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Chromium is a major industrial pollutant and its environmental level keeps increasing due to the extensive usage in leather tanning, stainless-steel production, and electroplating[1].Chromium can accumulate in human's food chain to impact human physiology, and causes many diseases. It may result in severe health problems ranging from simple skin irritation to lung carcinoma when contact with chromium[2]. The removal of highly toxic Cr(Ⅵ) has been proposed using membrane filtration[3], chemical agents and precipitation[4], ion exchange resins[5] among many others. However, these techniques are costly, energy-intensive and not efficient for removing Cr(Ⅵ) at low concentration. With the rapid development of nanotechnology, nanoparticles have been widely used in wastewater purification[6-9].
The studies on magnetite nanoparticles (MNPs) have shown good potential in the removal of metals via surface adsorption. This benefits its physicochemical properties, such as the high ratio of surface-to-volume that results in a better and more efficient on adsorption ability. Magnetite-supported adsorbents can be easily separated from the treated water using an external magnet. Therefore an efficient, economic, scalable synthesis of MNPs has been widely demanded in wastewater purification industry[10].
Although nanomaterials (NPs) have shown a profound impact on the practical applications, their potential biological and environmental toxicity have not been sufficiently studied yet. The adsorption of pollutants to nanoparticles alters the properties for both pollutant and nanoparticle, resulted adducts may cause an immediate threat to human health and ecology. For instance, the toxicity effects of titanium dioxide NPs + glucose to rats (Chen et al.[11]) showed that oral exposure of NPs + glucose induced more evident toxicity than NPs alone due to the effects of excessive glucose and the interactions between NPs and glucose. Wang et al.[12]have reported that vitamin C promoted the toxicity of ZnO NPs to gastric epithelial cell line and neural stem cells because the vitamin C accelerated uptake of Zn ions and the dissolution of ZnO NPs. Both indirect mechanisms and synergistic or inhibitory effects can enhance or suppress the expected responses from the specific classes of pollutants. Therefore, the biological impacts of nanoparticles-adducts need to be cautious evaluated. Recently, significant research efforts have been made toward the investigation of nanoparticle toxicity, very little attention has been paid to nanoadducts though the nanoadduct formations has been broadly used in environmental remediation which also has potentials to cause pollution in even broader areas. Thus, the impact of nanoadducts also needs to be thoroughly studied and it is just as important as the study of nanomaterials and pollutants.
The objective of the present study was to evaluate the effect of MNPs/Cr(Ⅵ) adducts on human embryonic kidney cell line HEK293 by assessing cell viability, apoptosis, oxidative stress induction, and cellular uptake. HEK293 was used as a model because Cr(Ⅵ) is known to induce nephrotoxicity[13]. they are essential in defining the toxicological response of the in vitro culture models to nanoparticle adducts exposure. Our results indicated that the cytotoxicity of the MNPs/Cr(Ⅵ) adducts was remarkably reduced compared to Cr(Ⅵ) anions. And the cellular uptake of MNPs/Cr(Ⅵ) adducts was rare. The particles were endocytosed from the extracellular fluid and could not enter into the cell nucleus. In this case, MNPs/Cr(Ⅵ) adduct formation significantly reduces its associated cytotoxicity.
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Size, shape, and chemical composition are the important properties in the nanoparticle toxicity investigation[16]. The particle size and appearance of MNPs and MNPs/Cr(Ⅵ) adducts were determined by SEM. The images clearly showed spherical morphology and the diameters of MNPs and MNPs/Cr(Ⅵ) ranged from 10 to 20 nm (Figure 1A-B). The hydrodynamic diameter of MNPs and MNPs/Cr(Ⅵ) in culture medium was 216 nm and 352 nm respectively, as determined by DLS (Table 1). The size of nanoparticles in aqueous suspension was much higher compared to SEM. This might be due to the particle agglomeration tendency in the aqueous state. This finding was consistent with our previous observation[17-19].The crystalline structure and magnetic properties of MNPs and MNPs/Cr(Ⅵ) adducts were verified by XRD patterns (Figure 1C).
Characterization MNPs Adducts Primary size/SEM (nm) 10-20 Hydrodynamic size/DLS (nm) 216 352 Absorption/K2Cr2O7μg/mg MNPs NA 13.4 Table 1. Characterization of MNPs and MNPs/ Cr(Ⅵ) Adducts
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Adsorption of Cr(Ⅵ) onto Fe3O4MNPs reduced the quantity of Cr(Ⅵ) that was readily taken up by cells. Cr(Ⅵ)-induced cytotoxicity was demonstrated by the optic microscopic observation in which the cell shrinkage was aggravated as increase of Cr(Ⅵ) concentration. MNPs/Cr(Ⅵ) adducts-induced cytotoxicity was significantly reduced as showed in the Figure 2A. We therefore expected that MNPs/Cr(Ⅵ) might result in less cytotoxicity compared to free Cr(Ⅵ). To verify this, we identified the viability of cells by analyzing the activity of mitochondria dehydrogenase, which is proportional to the number of live cells. Figure 2B showed the cell viabilities of different treatments. The Cr(Ⅵ) clearly induced a dose-dependent cytotoxic effect on HEK293 cells. As the Cr(Ⅵ) concentration increased to 30 mg/L, the cell viability was reduced to 55.3% compared to the controls. For the cells treated with both Cr(Ⅵ) and MNPs, the cell viabilities were not significantly different from the control cells and from the cells treated with MNPs only. Therefore, the Cr(Ⅵ) inducted cytotoxicity was markedly ameliorated after it was adsorbed onto the MNPs to form the MNPs/Cr(Ⅵ) adducts, which reduced the free-Cr(Ⅵ) level in the system.
Figure 2. Cells were treated with different concentrations of Cr(Ⅵ), MNPs and corresponding MNPS/Cr(Ⅵ) adducts for 24 h. (A) Changes of cell morphology in Cr(Ⅵ)-exposed cells observed by optic microscope, magnification is × 200. (B) Cell viability assessment by quantifying mitochondria dehydrogenase activity. Significant differences are indicated by *vs. control. a, b, cvs. the corresponding Cr(Ⅵ) groups without MNPs (*P < 0.05, **P < 0.01). (C) Cell apoptosis analysis by Flow cytometry.
To identify whether Cr(Ⅵ) and MNPs/Cr(Ⅵ) adducts induced apoptosis, the treated cells were stained with Annexin V-FITC/PI. The living cells were Annexin V -FITC and PI double-negative, whereas the late apoptotic or secondary necrotic cells were double-positive. The early apoptotic cells were only Annexin V -FITC positive, whereas the isolated nuclei or cellular debris were only PI positive. An increase in the FITC-conjugated Annexin V -positive cells is an early marker for apoptosis. PI staining was used to investigate the loss of cell membrane integrity. The rate of apoptosis in HEK293 cells (i.e., the proportion of AnnexinV+ cells) was shown in Figure 2C. Twenty-four hours after Cr(Ⅵ) exposure, higher percentages of early-and late-apoptotic cells were observed compared with the control. However, after MNPs/Cr(Ⅵ) adducts exposure, the percentage of apoptotic cells were significantly decreased.
Oxidative stress has been recommended as an ordinary pathway for nanoparticle-induced toxicity[20-21].This parameter was evaluated with three indicators, SOD, GSH, GSSG, and MDA in our study. The Indicator activities impacted by Cr(Ⅵ), MNPs, and MNPs/Cr(Ⅵ) adducts, were compared in Figure 3. Results indicated that the GSSG and MDA levels were significantly enhanced and the SOD and GSH concentrations were significantly inhibited in Cr(Ⅵ) treated HEK293 cells relative to the control group. Comparing the four parameters among all treated groups, the SOD and GSH concentrations were remarkably higher and both GSSG and MDA levels were significantly declined in HEK293 cells treated with MNPs and MNPs/Cr(Ⅵ) adducts relative to the groups treated with the high level of Cr(Ⅵ), but there is no significant differences between MNPs group and MNPs/Cr(Ⅵ) adducts group.
Figure 3. Effects of Cr(Ⅵ) (0-30 mg/L) with and without MNPs (4 g/L) on (A) superoxide dismutase (SOD), (B) malondialdehyde (MDA), (C) reduced glutathione (GSH), and (D) oxidized glutathione (GSSG) in HEK293 cells. Significant differences are indicated by *vs. control. a, b, cvs. the corresponding Cr(Ⅵ) groups without MNPs (*P < 0.05, **P < 0.01).
Our results are in accordance with those of previous studies in which the MNPs treatment caused an obvious increase in SOD and GSH levels and a significant reduction in MDA levels[22-25].
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To further evaluate the cell-specific cytotoxicity, we took a series of TEM images for HEK293 cell to observe the entire internalization process of MNPs and MNPs/Cr(Ⅵ) adducts into the HEK293 cell. (Representative images after 24 h incubation were showed in Figure 4) As it can be seen in the figures, after the cells were cultured with MNPs and MNPs/Cr(Ⅵ) adducts for 24 h, they were rarely detectable inside the HEK293 cells and the cell nucleus remained intact. To confirm the uptake of the MNPs and MNPs/Cr(Ⅵ) by the cells, elemental analysis was employed by ICP-MS to determine the iron content of the cells. The concentration of iron atoms in acid digesting solution was directly measured. The results were consistent with the TEM observations. The cellular iron levels of MNPs and MNPs/Cr(Ⅵ) adducts were less than 2.78 pg/cell and 1.85 pg/cell respectively, as determined by ICP-MS (Figure 4F).