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SUPPORTING INFORMATION

p38 MAPK activation, DNA damage, cell cycle arrest and apoptosis as
mechanisms of toxicity of silver nanoparticles in Jurkat T cells
HYUN-JEONG EOM and JINHEE CHOI*

School of Environmental Engineering and Graduate School of Energy and Environmental System
Engineering, College of Urban Science, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu,
Seoul 130-743, Korea

Author E-mail : canada321@daum.net


Title Running Head : Toxicity of silver nanoparticles in Jurkat T cells
*Corresponding Author :
Tel: 82-2-2210-5622; Fax: 82-2-2244-2245; E-mail : jinhchoi@uos.ac.kr

Total number of pages : 11


Total number of table : 1
Total number of figures : 6

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Materials and Methods


Cells and Silver nanoparticles. Cells were maintained as described in Table S1. AgNPs
(size <100 nm, Sigma-Aldrich Chemical, St. Louis, MO, USA) were homogenously dispersed in
tetrahydrofuran (THF, Sigma-Aldrich) by sonication for 3 h (Branson-5210 sonicator, Branson Inc.,
Danbury, CT), with stirring for 3 days to volatilize the THF, refilled with deionized water, and
filtered through a cellulose membrane (pore size 100 nm, Advantec, Toyo Toshi Kaisha, Japan) to
remove the AgNP aggregates. To check whether THF remains in cell culture media after stirring
process, nuclear magnetic resonance (NMR) Spectrometer (Bruker, Rheinstetten, Germany) analysis
was conducted and free THF in AgNPs solution was not detected (data not shown). To compare the
toxicities of AgNPs and Ag ions, aqueous AgNO3 (AG002, Next Chimica, Centurion, Republic of
South Africa) in deionized water was used, with the final concentrations of AgNPs and Ag ions
estimated using a multitype inductively coupled plasma emission spectrometer (ICPE-9000,
Shimadzu, Tokyo, Japan). The concentrations for AgNPs and Ag ions had equivalent Ag masses.
Energy filtering transmission electron microscopy (TEM) was used to examine the particle shape and
size of the AgNPs. 20 L of particle suspension were dried onto a 400 mesh carbon-coated copper
grid and imaged with a LIBRA 120 TEM (Carl Zeiss, Oberkochen, Baden-Wrttemberg, Germany)
at 80-120 kV. The size distribution of the AgNPs was evaluated using a Photal dynamic light
scattering (DLS) spectrometer, DLS-7000 (Otsuka Electronics Co., Inc., Osaka, Japan). Images of
cells exposed to AgNPs and Ag ions were investigated by observation under optical microscopy.
Flow cytometer (FCM, Cell Lab Quanta SC, Beckman Coulter, Fullertonm, CA, USA) was
performed for the analysis of the amount of particles taken up by the cells. The intensities of
forward-scattered light (FS) and side-scattered light (SS) are proportional to the size of cells and the
intracellular density, respectively.
Cell Viability. The cell viability was measured using trypan blue (Invitrogene, Grand Island,
NY, USA) reagent. Jurkat T cells were plated on 6-well plates and the cells were treated with 0.05 to
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1 mg/L of AgNPs and Ag ions for 24 h. Treated and control cells were stained with 0.4% trypan blue,
with the total numbers of stained and unstained cells counted using a hemocytometer. All
experiments were performed in triplicate.
Formation of ROS. Cells grown to confluence were treated with 0.2 mg/L of AgNPs and
Ag ions for 1, 2, 4, 12 and 24 h, and following incubation with 2, 7-dichlorofluoroscein diacetate
(DCFH-DA, 25 M, 30 min), the fluorescence of DCF, which is the oxidized product of DCFH-DA,
was visualized using a fluorescent microscope (Leica, Wetzlar, German), with excitation and
emission wavelengths of 485 and 530 nm, respectively. The fluorescence intensity was measured
with a FCM (Beckman Coulter) equipped with a 488-nm argon laser and a band pass filter of 530 nm.
Western Blotting. Cells were exposed to 0.05, 0.1 and 0.2 mg/L of AgNPs and Ag ions for
24 h and to 0.2 mg/L for 4, 12 and 24 h. Western blotting analysis was then performed, as described
previously (1), using an enhanced chemiluminescence western blotting detection kit (Amersham,
Little Chalfont, Buckinghamshire, UK). Anti- ERK antibody, anti-phospho- ERK antibody, anti-p38
MAPK antibody, anti-phospho- p38 MAPK antibody, anti- JNK antibody and anti-phospho- JNK
antibody were purchased from Cell Signaling (Beverly, MA, USA). Anti-Nrf-2 antibody was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-NF-B antibody from Assay
Designs (Ann Arbor, MI, USA) and anti-p-Histone H2A.X antibody from Bioworld Technology
(Minneapolis, MN, USA). Anti-rabbit and anti-mouse secondary antibodies were purchased from
Assay Designs. Three replicates for each treatment and a control were conducted for the Western blot
analysis. Following the Western blotting, the relative densities of the protein bands were determined
using an image analyzer, the Gel Documentation system (Vilber Lourmat TFX-20.M, Marne
laVallee, France), coupled to a Kodak 1D 3.6 camera (Kodak EDAS 290, Rochester, NY, USA).
Cell cycle analysis. Cells were exposed to 0.05, 0.1 and 0.2 mg/L of AgNPs and Ag ions for
24 h and to 0.2 mg/L for 4, 12 and 24 h, and then stained with propidium iodide (PI, Sigma-Aldrich)
for the cell cycle analysis. The fluorescence intensity was measured with a FCM (Beckman Coulter)
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at 488 nm, with an emission wavelength of 610 nm. Data collected for 1x104 cells was analyzed
using MultiCycle AV for Windows software (Phoenix Flow Systems, San Diego CA. USA).
Comet assay. Cells were exposed to 0.2 mg/L of AgNPs and Ag ions for 4, 12 and 24 h, and
the Comet assay then conducted, as described previously (1). Approximately 50 cells per slide (3
slides per treatment) were examined. DNA damage was expressed as the tail moment using an image
analysis computerized method (Komet 5.5, Kinetic Imaging Limited, Nottingham, UK).
Annexin V/PI, Hoechst 33342 and TMRE stainings. Jurkat T cells were plated on 6-well
plates, and the cells then treated with 0.2 mg/L of AgNPs and Ag ions for 0, 4, 12 and 24h.
Apoptosis was determined by FCM (Beckman Coulter) using the Annexin V-FITC apoptosis kit
(BioVision, Mountain View, CA, USA). Apoptotic body was identified using fluorescence
microscopy (Leica) after staining with Hoechst 33342 (Sigma-Aldrich). Analysis of mitochondrial
membrane potential (m) was carried out by using tetramethylrhodamine ethyl ester (TMRE,
ImmunoChemistry Technology, Bloomington, MN, USA). Stained cells were imaged using a
fluorescent microscope (Leica), with excitation and emission wavelengths of 550 or 488 nm,
respectively.
Data Analysis. Statistical differences between the control and treated cells were examined with the
aid of ANOVA test using SPSS 12.0KO (SPSS Inc., Chicago, Il, USA).

SI Literature Cited
(1) Park, S. Y.; Lee, S. M.; Ye, S. K.; Yoon, S. H.; Chung, M. H.; Choi, J. Benzo[a]pyreneinduced
DNA damage and p53 modulation in human hepatoma HepG2 cells for the identification of
potential biomarkers for PAH monitoring and risk assessment. Toxicol. Lett. 2006, 167, 27-33.

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Table S1. Culture media and maintain conditions of the cells used in this study
Cell lines
Jurkat T (human lymphoma cells)
HeLa (human cervical cancer cells)
MCF-7 (human breast cancer cells)
NCI-H460 (human lung cancer cells)

Beas-2B (human bronchial epithelial cells)

Media
RPMI 1640 (GIBCO, Rockville, MD, USA)
10% (v/v) fetal bovine serum
1% penicillin streptomycin

37 C
DMEM F/12 (GIBCO)

humidified

10% (v/v) fetal bovine serum

atmosphere

1% antibiotics

5% CO2.

MEM (GIBCO)
HepG2 (human liver carcinoma cells)

Maintain
condition

10% (v/v) fetal bovine serum


1% penicillin streptomycin

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25
***

0
0

***

50
25
0

0.05 0.1 0.5 1


Concentration (mg/L)

HepG2

100
Cell viability(%)

***
***

50

75

**

75
**

50
25

0.05 0.1 0.5 1


Concentration (mg/L)

75
50
25
0

0.05 0.1 0.5 1


Concentration (mg/L)

MCF7

100
75

**
**

50
25
0

HeLa

100
Cell viability(%)

***
***

NCI-H460

100

0.05 0.1 0.5 1


Concentration (mg/L)

Beas-2B

100
Cell viability(%)

75

** *

Cell viability(%)

Cell viability(%)

**

Cell viability(%)

Jukat

100

**

**

** **

75
50
25
0

0.05 0.1 0.5 1


Concentration (mg/L)

0.05 0.1 0.5 1


Concentration (mg/L)

Figure S1. Comparison of the cytotoxicities induced by AgNPs ( ) and Ag ions ( ) treatment.
Viability tests were conducted on cells exposed to 0.05, 0.1, 0.5 and 1mg/L of AgNPs and Ag ions
for 24 h. Results are presented in relative units compared to the control. Data represent the mean
standard error of the mean of three individual experiments. * p<0.05, ** p<0.01 and *** p<0.005
compared to the control group.

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Cell viability(%)

(A)

**

100

**

**

AgNPs
Ag ions
**
**

***

75

***

50
25
0
0

12

Time(h)

24

(B)
Control

50m

AgNPs 0.2 mg/L

50m

Ag ions 0.2 mg/L

50m

Figure S2. Cell viability (A) and morphology (B) of Jurkat T cells exposed to AgNPs and Ag ions.
Viability tests were conducted on Jurkat T cells exposed to 0.2 mg/L AgNPs and Ag ions for 4, 12
and 24 h. Results are presented in relative units compared to the control. Data represent the mean
standard error of the mean of three individual experiments. * p<0.05, ** p<0.01 and *** p<0.005
compared to the control group. The morphologies of cells exposed to 0.2 mg/L AgNPs and Ag ions
for 24 h were observed by optical microscopy.

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(A)

(B)
Number of particles(%)

50
40
30
20
10
0

20

28.5 35.1 43.3 53.4 65.8 81.1 100.0

Diameter(nm)

(C)

(D)
Control
AgNPs
Cell numbers

Cell Numbers

100

Cell viability(%)

Control
AgNPs

75
*

50
25
0

Side Scatter (SS)

*
Control THF Control AgNPs

Forward Scatter

Ag ions

Figure S3. Characterization of AgNPs in the cell culture medium using electron microscopy (TEM,
A), dynamic light scattering (DLS, B) and flow cytometry (FCM, C). Viability of Jurkat T cells
exposed to THF, to AgNPs (1mg/L) and to Ag ions (1mg/L, D). The particle shapes were analyzed
by TEM, with the size distribution in the test medium evaluated by DLS. Flow cytometric
histograms of the forward scatter (FS) and side scatter (SS) were observed in the cells exposed to
AgNPs. Cell viability results are presented in relative units compared to the control and THF control.
Data represent the mean standard error of the mean of three individual experiments. * p<0.005
compared to the control group.

S8

Control

1h AgNPs

2h AgNPs

4h AgNPs

12h AgNPs

H2 O2

1h Ag ions

2h Ag ions

4h Ag ions

12h Ag ions

24h AgNPs

24h Ag ions

Figure S4. Formation of ROS in Jurkat T cells exposed to 0.2 mg/L AgNPs and Ag ions for 1, 2, 4,
12 and 24 h. 100M H2O2 was used as the positive control. The cells were incubated with 25 M
DCFH-DA at 37C for 30 min, and observed under fluorescent microscopy.

S9

(A)

(B)
AgNPs (mg/L, 24 h)
0

0.05

0.1

0.2

Ag ions (mg/L, 24 h)
0

0.05

0.1

AgNPs (0.2 mg/L, h)

0.2

ERK

ERK

p-ERK

p-ERK

JNK
Cytosol

12

24

Ag ions (0.2 mg/L, h)


0

12

24

JNK

p-JNK

Cytosol

p-JNK

p38

p38

p-p38

p-p38

Actin

Actin

Nrf-2
Nucleat

Nrf-2

NF-B

Nuclear

Actin

NF-B
Actin

Figure S5. The expressions of ERK, p38 and JNK MAPK, as well as the transcription factors, Nrf-2
and NF-B, in Jurkat T cells exposed to 0.05, 0.1 and 0.2 mg/L AgNPs and Ag ions for 24 h (A) and
0.2 mg/L for 0, 4, 12 and 24 h (B). The expressions of ERK, p38 and JNK MAPK were measured in
cytosol; whereas, those of Nrf-2 and NF-B were measured in the nuclear fractions.

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(A)
0h

4 h AgNPs

12 h AgNPs

24 h AgNPs

H2O2

4 h Ag ions

12 h Ag ions

24 h Ag ions

0h

4 h AgNPs

12 h AgNPs

24 h AgNPs

H2O2

4 h Ag ions

12 h Ag ions

24 h Ag ions

(B)

Figure S6. Apoptosis investigated in the Jurkat T cells exposed to 0.2 mg/L AgNPs and Ag ions for 4,
12 and 24 h, using Hoechst 33342 staining (A) and TMRE staining (B) by fluorescent microscopy.
Apoptotic cells were detected by Hoechst 33342 staining (arrows indicate apoptotic cells showing
nuclear condensation) and changes in the mitochondrial membrane potential were examined by
TMRE staining in Jurkat T cells.

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