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Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins 1
secreted by HepG2 cells using proteomic analysis 2
3
4
Seonyoung Choi1, So-Young Park2, Ji Jeong1, Eunkyung Cho1, Sohee Phark1, Min Lee2, 5
Dongsub Kwak2, Ji-Youn Lim2, Woon-Won Jung3, Donggeun Sul1,2* 6
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1Graduate School of Medicine, Korea University, 126-1 Anam-Dong 5 Ka, Sungbuk-Ku, 8
Seoul, 136-705, Republic of Korea, 2Environmental Toxico-Genomic and Proteomic 9
Center, College of Medicine, Korea University, 126-1, Anam-Dong 5 Ka, Sungbuk-Ku, 10
Seoul, 136-705, Republic of Korea, 3College of Health Sciences, Korea University, San 11
1, Jeongreung-Dong, Seongbuk-Ku, Seoul, 136-703, Republic of Korea 12
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* Corresponding authors. Donggeun Sul, Ph.D. 14
Tel: + 82-2-920-6420 15
Fax: +82-2-929-6420 16
E-mail address: [email protected]. 17
© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 18
Received: September 24, 2009 / Revised: November 26, 2009 / Accepted: February 07, 2010 19
DOI: 10.1002/pmic200900674 20
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Abstracts 1
The effects of di(2-ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells 2
were studied using a proteomic approach. HepG2 cells were exposed to various 3
concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 µM) for 24 or 48 h. MTT 4
and comet assays were then conducted to determine the cytotoxicity and genotoxicity of 5
DEHP, respectively. The MTT assay showed that 10 µM DEHP was the maximum 6
concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells 7
exposed to DEHP was found to increase in a dose and time dependent fashion. 8
Proteomic analysis using two different pI ranges (4-7 and 6-9) and large size two 9
dimensional gel electrophoresis (2-DE) revealed the presence of 2776 protein spots. A 10
total of 35 (19 up- and 16 down-regulated) proteins were identified as biomarkers of 11
DEHP by ESI-MS/MS. Several differentiated protein groups were also found. Proteins 12
involved in apoptosis, transportation, signaling, energy metabolism, and cell structure 13
and motility were found to be up- or down-regulated. Among these, the identities of 14
cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf 15
kinase inhibitory protein, triose phosphate isomerase, cofilin-1, and HPR related protein 16
were confirmed by western blot assay. Therefore, these proteins could be used as 17
potential biomarkers of DEHP and human disease associated with DEHP.. 18
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Keywords: Biomarkers, DEHP, HepG2, secreted proteins, proteomics, two-dimensional 20
polyacrylamide gel electrophoresis, 21
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1 Introduction 1
Phthalate esters are among the most extensively used industrial chemicals and are 2
widely distributed in the environment due to their use as industrial solvents and 3
plasticizers [1]. Di(2-ethylhexyl) phthalate (DEHP), which is the most commonly used 4
phthalate ester, is found in a wide variety of consumer products, such as building 5
products, car products, clothing, food packaging, children’s products and some medical 6
devices made of polyvinyl chloride [2]. 7
DEHP is a lipophilic compound that can be absorbed through the skin, lungs and 8
orally by both human and rodents. Once taken in, DEHP is rapidly metabolized to 9
mono-(2-ethylhexyl)phthalate (MEHP) and 2-ethylhexanol via pancreatic lipases [2- 4]. 10
DEHP has been reported to have cytotoxic, immunotoxic, genotoxic and reproductive 11
toxic properties [5-15], and in long term feed toxicological studies it has been shown to 12
be carcinogenic in mice and rats [16, 17]. Specifically, DEHP is a known peroxisome 13
proliferator that has a hepatocarcinogenic potential in rodents [4, 6, 16]. In addition, 14
DEHP and MEHP have been shown to cause the oxidative stress and subsequent DNA 15
damage and lipid peroxidation in many species and cells [6, 10-14]. Furthermore, the 16
presence of high concentrations of DEHP in house dust has been found to induce a 17
human nasal immune response resulting in changes in the expression of cytokines [15]. 18
Recently, proteome analysis has emerged as an approach for the analysis of 19
differential gene expression at the protein level and the identification of biomarkers 20
based on comparison of the patterns of proteomes after exposure to compounds of 21
toxicological relevance [18-22]. Moreover, a wide range of immobilized pH gradients 22
(IPG) strips and more advanced two-electrophoresis or LC-MS/MS analysis has made it 23
possible to identify a number of proteins whose level significantly increased or 24
4
decreased after treatment with toxic compounds in cells, animals and humans. 1
Many proteomic studies have been conducted in vitro and in vivo to characterize 2
proteomes induced by environmental toxicants that has been useful as biomarkers or 3
target molecules for determination of the mechanism of their toxicity [23- 34]. However, 4
few proteomic studies of DEHP have been conducted in house dust mice allergic 5
subjects and rat pituitaries [15, 23]. 6
Recently, there has been a great deal of interest in characterization of proteins 7
secreted from cells and tissues because they have been used in the search for biomarkers, 8
which are good targets and sources for therapeutic and drug-based intervention as well 9
as useful tools for the diagnosis and prognosis of diseases like cancer [35]. 10
In present study, we evaluated the toxicological biomarkers of DEHP in secreted 11
proteins using a human hepatocyte cell, HepG2. The HepG2 cell line was chosen 12
because it is widely used as a model for human hepatocytes due to its suitability for 13
genotoxicitiy studies [36] and its ability to display a cellular morphology similar to that 14
of liver parenchymal cells, which play a role in the synthesis of major plasma proteins, 15
receptors for insulin, transferrin, epidermal growth factor and low density lipoprotein 16
[37, 38]. 17
For this proteomic study, we evaluated the lowest concentrations of DEHP that did 18
not cause cytotoxicity, but did cause genotoxicity in HepG2 cells during cytotoxic and 19
genotoxic assays because humans are exposed to low levels of DEHP in the 20
environment. The profiles of the secreted proteins were then determined using 2-DE 21
with the goal of identifying toxicological monitoring markers in HepG2 Cells exposed 22
to DEHP. The identified markers have the potential for use as biomarkers of human 23
diseases associated with DEHP exposure. 24
5
2 Materials and methods 1
2
2.1 Chemicals 3
4
Urea, thiourea, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 5
dithiothreitol (DTT), acrylamide, NN'-methylene-bisacrylamide, iodoacetamide, and 6
sodium thiosulfate were purchased from Sigma Chemical (St Louis, MO). Protease 7
inhibitor cocktail was purchased from Roche (Mannheim, Germany). High performance 8
liquid chromatography (HPLC) grade solvents including acetonitrile, acetic acid, and 9
methanol were purchased from Merck (Merck Co. Damstadt, Germany). DEHP was 10
purchased from Cerilliant CIL, Inc (Austin, TX). 11
12
2.2 Cell culture 13
14
Human hepatocyte cells (HepG2) were obtained from the American Type Culture 15
Collection (ATCC-HB 8065) and maintained in DMEM containing 10% fetal bovine 16
serum (FBS) (Gibco BRL, Grand Island, USA), penicillin (100 units/ml) and 17
streptomycin (100 µg/ml) at 37 C° under 5% CO2. Cells cultured in DMEM without 18
FBS for 24 h or 48 h with 0, 2.5, 5, 10, 25, 50, 100 and 250 µM DEHP were used for 19
the MTT and Comet assays. The final concentration of DMSO in the media did not 20
exceed 0.2%. 21
22
2.3 MTT assay 23
24
6
HepG2 cells (1 x 104) were incubated with different concentrations of DEHP in 96 well 1
plates and the cell viability was then determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-2
diphenyl tetrazolium bromide (MTT) assay (Sigma. Co, St Louis, MO, USA) 3
(Mosmann et al. 1983). Briefly, 20 μl of 5mg/ml MTT in PBS were added to each well 4
and the samples were then incubated for 3 h at 37°C. The media were then removed and 5
formazan crystals in the cells were dissolved in the presence of 100 μl of lysis buffer 6
(10% w/v of SDS in 0.01N HCl). The absorbance of the plates was then read at 570 nm 7
using an ELISA reader (Molecular Devices Co. Sunnyvale, CA, USA). The percentage 8
of cell proliferation and cytotoxicity was determined by comparing the optical densities 9
of cells treated with different concentrations of toxicants with that of the control. 10
11
2.4 Comet Assay 12
13
The comet assay was performed as previously described (39). In brief, normal melting 14
point agarose (Ameresco, NMA) and low melting point agarose (Ameresco, LMA) were 15
dissolved in PBS (Gibco, BRL) by heating in a microwave. Then, 100 μl of 1% NMA 16
was added to fully frosted slides that were precoated with 50 μl of 1% NMA for a firm 17
attachment, after which the slides were allowed to solidify with cover slips in the 18
refrigerator for 5 minutes. After solidification of the gel, the cover slips were removed 19
and 50 μl of lymphocytes mixed with 50 μl of 1% LMA were added. The cover slips 20
were added to the layer and the slides were again allowed to solidify in the refrigerator 21
for 5 minutes. After removing the cover slips, 100 μl of 0.5% LMA was added to the 22
third layer, and the slides were placed with cover slips in the refrigerator again for 5 23
minutes. The slides were submersed in the lysing solution (2.5 M NaCl, 100 mM 24
7
EDTA-2Na, 10 mM Tris-HCl, pH 10; 1% Triton X-100 and 10% DMSO, pH 10 were 1
added fresh) for 1 hour. The slides were then placed in unwinding buffer (1 mM EDTA 2
and 300 mM NaOH, pH 13) for 20 minutes and electrophoresis was carried out using 3
the same solution for 20 minutes at 25 V and 300 mA (0.8 V/cm). After electrophoresis, 4
the slides were neutralized via three washings with neutralization buffer (400 mM Tris-5
HCl, pH 7.4) 5 minutes each and were stained with 50 μl of 10 μg/ml ethidium bromide. 6
The slides were examined using a Komet 4.0 image analysis system (Kinetic Imaging, 7
Liverpool, UK) fitted with an Olympus BX50 fluorescence microscope equipped with 8
an excitation filter of 515-560 nm and a barrier filter of 590 nm. For each treatment 9
group, two slides were prepared and each 50 randomly chosen cells (total 100 cells) 10
were scored manually. The parameter, Olive tail moment (=(Tail.mean-11
Head.mean)*Tail%DNA /100), was calculated automatically using the Komet 4.0 image 12
analysis system, which was used for global comet description. 13
14
2.5 2-DE PAGE 15
16
2.5.1 Sample preparation 17
HepG2 cells were incubated with 5 µM DEHP for 24 h and 48 h (time dependent 18
experiment) and with 5 and 10 µM DEHP for 24 h (dose dependent experiment) in 19
DMEM media without FBS. The media were then collected and concentrated using a 20
molecular cut off column (3 kDa; Amicon, Millipore, Bradford, MA). Concentrated 21
secreted proteins were mixed with an equal volume of sample buffer containing 7 M 22
urea, 2 M thiourea, 40 mM Tris (0.5 M, pH 8.5), 4% CHAPS, 65 mM DTT, 1% IPG 23
buffer (pH 3-11) and 1% protease inhibitors. The sample mixtures were centrifuged 4 24
8
times adding sample buffer using a molecular cut off column at 3500 rpm for 1 h at 1
12°C. The resulting supernatants were then stored at -70°C for further experiments after 2
measuring the protein concentration by a Bradford assay. 3
4
2.5.2 Isoelectric focusing (IEF) and SDS-PAGE 5
The secreted protein (200ug) was mixed with a rehydration buffer containing 8 M urea, 6
2%CHAPS, 1% IPG buffer, 65mM DTT and a trace of bromophenol blue (BPB) to a 7
final volume of 450 ml per sample. IEF was conducted using commercially available 8
immobilized pH gradients (pH 4-7 and 3-10, 24 cm) in conjunction with the IPGphor 9
(Amersham Biosciences, Amersham, UK) apparatus. The gel was rehydrated in the 10
presence of the sample for 10 h, after which it was focused for 60kVh. After IEF, the 11
IPG gel strips were equilibrated twice for 15 min, under gentle shaking at room 12
temperature, first in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% 13
glycerol, 1%w/v SDS) containing 1% DTT, then in an equilibration buffer containing 14
2.5% iodoacetamide. In the second dimension SDS-PAGE, proteins were resolved 15
solely on the basis of their molecular masses in 16.5% tricine gel (35×45cm) using an 16
Owl separation system runner (Owl Separation System, Portamouth, NH, USA). The 17
running conditions were 1W/gel for 1 h and 20 W/total for 40 h until the BPB reached 18
the bottom of the gel in the cooling system. 19
20
2.5.3 Visualization and image analysis 21
Proteins were visualized using silver staining for image analysis. Briefly, the gels were 22
fixed in 50% methanol and 12% acetic acid, followed by washing three times for 20 23
min each in 50% ethanol. The gels were then sensitized by incubating in 0.02% sodium 24
9
thiosulfate followed by washing three times for 20 sec in double distilled water. Next, 1
the gels were immersed in 0.1% silver nitrate for 20 min, after which they were rinsed 2
two times for 20 sec each in double distilled water. The samples were developed in 6% 3
sodium carbonate and 0.05% formaldehyde (37%). Finally, the reaction was terminated 4
with 40mm EDTA-Na2. The preparative gels were fixed in 40% methanol and 5% acetic 5
acid for 60 min, then stained for at least 4 h in colloidal coomassie blue solution (0.1% 6
Brilliant Blue G-250, 34% methanol, 3% phosphoric acid, 17% ammonium sulfate). 7
Stained gels were washed with 1% acetic acid until the background of the gels was clear, 8
after which they were stored at 4°C in plastic foils until analysis. Silver-stained gels 9
were scanned using a 300 dpi instrument (Epson expression 10000XL) and the image 10
files were saved into Tagged Image File (TIF) format using gray. A calibration filter 11
using different shades of gray was then utilized to transform the pixel intensities into 12
optical density units. The images were then exported in TIF format and imported into 13
the Progenesis Discovery 2-D gel image analysis software (Nonlinear Dynamics, 14
Newcastle upon Tyne, UK) for analysis. This software automatically detects individual 15
protein spots within each image and matches identical protein spots across all images. 16
The gel that contains the most spots was chosen as the reference gel, and used for the 17
automatic matching and warping of spots in the other 2-D gels. This software also 18
removes noise from the measurements of spot volumes using a proprietary algorithm for 19
noise determination and correction. After automatic matching, manual review and 20
adjustment was conducted to confirm proper spot detection and matching. The intensity 21
of each protein spot was then normalized based on the total volume of each gel by 22
dividing the pixel intensity of each spot by the sum of all spots in the gel. 23
24
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2.5.4 In-gel digestion of silver stained protein spots 1
Protein spots on the gel were excised, and then destained according to the method 2
described by Guerreiro [40]. Briefly, 30–50 μl of working solution that contained 30 3
mM potassium ferricyanide and 100 mM sodium thiosulfate (1:1, v/v) were added to the 4
gel pieces, which were then occasionally vortexed until the brownish color of the gel 5
pieces disappeared or changed to yellow. Gel pieces were then washed several times 6
with distilled water, after which 100 μl of 50 mM ammonium bicarbonate was added 7
and the solution was incubated for 20 min. After centrifugation, the supernatant was 8
discarded and the gel pieces were dehydrated repeatedly with 100% ACN until their 9
color became opaque white. After destaining, the gel pieces were dried in a vacuum to 10
remove the solvent and then rehydrated in digestion buffer containing 50 mM 11
ammonium bicarbonate, 5 mM calcium chloride and 12.5 ng/μl trypsin. The gel pieces 12
were then incubated at 37°C for 12–16 h, after which the peptides were recovered by 13
two extractions with 100% ACN. The resulting peptide extracts were pooled, 14
lyophilized in a vacuum centrifuge, and then stored at 4°C for subsequent nano-LC-ESI-15
MS/MS experiments.. 16
17
2.5.5 Protein identification by nano-LC-ESI-MS/MS and Data Analysis 18
All MS/MS experiments for peptide identification were conducted using a nano LC/MS 19
system consisting of an HPLC system (Surveyor, USA) and an ESI-quadruple ion trap 20
MS (LCQ Deca XP-Plus, Thermo Finnigan, USA) equipped with a nano-ESI source. 21
Ten μl of sample were loaded by the autosampler (Surveyor, USA) onto a C18 trap 22
column (id 300 mm, length 5mm, particle size 5 mm; LC Packings) for desalting and 23
concentration at a flow rate of 20 μl/min. The trapped peptides were then back-flushed 24
11
and separated on a homemade microcapillary column [41] (length 150mm) packed with 1
C18 resin (particle size 5 mm) in 75-μm silica tubing (8-μm id orifice). Mobile phases 2
A and B were composed of 0% and 90% ACN, respectively, and each contained 0.05% 3
TFA and 0.1% acetic acid. The gradient began at 10% B for 15 min, was ramped to 4
20% B for 3 min, to 60% for 65 min, to 100% for 5 min, and then held at 95% B for 20 5
min. The column was equilibrated with 10% B for 10 min prior to the next run. The MS 6
and MS/MS spectra were obtained at a heated capillary temperature of 220°C, an ESI 7
voltage of 2.5 kV, and a collision energy setting of 35%. Data-dependent peak selection 8
of the three most abundant MS ions from the MS was used. Dynamic exclusion was 9
enabled with a repeat count of 2, a repeat duration of 0.5 min and a 3 min exclusion 10
duration. Mass spectrometer scan functions and HPLC solvent gradients were controlled 11
by the Xcalibur data system (Thermo Finnigan, USA). MS/MS mass peak lists were 12
analyzed for b and y ions using the Bioworks software (version 3.3.1, Thermo Electron 13
Corporation, USA). SEQUEST was used to match MS/MS spectra to peptides in the IPI 14
human database (IPI.- HUMAN.v.3.38, 70,757 entries) maintained by the European 15
Bioinformatics Institute (EBI) (http://www.ebi.ac.uk/). Two missed cleavages per 16
peptide were allowed, and modifications of the proteins were not taken into account. 17
The validity of the peptide/spectrum matches was hence assessed using the SEQUEST 18
defined parameters, cross-correlation score (XCorr), and normalized difference in cross-19
correlation scores (ΔCn). Matched peptide sequences must pass the following filters for 20
provisional identification: 1) the uniqueness scores of matches (ΔCn) must be at least 21
0.1 2) the minimum cross-correlation scores XCorr were 1.9, 2.2 and 3.75 for charge 22
states of +1, +2, and +3, respectively. SEQUEST automatically saves the search 23
results. SRF file including merging of proteins, filter and sort settings, ratios and protein 24
12
area/height values were used to select and sort peptide/spectrum matches passing this 1
set of criteria. When multiple proteins were identified, we selected one protein that had 2
a similar molecular weight and pI in 2-DE. In this study, SEQUEST match displayed 3
one protein, when proteins were identified with only one or more than two peptides. In 4
the case of proteins that were identified with only one peptide, a BLAST search was 5
performed and some peptide sequences were found to be shared among protein families. 6
7
2.6 Western Blotting 8
9
HepG2 secreted proteins were solubilized in lysis buffer (pH 7.4) containing a protease 10
inhibitor cocktail (Roche, Germany) on ice using a homogenizer. The lysates were then 11
centrifugated at 12,000 rpm for 15 min at 4 °C, after which the protein concentration of 12
the total lysate was determined using a Bradford protein assay (Bio-Rad Laboratory, 13
Richmond, CA, USA). Proteins (50µg) were loaded and separated on 12% gel by 14
electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore 15
Corporation, MA, USA) at 350 mA for 1 h using a transfer buffer (pH 8.3). The 16
membranes were then blocked with blocking buffer on PBS for 1 h at room temperature, 17
followed by incubation with primary antibodies overnight at 4°C. Primary antibodies 18
against cystatin C (Santa Cruz, 1: 500 dilution), DEK (Santa Cruz, 1:200 dilution), 19
gelsolin (Santa Cruz, 1:100 dilution), RKIP (Santa Cruz, 1:1000 dilution), RBP (Santa 20
Cruz, 1:1000 dilution), TIM (Santa Cruz, 1:500 dilution), CFL-1 (Santa Cruz, 1:1000 21
dilution), HPR (Santa Cruz, 1:1000 dilution) and Rho-GDI (Santa Cruz, 1:100 dilution) 22
were applied at the optimized concentrations. After washing the membranes with PBS 3 23
times for 10 min each, they were further incubated with horseradish peroxidase-24
13
conjugated secondary antibodies [anti-rabbit IgG or anti-goat IgG (1:2000, Santa Cruz, 1
CA, USA)] for 1 h at room temperature and then washed with PBS 3 times for 20 min. 2
The immune complexes were then detected using ECL and ECL Plus systems 3
(Amersham Pharmacia Biotech, Piscataway, NJ). Bands were visualized by 4
chemiluminescence and scanned using a flat-bed scanner. The digitalized images were 5
then analyzed using the Progenesis Discovery image analysis software (Nonlinear 6
Dynamics, Newcastle upon Tyne, UK). 7
8
2.7 Statistical Analysis 9
10
All statistic analyses were performed using SAS version 9.1. We used the analysis of 11
variance (ANOVA) method with Duncan’s and Tukey’s test to identify differences 12
between the exposure and control groups. A p<0.05 was considered to indicate 13
statistical significance in all cases. 14
15
3. Results 16
17
3.1 Cytotoxicity 18
19
Cells were incubated with various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100 20
and 250 µM) for 24 or 48 h. MTT assays were then conducted to investigate the 21
cytotoxicity (Fig. 1). The MTT assay of HepG2 cells exposed to lower concentrations of 22
DEHP (from 0.25 to 10 µM) for 24 h and 48 h revealed no significant difference in cell 23
growth (p>0.05). However, DEHP concentrations greater than 10 µM led to a reduction 24
14
in the number of cells (p<0.05). Based on the MTT assays, 10 µM DEHP was the 1
maximum concentration that did not cause cell death. 2
3
3.2 DNA Damage in HepG2 cells 4
5
The results of comet assays of the HepG2 cells are shown in Fig. 2. The mean value of 6
the Olive tail moment of the control HepG2 cells was 1.06 ± 0.02. After exposure to 7
DEHP for 24 and 48 h, DNA damage increased significantly with increasing 8
concentrations of DEHP (from 2.5 to 250 µM). The mean values of Olive tail moments 9
of HepG2 exposed to the highest concentration of DEHP (250 µM) for 24 and 48 h were 10
2.50 ± 0.02 (p=0.001) and 3.47 ± 0.05 (p=0.001), respectively. 11
12
3.3 2-DE analysis of secreted proteins expressed in a dose dependent manner by 13
HepG2 cells exposed to DEHP 14
15
Proteomic analysis was conducted using three different pI ranges (3.0-10, 4-7 and 6-9) 16
and a large size 2-DE system (Fig. 3). Treatment of HepG2 cells with two 17
concentrations of DEHP, 5 or 10 µM for 24 h was used to identify biological markers of 18
secreted proteins. As shown in Figure 3, which shows the 2DE-patterns of secreted 19
proteins of HepG2 cells exposed to DEHP using the two different ranges of pI strips (at 20
4-7 and 6-9), 1748 and 1523 protein spots were present in the gels, respectively. Thus, a 21
total of 2776 protein spots were resolved (Fig. 3). Of these, 25 secreted proteins were 22
found to be up- and down-regulated at the 4-7 and 6-9 pI ranges (Figs. 4 and 5). 23
Specifically, 14 and 11 protein spots were up and down-regulated in a dose dependent 24
15
manner using strips of pI 4-7 and 6-9 strips, respectively (Figures 4 and 5). 1
2
3.4 2-DE analysis of secreted proteins expressed in a time dependent manner by 3
HepG2 cell exposed to DEHP 4
5
Proteomic analysis was conducted using two different pI ranges (4-7 and 6-9) and a 6
large size 2-DE system. One concentration (5 µM) of DEHP applied for 24 and 48 h 7
was used to identify the biological markers of proteins secreted by HepG2 cells. A total 8
of 10 proteins were found to be up- and down-regulated in the proteins secreted by 9
exposed cells (Figs. 6 and 7). Specifically, one and four protein spots were up-regulated 10
in a dose dependent manner when strips with a pI 4-7 and 6-9 (Figs. 6 and 7) were used, 11
respectively, while four and one spots were down-regulated when strips with a pI of 4-7 12
and 6-9 strips were used, respectively (Figs. 6 and 7). 13
14
3.5 Identification and comparison of differentially expressed secreted proteins 15
16
Thirty-five differentially expressed secreted proteins were identified using ESI-MS/MS. 17
Among these proteins, 25 were identified as known proteins and 10 were unknown. The 18
identified proteins included those involved in apoptosis, transportation, metabolism, 19
signaling, and cellular reaction (Table 1-4). Of differentially expressed proteins, 1 was 20
revealed in time and dose dependent 2-DE pattern. 21
22
3.6 Confirmation of the Identities of Proteins by Western Blotting 23
24
16
Of the 35 differentially expressed secreted proteins, 25 identified by ESI-MS/MS were 1
subjected to Western blot analysis. Commercially available cystatin C, Rho GDP 2
inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, 3
triose phosphate isomerase, gofilin-1 and haptoglobin-related protein monoclonal 4
antibodies were purchased and used to conduct Western blot analysis to confirm their 5
identities. The expression of 5 proteins, Rho GDP inhibitor, gelsolin, Raf kinase 6
inhibitory protein, triose phosphate isomerase, and cofilin-1 were significantly up-7
regulated, while the expression of cystatin C, retinol binding protein 4, DEK protein and 8
haptoglobin-related protein was down-regulated in response to increasing DEHP 9
exposure time and concentration (Fig. 8). 10
11
4 Discussion 12
13
In present study, we evaluated the cytotoxic and genotoxic effects of DEHP on HepG2 14
cells before investigating the differential expressions of secreted proteins. An MTT 15
assay was conducted to determine the cytotoxicity and Comet assays were used for 16
genotoxicity evaluations. Two different concentrations of DEHP (5 and 10 µM) were 17
chosen because 10 µM DEHP did not cause cell death but did cause DNA damage and 18
25 µM DEHP, which caused significant cytotoxicity and genotoxicity, also induced 19
great changes in protein expression in HepG2 cells. It has been reported that DEHP and 20
MEHP caused DNA damage in human lymphocytes, mucosal cells, sperm and rat liver 21
(9-11). 22
New pharmaceuticals and chemicals should be evaluated for their genotoxicity to 23
test their possible health risk because genotoxic compounds induce genetic or DNA 24
17
damage, which could subsequently induce aberrant protein production and cell 1
proliferation, thereby leading to malignant transformation. In addition, genotoxic 2
compounds can induce reproductive system changes (42-44). Indeed, these previous 3
reports suggest that genotoxicity induced by DEHP could affect protein secretion by the 4
cells and cause the up or down-regulation of proteins, even though no cytotoxicity was 5
observed. 6
Previously, Randic et al. (45) and Balasubramanian et al. (46) reported the 7
mathematical characterization of 2D map data obtained by traditional methods and 8
provided 2D gel data obtained from the cells that were exposed to peroxisome 9
proliferators including DEHP. However, the data were converted using a mathematical 10
method; therefore, it is difficult to compare their data to ours. 11
For proteomic analysis, cells were treated with 5 and 10 µM of DEHP for 24 and 48 12
h, after which their secreted profiles were analyzed and identified by large 2-DE and 13
ESI-MS/MS. A total of 2776 secreted protein spots were detected in the 2-DE gel and 14
35 secreted proteins were up- and down-regulated, which showed dose and time 15
dependent expression by HepG2 cells. To confirm the identities, nine proteins 16
corresponding to , Rho GDP inhibitor, Retinol binding protein 4, gelsolin, DEK protein, 17
Raf kinase inhibitory protein, triose phosphate isomerase, cofilin-1, and haptoglobin-18
related protein were confirmed by western blot analysis 19
Gelsolin exists as a cytoplasmic or a secreted form originating from the alternative 20
splicing of a single gene in many cell types and in the plasma of vertebrates (47, 48). 21
Gelsolin is an actin binding protein that has multiple actin regulatory activities, 22
including cytoskeletal remodeling and ion channel regulation, and has both 23
antiapoptotic and proapoptotic functions (48, 49). In the present study, gelsolin was 24
18
significantly up-regulated in secreted proteins of HepG2 exposed to DEHP. It has been 1
reported that MCF-7 Gelmut cell, which contains a gelsolin gene that has been disrupted 2
by retroviral vector insertion in exon 2, shows strong resistance to tumor necrosis factor 3
(TNF)-induced apoptosis. Furthermore, exogenous expression of gelsolin restored the 4
sensitivity of MCF-7 Gelmut cells to TNF stimulation, indicating that gelsolin is 5
involved in TNF-induced apoptosis of MCF-7 cells (50). The results of another study 6
showed that increased expression of gelsolin may facilitate a subset of tumor cells with 7
increased motility, thereby enhancing its capability and probability of invading adjacent 8
tissues and metastasis to remote organ sites (51). 9
Cofilin is also an actin binding protein that plays a central role in regulating the 10
rapid cycling of actin assembly and disassembly, which is essential for cellular viability 11
(52). In this study, cofilin was significantly up-regulated in HepG2 exposed to DEHP. 12
Cofilin over expression enhances the motility of a variety of cell types in vitro and 13
increased cofilin expression has been detected in cells exhibiting invasive phenotypes 14
(53). 15
The Rho guanine nucleotide-dissociation inhibitors (RhoGDIs) are a major class of 16
regulators of Rho GTPases that have various functions in cell migration, epithelial cell 17
polarization, phagocytosis, and cell cycle progression (54) and play essential roles in 18
normal cell growth and malignant transformation (55). Additionally, RhoGDI has been 19
shown to function as a metastasis suppressor in bladder cancer, and it may play the 20
same role in other tumors (56, 57). 21
In the present study, gelsolin, cofilin and RhoGDI were up-regulated in a time and 22
dose dependent manner in the proteins secreted by HepG2 exposed to DEHP. Based on 23
this finding, DEHP significantly plays a significant role in the formation of cell 24
19
structure, such as remodeling of cytoskeleton and cell cycle progression, apoptosis and 1
tumor progression. Indeed, recent studies have shown that the over expression of these 2
proteins play essential roles in apoptosis and tumor progression. Therefore, these three 3
proteins may be involved in a protective response that enables HepG2 cells to survive 4
against the cytotoxicity of DEHP. 5
Other up-regulated proteins secreted by HePG2 exposed to DEHP included Raf 6
kinase inhibitory protein (RKIP) and triose-phosphate isomerase (TIM). RKIP has been 7
identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway and 8
loss of RKIP function promotes tumor metastasis in hepatoma cancer, prostate cancer, 9
breast cancer, colorectal cancer and melanoma (58-62). In this study, a low 10
concentration of DEHP led to up-regulation of RKIP in HepG2 cells, but a high 11
concentration of DEHP reduced the expression of RKIP. The over expression of RKIP 12
may have revealed protective effects against low concentrations of DEHP, but 13
increasing toxic effects of DEHP may suppress the expression of RKIP, thereby 14
increasing the risk of tumor progression in HepG2 cells. TIM is ubiquitously expressed 15
and catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and 16
glyceraldehydes-3-phosphate in glycolysis (63). TIM deficiency is a rare autosomal 17
recessive multisystem disorder, characterized by decreased enzyme activity in all tissues, 18
which is accompanied by elevation of DHAP level in erythrocytes (63, 64). However, in 19
this study, elevated expression of TIM may play a role in providing metabolic energy 20
for use in protection against the cytotoxic effects of DEHP. 21
Among the down-regulated proteins, retinol binding protein (RBP) was found to be 22
a transport protein (65). RBP has been known as a murine adipokine involved in the 23
development of insulin resistance since Tsutsumi et al. showed that RBP4 was also 24
20
secreted by adipocytes (66). In this study, RBP was significantly down-regulated in 1
HepG2 cells exposed to DEHP. DEHP may be involved in a RBP related metabolic 2
mechanism; however, further studies are required to identify any such relationships. 3
Haptoglobin-related protein (HRP) is a plasma protein with 91% sequence 4
homology with haptoglobin and a size of approximate 45-KD (67). However, few 5
studies have been conducted to evaluate HRP function. In the present study, DEHP 6
reduced the expression of HRP in HepGe2 cells. Further studies should be conducted to 7
elucidate the mechanism by which DEHP affects the expression of HRP. 8
DEK has been known as an abundant and highly conserved nuclear phosphoprotein 9
that has a strong association with critical human malignancies since this gene was 10
originally identified in the chromosomal translocation of a subset of acute myeloid 11
leukemia (68, 69). Conversely, in the nucleus, DEK is involved in a variety of DNA- 12
and RNA-dependent processes, such as DNA replication, splice site recognition, and 13
gene transcription. Moreover, DEK has been found to be involved in the repair of DNA 14
strand breaks and the protection of cells from genotoxic agents (69). Based on these 15
properties, the reduced expression of DEK in HepG2 cells observed in response to 16
DEHP was likely caused by DNA damage. 17
Cystatin C is a cysteine protease inhibitor that belongs to the type II cystatin gene 18
superfamily and has been identified as a biomarker of various diseases including 19
chronic kidney disease, cardiac disease, amyotrophic lateral sclerosis and Alzheimer’s 20
disease (70-73). However, it has been reported that cystatin C was significantly up-21
regulated in rat lung cells treated with arsenic trioxide (74). In addition, cystatin C has 22
protective effects against various oxidative stresses that induce cell death in PC 12 cells 23
(75). In the present study, DEHP reduced the expression of cystatin C, which resulted in 24
21
the loss of protective effects against oxidative stress in HepG2 cells. 1
Phthalates, including DEHP, are well-known endocrine-disruptors that are widely 2
used in household products such as children’s toys, baby care products, cosmetics and 3
polyvinyl chloride (PVC) tubing. The reduction of cystatin C and RBP, and the 4
induction of RhoGDI and gelsolin induced by treatment with DEHP could be associated 5
with reproductive toxicity. Cystatin C is a known regulator of conceptus development 6
and implantation (76), and RBP is important for implantation and maintenance of 7
pregnancy (77); therefore, it is possible that reduced expression of those proteins could 8
induce adverse effects on the implantation and pregnancy. In addition, suppression of 9
RhoGDI gene has been found to induce defects in the reproductive system in RhoGDI-/- 10
adult mice (78). Gelsolin, which is essential for proper ductal morphogenesis (79), was 11
reduced by treatment with DEHP, suggesting that DEHP may have adverse effects on 12
ductal development. Furthermore, the decreased expression of gelsolin could be 13
associated with ovarian cancer (80) and breast cancer (79). Moreover, the reduction of 14
RKIP could be related to the effects of DEHP on reduced reproduction rate (81). 15
Additionally, the reduced levels of , RBP, gelsolin and RKIP could be related with the 16
thyroid toxicity induced by DEHP. Patients diagnosed with hypothyroidism have been 17
showed to have reduced levels of cystatin C (82), whereas lower levels of gelsolin (83) 18
and RKIP (84) have been found to be related to increased tumor progression in thyroid 19
cancer. Finally, it is known that DEHP can induce inflammatory responses such as 20
asthma. Specifically, the levels of RhoGDI and gelsolin were increased in an asthmatic 21
murine model (85) and in the airways of asthma patients (86). 22
In addition, we compared proteins which up- or down-regulated by DEHP to those 23
that were altered in response to treatment with other endocrine disruptors such as 24
22
bisphenol A (BPA) and 2,3,7,8-TCDD to determine the specificity of the proteins to 1
DEHP. Prenatal exposure of BPA to mouse offspring up-regulated apolipoprotein A-I 2
precursor, dioeotidyl peptidase III and vesicle amine transport protein 1 in immune 3
organs such as spleens and thymuses (87). Conversely, rat livers exposed to TCDD 4
showed the alterations of protein expression such as apolipoprotein A-IV, α-1-5
macroglobulin, acidic ribosomal protein PO, Bal-647, endoplasmic protein 29, 6
proteosome subunit β type 3, and MAWD binding protein (88). In addition, treatment of 7
HepG2 cells with TCDD altered the expression of secreted proteins including UDP-8
glucose 6-hydrogenase, human homogentisate deoxygenase, aldo-keto reductase 1C3, 9
alcohol dehydrogenase, proteasome subunit beta type-5, peroxiredoxin-1, 10
lactoylglutathione lyase and proteasome subunit beta type-6 (unpublished data). These 11
comparisons suggest that DEHP-induced proteome alterations are different from those 12
observed in response to other endocrine disruptors such as BPA and TCDD, even 13
though there were some limitations in the comparisons due to the differences among 14
subjects that were exposed to the toxins. 15
In summary, evaluation of the cytotoxicity and genotoxicity induced by DEHP 16
using MTT assays and Comet genotoxic assays, respectively, revealed that these effects 17
increased as the DEHP levels increased. Proteomic analysis of the proteins secreted 18
from HepG2 exposed to DEHP using two different pI ranges and large size 2-DE 19
revealed 2776 protein spots, 19 of which were up-regulated and 16 that were down-20
regulated after exposure to DEHP. Of these 35 proteins, 25 were identified by ESI-21
MS/MS. The identities of nine proteins, cystatin C, Rho GDP inhibitor, retinol binding 22
protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate 23
isomerase, and cofilin-1 were confirmed by Western blot assays. These proteins are 24
23
related to cellular functions including cell structure, tumor progression, apoptosis, 1
energy metabolism and oxidative stress. Therefore, these proteins may be potential 2
biomarkers of DEHP and the human diseases associated with DEHP exposure. 3
4
Acknowledgements 5
6
This work was supported by the Ministry of Environment as "The Eco-Technopia 21 7
project" (No.2009-09001-0075-0) and by the ACE program through the National 8
Research Foundation of Korea(NRF) grant funded by the Korean Ministry of Education, 9
Science and Technology (MEST) (No. 2009-009-1414). 10
11
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335-343. 18
19
20
21
22
23
24
34
Figure legends 1
2
Figure 1. Cell proliferation of HepG2 cells exposed to various concentrations of DEHP 3
(0, 2.5, 5, 10, 25, 50, 100 and 250 µM) as determined by MTT assay. After 24 or 48 h, 4
the toxicity was determined by measuring the mitochondrial metabolism of MTT. Data 5
represent the means ±SD (n=7). * p<0.05 and ** p<0.01, compared to the controls. 6
7
Figure 2. DNA damage in HepG2 cells exposed to varying concentrations of DEHP (0, 8
2.5, .5, 10, 25, 50, 100, and 250 µM). After 24 or 48 h, genotoxicity was assessed by 9
DNA single strand breakage via a Comet assay. Data represent the means ± SD (n=7). * 10
p<0.05, compared to the controls. 11
12
Figure 3. 2-DE analysis using two different ranges of pI strips (3-10, 4-7 and 6-9). 13
Large size gels (35×45 cm) were used to analyze protein profiles and protein spots were 14
visualized by silver staining. 15
16
Figure 4. A. The 2-DE pattern obtained using a 4-7 pI strip to evaluate the secreted 17
proteins obtained from HepG2 cells exposed to DEHP (0, 5 and 10 µM) for 24 h. Gels 18
were visualized by silver staining. The images of protein spots were analyzed using the 19
Image Master 2-DE Progenesis Discovery Software program (Nonlinear Dynamics, 20
Newcastle upon Tyne, UK). The 2-DE image demonstrates the secreted proteome 21
pattern of untreated control cells. The images of each changed spot were compared at 22
increased DEHP concentrations. B. Spot volumes were calculated by normalization 23
against the total spot volumes. The quantity presented by each spot is expressed as a 24
35
relative intensity. 1
2
Figure 5. A. The 2-DE gel pattern obtained using a 6-9 pI strip to evaluate the secreted 3
proteins obtained from HepG2 cells exposed to DEHP (0, 5 and 10 µM) for 24 h. Gels 4
were visualized by silver staining. The image of protein spots was analyzed using the 5
Image Master 2-DE Progenesis Discovery Software program (Nonlinear Dynamics, 6
Newcastle upon Tyne, UK). The 2-DE image demonstrates the secreted proteome 7
pattern of untreated control cells. The images of each changed spot were compared at 8
increased DEHP concentration. B. Spot volumes were calculated by normalization 9
against the total spot volumes. The quantity presented by each spot is expressed as a 10
relative intensity. 11
12
Figure 6. A. The 2-DE pattern obtained using a 4-7 pI strip strip to evaluate the secreted 13
proteins obtained from HepG2 cells exposed to DEHP (0, and 5 µM) for 24 and 48 h. 14
Gels were visualized by silver staining. The images of protein spots were analyzed 15
using the Image Master 2-DE Progenesis Discovery Software program (Nonlinear 16
Dynamics, Newcastle upon Tyne, UK). The 2-DE image demonstrates the plasma 17
proteome pattern of untreated control rats. The images of each changed spot were 18
compared at increased DEHP exposure time. B. Spot volumes were calculated by 19
normalization against the total spot volumes. The quantity presented by each spot is 20
expressed as a relative intensity. 21
22
Figure 7. A. The 2-DE pattern obtained using a 6-9 pI strip to evaluate the secreted 23
proteins obtained from HepG2 cells exposed to DEHP (0, and 5 µM) for 24 h. Gels 24
36
were visualized by silver staining. The images of protein spots were analyzed using the 1
Image Master 2-DE Progenesis Discovery Software program (Nonlinear Dynamics, 2
Newcastle upon Tyne, UK). The 2-DE image demonstrates the secreted proteome 3
pattern of untreated control cells. The images of each changed spot were compared at 4
increased DEHP exposure time. B. Spot volumes were calculated by normalization 5
against the total spot volumes. The quantity presented by each spot is expressed as a 6
relative intensity. 7
8
Figure 8. A. Western blots of; (1) cystatin C, (2) Rho GDP inhibitor, (3) retinol binding 9
protein 4, (4) gelsolin, (5) DEK protein, (6) Raf kinase inhibitory protein, (7) triose 10
phosphate isomerase, (8) cofilin-1, (9) haptoglobin-related protein. Fifty microgram of 11
secreted proteins was loaded into each lane. B. Quantities represented by gel bands are 12
expressed as intensities relative to ß-actin. 13
14
15
16
17
37
Table 1. Dose dependent up-regulated protein spots in the proteins secreted by HepG2 cells exposed to 1
DEHP 2
3
Spot No. Accession no. Protein name Major
decision DB
Matched peptides no.
Sequence coverage (%)
Theoreticalvalue (Mr)
1757 IPI00007221.1 Plasma serine protease inhibitor (PAI-3) IPI human v3.38 7 18.23 45
2520 IPI00026314 Gelsolin IPI human v3.38 2 1.6 85
2853 IPI00217966.7 L-lactate dehydrogenase A chain (LDHA) IPI human v3.38 17 24.40 36
3007 Unidentified
3045 Unidentified
3321 IPI00451401 Triose phosphate isomerase (TIM) IPI human v3.38 1 16.3 26
3358 IPI00180956.6 Putative uncharacterized protein IPI human v3.38 1 10.19 48
3497 IPI00219446 Raf kinase inhibitory protein (RKIP) IPI human v3.38 1 17.2 21
3873 AAA51747 Proapolipoprotein IPI human v3.38 1 5.6 28
3874 AAA51747 Proapolipoprotein IPI human v3.38 1 5.6 28
3929 Unidentified
3930 Unidentified
4110 IPI00218693.8 Adenine phosphoribosyltransferase (APRT) IPI human v3.38 5 20.00 20
4135 IPI00003815 Rho GDP inhibitor (Rho-GDI) IPI human v3.38 1 7.3 23
HepG2 cells were exposed to 5 and 10 μM of DEHP for 24 hr. 4
5
38
Table 2. Dose dependent down-regulated protein spots in the proteins secreted by HepG2 1
cells exposed to DEHP 2
Spot No. Accession no. Protein name Major
decision DB
Matched peptides no.
Sequence coverage (%)
Theoreticalvalue (Mr)
2476 Unidentified
2338 Unidentified
2343 IPI00020021.3 DEK protein IPI human v3.38 1 4.3 42
2350 IPI00032258.4 Complement component 4-A (C4A) IPI human v3.38 23 10.03 192
2384 Unidentified
2608 IPI00303139.2 48 kDa protein IPI human v3.38 1 4.8 48
2718 IPI00783987 Complement C3 (C3) IPI human v3.38 26 34.10 188
3662 IPI00032293 Cystatin C IPI human v3.38 4 11.6 16
4075 IPI00022420 Retinol binding protein 4 (RBP) IPI human v3.38 2 13
4082 Unidentified
4333 IPI00290085 Cadherin-2 IPI human v3.38 4 6.40 100
HepG2 cells were exposed to 5 and 10 μM of DEHP for 24 hr. 3
4
5
6
39
Table 3. Time dependent up-regulated protein spots in the proteins secreted by HepG2 1
cells exposed to DEHP 2
3
Spot No. Accession no. Protein name Major
decision DB
Matched peptides no.
Sequence coverage (%)
Theoreticalvalue (Mr)
2201 Unidentified
3023 IPI00294398.1 Hydroxyacyl-coenzyme A dehydrogenase IPI human v3.38 5 20.06 34
3153 IPI00011290.3 Sulfotransferase 1C2 IPI human v3.38 1 4.00 35
3556 IPI00012011.6 Cofilin-1 (CFL-1) IPI human v3.38 2 6.63 184
3586 IPI00748705.1 Hypothetical protein IPI human v3.38 1 5.2 28
HepG2 cells were exposed to 5 μM of DEHP for 24 and 48 hr. 4
5
40
Table 4. Time dependent down-regulated protein spots in the proteins secreted by HepG2 1
cells exposed to DEHP 2
3
Spot No. Accession no. Protein name Major
decision DB
Matchedpeptides no.
Sequence coverage (%)
Theoreticalvalue (Mr)
1211 IPI00783987.2 Complement C3 (Fragment) IPI human v3.38 53 23.21 187
2233 IPI00027666.1 Cholecystokinin receptor (CCKAR) IPI human v3.38 2 4.0 47
3885 Unidentified
4168 IPI00477597.1 Haptoglobin-related protein (HPR) IPI human v3.38 3 4.31 39
4333 IPI00290085 Cadherin-2 IPI human v3.38 4 6.40 100
HepG2 cells were exposed to 5 μM of DEHP for 24 and 48 hr. 4
5
6
7
8
41
1
2
3
42
1
2
3
43
1
2
44
1
45
1
2
46
1
47
1
48
1
