Graphene Oxide Attenuates the Cytotoxicity and Mutagenicity of PCB

Feb 13, 2016 - Graphene toxicity as a double-edged sword of risks and exploitable opportunities: a critical analysis of the most recent trends and ...
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Graphene Oxide Attenuates the Cytotoxicity and Mutagenicity of PCB 52 via Activation of Genuine Autophagy Yun Liu,†,∥ Xinan Wang,†,∥ Juan Wang,† Yaguang Nie,† Hua Du,† Hui Dai,† Jingjing Wang,† Mudi Wang,† Shaopeng Chen,† Tom K. Hei,‡ Zhaoxiang Deng,§ Lijun Wu,† and An Xu*,† †

Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences and Anhui Province, Hefei, Anhui 230031, P. R. China ‡ Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, United States § Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China S Supporting Information *

ABSTRACT: Graphene oxide (GO), owing to its large surface area and abundance of oxygen-containing functional groups, is emerging as a potential adsorbent for polychlorinated biphenyls (PCBs), which accumulate over time and are harmful to both natural ecosystems and human health. However, the effect of GO against PCB-induced toxicity remains largely unexplored. The present study aimed to investigate the protective effect of GO against PCB 52 induced cytotoxic and genotoxic response in mammalian cells at various exposure conditions and clarify the protective role of autophagy. Pretreatment with GO dramatically decreased PCB 52 induced cytotoxicity and CD59 gene mutation in human− hamster hybrid (AL) cells. The toxic response in cells either pretreated with PCB 52 and then treated with GO or concurrently treated with GO and PCB 52 did not differ significantly from the toxic response in the cells treated with PCB 52 alone. Using autophagy inhibitors (3-methyladenine and wortmannin) and inducers (trehalose and rapamycin), we found that genuine autophagy induced by GO was involved in decreasing PCB 52 induced toxicity. These findings suggested that GO has an antagonistic effect against the toxicity of PCB 52 mainly by triggering a genuine autophagic process, which might provide new insights into the potential application of GO in PCB disposal and environmental and health risk assessment.

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INTRODUCTION Graphene oxide (GO), as a two-dimensional (2D) carbonbased nanomaterial, has promising applications in various fields owing to its unique structure and its mechanical, optical, and electronic properties.1 In general, GO comprises aromatic regions with unoxidized benzene rings and regions containing aliphatic six-membered rings.2 The epoxy (−O−) and hydroxyl (−OH) functionalities lie above and below these carbon layers, while carboxyl groups (−COOH), which have a strong acidic character, decorate the sheet edges. Owing to its high specific surface area and abundance of oxygen-containing groups, GO is expected to serve as a potential superior adsorbents in environmental remediation and water treatment.3 Recent studies have shown that GO is an excellent adsorption material for organic compounds and heavy metals,4,5 which afford a great opportunity for GO and pollutants to interact and exhibit combined toxicity in the natural environment. However, related toxicity studies focusing on GO and environmental pollutants are largely unknown. Polychlorinated biphenyls (PCBs) are a class of chlorinated aromatic hydrocarbon compounds, which are harmful to both © XXXX American Chemical Society

natural ecosystems and human beings at elevated concentrations.6 It has been reported that several wild aquatic species including the Chinese mystery snails, prawns, fish, and water snakes collected from a reservoir surrounded by several electronic waste-recycling regions accumulate high contents of PCB congeners. Compared to the PCB levels in reference samples (82.8 ng/g), the PCB levels in these wild aquatic species reach a maximum concentration of 25 958 ng/g.7 The contents of PCBs in the muscles of water bird species collected from an electronic waste-recycling region in the Pearl River Delta in south China were found to be as high as 1 400 000 ng/ g in lipid.8 Many PCB congeners have been detected in human milk, blood, and other tissues.9 As a kind of Group IIA carcinogen (determined by the International Agency for Research on Cancer (IARC)),10 PCBs have been shown to induce reproductive toxicity, immunotoxicity, neurotoxicity, Received: August 12, 2015 Revised: January 31, 2016 Accepted: February 13, 2016

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Environmental Science & Technology and endocrine disorders.11,12 The genotoxic potential of individual PCB congeners and their mixtures has been investigated in various cell lines. There was evidence that PCB quinine was cytotoxic to HepG2 cells and could cause oxidative DNA double-strand breaks via reactive oxygen species (ROS) induction.13 Aroclor 1254, a commercial mixture of PCBs, was reported to induce genotoxicity through oxidative DNA damage and the inhibition of DNA repair-related gene expression.14 For the efficient removal of PCBs from water and sediments, increasing efforts have been made to understand and evaluate the potential use of GO as a sorbent for the remediation of PCBs. Different types of GO and GO complexes, such as GO, Fe3O4 grafted GO, and zerovalent iron/iron oxide−oxyhydroxide/graphene complexes, have been employed to enrich PCBs in wastewater.15−17 Therefore, it is of great environmental and health importance to explore the underlying toxicity mechanisms of GO and PCBs in biological systems to ensure environmental safety and human health. Autophagy, an evolutionarily conserved and highly regulated intracellular process for degrading long-lived proteins, defective organelles, and invasive xenobiotics, exhibits important physiological and pathological roles in mammalian cells.18,19 Constitutive basal or moderate level of autophagy acts as a vital cellular mechanism to defend against various environmental stresses by degrading impaired components and xenobiotics, and prolonged stress and overstimulated autophagic effects are detrimental to cells, for the extreme environmental conditions may disrupt the normal autophagic flux and balance by impairing the lysosomal degradation system.20 Graphene oxide,21 graphene quantum dots,22 carbon nanotubes,23 and fulluence C6024 have all been shown to induce autophagic effects in different cell lines. However, autophagy induced by carbon nanomaterials has been suggested to act as a “doubleedged sword” and has different roles in modulating the cellular response to extrinsic stressors, which depends on the integrity of autophagic flux and the molecular mechanisms of autophagy.25 The autophagy enhanced by C60-pentoxifylline dyad nanoparticles could prevent the cytotoxic effects of βamyloid peptide through autophagic degradation.26 On the contrary, autophagy induced by fullerene C60 sensitized the chemotherapeutic killing of cancer cells in a ROS-dependent and photoenhanced manner.24 Therefore, it is significant to elucidate the role of autophagy in the toxicity response to PCB in combination with GO. Among all of the PCB congeners, 2,2,5,5-tetrachlorobiphenyl (PCB 52, an ortho-substituted, noncoplanar congener) is one of the most ubiquitously existing PCB congeners in the natural environment and has been defined as one of the World Health Organization (WHO) indicator congeners.27 Therefore, PCB 52, which has obvious cytotoxicity and genotoxicity, was used in the present study. Because ovarian tissue is one of the vital targets of PCBs,28 human−hamster hybrid (AL) cells, formed by fusion of human fibroblasts and the gly2A mutant of Chinese hamster ovary (CHO) cells,29 were employed in this study. To clarify the effect of GO against PCB 52-induced toxicity in mammalian cells, we exposed AL cells to GO and PCB in different ways. GO pretreatment could efficiently decrease the cytotoxicity and CD59 gene mutation of PCB 52. However, the toxic effects in cells either pretreated with PCB 52 and then with GO or concurrently treated with PCB 52 and GO did not differ from the toxic effects in cells treated with PCB 52 only. In addition, GO was proved to induce genuine autophagy in AL cells, which was further certified to play a vital

protective role in attenuating the toxicity of PCB 52. Our results clearly demonstrated that GO performed an antagonistic action in modulating the toxicity of PCB 52, which might be regulated by genuine autophagy.

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MATERIALS AND METHODS Chemical and Biological Reagents. 2,2′,5,5′-Tetrachlorobiphenyl (C-025N) was purchased from Accustandard. Alamar Blue Cell Viability Reagent (DAL1025) and Hoechst 33342 (H3570) were from Life Technologies. Dichlorofluorescein-diacetate (DCF-DA) (35845), FITC conjugated antirabbit antibody (F0382), and rapamycin (R117) were purchased from Sigma-Aldrich. MitoSOX Red Mitochondrial Superoxide Indicator (MitoSox) (40778ES50) was from Shanghai Yeasen Company. Annexin V-FITC/PI apoptotic detection kits (CA1020) were from Beijing Solarbio Company. LC3 antibody (NB100−2220) was from Novus. β-Actin antibody (YT0099) was from Immunoway. HRP-conjugated antirabbit (W4011) and HRP-conjugated antimouse (W4021) antibodies were purchased from Promega. Bafilomycin A1 (196000), 3-methyladenine (189490), wortmannin (681675) and rabbit serum complement (234400) were all purchased from Calbiochem. Trehalose (TB0966) was from Shanghai Sango Biotech. mTOR antibody (2972), phosphor-mTOR antibody (2971), p70 S6 kinase antibody (9202), and phosphor-p70 S6 kinase antibody (9205) were all purchased from Cell Signaling Technology. LysoTracker Red (L7528) was from Invitrogen. Atg5 siRNA (sc-41446), control siRNA (sc37007), and Atg5 antibody (sc-133158) were from Santa Cruz Biotechnology. PCB 52 was dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in culture medium was never greater than 0.1% (v/v). Nanomaterial Preparation and Characterization. Graphene oxide dispersion in water (SKU-HCGO-W-60) with a concentration of 5 mg/mL, a flake size of 0.5−5 μm, and a thickness of 1 atomic layer (at least 60%) was purchased from Graphene Supermarket. Because a wide range of diameter distribution may increase the nondeterminacy of the toxicology testing, GO was exposed to ultrasound (100W, 30 min) before characterization or incubation with cells. GO was characterized by atomic force microscopic (AFM) assessment using a DI MultiMode V scanning probe microscope (MultiMode V, Bruker) and transmission electron microscopy (H-7650, Hitachi). Dynamic light scattering (DLS) and ζ potential obtained by a laser-particle-size analyzer (Zetasized Nano-ZS, Malvern Instruments), were used for determining the hydrodynamic particle size and surface charge of the sonicated nanomaterials. Raman spectrometry (XploRA, HORIBA JOBIN YVON) with a 514 nm laser was employed to analyze the structure of GO. GO nanosheets were deposited onto a silicon wafer, followed by air-drying and then sending for X-ray photo electron spectroscopy (XPS) detection. Additionally, the Fourier transform infrared (FTIR) spectrum was employed for identifying the presence of functional groups on the surface of GO nanosheets. The spectrum was taken from 4000 to 1000 cm−1 on FTIR (Nicolet FT-SPR 100, Thermo Fisher). Cell Culture. The human−hamster hybrid AL cells, which show a standard set of Chinese hamster ovary (CHO)-K1 chromosomes and a single copy of the human chromosome 11, were employed in these experiments. Cells were cultured continuously at 37 °C and 5% CO2 in Ham’s F-12 medium (Hyclone) supplemented with 8% fetal bovine serum (FBS) (Hyclone), 2 × 10−4 M glycine, and 25 μg/mL gentamicin.30 B

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Figure 1. Characterization of GO. (A) Raman spectra of GO. (B) Representative AFM topographic images of GO. (C) Histogram of GO size distribution. The histograms were developed by counting 200 nanosheets. (D) TEM images of GO nanosheets. (E) Characterization of the surface composition of GO by XPS.

30 min (For ROS measurement) or 5 μM MitoSox probe for 10 min (For MitoSox measurement) at 37 °C. DCF-DA fluorescence was recorded at 525 nm using an excitation wavelength of 488 nm on a plate reader. MitoSox fluorescence was recorded at 580 nm using an excitation wavelength of 510 nm on a plate reader. Western Blot Analysis for Detecting the Transformation of Autophagic Marker Protein. Briefly, cells treated with GO and PCB 52 for 24 h were harvested and lysed in RIPA lysis buffer (Beyotime, China) on ice. Certain proteins in the cells lysates were separated on a 15% SDS/PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Roche, Swiss). After blocking with TBST (0.1% Tween-20) containing 5% nonfat dry milk for 1 h at room temperature, the PVDF membrane was incubated at 4 °C overnight with primary antibody at appropriate dilutions. The membrane was then washed six times with TBST (10 min each) and incubated with HRP-conjugated secondary antibodies at a dilution of 1:10 000 for 1 h at room temperature. Immunolabeling was visualized using an enhanced chemiluminescence (ECL) (BOSTER, China) kit. β-Actin was used as the internal control. Immunofluorescence for Detecting the Transformation of Autophagic Marker Protein and the Co-localization between Autophagosomes and Lysotracker Red. Cells treated with GO and PCB 52 were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. The cells were then permeabilized with 0.5% Triton-X 100 for another 10 min and blocked with 5% FBS for 1 h at room temperature. Cells were then incubated with LC3 antibody for 4 h followed by incubation with fluorescein isothiocyanate (FITC) conjugated secondary antibody for 2.5 h at room temperature. Nuclei were stained with Hoechst 33342 for 15 min. For the co-localization assay, cells

Cell Viability Assay for Detecting the Combined Cytotoxicity. Cells were seeded at a density of 1.5 × 104 cells per well in a 96-well plate and cultured for 24 h. Cells were then treated with various concentrations of GO or PCB 52 and incubated for 24 h. After treatment, the medium was removed, and the cells were washed thrice with PBS. Alamar blue cell viability reagent was added to the cells according to manufacturer’s instructions and incubated for another 3−4 h.31 The absorbance of each well was read at 570 nm with subtracting the average 600 nm absorbance values of the cell medium alone (background). Cell-Counting Assay for Detecting the Combined Cytotoxicity. Cells were seeded at a density of 7.5 × 104 cells per well in a 24-well plate and cultured for 24 h. Cells were then treated with various concentrations of GO or PCB 52 and incubated for 24 h. After treatment, medium was removed, cells were washed with PBS, and they were then harvested for the counting of the number of live cells. Apoptotic Assay for Detecting the Combined Cytotoxicity. Apoptosis detection was performed with the Annexin V-FITC/PI Apoptosis Detection Kit. Briefly, cells were collected and washed with ice-cold PBS twice and then resuspended in 200 μL of binding buffer. A total of 10 μL of Annexin V stock solution and 10 μL propidium iodide (PI) stock solution were added to the cells and incubated for 30 min at 4 °C. Cells were then immediately analyzed by FACS. Approximately 1 × 104 cells were analyzed in each of the samples. ROS and MitoSox Assay for Detecting the Oxidative Stress in Single and Co-exposure. Cells were cultured in 96-well plates and cultured for 24 h. Cells were then treated with GO and PCB 52 and incubated for 1, 3, and 24 h. After treatment, medium was removed, and cells were washed with PBS. Cells were then incubated with 10 μM DCF-DA probe for C

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Figure 2. Combined cytotoxicity of GO and PCB 52. (A) Schematic model illustrating the manners of coexposure in AL cells. (B) Cells were pretreated with GO (2 and 20 μg/mL) for 4 h, followed by exposure to PCB 52 (75 and 100 μM) for another 24 h. (C) Cells were pretreated with PCB 52 (75 and 100 μM) for 4 h, followed by exposure to GO (2 and 20 μg/mL) for another 24 h. (D) Cells were exposed to GO (2 and 20 μg/ mL) and PCB 52 (75 and 100 μM) simultaneously for 24 h. (The results are the mean values of triplicates from a representative of three experiments. Mean ± S.D., n = 3).

plated in six 60 mm dishes in 2 mL F12 medium as described33 and incubated for 2−3 h to allow cell attachment, after which 0.2% antiserum and 1.5% (v/v) freshly thawed complement were added to each dish. Cells were cultured for another 8 days and then fixed and stained, and the number of CD59 mutant colonies was scored. Negative control groups included identical sets of dishes containing antiserum or complement alone or neither. The mutant fraction of each group was calculated as the number of surviving colonies divided by the total number of cells plated after correction for any nonspecific cytotoxicity due to complement alone.34 Statistics. Data from three independent experiments were subjected to statistical analysis. All data were expressed as mean ± SD and analyzed by one-way analysis of variance (ANOVA). The results with p values of 85%) GO flakes ranged from 0 to 300 nm after sonication (Figure 1C). The morphology of GO sheets was also confirmed by TEM characterization (Figure 1D). Although DLS cannot support accurate results for nonspherical particles such as GO nanosheets in this study, it can provide valuable information D

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Figure 3. Combined apoptotic level of GO and PCB 52. Representative images of apoptosis after 24 h of treatment with GO, PCB 52, and coexposure, assessed by FACS analysis with Annexin V-FITC and PI staining. GO-PCB: Cells were pretreated with 10 μg/mL GO for 4 h, followed by the addition of 100 μM of PCB 52 for another 24 h. PCB-GO: Cells were pretreated with 100 μM of PCB 52 for 4 h, followed by adding with 10 μg/mL GO for another 24 h. GO+PCB: Cells were exposed to 10 μg/mL GO and 100 μM of PCB 52 for 24 h.

exposure processes, as shown in Figure 2A. In process one, cells were treated with indicated concentrations of GO (2 μg/mL, 20 μg/mL) for 4 h and then treated with PCB 52 (75 μM, 100 μM) for another 24 h. In process two, cells were treated with PCB 52 for 4 h, and then various concentrations of GO were added into medium for further 24 h. In process three, cells were treated with indicated concentrations of GO and PCB 52 simultaneously for 24 h. As shown in Figure 2B, the viability of cells reduced by 32.3% ± 7.97% and 66.2% ± 4.13% when the concentrations of PCB 52 were 75 and 100 μM, respectively. However, the viability of PCB 52 treated cells increased dosedependently upon pretreatment with GO. The viability of cells exposed to 75 μM PCB 52 increased from 67.7% ± 7.97% to 77.8% ± 7.38% and to 86.3% ± 5.67%, when the pretreated concentrations of GO was at 2 and 20 μg/mL, respectively. In contrast, GO had a minimal effect on the toxicity of PCB 52 in the other two types of exposure manners, including PCB 52 pretreatment and concurrent treatment with PCB 52 and GO (Figure 2C,D). Similar trends of combined cytotoxicity could also be displayed by cell morphology (Figure S3) and cellcounting assay (Figure S4). Apoptosis is also a typical endpoint for manifesting the cytotoxicity of toxicants. As shown in Figure 3, pretreatment with GO could obviously decrease the apoptotic level (early apoptotic cells shown in the lower-right quadrant and late apoptotic and necrotic cells shown in the upper-right quadrant) induced by 100 μM PCB 52, from 68.35% (PCB 52 treatment only) to 48.17% (coexposure). The other two manners of coexposure did not show this phenomenon. Taken together, these results indicated that pretreatment with GO could decrease the cytotoxicity caused by PCB 52. Pretreatment with GO Decreased the CD59 Gene Mutation Frequency Induced by PCB 52. PCB 52 had

about aggregation extents of the particles. The average hydrodynamic particle size of GO in water was 253.10 ± 10.78 nm. However, after GO was dispersed in F12 medium supplemented with 8% FBS for 24 h, the average size increased to 1342.00 ± 153.70 nm (Figure S1A−C), indicating that GO was prone to aggregation in culture medium. As shown in Figure S1A, GO was negatively charged in water (−46.00 ± 1.08 mV), while the ζ potential increased to −10.55 ± 0.54 mV in culture medium, suggesting that protein binding in the medium may affect the surface charge of GO nanosheets. X-ray photoelectron spectroscopy was performed for detecting the oxygen level and surface groups of the GO samples, with characteristic peaks at 284.7, 286.9, and 288.8 eV, representing C−C or CC, CO, and OC−OH (Figure 1E). Additionally, Fourier transform infrared spectroscopy was employed for further characterizing the GO nanosheets. The peak at 3290 cm−1 was attributed to residual O−H stretching vibration. The CO vibration band was located at 1635 cm−1, and the band corresponding to C−H was identified at 2850 and 2919 cm−1 (Figure S1D). Because GO aqueous solution was used for the FTIR detection through attenuated total reflection (ATR) method, there may be some differences in the peaks comparing with others’ spectrum. Pretreatment with GO Attenuated the Cytotoxicity of PCB 52. As shown in Figure S2A, GO essentially had no effect on the ability of colony formation of AL cells at the concentration range from 0 to 100 μg/mL. In contrast, treatment with PCB 52 decreased the viability of AL cells in a dose-dependent manner (Figure S2B). Thus, the nontoxic concentrations of GO were used in the present study to eliminate the toxic effect on cells exposed to PCB 52. To verify the influence of exposure order on the toxicity of PCB 52 in combination with GO, we employed three different E

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Environmental Science & Technology been reported to cause genotoxicity via increasing DNA breakage.35 To further investigate the influence of GO on the toxicity of PCB 52, we employed CD59 gene mutation in AL cells as a representative end point for genotoxicity determination. As shown in Figure 4, the average mutation background

Figure 5. Induction of autophagy by GO in AL cells. (A) Dosedependent manner of LC3 conversion induced by GO for 24 h. (B) Time-dependent manner of LC3 conversions induced by 10 μg/mL GO. (C) Cells untreated (control) or treated with 10 μg/mL GO for 24 h were analyzed for endogenous LC3 dots formation via immunofluorescence. HO (Hoechst 33342) represented the nuclei. (D) Co-localization between endogenous LC3 aggregates and LysoTracker (75 nM) in AL cells untreated or treated with 200 nM Rap for 24 h and 10 μg/mL GO for 24 h. HO represented the nuclei. (In A and B, β-actin served as loading control; in B, the asterisk indicates nonspecific bands.)

Figure 4. Combined mutagenicity of GO and PCB 52. GO-PCB: Cells were pretreated with 10 μg/mL GO for 4 h, followed by addition of 100 μM of PCB 52 for another 72 h. PCB-GO: Cells were pretreated with 100 μM of PCB 52 for 4 h, followed by adding with 10 μg/mL GO for another 72 h. GO+PCB: Cells were exposed to 10 μg/mL GO and 100 μM of PCB 52 for 72 h. (The results are the mean values of triplicates from a representative of three experiments. Mean ± S.D., n = 3, compared with the control of each group).

of AL cells at CD59 locus shown in this experiment was 74.6 ± 8.87 mutants per 105 survivors. The mutation yield at CD59 gene locus elevated to 99.9 ± 5.54 mutants per 105 survivors in cells treated with 75 μM PCB 52 for 3 days, while GO pretreatment could also significantly decrease the mutation yield to 85.5 ± 8.61 mutants per 105 survivors. In contrast, pretreatment with PCB 52 or simultaneously coexposure showed the same levels of mutation yield as compared with PCB 52 treatment alone. This finding indicated that pretreatment with GO may induce a moderate protective response to the cytotoxicity and genotoxicity of PCB 52 in AL cells. GO Treatment Alone Elicited Genuine Autophagy in AL Cells. The determination of MAP1LC3 levels (LC3) is one of the main parameters with which to monitor autophagy induction, where the detection of LC3 conversion into its lipidated form (LC3-II) by Western blotting or the presence of LC3 positive vesicles are mostly used to determine LC3 dots formation, which is a strong hallmark for autophagy activation. As shown in Figure 5A,B, GO treatment significantly increased the level of LC3-II in a dose-dependent and time-dependent manner, even at a low dose (1 μg/mL) or an early time-point (4 h). Compared with the dispersed green fluorescence in negative control cells, the endogenous LC3 protein revealed as multiple green punctuated dots in GO-treated AL cells because of accumulation on the autophagosomal membrane (Figure 5C). As shown in Figure 5D, The control cells showed a dispersed green fluorescence and could not overlap with the staining patterns of LysoTracker Red (a selective dye for labeling lysosome). However, the endogenous LC3 dots in cells treated with either rapamycin (a commonly used autophagy inducer) or 10 μg/mL GO for 24 h could both co-localize well with lysosomes. To shed light on more details of GO-induced autophagy, we first tracked the interaction between GO nanosheets and AL cells via labeling GO with FITC-BSA. As shown in Figure 6A, compared with control group, FITC-BSA labeled GO could be detected on the plasma membrane of AL cells after incubation with FITC-BSA-labeled GO for 24 h, emerging as obvious

bright big green puncta. Meanwhile, a few dispersed small green dots could also be seen in the cytosol, suggesting that a small amount of GO nanosheets were taken up into cells. Thereafter, the activity of the mechanistic target of rapamycin (mTOR) signal pathway was also detected after exposing to GO. Compared with rapamycin, which induce autophagy via inhibiting the activity of mTOR, GO treatment could not influence the phosphorylation level of mTOR and its substrate p70 S6 Kinase, indicating that GO-induced autophagy in a mTOR-independent manner (Figure 6B). Autophagy is a dynamic flux, and the downstream degradation is essential for maintaining its protective role in intracellular homeostasis. Bafilomycin A1 (BFA), which inhibits the vacuolar H+-ATPase and prevents the fusion between autophagosome and lysosomes, was first employed to detect the integrity of the autophagic flux.36 As shown in Figure 6C, the level of LC3-II increased by 10 μg/mL GO + BFA treatment was significantly higher than either GO or BFA treatment alone, suggesting that GO at 10 μg/mL could enhance a new formation of autophagosomes. Additionally, the degradation of SQSTM1/p62 (abbreviated as p62) was also monitored as another concrete proof of autophagic flux. Western blotting assay showed that the total expression of p62 decreased sharply after incubation with 10 μg/mL GO for 4 h, illustrating the powerful degradative capacity of autophagy (Figure 6D). Taken together, these data strongly suggested that 10 μg/mL GO elicited genuine autophagy with a functional degradative pathway downstream in AL cells. Genuine Autophagy Induced by GO Involvement in Attenuating the Toxicity of PCB 52. Oxidative stress is one of the most significant mechanisms underlying the toxicity for both GO and PCBs. Meanwhile, oxidative stress is also a vital factor for eliciting autophagy. Therefore, the overall cellular and mitochondrial oxidative stresses were measured by ROS and MitoSox assessing. As shown in Figure S5, after treating AL cells for 1, 3, and 24 h, there was no significant difference between F

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Figure 6. The mechanism of autophagy elicited by GO in AL cells. (A) Representative images of GO localization in AL cells through confocal laser scanning microscopy. FITC-BSA labeled GO is visualized in green. The cell membrane is stained in red with rhodamine−phalloidin; cell nuclei are stained in blue with Hochest 33342. (B) Cells treated with 10 μg/mL GO for 24 h were analyzed for mTOR activity by Western blotting for levels of total (T) and phosphor (P)-mTOR and p70 S6 Kinase. Rapamycin (Rap, 200 nM) was served as a positive control. (C) Cells were treated with 10 μg/mL GO for 24 h in the presence or absence of 400 nM bafilomycin A1 (BFA, added 4 h before cell harvest) and then subjected to Western blotting using anti-LC3 antibodies. (D) Cells were treated with 10 μg/mL GO for 4, 8, and 24 h. Endogenous SQSTM1/p62 levels were detected by Western blotting using anti-SQSTM1/p62 antibodies. (In panels C and D, β-actin was served as loading control. LC3-II levels were quantified by densitometric analysis relative to β-actin. Mean ± S.D., n = 3, compared with the control of each group).

Figure 7. Autophagy induced by GO was involved in attenuating the toxicity of PCB 52. Cell viability assay (A) and CD59 gene mutation assay (B) of AL cells untreated or treated with 2.5 mM of 3-MA; 500 nM of wortmannin (WM); 10 μg/mL GO; and 100 μM of PCB 52, GO−PCB, or 3-MA (WM) + Mixture (GO−PCB co-treated with 3-MA or WM). Cell viability assay (C) and CD59 gene mutation assay (D) of AL cells untreated or treated with 100 mM of trehalose (Tre), 200 nM of rapamycin (Rap), 10 μg/mL GO, 100 μM PCB 52, and GO−PCB, Tre−PCB, and Rap−PCB (100 μM PCB 52 pretreated with GO, trehalose, or rapamycin). (The results are the mean values of triplicates from a representative of three experiments. Mean ± S.D., n = 3).

GO treatment and combined treatment in ROS generation in cellular and mitochondrial level, which were both higher than PCB 52 treatment only. These results indicated that global ROS generation may not be the main reason for interpreting the combined toxicity.

As shown in Figure S6, pretreatment with GO could induce similar expression of LC3-II protein in cells exposed to PCB 52, as compared to GO treatment alone. PCB 52 itself could only slightly increase the expression of LC3-II. In contrast, the expression of LC3-II in cells either with pretreatment with PCB G

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up to 140 Mbp of DNA can be detected. Therefore, AL cells show sensitivity in detecting mutagens that induce mostly large, multilocus deletions such as particulate matters, nanomaterials, POPs, and certain heavy metals.39,40 Meanwhile, a 50-fold increase in mutations occurred at the CD59 locus in comparison to that at the HPRT locus in crocidolite-treated AL cells.32 With the help of this sensitive model, we demonstrated that pretreatment with GO could significantly attenuate the cytotoxicity and CD59 gene mutation caused by PCB 52 (Figures 2B, 3, and 4). Because the present study focused on the influence of GO on the toxicity of PCB 52, the concentrations of GO chosen herein had nearly no toxic effects on the cells to minimize the impact of GO in the combined toxicity; however, a relatively high concentration of PCB 52 (75 μM, 100 μM) was used to better clarify the antagonistic interaction between the complex and mammalian cells. The influence of GO on the toxicity of PCB 52 may vary depending on the exposure processes in the environment. The differential toxicity observed upon changing the order of treatment could do a great help for interpreting the mechanism of the combined effect. It was reported that pretreatment with silver nanoparticles followed by UVA irradiation significantly enhanced the antibacterial effect, while other exposure methods did not show a significant phenomenon, illustrating that the surface oxidation of silver nanoparticles induced by UVA irradiation mainly contributed to the improved antibacterial effect.41 Pretreatment with C60(Nd) for 24 h, which is characterized by a neodymium (Nd) atom wrapped in the C60 spherical cage, could obviously enhance the cytotoxicity of chemotherapeutic agents and reduce drug resistance through the induction of pro-death autophagy.42 Thus, it is of great interest to determine the toxicity interactions between GO and PCB 52 and the underlying mechanisms. Here, our data provided direct evidence that pretreatment with GO could significantly decrease the toxicity of PCB 52, while there was no significant difference in cytotoxicity and mutagenicity when the cells were exposed to PCB 52 via the other two ways (pretreatment with PCB 52 or adding simultaneously with GO and PCB 52) (Figures 2−4). Therefore, we hypothesized that a protective mechanism may be stimulated by GO pretreatment. Autophagy is a cyclic and dynamic process, with LC3 dot accumulation owing to either autophagy induction or the blockade of autophagic flux. Therefore, it is of great importance to determine the mechanism of GO-elicited autophagy involving the integrity of the autophagic flux. GO was suggested to induce autophagosome accumulation and lysosome impairment in cultured primary murine peritoneal macrophages.43 In CT26 colon cancer cells, GO was proved to divert the autophagic flux, enhance nuclear import, elevate necrosis, and improve antitumor effects, thus acting as a chemosensitizer.44 In our current study, the adhesion and uptake of GO by AL cells may first elevate the production of ROS. The uptake of nanosheets and the oxidative stress were both responsible for the followed induction of autophagic effect (Figure 6A, S5). By inhibiting the maturation of autolysosomes through BFA, detecting the ability of degrading universal autophagic cargo, and comparing the colocalization between autophagosome and lysosome, GO was confirmed to induce authentic autophagy with valid degradative capacity (Figure 6C−D). The discrepancy between these two results on autophagic flux may depend on the type of cell lines, the incubation time, the concentration of nanomaterials, and possible slice differences in physiochemical properties of GO. It was noteworthy that GO treatment led

or concurrently treated with PCB 52 and GO was significant lower than that of GO treatment alone. With the help of 3-methyladenine (3-MA) and wortmannin, two well-known autophagy inhibitors, we further determined the role of autophagy in PCB 52-induced toxicity in cells pretreated with GO. AL cells with or without pretreatment with autophagy inhibitors for 3 h were treated with GO for 4 h and further treated with PCB 52 for another 24 h. As shown in Figure S7A,B, both inhibitors decreased the LC3-II level elicited by GO treatment alone. GO pretreatment evidently restored the cell viability of PCB 52 to 64.7% ± 4.78%. In the presence of either 3-MA or wortmannin, the cell viability of AL cells pretreated with GO and then with PCB 52 was decreased from 64.7% ± 4.78% to 42.9% ± 4.34% and 37.9% ± 3.49%, respectively. 3-MA or wortmannin alone at the same concentration had no effect on AL cell viability (Figure 7A). Similarly, the presence of 3-MA and wortmannin significantly increased mutation yields caused by PCB 52 in cells pretreated with GO, respectively, as compared to those in cells pretreated with GO and followed by PCB 52 exposure (Figure 7B). In addition, using ATG5 siRNA (an essential gene in autophagy induction), which could effectively knock down the expression level of ATG5 protein and the autophagic process, the protective role of GO in decreasing the cytotoxicity of PCB 52 could also be diminished to some extent (Figure S8). Furthermore, we also employed autophagy inducers to confirm the relationship between autophagy and the toxicity of PCB 52 regulated by GO. Trehalose and rapamycin are two widely used autophagy inducers that could increase the level of LC3-II significantly (Figure S7C−D). AL cells were treated with autophagy inducers for 3 h and then treated with PCB 52 for 24 h. Consistent with the function of GO, pretreatment with trehalose and rapamycin could attenuate the cytotoxicity of PCB 52 by 44.12% and 39.8%, respectively (Figure 7C). Similarly, autophagy inducer also decreased the mutation frequencies at CD59 gene locus by 26.01% (trehalose) and 18.61% (rapamycin) (Figure 7D). These data provided direct evidence that autophagy induced by GO pretreatment was involved in down-regulating the cytotoxicity and genotoxicity caused by PCB 52.

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DISCUSSION GO had been reported to adsorb and enrich various organic and inorganic pollutants via a critical physiochemical process at the GO−water interface owing to its large surface area and surface-active properties. π−π interaction is suggested to be one of the most significant mechanisms contributing to GO interaction with aromatic compounds, macromolecules, and graphitic particles. The behavior of adsorption on GO can not only affect the mobility and fate of pollutants but also alter the surface characteristics of GO itself through coating and modification.37 However, the influence of GO on the toxicity of pollutants is still largely unknown. Recently, GO had been reported to amplify the phytotoxicity of As[V] in wheat through co-transporting GO-loading As and transforming As[V] to high-toxicity As[III].38 In the present study, human−hamster hybrid AL cells, derived from ovarian tissue (a vital target organ of PCBs), were employed for investigating the influence of GO on the toxicity of PCB 52. The AL cell line is characterized by a complete set of CHO-K1 chromosomes and a single copy of human chromosome 11. Because only a small part of region 11p15.5 is required for the viability of AL cells, mutations in the human chromosome 11 ranging in size H

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to a sharp decrease of p62 after 4 h, while the level of p62 increased as time went by. Sahani et al. explained that the expression level of p62 did not always inversely correlate with autophagic activity, while p62 protein levels could be restored to basal levels during prolonged starvation (a common and basal inducer of autophagy) in certain cell lines. They further demonstrated that the level of total p62 is determined by at least three aspects: autophagic degradation, transcriptional upregulation, and the availability of lysosomal-derived amino acids.45 Therefore, our results displayed a natural time changing trend of p62 under a functional autophagic degradation pathway (Figure 6D). Through degradation of damaged organelles, xenobiotics, and long-lived proteins, autophagy plays an essential protective role in defending the cell from exogenous material invasion and conserving cell function. In most cases, complete autophagic flux possesses the ability to maintain intracellular homeostasis for cell survival, while disturbed autophagic flux displays a prodeath role because of disruption in the balance of cellular circulation. In the present study, autophagy induced by GO was moderate and complete, with a functional degradative downstream pathway. Hence, through accelerating the degradation of impaired organelles, proteins, and PCB itself, pretreatment with GO seemed to establish a protective barrier and improve the capacity of resistance to toxicants before PCBs invasion. On the basis of these theories, the conspicuous protection caused by GO pretreatment could be further validated via employing the inhibitors and inducers of autophagy (Figure 7). A similar protective phenomenon of autophagy was also observed by other researchers. Silver nanoparticles were discovered to induce cytoprotective autophagy, and the inhibition of autophagic effect could improve the efficacy of anticancer therapy.46 Nanosized paramontroseite VO2 nanocrystals (PVO2) was also reported to induce cytoprotective autophagy through increasing the expression of heme oxygenase-1 (HO1).47 A synergistic tumor-killing effect of curcumin and temozolomide was not achieved due to activated protective autophagy, and the inhibition of autophagy rendered cells susceptible to curcumin−temozolomide combination therapy by increasing apoptosis.48 In addition, as shown in Figures 7A and S8, the inhibition of autophagy could not restore the toxicity of PCB 52 to the levels induced by PCB 52 treatment only, indicating that the induction of autophagy may not be the only factor responsible for the protective effect. Coating the cell surface and blocking the contact of PCB 52 to the receptors on cellular membrane could also be involved in attenuating the toxicity of PCBs.49,50 In summary, our present data illustrated that GO pretreatment attenuated the cytotoxicity and genotoxicity of PCB 52, which were closely related to the induction of authentic autophagic effect. Because GO has been considered and researched as an attractive adsorbent for enriching and removing PCBs, it is valuable to investigate the interaction between GO and PCB and the influence of GO on PCBs’ toxicity to ecosystem and human beings. Another noteworthy point is that the antagonistic interaction between GO and PCB 52 marks a promising possibility for controlling the hazard caused by PCBs pollution. It is still necessary to further elucidate the underlying mechanisms of the antagonistic interaction both in vitro and in vivo.

Article

ASSOCIATED CONTENT

S Supporting Information *

. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03895. Figures showing dynamic light scattering and ζ potential of GO in water and in culture medium; the FTIR spectrum of GO nanosheets; cytotoxicity of GO and PCB 52; cell morphology; cell-counting assay of GO, PCB 52, and different manners of combined exposure; oxidative stress induced by GO, PCB 52, and coexposure; combined autophagic effect caused by GO, PCB 52, and different manners of combined exposure; autophagy induced by GO could be inhibited by 3-MA and wortmannin; autophagy elicited by rapamycin and trehalose; and combined cytotoxicity under the condition of knocking down the expression of ATG5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 551 65593336; fax: +86 551 65595670; e-mail: [email protected]. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. Qing Huang, Prof. Sijin Liu, and Prof. Wei Chen for their supports in this work. This work was supported in part by grants from Major National Scientific Research Projects, 2014CB932002; Strategic Leading Science & Technology Program (B), XDB14030502; National Natural Science Foundation of China grants U1232144 and 21177133; the Major/Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology, 2014FXCX010; CASHIPS director’s fund, YZJJ201501; and the CAS/SAFEA International Partnership Program for Creative Research Teams.

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REFERENCES

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