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Article Cite This: ACS Omega 2019, 4, 573−581
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Zinc Oxide Particles Induce Activation of the Lysosome−Autophagy System Lauren Popp† and Laura Segatori*,†,‡,§ †
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Department of Chemical and Biomolecular Engineering, Rice University, MS-362, 6100 Main Street, Houston, Texas 77005, United States ‡ Department of Bioengineering, Rice University, MS-142, 6100 Main Street, Houston, Texas 77005, United States § Biochemistry and Cell Biology Graduate Program, Rice University, MS-180, 6100 Main, Houston, Texas 77005, United States S Supporting Information *
ABSTRACT: Metal-oxide-based materials are highly versatile and used in a wide variety of applications ranging from medical technology to personal care products. Generally recognized as safe by the US Food and Drug Administration, zinc oxide (ZnO) has been increasingly used in pharmaceutical, cosmetic, food, and commodity chemical industries. As a result, exposure to nano- and micron-sized ZnO particles through occupational processes and consumer products is increasing and has raised concerns over the health effects associated with the large-scale production and commercialization of ZnO-based materials. It is therefore important to investigate the interaction of ZnO particles with biological systems and elucidate the consequent effect on cell physiology. Of particular interest is the autophagic response to zinc oxide particles, as autophagy is the first line of defense activated in response to the uptake of foreign materials. As the main cellular catabolic pathway, the lysosome−autophagy system plays an important homeostatic function and defects or deficiency of this degradation system is associated with the cellular pathogenesis of a number of human diseases, ranging from neurodegenerative disorders to cancer. In this study, we investigated the response of the lysosome−autophagy system to three relevant types of ZnO particles, namely, a polydisperse mixture of bare, micron-sized particles (100−1000 nm) and monodisperse, bare, and coated (with triethoxycaprylylsilane) ZnO nanoparticles (85 nm). To investigate the molecular mechanisms mediating the response of the lysosome−autophagy system to these ZnO particles, we examined a complete set of markers of this pathway and characterized each step, from transcriptional activation to clearance of autophagic cargo. To evaluate the effect of the different types of ZnO particles on the lysosome−autophagy system, biological assays were conducted under conditions that do not cause considerable cytotoxicity. All three types of ZnO particles were found to result in activation of the transcription factor EB, a master regulator of autophagy and lysosomal biogenesis. Cellular exposure to bare and coated nano-sized ZnO enhanced the formation and turnover of autophagosomes and cellular clearance. Cellular exposure to the polydisperse mixture of ZnO particles, however, resulted in enhancement of autophagosome formation, but also in blockage of the autophagic flux. Results from this study underscore the importance of characterizing the autophagic response to ZnO-based materials and contribute significant engineering principles for the future design of nano- and micron-sized ZnO materials with the desired autophagy-modulating properties.
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INTRODUCTION The global production of zinc oxide nanoparticles (1−100 nm in diameter) is expected to exceed 40 000 tons per year by the year 2020, with about 80% of this production predicted to be used in the cosmetics industry and, particularly, for the production of sunscreens.1 More recently, zinc oxide nanoparticles have also been used in a variety of biomedical and pharmaceutical applications, including imaging devices and drug and gene delivery systems.2 The production of micronsized zinc oxide particles (ZnOPs) (0.1−10 μm in diameter) has also dramatically increased3 because of their application in the manufacturing of rubber and ceramics.4 The growth in production and use of zinc oxide particles (ZnOPs) has © 2019 American Chemical Society
paralleled a rise in concern over the health effects associated with accidental exposure to nano- and micron-sized ZnO materials. In fact, pulmonary inflammation and metal fume fever in association with inhalation of micron-sized zinc oxide fumes have been documented in occupational settings for decades.5 In vitro studies revealed that cell exposure to high doses of ZnOPs can cause production of reactive oxygen species,6 actin depolymerization,7 and DNA damage,8 ultimately affecting cell viability. 9−11 Interestingly, cell Received: June 29, 2018 Accepted: December 11, 2018 Published: January 8, 2019 573
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Figure 1. Characterization of ZnOPs. (A) Transmission electron microscopy (TEM) images of ZnOPs dispersed in water. Scale bar is 500 nm. (B, C) Dynamic light scattering analyses of the (B) average hydrodynamic diameter and (C) average polydispersity of ZnOPs dispersed in DMEM. Data are presented as mean ± s.d. (D−F) Dynamic light scattering analyses of the distribution of hydrodynamic diameters of (D) micro/poly, (E) nano, and (F) nano/coated ZnOPs dispersed in DMEM. The average value for sizing measurements is represented as a vertical dashed line.
system, which has been reported in association with cellular internalization of some nano-sized materials,26−28 typically results in blockage of autophagic flux and, possibly, accumulation of intracellular autophagic substrates.19,21,29 Biopersistent nanomaterials, for instance, may induce transcriptional upregulation of the lysosome−autophagy system, but may also affect lysosomal integrity, disrupting the formation of autolysosomes and, ultimately, impairing clearance of autophagic cargo.26 The autophagic response to engineered nanomaterials seems to vary dramatically depending on the material’s physicochemical properties such as size21 and surface charge.19 In this study, we investigated the effect of three commercially relevant ZnOPs with different physicochemical properties on the response of the lysosome−autophagy system, using conditions that do not cause cytotoxicity, which would preclude an accurate assessment of the causative relationship between uptake of ZnOPs and activation of cellular clearance mechanisms. We tested a polydisperse mixture of micronsized particles ranging in diameter from 100 to 1000 nm (micro/poly-ZnOPs), which are representative of ZnOPs that may be found in the air as a result of combustion or industrial processes such as rubber production,30 and a monodisperse sample of nanoparticles of 85 nm diameter (nano-ZnOPs), which are representative of ZnOPs with high volume applications as UV filters in cosmetics and antimicrobial agents in deodorants or medical devices.1 These particles are broadly used across a number of industries, thus presenting potential risk of exposure for employees as well as consumers. We also tested the effect of a commonly used ZnOP coating material by comparing uncoated nano-ZnOPs to the same monodisperse sample of nanoparticles of 85 nm diameter coated with triethoxycaprylylsilane (nano/coated ZnOPs). Triethoxycaprylylsilane is typically used as a coating on ZnOPs to improve their suspension and stability in the oil phase of cosmetics formulations.31,32 To characterize the
exposure to ZnOPs seems to affect cell physiology even at subtoxic doses,10,12 pointing to the critical need for a detailed characterization of the cellular response to the uptake of ZnOPs. Of particular interest is the interaction of nano- and micronsized ZnOPs with autophagy, the main catabolic pathway in mammalian cells that mediates the clearance of cytoplasmic material.13 Autophagy is an intracellular degradation system that delivers cytoplasmic components to the lysosome: the process is initiated upon the formation of an insolation membrane near the intracellular cargo material and proceeds through the expansion of the isolation membrane and sequestration of autophagic cargo into autophagosomes. The autophagosomes fuse with lysosomes, which are membranebound organelles containing hydrolytic enzymes, resulting in the formation of autolysosomes where degradation occurs. A variety of materials have been reported to induce activation of the lysosome−autophagy system, likely in response to what the cell perceives as foreign or toxic.14,15 For instance, transcriptional activation of the lysosome−autophagy system was observed upon cell exposure to 2-hydroxypropyl-β-cyclodextrin, quantum dots, nanoceria, and polystyrene nanoparticles.16−19 Inorganic metal nanomaterials, such as gold and silver nanoparticles, were reported to accumulate within autophagosomes,20,21 suggesting upregulation of cellular clearance pathways. Micron-sized materials may also be recognized as autophagic cargo and transferred to lysosomes for processing.22 Transcriptional activation of the lysosome− autophagy system in response to internalization of foreign materials may lead to enhancement of the cellular clearance capacity, resulting in an increase in the extent of degradation of intracellular constituents normally degraded by autophagy.23−25 Autophagic clearance, however, depends on the cooperation of multiple processes that culminate in the formation of autolysosomes and degradation of cargo. Impairment of different factors of the lysosome−autophagy 574
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Figure 2. Viability of HeLa cells exposed to ZnOPs. (A, B) Flow cytometry analyses of (A) early apoptotic [Annexin V-FITC (+)/PI (−)] and (B) late apoptotic/necrotic [Annexin V-FITC (+)/PI (+)] HeLa cells incubated with ZnOPs (24 h). Data are presented as mean ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Flow cytometry analyses of FluoZin-3 fluorescence in HeLa cells exposed to ZnOPs (24 h). Relative fluorescence values were obtained by normalizing the fluorescence of ZnOP-treated cells to that of untreated cells. Data are presented as mean ± s.d. *p < 0.05, **p < 0.01. (D) Flow cytometry analyses of the side scattering parameter (SSC) of HeLa cells exposed to ZnOPs (24 h). Relative SSC values were obtained by normalizing the SSC of ZnOP-treated cells to that of untreated cells. Data are presented as mean ± s.d. *p < 0.05, **p < 0.01.
ZnOPs of different shapes and sizes and presenting different surface functionalities have been reported to cause cytotoxicity in a variety of cells types.9,11,33,34 To assess the cytotoxic effect of the ZnOPs used in this study and to determine the experimental conditions that are not associated with the induction of excessive cytotoxicity, which would preclude accurate characterization of the response of the lysosome− autophagy system to ZnOPs, we analyzed markers of apoptosis in HeLa cells cultured in the presence of ZnOPs (0−25 μg/ mL; 24 h) (Figure 2). Specifically, we monitored membrane rearrangement, which is characteristic of early apoptosis, by quantifying binding of Annexin V-FITC (Figure 2A), and membrane fragmentation, which is characteristic of late apoptosis and cell death, by quantifying staining of propidium iodide (PI) (Figure 2B). Induction of apoptosis was observed upon cell exposure to the ZnOPs at media concentrations of at least 25 μg/mL, similar to previously published studies,35,36 with a small population of cells exposed to 20 μg/mL of nano/ coated ZnOPs undergoing early apoptosis. Previous studies suggest a correlation between the effects of ZnOPs on cell physiology and the intracellular dissolution of Zn2+ ions from the ZnOPs.34,36,37 To monitor intracellular levels of Zn2+ under the conditions used in this study, we measured FluoZin-3 fluorescence in HeLa cells cultured in the presence of ZnOPs (0−25 μg/mL; 24 h) by flow cytometry (Figure 2C). The intracellular levels of Zn2+ were found to depend on the media concentration of ZnOPs for all three ZnOPs, as expected.36 FluoZin-3 fluorescence measurements indicate that cellular treatment with the micro/poly- and nanoZnOPs leads to low levels of intracellular Zn2+ (similar to what was previously reported by Song et al.),36 whereas significantly higher levels of Zn2+ were detected upon cell treatment with high media concentrations of nano/coated ZnOPs. The differences in intracellular Zn2+ levels observed in cells treated
lysosome−autophagy system in response to the cellular uptake of these materials, we measured relevant markers of transcriptional regulation, formation and turnover of autophagic vesicles, and autophagic clearance. Results from this study provide important design rules for engineering ZnOPs with desired effects on the lysosome−autophagy system and for the production of safe materials containing ZnOPs.
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RESULTS Cytotoxicity and Release of Zn2+ Ions from ZnOPs. The cellular response to exposure to ZnOPs was investigated using representative, commercially available zinc oxide powders. Namely, a mixture of polydisperse ZnOPs ranging in size from 100 to 1000 nm in diameter (micro/poly-ZnOPs) and monodisperse powders consisting of bare zinc oxide nanoparticles of 85 nm diameter (nano-ZnOPs) and zinc oxide nanoparticles of 85 nm diameter coated with a silicone derivative (triethoxycaprylylsilane) (nano/coated ZnOPs) (Figure 1A). To determine the behavior of these particles in the solution, we measured the average hydrodynamic diameter of each particle type in cell culture media (Dulbecco’s modified Eagle’s medium, DMEM) using dynamic light scattering (Figure 1B−F). Light scattering analyses revealed that the micro/poly-ZnOPs form a polydisperse suspension with an average particle hydrodynamic diameter of 361.5 ± 95.8 nm (Figure 1B) and a polydispersity index of 0.5 ± 0.1 (Figure 1C), with the majority of particles (>80%) ranging in size from 100 to 1000 nm in diameter (Figure 1D). Analyses of the suspensions of nano-ZnOPs and nano/coated ZnOPs revealed that these particles present average hydrodynamic diameters of 157.0 ± 8.1 and 143.7 ± 5.9 nm (Figure 1B), respectively, and a low polydispersity index (0.3 ± 0.0) (Figure 1C,E,F). Sonication conditions did not affect the results of light scattering analyses (not shown). 575
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Figure 3. TFEB activation in HeLa/TFEB cells exposed to ZnOPs. Confocal microscopy analyses of TFEB in HeLa/TFEB cells exposed to ZnOPs (24 h). (A) Average fraction of nuclear TFEB. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Fraction of HeLa/ TFEB cells with TFEB nuclear localization greater than the average fraction of nuclear TFEB in untreated cells (0.16 ± 0.02). Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, n ≥ 3. Each sample was analyzed by imaging on average 100 cells.
greater than that of untreated cells. Cell exposure to the micro/ poly-, nano-, and nano/coated ZnOPs resulted in similar extents of TFEB activation. ZnOPs Affect the Autophagic Flux. To determine whether activation of the lysosome−autophagy system at the transcriptional level mediated by TFEB in the cells exposed to ZnOPs parallels an increase in the biogenesis of autophagic vesicles, we monitored the processing of microtubuleassociated light chain 3 (LC3).41 Under basal conditions, LC3 is a cytoplasmic soluble protein (LC3-I). Upon activation of autophagy, LC3-I is conjugated to phosphatidylethanolamine to form an LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to the membrane of autophagosomes.42 Fusion of autophagosomes with lysosomes leads to the formation of autolysosomes and degradation of autophagosomal components, including LC3-II, by lysosomal hydrolases. The LC3 levels in HeLa cells exposed to ZnOPs (20 μg/ mL; 24 h) were first monitored by Western blot (Figures 4A and S2). Cell exposure to all three ZnOPs was found to induce an increase in the ratio of LC3-II/LC3-I, indicating an increase in the formation of autophagic vesicles. Addition of Bafilomycin A1, a known inhibitor of autophagy that prevents fusion of autophagosomes and lysosomes,43 resulted in further increase in the LC3-II/LC3-I ratio in cells cultured in the presence of nano- or nano/coated ZnOPs, but not in cells exposed to micro/poly-ZnOPs. The increase in LC3-II/LC3-I ratio observed upon the addition of Bafilomycin A1 is indicative of an increase in autophagy flux. These results suggest that although all three ZnOPs cause an increase in the formation of autophagosomes, exposure to the micro/polyZnOP also inhibits turnover of autophagic vesicles. The accumulation of autophagic vesicles was also investigated using HeLa cells stably transfected to express LC3 fused to GFP (HeLa/GFP-LC3) (Figure 4B). HeLa/GFP-LC3 cells were incubated with ZnOPs (20 μg/mL; 24 h) and Bafilomycin A1 (10 nM; 1 h) and treated with saponin prior to flow cytometry analyses. Saponin treatment allows extraction of the soluble, cytoplasmic form of LC3 (LC3-I) and enables measurement of autophagosome-associated LC3-II.44 Cell treatment with ZnOPs and Bafilomycin A1 was first confirmed not to affect the expression of GFP-LC3 (Figure S3). Cell exposure to all three ZnOPs induced an increase in GFP fluorescence compared to untreated cells, indicating an increase in the amount of GFP associated with autophagosomes upon ZnOP treatment. A further increase in GFP fluorescence was detected upon the addition of Bafilomycin A1 to cells exposed to nano- or nano/coated ZnOPs, indicating
with nano/coated ZnOPs compared to cells treated with micro/poly- and nano-ZnOPs may explain the differences in the cytotoxic effect of the ZnOPs observed from the results of the apoptosis assays (Figure 2A), with cell exposure to nano/ coated ZnOPs resulting in higher levels of intracellular Zn2+ and induction of apoptosis at slightly lower media concentrations than micro/poly- and nano-ZnOPs. Biological assays of different steps of the lysosome−autophagy system were thus conducted by exposing cells to media containing 20 μg/mL of ZnOPs. To determine the level of cellular uptake of the ZnOPs used in this study and to evaluate whether the effect of ZnOPs on cell viability and the lysosome−autophagy system depend on the extent of cellular internalization, we also measured ZnOP internalization by quantifying the side scattering parameter (SSC) of HeLa cells cultured in the presence of ZnOPs using flow cytometry (Figure 2D). Exposure to micro/poly-, nano-, and nano/coated ZnOPs resulted in similar uptake levels at all concentrations tested (0−25 μg/mL; 24 h). Cell Exposure to ZnOPs Results in Activation of Transcription Factor EB (TFEB). To investigate whether ZnOPs activate autophagy, we investigated the transcriptional regulatory network that controls autophagy activation in cells exposed to ZnOPs. Specifically, we monitored the activation of the transcription factor EB (TFEB),38,39 which regulates the autophagic response by coordinating expression of genes involved in the biogenesis of autophagosomes and lysosomes.38−40 TFEB is primarily found in the cytoplasm of resting cells and translocates to the nucleus upon activation, under conditions that result in upregulation of the lysosome− autophagy system.39 The TFEB subcellular localization was evaluated in a model system of TFEB activation consisting of HeLa cells stably transfected for the expression of TFEB3XFLAG (HeLa/TFEB cells). The HeLa/TFEB cells were cultured in the presence of ZnOPs (0−25 μg/mL; 24 h) and the subcellular localization of TFEB was quantified by immunofluorescence confocal microscopy evaluating the colocalization of Hoechst nuclear stain and an anti-FLAG antibody for TFEB detection (Figures 3 and S1). The TFEB activation was quantified by evaluating the Mander’s overlap coefficient of the TFEB (anti-FLAG) and nuclear (Hoechst stain) signals (Figure 3A) and by calculating the fraction of cells with TFEB activation greater than that of untreated cells (Figure 3B). Colocalization analyses revealed that cells exposed to at least 10 μg/mL of ZnOPs present an increase in both the fraction of TFEB that localizes in the nucleus and in the number of cells with nuclear translocation of TFEB 576
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exposure to the micro/poly-ZnOP also blocks the turnover of autophagosomes, suggesting a blockage of autophagic flux. To investigate whether the accumulation of autophagosomes observed in cells exposed to micro/poly-ZnOPs is due to a blockage of autophagic flux, we evaluated the fusion of autophagosomes with lysosomes and formation of autolysosomes. Specifically, we quantified the colocalization of LC3 and lysosomal membrane associated protein LAMP-2, which is monitored here as representative proteins that localize on the membranes of autophagosomes and lysosomes, respectively. HeLa cells were treated with ZnOPs (20 μg/mL; 24 h) and the colocalization of LC3 and LAMP-2 was measured by immunofluorescence confocal microscopy using anti-LC3 and anti-LAMP-2 antibodies (Figures 4C and S4). All three ZnOPs were found to increase the levels of LC3−LAMP-2 colocalization, as determined by evaluating the Mander’s overlap coefficient of LC3 and LAMP-2 signals, with the micro/poly-ZnOP inducing the lowest levels of colocalization. These results suggest that the increase in transcriptional activation of autophagy and formation of autophagosomes observed upon exposure to the micro/poly-ZnOP may not parallel efficient autophagosome−lysosome fusion, resulting in suboptimal turnover of autophagosomes compared to what is observed in cells treated with the nano- or nano/coated ZnOPs. These results, taken together, suggest that cell exposure to the nano or nano/coated ZnOPs enhances the formation of autophagosomes, fusion of autophagosomes with lysosomes, and, finally, autophagosome turnover. Cell exposure to the nano or nano/coated ZnOPs did not result in significant differences in autophagosome formation and turnover under the conditions tested, suggesting that the triethoxycaprylylsilane coating on the nano/coated ZnOPs does not significantly affect the autophagic response to nano-sized (85 nm) ZnOPs under the conditions tested in this study. Cell exposure to the micro/poly-ZnOP, on the other hand, enhanced the formation of autophagosomes, but was also observed to cause blockage of autophagic flux, ultimately leading to an increase in the number of autophagosomes. Nature of Autophagic Response to ZnOP Determines Intracellular Clearance. To test whether transcriptional upregulation of the lysosome−autophagy system induced by ZnOPs is associated with autophagic clearance, we monitored the degradation of intracellular autophagic cargo. Specifically, we used fibroblasts derived from a patient with late infantile neuronal ceroid lipofuscinosis (LINCL), which are characterized by accumulation of ceroid lipopigment, an autofluorescent lipofuscin-like material that is normally degraded through autophagy.45 The effect of ZnOPs on autophagic degradation was assessed by monitoring the total amount of ceroid lipopigment fluorescence in LINCL cells treated with ZnOPs (20 μg/mL; 24 h) by confocal microscopy (Figure 5). Microscopy analyses showed that the accumulation of ceroid lipopigment was reduced by approximately 25% in cells treated with nano- or nano/coated ZnOPs, suggesting that TFEBmediated activation of the lysosome−autophagy system and upregulation of autophagosome biogenesis and turnover lead to enhancement of cellular clearance. Removal of lipofuscin from cells has previously been demonstrated to enhance cell viability,46 suggesting that the autophagic response to nano- or nano/coated ZnOPs may have beneficial effect on cell physiology and enhance cell survival. Cellular treatment with micro/poly-ZnOPs under the same conditions, however, did
Figure 4. Autophagosome formation and turnover in HeLa cells exposed to ZnOPs. (A) Western blot analyses of LC3 levels in HeLa cells incubated with ZnOPs (20 μg/mL; 24 h) and Bafilomycin A1 (Baf; 10 nM; 1 h). LC3-II/LC3-I ratios relative to untreated cells (UT) are reported. Data are presented as mean ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Flow cytometry analyses of HeLa/GFPLC3 cells incubated with ZnOPs (20 μg/mL; 24 h) and Bafilomycin A1 (Baf; 10 nM; 1 h) and treated with saponin. Relative GFP fluorescence values were obtained by normalizing the GFP signal of cells treated with ZnOPs to that of untreated cells (UT). Data are presented as mean ± s.d. *p < 0.05, **p < 0.01. (C) Quantitative analyses of confocal microscopy images of LC3−LAMP colocalization in HeLa cells cultured in the presence of ZnOPs (20 μg/mL; 24 h). Untreated, UT. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001; n ≥ 3. Each sample was analyzed by imaging on average 50 cells.
that cell treatment with nano- or nano/coated ZnOPs enhances the formation and turnover of autophagosomes. Addition of Bafilomycin A1 was found not to affect the GFP signal of cells exposed to micro/poly-ZnOPs, indicating that cell treatment with micro/poly-ZnOP causes accumulation of autophagosomes. Taken together, the measurements of LC3 levels obtained from Western blot analyses (Figure 4A) and flow cytometry experiments (Figure 4B) indicate that whereas exposure to all three ZnOPs tested causes an increase in the formation of autophagosomes, consistent with the activation of TFEB, 577
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autophagic clearance. The triethoxycaprylylsilane coating was found not to alter the autophagic response to nano-sized ZnOPs under the conditions of this study. Cell treatment with micro/poly-ZnOPs, on the other hand, was found to induce accumulation of autophagosomes and blockage of autophagic flux. Although disruption of lysosomal stability and trafficking are typically the cause of defects in autophagic clearance, it remains to be determined whether other mechanisms, such as disruption of the cytoskeleton,26 may be involved in mediating blockage of autophagic flux in cells treated this polydisperse, micron-sized mixture of ZnOPs. Interestingly, it was previously determined that cell exposure to pharmacologic inhibitors of autophagy prevents the induction of apoptosis associated with internalization of ZnOPs,37 suggesting that the toxic effect of ZnOPs is likely due to autophagy-associated cell death. In summary, results from this work provide important insights into the nature of the autophagic response to cellular uptake of three commercially relevant types of ZnOPs. Differing physicochemical properties of these materials may play a role in determining the nature of the autophagic response that manifests upon cellular exposure to ZnOPs. These results point to the need to evaluate the response of cellular clearance pathways as part of the design of ZnOPs that are expected come into contact with biological systems.
Figure 5. Accumulation or clearance of autophagic substrates in LINCL cells exposed to ZnOPs. Confocal microscopy analyses of accumulation of ceroid lipopigment in LINCL cells incubated with ZnOPs (20 μg/mL; 24 h). (A) Representative images of ceroid lipopigment (green) and nuclei (blue). Untreated, UT. Scale bar is 50 μm. (B) Total ceroid lipopigment fluorescence signal detected in LINCL cells. Untreated, UT. Data are reported as mean ± s.e.m. *p < 0.05, **p < 0.01; n ≥ 3. Each sample was analyzed by imaging on average 100 cells.
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METHODS
Nanomaterial Characterization Studies. ZnOPs (micro/poly, nano, and nano/coated) were purchased from Making Cosmetics. The micro/poly (100−1000 nm) and nano (85 nm) ZnOPs consist of super purity zinc oxide, and the nano/coated ZnOPs consist of ∼98% super purity zinc oxide and 2% hydrophobic coating material (triethoxycaprylylsilane) (85 nm). ZnOPs were suspended in filtered dimethyl sulfoxide (micro/poly and nano) or methanol (nano/coated) to prepare 20 mg/mL stock solutions, which were then sonicated immediately prior to use in a bath sonicator for 15 min and diluted in cell culture media. Transmission electron microscopy (TEM) samples were prepared by dropping ZnOP suspensions onto carbon type A 300 mesh copper grids. The TEM micrographs were taken using a JEOL 2010 TEM operated at 100 kV with a single tilt holder. Particle hydrodynamic diameter measurements were obtained using a Malvern Instruments Zen 3600 Zetasizer system. Cell Cultures. HeLa cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin− streptomycin−glutamine (PSQ). HeLa/TFEB cells (a gift from Marco Sardiello (Baylor College of Medicine))39 were cultured in DMEM supplemented with 10% FBS and 1% PSQ and selected using G418 (1 mg/mL). HeLa/GFP-LC3 cells were created by transfecting HeLa cells with pBABEpuro GFPLC3 (Addgene) using jetPRIME (Polyplus Transfection) according to the manufacturer’s instructions. The transfection media were replaced with fresh media 24 h after transfection. The HeLa/GFP-LC3 cells were cultured in DMEM supplemented with 10% FBS and 1% PSQ and selected using puromycin (0.5 μg/mL). Fibroblasts derived from a patient with late infantile neuronal ceroid lipofuscinosis (LINCL cells) were from Coriell Cell Repositories (GM16486) and were grown in DMEM supplemented with 20% FBS, 1% PSQ, and nonessential amino acids. Cells were
not lead to a reduction of ceroid lipopigment autofluorescence, suggesting that transcriptional upregulation of the lysosome− autophagy system mediated by TFEB in the cells treated with micro/poly-ZnOPs does not result in enhancement of autophagic clearance. These results are consistent with the evidence of blockage of autophagic flux in cells treated with micro/poly-ZnOPs, as detected by monitoring the extent of turnover of autophagosomes (Figure 4A,B) and formation of autolysosomes (Figure 4C).
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DISCUSSION The increasing production and use of ZnOPs in commercial products have raised concerns regarding the potentially deleterious consequences of human exposure to nano- and micron-sized ZnOPs. Cellular uptake of ZnOPs through endocytosis has been amply documented.47,48 In this study, we characterized the autophagic response to three representative ZnOPs commonly used in commercial applications, namely, polydisperse micron-sized ZnOPs (micro/poly) and monodisperse nano-sized ZnOPs uncoated and coated with triethoxycaprylylsilane, a binding and emulsifying agent (nano and nano/coated). The autophagic response to these ZnOPs was characterized by analyzing markers of the lysosome− autophagy system, from transcriptional activation to clearance of a model autophagic substrate. Whereas the specific mechanism of autophagy activation remains to be characterized, we discovered that the autophagic response to ZnOPs is mediated by the transcription factor EB. Particularly, cell exposure to micro/poly-, nano-, and nano/coated ZnOPs was found to cause transcriptional upregulation of the lysosome− autophagy system. Transcriptional upregulation of this pathway was paralleled by enhancement in the formation and turnover of autophagic vesicle in cells treated with either nanoor nano/coated ZnOPs, eventually leading to an increase in 578
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cultured at 37 °C in 5% CO2 and passaged using TrypLE Express. Cell Viability Analyses. The analyses of cell viability were conducted as previously described.49 Briefly, HeLa cells were plated in 12-well plates (8 × 104 cell/well) and cultured in the presence of ZnOPs for 24 h. Cell toxicity was tested using the Dead Cell Apoptosis Kit with Annexin V and propidium iodide (Life Technologies) according to the manufacturer’s instructions and analyzed by flow cytometry using a BD FACSCanto II Flow Cytometer with the 488 nm Argon laser. Nanoparticle Uptake Studies. Cellular uptake of ZnOPs was evaluated as previously described.50,51 Briefly, HeLa cells were plated in 12-well plates (8 × 104 cell/well) and cultured in the presence of ZnOPs for 24 h. The cells were washed with phosphate buffered saline (PBS) and the side scattering parameter was measured using a BD FACSCanto II Flow Cytometer with the 488 nm Argon laser to quantify ZnOP uptake. Intracellular Zinc Ion Measurements. Analyses of intracellular levels of Zn2+ ions were conducted using the cell permeant form of FluoZin-3 AM (Life Technologies) according to the manufacturer’s instructions. HeLa cells were plated in 12-well plates (8 × 104 cell/well) and cultured in the presence of ZnOPs for 24 h. The ZnOP-containing media were replaced with serum-free media containing 500 nM FluoZin-3 AM and cells were incubated for 30 min at room temperature. The cells were washed once with serum-free media to remove any dye associated with the cell membrane and then incubated in serum-free media for another 30 min to allow complete deesterification of the dye molecules. The cells were then collected in PBS and analyzed by flow cytometry using a BD FACSCanto II Flow Cytometer with the 488 nm Argon laser. Immunofluorescence Studies. Immunofluorescence studies were conducted as previously described.24 Briefly, cells were seeded on glass coverslips in 12-well plates (2 × 104 cell/well) and incubated in a medium supplemented with ZnOPs for 24 h. The cells were washed with PBS, fixed with 4% paraformaldehyde (15 min), permeabilized with 0.1% Triton X-100 (10 min), and blocked in 8% bovine serum albumin−PBS (30 min). All the cells were visually inspected under brightfield prior to image acquisition. TFEB subcellular localization studies were conducted as previously described.24 HeLa/TFEB cells were incubated with an anti-FLAG antibody (1:1000, F7425, Sigma) for 1 h, washed, incubated with a fluorescent secondary antibody (1:500, 611-142-002, Rockland) for 1 h, washed, and then incubated with Hoechst nuclear stain for 10 min. Images were obtained using a Nikon A-1 confocal microscope and the Nikon NIS Elements C imaging software. Imaging analyses and colocalization studies were done using the JACoP plugin in ImageJ.52 The Mander’s overlap coefficient of each image was calculated to quantify the fraction of TFEB that localizes in the nucleus. The number of cells with Mander’s overlap coefficient indicting nuclear localization of TFEB greater untreated cells plus one standard deviation was determined to obtain the percentage of cells presenting TFEB nuclear localization. LC3−LAMP colocalization studies were conducted as previously described.19 The cells were incubated with antiLAMP-2 antibody (1:2000, 354301, BioLegend) and anti-LC3 antibody (1:1000, NB100-2220, Novus Biologics) for 1 h, washed, and incubated with appropriate fluorescent secondary antibodies (1:500, 611-143-002, Rockland and 1:500, 610-142002, Rockland) for 1 h. Images were obtained using a Nikon A-
1 confocal microscope and the Nikon NIS Elements C imaging software. Imaging analyses and colocalization studies were conducted using the JACoP plugin in ImageJ.52 The Mander’s overlap coefficient of each image was calculated to quantify colocalization of LC3 and LAMP. Western Blot Analyses. Cells were plated in 10 cm dishes (8 × 105 cell/dish) and cultured in the presence of ZnOPs for 24 h, collected, and lysed with Complete Lysis-M buffer (Roche) containing a protease inhibitor cocktail. Total protein concentrations were determined using the Bradford assay and aliquots of 40 μg from each sample were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were incubated with primary antibodies (LC3, 1:1000, NB-100-2220, Novus Biologicals; GAPDH, 1:10 000, NB-300-320, Novus Biologicals) and appropriate secondary antibody (1:12 000, sc-2004, Santa Cruz Biotechnology; 1:12 000, sc-2020, Santa Cruz Biotechnology). Imaging was conducted using an LAS 4000 Imager and band densities quantified using ImageJ. GFP-LC3 Measurements. HeLa/GFP-LC3 cells were plated into 12-well plates (8 × 104 cell/well) and cultured in the presence of ZnOPs for 24 h. Cells were collected in PBS, pelleted, and washed with either PBS or saponin (0.05% in PBS).44 Cells were pelleted and resuspended in PBS for the analysis by flow cytometry using a BD FACSCanto II Flow Cytometer with the 488 nm Argon laser. Ceroid Lipopigment Accumulation Studies. The accumulation of ceroid lipopigment in LINCL cells was conducted as previously described.18 Briefly, cells were plated on coverslips in 12-well plates (2 × 104 cell/well) and incubated with ZnOPs for 24 h, washed three times with PBS, and fixed with 10% formaldehyde (10 min). Cell nuclei were stained using a Hoechst nuclear stain (10 min). Coverslips were then mounted onto glass slides, imaged using a Nikon A1 confocal microscope, and analyzed using the Nikon NIS Elements C imaging software. Image postprocessing was conducted using ImageJ. The average ceroid lipopigment signal was evaluated by determining the corrected total cell fluorescence (CTCF = integrated density − (area of selected cell × mean fluorescence of background reading)) for individual cells in each image. Statistical Analyses. The statistical significance was calculated using a two-tailed Student’s t-test unless stated otherwise and determined as p < 0.05. All the data are presented as mean ± s.d.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01497. TFEB activation in HeLa/TFEB cells exposed to ZnOPs (Figure S1); LC3 levels in HeLa cells exposed to ZnOPs (Figure S2); GFP fluorescence of HeLa/GFP-LC3 cells exposed to ZnOPs (Figure S3); LC3−LAMP colocalization in HeLa cells exposed to ZnOPs (Figure S4) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Lauren Popp: 0000-0002-6383-0357 579
DOI: 10.1021/acsomega.8b01497 ACS Omega 2019, 4, 573−581
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Laura Segatori: 0000-0001-5749-5479 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was funded by the National Science Foundation (CBET-1805317 and CBET-1615562) and the Welch Foundation (C-1824). We greatly appreciate Santiago Martinez Legaspi (Chemical and Biomolecular Engineering, Rice University) for providing TEM images and expertise on dynamic light scattering techniques.
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