chromium(iii) oxide nanoparticles induced remarkable oxidative stress and apoptosis on culture cells

Chromium(III) Oxide Nanoparticles Induced RemarkableOxidative Stress and Apoptosis on Culture Cells

Masanori Horie,1 Keiko Nishio,1 Shigehisa Endoh,2 Haruhisa Kato,3 Katsuhide Fujita,4

Arisa Miyauchi,2 Ayako Nakamura,3 Shinichi Kinugasa,3 Kazuhiro Yamamoto,5

Etsuo Niki,1 Yasukazu Yoshida,1 Hitoshi Iwahashi1

1Health Research Institute (HRI), National Institute of Advanced Industrial Science and Technology(AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan

2Research Institute for Environmental Management Technology (EMTECH), AIST, PolymerStandards Section Japan (PSSJ), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan

3National Metrology Institute of Japan (NMIJ), AIST, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan

4Research Institute of Science for Safety and Sustainability (RISS), AIST, 16-1 Onogawa, Tsukuba,Ibaraki 305-8569, Japan

5Research Institute of Instrumentation Frontier (RIIF), AIST, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8565, Japan

Received 28 August 2010; revised 11 December 2010; accepted 18 December 2010

ABSTRACT: Chromium(III) oxide (Cr2O3) is used for industrial applications such as catalysts and pigments.In the classical form, namely the fine particle, Cr2O3 is insoluble and chemically stable. It is classified as alow-toxicity chromium compound. Recently, industrial application of nanoparticles (a new form composed ofsmall particles with a diameter of �100 nm, in at least one dimension) has been increasing. Cellular effectsinduced by Cr2O3 nanoparticles are not known. To shed light upon this, the release of soluble chromium fromCr2O3 nano- and fine-particles in culture medium was compared. Fine Cr2O3 particles were insoluble in theculture medium; on the contrary, Cr2O3 nanoparticles released soluble hexavalent chromium into the culturemedium. Cr2O3 nanoparticles showed severe cytotoxicity. The effect of Cr2O3 nanoparticles on cell viabilitywas higher than that of fine particles. Cr2O3 nanoparticles showed cytotoxicity equal to that of hexavalentchromium (K2Cr2O7). Human lung carcinoma A549 cells and human keratinocyte HaCaT cells showed anincrease in intracellular reactive oxygen species (ROS) level and activation of antioxidant defense systems onexposure to Cr2O3 nanoparticles. Exposure of Cr2O3 nanoparticles led to caspase-3 activation, showing thatthe decrease in cell viability by exposure to Cr2O3 nanoparticles was caused by apoptosis. Cellularresponses were stronger in the Cr2O3 nanoparticles-exposed cells than in fine Cr2O3- and CrCl3-exposedcells. Cellular uptake of Cr2O3 particles were observed in nano- and fine-particles. The cellular influence ofthe extracellular soluble trivalent chromium was lower than that of Cr2O3 nanoparticles. Cr2O3 nanoparticlesshowed cytotoxicity by hexavalent chromium released at outside and inside of cells. The cellular influencesof Cr2O3 nanoparticles matched those of hexavalent chromium. In conclusion, Cr2O3 nanoparticles have ahigh cytotoxic potential. # 2011 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2011.

Keywords: chromium oxide; nanoparticle; oxidative stress; hexavalent chromium; apoptosis

Correspondence to:M. Horie; e-mail: [email protected]

Contract grant sponsor: New Energy and Industrial Technology Devel-

opment Organization (NEDO); Contract grant number: P06041 (entitled

‘‘Evaluating risks associated with manufactured nanomaterials’’)

Published online in Wiley Online Library (wileyonlinelibrary.com).

DOI 10.1002/tox.20695

�C 2011 Wiley Periodicals, Inc.

1

INTRODUCTION

Among chromium oxide compounds, Cr2O3, trivalent chro-

mium (Cr(III)) is an important industrial material. It has

been used for catalysis, as abrading agents, and as pigments

(e.g., Viridian). Cr2O3 is chemically stable and insoluble in

acidic and basic solutions. Trivalent chromium is an essen-

tial element for human health and has a role in maintaining

the appropriate metabolism of carbohydrates and lipids by

enhancing insulin signaling (Vincent, 2000, 2004).

The toxicity of Cr2O3 is lower than that of hexavalent

chromium (Cr(VI)) compounds such as CrO3, which is

water soluble and a strong oxidant (Barceloux, 1999;

Hininger et al., 2007; Urrutia et al., 2008). There have been

many investigations focusing on the carcinogenicity of hex-

avalent chromium (De Flora and Wetterhahn, 1989;

Holmes et al., 2008). Although the mechanism of chro-

mate-induced lung cancer is incompletely understood, it is

thought that genomic instability is involved (Holmes et al.,

2008).

A carcinogenesis mechanism for hexavalent chromium

has been proposed (Holmes et al., 2008). Briefly, the cyto-

toxic effect of particulate hexavalent chromium such as

lead chromate (PbCrO4) is dependent on the particle solu-

bility. The particulate chromium internalizes to the cell by

phagocytosis, but the ‘‘particle’’ does not affect the cell

(Xie et al., 2004). The particulate hexavalent chromium dis-

solved in the extracellular fluid and soluble Cr(VI) enter the

cell by crossing the cell membrane via an anion transporter

(De Flora and Wetterhahn, 1989; Wise et al., 2004). The in-

tracellular Cr(VI), which enters via ion channels or is

released from internalized particulate hexavalent chro-

mium, is rapidly reduced to Cr(III) (De Flora and Wetter-

hahn, 1989). The reduction of hexavalent chromium to tri-

valent chromium produces reactive oxygen species (ROS),

resulting in oxidization of lipids and proteins. The lipid per-

oxide and oxidized proteins induce metabolic dysfunction

and chromosomal variation (Bagchi et al., 2002; Mattagaja-

singh et al., 2008). Compared with hexavalent chromium,

the cellular influences of trivalent chromium are less signif-

icant because trivalent chromium is more chemically stable

than hexavalent chromium. Additionally, extracellular

Cr(III) has more difficulty in passing through the cell mem-

brane than Cr(VI) (De Flora and Wetterhahn, 1989; Barce-

loux, 1999). Therefore, trivalent chromium compounds

such as Cr2O3 are less cytotoxic than hexavalent chromium

(Hininger et al., 2007).

Nevertheless, some of the biological effects of trivalent

chromium are known. Continuous exposure of human la-

ryngeal carcinoma Hep-2 cells to trivalent chromium

induced cell membrane injury and subsequent apoptosis

(Rudolf and Cervinka, 2003). It has been reported that tri-

valent chromium induced oxidative stress in the brain, liver,

and kidney of goldfish (Lushchak et al., 2009a,b). Cr(III) is

involved in the formation of a Cr-DNA adduct and inhibits

DNA replication (Zhitkovich et al., 2001; Zhitkovich,

2005). Although the biological effect of trivalent chromium

is lower than that of Cr(VI), attention to the toxic effect of

the former is necessary (Levina and Lay, 2008).

Similar to chromium compounds, the toxicity of nickel

compounds is affected by their solubility. In general,

among nickel compounds, nickel oxide (NiO) is classified

as having low toxicity because it is insoluble in water.

Additionally, black NiO has a higher solubility and toxicity

than green NiO (Takahashi et al., 1992).

Nanoparticles (particles with a diameter of �100 nm, in

at least one dimension) have larger surface area per weight

than fine particles (classical larger form). Therefore, the

chemical and physical reactivities of nanoparticle per

weight are larger than that of fine particles. For example,

although NiO is classified as insoluble material in water,

green NiO nanoparticles have higher solubility in water and

in culture media, and NiO nanoparticles show strong cyto-

toxicity than fine particles (Horie et al., 2009b). These

properties induce not only industrial benefits, but also novel

biological effects. Precise assessment of the biological

effects of nanoparticles is, therefore, important for the pre-

cise use of such materials. Understanding the properties of

the cellular response and the response mechanism induced

by nanoparticles are indispensable.

Details of properties of the soluble chromium release from

Cr2O3 nanoparticle in biological fluids and subsequent bio-

logical effects induction are not known. In this study, the bio-

logical effects induced by Cr2O3 nanoparticles were exam-

ined using cultured cells. In particular, we focused on the

soluble chromium release in culture medium and the induc-

tion of oxidative stress. The cellular responses of Cr2O3 nano-

particles were compared with those of fine Cr2O3 particles

and hexavalent chromium compound (K2Cr2O7).

MATERIALS AND METHODS

Cell Culture

Human keratinocyte HaCaT cells were purchased from the

German Cancer Research Center (DKFZ, Heidelberg, Ger-

many). Human lung carcinoma A549 cells were purchased

from the Riken BioResource Center (Tsukuba, Ibaraki, Ja-

pan). These cells were cultured in Dulbecco’s modified

Eagle’s medium (DMEM; Gibco, Invitrogen, Grand Island,

NY) supplemented with 10% heat-inactivated fetal bovine

serum (FBS; CELLect GOLD; MP Biomedicals, Solon,

OH), 100 units/mL of penicillin, 100 lg/mL of streptomy-

cin, and 250 ng/mL of amphotericin B (Nacalai Tesque,

Kyoto, Japan). In this study, this ‘‘DMEM cocktail’’ was

called ‘‘DMEM-FBS.’’

DMEM cultures were placed in a 75-cm2 flask (Corning,

Corning, NY) and incubated at 378C in an atmosphere of

5% CO2. For cellular examinations, cells were seeded in

2 HORIE ET AL.

Environmental Toxicology DOI 10.1002/tox

96-well or six-well plates (Corning) at 2 3 105 cells/mL

and incubated for 24 h. Subsequently, the culture medium

was removed, and cells were subjected to metal-oxide dis-

persion and incubated for a further 24 h.

Cr2O3 Particles and Preparation of Cr2O3

Dispersions in DMEM-FBS

Cr2O3 nanoparticles were purchased from Nanostructured &

Amorphous Materials (Houston, TX). According to the man-

ufacturer’s data sheet, the primary particle size was 60 nm

and was obtained from transmission electron microscopy

(TEM) images. Fine Cr2O3 particles and chromium(III) chlo-

ride (CrCl3) were purchased from Kanto Chemical Co. (To-

kyo, Japan). Potassium dichromate (K2Cr2O7) was purchased

fromWako Pure Chemical Industries (Osaka, Japan).

Specific surface area was measured by the Brunauer,

Emmett, and Teller (BET) method. Primary particle size

(Sauter mean diameter) was also estimated by the BET

method. For the cytotoxicity assays described in Figures 1–3,

Cr2O3 powder was dispersed directly in DMEM-FBS at

0.1–10 mg/mL without a dispersant. For some examina-

tions described in Figures 4–9, stable and uniform

Fig. 1. Effects of fine- and nano-Cr2O3 particles on cell viabil-ity. Cr2O3 particles were dispersed in DMEM-FBS without cen-trifugal fractionation. NiO-DMEM-FBS dispersions wereapplied to HaCaTcells and A549 cells. After incubation for 24h, the viability and proliferation of cells weremeasured. Cell via-bilities based on mitochondrial activity and cell membranedamage were measured by the MTT assay and LDH assay,respectively. The calculation for the cytotoxicity is described intheMaterials andMethods section. The black bars indicate thenanoparticle and the white bars indicate fine particles. **P\0.01 indicates significant differences against nonexposedcells. #P\0.05 and ##P\0.01 indicate significant differencesbetween the fine- and nano-Cr2O3 particles (ANOVA, Tukey).

Fig. 2. Effects of soluble components released from fine-and nano-Cr2O3 particles dispersions on cytotoxicity. Thecentrifugal supernatants of NiO-DMEM-FBS dispersionswere applied to HaCaTcells and A549 cells. After incubationfor 24 h, cell viabilities were measured. Cytotoxicities basedon mitochondrial activity and cell membrane damage weremeasured by the MTT assay and LDH assay, respectively.The calculation for the cytotoxicity is described in the Mate-rials and Methods section. The black bars indicate nanopar-ticles, and the white bars indicate fine particles. *P \ 0.05and **P\ 0.01 indicate significant differences against non-exposed cells. ##P \ 0.01 indicates significant differencesbetween the fine- and nano-Cr2O3 particles (ANOVA, Tukey).

3CYTOTOXICITY OF Cr2O3 NANOPARTICLE


dispersions were prepared using a previously described pre-

adsorption and centrifugation method (Horie et al., 2009a).

Nanoparticles induce artificial cellular influences by me-

dium depletion because they can adsorb protein (Casey et al.,

2008; Horie et al., 2009a). To prevent cell starvation caused

by adsorption of medium components onto the particle sur-

face, Cr2O3 particles were initially dispersed in FBS at 80

mg/mL. The dispersion was subsequently centrifuged at

16,000 3 g for 20 min. Precipitated Cr2O3 particles were

washed once with FBS-free DMEM and re-dispersed in an

equivalent volume of fresh DMEM-FBS. This dispersion of

nanoparticles in DMEM-FBS was centrifuged at 8000 3 gfor 20 min. After discarding the supernatant, the precipitate

was resuspended in an equal volume of fresh DMEM-FBS,

and the resulting Cr2O3 dispersion was centrifuged at 40003

g for 20 min. The supernatant was collected as a Cr2O3-

DMEM-FBS dispersion. The Cr2O3-DMEM-FBS dispersions

used in this study did not lead to medium depletion. Adsorp-

tion effects were verified by measuring the concentrations of

protein and calcium in the DMEM-FBS dispersions.

Characterization of Cr2O3-DMEM-FBSDispersions

In this study, we defined ‘‘secondary particles’’ as complex

aggregates of primary particles, proteins from FBS, and

other medium components such as Ca21. The Cr2O3-

DMEM-FBS dispersions prepared by the methods stated

above were divided into three parts and used for simultane-

ous biological examinations, chromium concentration

measurements, and particle-size measurements.

Secondary particle sizes in the Cr2O3-DMEM-FBS dis-

persions were measured by dynamic light scattering (DLS),

as described previously (Kato et al., 2009). Estimated diam-

eters of the secondary particles were calculated as the mean

of three measurements at different wavelengths taken with

the following devices: a DLS-7000 spectrophotometer (633

nm; Otsuka Electronics Co., Hirakata, Japan), an FPAR-

1000 fiberoptic particle analyzer (660 nm; Otsuka Electron-

ics Co.), and a Nanotrac machine (780 nm; Nikkiso Co.,

Tokyo, Japan). Undiluted dispersions were used in these

measurements. Measurements were carried out at 25.08C 60.18C with sample concentrations of 50–80 lg/mL. The

samples for particle-size measurements and cytotoxicity

assays were obtained 1 cm from the surface of the solutions

in static 15-mL tubes. Viscosities of the dispersions were

measured using an Ubbelohde viscometer number 0C

(Sibata Scientific Technology, Tokyo, Japan). DLS can be

used to calculate particle size but has low reliability in cal-

culating particle-size distribution.

The total concentration of chromium was measured by

X-ray fluorescence (XRF). Briefly, 13 mL of the Cr2O3-

DMEM-FBS dispersion was added to 13 mL of a standard

solution containing 0.1 mg/mL of iron (internal standard)

and mixed well. Next, 5 mL of the mixture was dried in an

oven at 2008C for 24 h. The dried sample was ground in an

agate mortar and subjected to XRF analyses using an

energy-dispersive XRF spectrometer (JSX-3201; Jeol, To-

kyo, Japan). The amount of chromium was estimated from

the molar ratio of chromium and the internal standard.

The concentration of soluble chromium was measured

by inductively coupled plasma atomic emission spectrome-

try (ICP-AES, SPS-4000; Seiko Instruments, Chiba, Japan)

in conjugation with ultrafiltration. To completely remove

particulate Cr2O3, the dispersion was kept standing for 24

h. Then, 10 mL of the dispersion was applied to the ultrafil-

tration membrane (molecular weight cutoff was 5000:

Vivaspin 20–50 k, GE Healthcare, Buckinghamshire, UK)

and centrifuged at 6,000 3 g for 50 min. After the mem-

brane was washed thrice with 5 mL of Milli-Q water, the

Fig. 3. Soluble chromium-releasing abilities of fine- andnano-Cr2O3 particles in DMEM. (A) Various amounts ofCr2O3 particles were dispersed in DMEM-FBS and kept for24 h. The dispersion was then separated to particle andsoluble chromium by ultrafiltration. The amount of chromiumin the filtrate (soluble chromium) was detected by ICP-massspectrometry (MS). The black circles indicate Cr2O3 nano-particles, and the white squares indicate fine Cr2O3 par-ticles. (B) Determination of oxidation state of soluble chro-mium released from Cr2O3 nanoparticles. The oxidationstate of soluble chromium was determined by diphenylcar-bazide color comparison method. The value was indicatedas K2Cr2O7 equivalence.

4 HORIE ET AL.


Fig. 4. Comparison of the effect of Cr2O3 nanoparticles, Cr2O3 fine particles, CrCl3, andK2Cr2O7 on mitochondrial activity. (A) The influence of chromium compounds on cell via-bility. HaCaT cells and A549 cells were exposed to the Cr2O3 nanoparticle, the mixture ofCr2O3 fine particle and CrCl3, 1 mM CrCl3 solution, and 0.1 and 1 mM K2Cr2O7 solution for6 and 24 h. Mitochondrial activity was then measured by the MTT assay. The Cr2O3 nano-particles dispersion contained 88 lg/mL of Cr2O3 particles and 1 mM soluble chromium.The fine Cr2O3 dispersion contained 88 lg/mL of Cr2O3 particles and 1 mM CrCl3 solution*P\0.05 and **P\ 0.01 versus unexposed cells (ANOVA, Dunnett). (B) Effect of mediumchange on the cytotoxicities of Cr2O3 and K2Cr2O7. Cr2O3 nanoparticles and K2Cr2O7 so-lution was applied to HaCaT and A549 cells. Each dispersion included the same concen-tration of soluble chromium (�1 mM). The cells were exposed to the Cr2O3 nanoparticlesdispersion or K2Cr2O7 solution and then changed to a chromium-free culture medium after2 h. After incubation for additional 22 h, the mitochondrial activities were measured byMTT assay. The MTT conversion was estimated as relative values against the unexposedcells (100%). **P\0.01 versus unexposed cells (ANOVA, Dunnett).



amount of chromium in the filtrate was measured by ICP-

AES. DLS was carried out on the filtrate, and light intensity

was not observed. This observation indicated that the par-

ticles were completely removed by ultrafiltration.

Hexavalent chromium concentration was determined by

diphenylcarbazide color comparison method using simpli-

fied assay kit ‘‘PACKTEST’’ (Kyoritsu Chemical-Check

Laboratory, Corp., Tokyo, Japan). To remove the particles,

Cr2O3 DMEM-FBS dispersion (10 mg/mL) was centrifuged

at 16,000 3 g for 20 min. Then, the supernatant or CrCl3solution was mixed with PACKTEST reagent. Subse-

quently, optical density at 540 nm (OD540) was measured.

K2Cr2O7 was used as standard of Cr(VI). The values were

indicated as K2Cr2O7 equivalence.

Measurement of the Cytotoxicity andColony-Forming Ability of Cells

For the cell viability assay, cells were seeded in a 96-well

plate (Corning) at 2 3 105 cells/well. They were incubated

for 24 h, and the culture medium was removed. Subse-

quently, Cr2O3-DMEM dispersion or chromium compound

solution was applied, and the cells were incubated for a fur-

ther 6 and 24 h. In some experiments, the cells were pre-

treated with N-acetylcysteine (NAC; Sigma-Aldrich, St.

Louis, MO). The cells were seeded at 1 3 105 cells/mL and

incubated for 24 h. Subsequently, the medium was changed

to fresh medium containing 2 mM NAC and incubated for

another 24 h. Then, the medium was removed and exposed

to the Cr2O3 dispersion or K2Cr2O7 solution containing 2

mM NAC and incubated for another 2 h. Then, the disper-

sion or solution was changed to fresh medium containing 2

mM NAC and incubated for another 22 h.

For the determination of mitochondrial activity, the 3-

(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bro-

mide (MTT) assay was used. Cells were incubated with 0.5

mg/mL MTT (Nacalai Tesque) at 378C for 2 h. Isopropyl

alcohol containing 40 mM HCl was added to the culture

medium (3:2 by volume). These components were mixed

by pipette until the formazan was completely dissolved.

The optical density of formazan was measured at OD570

using a Multiskan Ascent plate reader (Thermo Labsys-

tems, Helsinki, Finland). In the lactate dehydrogenase

(LDH) assays, LDH release was measured with a tetrazo-

lium salt using a Cytotoxicity Detection Kit PLUS (LDH)

(Roche Diagnostics GmbH, Mannheim, Germany) accord-

ing to the manufacturer’s protocol. The amount of forma-

zan salt formed was measured at 492 nm using the Multis-

kan Ascent plate reader. The maximum amount of LDH

released was determined by incubating the cells with a lysis

solution provided in the kit. Cytotoxicity was calculated

using the following equation:

Cytotoxicity ð%Þ ¼ ðexperimental value � low controlÞ=ðhigh control � low controlÞ3100

where the low control (refers to spontaneous LDH release)

was determined as the LDH released from untreated normal

cells and the high control (refers to the maximum release of

LDH) was determined as the LDH released from cells lysed

by surfactant treatment.

Colony-forming ability was examined by a clonogenic

assay based on the methods described by Herzog et al. (2007)

and Franken et al. (2006). Cells were seeded in six-well plates

(Corning) at 300 cells/well. Each well contained 2 mL of the

cell culture medium. Cells were allowed to attach for �14 h

before they were washed with phosphate-buffered saline

(PBS) and treated with 2 mL of centrifuged supernatant. Cells

were cultured over the time period needed by the control cells

to form colonies, whereby one colony was defined as �50

cells. Here, the cells were cultured for 7 days. After the cul-

ture procedure was completed, supernatants were removed

and cells were washed once with 2 mL of PBS. Cells were

then fixed by 100% methanol for 15 min. After the methanol

was removed, cells were stained by Giemsa’s staining solu-

tion (Nacalai Tesque) diluted 50 times with water for 15 min

and rinsed once with distilled water. The number of colonies

was then counted.

Measurement of Intracellular Oxidative Stress

Intracellular ROS levels were detected using 20,70-dichloro-fluorescin diacetate (DCFH-DA; Sigma-Aldrich). DCFH-

DA was dissolved in dimethyl sulfoxide (DMSO) at 5 mM

Fig. 5. Comparison of the effect of Cr2O3 nanoparticles,Cr2O3 fine particles, CrCl3, and K2Cr2O7 on colony-formingability. The Cr2O3 nanoparticles dispersion, the mixture offine Cr2O3 particles and CrCl3, 1 mM of CrCl3 solution, and0.1 and 1 mM K2Cr2O7 solution were applied to HaCaT cellsand A549 cells. Colony-forming ability was measured by aclonogenic assay. The black bars indicate HaCaT cells, andthe white bars indicate A549 cells. The Cr2O3 nanoparticlesdispersion contained 88 lg/mL of Cr2O3 particles and 1 mMsoluble chromium. The fine Cr2O3 dispersion contained 88lg/mL of Cr2O3 particles and 1 mM CrCl3 solution. **P \0.01 versus unexposed cells (ANOVA, Dunnett).

6 HORIE ET AL.


as a stock solution and stored at 2208C; when used in an

experiment, it was diluted 500 times with serum-free me-

dium. Cells were exposed to Cr2O3 dispersion for 2, 6, 12,

and 24 h. The dispersion was then changed to serum-free

DMEM that included 10 lM of DCFH-DA and incubated

for 30 min at 378C. Cells were then washed once with PBS,

collected by 0.25% trypsinization, washed once again with

PBS, and resuspended in 500 lL of PBS. Cell samples in

PBS were excited with a 488-nm argon ion laser in a

Cytomics FC500 flow cytometry system, and the emission

of 20,70-dichlorofluorescein (DCF) was recorded at 525 nm.

Data were collected from at least 5000 gated events.

Fig. 6. Comparison of the effect of Cr2O3 nanoparticles, Cr2O3 fine particles, CrCl3, andK2Cr2O7 on the intracellular ROS level. (A) Effects of NAC on mitochondrial activity. Thecells were pretreated with 2 mM NAC for 24 h. Then, the cells were exposed to the Cr2O3

dispersion or K2Cr2O7 and changed to a chromium-free culture medium containing 2 mMNAC after 2 h. After incubation for additional 22 h, the mitochondrial activities were meas-ured by MTT assay. Significant differences are shown in the figure (ANOVA, Tukey). **Sig-nificantly higher than unexposed cells (P\ 0.01). (B) The Cr2O3 nanoparticle dispersion,the mixture of fine Cr2O3 particle and CrCl3, 1 mM of CrCl3 solution, and 0.1 and 1 mMK2Cr2O7 solution were applied to HaCaT cells and A549 cells. The intracellular ROS levelwas measured by the DCFH method after exposure for 2, 6, 12, and 24 h. The intracellularROS level is shown by a relative value to unexposed cells. The Cr2O3 nanoparticle disper-sion contained 88 lg/mL of Cr2O3 particles and 1 mM soluble chromium. The fine Cr2O3

dispersion contained 88 lg/mL of Cr2O3 particles and 1 mM CrCl3 solution. The intracellu-lar ROS level of HaCaT cells exposed to Cr2O3 nanoparticle dispersion for 24 h could notbe measured. Significant differences are shown in the figure (versus unexposed cells,ANOVA, Tukey). **Significantly higher than unexposed cells (P\0.01).



Determination of Glutathione Content

Intracellular glutathione (GSH) content was determined

enzymatically using 5,50-dithiobis-(2-nitrobenzoic acid)

(DTNB), according to the method of Anderson (1985).

Treated cells (1.7 3 105 cells) were harvested by trypsini-

zation, washed with cold PBS, and resuspended in 190 lLof PBS. An equal volume of ice-cold 10% trichloroacetic

acid (TCA) was added to 150 lL of the cell suspension.

Then, cell samples were vortex-mixed for 1 min and centri-

fuged at 1000 3 g for 15 min. The treated soluble superna-

tant was mixed with 0.2 M phosphate buffer (pH 7.5) con-

taining 5 mM EDTA, 0.2 mM NADPH, and 1.0 U/mL GSH

reductase, and maintained at 378C. Absorbance at 412 nm

followed the addition of 1.0 mM DTNB. GSH content was

calculated using reduced GSH (Nacalai Tesque) as the

standard.

Caspase Activity Assay

To obtain total cell extracts, chromium compound-treated

cells were collected by 0.25% trypsinization, washed with

cold PBS, and resuspended on ice for 10 min in lysis buffer

(150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 50 mM NaF, 5

mM EDTA, 0.5% Triton X-100, and 1 mM Na3VO4, along

with Complete; a protease inhibitor cocktail tablet (Roche

Diagnostics)). Nuclei and nonlysed cellular debris were

removed by centrifugation at 10,000 3 g for 1 min. The

protein concentration was determined using a bicinchoninic

acid (BCA) protein assay kit (Thermo Fisher Scientific,

Rockford, IL) with bovine serum albumin (BSA) as the

standard. Caspase enzyme activity was measured by cleav-

age of the Asp-Glu-Val-Asp (DEVD) peptide-conjugated

7-amino-4-trifluoromethyl coumarin (AFC), according to

the protocol outlined by the manufacturer of the caspase-3

fluorometric protease assay kit (Medical & Biological Lab-

oratories Co., Nagoya, Japan). Substrate cleavage, which

resulted in the release of AFC fluorescence (excitation, 400

nm; emission, 505 nm), was measured using a Fluoroskan

Ascent CF Plate Reader (Thermo Labsystems).

Western Blotting Analysis

Protein samples from cells were solubilized with sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) sample buffer (200 lL of 10% SDS, 60 lL of 1.0

M Tris–HCl (pH 6.8), 50 lL of 2-mercaptoethanol, 100 lLof glycerol, 1 mg of bromophenol blue, and 590 lL of dis-

tilled water) and heated at 1008C for 5 min. Proteins were

separated by 12.5% SDS-PAGE and transferred to a polyvi-

nylidene difluoride (PVDF) membrane using the iBlotTM

gel transfer system (Invitrogen). Blots were blocked for 1 h

at room temperature in fresh blocking buffer (0.1% Tween

20 in Tris-buffered saline, pH 7.4, containing 5% skimmed

milk). The membrane was incubated overnight at 48C with

anti-caspase-3 antibody (Cell Signaling Technology, Dan-

vers, MA), anti-poly(ADP-ribose) polymerase (PARP) anti-

body (Cell Signaling Technology), or anti-actin antibody

(Chemicon; Millipore Corp., Billerica, MA) in Tris-buf-

fered saline containing 0.1% Tween 20 (TBST). After

Fig. 7. Comparison of intracellular GSH level on Cr2O3 nanoparticles-, Cr2O3 fine particles-,CrCl3-, and K2Cr2O7-exposed cells The Cr2O3 nanoparticle dispersion, the mixture of fineCr2O3 particle and CrCl3, 1 mM of CrCl3 solution, and 0.1 and 1 mM K2Cr2O7 solution wereapplied to HaCaTcells and A549 cells. The intracellular GSH level was measured after 6 h ofexposure. The Cr2O3 nanoparticle dispersion contained 88 lg/mL of Cr2O3 particles and 1mM soluble chromium. The fine Cr2O3 dispersion contained 88 lg/mL of Cr2O3 particles and1 mM CrCl3 solution. Significant differences are shown in the figure (versus unexposed cells,ANOVA, Dunnett). *Significantly higher than unexposed cells (**P\0.01).

8 HORIE ET AL.


incubation, the membrane was washed thrice for 5 min

with TBST and incubated for 1 h at room temperature with

peroxidase-conjugated anti-mouse IgG antibody (Jackson

Immuno Research Laboratories, West Grove, PA) or perox-

idase-conjugated anti-rabbit IgG antibody (Jackson

Immuno Research) and then washed again with TBST. Per-

oxidase-active species on the membrane were detected

using a Chemi-Lumi One Kit (Nacalai Tesque) and

Lumino-Image Analyzer LAS-3000 mini (Fujifilm Corp.,

Tokyo, Japan). Densitometric analysis was performed using

Multi Gauge Version 3.0 (Fujifilm Corp.).

Determination of DNA Damage

DNA damage by Cr2O3, CrCl3, and K2Cr2O7 was detected

by the comet assay (1988) using the CometAssayTM Silver

kit (Trevigen, Gaithersburg, MD) according to the protocol

outlined by the manufacturer. The kit included LMAgarose,

Fig. 8. Effect of Cr2O3 nanoparticles on caspase activityand PARP activation. The Cr2O3 nanoparticles dispersioncontained 88 lg/mL of Cr2O3 particles and 1 mM solublechromium. (A) Caspase enzyme activity. Cell lysate sampleswere taken after cells were exposed to the Cr2O3 nanopar-ticles dispersion for 2 and 6 h. The cell lysate was thenapplied to the caspase activity assay. Caspase activity wasmeasured using DEVD-AFC as the substrate. Caspase ac-tivity is shown as a relative value to unexposed cells. *P \0.05 (versus control, Dunnett, ANOVA). (B) Activated cas-pase-3 and PARP were detected by Western blotting. Lane1: untreated cells; lane 2: Cr2O3 nanoparticle-exposed cells.Relative densitometric intensity of each band showeduntreated cell as 1.0.

Fig. 9. Comparison of DNA damage to cells exposed to theCr2O3 nanoparticles, fine Cr2O3 particles, CrCl3, andK2Cr2O7. Cells were exposed to the Cr2O3 nanoparticlesdispersion, the mixture of fine Cr2O3 particles and CrCl3, 1mM of CrCl3 solution, and 0.1 and 1 mM K2Cr2O7 solutionfor 6 h, and the DNA damage was measured by the cometassay. The Cr2O3 nanoparticles dispersion contained 88 lg/mL of Cr2O3 particles and 1 mM soluble chromium. The fineCr2O3 dispersion contained 88 lg/mL of Cr2O3 particles and1 mM CrCl3 solution. Approximately 200 cells were counted,and DNA damage classified according to the classificationdescribed by Tayama and Nakagawa (2004). A typical imageat each class is shown under the graph.



Fig. 10. Transmission electron microscopic observations of cells exposed to Cr2O3. (A–D)HaCaT cells exposed to the nano- and fine-Cr2O3 dispersion for 6 h. (E–H) A549 cellsexposed to the nano- (A, C, E, and G) and fine- (B, D, F, and H) Cr2O3 dispersion for 6 h.The bar in panels A, B, E, and F indicates 3.0 lm, and the bar in panels C, D, G, and H indi-cates 306 nm.

10 HORIE ET AL.


CometSlide, and Lysis Solution. HaCaT cells were exposed

by a TiO2-DMEM-FBS dispersion for 24 h, harvested by

trypsinization, and prepared at 1 3 105 cells/mL with PBS.

Cells were mixed with molten LMAgarose at 1:10 (v/v),

and 75 lL of the mixture was immediately applied onto the

CometSlide. After the application of agarose gel, slides

were immersed in prechilled Lysis Solution and left on ice

for 30 min. After removing excess buffer from the slides,

slides was immersed in alkaline solution (300 mM NaOH,

1 mM EDTA, pH[13) and left for 20 min at room temper-

ature in the dark. Slides were washed with Tris-Borate-

EDTA (TBE) twice, and electrophoresis was carried out

using a Mupid submarine electrophoresis system (Advance

Co., Tokyo, Japan) with 50 V for 2 min. Slides were then

rinsed by dipping several times in dH2O. After the slides

were soaked in 70% ethanol for 5 min, electrophoresed

DNAs were stained by silver staining and counted under a

microscope. Determination of the level of DNA damage

was as described by Tayama and Nakagawa (2004).

TEM Observations

Cell specimens exposed to Cr2O3 were observed using a

transmission electron microscope. Briefly, cells were fixed

using 1.2% glutaraldehyde for 1 h at 48C and 1% OsO4 so-

lution for 1 h at 48C, dehydrated in ethanol, and embedded

in epoxy resin. The resulting sample was then cut into ultra-

thin sections suitable for TEM observations by diamond-

knife ultramicrotomy. TEM observations were then carried

out using an H-7000 machine (Hitachi, Tokyo, Japan). The

acceleration voltage in the TEM was 75 kV.

Statistical Analyses

Data are the mean 6 standard deviation (SD) for at least

three separate experiments. Statistical analyses were done

by the analysis of variance (ANOVA) using the Dunnett or

Tukey tests for multiple comparisons. The calculation

method has been described in each figure legend.

RESULTS

Comparison of Cytotoxicity Induced by Cr2O3

Fine and Nanoparticles

Specific surface area of Cr2O3 fine and nanoparticles were

2.63 and 43.41 m2/g, respectively. Primary particle size of

Cr2O3 fine and nanoparticles were estimated by BET results

were 440 and 26.5 nm, respectively.

Cr2O3 fine and nanoparticles were applied to A549 cells

and HaCaT cells at concentrations of 0.1–10 mg/mL. After

24 h exposure, cytotoxicities were detected by the MTT

assay and LDH assay. Cell viability was dose-dependently

decreased by Cr2O3 exposure in the MTT assay and LDH

assay (Fig. 1). The cytotoxicity of nanoparticles was stron-

ger than that of fine particles. HaCaT cells showed a greater

reduction in cell viability by Cr2O3 exposure than A549

cells. In particular, the cytotoxicity of nanoparticles was

higher than that for fine particles at a high concentration of

Cr2O3 (0.5 mg/mL). In NiO, if the particle size became

nano-scale (1–100 nm), solubility increased. The released

Ni21 affects cytotoxicity (Horie et al., 2009b). Some previ-

ous studies suggest that metal ion release from metal oxide

nanoparticles was involved in their cytotoxicity (Horie

et al., 2009b; Studer et al., 2010).

Therefore, next, the cellular influences of soluble compo-

nents from Cr2O3 particles were examined. The Cr2O3 me-

dium dispersion used in the examination shown in Figure 1

was centrifuged at 16,0003 g for 20 min to remove the parti-

cle. The supernatant was applied to the cells, and the influ-

ence on cell viability was examined (Fig. 2). The centrifugal

supernatant of the fine Cr2O3 dispersion did not show cyto-

toxicity. On the other hand, the centrifugal supernatant of

Cr2O3 nanoparticle dispersion had cytotoxic activity. These

results suggest that the soluble component released from

Cr2O3 is involved in the cytotoxicity induced by Cr2O3 nano-

particles. The specific gravity of fine and nanoparticles is dif-

ferent; so, the cytotoxic effect of Cr2O3 nanoparticles in the

supernatant may not be completely removed by centrifuga-

tion. The smaller nanoparticle might be remaining in the su-

pernatant. Therefore, further tests were carried out.

Release of Soluble Chromium from Cr2O3 Fineand Nanoparticles to the Culture Medium

The release of soluble chromium from fine and nanoparticles

in DMEM-FBS was measured (Fig. 3A). Fine Cr2O3 particles

were insoluble in DMEM-FBS. The soluble chromium

released from fine Cr2O3 particles was not detected by ICP-

AES. In contrast, the Cr2O3 nanoparticles dissolved in the

DMEM-FBS. The rate of soluble chromium from Cr2O3

nanoparticles against charged Cr2O3 particles was estimated

as 1–1.5%. When the initial concentration of Cr2O3 was 0.1,

0.5, 1.0, 5.0, and 10 mg/mL, the concentration of soluble

chromium from Cr2O3 nanoparticles was estimated to be

0.24, 0.98, 4.76, 19.49, and 51.23 lg/mL, respectively. Cr2O3

did not influence the salts (P, Na, and Ca) contained in the

medium. Concentration of P, Na, and Ca in the Cr2O3 nano-

particle dispersion tended to be higher than the concentration

of these elements in the fine Cr2O3 dispersion. Although the

concentration of these elements was slightly low when Cr2O3

concentration was high, there was no noted influence of

Cr2O3 concentration on the concentration of P, Na, and Ca.

Oxidation state of soluble chromium released from

Cr2O3 nanoparticles was determined (Fig. 3B). Cr2O3 nano-

particle-DMEM-FBS dispersion at a concentration of 10

mg/mL included �0.4 mM K2Cr2O7 equivalence. This

result indicated that Cr2O3 nanoparticle released hexavalent

chromium in medium.



Influence of Released Soluble Chromium fromCr2O3 Particles on the Viability andProliferation of Cells

The cytotoxicity induced by Cr2O3 nanoparticles could

have been caused by released soluble Cr(VI). Therefore, we

compared the cellular influences of Cr2O3 nanoparticles,

fine Cr2O3 particles, CrCl3 as soluble Cr(III), and K2Cr2O7

as soluble Cr(VI).

The Cr2O3 nanoparticle-DMEM-FBS dispersion includ-

ing soluble chromium compound was prepared by centrifu-

gation. The secondary particle size of Cr2O3 in the DMEM-

FBS dispersion was measured by DLS. Secondary particle

size on the day after preparation of the dispersion was

295.1 and 124.6 nm as light intensity particle size and num-

ber average particle size, respectively. However, precipita-

tion of aggregate could be observed by the naked eye in the

Cr2O3 nanoparticle dispersion. The light intensity of DLS

measurement decreased with time. Therefore, the second-

ary particles of Cr2O3 nanoparticles in DMEM-FBS were

very unstable. The total chromium concentration in the

Cr2O3 nanoparticle-DMEM-FBS dispersion was 163.36 66.09 lg/mL (mean 6 SD). The concentrations of Cr2O3

particles and soluble chromium in the dispersion were

88.37 lg/mL (1.16 mM) and 75 lg/mL (0.99 mM), respec-

tively. Therefore, cellular influences induced by the Cr2O3

nanoparticle dispersion, the mixture of 88 lg/mL of fine

Cr2O3 particles and 1.0 mM CrCl3, 1.0 mM CrCl3 solution,

and 0.1 and 1.0 mM K2Cr2O7 solution were compared.

However, the sedimentation occurred in both Cr2O3 nano-

particle and Cr2O3 fine particle dispersion because these

dispersions were unstable. Because the particle accumu-

lated on the cell, the concentration of dispersion might not

reflect the cellular exposure concentration accurately.

Therefore, an accurate evaluation of concentration depend-

ency is not suitable.

The influence on cell viability was initially examined

(Fig. 4A). Each dispersion or solution was applied to

HaCaT cells and A549 cells and exposed for 6 and 24 h.

Cell viabilities were then measured by the MTT assay. Cell

viability was drastically reduced in cells exposed to the

Cr2O3 nanoparticle dispersion and K2Cr2O7 solution. The

exposure of the mixture of fine Cr2O3 particle and CrCl3led to a slight decrease in cell viability; cell viability after

24 h exposure was 80–90% in A549 cells. CrCl3 solution

did not induce a reduction in cell viability. Furthermore, we

examined the relationship between the extracellular soluble

chromium and cytotoxicity of Cr2O3 nanoparticles (Fig.

4B). The cells were exposed to the Cr2O3 nanoparticle dis-

persion or K2Cr2O7 solution and then changed to a chro-

mium-free culture medium after 2 h. After incubation for

additional 22 h, the mitochondrial activities were measured.

Both the Cr2O3 nanoparticles and the K2Cr2O7 solution

reduced mitochondrial activity. There were no significant

differences between the Cr2O3 nanoparticles- and the

K2Cr2O7 solution-exposed cells. This result indicates that

extracellular soluble chromium is critical for cytotoxicity

of Cr2O3 nanoparticle.

The influence on cell proliferation was examined by a

clonogenic assay (Fig. 5). An inhibitory effect on colony-

forming ability was shown in the Cr2O3 nanoparticle dis-

persion, the mixture of fine Cr2O3 particle and CrCl3, and

K2Cr2O7 solution.

Cellular Effects Induced by Cr2O3

Nanoparticles

The cellular influences induced by Cr2O3 nanoparticles

were examined. The decrease in mitochondrial activity was

reduced by pretreating the HaCaT cells with NAC (Fig.

6A). In A549 cells, NAC treatment did not significantly

affect the mitochondrial activity. However, pretreatment of

the A549 cells with NAC tended to reduce mitochondrial

damage. NAC is readily taken up by cells and, subse-

quently, acts as a source of cellular GSH. This result sug-

gests that oxidative stress is involved in the cytotoxicity of

Cr2O3 nanoparticles.

Levels of intracellular ROS in Cr2O3-, CrCl3-, or

K2Cr2O7-exposed cells over time were measured (Fig. 6B).

The intracellular ROS level was increased in cells exposed

to Cr2O3 nanoparticles and K2Cr2O7 solution. Exposure of

fine Cr2O3 particles and CrCl3 did not induce an increase in

the intracellular ROS level. The increase in intracellular

ROS level induced by exposure to Cr2O3 nanoparticles was

time-dependent. After cells were exposed to Cr2O3 nano-

particles for 6 h, intracellular ROS levels compared with

nonexposed cells were 11 and 7.8 times than that seen in

HaCaT cells and A549 cells, respectively. In Cr2O3 nano-

particles-exposed HaCaT cells, the intracellular ROS level

was increased to 27 times than that in nonexposed cells at

12 h exposure. Exposure of HaCaT cells to Cr2O3 nanopar-

ticles for 24 h led to cell death; thus, the intracellular ROS

level at 24 h exposure could not be measured. In A549

cells, intracellular ROS levels in Cr2O3 nanoparticles-

exposed cells at 12 and 24 h exposure were 10 and 16 times

of those of nonexposed cells, respectively. The induction of

intracellular ROS level by Cr2O3 nanoparticle was similar

to induction by K2Cr2O7.

The intracellular level of GSH was then measured

(Fig. 7). GSH level was reduced by exposure to Cr2O3 nano-

particles and K2Cr2O7. Compared with the Cr2O3 nanopar-

ticle, exposure of the fine Cr2O3 particle and CrCl3 did not

lead to GSH decrease in HaCaT cells. In A549 cells, expo-

sure of the CrCl3 led to a slight decrease in GSH after 12 h

exposure. Increase in the intracellular ROS level is related to

the induction of apoptosis (Annunziato et al., 2003). There-

fore, the caspase activity, which is apoptosis-related cysteine

peptidase activity, of Cr2O3-exposed cells was measured.

Caspase activity was increased in Cr2O3 nanoparticles-

12 HORIE ET AL.


exposed cells after 6 h (Fig. 8A). Further, activation of cas-

pase-3, which is caspase at downstream of caspase-related ap-

optosis pathway, was detected by Western blotting (Fig. 8B).

Cleavage of PARP was also detected by Western blotting.

PARP cleavage is an important marker of caspase-3-mediated

apoptosis. The cleavage of 116-kDa PARP by caspase-3

forms 85-kDa fragments (Nicholson et al., 1995; Robertson

et al., 1997). PARP cleavage was detected in Cr2O3 nanopar-

ticles-exposed cells. DNA injury was detected by comet

assay (Fig. 9). Exposure to Cr2O3 nanoparticles and K2Cr2O7

solution induced considerable DNA injury. These results sug-

gest that exposure to Cr2O3 nanoparticles led to increased

levels of intracellular ROS and subsequent apoptosis and

DNA injury.

Cellular Uptake of Cr2O3 Particles

Cellular uptake of Cr2O3 particles was observed by TEM

because Cr2O3 nanoparticles showed stronger cytotoxicity

than fine Cr2O3 particles and CrCl3 (Fig. 10). Cellular

uptake of nanoparticles and fine particles was observed in

HaCaT cells and A549 cells. Internalized Cr2O3 formed

aggregates and was present in the cytosol. Entry of particles

to the nucleus as particles was not observed. Fine particles

tended to adhere to the cell surface.

DISCUSSION

In general, trivalent chromium has lower toxicity than hex-

avalent chromium. Hexavalent chromium shows toxicity

due to reduction from Cr(VI) to Cr(III) (e.g., formation of

Cr(V) by reduction of Cr(VI) generates the hydroxyl radical

(Shi and Dalal, 1989). Although Cr(VI) passes through the

cell membrane via an ion channel, Cr(III) permeability of

the cell membrane is lower than for Cr(VI) (De Flora and

Wetterhahn, 1989; Barceloux, 1999). Thus, the potential

cytotoxic activity of Cr(III) is lower than for Cr(VI) (Bie-

dermann and Landolph, 1990; Bagchi et al., 2002; Hininger

et al., 2007).

If Cr2O3 was insoluble, the cellular effects induced by

Cr2O3 would be limited, but Cr2O3 nanoparticles were solu-

ble in the cell culture medium. Cr2O3 nanoparticles used in

this study released hexavalent chromium in culture media.

Moreover, Cr2O3 nanoparticles showed remarkable cyto-

toxicity at a low concentration. In our study, cellular influ-

ences induced by Cr2O3 nanoparticles matched to that of

K2Cr2O7.

This study showed that extracellular soluble chromium

was critical for cytotoxicity of Cr2O3 nanoparticles. Extrac-

ellular Cr(VI) enters the cell via ion channel and shows

strong oxidizability outside and inside of cell (De Flora and

Wetterhahn, 1989). On the other hand, TEM observations

of Cr2O3-exposed cells showed cellular uptake of Cr2O3

nano- and fine-particles. Subordinately, internalized Cr2O3

particles will release soluble chromium inside the cell. This

intracellular soluble Cr(VI) release also is an important fac-

tor for Cr2O3 nanoparticles cytotoxicity. Additionally, in-

tracellular dissolution of Cr2O3 nanoparticles leads to a

high concentration of Cr(VI) inside the cell. Although the

cellular uptake of fine Cr2O3 particles was also observed,

the cellular effects induced by fine Cr2O3 particles were

small compared with the same weight of Cr2O3 nanopar-

ticles. We concluded that cellular uptake of Cr2O3 nanopar-

ticles and subsequent cytotoxicity was due to released

Cr(VI) inside and outside the cell. It has been reported that

cellular uptake of metal oxide nanoparticles and subsequent

release of metal ions induce cytotoxicity due to NiO (Horie

et al., 2009b). Cellular uptake of nanoparticles was also

observed in TiO2 particles (Horie et al., 2010). However,

taken into account that TiO2 nanoparticles were insoluble

in the medium, the cellular influence of TiO2 was lower

than for NiO and Cr2O3. These observations indicate that

the cellular uptake of nanoparticles is a prerequisite for its

cellular effects. However, pre-eminent cytotoxicity is not

induced only by cellular uptake. The increase in solubility

in the biological fluid such as a culture medium is one of

the important factors for the cytotoxicity of Cr2O3 nanopar-

ticles. Even though Cr2O3 is classified as trivalent chro-

mium, why Cr2O3 nanoparticles release hexavalent chro-

mium is still unclear. The composition or purity of Cr2O3

nanoparticles powder was not examined in this study, but it

can be said that at least the nanoparticles that were sold as

Cr2O3 released hexavalent chromium.

Understanding the properties of nanoparticle, such as

solubility and oxidation state, is essential for industrial use.

This fact is also important for environmental burden caused

by disposed Cr2O3 nanoparticles. In our examinations of

the cellular influences of Cr2O3, strong oxidative stress and

apoptosis were induced. On theoretical grounds, Cr(VI)

will be reduced to Cr(III) in the cell; hence, toxicity of

Cr(III) is lower than that of Cr(VI). Nevertheless, there are

some investigations that Cr(III) also showed cellular influ-

ences. Oral administration of Cr(III) induced an increase in

lipid peroxidation in urine, though; the increase in the lipid

peroxidation level was lower than for Cr(VI) (Bagchi et al.,

2002). Several investigations suggest that Cr(III) induces

an increase in intracellular ROS level and apoptosis (Bala-

murugan et al., 2002; Rudolf and Cervinka, 2003; Rudolf

and Cervinka, 2009). Oxidative stress induced by Cr(III)

has also been reported in vivo. Oxidative stress was

increased in the brain, liver, and kidney of goldfish exposed

to Cr(III) (Lushchak et al., 2009a,b). In these investigations,

soluble chromium compounds such as chromium chloride

and chromium acetate were used. Therefore, the toxicity

would be due mainly to extracellular soluble Cr(III). It is

important for the cytotoxicity that Cr2O3 nanoparticles

become source of Cr(VI) or Cr(III) inside of cells after cel-

lular uptake. This study showed that Cr2O3 fine particles



did not dissolve in the culture medium. Hence, Cr2O3 solu-

bility was not sufficient for cytotoxicity. In contrast, Cr2O3

nanoparticles showed high solubility in the medium, and

cellular uptake of Cr2O3 nanoparticles was observed. The

extracellular and the intracellular dissolution of Cr2O3

nanoparticles induces drastic effects such as oxidative

stress, apoptosis, and DNA injury.

These cellular influences induced by Cr2O3 nanopar-

ticles were equivalent to cellular influences induced by

K2Cr2O7. This suggests that Cr2O3 nanoparticle has equiva-

lent risks as that of Cr(VI). Increased solubility, compared

with its larger form, is one of the features of metal oxide

nanoparticles, and this property is of great importance for

the toxicity of nanoparticles.

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