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Assessing the in vitro and in vivo toxicity of ultrafine carbon black to mouse liver
Science of the Total Environment 655 (2019) 1334–1341
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Assessing the in vitro and in vivo toxicity of ultrafine carbon black to mouse liver Rui Zhang, Xun Zhang, Sichen Gao, Rutao Liu ⁎ School of Environmental Science and Engineering, Shandong University, China–America CRC for Environment & Health, Shandong Province, 72# Jimo Binhai Road, Qingdao, Shandong 266237, PR China
H I G H L I G H T S
G R A P H I C A L
• UFCB (ultrafine carbon black) aggregated in complete medium and had a wide range of size distribution. • UFCB decreased the viability of hepatocytes. • UFCB increased the intracellular CAT activity through stimulating ROS generation. • UFCB caused a significant inflammation in the mouse liver. • This study revealed the negative effects of UFCB in vitro and in vivo.
UFCB dispersed in complete medium and had a wide range of size distribution. UFCB particles invaded into the liver cell.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 22 September 2018 Received in revised form 16 November 2018 Accepted 19 November 2018 Available online 22 November 2018 Editor: Jay Gan Keywords: Carbon black Hepatocytes Histopathology Oxidative stress Nanotoxicology
A B S T R A C T
The increasing presence of nanomaterials in commercial products makes large quantities of nanoparticles reach the environment intentionally or accidentally. Their ability to be cleared from lung to stomach and then translocate into blood circulation suggests they may cause effects on the organs and cells of the organism. In this study, we characterized the dispersity of UFCB (ultrafine carbon black, FW200) in the complete medium and investigated the toxicity of FW200 to mouse hepatocytes and the liver both in vitro and in vivo. FW200 dispersed homogeneously in the complete medium with an average size at around 100 nm. In vitro, FW200 induced apparent cytotoxicity in the hepatocytes with the level of oxidative stress, apoptosis and the viability of hepatocytes changed by approximately 30%. The intracellular catalase (CAT) activity was stimulated by FW200 to a higher level than the control group. In vivo, the 7-week mice were exposed to FW200 (10 mg/kg body weight) by oral administration for six days. The liver was collected and used for histopathological analysis. In our findings, the 13 nm carbon black nanoparticle was proved to induce acute inflammation and apoptosis in the liver. The particles were also proved to have a damage to central veins and architecture of the hepatocytes. These findings suggest that the carbon black nanoparticle could cause a negative effect at both the cellular and organism level and unearthed the potential effects of carbon black nanoparticles on animals and human. © 2018 Elsevier B.V. All rights reserved.
1. Introduction ⁎ Corresponding author. E-mail address: [email protected] (R. Liu).
https://doi.org/10.1016/j.scitotenv.2018.11.295 0048-9697/© 2018 Elsevier B.V. All rights reserved.
Ultrafine carbon black (UFCB) is produced by thermal decomposition of hydrocarbons and consists of primary particles with the
R. Zhang et al. / Science of the Total Environment 655 (2019) 1334–1341
diameter smaller than 100 nm in all three dimensions. It is a kind of nearly pure amorphous carbon with a high surface area to volume ratio (Zhang et al., 2014). Due to its characters of sluggishness and stability, UFCB has been a high production volume chemical used in the manufacturing of rubber, printing inks, paints, and catalyst (Niranjan and Thakur, 2017; Yu et al., 2011). Scientists and the general public have raised concerns about the use of UFCB which is classified as a possible carcinogen to human by International Agency for Research on Cancer (IARC) (Chen et al., 2014; Kyjovska et al., 2015). The unique physical and chemical properties of nanoparticles endow them with distinct toxic effects compared with their fine counterparts (Hou et al., 2017a). The toxicity of carbon nanoparticles are higher than those of their fine counterparts has been proved by massive works (Hou et al., 2017b; Nel et al., 2006; Oberdörster et al., 2005). Brown et al. and Li et al. also proved that nanoparticles have a higher ability to trigger the generation of reactive oxygen species (ROS) than that of larger particles of the same material (Brown et al., 2001; Li et al., 2003). The CB exposure mostly happens in its production, collection, and handling process. In addition to pulmonary exposure, the gastrointestinal tract is another important route of exposure to nanoparticles, although it has been considerably less investigated than pulmonary exposure. First, carbon black has been used as a food coloring agent and a remedy for a long time (Folkmann et al., 2012). Moreover, SPECT/CT imaging studies demonstrated that 8.3–18.7% and 23.3–50.6% of the deposited dose of particles after short-term inhalation was cleared to the stomach in rats and mice (Kuehl et al., 2012). Therefore, studying the toxicity of nanoparticles to organisms through the gastrointestinal tract exposure is emergent. Nanomaterials are able to translocate from their portal of entry like stomach to systemic circulation and thus to the organs such as spleen, kidney, and liver (Mills et al., 2006; Pietroiusti, 2012; Zhao and Liu, 2012). As such, the possibility of cellular interactions of nanoparticles is extremely high and it is of great importance to know the nanomaterial-mediated cytotoxicity. Our present work aimed to investigate the toxicity of FW200 to the hepatocytes in vitro and to the liver in vivo (Scheme 1). In vitro, we studied the cytotoxicity of FW200 to the mouse hepatocytes. In vivo, we performed the histopathological analysis on the mouse liver tissue after six-day FW200 gavage. To our best knowledge, this is the first time to investigate the toxicity of FW200 at the cellular and organism
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level simultaneously. The in vitro study of FW200 explained the deep mechanism of its in vivo toxicity and revealed the interaction between FW200 and hepatocytes that cannot be reflected by in vivo study. On the other hand, the in vivo study of FW200 further confirmed the results of FW200's in vitro study. We hypothesized that FW200 could cause negative effects at both levels. To date, most investigations on the toxicity of nanomaterials employed a mass-basis approach to calculate the dose of nanomaterials with its concentrations ranging from 0.001 μg mL−1 to 400 μg mL−1 (Gojova et al., 2007; Gratton et al., 2008; Schrand et al., 2008; Schrand et al., 2010; Verma et al., 2008). In this study, we used FW200 in a concentration ranging from 20 μg mL−1 to 50 μg mL−1. 2. Experiment 2.1. Materials Ca2+ and Mg2+-free Hank's balanced salt solution (HBSS) was obtained from Beijing Solarbio Ltd. (Beijing, China). Dulbecco's Modified Eagles Medium (DMEM), fetal bovine serum and penicillin/streptomycin were all obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). N-acetyl-L-cysteine (NAC), and 3-amino-1,2,4-triazole (3-AT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FW200 UFCB particles purchased from Evonik Degussa Corporation (Beijing, China) were used throughout the tests. The manufacturer reported an average primary particle size of 13 nm and a low organic impurity content (Fig. S4 and Table S1). 2.2. Particle preparation and characterization FW200 was heated at 200 °C for 120 min in an electric heater to eliminate endotoxins, then suspended by sonication in complete medium (DMEM containing 10% fetal bovine serum and 1% penicillin/ streptomycin). The particle suspensions (50 μg mL−1) were sonicated using a 250 W Scientz Sonifier SB-5200 DTD (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, Zhejiang, China) for 10mins to mix and form a homogeneous dispersion. Particle suspensions were continuously cooled on ice during the sonication procedure and then diluted as required before exposure procedure. Complete medium was used as vehicle control solution for the in vitro assay. The dispersion effect of FW200 in complete medium was visualized with a transmission electron microscope (TEM, HRTEM; JEOL, Japan) and scanning electron microscope (SEM, SU8010, Hitachi, Japan). The zeta potential and hydrodynamic diameter of FW200 in the complete medium were characterized by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (ZEN3600, Malvern, Worcestershire, UK) and the data were analyzed using the Dispersion Technology Software (DTS) version 5.0 (Malvern Instruments Ltd). The 0.8 μm and 0.22 μm syringe filters (Jin Long, Tianjin Branch billion Lung Experimental Equipment Co., Ltd.; Tianjin, China) were used in the experiment. 2.3. Animals Female C57BL/6 mice aged 5 weeks were purchased from the Experimental Animal Center of Shandong University and acclimatized for 2 weeks before the experiment. All mice were given Laboratory Rodent Diet and water ad libitum and housed in polypropylene cages with sawdust standard laboratory conditions. All mice were fasted for 18 h prior to the animal sacrifice. All experiments were approved by the Institutional Animal Ethics Committee for Experimentation on Animals of Shandong University. 2.4. Cell isolation and treatment
Scheme 1. Schematic presentation of the investigation on the toxicity of FW200 in vitro and in vivo.
We isolated the mouse hepatocytes according to rules described in Seglen (1976). Obtained cells were then centrifuged at 150g for 5 min
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at 4 °C. We washed the cells three times with cold HBSS and the final concentration of the cell was 1 × 107/ml. The hepatocytes were incubated with FW200 in a cell incubator for 24 h after being pretreated with or without 0.1 mM NAC (ROS scavenger) or 20 mM 3-AT (catalase (CAT) inhibitor) for 1 h. The temperature of the cell incubator and concentration of CO2 were set at 37 °C and 5%, respectively. Cells were incubated with 100 μM H2O2 for 15 min as the positive control (data not shown) in the cell viability, apoptosis, and ROS detection experiments.
method was employed to determine the content of the protein by measuring the change of the absorbance at 595 nm (A595) (Bradford, 1976). We measured A240 and A595 on a UV-2450 spectrophotometer (Shimadzu, Japan). The measurement of hepatocyte apoptosis was carried out on ACEA NovoCyte™ flow cytometer and BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit was employed to quantitatively analyze the live, necrotic, and early and late apoptotic cells after 24 h FW200 treatment.
2.5. Cell viability and ROS detection assay
2.7. GSSG, GSH, and MDA measurement
Cell viability of hepatocytes was investigated using a CCK-8 assay (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan). After incubation with FW200 for 24 h, 10 μL of the CCK-8 solution was added to each well of the 96-well plate. The cells were then incubated at 37 °C for 2 h. Absorbance at 450 nm was recorded on a microplate reader (GFM3000, Rainbow, China). Results were expressed as a percentage of the blank control. The measurement of hepatocytes ROS level was performed on an ACEA NovoCyte™ flow cytometer (Novo Express™, ACEA Bioscience. Inc., USA) by using a Reactive Oxygen Species Assay Kit after 24 h FW200 treatment.
The measurements of the GSH, GSSG, and MDA content in the mouse liver were conducted according to the protocol provided by the kits (Nanjing Jiancheng Bioengineering Institute, China). Briefly, the mouse liver tissue was homogenized in PBS at 4 °C and centrifuged at 13,000g for 30 min. The GSH and GSSG contents were measured based on their absorbance at 405 nm and the DTNB-GR recycling reaction (Rahman et al., 2006). MDA measurement was conducted using the thiobarbituric acid (TBA) colorimetric method (532 nm) (Janero, 1990). Absorbance at 405 nm and 532 nm were measured on Microplate Reader (Thermo Scientific) and UV-2450 spectrophotometer (Shimadzu, Japan) respectively. Results are expressed as the percentage of the control group which was set as 100%.
2.6. Intracellular CAT activity assay and apoptosis measurement 2.8. Histopathological studies After being treated with FW200 for 24 h, the hepatocytes were washed twice with HBSS, then lysed by sonication using an ultrasonic cell disruptor (Microson™, VCX150PB, SONICS & MATERIALS INC.) at 5 W for 5 s on ice. The homogenate was centrifuged at 10000g for 10 min. The obtained supernatant was used for CAT activity. Decreasing rate of absorbance at 240 nm (A240) in 3 mL HBSS with 10 mM H2O2 and 200 μL supernatants was investigated to figure out the activity of CAT. Results were expressed as relative activity of the blank control. Bradford
In the histopathological studies, as shown in Fig. S1, all mice were randomly assigned to three groups: a control group (n = 6) and two FW200 group (n = 6). Mice were treated one time per day with either FW200 solution (5 or 10 mg/kg/d, dispersed by deionized water) or with deionized water by oral gavage. The oral administration time axis is shown in Table S2. Then they were subsequently sacrificed at the indicated times after six-day exposure. The liver was sectioned and fixed
Fig. 1. Scanning electron microscope (A-B) and transmission electron microscope (C-D) images of FW 200. Complete medium was used to disperse FW 200 before microscope analysis.
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in phosphate-buffered 10% formaldehyde for histopathological analysis. Hematoxylin and eosin were used to stain the five-micrometer sections which were observed under light microscopy. 2.9. Statistics analysis All statistical analyses were two-sided and results are presented as the mean ± standard error of the mean (SEM) of three independent assays. Dunnett's one-way analysis of variance (ANOVA) was used to evaluate the multiple comparisons among control and exposure groups. The level of significance in all of the tests was 0.05. 3. Results and discussion 3.1. Particle characterization The FW200 particle characterization was conducted by SEM, TEM, and DLS. As shown in Figs. 1 and 2, the hydrodynamic radius of FW200 in complete medium was larger than its respective diameter (50 nm), which indicated an aggregation of the particles in the complete medium. Various concentrations of FW200 dispersed in a complete medium is shown in Fig. S1, which demonstrates that FW200 dispersed in the medium homogeneously. Fig. 1A, B and C indicate that FW200 aggregated mildly in the complete medium. The nano-onion structure of FW200 which was confirmed by our previous work was also observed in this work (Fig. 1D) (Zhang et al., 2017). As can be seen from the DLS analysis results, the unfiltered FW 200 suspended in complete medium agglomerated highly. The low peak
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zeta-potential (−9 mV, phase plot shown in Fig. S3) of FW 200 could be one of the reasons for its agglomeration propensity (Bourdon et al., 2012). Numerous papers have reported the characterization of carbon nanoparticles by DLS (Canesi et al., 2008; Saber et al., 2012), however, little tried to detect the smaller particles existing in the solution. Herein, we investigated the size distribution by DLS and presented data in detail (Fig. 2A). The high polydispersity index (PDI = 0.75) of the raw FW 200-complete medium dispersion indicated that the size distribution of FW 200 was not concentrated and the particles agglomerated highly as shown in the electron microscopy analysis. The hydrodynamic number size-distribution curves indicated a major peak at approximately 580 nm, which corresponds to the peak-size (630 nm) in the volumesize-distribution. Due to the presence of a huge number of large agglomerates, smaller particles may not be detected. Therefore, the raw FW 200-complete medium dispersion was filtered through 0.8 and 0.22 μm filter and the filtrate was used for further analysis. The filtrate obtained from 0.8 μm filtration indicated the presence of particles whose number and volume peak sizes were both about 100 nm and the polydispersity index was a little high (PDI = 0.51), which reveals a mildly broad size-distribution of FW 200. Filtration through the 0.22 μm filter demonstrated the presence of much smaller particles with the number and volume peak sizes at approximately 30 nm. The filtration led to a more consistent system (PDI = 0.232) and the size of particles mainly concentrated at 30 nm. In order to investigate the potential disturbance caused by protein agglomerates to the DLS analysis of FW200, DLS analysis of the pure complete medium was conducted. As can be seen in Fig. 2B, the diameter of protein agglomerates in the pure complete medium was b20 nm, which is out of the sizedistribution range of FW200 in complete medium. This indicates that the particles shown in Fig. 2A are ascribed to the FW200 nanoparticle rather than the protein agglomerates in complete medium. 3.2. Effects of FW200 on cell viability After various concentrations of FW200 treatment for 24 h, cck-8 assay was employed to evaluate the viability of the hepatocytes. In Fig. 3, the results of cck-8 assay demonstrated that the cell viability of hepatocytes decreased in response to FW200 exposure at 10, 20, 30, 40, and 50 μg mL−1. The cell viability at 50 μg mL−1 FW200 was about 71% of the blank control after 24 h incubation, indicating that FW200
Fig. 2. Dynamic light scattering analysis of complete medium: A: 50 μg mL−1 FW200 and complete medium; B: complete medium alone.
Fig. 3. The viability of hepatocytes after 24 h exposure to FW200. Cell viability was expressed as percentages of the control. The values are presented as the mean ± SEM of independent experiments (n = 3). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h and pretreated with or without 0.1 mM NAC or 20 mM 3-AT for 1 h before the nanoparticle incubation. Differences are considered statistically significant at: **p b 0.01 and ***p b 0.001, compared with the control. #p b 0.05 and ##p b 0.01, compared with samples treated with same FW200 concentration alone.
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Fig. 4. Relative ROS levels in hepatocytes treated with FW200 after 24 h (A-F). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h and pretreated with or without 0.1 mM NAC or 20 mM 3-AT for 1 h before FW200 incubation. Differences are considered statistically significant at: *p b 0.05 and **p b 0.01, compared with the control. #p b 0.05, compared with samples treated with same FW200 concentration alone.
caused a cytotoxicity to the hepatocytes. In order to investigate the relationship between CAT, ROS and the cell viability, we conducted our study by pretreating the cells with 3-AT and NAC. The cell viability of the group pretreated with NAC was higher than those treated with FW200 alone, while the cell viability of the group pretreated with 3AT was lower than the group treated with FW200 alone. The results suggested the inhibition of CAT activity decreased the viability of hepatocytes and the reduction of ROS level increased that of the hepatocytes. Thus we can conclude that FW200 might inhibit the cell viability through stimulating the ROS generation in the hepatocytes and CAT was crucial in attenuating the cytotoxicity caused by FW200.
CAT variation was quantified after the hepatocytes being exposed to FW200 nanoparticles. After the hepatocytes being treated with FW200 for 24 h, the activity of CAT finally increased to 133% of the control and changed slightly after the FW200 reached its maximum concentration (Fig. 5). This phenomenon could be caused by the ROS generation in the hepatocytes or the direct interaction between FW200 and CAT molecule. To figure out this issue, NAC was employed to pretreat the cell, the groups pretreated with NAC exhibited a much lower CAT activity than those treated with FW200 alone. This preliminarily indicated that FW200 increased the activity of CAT by inducing the generation of ROS in the cell. Furthermore, our previous study on the interaction
3.3. The effects of FW200 on ROS level in hepatocytes ROS was stimulated in hepatocytes after being incubated with FW200 for 24 h and the increase of the ROS followed a dosedependent manner (Fig. 4). Similar to the results of the cell viability assay, the ROS level of hepatocytes changed gradually with the addition of FW200. The ROS level of hepatocytes increased to approximately 127% of the blank control, suggesting that FW200 could lead to a more oxidizing environment by inducing oxidative stress in hepatocytes. In this part, NAC was confirmed as an efficient ROS scavenger and 3-AT was used to prove the efficiency of CAT in eliminating ROS. In Fig. 4, the ROS level of the hepatocytes was significantly increased by 3-AT and decreased by NAC compared to the cells treated with FW200 alone, which suggested that the CAT played a similar role with NAC in scavenging ROS in the hepatocytes. 3.4. Measurement of CAT activity in hepatocytes The intracellular CAT activity of hepatocytes has a close relationship with the cell oxidation-reduction equilibrium which determines the cell viability (Nyska and Kohen, 2002; Shao et al., 2012; Wang et al., 2016). CAT also plays a key role in extending the lifespan of the organism (Melov et al., 2000; Schriner et al., 2005; Weydert and Cullen, 2010). To determine the effects of FW200 on the CAT activity, the activity of
Fig. 5. CAT activity in hepatocytes after 24 h exposure to FW200. Data are expressed as relative activity and compared to each control. Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h and pretreated with or without 0.1 mM NAC for 1 h. Differences are considered statistically significant at: *p b 0.05 and **p b 0.01, compared with the control. #p b 0.05, compared with samples treated with same FW200 concentration alone.
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Fig. 6. Percentage of apoptotic cells of hepatocytes after 24 h incubation with FW200 (A-F). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h. Differences are considered statistically significant at: ⁎⁎p b 0.01 and ⁎⁎⁎pb0.001, compared with the control.
of FW200 and CAT molecule proved that the direct interaction of FW200 and CAT molecule would reduce the activity of CAT (Zhang et al., 2017). Therefore, it was the ROS generation rather than the FW200-CAT interaction that stimulated the increase of CAT activity.
3.5. Detection of apoptotic cells After the hepatocytes being treated with FW200 for 24 h, massive apoptosis was induced in hepatocytes. The viable cells, apoptotic cells and necrotic cells of the hepatocytes were quantitated to calculate the ratio of apoptotic cells in the total dead cells. In Fig. 6, the number of apoptotic cells increased markedly (about 127% of the blank control) with the increasing amount of FW200 in the cell suspension, while that of necrotic cells increased mildly without a significant difference. This indicated that FW200 could cause a damage to the hepatocytes by inducing apoptosis in it.
3.6. MDA and GSH measurement As one of the most important antioxidants in the cell, GSH (reduced oxidized) plays a significant role in eliminating peroxides in the enzymatic reactions by transforming the peroxides into GSSG (oxidized glutathione) (Jones, 2002). The steady-state balance of GSH and GSSG is key to keep the ROS in the cell at a normal level. The amount of GSH present could reflect the antioxidant potential of an organelle. In the present study, we observed that the UFCB exposure to hepatocytes led to a remarkable concentration-dependent decrease of GSH in the mouse hepatocytes (Fig. 7). As the hepatocytes was exposed to UFCB of which the concentration was b20 μg/mL, the GSH level of hepatocytes changed little. However, when the concentration of UFCB was higher than 20 μg/mL, the GSH level decreased by 41%. The result demonstrated that the number of peroxides initially stimulated by UFCB was very little and could be eliminated by the GSH newly synthesized in the cell, however, as the concentration of the UFCB was higher than
Fig. 7. GSH, GSSG and MDA changes of hepatocytes after 24 h incubation with FW200 (A-F). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h. Differences are considered statistically significant at: *p b 0.05 and ⁎⁎p b 0.01, compared with the control.
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Fig. 8. The effects of FW200 on the mouse liver histopathology. A: Normal group liver section; B: liver of the mice treated with FW200 showing centrilobular necrosis and acute inflammation (arrow); C and D are the magnification of A and B, respectively.
20 μg/mL, the newly synthesized GSH was not enough to eliminate excess peroxides and then decreased distinctly. On the other hand, the MDA contents increased by 38% with the concentration of FW200 ranging from nil to 50 μg/mL. The increase of MDA in the cell indicated that the lipid peroxidation was induced by FW200 after 24 h exposure. These results are in line with the ROS measurement. 3.7. Histopathological analysis Analysis of histopathological change of the mouse liver treated with FW200 or deionized water was characterized in Fig. 8. As can be seen in the images, the histological characterization revealed no pathological abnormities in the mouse liver of the control. The liver section of the mice treated with 5 mg/kg/d FW200 showed no pathological abnormities either (Fig. S6). However, histopathological analysis of the liver sections of FW200-treated mice showed slight centrilobular necrosis, hepatocyte ballooning and infiltration of inflammatory cells, indicating the cellular damage caused by FW200. In the perivenular zone extending to the central region of the liver cell, diffused areas of hepatitis with a slight loss of hepatic architecture were observed after FW200 treatment (Fig. 8B). Moreover, central veins/portal triads were also severely damaged in the FW200-exposed liver tissue. However, we did not detect the UFCB particles accumulation in the liver. 4. Conclusions In this work, we characterized the dispersity of FW200 in the complete medium. FW200 formed a homogeneous solution in the complete medium with an average size at around 100 nm. Our study mainly demonstrated the toxicity of FW200 to mouse hepatocytes in vitro and to the liver tissue in vivo. In vitro, FW200 induced intracellular ROS and then stimulated the increase of the intracellular CAT activity, resulting in significant cytotoxicity to the hepatocytes. As the ROS level of mouse hepatocytes increased dose-dependently, its cell viability decreased by 29% and its apoptosis level increased by 27%, indicating
that FW200 caused injury to the cell through inducing ROS in it. FW200 could also cause damage to the mouse hepatocytes by invading into the cell, which was confirmed by the TEM image of the liver cell exposed to FW200 (50 μg mL−1) for 24 h (Fig. S5). The increase of the CAT activity induced by FW200 exposure suggested the important role of CAT in defending hepatocytes from the oxidative stress. In vivo, symptoms of centrilobular necrosis, hepatocyte ballooning, and infiltration of inflammatory cells revealed that FW200 nanoparticles had serious toxic effects on the mouse liver tissue. The loss of hepatic architecture of the liver cell demonstrated the cellular damage caused by FW200, which is in accordance with the effects of FW200 on the mouse hepatocytes in vitro. This information could be used to better understand the negative effects of FW200 both on the cellular and organism level, and further proved the potential toxicity of UFCB after they invade into the blood circulation system. Conflicts of interest There are no conflicts to declare. Acknowledgements This work is supported by NSFC (21277081, 21477067, 21777088), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education and Ministry of Education of China (708058, 20130131110016) and independent innovation program of Jinan (201202083), Science and Technology Development Plan of Shandong Province (2014GSF117027) are also acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.11.295.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Assessing the in vitro and in vivo toxicity of ultrafine carbon black to mouse liver Rui Zhang, Xun Zhang, Sichen Gao, Rutao Liu ⁎ School of Environmental Science and Engineering, Shandong University, China–America CRC for Environment & Health, Shandong Province, 72# Jimo Binhai Road, Qingdao, Shandong 266237, PR China
H I G H L I G H T S
G R A P H I C A L
• UFCB (ultrafine carbon black) aggregated in complete medium and had a wide range of size distribution. • UFCB decreased the viability of hepatocytes. • UFCB increased the intracellular CAT activity through stimulating ROS generation. • UFCB caused a significant inflammation in the mouse liver. • This study revealed the negative effects of UFCB in vitro and in vivo.
UFCB dispersed in complete medium and had a wide range of size distribution. UFCB particles invaded into the liver cell.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 22 September 2018 Received in revised form 16 November 2018 Accepted 19 November 2018 Available online 22 November 2018 Editor: Jay Gan Keywords: Carbon black Hepatocytes Histopathology Oxidative stress Nanotoxicology
A B S T R A C T
The increasing presence of nanomaterials in commercial products makes large quantities of nanoparticles reach the environment intentionally or accidentally. Their ability to be cleared from lung to stomach and then translocate into blood circulation suggests they may cause effects on the organs and cells of the organism. In this study, we characterized the dispersity of UFCB (ultrafine carbon black, FW200) in the complete medium and investigated the toxicity of FW200 to mouse hepatocytes and the liver both in vitro and in vivo. FW200 dispersed homogeneously in the complete medium with an average size at around 100 nm. In vitro, FW200 induced apparent cytotoxicity in the hepatocytes with the level of oxidative stress, apoptosis and the viability of hepatocytes changed by approximately 30%. The intracellular catalase (CAT) activity was stimulated by FW200 to a higher level than the control group. In vivo, the 7-week mice were exposed to FW200 (10 mg/kg body weight) by oral administration for six days. The liver was collected and used for histopathological analysis. In our findings, the 13 nm carbon black nanoparticle was proved to induce acute inflammation and apoptosis in the liver. The particles were also proved to have a damage to central veins and architecture of the hepatocytes. These findings suggest that the carbon black nanoparticle could cause a negative effect at both the cellular and organism level and unearthed the potential effects of carbon black nanoparticles on animals and human. © 2018 Elsevier B.V. All rights reserved.
1. Introduction ⁎ Corresponding author. E-mail address: [email protected] (R. Liu).
https://doi.org/10.1016/j.scitotenv.2018.11.295 0048-9697/© 2018 Elsevier B.V. All rights reserved.
Ultrafine carbon black (UFCB) is produced by thermal decomposition of hydrocarbons and consists of primary particles with the
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diameter smaller than 100 nm in all three dimensions. It is a kind of nearly pure amorphous carbon with a high surface area to volume ratio (Zhang et al., 2014). Due to its characters of sluggishness and stability, UFCB has been a high production volume chemical used in the manufacturing of rubber, printing inks, paints, and catalyst (Niranjan and Thakur, 2017; Yu et al., 2011). Scientists and the general public have raised concerns about the use of UFCB which is classified as a possible carcinogen to human by International Agency for Research on Cancer (IARC) (Chen et al., 2014; Kyjovska et al., 2015). The unique physical and chemical properties of nanoparticles endow them with distinct toxic effects compared with their fine counterparts (Hou et al., 2017a). The toxicity of carbon nanoparticles are higher than those of their fine counterparts has been proved by massive works (Hou et al., 2017b; Nel et al., 2006; Oberdörster et al., 2005). Brown et al. and Li et al. also proved that nanoparticles have a higher ability to trigger the generation of reactive oxygen species (ROS) than that of larger particles of the same material (Brown et al., 2001; Li et al., 2003). The CB exposure mostly happens in its production, collection, and handling process. In addition to pulmonary exposure, the gastrointestinal tract is another important route of exposure to nanoparticles, although it has been considerably less investigated than pulmonary exposure. First, carbon black has been used as a food coloring agent and a remedy for a long time (Folkmann et al., 2012). Moreover, SPECT/CT imaging studies demonstrated that 8.3–18.7% and 23.3–50.6% of the deposited dose of particles after short-term inhalation was cleared to the stomach in rats and mice (Kuehl et al., 2012). Therefore, studying the toxicity of nanoparticles to organisms through the gastrointestinal tract exposure is emergent. Nanomaterials are able to translocate from their portal of entry like stomach to systemic circulation and thus to the organs such as spleen, kidney, and liver (Mills et al., 2006; Pietroiusti, 2012; Zhao and Liu, 2012). As such, the possibility of cellular interactions of nanoparticles is extremely high and it is of great importance to know the nanomaterial-mediated cytotoxicity. Our present work aimed to investigate the toxicity of FW200 to the hepatocytes in vitro and to the liver in vivo (Scheme 1). In vitro, we studied the cytotoxicity of FW200 to the mouse hepatocytes. In vivo, we performed the histopathological analysis on the mouse liver tissue after six-day FW200 gavage. To our best knowledge, this is the first time to investigate the toxicity of FW200 at the cellular and organism
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level simultaneously. The in vitro study of FW200 explained the deep mechanism of its in vivo toxicity and revealed the interaction between FW200 and hepatocytes that cannot be reflected by in vivo study. On the other hand, the in vivo study of FW200 further confirmed the results of FW200's in vitro study. We hypothesized that FW200 could cause negative effects at both levels. To date, most investigations on the toxicity of nanomaterials employed a mass-basis approach to calculate the dose of nanomaterials with its concentrations ranging from 0.001 μg mL−1 to 400 μg mL−1 (Gojova et al., 2007; Gratton et al., 2008; Schrand et al., 2008; Schrand et al., 2010; Verma et al., 2008). In this study, we used FW200 in a concentration ranging from 20 μg mL−1 to 50 μg mL−1. 2. Experiment 2.1. Materials Ca2+ and Mg2+-free Hank's balanced salt solution (HBSS) was obtained from Beijing Solarbio Ltd. (Beijing, China). Dulbecco's Modified Eagles Medium (DMEM), fetal bovine serum and penicillin/streptomycin were all obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). N-acetyl-L-cysteine (NAC), and 3-amino-1,2,4-triazole (3-AT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FW200 UFCB particles purchased from Evonik Degussa Corporation (Beijing, China) were used throughout the tests. The manufacturer reported an average primary particle size of 13 nm and a low organic impurity content (Fig. S4 and Table S1). 2.2. Particle preparation and characterization FW200 was heated at 200 °C for 120 min in an electric heater to eliminate endotoxins, then suspended by sonication in complete medium (DMEM containing 10% fetal bovine serum and 1% penicillin/ streptomycin). The particle suspensions (50 μg mL−1) were sonicated using a 250 W Scientz Sonifier SB-5200 DTD (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, Zhejiang, China) for 10mins to mix and form a homogeneous dispersion. Particle suspensions were continuously cooled on ice during the sonication procedure and then diluted as required before exposure procedure. Complete medium was used as vehicle control solution for the in vitro assay. The dispersion effect of FW200 in complete medium was visualized with a transmission electron microscope (TEM, HRTEM; JEOL, Japan) and scanning electron microscope (SEM, SU8010, Hitachi, Japan). The zeta potential and hydrodynamic diameter of FW200 in the complete medium were characterized by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (ZEN3600, Malvern, Worcestershire, UK) and the data were analyzed using the Dispersion Technology Software (DTS) version 5.0 (Malvern Instruments Ltd). The 0.8 μm and 0.22 μm syringe filters (Jin Long, Tianjin Branch billion Lung Experimental Equipment Co., Ltd.; Tianjin, China) were used in the experiment. 2.3. Animals Female C57BL/6 mice aged 5 weeks were purchased from the Experimental Animal Center of Shandong University and acclimatized for 2 weeks before the experiment. All mice were given Laboratory Rodent Diet and water ad libitum and housed in polypropylene cages with sawdust standard laboratory conditions. All mice were fasted for 18 h prior to the animal sacrifice. All experiments were approved by the Institutional Animal Ethics Committee for Experimentation on Animals of Shandong University. 2.4. Cell isolation and treatment
Scheme 1. Schematic presentation of the investigation on the toxicity of FW200 in vitro and in vivo.
We isolated the mouse hepatocytes according to rules described in Seglen (1976). Obtained cells were then centrifuged at 150g for 5 min
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at 4 °C. We washed the cells three times with cold HBSS and the final concentration of the cell was 1 × 107/ml. The hepatocytes were incubated with FW200 in a cell incubator for 24 h after being pretreated with or without 0.1 mM NAC (ROS scavenger) or 20 mM 3-AT (catalase (CAT) inhibitor) for 1 h. The temperature of the cell incubator and concentration of CO2 were set at 37 °C and 5%, respectively. Cells were incubated with 100 μM H2O2 for 15 min as the positive control (data not shown) in the cell viability, apoptosis, and ROS detection experiments.
method was employed to determine the content of the protein by measuring the change of the absorbance at 595 nm (A595) (Bradford, 1976). We measured A240 and A595 on a UV-2450 spectrophotometer (Shimadzu, Japan). The measurement of hepatocyte apoptosis was carried out on ACEA NovoCyte™ flow cytometer and BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit was employed to quantitatively analyze the live, necrotic, and early and late apoptotic cells after 24 h FW200 treatment.
2.5. Cell viability and ROS detection assay
2.7. GSSG, GSH, and MDA measurement
Cell viability of hepatocytes was investigated using a CCK-8 assay (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan). After incubation with FW200 for 24 h, 10 μL of the CCK-8 solution was added to each well of the 96-well plate. The cells were then incubated at 37 °C for 2 h. Absorbance at 450 nm was recorded on a microplate reader (GFM3000, Rainbow, China). Results were expressed as a percentage of the blank control. The measurement of hepatocytes ROS level was performed on an ACEA NovoCyte™ flow cytometer (Novo Express™, ACEA Bioscience. Inc., USA) by using a Reactive Oxygen Species Assay Kit after 24 h FW200 treatment.
The measurements of the GSH, GSSG, and MDA content in the mouse liver were conducted according to the protocol provided by the kits (Nanjing Jiancheng Bioengineering Institute, China). Briefly, the mouse liver tissue was homogenized in PBS at 4 °C and centrifuged at 13,000g for 30 min. The GSH and GSSG contents were measured based on their absorbance at 405 nm and the DTNB-GR recycling reaction (Rahman et al., 2006). MDA measurement was conducted using the thiobarbituric acid (TBA) colorimetric method (532 nm) (Janero, 1990). Absorbance at 405 nm and 532 nm were measured on Microplate Reader (Thermo Scientific) and UV-2450 spectrophotometer (Shimadzu, Japan) respectively. Results are expressed as the percentage of the control group which was set as 100%.
2.6. Intracellular CAT activity assay and apoptosis measurement 2.8. Histopathological studies After being treated with FW200 for 24 h, the hepatocytes were washed twice with HBSS, then lysed by sonication using an ultrasonic cell disruptor (Microson™, VCX150PB, SONICS & MATERIALS INC.) at 5 W for 5 s on ice. The homogenate was centrifuged at 10000g for 10 min. The obtained supernatant was used for CAT activity. Decreasing rate of absorbance at 240 nm (A240) in 3 mL HBSS with 10 mM H2O2 and 200 μL supernatants was investigated to figure out the activity of CAT. Results were expressed as relative activity of the blank control. Bradford
In the histopathological studies, as shown in Fig. S1, all mice were randomly assigned to three groups: a control group (n = 6) and two FW200 group (n = 6). Mice were treated one time per day with either FW200 solution (5 or 10 mg/kg/d, dispersed by deionized water) or with deionized water by oral gavage. The oral administration time axis is shown in Table S2. Then they were subsequently sacrificed at the indicated times after six-day exposure. The liver was sectioned and fixed
Fig. 1. Scanning electron microscope (A-B) and transmission electron microscope (C-D) images of FW 200. Complete medium was used to disperse FW 200 before microscope analysis.
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in phosphate-buffered 10% formaldehyde for histopathological analysis. Hematoxylin and eosin were used to stain the five-micrometer sections which were observed under light microscopy. 2.9. Statistics analysis All statistical analyses were two-sided and results are presented as the mean ± standard error of the mean (SEM) of three independent assays. Dunnett's one-way analysis of variance (ANOVA) was used to evaluate the multiple comparisons among control and exposure groups. The level of significance in all of the tests was 0.05. 3. Results and discussion 3.1. Particle characterization The FW200 particle characterization was conducted by SEM, TEM, and DLS. As shown in Figs. 1 and 2, the hydrodynamic radius of FW200 in complete medium was larger than its respective diameter (50 nm), which indicated an aggregation of the particles in the complete medium. Various concentrations of FW200 dispersed in a complete medium is shown in Fig. S1, which demonstrates that FW200 dispersed in the medium homogeneously. Fig. 1A, B and C indicate that FW200 aggregated mildly in the complete medium. The nano-onion structure of FW200 which was confirmed by our previous work was also observed in this work (Fig. 1D) (Zhang et al., 2017). As can be seen from the DLS analysis results, the unfiltered FW 200 suspended in complete medium agglomerated highly. The low peak
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zeta-potential (−9 mV, phase plot shown in Fig. S3) of FW 200 could be one of the reasons for its agglomeration propensity (Bourdon et al., 2012). Numerous papers have reported the characterization of carbon nanoparticles by DLS (Canesi et al., 2008; Saber et al., 2012), however, little tried to detect the smaller particles existing in the solution. Herein, we investigated the size distribution by DLS and presented data in detail (Fig. 2A). The high polydispersity index (PDI = 0.75) of the raw FW 200-complete medium dispersion indicated that the size distribution of FW 200 was not concentrated and the particles agglomerated highly as shown in the electron microscopy analysis. The hydrodynamic number size-distribution curves indicated a major peak at approximately 580 nm, which corresponds to the peak-size (630 nm) in the volumesize-distribution. Due to the presence of a huge number of large agglomerates, smaller particles may not be detected. Therefore, the raw FW 200-complete medium dispersion was filtered through 0.8 and 0.22 μm filter and the filtrate was used for further analysis. The filtrate obtained from 0.8 μm filtration indicated the presence of particles whose number and volume peak sizes were both about 100 nm and the polydispersity index was a little high (PDI = 0.51), which reveals a mildly broad size-distribution of FW 200. Filtration through the 0.22 μm filter demonstrated the presence of much smaller particles with the number and volume peak sizes at approximately 30 nm. The filtration led to a more consistent system (PDI = 0.232) and the size of particles mainly concentrated at 30 nm. In order to investigate the potential disturbance caused by protein agglomerates to the DLS analysis of FW200, DLS analysis of the pure complete medium was conducted. As can be seen in Fig. 2B, the diameter of protein agglomerates in the pure complete medium was b20 nm, which is out of the sizedistribution range of FW200 in complete medium. This indicates that the particles shown in Fig. 2A are ascribed to the FW200 nanoparticle rather than the protein agglomerates in complete medium. 3.2. Effects of FW200 on cell viability After various concentrations of FW200 treatment for 24 h, cck-8 assay was employed to evaluate the viability of the hepatocytes. In Fig. 3, the results of cck-8 assay demonstrated that the cell viability of hepatocytes decreased in response to FW200 exposure at 10, 20, 30, 40, and 50 μg mL−1. The cell viability at 50 μg mL−1 FW200 was about 71% of the blank control after 24 h incubation, indicating that FW200
Fig. 2. Dynamic light scattering analysis of complete medium: A: 50 μg mL−1 FW200 and complete medium; B: complete medium alone.
Fig. 3. The viability of hepatocytes after 24 h exposure to FW200. Cell viability was expressed as percentages of the control. The values are presented as the mean ± SEM of independent experiments (n = 3). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h and pretreated with or without 0.1 mM NAC or 20 mM 3-AT for 1 h before the nanoparticle incubation. Differences are considered statistically significant at: **p b 0.01 and ***p b 0.001, compared with the control. #p b 0.05 and ##p b 0.01, compared with samples treated with same FW200 concentration alone.
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Fig. 4. Relative ROS levels in hepatocytes treated with FW200 after 24 h (A-F). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h and pretreated with or without 0.1 mM NAC or 20 mM 3-AT for 1 h before FW200 incubation. Differences are considered statistically significant at: *p b 0.05 and **p b 0.01, compared with the control. #p b 0.05, compared with samples treated with same FW200 concentration alone.
caused a cytotoxicity to the hepatocytes. In order to investigate the relationship between CAT, ROS and the cell viability, we conducted our study by pretreating the cells with 3-AT and NAC. The cell viability of the group pretreated with NAC was higher than those treated with FW200 alone, while the cell viability of the group pretreated with 3AT was lower than the group treated with FW200 alone. The results suggested the inhibition of CAT activity decreased the viability of hepatocytes and the reduction of ROS level increased that of the hepatocytes. Thus we can conclude that FW200 might inhibit the cell viability through stimulating the ROS generation in the hepatocytes and CAT was crucial in attenuating the cytotoxicity caused by FW200.
CAT variation was quantified after the hepatocytes being exposed to FW200 nanoparticles. After the hepatocytes being treated with FW200 for 24 h, the activity of CAT finally increased to 133% of the control and changed slightly after the FW200 reached its maximum concentration (Fig. 5). This phenomenon could be caused by the ROS generation in the hepatocytes or the direct interaction between FW200 and CAT molecule. To figure out this issue, NAC was employed to pretreat the cell, the groups pretreated with NAC exhibited a much lower CAT activity than those treated with FW200 alone. This preliminarily indicated that FW200 increased the activity of CAT by inducing the generation of ROS in the cell. Furthermore, our previous study on the interaction
3.3. The effects of FW200 on ROS level in hepatocytes ROS was stimulated in hepatocytes after being incubated with FW200 for 24 h and the increase of the ROS followed a dosedependent manner (Fig. 4). Similar to the results of the cell viability assay, the ROS level of hepatocytes changed gradually with the addition of FW200. The ROS level of hepatocytes increased to approximately 127% of the blank control, suggesting that FW200 could lead to a more oxidizing environment by inducing oxidative stress in hepatocytes. In this part, NAC was confirmed as an efficient ROS scavenger and 3-AT was used to prove the efficiency of CAT in eliminating ROS. In Fig. 4, the ROS level of the hepatocytes was significantly increased by 3-AT and decreased by NAC compared to the cells treated with FW200 alone, which suggested that the CAT played a similar role with NAC in scavenging ROS in the hepatocytes. 3.4. Measurement of CAT activity in hepatocytes The intracellular CAT activity of hepatocytes has a close relationship with the cell oxidation-reduction equilibrium which determines the cell viability (Nyska and Kohen, 2002; Shao et al., 2012; Wang et al., 2016). CAT also plays a key role in extending the lifespan of the organism (Melov et al., 2000; Schriner et al., 2005; Weydert and Cullen, 2010). To determine the effects of FW200 on the CAT activity, the activity of
Fig. 5. CAT activity in hepatocytes after 24 h exposure to FW200. Data are expressed as relative activity and compared to each control. Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h and pretreated with or without 0.1 mM NAC for 1 h. Differences are considered statistically significant at: *p b 0.05 and **p b 0.01, compared with the control. #p b 0.05, compared with samples treated with same FW200 concentration alone.
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Fig. 6. Percentage of apoptotic cells of hepatocytes after 24 h incubation with FW200 (A-F). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h. Differences are considered statistically significant at: ⁎⁎p b 0.01 and ⁎⁎⁎pb0.001, compared with the control.
of FW200 and CAT molecule proved that the direct interaction of FW200 and CAT molecule would reduce the activity of CAT (Zhang et al., 2017). Therefore, it was the ROS generation rather than the FW200-CAT interaction that stimulated the increase of CAT activity.
3.5. Detection of apoptotic cells After the hepatocytes being treated with FW200 for 24 h, massive apoptosis was induced in hepatocytes. The viable cells, apoptotic cells and necrotic cells of the hepatocytes were quantitated to calculate the ratio of apoptotic cells in the total dead cells. In Fig. 6, the number of apoptotic cells increased markedly (about 127% of the blank control) with the increasing amount of FW200 in the cell suspension, while that of necrotic cells increased mildly without a significant difference. This indicated that FW200 could cause a damage to the hepatocytes by inducing apoptosis in it.
3.6. MDA and GSH measurement As one of the most important antioxidants in the cell, GSH (reduced oxidized) plays a significant role in eliminating peroxides in the enzymatic reactions by transforming the peroxides into GSSG (oxidized glutathione) (Jones, 2002). The steady-state balance of GSH and GSSG is key to keep the ROS in the cell at a normal level. The amount of GSH present could reflect the antioxidant potential of an organelle. In the present study, we observed that the UFCB exposure to hepatocytes led to a remarkable concentration-dependent decrease of GSH in the mouse hepatocytes (Fig. 7). As the hepatocytes was exposed to UFCB of which the concentration was b20 μg/mL, the GSH level of hepatocytes changed little. However, when the concentration of UFCB was higher than 20 μg/mL, the GSH level decreased by 41%. The result demonstrated that the number of peroxides initially stimulated by UFCB was very little and could be eliminated by the GSH newly synthesized in the cell, however, as the concentration of the UFCB was higher than
Fig. 7. GSH, GSSG and MDA changes of hepatocytes after 24 h incubation with FW200 (A-F). Cells were exposed to 0, 10, 20, 30, 40, 50 μg mL−1 FW200 (1–6) for 24 h. Differences are considered statistically significant at: *p b 0.05 and ⁎⁎p b 0.01, compared with the control.
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Fig. 8. The effects of FW200 on the mouse liver histopathology. A: Normal group liver section; B: liver of the mice treated with FW200 showing centrilobular necrosis and acute inflammation (arrow); C and D are the magnification of A and B, respectively.
20 μg/mL, the newly synthesized GSH was not enough to eliminate excess peroxides and then decreased distinctly. On the other hand, the MDA contents increased by 38% with the concentration of FW200 ranging from nil to 50 μg/mL. The increase of MDA in the cell indicated that the lipid peroxidation was induced by FW200 after 24 h exposure. These results are in line with the ROS measurement. 3.7. Histopathological analysis Analysis of histopathological change of the mouse liver treated with FW200 or deionized water was characterized in Fig. 8. As can be seen in the images, the histological characterization revealed no pathological abnormities in the mouse liver of the control. The liver section of the mice treated with 5 mg/kg/d FW200 showed no pathological abnormities either (Fig. S6). However, histopathological analysis of the liver sections of FW200-treated mice showed slight centrilobular necrosis, hepatocyte ballooning and infiltration of inflammatory cells, indicating the cellular damage caused by FW200. In the perivenular zone extending to the central region of the liver cell, diffused areas of hepatitis with a slight loss of hepatic architecture were observed after FW200 treatment (Fig. 8B). Moreover, central veins/portal triads were also severely damaged in the FW200-exposed liver tissue. However, we did not detect the UFCB particles accumulation in the liver. 4. Conclusions In this work, we characterized the dispersity of FW200 in the complete medium. FW200 formed a homogeneous solution in the complete medium with an average size at around 100 nm. Our study mainly demonstrated the toxicity of FW200 to mouse hepatocytes in vitro and to the liver tissue in vivo. In vitro, FW200 induced intracellular ROS and then stimulated the increase of the intracellular CAT activity, resulting in significant cytotoxicity to the hepatocytes. As the ROS level of mouse hepatocytes increased dose-dependently, its cell viability decreased by 29% and its apoptosis level increased by 27%, indicating
that FW200 caused injury to the cell through inducing ROS in it. FW200 could also cause damage to the mouse hepatocytes by invading into the cell, which was confirmed by the TEM image of the liver cell exposed to FW200 (50 μg mL−1) for 24 h (Fig. S5). The increase of the CAT activity induced by FW200 exposure suggested the important role of CAT in defending hepatocytes from the oxidative stress. In vivo, symptoms of centrilobular necrosis, hepatocyte ballooning, and infiltration of inflammatory cells revealed that FW200 nanoparticles had serious toxic effects on the mouse liver tissue. The loss of hepatic architecture of the liver cell demonstrated the cellular damage caused by FW200, which is in accordance with the effects of FW200 on the mouse hepatocytes in vitro. This information could be used to better understand the negative effects of FW200 both on the cellular and organism level, and further proved the potential toxicity of UFCB after they invade into the blood circulation system. Conflicts of interest There are no conflicts to declare. Acknowledgements This work is supported by NSFC (21277081, 21477067, 21777088), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Research Fund for the Doctoral Program of Higher Education and Ministry of Education of China (708058, 20130131110016) and independent innovation program of Jinan (201202083), Science and Technology Development Plan of Shandong Province (2014GSF117027) are also acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.11.295.
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