Effects of fullerene nanowhiskers on cytotoxicity and gene expression

doi:10.4236/health.2010.212216

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Vol.2, No.12, 1456-1459 (2010) Health

doi:10.4236/health.2010.212216

Effects of fullerene nanowhiskers on cytotoxicity and

gene expression

Junko Okuda-Shimazaki1, Shinichi Nudejima2, Saiko Takaku1, Koki Kanehira3, Shuji Sonezaki3

Akiyohshi Taniguchi1*

1Advanced Medical Materials Group, Biomaterials Center, National Institute for Materials Science, Namiki, Tsukuba, Ibaraki, Japan;

2Advanced Nanomaterials Laboratory, National Institute for Materials Science, Namiki, Tsukuba, Ibaraki, Japan;

3TOTO Ltd. Research Institute, Nakashima, Kokurakita, Kitakyushu, Japan; *Corresponding Author: taniguchi.akiyoshi@nims.go.jp

Received 23 June 2010; revised 10 July 2010; accepted 14 July 2010

ABSTRACT

With recent developments in nanoscience and

nanotechnology, carbon-based nanomaterials, such

as carbon nanotub es or Fullerene nano whiskers

(FNWs), are attracting a great deal of interest.

However, nanomaterials have novel properties

that may cause safety problems in biological

systems. Although the toxic effects of multiwall

carbon nanotubes (MWCNTs) in vitro and in vivo

have been well described, the effect of FNWs on

cells remains unclear. In the present study, we

analyzed the gene expression and cytotoxicity

of FNWs-treated cells. The results of cell viabil-

ity and gene expression analysis indicate that

FNWs has a relatively smaller ability to induce

cellular gene expression as compared with

MWCNTs or titania nanoparticles. Our results

suggest that FNWs have weak cytotoxic effects

as compared to the effects of MWCNTs and tita-

nia nanoparticles.

Keywords: Nanomaterials; Cytotoxicity; Gene

Expression; Fullerene Nanowhisker

1. INTRODUCTION

Nanomaterials are currently being investigated and

have potential applications in various fields. With recent

developments in nanoscience and nanotechnology, car-

bon-based nanomaterials, such as carbon nanotubes

(CNTs) and Fullerene nanowhiskers (FNWs), are at-

tracting a great deal of interest. Because of their size,

chemical composition, surface structure, shape, and ag-

gregation properties, nanomaterials are expected to have

novel physicochemical properties. The small size of na-

nomaterials allows them to penetrate into the body [1].

These novel properties raise safety concerns for the use

of nanomaterials in biological systems. Recent studies

suggest that nanomaterials affect biological behavior and

have the potential to be toxic [2-6]. The toxic effects of

multiwall carbon nanotubes (MWCNTs) in vitro and in

vivo have been well described [5,7-11]. FNWs also have

the potential to be used as carbonaceous engineered na-

nomaterials in the future. However, the effect of FNWs

on cells remains unclear.

In the present study, to understand the biological ac-

tivity of FNWs, we analyzed the gene expression and

cytotoxicity of FNWs-treated cells. Our results suggest

that FNWs have a very weak effect on cytotoxic activity

and the induction of gene expression as compared to the

effects of MWCNTs and titania nanoparticles.

2. MATERIALS AND METHODS

2.1. Nanomaterials

FNWs, which were formed by the liquid-liquid inter-

facial precipitation method [12-14], were a kind gift

from Dr K. Miyazawa (National Institute for Materials

Science). MWCNTs (MITSUI MWNT-7; Lot. 061220-

02) were a kind gift from Dr. S. Tsuruoka (Mitsui &

Co.,Ltd.). Small aggregated titania nanoparticles were

prepared by the centrifuge method [6].

2.2. Cell Cultures

A human acute monocytic leukemia cell line, THP-1

[14,15], was cultured in RPMI 1640 medium supple-

mented with 10% fetal bovine serum, 100 U/mL penicil-

lin, and 100 μg/mL streptomycin under 5% CO2 with

100% humidity at 37°C. For the exposure experiments,

THP-1 cells were treated with 200 nM phorbol 12- my-

ristate 13-acetate (PMA) for 48 h. To expose the cells,

PMA-treated THP-1 cells that had been seeded 24 h

prior were exposed to titania particles for 24 h.

J. Okuda-Shimazaki et al. / Health 2 (2010) 1456-1459

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2.3. Microscopic Observation

Cells exposed to nanomaterials were fixed with para-

formaldehyde and stained with Hoechst 33342 (nucleus

marker) and rhodamine-phalloidin (F-actin marker). Mi-

croscopic images of fixed cells were obtained by laser

scanning microscopy.

2.4. Cell Viability Test

Cell viability was measured by using a CellTiter-Glo

Luminescent Cell Viability Assay kit (Promega). THP-1

cells (5.0 × 104 per well) were seeded in a 96-well cell

culture plate. Titania particle suspensions were prepared

at final concentrations from 0.00001% w/v (0.1 μg/mL)

to 0.001%w/v (10 μg/mL). Each concentration of titania

particle suspension was added to the cell culture medium

at a 1/100 volume, and the cells were cultured for 24 h

prior to adding a reagent mixture containing cell lysis

solution, luciferase, and luciferase substrate to the wells.

The luminescence of the luciferase reaction, which de-

pends on the cytoplasmic ATP concentration, was then

analyzed.

2.5. Gene Expression Analysis

For mRNA expression analysis, THP-1 cells (1.4 ×

104/cm2) were seeded in cell culture dishes. Nanomate-

rials were prepared at a final concentration of 100 μg/mL.

Following 24 h of exposure to nanomaterials, cells were

detached by mechanical dissociation and utilized for

gene expression analysis.

The expression levels of marker genes were deter-

mined by quantitative real-time RT-PCR as described

previously [6]. Total cellular RNA was extracted from

titania-exposed cells by using an RNeasy Kit (Qiagen).

Extracted RNA was treated with DNaseI. Total cellular

RNA (2 μg) was reversibly transcribed with a random

hexamer primer by using the SuperScript III First-Strand

Synthesis System for RT-PCR (Invitrogen). The cDNA

(2 μL) was mixed with 10 μL of 2x Master Mix from the

qPCR Mastermix Plus for SYBR Green I kit (Takara,

Japan) and with 10 pmol of each specific primer. The

PCR was performed as previously described [6]. Ex-

pression levels of marker genes were normalized to that

of a housekeeping gene, glyceraldehyde-3-phosphate

dehydrogenase (GAPDH), which was used as an endo-

genous control in the same reaction as the gene of inter-

est. The primers for qPCR were as follows: for GAPDH,

forward 5’-CCCCCACCACACTGAATCTC- 3’ and

reverse 5’-GCCCCTCCCCTCTTCAAG-3’; and for Inter-

leukin 6 (IL6), forward 5’-TGAGTACAAAAGTCCT

GA-3’ and reverse 5’-TCTGTGCCTGCAGCTTCGT-3’.

The results from at least three independent tests were

evaluated by using Dunnett’s multiple comparison test.

2.6. DNA Microarray Analysis

One microgram of total RNAs was amplified and la-

beled by using an Amino Allyl MessageAmp aRNA kit

(Ambion, Austin, TX). The DNA microarray, AceGene- 1

Chip Version-Mouse (Hitachi Software Engineering,

Tokyo, Japan) with about 30,000 oligo-sense-nucleotides

including 22,917 unique genes on the basis of GeneID,

was covered with a gap cover glass (Takara), the solution

was injected from the edge of the cover glass, and the

microarray was placed in a hybridization cassette (TaKa-

Ra Bio). The DNA microarray was dried by centrifugation

and scanned by using GenePix4000 (Axon Instruments,

Silicon Valley, CA) to detect the array image.

3. RESULTS

3.1. Microscopic Images of FNWs-Exposed

Cells

Exposure tests were performed with a human mono-

cytic cell line, THP-1. THP-1 cells were differentiated,

before FNWs exposure, by the addition of PMA for

phagocytosis. Microscopic images of FNWs-exposed

cells suggested that the FNWs were taken up by THP-1

cells and localized in the cytoplasmic space (Figure 1).

3.2. Viability of FNWs-Exposed Cells

We measured the viability of cells exposed to FNWs

or MWCNTs (as a control) based on the quantification

of the cytoplasmic ATP concentration, which signals the

presence of metabolically active cells. Ninety percent of

FNWs-and MWCNTs-exposed THP-1 cells were viable

Figure 1. Microscopic images of FNWs-exposed THP-1 cells.

FNWs-exposed cells were fixed with PFA and stained with

Hoechst 33342 (a) and rhodamine-phalloidin (b) FNWs were

observed in differential interference images (c) Images

represent the nucleus (a), F-actin (b), differential interference

images (c), merged images (d), and Z-section images of

FNWs- exposed cells (e).

(a) (b)

(e)

J. Okuda-Shimazaki et al. / Health 2 (2010) 1456-1459

1458

Figure 2. Cell viability test based on cytoplasmic

ATP concentrations. Cell viability was measured by

using a CellTiter-Glo Luminescent Cell Viability

Assay kit (Promega). THP-1 cells were exposed to

Fullerene as a control (closed circle) or FNWs (open

circle) or MWCNTs (closed triangle).

at high concentrations of FNWSS and MWCNTs (Fig-

ure 2). There were no significant differences between

the viability of FNWs-exposed cells and MWCNTs-

exposed cells. Thus, FNWs had very weak cytotoxic

activity at these concentrations.

3.3. IL-6 mRNA Expression in FNWs-exposed

Cells

We next investigated the mRNA expression of the

IL-6 gene in FNWs-exposed cells. We previously

showed that the IL-6 gene is a good marker for detecting

the cytotoxicity of nanomaterials [6]. The expression of

IL6 mRNA was induced in THP-1 cells exposed to

MWCNTs (Figure 3). There was no apparent change in

expression in FNWs-exposed cells. These results indi-

cate that FNWs has a relatively smaller ability to induce

cellular gene expression as compared to MWCNTs.

3.4. DNA Microarray Analysis

We used DNA microarray analysis to compare the

gene expression profiles of THP-1 cells exposed to

FNWs and titania nanoparticles (Table 1). A total of 576

genes were induced by more than 4-fold after titania

Table 1. Number of up-or down-regulated genes by nanomate-

rials using DNA microarray analysis.

Fold induction Titania vs control ENW vs control

4< 8 1

2< <4 568 2

1.5< <2 729 22

–1.5< <1.5 18087 20869

–2> >–4 78 254

–2> >–4 42 17

–4> 2 0

Figure 3. IL-6 mRNA expression of nanomaterials-exposed

THP-1 cells. The expression levels of IL-6 gene was de-

termined by quantitative real-time RT-PCR method.

PMA-activated THP-1 cells were exposed to 100 μg/mL

Fullerene (solid bar) or 100 μg/mL FNWs (gray bar) or

100 μg/mL MWCNTs for 24 h. mRNA expression was

standardized by internal GAPDH (glyceraldehyde-3-pho-

sphate dehydrogenase) expression, and the relative expres-

sion level versus control (isopropyl alcohol was added in-

stead of nanomaterials) is shown.

nanoparticle exposure. On the other hand, only three

genes were induced by more than 4-fold after FNWs

exposure. These results indicate that FNWs has a rela-

tively smaller ability to induce cellular gene expression

as compared with titania nanoparticles.

4. DISCUSSION

In the present study, we analyzed the biological activ-

ity of FNWs. The physicochemical properties of FNWs,

such as size and chemical composition, were very simi-

lar to those of MWCNT. However, our results suggest

that FNWs had a very weak effect on cytotoxic activity

and the induction of gene expression as compared to

MWCNTs.

We previously studied the phagocytosis of FNWs by

PMA-treated THP1 cells [14,16]. Our results suggested

that PMA-treated THP1 cells might be able to decom-

pose FNWs into fullerene molecules. The weak effects

of FNWs on cytotoxic activity and the induction of gene

expression might be due to the biodegradable property of

FNWs in cells.

5. ACKNOWLEDGMENT

We thank the Bio-Organic Materials Facility at the Nanotechnology

Innovation Center for technical assistance. Thanks Dr. K. Miyazawa

(National Institute for Materials Science) and Dr. S. Tsuruoka (Mitsui

& Co.,Ltd.) for kind gift FNWs and MWCNTs.

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