Plasma Protein Level Changes in Waste Incineration Workers

Plasma Protein Level Changes in Waste Incineration Workers Exposed to 2,3,7 ... TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a chemical compound whic...
0 downloads 0 Views 553KB Size
Download PDF
Plasma Protein Level Changes in Waste Incineration Workers Exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Mee Jeong Kang, Do-Youn Lee, Won-A Joo, and Chan-Wha Kim* School of Life Sciences and Biotechnology, Korea University, Seoul, Korea Received December 22, 2004

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a chemical compound which is known to induce severe reproductive and developmental problems, immune system damage, and interference with regulatory hormones. To characterize changes in the expression of plasma proteins caused by exposure to TCDD, we analyzed plasma samples from workers at municipal incinerators using two-dimensional gel electrophoresis (2-DE). Proteins exhibiting differences in expression were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF mass spectrometry. One newly expressed protein was identified as the adrenomedulin binding protein (AMBP). Seven overexpressed proteins were identified in this study, and the most overexpressed protein was identified as R-fetoprotein (AFP). In addition, we cultured HepG2 cells in the presence of TCDD, to determine the effects of TCDD on the AFP and albumin expression in mRNA and protein levels, via RT-PCR and Western blotting, respectively. TCDD treatment resulted in an increase in the mRNA and protein expression levels of AFP, but reduced albumin expression. According to our results, exposure to TCDD may induce liver disease or cancer, and the proteins identified in this study could help reveal the mechanisms underlying TCDD toxicity. Keywords: R-fetoprotein (AFP) • albumin • adrenomedulin binding protein • human plasma • 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) • two-dimensional gel electrophoresis (2-DE) • quantative RT-PCR • Western blotting

Introduction 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a well-known highly toxic compound, which is present in nearly all components of the global ecosystem, including the air, soil, and sediment. The compound has also been isolated in both fish and humans. TCDD is also known to be one of the primary dioxin compounds generated by incineration.1 Many individuals have been exposed to TCDD, primarily through diet, occupational and accidental exposures. Epidemiological study of populations exposed to high levels of TCDD or dioxin-like compounds, whether occupationally or accidentally, will prove to be invaluable with regard to the determination of relationships between human exposure to these compounds and adverse health outcomes, including chloracne and cancer.2-3 In humans, TCDD produces skin cancers, endocrine alterations, immunological changes, and/or birth defects (as it does in animals). TCDD also sometimes induces xenobiotic-metabolizing enzymes, which can catalyze the metabolic processing of lipophilic chemicals into water-soluble derivatives in blood, which means that TCDD is able to affect a variety of proteins in human plasma. The structure of TCDD is related to a compound which involves initial binding to a cytosolic protein known as AHR. AHR affects the activation of several genes, including the * To whom correspondence should be addressed. Prof. Chan-Wha Kim, School of Life Sciences and Biotechnology, Korea University, 1-5 Anam-dong, Sungbuk-ku, Seoul 136-701, Korea. Tel: +82-2-3290-3439. Fax: +82-2-32903957. E-mail: [email protected].

1248

Journal of Proteome Research 2005, 4, 1248-1255

Published on Web 06/16/2005

cytochrome p450 genes, CYP1A1 and CYP1B1. The specific deleterious effects associated with dioxin exposure are believed to be due to the species- and tissue-specific expression of dioxin-regulated proteins. The analysis of the expression of these dioxin-inducible proteins in human populations may facilitate the determination of dose-response relationships for human exposure to dioxins in vivo, as well as assessments of the variability of human response, and cross-species comparisons of the effects of dioxin. The interindividual variability of response is crucial to assessments of human health risk, as a high degree of variability may indicate the presence of subpopulations that are more sensitive to chemical exposure. In this study, we analyzed the levels of plasma proteins in workers at municipal incinerators, and compared them with those isolated from unexposed human plasma, via 2-DE analysis. The proteins exhibiting differences in expression were identified via MALDI-TOF and ESI-Q-TOF mass spectrometry. Among the proteins identified, we confirmed the expression levels of R-fetoprotein (AFP) and fibronectin via Western blotting. In addition, to determine the hepatotoxicity of TCDD, we cultured hepatocellular carcinoma cells (HepG2 cells) in the presence of TCDD. The levels of albumin and AFP expression were measured via Western blotting, and the mRNA levels of albumin and AFP were determined via quantitative RT-PCR.

Experimental Procedures Materials and Apparatus. For our 2-DE analyses, we used the IPGphor IEF system and the Ettan DALT II SDS system 10.1021/pr049756d CCC: $30.25

 2005 American Chemical Society

Effect of TCDD Exposure on Human Plasma Proteins

(Amersham Pharmacia Biotech, Uppsala, Sweden), with 24 cm Immobiline DryStrips (pH 4-7). Urea, immobilized pH gradient (IPG) buffer (pH 4-7), and dithiothreitol (DTT) were also obtained from Amersham Pharmacia Biotech. DryStrip cover fluid, CHAPS, Tris, glycine, acrylamide, piperazine diacrylamide (PDA), SDS, and ammonium persulfate were purchased from Bio-Rad (Hercules, CA). Iodoacetamide (IAA), TEMED, glycerol, bromophenol blue (BPB), silver nitrate, thiourea, acetone, and ammonium bicarbonate were all obtained from the Sigma Chemical Co. (St. Louis, MO). The protease inhibitor cocktail (PIC) was obtained from Roche (Indianapolis, IN). All chemicals used in 2-DE, MALDI-TOF, and ESI-Q-TOF-MS/MS analysis were of either electrophoresis grade or analytic grade. All buffers were prepared with Mili-Q water. Blood Samples. TCDD-exposed blood samples were obtained from 31 local waste incinerator workers, and unexposed control blood samples were obtained from 33 individuals who had received an annual check-up at the Soon Chun Hyang University Hospital (Seoul, Korea). All study subjects completed a questionnaire, which included questions regarding smoking, drinking, age, working period, and medication. The ages of the waste incineration workers and control subjects were not significantly different. The waste incinerator workers had worked in that capacity for an average of 7 years (data not shown). The concentration of dioxin in the air at the waste incineration facility, as determined by High-Resolution Gas Chromatography (31.17 ng-TEQ Sm3) was 100 times higher than that measured in the air in the generalized area of Seoul, Korea (2002, 0.32 ng-TEQ Sm3, data not shown), as reported in the annual study of the Korean EPA. Presently, specific method has been established for the evaluation of human dioxin exposure in vivo. Therefore, we determined the levels of urinary metabolites of the polyaromatic hydrocarbons, 1-hydroxypyrene (1-OHP) and 2-naphthol, to infer the degree to which given individuals had been exposed to dioxin. We determined that the levels of these urinary metabolites were significantly different between the waste incineration workers and the control subjects. The mean 1-OHP values in the waste incineration workers and control subjects were 0.57 ( 0.46 and 0.04 ( 0.03 mol/mol creatinine, respectively. In the waste incineration workers, we recorded a mean 2-naphthol level of 8.30 ( 4.79, and a mean creatinine level of 0.254 ( 4.53 mol/mol. This indicated that the waste incineration subjects in this study had been exposed to dioxins.4 Blood samples, consisting of 4∼5 mL of heparinized whole blood, were collected by venipuncture from all study subjects, and were then centrifuged at 2200 × g for 5 min, to isolate the plasma fraction. Plasma Preparation for 2DE. Plasma samples for 2-DE were prepared as has been previously described.5-6 Diluted plasma and PBS were mixed with equal volumes of sample buffer which contained 7 M urea, 2 M thiourea, 0.5 M Tris-HCl, 4% CHAPS, 65 mM DTT, 0.5% IPG buffer, 0.5 M EDTA, and 1 mM Protease Inhibitor Cocktail (PIC), at a pH of 8.5. This mixture was then centrifuged for 1 h at 3500 rpm at 12 °C using a centrifugal filter device (Centricon, 3 kDa cutoff, Millipore, MA). After delipidation and desalting, the protein concentrations in the samples were measured by a modified version of the Bradford method.5 2-D Gel Electrophoresis. Immobiline DryStrips (24 cm, pH 4-7) were used to perform IEF. DryStrips were rehydrated with the samples (40 µg of protein) in 450 µL of solubilization solution, which consisted of 8 M urea, 2% CHAPS, 1% IPG buffer (pH 4-7), 13 mM DTT, and a trace of bromophenol blue.

research articles Rehydration proceeded for 5 h without electrical current, and was continued for an additional 5 h with an applied 50 V current. IEF was conducted using the IPGphor IEF system, for 146 000 Vhr. The second dimension was then conducted on 11-16% SDS-PAGE, using an Ettan DALT II system. 2-D Gel Image Analysis. Proteins were visualized via silver staining.5 ImageMaster 2D Elite Software (AP Biotech) was used to conduct computer analyses of the 2-DE images. Spot expression levels were determined by the comparing the relative protein volume of the spots to the total volume of the spots (total spot normalization), and was expressed as relative intensity. For each spot, relative intensity was averaged and expressed as the mean ( SE. We also performed Student’s T-test analyses (p < 0.001) in order to compare the means of the relative intensity of each spot between the TCDD-exposed and control individuals. The newly expressed spots, which appeared only in the TCDD-exposed group, and the overexpressed spots, which were more than five times as prominent in the TCDD-exposed group as in the controls, were selected and identified via MALDI-TOF-MS and ESI-Q-TOF-MS/MS. Protein Identification. To conduct peptide mapping fingerprinting via MALDI-TOF-MS, we first excised the target spot from the gel and destained it. To remove the silver ions, we carried out in-gel digestion and peptide extraction, according to the methods described by Joo et al.,6 with some minor modifications. The extracted peptides were then desalted with Zip tips C18 (Millipore), mixed with a fast evaporation matrix (CHCA in NC wetted with 0.1% TFA acid on the metal MALDITOF target), and dried for 10 min. A PerSeptive Biosystems MALDI-TOF Voyager DE-STR Mass Spectrometer (Framingham, MA) in reflectron mode was used to analyze the peptide mass fingerprinting, and we conducted internal mass calibrations using trypsin autodigestion products (842.5099 and 2211.1046 Da). Using the ProFound, MS-Fit, Mascot, and Peptident search programs, the interpreted tandem mass spectra of the tryptic digested peptides were searched against the databases of the National Center for Biotechnology Information (NCBI) and Swissprot, which are accessible at http:// www.ncbi.nlm.nih.gov/ and http://www.expasy.com/. To perform protein identification with peptide amino acid sequencing using ESI-Q-TOF-MS/MS, we conducted MS/MS analysis according to the protocols described in the previous study.4 The destained gel spots were digested overnight with 0.2 µg of modified trypsin in 20 µL of 50 mM ammonium bicarbonate at 37 °C. The peptide mixture was then desalted and concentrated with custom-made chromatographic columns consisting of 0.1-0.3 µL of porous reverse phase R2 material (20-30 µm bead size, PerSeptive Biosystems, USA) packed into a constricted GELoader tip (Eppendorf, Hamburg, Germany). A 10 µL syringe was used to force liquid through the column, by the application of gentle air pressure. Thirty microliters of the desalted peptide mixture from the digested supernatant were then diluted with 30 µL of 5% formic acid, loaded onto the column, and washed with 30 µL of 5% formic acid. In the MS/MS analyses, the peptides were eluted using 1.5 µL 50% methanol, 49% H2O, and 1% formic acid, which was run directly into a precoated borosilicate nanoelectrospray needle (Micromass, Manchester, UK). The MS/MS analyses of the peptides generated by in-gel digestion were conducted via nano-ESI on a Q-TOF mass spectrometer (Micromass Manchester, UK). These procedures were run at a source temperature of 80 °C. A 1 kV potential was applied to the precoated borosilicate nanoelectrospray needles (EconoTip, New ObjecJournal of Proteome Research • Vol. 4, No. 4, 2005 1249

research articles tive, USA) in the ion source, combined with a nitrogen backpressure of 0-5 psi, to ensure a stable flow rate (10-30 nL/ min). The cone voltage used was 40 V. The quadrupole analyzer was utilized in the selection of select precursor ions for fragmentation in the hexapole collision cell. The collision gas used was argon at a pressure of (6-7) × 105 millibars, and the collision energy was 20-30 V. Product ions were analyzed with an orthogonal TOF analyzer, which had been fitted with a reflector, a microchannel plate detector, and a time-to-digital converter (Micromass). Data were processed with a Mass Lynx Windows NT PC system (Micromass). To identify the protein, all MS/MS spectra recorded on tryptic peptides derived from the spots were searched against protein sequences from the NCBInr databases, using the MASCOT search program (www.matrixscience.com). Detection of R-Fetoprotein and Fibronectin by Western Blotting. Forty micrograms of plasma proteins were diluted to 1/10 with PBS buffer, and then subjected to SDS-PAGE. 10% SDS gel was blotted onto nitrocellulose membranes. The membranes were incubated with blocking solution, containing a 1:1000 dilution of anti-fibronectin antibody (Neomarkers, CA), then incubated with blocking solution which contained a 1:10 000 dilution of goat anti-mouse IgG (H+L) horseradish peroxidase conjugate secondary polyclonal antibody (ZYMED Laboratories, CA). The AFP (Santa Cruz Biotechnology, CA) antibodies were diluted to 1:1000 with blocking solution, and the anti-mouse secondary polyclonal antibodies were utilized in the detection of AFP. An ECL system (Pierce Biotechnology Inc., IL) was used for signal detection. Western blot bands were scanned with a flat-bed scanner, and were digitized using Scion image analysis software (Scion Corp., MD). Values are expressed as the means ( standard errors of the mean (SEM). Statistical significance was assessed via unpaired Student’s t tests. TCDD Treatment to HepG2 Cell. The HepG2 human hepatoma cell line was obtained from the American Type Culture Collection (Rockville, MD). The HepG2 cells were grown as monolayers in R-minimal essential medium (R-MEM), containing 10% fetal bovine serum (without antibiotics), and were maintained in an atmosphere containing 5% CO2 and 95% room air, at 37 °C. The cells (1 × 105 cells/ml) were then treated with 0.5, 2, and 7.5 nM TCDD in R-MEM, plus 10% fetal bovine serum, for 15 h. Up to a concentration of 7.5 nM TCDD, no significant reductions in cell viability were noted (Data not shown). Each concentration of TCDD was dissolved in DMSO before being added to the R-MEM media, and the control HepG2 cells were treated only with DMSO. Quantitative RT-PCR. Total RNA was isolated from the cells with Trizol reagent (Gibco-BRL), as has been previously described.7 For reverse transcription, 2 µg of total RNA from each HepG2 cell sample was heated at 70 °C for 10 min, with 1 µg of random primers. Reverse transcription was then conducted using Superscript II Reverse Transcriptase (Gibco BRL) at 42 °C for both the control and TCDD exposed HepG2 cell RNA, followed by inactivation of the enzymes at 70 °C for 10 min, and storage at -20 °C until later use. First-strand cDNA was synthesized from 2 µg of total RNA in a 20 µL reaction mixture. PCR was carried out for albumin and AFP cDNA, using genespecific primers located within different exons. The primers for GAPDH, albumin, and AFP used in this study have previously been described.7 In the alb cDNA amplification, we used the following sense and antisense primers: 5′-TGCTTGAAT1250

Journal of Proteome Research • Vol. 4, No. 4, 2005

Kang et al.

GTGCTGATGACAGGG-3′ and 5′-AAGGCAAGTCAG CAGGCATCTCATC-3′. In the afp cDNA amplification, the sense and antisense primers were as follows: 5′-TGCAGCCAAAGTGAAGAGGGAAGA-3′ and 5′-CATAGCGAGCAGCCCAAAGA AGAA-3′. GAPDH was used as an internal control, to ensure that an exact amount of high-integrity RNA was being used. The primers were then synthesized from Genotech (Daejeon, Korea). PCR amplifications contained 100 ACUs, 10× reaction buffer, 2.5 nM MgCl2, 300 µM of each dNTP, 2.5 units of Taq DNA polymerase (Promega), 0.3 µM of each primer, and 8% DMSO in a 50 µL volume. PCR reactions were conducted on the GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA), with cycling conditions set as follows: one cycle of 94 °C for 2 min, 30 cycles of 94 °C for 1 min, 55 °C for 50 s, and 72 °C for 1 min, followed by one cycle of 72 °C for 10 min. Each sample was analyzed in duplicate. The resultant PCR products were then mixed with 5× Orange G loading dye (0.4% in 10% Ficoll, 10 mM Tris-Cl, pH 7.5, 50 mM EDTA; from Sigma), and were run on 2% agarose gel containing ethidium bromide, and visualized under UV light. To analyze and quantify the DNA agarose gels, we used the GelDoc analysis system (BioRad, Hercules, CA). Western Blot Analysis of Albumin and AFP from HepG2 Cell. Control and TCDD-exposed HepG2 cells were washed with PBS, then scrapped. Each pellet was collected after 5 min of centrifugation at 1200 rpm at 4 °C. The cell pellets were resuspended in lysis solution, which contained 5 mM NaPO4 (pH 7.4), 5 mM EDTA, 0.32 M sucrose, and 1 mM 2-mercaptoethanol, together with protease inhibitor cocktail. The cell suspension was then rapidly frozen with liquid nitrogen and thawed. This lysis step was repeated five times. After 45 min of centrifugation at 100 000 × g for the removal of solid tissue, the supernatants were stored at -80 °C until needed. The protein concentrations in each supernatant were determined via a modified version of the Bradford assay.6 50 micrograms of protein from each of the solubilized HepG2 cells obtained from the control and TCDD treatments were subjected to SDSPAGE. Our Western blotting and the evaluation of bands from the Western blotting were carried out as was described in section 2.4.1, with the exception of the antibodies used in these protocols. Anti-albumin antibody (1:1000 dilution, ABcam, MA) and AFP antibody (1:1,000 dilution, Santa Cruz Biotechnology, CA) were diluted with blocking solution, and anti-mouse secondary polyclonal antibodies (1:10 000, ZYMED Laboratories, CA) were used for detection.

Results Comparative Analysis of Protein Expression in Human Plasma Exposed to TCDD. 2-DE was performed in order to analyze 31 plasma samples from the TCDD-exposed workers and 33 plasma samples from unexposed (control) subjects. To compare protein expression levels, the obtained 2-DE gels were analyzed with a 2-D gel image analyzer. When the expression level of a spot from the TCDD plasma was more than 5 times as pronounced as the corresponding spot from the control group, the spot was selected as an overexpressed spot. Figure 1 shows the 2-DE pattern of the human plasma (with loading of 40 µg protein), indicating the overexpressed protein spots from the TCDD-exposed samples compared to the corresponding spots from the control plasma. Seven spots were overexpressed in the TCDD-exposed plasma, and one spot was newly expressed. The new and overexpressed proteins are listed in Table 1. The identified proteins appeared to belong to a group

research articles

Effect of TCDD Exposure on Human Plasma Proteins

Figure 1. Ultra-zoom 2D gel of seven protein spots and one neo expressed protein spot. Blow-up images of spots were compared between the TCDD-exposed and nonexposed control gels. 2-DE was performed with 40 µg of plasma proteins, using 24 cm pH 4-7 IPG strips, and 11-16% SDS-PAGE. The gels, which were visualized by silver staining and 2D gel images, were analyzed with the Image Master Software. The 2-D gel images of the seven overexpressed spots and the one neo expressed spot were magnified in the left and right lanes. Table 1. Identification of Differentially Expressed Spots on TCDD Exposed Gels by MALDI/TOF-MS and/Or ESI-Q-TOF-MS/MS spot no.

MW (kDa)

pI

classification

identification

match(%)a & scoreb

150 540c 553c 713 1755 2525 2970 5199

57 59 52 33 51 38 42 40

6.0 5.7 5.5 5.8 6.1 5.5 6.4 6.0

overexpressed spot overexpressed spot overexpressed spot overexpressed spot overexpressed spot overexpressed spot overexpressed spot new spot

R-feto protein albumin precursor fibrinogen γ_A chain precursor XAP-5 human rab GDI follistatin fibronectin AMBP

33 (%) 102 119 18 (%) 13 (%) 14 (%) 96.5 9 (%)

a Match (%) is based on number of peptide masses matched in NCBInr databases using MS-Fit searching program with MALDI-TOF/MS data. b Score is -10*Log(P), where P is the probability that the observed match is a random event, it is based on NCBInr database using MASCOT searching program as MALDI-TOF-MS or ESI-Q-TOF-MS/MS data. c Spot 540 and 553 were identified with ESI-Q-TOF-MS/MS and the others were identified with MALDITOF-MS.

of proteins related to cellular proliferation during cancer promotion, or to liver disease. This group of proteins includes

R-fetoprotein (AFP), fibronectin, pre-albumin, fibrinogen γ A chain precursor, and XAP-5. Journal of Proteome Research • Vol. 4, No. 4, 2005 1251

research articles

Kang et al.

Figure 2. R-Fetoprotein (A) and fibronectin (B) confirmed by Western blotting. Western blot of plasma protein (40 µg) from unexposure and TCDD exposed workers was carried out. The gel bands in upper lane are western blot results, the graphs in bottom lane is the comparison of quantitative density in each band of two groups. Two-tailed student’s test showed significant difference (/p < 0.01 //p < 0.001) between control and TCDD exposed plasma. The bars represent the mean ( SEM of density of gel bands determined from between control (n ) 3) and TCDD exposure (n ) 3).

Figure 3. Decrease of albumin region in some TCDD exposed workers. Albumin region of some TCDD exposed workers significantly decreased in over 15 gels of 31 gels.

TCDD Effects on AFP and Fibronectin Proliferation. To confirm the identified spots via proteomics techniques, we analyzed the AFP and fibronectin expression levels in the plasma samples by Western blotting with the appropriate antibodies. AFP and fibronectin were the proteins identified in the most overexpressed spots from the TCDD exposed plasma, and the coverage and match scores from MS analysis were highest in association with these proteins. Therefore, the Western blotting of those proteins was conducted with 3 plasma samples from the control group, and 3 samples from the TCDD group. Each of the samples was selected randomly from their respective group. 1252

Journal of Proteome Research • Vol. 4, No. 4, 2005

As shown in Figure 2A, AFP (57 kDa) expression in the TCDD-exposed workers was shown to increase by more than 3-fold (/p < 0.01) as compared to that observed in the control group. In Figure 2B, fibronectin (42 kDa) expression was elevated 3-fold (//p < 0.001) in the plasma obtained from the TCDD-exposed workers. Decreased Albumin Expression in TCDD Exposed Plasma Samples. Interestingly, as was shown in Figure 3, albumin concentrations were severely depressed in some of the plasma samples from the TCDD exposed group (15 out of 31 gels). As albumin is widely distributed on 2-DE gel, forming a constellation of spots, it is quite difficult to determine the precise

Effect of TCDD Exposure on Human Plasma Proteins

research articles

volume intensity of all of the albumin spots, using 2-DE gel image analysis. However, it was quite clear that the albumin expression levels in the TCDD-exposed samples had been drastically lowered. Therefore, an in vitro study with HepG2 cells was performed, to verify the decrease in albumin expression that had occurred as the result of exposure to TCDD. HepG2 Cell Exposed to TCDD. To determine whether AFP and albumin expression is affected by exposure to TCDD, we cultured HepG2 cells in the presence of TCDD. Significant cell death was induced as the result of TCDD treatment of over 7.5 nM, for at least 15 h (data not shown). The AFP and albumin mRNA levels were measured with quantitative RT-PCR, and the AFP and albumin expression levels were determined by Western blotting. AFP mRNA expression was robustly induced by TCDD in the HepG2 cells; TCDD treatment also induced AFP mRNA expression in a dose-dependent manner at TCDD concentrations of up to 2 nM. At TCDD concentrations over 2 nM, AFP mRNA expression was observed to decrease (Figure 4A). As expected, AFP protein expression in the HepG2 cells increased directly TCDD concentration, within a TCDD concentration range from 0.5 to 7.5 nM (Figure 4B). In Figure 5, a minor decrease can be seen in the mRNA level of albumin, concurrent with the application of 0.5 nM TCDD. This decrease, however, was also noticeable at a TCDD concentration of more than 2 nM (Figure 5A). Albumin protein expression levels in the HepG2 cells varied inversely with TCDD concentration within a TCDD concentration range of 0.5∼7.5 nM (Figure 5B).

Discussion Recently, interest has grown with regard to the effects of TCDD exposure in humans. However, due to difficulties inherent to human in vivo studies of TCDD exposure, no definitive data on this subject has yet been published. Therefore, our current investigation relies primarily on the results of in vitro cell cultures and in vivo animal studies.8 In earlier studies concerning TCDD, some proteins, including proliferin,9 CYP1 class,10 TEC,11 IgE-dependent histamine,12 green fluorescent protein (GFP),13 and CK914 have been proposed for use as biomarkers for TCDD exposure in animal studies. Proteomics is one of the most effective tools in our understanding of the toxicity of compounds and drugs. 2-DE is a highly sensitive means for the screening of proteins related to toxicity, and has also furnished the scientific community with a great deal of information regarding toxic mechanisms. In this study, we analyzed plasma protein profiles from 31 waste incinerator workers by 2-DE, to characterize the toxic effects of TCDD in humans. When we compared our results between the waste incineration workers (TCDD-exposed group) and the control group, we were able to identify seven overexpressed spots and one spot which was uniquely expressed in the TCDD group (Figure 1). In one of our earlier studies, we were able to detect pre-albumin and fibrinogen gamma A chain precursors among the identified proteins harbored by PAH-exposed workers at automobile inspection stations.15 This may be attributable to the variability inherent to the material burned in waste incinerators, including oils and plastics. Fibronectin, which is generally located in the interstitial matrix and the plasma, was identified as an overexpressed protein in the plasma of the TCDD-exposed group (Table 1). Fibronectin has been implicated in a host of cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. The principal function of fibronectin

Figure 4. mRNA and protein level of AFP in the HepG2 cell exposed to various level of TCDD for 15 h. HepG2 cells (106 cells/ ml) were treated with 0.5 nM, 2 nM and 7.5 nM of TCDD for 15 h. Total HepG2 cell RNA (2 µg) was reverse transcribed and amplified by PCR (20 cycles). PCR products were separated on 2% agarose gel and subjected to Scion image analysis software. A is the result of RT-PCR; 1st and 2nd lane show representative images of PCR products and GAPDH, respectively. The control underwent no TCDD treatment, and the 0.5 nM, 2 nM and 7.5 nM indicate the TCDD treated cells. The third lane displays the quantitative results of AFP mRNA expression, normalized to GAPDH. B is the results of Western blots, which were performed with the protein extracts (50 µg) from the cells. Each bar in both graphs represents the mean ( SD of the relative intensity of AFP expression. Two-tailed Student’s tests revealed significant differences (/p < 0.001) between the TCDD-treated and control groups.

appears to be in the formation of an extracellular matrix for the adhesion of cells during wound healing and development. Fibronectin and its integrin receptors perform important functions in several stages of tumor development.16-17 In recent studies, fibronectin has been detected in the hearts of TCDDtreated animals,18 and has thus been proposed for use as a biomarker for the diagnosis of TCDD exposure. Adrenomedulin binding protein (AMBP) was uniquely expressed in the plasma of the TCDD exposure group. Adrenomedulin is a pluripotent hormone, which exhibits a high degree of structural similarity to a calcitonin gene-related peptide. Adrenomedulin is expressed in a variety of tissues, and has been implicated in a myriad of diseases, including cardiovascular and renal disorders, sepsis, inflammation, diabetes, and cancer. Adrenomedulin has also been identified as an apoptosis survival factor for cancer cells, and is an indirect immune Journal of Proteome Research • Vol. 4, No. 4, 2005 1253

research articles

Figure 5. mRNA and protein level of ALB in the HepG2 cell exposed to various level of TCDD for 15 h. The conditions of qRTPCR and Western blotting of ALB in HepG2 cells are identical to the previously described conditions in Figure 4. A is the result of RT-PCR; the 1st and 2nd lane show representative images of the PCR products and GAPDH, respectively. The third lane is the quantitative results of ALB mRNA expression. B is the result of Western blotting; the 1st lane shows the representative gel image, and the 2nd lane is the quantitative analysis of ALB protein expression. Each bar in both graphs represents the mean ( SD (n ) 3) of relative intensity of ALB expression. Two-tailed Student’s tests revealed significant differences (/p < 0.001) between the TCDD treated cells and the controls.

response suppressor, by virtue of its binding protein, AMBP. Therefore, TCDD exposure may well induce the expression of AMBP. In addition, AFP expression was increased as the result of TCDD exposure, and albumin expression was severely reduced (in some cases). These results strongly suggest that TCCD exerts a hepatotoxic effect. Elevated AFP levels would tend to suggest the presence of either a primary liver cancer, or a germ cell tumor. AFP mRNA levels have been shown to be 30-1000-fold higher in human hepatocarcinoma cells (HCC cells) than in nontumor cells. AFP has been identified as an onco-development gene product. In adults, AFP is abundantly expressed during both liver regeneration and hepatocarcinomagenesis, and can be employed as a biomarker in the diagnosis of hepatocellular carcinoma.19-21 A series of investigations into the regulation and activation of AFP gene expression have been conducted previously.22-24 AFP expression increases in human plasma exposed to TCDD, as is shown in Figure 2. As expected, 1254

Journal of Proteome Research • Vol. 4, No. 4, 2005

Kang et al.

in the in vitro study using HepG2 cells, we were able to determine that both AFP mRNA levels and protein expression were augmented as the result of TCDD treatment (Figure 4). This may be attributable to the toxicity exerted by the AFP: TCDD complex. As TCDD has been demonstrated to form a stable complex with AFP, in a 2:1 (TCDD:AFP) ratio,25 the apparent solubility of TCDD in water increases 5-fold due to the formation of a complex. Once TCDD is exposed, AFP may facilitate TCDD transport through tissues and enhance its toxic effects. Therefore, TCDD may contribute to proliferation of tumorigenic processes in other tissues. Our quantitative assay of the TCDD-exposed HepG2 cells indicated that the average AFP mRNA levels had increased 2-fold, as compared to the controls. AFP mRNA may be present in the blood only for a short period of time, as the shed cells appear to be cleared by capillary systems, especially in the bone marrow. At the beginning of carcinoma formation, rapid necrosis and low numbers of tumor cells are apparent in the bloodstream. Another remarkable result in our study involved the significant decrease observed in albumin expression in some of the members of the TCDD-exposed group. Although albumin expression had been reduced in some of the gels, albumin remained dominant in the plasma, and albumin produced a masking effect on the 2-DE gels. To confirm these reductions in albumin expression, we cultured HepG2 cells in the presence of low TCDD concentrations. As expected from the 2-DE gels, both albumin mRNA and protein expression were reduced. Reduced albumin levels in the blood denote chronic liver or kidney disease, systemic lupus erythematosus (SLE), rheumatoid arthritis, or cancer.26 In this study, to measure the albumin levels in the in vitro HepG2 cell cultures, the cells were recovered and disintegrated. Then, we recovered the total soluble proteins from the cell lysates. 50 micrograms of recovered proteins were then loaded onto SDS-PAGE gels for Western blotting. Therefore, the observed reductions in albumin expression in the HepG2 cell cultures appear to be solely attributable to reductions in the expression ability of the HepG2 cells. According to our results, it appears that TCDD exposure may indeed cause serious health effects in humans, including liver disease and cancer. In particular, the workers exhibiting reduced albumin levels had worked at the municipal incinerator facility for more than 7 years, and had clearly been exposed to higher concentrations of TCDD than many of the other workers. Although cancer was not detected in any of the workers at the time of our study, we did find evidence to suggest that the livers of many of the workers were undergoing reductions in the ability to produce albumin.

Concluding Remarks To determine the effects of TCDD exposure on protein expression in human blood, we analyzed plasma samples from workers at municipal incinerators using 2-DE and MS/MS. Adrenomedulin binding protein (AMBP) was uniquely expressed in the TCDD samples, and R-fetoprotein (AFP), fibronectin, pre-albumin, fibrinogen gamma A chain precursor, XAP-5, human rab GDI and follistatin were overexpressed. Also, albumin expression was found to have been significantly reduced in 15 of the 31 TCDD-exposed human plasma samples, and this was verified in an in vitro study with HepG2 cells. TCDD treatment of the HepG2 cells resulted in increases in the mRNA level and protein expression of AFP, but reduced albumin expression. Therefore, TCDD exposure may induce

research articles

Effect of TCDD Exposure on Human Plasma Proteins

liver-associated diseases or cancer, and the proteins identified in this study may contribute to understanding of the toxic mechanisms inherent to TCDD. Abbreviations: AFP, R-fetoprotein; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AMBP, adrenomedulin binding protein; 2-DE, two-dimensional gel electrophoresis; MALDI-TOF, matrixassisted laser desorptiion inoization time-of flight

Acknowledgment. This study was supproted by a grant from the Korean Ministry of Commerce, Industry and Energy. We appreciate the help given by Jong-Bok Seo (Korea Basic Science Institute) in regard to the analysis of the MALDI-TOF MS data. References (1) Lim, Y.; Yang, J.; Kim, Y.; Chang, Y.; Shin, D. Assessment of human health risk of dioxin in Korea. Environ. Monit. Assess. 2004, 92, 211-228. (2) Yamada, R.; Sato, M.; Kawabata, M.; Nakatsuka, H.; Nakamura, K.; Takashima, S. Hepatic artery embolization in 120 patients with unresectable hepatoma. Radiology 1983, 148, 397-401. (3) Kim, P.; Prapong, W.; Sze, D. Y.; So, S. K.; Razavi, M. K. Treatment of hepatocellular carcinoma with sub-selective transcatheter arterial oily chemoinfusion. Gastroenterology 1989, 97, 965-971. (4) Sul, D.; Oh, E.; Im, H.; Yang, M.; Kim, C. W.; Lee, E. DNA damage in T- and B-lymphocytes and granulocytes in emission inspection and incineration workers exposed to polycyclic aromatic hydrocarbons. Mutation Res. 2003, 538, 109-119. (5) Joo, W. A.; Kang, M. J.; Son, W. K.; Lee, H. J.; Lee, D. Y.; Lee, E.; Kim, C. W. Monitoring protein expression by proteomics: human plasma exposed to benzene. Proteomics 2003, 3, 2402-2411. (6) Joo, W. A.; Lee, D. Y.; Kim, C. W. Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D gel electrophoresis. Biosci. Biotechnol. Biochem. 2003, 67, 1574-1577. (7) Shin, J. S.; Kwon, Y. S.; Lee, J. J.; Kim, C. W. Isolation of ethanolinduced genes in pancreatic beta-cells by representational difference analysis (RDA). Exp. Mol. Med. 2004, 36, 36-42. (8) Lee, S. H.; Lee, D. Y.; Son, W. K.; Joo, W. A.; Kim, C. W. Proteomic characterization of rat liver exposed to 2,3,7,8-tetrachlorobenzop-dioxin. J. Proteome Res. 2005, in press. (9) Parfett, C. L. Combined effects of tumor promoters and serum on proliferin mRNA induction: a biomarker sensitive to saccharin, 2,3,7,8-TCDD, and other compounds at minimal concentrations promoting C3H/10T1/2 cell transformation. J. Toxicol. Environ. Health A 2003, 66, 1943-1966. (10) van Duursen, M. B.; Sanderson, J. T.; van der Bruggen, M.; van der Linden, J.; van den Berg, M. Effects of several dioxin-like compounds on estrogen metabolism in the malignant MCF-7 and nontumorigenic MCF-10A human mammary epithelial cell lines. Toxicol. Appl. Pharmacol. 2003, 190, 241-250. (11) Riecke, K.; Grimm, D.; Shakibaei, M.; Kossmehl, P. Low doses of 2,3,7,8-tetrachlorodibenzo- p-dioxin increase transforming growth factor beta and cause myocardial fibrosis in marmosets (Callithrix jacchus). Arch. Toxicol. 2002, 76, 360-366.

(12) Oikawa, K.; Ohbayashi, T.; Mimura, J.; Fujii-Kuriyama, Y.; Rokutan, K.; Mukai, K.; Kuroda, M. Dioxin stimulates synthesis and secretion of IgE-dependent histamine-releasing factor. Biochem. Biophys. Res. Commun. 2002, 290, 984-7. (13) Mattingly, C. J.; McLachlan, J. A.; Toscano, W. A., Jr. Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio). Environ. Health Perspect. 2001, 109, 845-9. (14) Son, W. K.; Lee, D. Y.; Lee, S. H.; Joo, W. A.; Kim, C. W. Analysis of proteins expressed in rat plasma exposed to dioxin using 2-dimensional gel electrophoresis. Proteomics 2003, 3, 23932401. (15) Noh, K. S.; Lee, D. Y.; Cha, J. H.; Joo, W. A.; Lee, E.; Kim, C. W. Protein biomarkers in the plasma of workers occupationally exposed to polycyclic aromatic hydrocarbons. Proteomics 2004, 4, 3505-3513. (16) Ruoslahti, E. Fibronectin and its integrin receptors in cancer. Adv. Cancer Res. 1999, 76, 1-20. (17) Alexandra, K. K.; Kyoko, K.; Margaret, P. Q. Identification of genes involved in epithelial-mesenchymal transition and tumor progression. Oncogene 20, 6679-6688. (18) Woodward, T. L.; Mienaltowski, A. S.; Modi, R. R.; Bennett, J. M.; Haslam S. Z. Fibronectin and the R(5)β(1) integrin are under developmental and ovarian steroid regulation in the normal mouse mammary gland. Endocrinology 2001, 142, 3214-3222. (19) Tu, D. G.; Wang, S. T.; Chang, T. T.; Chiu, N. T.; Yao, W. J. The value of serum tissue polypeptide specific antigen in the diagnosis of hepatocellular carcinoma. Cancer 1999, 85, 1039-1043. (20) Yuasa, T.; Yoshiki, T.; Ogawa, O.; Tanaka, T.; Isono, T.; Mishina, M.; Higuchi, K.; Okada, Y.; Yoshida, O. Detection of alphafetoprotein mRNA in seminoma. J. Androl. 1999, 20, 336-340. (21) Chen, H.; Egan, J. O.; Chiu, J. F. Regulation and activities of alphafetoprotein. Crit. Rev. Eukaryot. Gene Expr. 1997, 7, 11-41. (22) Ishikawa, H.; Nakata, K.; Tsuruta, S.; Nakao, K.; Kato, Y.; Tamaoki, T.; Eguchi, K. Differential regulation of albumin gene expression by heparin-binding epidermal growth factor-like growth factor in alpha-fetoprotein-producing and -nonproducing human hepatoma cells. Tumour Biol. 1999, 20, 130-138. (23) Abelev, G. I.; Eraiser, T. L. Cellular aspects of alpha-fetoprotein reexpression in tumors. Semin. Cancer Biol. 1999, 9, 95-107. (24) Magee, T. R.; Cai, Y.; El-Houseini, M. E.; Locker, J.; Wan, Y. J. Retinoic acid mediates down-regulation of the alpha-fetoprotein gene through decreased expression of hepatocyte nuclear factors. J. Biol. Chem. 1998, 273, 30024-30032. (25) Sotnichenko, A. I.; Severin, S. E.; Posypanova, G. A.; Feldman, N. B.; Grigor’ev, M. I.; Severin, E. S.; Petrov, R. V. Water-soluble 2,3,7,8-tetrachlorodibenzo-p-dioxin complex with human alphafetoprotein: properties, toxicity in vivo and antitumor activity in vitro. FEBS Lett. 1999, 450, 49-51. (26) Wang, Y. Y.; Lo, G. H.; Lai, K. H.; Cheng, J. S.; Lin, C. K.; Hsu, P. I. Increased serum concentrations of tumor necrosis factor-alpha are associated with disease progression and malnutrition in hepatocellular carcinoma. J. Chin. Med. Assoc. 2003, 66, 593-8. (27) Ohguchi, S.; Nakatsukasa, H.; Higashi, T.; Ashida, K.; Nouso, K.; Ishizaki, M.; Hino, N.; Kobayashi, Y.; Uematsu, S.; Tsuji, T. Expression of alpha-fetoprotein and albumin genes in human hepatocellular carcinomas: limitations in the application of the genes for targeting human hepatocellular carcinoma in gene therapy. Hepatology 1998, 27, 599-607.

PR049756D

Journal of Proteome Research • Vol. 4, No. 4, 2005 1255