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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
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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)
Media
RPMI 1640 (GIBCO, Rockville, MD, USA)
10% (v/v) fetal bovine serum
1% penicillin streptomycin
37 C
DMEM F/12 (GIBCO)
humidified
atmosphere
1% antibiotics
5% CO2.
MEM (GIBCO)
HepG2 (human liver carcinoma cells)
Maintain
condition
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25
***
0
0
***
50
25
0
HepG2
100
Cell viability(%)
***
***
50
75
**
75
**
50
25
75
50
25
0
MCF7
100
75
**
**
50
25
0
HeLa
100
Cell viability(%)
***
***
NCI-H460
100
Beas-2B
100
Cell viability(%)
75
** *
Cell viability(%)
Cell viability(%)
**
Cell viability(%)
Jukat
100
**
**
** **
75
50
25
0
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
50m
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
Diameter(nm)
(C)
(D)
Control
AgNPs
Cell numbers
Cell Numbers
100
Cell viability(%)
Control
AgNPs
75
*
50
25
0
*
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.
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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.
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(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
0.2
ERK
ERK
p-ERK
p-ERK
JNK
Cytosol
12
24
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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