conditions by treating cells with NAC (N-acetyl cysteine, a precursor of GSH) and BSO (buthionine-[S, R]-sulfoximine, an inhibitor of GSH synthesis pathway). H2O2 was used as exogenous oxidant whenever necessary. The role of autophagy is increasingly recognized as a cell survival strategy and failure of which culminates in the death of cells by different mode of cell death [14–18]. In this study, autophagosome formation, an early event in autophagy, was determined using a kit (Sigma, MAK-138) in cells at lethal and sub-lethal concentrations of MgO NPs in order to understand the potential role of autophagy activation on cell survival and death. Physicochemical properties of MgO NPs were characterized by field emission transmission electron microscopy (FETEM) and energy dispersive spectrum (EDS) analysis respectively. Dynamic light scattering (DLS) and zeta potential of NPs in distilled water, phosphate buffer saline, serum free culture medium and complete culture media were measured to see the potential differences caused by serum and
Corresponding author at: King Abdullah Institute for Nanotechnology (KAIN) King Saud University, P.O. Box 2454, Riyadh 11451, Saudi Arabia. E-mail address: [email protected] (M.J. Akhtar).
https://doi.org/10.1016/j.jtemb.2018.07.016 Received 5 June 2018; Received in revised form 17 July 2018; Accepted 20 July 2018 0946-672X/ © 2018 Elsevier GmbH. All rights reserved.
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2. Materials and methods
viable cells were solubilized and absorbance taken at 570 nm using a plate reader (Biotek Synergy HT). Cell viability of treated and control cells is given as cell viability in percentage of control.
2.1. Chemicals and reagents
2.8. Neutral red uptake assay
Fetal bovine serum, penicillin–streptomycin were purchased from Invitrogen Co. (Carlsbad, CA, USA). DMEM F-12, Neutral red dye, MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], Bradford reagent, GSH, H2O2, o-phthalaldehyde (OPT), TBA, NADPH, pyruvic acid, NAC, BSO, H2O2 measuring kit were obtained from Sigma–Aldrich (Sigma–Aldrich, MO, USA). Ultrapure deionized-water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals used were of reagent grade.
The neutral red uptake (NRU) assay is based on the method described by Repetto et al. [22]. A549 cells per well were seeded in 96 well plates in 100 μL of culture medium as for MTT. Before 3 h treatment period to complete, 50 μL of NR dye (0.6 mg/ml in PBS, filtered with 0.22 μm filter) was added to each 96 well and incubated for the remaining 3 h. After NR dye was extracted in an elution solution, it was centrifuged and transferred to new plate. Absorbance was taken at 540 nm using a plate reader (Biotek Synergy HT) and data is given as cell viability in percentage of control.
media components.
2.2. Magnesium oxide NPs 2.9. Lactate dehydrogenase assay NPs of MgO was commercially obtained from Sigma. As per the information provided by supplier, TEM size of MgO was below 50 nm. Color of nanopowder was white.
The activity of cytoplasmic LDH released into the culture media was determined with the method described by Welder et al. [23]. A 100 μL sample from the centrifuged culture media was collected after the cells were treated for 24 h. The LDH activity was assayed by determining the rate of NADH oxidation at 340 nm for 3 min at 30 s interval at 25 ℃ using a spectrophotometer (Thermo-Spectronic Genesys, USA). The amount of LDH released is represented as LDH activity (IU/L) in culture media.
2.3. Transmission electron microscopy of MgO NPs Size of MgO NPs was determined by field emission transmission electron microscopy (FETEM) (JEM-2100 F, JEOL Inc,) using an accelerated voltage of 200 kV [19]. Normal and high resolution (HR) TEM images was also taken to observe the crystallinity of NPs as it happens in HR-TEM texture of typical crystals. Suspension of ultra-sonicated MgO NPs was placed onto a carbon-coated copper grid, air dried and observed with FETEM. Purity of MgO NPs was determined by energy dispersive spectrum (EDS) analysis.
2.10. Determination of lipid peroxidation LPO was assessed by the thiobarbituric acid reactive substances (TBARS) assay, which detects mainly malondialdehyde (MDA), a product of the peroxidation of polyunsaturated fatty acids and related esters. MDA was measured by slight modification of the method of Ohkawa et al. [24]. A 200 μL aliquot of cell suspension was subsequently mixed with 800 μL of LPO assay cocktail containing 0.4% (w/v) thiobarbituric acid, 0.5% (w/v) SDS, 5% (v/v) acetic acid, pH 3.5 and, incubated for 60 min at 95 ℃. The absorbance of the supernatants was read at 532 nm (Thermo-Spectronic Genesys, USA). Results were calculated as nmol TBARS/mg of cellular protein using 1.56 × 105 M−1 as molar extinction of MDA-TBA.
2.4. Agglomeration and zeta-potential of MgO NPs Agglomeration behavior and zeta potential of MgO NPs in water, phosphate buffer saline (PBS) and complete cell culture medium was determined by dynamic light scattering (DLS) system (Nano-ZetaSizerHT, Malvern Instruments, Malvern, UK) as described by Murdock et al. [20]. 2.5. Dissolution measurement of MgO NPs in aqueous solutions
2.11. Measurement of total reactive oxygen species by DCFH-DA and H2O2 specific sensor
MgO NPs were suspended in culture media and phosphate buffer saline (PBS) and left for 1 week at 37 ℃ with a gentle shake for 1 h per day using a shaker. A concentration of 1 mg/mL of MgO NPs was taken in each solution. After the incubation period, tubes were centrifuged at 1000x g for 10 min and supernatant was carefully collected for the estimation of free ions by atomic absorption spectroscopy (AAS) at 285.2 nm that was already calibrated with four standard concentrations of Mg2+ using MgCl2.
The generation of intracellular ROS was measured using 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) probe [25]. Cells were seeded and treated with NPs and salt as for MTT but in black plates with clear bottom. When the treatment period was over the medium was aspirated off and 100 μL of DCFH-DA working solution was incubated for 45 min. Then each well was washed twice with cold PBS and DCF fluorescent intensity was measured at emission from 528 nm band pass of plate reader (Biotek Synergy HT). A specific H2O2 sensor was employed for the detection of intracellular H2O2 according manufacturer’s protocol (Sigma, MAK164). A standard of H2O2 was run and measured similarly using H2O2 sensor.
2.6. Cell culture Human lung carcinoma (A549) cells (ATCC, USA) were maintained in DMEM-F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, at 37 ℃ in a humidified incubator with 5% CO2. The cells were passaged for every 3–4 days before reaching confluence level.
2.12. Measurement of mitochondrial membrane potential by JC-10 Mitochondrial membrane potential in cells treated with NPs and salt was determined by kit obtained from Sigma (#MAK159). According to manufacturer protocol, in healthy cells with high mitochondrial membrane potential, JC-10 spontaneously forms complexes known as J-aggregates with intense red fluorescence the ex/em intensity of which can be quantified at 560/595 nm. On the other hand, in apoptotic or unhealthy cells with low membrane potential, JC-10 remains in the monomeric form showing only green fluorescence the ex/em intensity
2.7. MTT assay MTT assay was carried out according to the protocol described by Mosmann [21] with minor modifications. Briefly, around 20,000 A549 cells per well were seeded in 96 well plates in 100 μL of culture medium. The next day, NPs at various concentration, suspended in equal volume of fresh media were exposed. Blue formazan formed in 284
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Fig. 1. Size confirmation of MgO NPs by TEM microscopy at the resolution of 20 nm (A) and 10 nm (B). TEM calculated MgO NPs to range from 40 to 50 nm. Shape of MgO NPs looked cuboidal. Regular crystal plane observed in high resolution (HR)-TEM image (B) is indicative of crystal structure of MgO NPs. Elemental dispersive spectrum analysis (C) detected no element other than Mg and O2.
cycles of freeze–thaw and centrifuged at 10,000 × g for 10 min at 4 ℃. For intracellular GSH, supernatant was precipitated in 1% perchloric acid and centrifuged at 10,000 × g for 5 min at 4 ℃. A 20 μL from the protein precipitated sample was mixed with 160 μl of 0.1 M phosphate–5 mM EDTA buffer, pH 8.3 and 20 μL o-phthalaldehyde (OPT, 1 mg/mL in methanol) in a black 96 well plate. After 2.5 h of incubation at room temperature in the dark, fluorescence was measured at an emission wavelength of 460 nm (Biotek Synergy HT) along with similarly prepared standards of GSH. Results are expressed as GSH nmol/mg protein.
of which can be quantified at 485/535 nm. Cells were seeded at appropriate densities in 96 well black plate. When treatment period was over, media was aspirated off from each well and 5 μM JC-10 in PBS was added for 45 min. Respective fluorescence ratio of the two fluorescent intensities obtained by plate reader (Biotek Synergy HT) was used as an indicator of cell health. 2.13. Caspase-3 activity Activity of caspase-3 enzyme was determined using a standard fluorometric microplate assay. In brief, 5 × 104 A549 cells/well were seeded in T25 flasks and exposed to NPs at the concentration of 50, 100, 200 and 400 μg/mL for 24 h. After the exposure was complete, cells were harvested in ice cold phosphate buffer saline and prepared cell lysate. Further, a reaction mixture containing 30 μL of cell lysate, 20 μL of Ac-DEVDAFC (caspase-3 substrate), and 150 μL of protease reaction buffer (50 mM Hepes, 1 mM EDTA, and 1 mM DTT) (pH 7.2) was incubated for 15 min in 96 well plates. The fluorescence of the reaction mixture was measured at 5 min intervals for 15 min at excitation/ emission wavelengths of 430/535 nm using a microplate reader (Biotek Synergy HT). 7-Amido-4- trifluoromethylcoumarin (AFC) standard ranging from 5 μM to 15 μM was prepared, and its fluorescence was recorded for calculation of caspase-3 activity in pmol AFC released/ minute/mg protein.
2.15. Detection of autophagosome formation Briefly, around 20,000 A549 cells per well were seeded in 96 well plates in a 100 μL of culture medium. The next day, NPs and salt at various concentration, suspended in equal volume of fresh media, were added to A549 cells. The autophagy assay kit (Sigma, MAK-138) provides a simple and easy detection of autophagosome using an autophagosome detection fluorescent probe. Fluorescence was measured at 520 nm emission using a plate reader (Biotek Synergy HT). Autophagosome formation data is given as percent of control as in [27].
2.16. Estimation of protein
2.14. Determination of intracellular glutathione
The total protein concentration was measured at 595 nm (ThermoSpectronic Genesys, USA) by Bradford method [28] using a ready-touse Bradford reagent with bovine serum albumin as protein standard.
The cellular content of GSH was quantified by fluorometric measurement [26]. After exposure, cell lysate was prepared in distilled water containing 0.1% deoxycholic acid plus 0.1% sucrose by four 285
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3.3. MgO NPs induced membrane damage poorly correlated with lipid peroxidation and ROS induction
Table 1 MgO NP’s properties in powder form and its behavior in relevant aqueous media.
MgO NPs did not cause significant loss of cell membrane integrity in cells at any of MgO NPs concentration. Amount of lactate dehydrogenase (LDH) release from cells into extracellular culture media confirmed the integrity in cell membrane up to 100 μg/mL of MgO NPs (Fig. 3A). Membrane damage is often considered as the result of peroxidation reactions in lipid bilayer constituent of polyunsaturated fatty acids due to ROS. MgO NPs did not induced significant lipid peroxidation (LPO) up to 200 μg/mL of MgO NPs (Fig. 3B). Significant LPO occurred only at 400 μg/ml. Like LPO, MgO NPs did not cause ROS generation in correlation of cytotoxicity. The ability of MgO NPs in general oxidant production was assessed by measuring DCF fluorescence as a reporter of total ROS generation. A concentration of 50, 100, and 200 μg/mL of MgO NPs exposed to 24 h did not cause a significant induction in ROS except at 400 μg/mL of NPs (Fig. 3C). Similarly, a sensor specific to H2O2 measured H2O2 production in cells treated with MgO NPs, and H2O2 as a positive control. Intracellular level of H2O2 in cells treated with MgO NPs for 24 h was not significantly different from that of control cells except at 400 μg/mL of MgO NPs (Fig. 3D). Cells that were treated with 100 μM H2O2 for 1 h before measurement were found to contain 14.9 ± 1.40 μM H2O2. In control cells, concentration of H2O2 was calculated to be 5.3 ± 0.5 μM from the standards of H2O2 measured similarly using H2O2 sensor. Measurements and calculations by several methods in cells have determined the intracellular concentration of H2O2 in the range of low-nanomolar to low-micromolar [29,30]. In essence, MgO NPs did not generate significant ROS at MgO NPs concentrations (i.e. 100 and 200 μg/mL of MgO NPs) that caused significant toxicity in cells.
MgO NPs Physico-chemical properties TEM size Color HR-TEM TEM shape EDS
40–50 nm White Crystallite texture Mostly cubic Elemental impurities not detected
Agglomeration and zeta potential in aqueous media Water Hydrodynamic size 300–350 nm Zeta potential −26.0 ± 4.1 mV PBS Hydrodynamic size Zeta potential
400–470 nm −16.0 ± 2.5 mV
Serum free culture media Hydrodynamic size Zeta potential
380–4300 nm −20.0 ± 3.0 mV
Complete culture media Hydrodynamic size Zeta potential
190–280 nm −34.0 ± 2.2 mV
NPs dissolution Complete culture media Serum free culture media PBS Water
27.2 16.5 15.2 17.5
± ± ± ±
7.4% 4.8% 5.5% 5.4%
2.17. Statistics Data were expressed as the mean ± SD from three independent experiments using four replicates in plate reader (Biotek Synergy HT) and three replicates in cuvette reader (Thermo-Spectronic Genesys, USA). One-way ANOVA and Dunnett’s Multiple Comparison Test was applied using Graph Pad prism (Version 6.0) software for significance testing, using a p value ≤ 0.05 as an indicator for statistical significance
3.4. MgO NPs mediated cellular GSH depletion correlated well with cytotoxicity MgO NPs caused GSH depletion in A549 cells in concentration-dependent that started from 100 μg/ml of MgO NPs and onwards (Fig. 4A). It is notable that non-cytotoxic concentration (i.e. 50 μg/mL) of MgO NPs significantly increased cellular GSH. In the presence of NAC, the significant GSH depletion in A549 cells caused by MgO NPs exposure was restored to that of control level (4B). NAC significantly restored GSH in A549 cells treated with MgO NPs. BSO, an inhibitor of GSH synthesizing enzyme, aggravated further GSH depletion in MgO NPs treated cells (Fig. 4B). Cell viability data correlated well in accordance of GSH level in cells treated under various co-culture conditions (Fig. 4C).
3. Results 3.1. MgO NP characterization Shape of MgO NPs was found to be cuboidal in shape as shown in Fig. 1. Size of MgO NPs was found to 40–50 nm as determined by TEM. Texture in HR TEM image confirms the crystalline nature of MgO NPs (Fig. 1B). EDS spectrum of MgO NPs is given in Fig. 1C. EDS result shows that there are no other elemental impurities present in MgO NPs other than Mg and O2. DLS data shows MgO NPs to be agglomerated potentially due to magnetic property of this NPs. Table 1 summarizes the physico-chemical characterization data of MgO NPs.
3.5. MgO NPs induced autophagy and apoptotic markers at high concentration
3.2. MgO NPs induced cytotoxicity in a concentration dependent manner Mitochondrial outer membrane permeabilization is an early step found in cells undergoing apoptosis. As given in Fig. 5A, MgO NPs induced this early event only at 400 μg/mL of MgO NPs. Recall, excess ROS induction has been suggested to play an important role in the mechanism of toxicity of a number of compounds by causing oxidation of proteins, lipid and DNA. In the present study, however, MgO NPs induced caspase-3 activation (Fig. 3B) at concentration that significantly induced ROS (and LPO). Autophagy is a conserved cell survival mechanism that allows a starving cell, or a cell that is deprived of growth factors, to survive [31]. Major inducing factor of autophagy in cells is the depletion nutrients and antioxidants such as ATP and GSH [32]. In this study, autophagy initiation was ensued at all GSH depleting concentrations of MgO NPs (Fig. 5C).
A549 cells were treated from 50, 100, 200 and 400 μg/mL of MgO NPs after which linearity in cell viability was lost abruptly. A concentration of 50 μg/mL of NPs did not cause significant reduction in cell viability when measured by MTT and NRU. As the concentrations of MgO NPs increased to 100, 200 and 400 μg/mL the cell viability significantly decreased (Fig. 2A). For example, at 400 μg/ml the cell viability decreased to 68% (MTT) and 69% (NRU). The cell viability for positive control was 50%. In summary, cell viability was found to be 101, 88, 76 and 68% respectively at 50, 100, 200 and 400 μg/mL of MgO NPs concentration when determined by MTT. Similar was the cell viability trend observed for NRU. Cell morphological studies (Fig. 2B), a direct evidence, also confirmed the cell viability findings determined by biochemical methods of MTT and NRU. 286
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Fig. 2. Cell viability due to MgO NPs (A) measured by MTT and NRU due to 24 h treatment of MgO NPs in cells. Concentration of NP is taken in μg/mL. Photomicrograph (×20) of control and treated cells (B) correlates well with the cell viability observed by biochemical methods. Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05).
Fig. 3. Membrane integrity, thought to be modulated by oxidative stress, was assessed by measuring LDH release (A), TBARS formation (B), total ROS induction (C) and cellular H2O2 production (D) due to 24 h treatment of MgO NPs at indicated concentration. It should be noted that significant LDH release, TBARS formation, cellular ROS and H2O2 induction occurred only at 400 μg/mL of MgO NPs. Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05).
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Fig. 4. Antioxidant GSH content in cells. Intracellular GSH level was significantly depleted in cell treated with MgO NPs in a concentration dependent manner except at 50 μg/mL of MgO NPs (A). Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05). α, β, and γ denotes a significant GSH changes with MgO NPs (200 μg/mL) when co-treated separately with NAC (2 mM), H2O2 (at IC50 = 680 μM) and BSO (200 μM) respectively when compared with GSH changes caused by NAC, H2O2, and BSO alone (without MgO NPs) (B). A concentration of MgO at 200 μg/mL was chosen because this concentration caused a mild cytotoxicity and GSH depletion. Photomicrograph (×20) of MgO treated cells in conjunction with NAC, H2O2, and BSO correlates well with the cell viability observed by biochemical methods (C).
4. Discussion
spherical NPs tend to dissolute at higher rate than NPs that are large in size and non-spherical in shape [35]. Ge et al. found that once the concentration of MgO NPs was higher than 500 μg/mL, the relative growth rate was lower in treated human umbilical vein endothelial cells than the control cells [37]. Among NPs of ZnO, TiO2 NPs and MgO, NPs of MgO were found to be the least cytotoxic in human astrocytes-like astrocytoma (U87) cells [38]. Toxic potentials of MgO NPs were investigated on liver (HepG2), kidney (NRK-52E), intestine (Caco-2), and lung (A549) cell lines and it was observed that MgO NPs induced cell death was not due to apoptosis [39]. Metal oxide NPs' antibacterial effect might be due to solely by dissoluted ions or by a combined effect of NPs and dissoluted ions or by only NPs [40]. Similarly, bio-response of metal oxide NPs to
Magnesium (Mg2+) is the second most abundant intracellular cation after potassium [33]. Using scanning electron microscopy, transmission electron microscopy, and X-ray diffraction, it was found that MgO NPs less than 10 nm dissoluted within few minutes while MgO NPs within a size range of 10–1000 nm dissolved with a significantly smaller dissolution rate in water [34]. For the cubic isomers, increasing the size of the MgO cluster in any dimension improved the nanostructure stability [35]. In this study, significantly, higher dissolution of MgO NPs occurred both in culture media and PBS as compared with other metal oxide NPs reported previously [36]. Dissolution and, thus, stability of MgO NPs is strongly dependent on its size and shape; low size and
Fig. 5. Effect of MgO NPs on apoptosis and autophagy, the two interrelated mode of cell death. Significant alteration of MMP (A) and increase in caspase activity (B) was found at only high (400 μg/mL) concentration of MgO NPs. Autophagy initiation, however was found to occur at all concentrations of MgO NPs except for 50 μg/ mL (C). Values are mean ± SD from three independent experiments. *Denotes a significant difference from the control (p < 0.05). 288
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the first report that find MgO NPs induced toxicity happened to be ROS independent but strongly GSH dependent in A549 cells.
mammalian cells is broad and vague [41,42]. Cell viability was found to decrease with increasing concentration of NPs. ROS is considered as being the main underlying chemical process in nanotoxicology, leading to secondary processes such as inflammations that can ultimately cause cell damage and even cell death. However, in this study sign of MgO NPs mediated ROS induction was not observed in correlation of MgO NPs induced cytotoxicity. Moreover, toxicity caused by Fe3O4 NPs in yeast cells was also ROS-independent [43]. Dihydroethidium (DHE), a specific probe to measure superoxide anion, suggested that 5 mM exogenous Mg2+ (corresponding to 601.83 μg/mL using MgSO4) had no significant effect on intracellular ROS induction in rat cardiomyocytes and decreased LDH release during re-oxygenation of hypoxic cardiomyocytes [44]. In this study, no significant reduction in cell viability was found when cells were treated with 100–800 μg/mL of MgCl2 for 24 h (data not shown). In a recent study, however, alteration in mitochondrial Mg2+ homeostasis lead to dysfunction of mitochondria with disrupted ATP production [45]. Now we are not sure about MgO NPs uptake inside cells, but it is safe to conclude that dissoluted Mg2+ might have primed cells towards cyto-protection [46,47]. Since toxicity was found significantly higher for NPs of Mg when compared to same amount of Mg salt, it is clear that toxicity is attributable to NP form of Mg. Majority of particulate toxicants exhibit a common phenomenon of ROS dependency in the core of toxicity mechanism [48,49]. However, Leung et al found that significant toxicity due to NPs of ZnO and TiO2 to Escherichia coli cells does not necessarily correlate with up-regulation of ROS and ROS-related proteins [50]. A novel ROS-independent toxicity mechanism of Fe3O4 nanoparticles to eukaryotic cells is recently reported [43]. Therefore, it is not surprising that relatively low toxic MgO NPs cause cell death independently of ROS. In the present study MgO NPs started GSH depletion at concentrations that were not cytotoxic and ROS inductive. When treated with BSO, cells were found to be strongly exhausted with GSH but there occurred no cytotoxicity. When cells were simultaneously treated with non-toxic concentration of MgO NPs and BSO, intracellular GSH depletion was apparently observed that was translated into significant cytotoxicity. GSH depletion caused by NPs and BSO was significantly robust than GSH depletion caused by combined action of NPs and H2O2. GSH precursor antioxidant NAC completely abolished the GSH depletion as well cytotoxicity caused by toxic concentration of MgO NPs. Our study concludes the involvement a mechanism of toxicity caused by MgO NPs that is primarily dependent on cellular antioxidant depletion that might be ROS independent particularly at MgO NPs concentration that cause cell viability around 75% or above. Interestingly supporting this mechanism of toxicity in bacteria, MgO NPs exhibited robust toxicity towards Escherichia coli bacterial cells in the absence of ROS [51]. Moreover, Proteomics data also clearly demonstrated the absence of oxidative stress and indicated that the primary mechanism of cell death might be due to cell membrane damage that occurred in the absence of lipid peroxidation [51]. Recently, biodegradable Mg-based alloy (Mg-Nd-Zn-Zr) extract induced necrosis and complete damage of cell function at high-concentration whereas middle-concentration extract induced cell apoptosis and partially impaired cell function in THP-1 cells and THP-1 macrophages [52]. There is ample evidence that nanomaterials can cause surviving autophagic failures and lysosomal dysfunctions resulting in toxicological consequences [53]. Autophagosome accumulation has been found to be associated with the exposure of NPs of Tantalum (Ta) in osteoblasts [54], CuO in A549 cells [55], and CNTs in murine peritoneal macrophages [56]. Further, failure of autophagic survival can activate apoptotic-dependent [57] or apoptotic-independent cell death [58]. In this study, GSH depletion mediated autophagy failure lead to MgO NPs induced death at higher concentrations that might have potentiated by induced ROS. This study also suggest a ROS-independent cytotoxicity by MgO NPs particularly at low concentrations. There are few evidences of ROS-independent toxicity caused by MgO NPs in prokaryotic cells [50,51] and other NPs in eukaryotic cells [43]. This is
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