Ultrafine NiO Particles Induce Cytotoxicity in Vitro by Cellular Uptake

Chem. Res. Toxicol. 2009, 22, 1415–1426

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Ultrafine NiO Particles Induce Cytotoxicity in Vitro by Cellular Uptake and Subsequent Ni(II) Release Masanori Horie,*,† Keiko Nishio,† Katsuhide Fujita,† Haruhisa Kato,‡ Ayako Nakamura,‡ Shinichi Kinugasa,‡ Shigehisa Endoh,§ Arisa Miyauchi,§ Kazuhiro Yamamoto,| Hideki Murayama,⊥ Etsuo Niki,† Hitoshi Iwahashi,† Yasukazu Yoshida,† and Junko Nakanishi⊥ Health Technology Research Center (HTRC), National Institute of AdVanced Industrial Science and Technology (AIST), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan, National Metrology Institute of Japan (NMIJ), AIST, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8565, Japan, Research Institute for EnVironmental Management Technology (EMTECH), AIST, Particle Measurement Group, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan, Research Institute of Instrumentation Frontier (RIIF), AIST, 1-1-1, Umezono, Tsukuba, Ibaraki 305-8565, Japan, and Research Institute of Science for Safety and Sustainability (RISS), AIST, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan ReceiVed February 13, 2009

Nickel oxide (NiO) is one of the important industrial materials used in electronic substrates and for ceramic engineering. Advancements in industrial technology have enabled the manufacture of ultrafine NiO particles. On the other hand, it is well-known that nickel compounds exert toxic effects. The toxicity of nickel compounds is mainly caused by nickel ions (Ni2+). However, the ion release properties of ultrafine NiO particles are still unclear. In the present study, the influences of ultrafine NiO particles on cell viability were examined in vitro to obtain fundamental data for the biological effects of ultrafine green NiO and ultrafine black NiO. Ultrafine NiO particles showed higher cytotoxicities toward human keratinocyte HaCaT cells and human lung carcinoma A549 cells than fine NiO particles and also showed higher solubilities in culture medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum) than fine NiO particles. In particular, the concentration of Ni2+ released into the culture medium by ultrafine green NiO was 150-fold higher than that released by fine green NiO. The concentrations of Ni2+ released by both types of NiO particles in an aqueous solution containing amino acids were remarkably higher than those released by NiO particles in water. Moreover, we prepared a uniform and stable dispersion of ultrafine black NiO in culture medium and examined its influence on cell viability in comparison with that of NiCl2, a soluble nickel compound. A medium exchange after 6 h of exposure resulted in a loss of cytotoxicity in the cells exposed to NiCl2, whereas cytotoxicity was retained in the cells exposed to NiO. Transmission electron microscope observations revealed uptake of both ultrafine and fine NiO particles into HaCaT cells. Taken together, the present results suggest that the intracellular Ni2+ release could be an important factor that determines the cytotoxicity of NiO. Ultrafine NiO is more cytotoxic than fine NiO in vitro. Introduction Nickel oxide (NiO) is one of the important materials used for various industrial purposes, including ceramic engineering and the manufacture of electronic components, condensers, and varistors. On the other hand, nickel compounds are known to exhibit toxicity and carcinogenicity (1-3). Recently, the production of ultrafine NiO particles as functional materials has increased because ultrafine particles have higher physical and chemical activities than fine particles. Although these higher physical and chemical activities are beneficial for industrial applications, they also lead to increased biological activities. Consequently, the increased production of ultrafine NiO particles leads to increased risks for workers, although the potential toxicity of ultrafine NiO particles remains unclear. Because the * To whom correspondence should be addressed. Tel: +81-72-751-9693. Fax: +81-72-751-9964. E-mail: [email protected]. † HTRC. ‡ NMIJ. § EMTECH. | RIIF. ⊥ RISS.

toxicities of nickel compounds depend on nickel ions (Ni2+) (4, 5), assessment of solubility (i.e., nickel ion release) is important for evaluating nickel compounds. In general, particulate nickel compounds, such as Ni3S2 and NiO, show higher toxicities than soluble nickel compounds, such as NiCl2 and NiSO4. Nickel compound particles enter cells by endocytosis and subsequently release Ni2+ inside the cells (6). On the other hand, soluble nickel compounds become ionized outside of the cell. In other words, soluble nickel compounds attack cells from the outside, whereas particulate nickel compounds attack cells from the inside. Consequently, particulate nickel compounds act as intracellular sources of Ni2+. The cellular responses induced by soluble and particulate nickel compounds are essentially the same (7-9). For example, NiCl2 (soluble) and NiO particles (insoluble) both inhibit the repair of DNA adducts formed with benzo[R]pyrene (10). However, because extracellular Ni2+ cannot easily pass through the cell membrane via ion channels, larger amounts of soluble nickel compounds are required to induce the same levels of cell responses to those caused by particulate nickel compounds (11). On the other hand, among the particulate nickel compounds, the cellular influences

10.1021/tx900171n CCC: $40.75  2009 American Chemical Society Published on Web 07/27/2009

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Table 1. List of the NiOs Used in This Studya material black NiO green NiO a

code in this study

average particle size (nm)

specific surface area (m2/g)

purity (%)

vendor

UF-B F-B UF-G F-G

20 1000-2000 100 600-1400

50-80 60-100 >6 3.44

99.8 unknown 99.0 >99.0

Nanostructured & Amorphous Materials (Houston, TX) INCO Special Products (Toronto, Ontario, Canada) Nanostructured & Amorphous Materials (Houston, TX) Sumitomo Metal Mining (Tokyo, Japan)

The data are taken from the manufacturers’ data sheets.

differ depending on their solubilities. For example, the toxicity of NiO is quite weak as compared with nickel subsulfide (Ni3S2), which is soluble and exerts strong carcinogenicity (12). Moreover, there are two types of NiO with different calcination temperatures, namely, black NiO (low calcination temperature) and green NiO (high calcination temperature). Black NiO and green NiO have different solubilities in water and culture media (13). Green NiO hardly dissolves in biological fluids (14). Because black NiO is more soluble than green NiO, it shows stronger cytotoxicity toward macrophages than green NiO. Moreover, the growth of Chinese hamster ovary (CHO) cells is inhibited by black NiO (13). Therefore, solubility is very important for risk assessments of nickel compounds. In addition, biological fluids such as serum, pulmonary surfactant, and culture medium contain proteins, lipids, salts, amino acids, etc., and these components are also important for understanding the mechanism of nickel toxicity. When Ni2+ is dissolved in serum, it binds to serum proteins, thereby preventing Ni2+-related DNA damage (15). It has also been reported that the solubility of NiO in serum-supplemented medium is lower than its solubility in water (14). These observations suggest that the components of the dispersion medium are also important for evaluating NiO toxicity. However, the above findings regarding the toxicity of NiO were obtained using fine particles. As mentioned above, ultrafine NiO particles have the potential for a higher toxic activity than fine particles. For example, ultrafine NiO particles produce greater amounts of reactive oxygen species in culture medium than fine NiO particles (16). Analysis of the gene expression profiles in rat lungs after inhalation exposure to ultrafine black (UF-B) NiO revealed the induction of acute inflammation (17). However, the relationships between the toxicity and the solubility of ultrafine NiO particles are still unclear. In the present study, we compared the Ni2+-releasing abilities of NiO particles and the cytotoxicities of ultrafine and fine NiO particles in vitro. In particular, we focused on the cellular uptake of NiO particles and the effects of internalized NiO particles on the cytotoxicity. Two kinds of cultured cells were examined in this study, namely, human keratinocyte HaCaT cells as a skin exposure model and human lung carcinoma A549 cells as an inhalation model.

Materials and Methods Cell Culture and Determinations of Cell Viability and Colony-Forming Ability. Human keratinocyte HaCaT cells were purchased from the German Cancer Research Center (DKFZ, Heidelberg, Germany). Human lung carcinoma A549 cells were purchased from the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM;1 Gibco, Invitrogen Corp., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS; 1 Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; TEM, transmission electron microscope; EF-TEM, energy-filtering transmission electron microscope; MTT, 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; DLS, dynamic light scattering; XRF, X-ray fluorescence.

CELLect GOLD; MP Biomedicals Inc., Solon, OH), 100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B (Nacalai Tesque Inc., Kyoto, Japan). In the present paper, this DMEM preparation is referred to as “DMEM-FBS”. The cells in DMEM-FBS were placed in 75 cm2 flasks (Corning Inc., Corning, NY) and cultured at 37 °C in a 5% CO2 atmosphere. For cell viability assays, the cells were seeded in 96 well plates (Corning Inc.) at 1 × 105 cells/mL and incubated for 24 h. Subsequently, the culture medium was exchanged for NiO-DMEM-FBS dispersion medium, and the cells were cultured for another 24 h. For cytotoxicity determinations, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were conducted. In the MTT assays, the cells were incubated with 0.5 mg/mL MTT (Nacalai Tesque Inc.) at 37 °C for 2 h. Next, isopropyl alcohol containing 40 mM HCl was added to the culture medium (3:2 v/v) and mixed with a pipet until the formazan had completely dissolved. The optical density of the formazan was measured at 570 nm using a Multiskan Ascent plate reader (Thermo Labsystems, Helsinki, Finland). In the LDH assays, LDH release was measured with a tetrazolium salt using a Cytotoxicity Detection KitPLUS (LDH) (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocol. The amount of formazan salt formed was measured at 492 nm using the Multiskan Ascent plate reader. The maximum amount of LDH released was determined by incubating the cells with a lysis solution provided in the kit. The cytotoxicity was calculated as follows: cytotoxicity (%) ) (experimental value - low control)/(high control - low control) × 100. The low control, which refers to spontaneous LDH release, was determined as the LDH released from untreated normal cells. The high control, which refers to the maximum LDH release, was determined as the LDH released from cells lysed by surfactant treatment. The colony-forming ability, namely, cell proliferation, was detected by a clonogenic assay that was based on the methods of Herzog et al. (18) and Franken et al. (19). Briefly, cells were seeded in six well microplates (Corning Inc.) at a density of 300 cells/well. Each well contained 2 mL of cell culture medium. The cells were allowed to attach for approximately 14 h, before they were washed with phosphate-buffered saline (PBS) and treated with 2 mL of the NiO DMEM-FBS dispersions or centrifugation supernatants from the dispersions. The cells were then cultured for the time period required for the control cells to form colonies (the colony was defined as including at least 50 cells), namely, 7 days. After completion of the culture procedure, the dispersions or the supernatants were removed, and the cells were washed once with 2 mL of PBS. After fixation with 100% methanol for 15 min, the cells were stained with Giemsa solution (Nacalai Tesque Inc.) diluted 1:50 in water for 15 min and then rinsed once with distilled water. The numbers of colonies were counted. NiO Particles and Preparation of NiO Dispersions in DMEMFBS. Table 1 lists the manufacturers of the ultrafine and fine NiO particles used in this study, along with the physical characteristics of each type of particle. NiCl2 was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). For the cytotoxicity assays described in Figures 1-3, NiO powder was dispersed directly in DMEM-FBS at a concentrations ranging from 0.1 to 10 mg/mL without a dispersant. For some examinations described in Figures 6 and 7, stable and uniform dispersions were prepared using a previously described preadsorption and centrifugation method (20). Ultrafine particles induce artificial cellular influences by medium depletion because of their protein adsorption ability (20, 21). To prevent cell starvation caused by adsorption of the medium

Cytotoxicity of NiO Particles by Ni(II) Release components onto the particle surface, the NiO particles were initially dispersed in FBS at a concentration of 80 mg/mL. Subsequently, each dispersion was centrifuged at 16000g for 20 min. The precipitated NiO particles were washed once with FBS-free DMEM and redispersed in an equivalent volume of fresh DMEM-FBS. This dispersion of ultrafine particles in DMEM-FBS was centrifuged at 8000g for 20 min. After the supernatant was discarded, the precipitate was resuspended in an equal volume of fresh DMEMFBS, and the resulting NiO dispersion was centrifuged at 4000g for 20 min. The above process was repeated until the supernatant was collected as a “uniform and stable NiO dispersion”. However, the centrifugal force was gradually reduced from 2000g to 1000g. The 4000g and the 1000g fraction were used for cytotoxicity assay. The NiO-DMEM-FBS dispersions used in this study did not lead to medium depletion. The adsorption effects were verified by measuring the protein and calcium concentrations in the DMEMFBS dispersions. Characterization of NiO-DMEM-FBS Dispersions. In this study, we defined “secondary particles” as complex aggregates of primary particles, proteins from FBS, and other medium components such as Ca2+. In addition, the “average particle size” was defined as the size of the secondary particles estimated from light scattering intensity measurements made under the assumption that the aggregates were globular. The NiO-DMEM-FBS dispersions prepared by the above-mentioned methods were divided into three parts that were used for simultaneous biological examinations, nickel concentration measurements, and particle size measurements. The secondary particle sizes in the NiO-DMEM-FBS dispersions were measured by dynamic light scattering (DLS), as described previously (22). The estimated diameters of the secondary particles were calculated as the average of three measurements at different wavelengths taken with the following devices: a DLS-7000 spectrophotometer (633 nm; Otsuka Electronics Co. Ltd., Hirakata, Japan), an FPAR-1000 fiber optics particle analyzer (660 nm; Otsuka Electronics Co. Ltd.), and a Nanotrac (780 nm; Nikkiso Co. Ltd., Tokyo, Japan). Undiluted dispersions were used for the above measurements. The measurements were performed at a temperature of 25.0 ( 0.1 °C with sample concentrations of 50-80 µg/mL. The samples for the particle size measurements and cytotoxicity assays were obtained at the position of 1 cm from the surface of the solutions in static 15 mL tubes. The viscosities of the dispersions were measured using a Ubbelohde viscometer No. 0C (Sibata Scientific Technology Ltd., Tokyo, Japan). Although, in principle, the particle size distribution can be calculated by DLS, DLS was not used to compute the particle sizes because of its low reliability. The total nickel concentration was measured by X-ray fluorescence (XRF) analysis. For this analysis, 13 mL of NiO-DMEMFBS dispersion was added to 13 mL of a standard solution containing 0.1 mg/mL of iron as an internal standard element and mixed well. Next, 5 mL of the mixture was dried in an oven at 200 °C for 24 h. The dried sample was ground in an agate mortar and subjected to XRF analysis using a dispersive XRF spectrometer (JSX-3201; JEOL Ltd., Tokyo, Japan). The amount of nickel was estimated from the molar ratio of nickel and the internal standard. Determination of Ni2+ Concentrations. For measurement of the Ni2+ concentrations, the fine and ultrafine NiO powders were dispersed directly in DMEM-FBS at concentrations of 0.1, 0.5, 1.0, 5.0, 10, and 50 mg/mL without a dispersant. For some measurements, NiO particles (10 mg/mL) were dispersed in pure water or water containing 10% FBS, 2 mM CaCl2, or 1× MEM amino acid solution (Invitrogen Corp., Carlsbad, CA) instead of DMEM-FBS. The 1× MEM amino acid solution included the following 12 amino acids: L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. The above-mentioned dispersions were incubated at 37 °C for 24 h and then centrifuged at 16000g for 20 min. The Ni2+ concentrations in the resulting supernatants were determined by colorimetry using 2-(5-bromo-2-pyridylazo)-5-[Nn-propyl-N-(3-sulfopropyl)amino]aniline (5-Br-PSAA; Dojindo Laboratories, Kumamoto, Japan) according to the procedure of Ohno et

Chem. Res. Toxicol., Vol. 22, No. 8, 2009 1417 al. (23). The NiO dispersions and cell culture supernatants were centrifuged at 16000g for 20 min, and the supernatants were carefully collected. After suitable dilution, 50 µL of each supernatant and 50 µL of 1 mM 5-Br-PSAA were added to 2.9 mL of a buffer (60 mM KH2PO4, 20 mM Na2B4O7·10H2O, 0.2% Tiron, pH 6.9). The resulting solutions were mixed well and measured for their absorbances at 568 nm using a DU530 spectrophotometer (Beckman Coulter Inc., Miami, FL). The Ni2+ concentrations were estimated from a standard curve based on a nickel standard solution (Wako Pure Chemical Industries). Transmission Election Microscope (TEM) Observations. Microstructural characterization of the NiO particles in the DMEMFBS dispersions was carried out using an energy-filtering transmission electron microscope (EF-TEM) (EM-922; Carl Zeiss NTS, Oberkochen, Germany). The microscope was equipped with an OMEGA-type energy filter that allowed the selection of electrons undergoing certain energy transitions during their transmission through the sample. Zero-loss imaging, which reduced the energy loss in electrons, was used to increase both the scattering of the electrons and the phase contrast of the TEM images. The energy window width of the filter was 20 eV, and the acceleration energy of the electrons was 200 kV. For TEM observations, the NiO particles were held on carbon films supported by a copper grid. The cell specimens exposed to NiO were subjected to TEM observations after appropriate preparation. Briefly, the cells were fixed with glutaraldehyde and OsO4 solution, dehydrated in ethanol, and embedded in epoxy resin. The resulting samples were cut into ultrathin sections suitable for TEM observations by diamond-knife ultramicrotomy. TEM observations were carried out using an H-7000 TEM (Hitachi, Tokyo, Japan), with an acceleration voltage of 75 kV. Statistics Analysis. Data are expressed as means ( SDs for at least three separate experiments. Statistical analyses were performed by the analysis of variance (ANOVA) using Dunnett’s or Tukey’s test for multiple comparisons. The calculation method has been described in each figure legend.

Results Cytotoxicities of Ultrafine and Fine NiO Particles toward Cultured Cells. To compare the cytotoxic activities of ultrafine and fine NiO particles, the viabilities and proliferation rates of NiO-exposed HaCaT and A549 cells were measured. Both the mitochondrial activity measured by the MTT assay and the cell membrane injury measured by the LDH assay indicated that ultrafine NiO particles were more cytotoxic toward the cells than fine NiO particles (Figure 1A,B). Similarly, the cell proliferation was more strongly inhibited by exposure to ultrafine NiO particles than by exposure to fine particles (Figure 1C). The UF-B NiO and ultrafine green (UF-G) NiO particles showed stronger cytotoxicities than the corresponding fine NiO particles. Among the fine particles, the cytotoxicity of fine black (F-B) NiO particles was stronger than that of fine green (F-G) NiO particles. This order corresponded to the cytotoxicity strengths in a previous study (14). In contrast, the cytotoxicity of the UF-G NiO particles was stronger than that of the UF-B NiO particles. The general cytotoxic effects of the different NiO particle types were in the following order: UF-G > UF-B > F-B > F-G. In particular, exposure to UF-G NiO particles caused a strong decrease in cell viability. To examine the association of the soluble material, namely, Ni2+, with the cytotoxicity, almost all of the NiO particles in the dispersions were removed by centrifugation at 16000g for 20 min, and the supernatants were applied to the cells (Figure 2). The supernatants only contained soluble nickel (Ni2+) already released from NiO because the NiO particles were almost completely removed by the centrifugation. Cytotoxicity was also observed with these supernatants since the viabilities were decreased in the supernatant-exposed

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Figure 1. Influences of fine and ultrafine NiO particles on the viability and proliferation of cultured cells. NiO particles were dispersed in the DMEM-FBS without a centrifugal fractionation process. The NiO DMEM-FBS dispersions were applied to HaCaT and A549 cells. After incubation for 24 h, the cell viability and proliferation were measured. (A) Cell viabilities based on mitochondrial activity measured by the MTT assay. (B) Cell viabilities based on cell membrane damage measured by the LDH assay. The calculation procedure for the cytotoxicity is described in the Materials and Methods. (C) Colony-forming ability measured by clonogenic assays. The black bar indicates the ultrafine particles, and the white bar indicates fine particles. The cell viabilities (A) and colony-forming ability rates (C) of all of the NiO-treated cells are significantly decreased as compared with untreated cells (P < 0.01). The cytotoxicity (B) of several NiO-treated cells is significantly decreased as compared with untreated cells (*P < 0.05 and **P < 0.01). #P < 0.05 and ##P < 0.01 indicate significant differences between the fine and the ultrafine NiO (ANOVA, Tukey).

cells. In comparisons of the cytotoxicity with the NiO concentration, the strength of the cytotoxic activity of the supernatant depended on the type of NiO, in the same order as the general cytotoxic effects shown in Figure 1, namely, UF-G > UF-B > F-B > F-G. Next, the influence of Ni2+ on the cell proliferation was examined. The cell proliferation was completely inhibited by 50 µg/mL Ni2+ (Figure 2C). Furthermore, the effects of particulate NiO on the cytotoxicity were examined. The Ni2+ concentrations in the supernatants

differed depending on the type of NiO and the Ni2+ concentration that corresponded to the cytotoxicity. However, even when the Ni2+ concentration in the medium was the same, the cytotoxic effect of NiO was stronger than that of NiCl2 (Figure 3). These results indicated that not only Ni2+ but also particulate NiO affected the cytotoxic activity. Furthermore, the differences in the cytotoxicity depending on the type of NiO particles, namely, influence of difference of solubility, were examined.

Cytotoxicity of NiO Particles by Ni(II) Release

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Figure 2. Influences of the soluble components released from fine and ultrafine NiO dispersion on the viability and proliferation of cultured cells. The centrifugal supernatants of NiO DMEM-FBS dispersions were applied to HaCaT and A549 cells. After incubation for 24 h, the cell viability and proliferation were measured. (A) Cell viability based on mitochondrial activity was measured by the MTT assay. (B) Cell viability based on cell membrane damage was measured by the LDH assay. The calculation procedure for the cytotoxicity is described in the Materials and Methods. (C) Colony-forming ability measured by clonogenic assays. The black bar indicates the ultrafine particles, and the white bar indicates fine particles. *P < 0.05 and **P < 0.01 vs untreated cells (ANOVA, Tukey). #P < 0.05 and ##P < 0.01 (ANOVA, Tukey).

Release of Ni2+ from Ultrafine and Fine NiO Particles in Culture Medium. The concentrations of Ni2+ released from NiO particles in DMEM-FBS were measured. NiO particles of each type (UF-B, F-B, UF-G, and F-G) were directly dispersed in DMEM-FBS at concentrations ranging from 0.1 to 50 mg/ mL. The dispersions were maintained at 37 °C for 24 h to replicate the conditions in the cell cultures or those inside the body. Under these conditions, the UF-B and UF-G particles released greater amounts of Ni2+ into the DMEM-FBS than the fine particles (Figure 4A). The order of the amounts of Ni2+ released from the NiO particles in DMEM-FBS was UF-G >

UF-B > F-B > F-G. This order agrees well with the order of the cytotoxicities exhibited by the NiO particles shown in Figures 1 and 2. The relationship between the initial concentration and the solubility of NiO was estimated (Figure 4B). For the ultrafine NiO particles, lower NiO concentrations were associated with higher NiO solubilities. In particular, when the initial concentration of UF-G particles was 5 mg/mL or less, the solubility was dramatically increased. For UF-G NiO particles, the rate of dissolution was largest for an initial NiO concentration of 50 µg/mL. The time course of dissolution of NiO particles in the DMEM-FBS was also measured (Figure

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Figure 3. Comparison of the effects of NiO and NiCl2 on cell viability based on the Ni2+ concentration. The cytotoxicities of NiO and NiCl2 based on the Ni2+ concentrations were examined. The data were estimated from the data shown in Figures 1 and 2. Circles, black NiO; triangles, green NiO; and squares, NiCl2. Solid lines, ultrafine particles; and broken lines, fine particles.

4C). The UF-B, F-B, and UF-G NiO particles rapidly released Ni2+ after they were dispersed in the DMEM-FBS. The released Ni2+ concentration reached equilibrium 1 day after preparation. Actually, in this study, because the NiO dispersions were used for the experiments 1 day after preparation, the extracellular Ni2+ concentration had not been changed for the experimental period. On the other hand, F-G NiO hardly released Ni2+. Furthermore, the effects of the medium components on the solubilities of the ultrafine NiO particles were examined. For all types of NiO particles, more Ni2+ was released into DMEM than into water. When amino acids were present in the dispersant, the release of Ni2+ from NiO particles was very efficient (Figure 5). On the other hand, the NiO particles dispersed in water or 2 mM CaCl2 aqueous solution hardly released any Ni2+. Because the amino acid solutions were acidic, we also examined whether the pH affected the solubility of NiO. Although the NiO particles released Ni2+ in an acidic solution of pH < 2.0 (aqueous solution adjusted with HCl), the NiO solubility was unaffected in the pH range used in this study (data not shown). In fact, the pH of each NiO dispersion was basic. Effects of a Uniform and Stable UF-B NiO Dispersion on the Cell Viability. NiO particles dispersed in DMEM-FBS immediately formed aggregates with proteins, and these large aggregates accumulated on cells because of gravity. The sedimentation of large aggregates influenced the “exposure concentration” for the cells (24). In other words, the “concentration of administration” and the “exposure concentration” were different. Therefore, to avoid overloading of the cell surface

by NiO particles because of gravity sedimentation, we prepared stable and uniform NiO dispersions by a preadsorption and centrifugation method (20). About only UF-B NiO, the stable and uniform NiO DMEM-FBS dispersions could be prepared. The characteristics of the NiO-DMEM-FBS dispersion fractions are shown in Table 2. The particle sizes in the two fractions of the UF-B NiO-DMEM-FBS dispersion after centrifugation at 4000g and 1000g were stable, and the number-averaged diameters of the aggregates in the two fractions were 68.4 and 107.7 nm, respectively. During the examination period, the light scattering intensity used to measure the particle sizes in the 4000g fraction did not change. On the other hand, the light scattering intensity in the 1000g fraction was decreased. Because the number-averaged particle size in the 1000g fraction did not change, the decrease in the light scattering intensity indicated the sedimentation of large aggregates. Although the dispersion stability of the 1000g fraction of the NiO dispersions was not entirely satisfactory, the UF-B NiO dispersion was reasonably uniform and stable. Therefore, we examined the influence of the uniform and stable NiO dispersions on cell viability. The dependence of the cytotoxicity of the UF-B NiO particles on the NiO concentration was investigated by diluting the NiODMEM-FBS dispersion with the culture medium (Figure 6). The aggregate sizes did not change even after the dispersion was diluted twice with DMEM-FBS. However, the sizes of the aggregates decreased when the dispersion was diluted 5 and 10 times. These observations indicate that high dilution leads to a change in the dispersion state and affects the particle size distribution. As shown in Figure 6 and Table 2, when the total



Figure 4. Ni2+-releasing abilities of fine and ultrafine NiO particles in DMEM. (A) Various amounts of the NiO particles were dispersed in DMEMFBS and incubated for 24 h at 37 °C. The dispersions were then centrifuged at 16000g for 20 min, and the Ni2+ concentrations in the supernatants were measured by 5-Br-PSAA. Filled symbols, ultrafine particles; and open symbols, fine particles. (B) Relationships between the initial concentration and the solubility of NiO particles. The solubilities of the NiO particles were estimated from the data shown in panel A. (C) The time course of dissolution of NiO particles in DMEM-FBS. Ten mg/mL of NiO particles was dispersed in the DMEM-FBS and incubated for 7 days at 37 °C. The concentration of Ni2+ was measured by the same way as described above after 3, 6, and 12 h and 1-7 days of incubation. The Ni2+ dissolution ratios of NiO are indicated as percentages as the moles of released Ni2+ against the moles of initial NiO.

nickel concentration was approximately 65 µg/mL, the 4000g and 1000g fractions included secondary particles of 48.4 and 103.4 nm, respectively. There was no significant difference between these dispersions regarding the decreases in the cell viability. These findings suggest that the decrease in the cell viability of the cultured cells exposed to the UF-B NiO particles was dependent on the total nickel concentration and not on the size of the secondary particles. Furthermore, we examined the relationship between the extracellular Ni2+ concentration and the cytotoxicity of UF-B NiO. The cells were exposed to the UF-B NiO dispersion or

NiCl2 solution and then changed to a nickel-free culture medium after 6 h. After incubation for an additional 18 h, the cell viabilities were measured. We used two types of UF-B NiO dispersions, containing secondary particles of 68.4 and 107.7 nm and total nickel concentrations of 328.6 and 129.9 µg/mL, respectively (see Table 2). All of the dispersions and solutions contained 50 µg/mL of soluble Ni2+. Exposure of HaCaT cells to the NiCl2 solution for 24 h resulted in a reduction in the cell viability to 70% (Figure 7). On the other hand, the cell viability was restored to the level of the control cells when the exposed cells were transferred from the NiCl2 solution to a Ni-free

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Figure 5. Ni2+-releasing abilities of fine and ultrafine NiO particles in various solutions. NiO particles (10 mg/mL) were dispersed in various solutions and incubated for 24 h at 37 °C. The dispersions were then centrifuged at 16000g for 20 min, and the Ni2+ concentrations in the supernatants were measured by 5-Br-PSAA. “DW + a.a.” indicates distilled water containing 1× MEM amino acid solution. The components of the amino acid solution are described in the Materials and Methods.

Table 2. Characterization of UF-B NiO Dispersions in DMEM-FBSa sample

dilution ratio

total nickel concn (µg/mL)

1

328.6 ( 6.5

2

164.3

4000g 5

65.72

10

32.86

UF-B NiO 1

129.9 ( 2.1

2

64.95

5

25.98

10

12.99

1000g

size (nm) dl dn dl dn dl dn dl dn dl dn dl dn dl dn dl dn

129.6 68.4 132.2 61.7 127.8 48.4 113.0 38.1 177.3 107.7 175.3 103.4 178.4 99.1 180.2 88.8

u (nm)

utime (nm)

uapp (nm)

umethod (nm)

4.2 11.0 6.0 14.4 6.4 18.9 7.7 15.9 8.7 7.6 8.4 8.0 5.3 15.0 7.6 21.2

1.7 3.6 3.1 1.4 0.9 0.5 3.1 1.1 2.9 7.1 0.8 4.2 2.0 3.6 1.5 3.4

2.5 10.3 4.5 14.3 5.8 18.0 4.7 14.2 7.6 2.6 7.6 4.6 3.2 13.9 5.9 20.6

3.0 1.5 2.7 1.0 2.4 5.8 5.2 7.0 3.2 1.1 3.5 5.1 3.8 4.3 4.6 3.7

a

dl, light scattering intensity-averaged diameter; and dn, number-averaged diameter. The total nickel concentration of a diluted solution was calculated from the measured value of an undiluted solution.

medium after 6 h of exposure. However, the viabilities of the cells exposed to NiO particles did not recover to the level of the control cells. Despite the removal of Ni2+ from the supernatant, the viability of the cells exposed to ultrafine NiO particles for 24 h became lower than that of cells exposed to the dispersion for 6 h. In other words, although the cytotoxicity of soluble nickel was lost after transfer of the cells to a nickelfree culture medium after 6 h, the cytotoxicity of the NiO particles remained unaffected. TEM Observations of NiO-DMEM-FBS Dispersions and HaCaT Cells Exposed to NiO. The secondary particles of NiO in the NiO-DMEM-FBS dispersions were observed using a TEM (Figure 8). The sizes of the secondary UF-B NiO particles agreed well with those estimated using DLS measurements. The outlines of the secondary UF-B NiO particles were unclear because of the adsorption of proteins onto the NiO particle surfaces. Moreover, the void between the primary particles was filled with proteins. The secondary UF-B NiO particles could represent a complex arrangement of primary NiO particles and proteins. The secondary UF-G NiO particles showed less clear

outlines and had smaller sizes than the secondary UF-B NiO particles. The degree of aggregation was lower, and the outline was clearer for fine NiO particles than for ultrafine NiO particles. HaCaT cells exposed to all types of NiO particles were observed using a TEM (Figure 9). The uptake of NiO particles into the cells after 6 h of exposure to UF-B, F-B, UF-G, and F-G was also observed. NiO particles were detected in the cytoplasm of the cells. However, no invasion of NiO particles into the nucleus was observed. In the case of UF-B NiO, intracellular NiO particles existed as aggregates within phagosome-like structures. Because the sizes of the intracellular UF-B NiO aggregates corresponded to those estimated from the DLS measurements, there was a very low probability of redistribution of the aggregates within a cell. The cells exposed to the UF-G NiO particles were considerably damaged, since the number of intracellular NiO particles was lower than those for the other types of NiO particles. Because of their high solubility in biological fluids, the UF-G NiO particles possibly dissolved in the cells after uptake, resulting in high concentrations of intracellular Ni2+ and drastic cell injury. Although the cells were



Figure 6. Influences of a stable ultrafine NiO dispersion on cell viability. A uniform and stable UF-B DMEM-FBS dispersion prepared by a preadsorption and centrifugation method was applied to HaCaT and A549 cells. After incubation for 24 h, the cell viabilities were measured by the MTT assay. The total nickel concentration and the secondary particle sizes were simultaneously measured with the MTT assay. The numbers and arrows in the top panel indicate the number-averaged diameters (nm) of the UF-B NiO secondary particles. The 100% cell viability value indicates the viability of the cells that were treated with DMEM-FBS without nickel compounds.

exposed to NiO dispersions at the same concentration (w/v), the fine NiO particles immediately sank and accumulated on the cells, consequently leading to differences in the numbers of NiO particles in the cells for different types of NiO particles. Therefore, comparisons between the cellular uptakes of the different types of NiO particles using TEM observations were not suitable for comparing the cytotoxicities of the different types of NiO particles.

Discussion According to previous in vitro studies, soluble nickel compounds are less toxic and carcinogenic than particulate nickel compounds (1, 25, 26). Although soluble nickel compounds (e.g., NiCl2) and insoluble nickel compounds (e.g., Ni3S2) basically induce the same cellular responses, soluble nickel compounds need to be present at higher intracellular concentrations than insoluble nickel compounds to induce the same intensity of toxicity (8). This difference in toxicity between soluble and insoluble nickel compounds occurs because it is difficult for soluble nickel to enter a cell. Therefore, soluble nickel compounds require a greater quantity of the parent compound than poorly soluble nickel compounds to produce the same concentration of intracellular Ni2+. Moreover, the intranuclear Ni2+ concentration in CHO cells was found to increase after exposure to particulate nickel compounds but not after exposure to soluble nickel compounds (27). Although extracellular Ni2+ blocks the influx

Figure 7. Effects of medium exchange on the cytotoxicities of NiO and NiCl2. The uniform and stable UF-B DMEM-FBS dispersion or NiCl2 solution was applied to HaCaT and A549 cells. Each dispersion included the same concentration of Ni2+ (approximately 54 µg/mL). After incubation for 6 (A) and 24 h (B), the cell viabilities were measured by the MTT assay. In other experiments, the cells were incubated for 6 h, before the medium including nickel compounds was exchanged for fresh medium without any nickel compounds (C). The cell viabilities were estimated as relative values against the untreated cells (100%). The numbers in the middle and bottom panels indicate the number-averaged diameters (nm) of the UF-B NiO secondary particles. The 100% cell viability value indicates the viability of the cells that were treated with DMEM-FBS without nickel compounds. *P < 0.025 and **P < 0.001 (Tukey, ANOVA). Except for the effect of NiCl2 (C) on HaCaT cells, the cell viability is significantly decreased against untreated cells by all of the treatments (P < 0.05, Dunnett, and ANOVA).

of Ca2+ into a cell, it cannot enter the cell through ion channels (11). Therefore, extracellular Ni2+ will influence the intracellular Ca2+ balance, and an imbalance in the intracellular Ca2+ concentration can cause cellular damage. However, the cytotoxicity caused by extracellular Ni2+ is evanescent, especially in vivo. In contrast to in vitro experiments in which Ni2+ is not cleared, clearance of Ni2+occurs rapidly in vivo. Moreover, medium exchange after 6 h of exposure to NiCl2 resulted in restoration of the cell viability to a similar level to that observed in control cells.

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Figure 8. TEM observations of NiO-DMEM-FBS dispersions. NiO particles in DMEM-FBS dispersions were observed by TEM. The diameters of the secondary UF-B NiO particles measured by DLS are shown in Table 2. The diameters of the other particles could not be detected by DLS because the dispersions were unstable.

Figure 9. TEM observations of NiO-exposed HaCaT cells. HaCaT cells were exposed to NiO DMEM-FBS dispersions for 6 h.

In contrast, even though extracellular Ni2+ was removed by the medium exchange, UF-B NiO exposure decreased the cell viability. These results indicate that the cellular responses induced by NiO are essentially caused by the Ni2+ released inside the cells. The difference in cytotoxicity between the particulate nickel compounds, including NiO, and the soluble nickel compounds is caused by cellular uptake and subsequent retention within the cell. Internalized NiO acts as an intracellular Ni2+ source. All four types of NiO particles (UFG, UF-B, F-G, and F-B) examined were internalized into the cells. The internalized ultrafine NiO particles probably released Ni2+ inside the cells, which subsequently attacked biological molecules such as enzymes and nucleic acids. The uptake of NiO particles into cells is a necessary, but not sufficient, condition for the cytotoxicity of NiO. If the dissolubility of NiO is very low, the cytotoxic activity may also be very weak even after particle uptake into a cell. For example, although F-G NiO particles were internalized in

the cells as efficiently as the other types of NiO particles, their cytotoxicity was weaker than those of the other particle types. This low toxicity of F-G NiO is caused by its low solubility. The F-G NiO particles hardly dissolved, even in culture medium. Therefore, the solubility of particulate nickel compounds affects their cytotoxicity and the induced cellular responses. Generally, NiO has been classified as an insoluble and weakly toxic material among particulate nickel compounds (1). Moreover, black NiO has higher solubility than green NiO among the NiO crystalline phases, leading to stronger cytotoxicity of black NiO as compared with green NiO (25, 27). In the present study, the intensities of the cytotoxicities obtained for the fine NiO particles were similar to those obtained in previous investigations. However, the intensities of the cytotoxicities obtained for ultrafine NiO particles were contrary to those obtained for fine particles. Specifically, UF-G NiO exhibited higher solubility and stronger cytotoxicity than UF-B NiO in vitro. The order of


the intensity of cytotoxicity conformed to the order of the solubility. These results indicate that the cytotoxicity of NiO particles is determined by their solubility. Surprisingly, the solubilities of NiO particles increased drastically when they were nanosized particles. These findings suggest that ultrafine NiO particles have stronger cytotoxic potential than fine particles. The cytotoxicity of nickel compounds is induced by the concentrations of both extracellular and intracellular Ni2+. Intracellular Ni2+ is generated from internalized NiO particles that are moderately soluble. The present study has demonstrated that ultrafine NiO particles have higher solubility in culture medium than fine NiO particles. A low initial concentration of ultrafine NiO particles led to an increase in their solubility. Therefore, internalized ultrafine NiO particles dissolve to greater extents within the cells. Because of their higher solubility at low concentrations, UF-G NiO particles were not detected in the cells. These results suggest that UFNiO particles dissolve promptly in cells and subsequently induce cytotoxicity. Moreover, UF-G and UF-B NiO showed higher solubilities in solutions containing amino acids than in distilled water. In particular, UF-G NiO showed remarkable solubility in DMEM-FBS. UF-NiO released Ni2+ in the solution containing amino acids because nickel molecules on the surface of the NiO particles formed complexes with the amino acids. Small amounts of Ni2+ were also released in the BSA solution. Kasprazak and Sunderman (28) reported that Ni2+ from Ni3S2 in serum formed complexes with albumin and free amino acids. In the present study, although the details of the NiO solubilization mechanism in biological fluids remain unclear, the solubility of ultrafine NiO particles in biological fluids is important for evaluation of ultrafine NiO toxicity. Many free amino acids and albumin exist in the human body. For example, 40% of the primary contents of skin moisturizers are free amino acids (29). Saliva, oral mucosa, and alveolar surfactants also contain proteins including albumin (30, 31). Although ultrafine metal oxide particles did not reach the dermis after skin exposure (32, 33), it is possible that Ni2+ released from ultrafine NiO particles adheres to the skin or respiratory tract and subsequently penetrates the deep tissues. However, even highly soluble particles such as UF-G NiO are rapidly cleared in vivo, and such particles possibly induce only acute and evanescent toxicity. In fact, some in vivo experiments have indicated that Ni2+ is removed by rapid lung clearance in experimental animals and humans (34, 35). Therefore, Ni2+ does not trigger severe reactions in vivo because of its rapid clearance but does induce inflammatory reactions, which are transient responses. Ultrafine NiO particles are more toxic than fine NiO particles because they can release greater amounts of Ni2+ than fine NiO particles. From the results stated above, we conclude that UF-NiO particles have higher potentials for biological activity than F-NiO particles. One of the causes of the toxic activity of ultrafine particles is their increased solubility. For example, green NiO fine particles are insoluble and/or poorly soluble, whereas green NiO ultrafine particles are readily soluble. Therefore, understanding of solubility is very important for evaluating ultrafine metal oxide particles such as NiO. In the present study, the threshold level of cytotoxicity induced by Ni2+ was estimated to be 50 µg/mL. If the solubility is very low, such as that of the F-G NiO particles examined in the present study, the cytotoxicity of these “insoluble” particles will be lower than those of soluble nickel compounds such as NiCl2. The intensities of the toxicities of the nickel


compounds examined were in the order of poorly soluble particles such as UF-G NiO, UF-B NiO, and F-B NiO followed by soluble compounds such as NiCl2 followed by insoluble particles such as F-G NiO. In the present study, we have clarified an interaction between the solubility and the cytotoxicity of ultrafine NiO particles. However, knowledge of solubility is still insufficient for other ultrafine metal oxide particles. Therefore, it will be necessary to clarify the solubilities of metal oxide nanoparticles in biological fluids in future studies. Moreover, further analyses of the effects of Ni2+ clearance are essential for estimating the in vivo toxicity of NiO. Acknowledgment. This work was funded by a grant from the New Energy and Industrial Technology Development Organization of Japan (NEDO) entitled “Evaluating risks associated with manufactured nanomaterials (P06041)”. We thank Dr. Yoshiro Saito (Doshisha University) for excellent advice.

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TX900171N

Ultrafine NiO Particles Induce Cytotoxicity in Vitro by Cellular Uptake

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