Article

Silica Nanoparticles Induce Oxidative Stress and Autophagy but Not Apoptosis in the MRC-5 Cell Line Sorina Nicoleta Petrache Voicu 1,2 , Diana Dinu 1 , Cornelia Sima 3 , Anca Hermenean 2,4 , Aurel Ardelean 2 , Elena Codrici 5 , Miruna Silvia Stan 1 , Otilia Z˘arnescu 1 and Anca Dinischiotu 1, * Received: 21 October 2015; Accepted: 30 November 2015; Published: 10 December 2015 Academic Editor: Bing Yan 1

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Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91–95 Splaiul Independentei, Bucharest 050095, Romania; [email protected] (S.N.P.V.); [email protected] (D.D.); [email protected] (M.S.S); [email protected] (O.Z.) Department of Experimental and Applied Biology, Institute of Life Sciences, Vasile Goldis Western University of Arad, 86 Rebreanu, Arad 310414, Romania; [email protected] (A.H.); [email protected] (A.A.) Laser Department, National Institute of Laser, Plasma and Radiation Physics, 409 Atomistilor, Bucharest-Magurele 077125, Romania; [email protected] Department of Histology, Faculty of Medicine, Pharmacy and Dentistry, Vasile Goldis Western University of Arad, 1 Feleacului, Arad 310396, Romania Biochemistry Proteomics Department, Victor Babes National Institute of Pathology, 99-101 Splaiul Independentei, Bucharest 050096, Romania; [email protected] Correspondence: [email protected]; Tel./Fax: +40-21-318-1575 (ext. 103)

Abstract: This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO2 NPs) on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The nanoparticles’ morphology, composition, and structure were investigated using high resolution transmission electron microscopy, selected area electron diffraction and X-ray diffraction. Our study showed a decreased cell viability and the induction of cellular oxidative stress as evidenced by an increased level of reactive oxygen species (ROS), carbonyl groups, and advanced oxidation protein products after 24, 48, and 72 h, as well as a decreased concentration of glutathione (GSH) and protein sulfhydryl groups. The protein expression of Hsp27, Hsp60, and Hsp90 decreased at all time intervals, while the level of protein Hsp70 remained unchanged during the exposure. Similarly, the expression of p53, MDM2 and Bcl-2 was significantly decreased for all time intervals, while the expression of Bax, a marker for apoptosis, was insignificantly downregulated. These results correlated with the increase of pro-caspase 3 expression. The role of autophagy in cellular response to SiO2 NPs was demonstrated by a fluorescence-labeled method and by an increased level of LC3-II/LC3-I ratio. Taken together, our data suggested that SiO2 NPs induced ROS-mediated autophagy in MRC-5 cells as a possible mechanism of cell survival. Keywords: SiO2 nanoparticles; heat shock proteins; oxidative stress; apoptosis; autophagy; MRC-5 cell line

1. Introduction Silica (SiO2 ) represents one of the most common minerals on earth and a basic component of soil, sand, and rocks, including granite and quartzite. It can be found in both crystalline and amorphous forms. Silica nanoparticles (SiO2 NPs) are easy to prepare, inexpensive to produce and are used as additives or rheological modifier in the formulation of paints, plastics, and synthetic rubber. During the utilization of these materials, SiO2 NPs can be released in a time-dependent manner. Int. J. Mol. Sci. 2015, 16, 29398–29416; doi:10.3390/ijms161226171

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Int. J. Mol. Sci. 2015, 16, 29398–29416

Occupational exposure to crystalline silica dust is associated with a high risk for pulmonary diseases including silicosis, chronic bronchitis, and lung cancer [1,2], whereas amorphous silica is considered relatively safe. Nanoparticles (NPs) are of great interest for a wide variety of potential applications in the fields of electronics, energy, and environmental and medical technology. The amorphous silica nanoparticles (SiO2 NPs) are among the most widely used nanomaterials, due to their advantages such as large surface area for loading biomacromolecules, biocompatibility, storage stability, and low cost production [3]. In the recent years, the use of silica nanoparticles for fundamental biomedical applications, including imaging [4], controllable drug delivery [5,6] and theranostics [7], as well as additives for food and cosmetics [8] has increased considerably. Despite their widespread use, SiO2 NPs toxicity and safety to the human body and the environment have not been extensively investigated. To date, several in vivo and in vitro studies of the toxicity of SiO2 NPs have been performed. Previous in vivo studies suggested that the size of SiO2 NPs was critical for their toxicity. To be more specific, the treatment of mice with 70, 300, and 1000 nm SiO2 NPs revealed no hematological, histopathological or biochemical alterations in various organs suggesting these NPs can be employed in food production [9]; by contrast, exposure to 10–15 nm NPs resulted in toxic effects [10]. Similarly, cytotoxic effects induced by SiO2 NPs were reported in various cell lines such as HaCaT [11,12], H9c2(2-1) [13], Hek293 [14,15], EAHY926 [16], HepG2 [17,18], and A549 [19,20], with these effects being size- and dose-dependent, as well as highly cell type-dependent [21,22]. The mechanism by which SiO2 NPs induce toxicity is not clear. The formation of reactive oxygen species (ROS) can be considered as a possible mechanism for SiO2 NPs taking into consideration that crystalline silica has been shown to cause oxidative and inflammatory responses [23]. In addition, a significant increase in ROS production after SiO2 NPs exposure has been reported in several cell types [14,24–26]. It is generally accepted that increased quantities of ROS initiate lipid peroxidation in the cellular, mitochondrial, and nuclear membranes resulting in the degradation of cytosolic proteins and DNA damage [27]. So far, very few studies have investigated the effects of SiO2 NPs on cellular components. Lipid peroxidation occurrence [28], reduced glutathione (GSH) depletion [19], and DNA damage [25,29] have been previously reported, while the possible effects of SiO2 NPs on proteins have not been studied. Recently, SiO2 NPs were shown to induce ROS-mediated apoptosis in the human liver HepG2 cell line [30] and in the human lung epithelial A549 cell line [31]. Cell survival during stress requires the induction of the heat shock response. High levels of heat shock proteins (Hsps) can be triggered after exposure to various environmental stress conditions, such as increased temperature, presence of environmental pollutants and free radicals [32]. Thus, studying the relation between oxidative stress and cell death in SiO2 NPs exposure may be an important goal in order to elucidate the effects of these nanoparticles on cells. Our study aimed to highlight the biochemical mechanisms responsible for the toxic effects of 7 nm size SiO2 NPs on a human lung fibroblast cell line (MRC-5). Mechanisms of ROS production and their effects on proteins, as well as the modulation of heat shock proteins’ expressions, were investigated. Apoptosis and autophagy, two processes by which damaged cells or organelles are eliminated, were also analyzed. 2. Results 2.1. Physico-Chemical Characterization of SiO2 Nanoparticles (NPs) The characterization of any type of NPs as far as their physicochemical properties are concerned is imperative for any nanotoxicological study [33]. The XRD spectrum (Figure 1A) showed that SiO2 NPs are amorphous, a result which was supported by the selected area electron diffraction (SAED) image (Figure 1B(d)). The spherical aspect of the SiO2 NPs can be observed in the high

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Int. J. Mol. Sci.transmission 2015, 16, page–page resolution electron microscopy (HRTEM) images presented in Figure 1B(a–c). Also, it can be observed that the particles are agglomerated in clusters. Based on a statistics from electron electron microscopy images, the size distribution was obtained. The NP size distribution, which is a microscopy images, the size distribution was obtained. The NP size distribution, which is a lognormal lognormal function, is shown in Figure 1C. The primary sizes of nanoparticles are between and an 13 function, is shown in Figure 1C. The primary sizes of nanoparticles are between 4 and 13 nm4with nm withvalue an average value of approximately 7 nm. average of approximately 7 nm. The hydrodynamic size of SiO 2 NPs was measured in different suspensions (ultrapure water, The hydrodynamic size of SiO2 NPs was measured in different suspensions (ultrapure water, cell cell culture media—Minimum Essential Medium (MEM) with/no bovine serum). Compared to culture media—Minimum Essential Medium (MEM) with/no fetalfetal bovine serum). Compared to the the primary size (7the nm) the nanoparticles a tendency to aggregate in media, aqueousreaching media, primary size (7 nm) nanoparticles showedshowed a tendency to aggregate in aqueous reaching diameters of 156 ± 1.55 nm in ultrapure water; 157 ± 2.66 nm in culture medium diameters of 156 ˘ 1.55 nm in ultrapure water; 157 ˘ 2.66 nm in culture medium MEM withoutMEM fetal withoutand fetal serum, and ± 2.85 nm in culture medium MEM with 10% The fetaleffect serum. effect of serum, 159 ˘ 2.85 nm159 in culture medium MEM with 10% fetal serum. of The aggregation aggregation by highlighted by Dynamic Light Scattering (DLS) were measurements confirmed by highlighted Dynamic Light Scattering (DLS) measurements confirmed were by HRTEM images HRTEM images (Figure 1B(b,c)). Considering that the values obtained were almost equal, this (Figure 1B(b,c)). Considering that the values obtained were almost equal, this suggests that there suggests that there were no differences between the dispersion media used. were no differences between the dispersion media used.

Figure (A) XRD XRD spectrum spectrum of nanoparticles (NPs); (NPs); (B) (B) high high resolution resolution transmission Figure 1. 1. (A) of SiO SiO22 nanoparticles transmission electron electron microscopy (HRTEM) (a–c) and selected area electron diffraction (SAED) (d) NPs; microscopy (HRTEM) (a–c) and selected area electron diffraction (SAED) (d) images images of of SiO SiO22 NPs; (C) Size distribution of SiO2 NPs. (C) Size distribution of SiO2 NPs.

2.2. 2.2. Cell Cell Viability Viability The The cell cell viability viability after after the the exposure exposure of of MRC-5 MRC-5 fibroblasts fibroblasts to to different different concentrations concentrations of of SiO SiO22 NPs (12.5, 31.25 and 62.5 µg/mL) for 24, 48, and 72 h are presented in Figure 2. No significant NPs (12.5, 31.25 and 62.5 µg/mL) for 24, 48, and 72 h are presented in Figure 2. No significant changes NPs after after 24, changes were were observed observed in in the the cells cells exposed exposedto to12.5 12.5and and31.25 31.25µg/mL µg/mL SiO SiO22 NPs 24, 48, 48, and and 72 72 h, h, respectively. respectively. Nevertheless, Nevertheless, aa 35% 35% decrease decrease in in viability viability was was observed observed when when cells cells were were exposed exposed to to the the highest forfor this dose after 24 24 and highest concentration concentration of of SiO SiO22 NPs NPs for for72 72h,h,with withno nochange changebeing beingrecorded recorded this dose after h 48 h. This result indicates that the viability of MRC-5 cells decreased in a timeand dose-dependent and 48 h. This result indicates that the viability of MRC-5 cells decreased in a time- and manner. Taking manner. into account these the these final data, concentration of SiO2 NPs used subsequent dose-dependent Taking intodata, account the final concentration of SiOin2 NPs used in experiments was 62.5 µg/mL. subsequent experiments was 62.5 µg/mL. 2.3. NPs in in MRC-5 MRC-5 Human Human Lung Lung Fibroblast 2.3. Morphological Morphological Changes Changes Induced Induced by by SiO SiO22 NPs Fibroblast Cells Cells A A marked marked vacuolization vacuolization of of the the cytoplasm cytoplasm was was observed observed after after the the treatment treatment of of MRC-5 MRC-5 cells cells with with 62.5 µg/mL SiO NPs (Figure 3). This vacuolization was not observed in the controls. 62.5 µg/mL SiO22NPs (Figure 3). This vacuolization was not observed in the controls. 29400

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2.4.SiO SiO22NPs NPsInduce InduceReactive ReactiveOxygen OxygenSpecies Species(ROS) (ROS)Generation GenerationininMRC-5 MRC-5Cells Cells 2.4. Int. J. Mol. Sci. 2015, 16, page–page Asignificant significantincrease increaseofofboth both intraextracellular levels observed in MRC-5 A intraandand extracellular ROSROS levels werewere observed in MRC-5 cells cells 2.4. treated with 62.5 µg/mL NPs. After 24 h of exposure, the fluorescence intensity of treated with 62.5 µg/mL SiO 2 SiO NPs. After 24 h of exposure, the fluorescence intensity of SiO2 NPs Induce Reactive Oxygen2 Species (ROS) Generation in MRC-5 Cells 1 1 2’,7’-dichlorodihydrofluorescein 2 ,7 -dichlorodihydrofluorescein (DCF) (DCF) was was unchanged, unchanged, but but itit became became higher higher in in SiO SiO22 NPs NPs treated treated A significant increase of both intra- and extracellular ROS levels were observed in MRC-5 cells cells, cells, by by 12% 12% and and 26% 26% after after 48 48 and and 72 72 h,h, respectively, respectively, suggesting suggesting an an increase increase in in ROS ROS generation generation treated with 62.5 µg/mL SiO2 NPs. After 24 h of exposure, the fluorescence intensity of (Figure (Figure 4A). 4A). The The extracellular extracellular ROS ROS concentration concentration increased increased by by 32%, 32%, 54%, 54%, and and 104% 104% after after 24, 24, 48, 48, and and 2’,7’-dichlorodihydrofluorescein (DCF) was unchanged, but it became higher in SiO2 NPs treated 72 respectively (Figure 72h, h,cells, respectively (Figure 4B). 4B). by 12% and 26% after 48 and 72 h, respectively, suggesting an increase in ROS generation

(Figure 4A). The extracellular ROS concentration increased by 32%, 54%, and 104% after 24, 48, and 72 h, respectively (Figure 4B).

Figure 2.2. The Theviability viabilityofofMRC-5 MRC-5human humanlung lungfibroblast fibroblast cells cells exposed exposed to to SiO SiO22 NPs, NPs, atat different different Figure Figure 2. The viability of MRC-5 humanare lung fibroblastascells exposed to(nSiO 2 NPs, at different concentrations, for 24, 48, and 72 h. Values calculated means ˘ SD = 3) and are expressed as concentrations, for 24, 48, and 72 h. Values are calculated as means ± SD (n = 3) and are expressed as concentrations, for 24, 48,vs. andcontrols. 72 h. Values are calculated as means ± SD (n = 3) and are expressed as % from controls. ** p < 0.01 % from controls. ** p < 0.01 vs. controls. % from controls. ** p < 0.01 vs. controls.

Figure 3. Cellular morphologyofofMRC-5 MRC-5 cells cells untreated exposed to 62.5 µg/mL SiO2 SiO2 Figure 3. Cellular morphology untreated(control) (control)and and exposed to 62.5 µg/mL for 48 24, and 48 and Thearrows arrowsindicate indicate the of of thethe cytoplasm during the exposure NPs NPs for 24, 72 72 h. h. The the vacuolization vacuolization cytoplasm during the exposure 2 NPs. Scale bars = 20 µm. to SiO to SiO Scalemorphology bars = 20 µm.of MRC-5 cells untreated (control) and exposed to 62.5 µg/mL SiO2 23.NPs. Figure Cellular

4 NPs for 24, 48 and 72 h. The arrows indicate the vacuolization of the cytoplasm during the exposure to SiO2 NPs. Scale bars = 20 µm.

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(A)

(A)

(B) (B)

Figure 4. Figure Reactive oxygen species production in MRC-5 cells to exposed SiO2 NPs: 4. Reactive oxygen species (ROS) (ROS) production in MRC-5 cells exposed SiO2 NPs:to(A) (A) intracellular and (B) extracellular ROS concentrations. Values are calculated as means ˘ SD intracellular and (B) extracellular ROS concentrations. Values are calculated as means ± SD (n = 3) Figure 4. Reactive oxygen species (ROS) production in MRC-5 cells exposed to SiO2 NPs: (A) and are expressed as % from controls. * p < 0.05 vs. controls; ** p < 0.01 vs. controls; *** p < 0.001 vs. (n = 3) and are expressed as % from controls. * p < 0.05 vs. controls; ** p < 0.01 vs. controls; *** p < 0.001 intracellular and (B) extracellular ROS concentrations. Values are calculated as means ± SD (n = 3) controls. and are expressed as % from controls. * p < 0.05 vs. controls; ** p < 0.01 vs. controls; *** p < 0.001 vs. vs. controls. controls.

2.5. Glutathione (GSH) Concentration

2.5. Glutathione (GSH) (GSH) Concentration 2.5. Glutathione Concentration A time-dependent decrease of the GSH level was observed in the MRC-5 cells treated with SiO 2 NPs. In more detail, GSH was diminished by 36%, 50%, and 78% after 24, 48, and 72 h, A time-dependent decrease of the GSH levelwas wasobserved observed ininthe cellscells treated with with SiO2 A time-dependent decrease of the GSH level theMRC-5 MRC-5 treated respectively (Figure 5). SiO 2 NPs. In more detail, GSH was diminished by 36%, 50%, and 78% after 24, 48, and 72 h, NPs. In more detail, GSH was diminished by 36%, 50%, and 78% after 24, 48, and 72 h, respectively respectively (Figure 5). (Figure 5).

Figure 5. Glutathione (GSH) level in MRC-5 cells treated with SiO2 NPs. Values are calculated as means (n =(GSH) 3) and are expressed as % from controls. *** pwith < SiO 0.001 vs. 2controls. Figure 5. Figure Glutathione level in inMRC-5 cells treated NPs. are Values are calculated as 5.± SD Glutathione (GSH) level MRC-5 cells treated with 2SiO NPs. Values calculated as means = 3)are and expressed are expressedasas% % from from controls. *** p*** < 0.001 vs. controls. means ˘ SD (n ±=SD 3)(nand controls. p < 0.001 vs. controls.

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2.6. Protein Oxidative Modifications

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The effects of the SiO2 NPs exposure on protein oxidation in MRC-5 cells are summarized in Table 1. In our experiment, advanced oxidation protein products (AOPP) levels were increased by 10%, 32% and 77% after 24, 48, and 72 h, respectively (Table 1). In addition, the protein thiol (PSH) levels were reduced by 20% and 35%, after 48 and 72 h, respectively, with no significant changes being recorded for the 24 h time point (Table 1). Furthermore, the protein carbonyl groups, another index

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2.6. Protein Oxidative Modifications The effects of the SiO2 NPs exposure on protein oxidation in MRC-5 cells are summarized in of protein oxidative modification, was increased by 11% and significantly elevated by 51%, after 48 Table 1. In our experiment, advanced oxidation protein products (AOPP) levels were increased by and 72 h of 10%, treatment SiO224, NPs (Table 32% andwith 77% after 48, and 72 h,1). respectively (Table 1). In addition, the protein thiol (PSH) levels were reduced by 20% and 35%, after 48 and 72 h, respectively, with no significant changes

Table 1.being Advanced products protein carbonyl (PCG)groups, and protein recorded oxidation for the 24 hprotein time point (Table 1).(AOPP), Furthermore, the protein carbonyl anotherthiol index of protein oxidative modification, was increased by 11% and significantly elevated by 51%, are (PSH) groups in MRC-5 cells after 24, 48 and 72 h of SiO2 nanoparticles (NPs) exposure. Results after 48 h and 72 h of treatment with SiO 2 NPs (Table 1). means ˘ SD (n = 3) and are expressed as % from controls.

Time (h) 24 48 72

Table 1. Advanced oxidation protein products (AOPP), protein carbonyl (PCG) and protein thiol AOPP (nmoles/mg) PSH (nmoles/mg) PCG (nmoles/mg) (PSH) groups in MRC-5 cells after 24, 48 and 72 h of SiO2 nanoparticles (NPs) exposure. Results are Control Exposed Cells as %Control Cells Exposed Cells Control Cells Exposed Cells means ±Cells SD (n = 3) and are expressed from controls. 100 ˘ 2.27

109.39 ˘ 2.55

AOPP (nmoles/mg) 100 ˘ 2.44 131.66 ˘ 11.66 *** Time 100(h) ˘ 2.13 176.47 ˘ 2.96 *** Control Cells Exposed Cells 24

100 ± 2.27

48 72

100 ˘ 1.79 98.4 ˘ 1.85 PSH 100 ˘ 1.80(nmoles/mg) 83.61 ˘ 4.33 *** 100 ˘ 6.10 67.02 ˘ 4.42 ***

Control Cells

Exposed Cells

100 ˘ 3.78 PCG100 (nmoles/mg) ˘ 2.54

100 ˘ 5.54

Control Cells

** p < 0.01 vs. controls; *** p < 0.001 vs. controls.

97.18 ˘ 9.69 111.18 ˘ 4.56 ** 150.98 ˘ 8.78 ***

Exposed Cells

109.39 ± 2.55

100 ± 1.79

98.4 ± 1.85

100 ± 3.78

97.18 ± 9.69

100 ± 2.44

131.66 ± 11.66 ***

100 ± 1.80

83.61 ± 4.33 ***

100 ± 2.54

111.18 ± 4.56 **

100 ± 2.13

176.47 ± 2.96 ***

100 ± 6.10

67.02 ± 4.42 ***

100 ± 5.54

150.98 ± 8.78 ***

2.7. Heat Shock Proteins Expression

** p < 0.01 vs. controls; *** p < 0.001 vs. controls.

Western blot studies showed that Hsp27 expression was strongly inhibited in the SiO2 NPs 2.7. Heat Shock Proteins Expression treated cells, by 80%, 83%, and 89%, after 24, 48, and 72 h, respectively (Figure 6A). A significant Western blot studies showed that Hsp27 expression was strongly inhibited in the SiO2 NPs decrease in Hsp60 expression, by approximately 74%, was registered in MRC-5 cells after 24 h of treated cells, by 80%, 83%, and 89%, after 24, 48, and 72 h, respectively (Figure 6A). A significant treatment. decrease For longer timeexpression, exposures, Hsp60 expression appeared restored as it24increased by in Hsp60 by approximately 74%, was registeredto in be MRC-5 cells after h of treatment. longer time exposures, Hsp60 expression appeared restored as not it increased 56% and 36% after 48For and 72 h, respectively (Figure 6B). The levels to ofbe Hsp70 did changebyin MRC-5 56% NPs and 36% after 48 and 72 h, respectively (Figure 6B). The levels of Hsp70 not changefor in MRC-5 cells after SiO exposure (Figure 6C). A down-regulation of 30% wasdid registered Hsp90 during 2 cells after SiO2 NPs exposure (Figure 6C). A down-regulation of 30% was registered for Hsp90 the entire 72 h exposure to SiO2 NPs (Figure 6D). Furthermore, the appearance of an additional Hsp90 during the entire 72 h exposure to SiO2 NPs (Figure 6D). Furthermore, the appearance of an protein band in MRC-5 cells treated NPscells was noticed 6D). (Figure 6D). additional Hsp90 protein band with in MRC-5 treated with (Figure NPs was noticed

Int. J. Mol. Sci. 2015, 16, page–page

Figure 6. Cont.

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Figure Western blotblot images and quantitative analyses of Hsps in MRC-5 Figure6.6.Representative Representative Western images and quantitative analyses ofexpression Hsps expression in cells after the treatment with 62.5 µg/mL of SiO NPs for 24, 48, and 72 h. (A) Hsp27; (B) Hsp60; 2 of SiO2 NPs for 24, 48, and 72 h. (A) Hsp27; (B) MRC-5 cells after the treatment with 62.5 µg/mL (C) Hsp70 (D) Hsp90 and Hsp90and proteolysis form (p.f). The expression normalized Hsp60; (C)and Hsp70 and (D) Hsp90 Hsp90 proteolysis formprotein (p.f). The proteinwas expression was to β-actin. Results are expressed as means ˘ SD (n = 3) and are represented as % from normalized to β-actin. Results are expressed as means ± SD (n = 3) and are represented ascontrols. % from **controls. p < 0.01** and p

Silica Nanoparticles Induce Oxidative Stress and Autophagy but Not Apoptosis in the MRC-5 Cell Line.

This study evaluated the in vitro effects of 62.5 µg/mL silica nanoparticles (SiO NPs) on MRC-5 human lung fibroblast cells for 24, 48 and 72 h. The n...
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