Evaluation of Toxicological Monitoring Markers ... - ACS Publications

Evaluation of Toxicological Monitoring Markers Using Proteomic Analysis in Rats Exposed to Formaldehyde Hosub Im,† Eunha Oh,‡ Joohee Mun,† Jin-Young Khim,† Eunil Lee,‡,§ Hyung-Sik Kang,| Eunmi Kim,| Hyunsuk Kim,⊥ Nam-Hee Won,‡,# Young-Hwan Kim,∧ Woon-Won Jung,*,⊥ and Donggeun Sul*,‡,O School of Public Health, Korea University, Anamdong 5, Sungbukku, Seoul, 136-705, Korea, Environmental Toxico-Genomic and Proteomic Center, College of Medicine, Korea University, Anamdong 5, Sungbukku, Seoul, 136-701, Korea, Department of Preventive Medicine, School of Medicine and Institute for Environmental Health, Medical Science Research Center, Korea University, Anamdong 5, Sungbukku, Seoul, 136-705, Korea, School of Biological Sciences and Technology, Chonnam University, 300 Yongbongdong, Bukku, Kwangju, 500-757, Korea, MyGene Bioscience Institute, 202-16, Nonhyundong, Sungok Bldg., Fifth floor, Kangnamku, Seoul, Korea, Department of Pathology, College of Medicine, Korea University, Anamdong 5, Sungbukku, Seoul, 136-705, Korea, Department of Environmental Health, College of Health Science, Korea University, San 1, Jeongreungdong, Seongbukku, Seoul, 136-703, Korea, and Graduate School of Medicine, Korea University, Anamdong 5, Sungbukku, Seoul, 136-701, Korea Received December 3, 2005

Formaldehyde (FA) is known as a low molecule weight organic compound and one of major components that causes sick building syndrome (SBS), and it has been reported that FA has cytotoxic, hemotoxic, immunotoxic, and genotoxic properties. The International Agency for Research on Cancer (IARC) has characterized FA as a carcinogen. In this study, we investigated the effects of FA on rat plasma proteins by using proteomic approach. Rats were exposed to three different concentrations of FA (0, 5, 10 ppm) for 2 weeks at 6 hours/day and 5 days/week in an inhalation chamber. Malondialdehyde (MDA) assay and carbonyl spectrometric assay were conducted to determine lipid peroxidation and protein oxidation levels and Comet assays were used for genotoxicity evaluation. Level of MDA, carbonyl insertion and DNA damage in plasma, livers, and in the lymphocytes of rats exposed to FA were found to be dose dependently increased. Proteomic analysis using three different pI ranges (3.5-5.6, 5.3-6.9, 6-9) and large size two-dimensional gel electrophoresis (2-DE) showed the presence of 3491 protein spots. A total of 32 (19 up- and 13 down-regulated) proteins were identified as biomarkers of FA, all showed dose dependent expressions in the plasma of rats exposed to FA and of these, 27 protein spots were identified by MALDI-TOF/MS. Several differentiated protein groups were found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of SNAP 23, apolipoprotein A-1 and E, clusterin, kinesin, and fibrinogen γ were confirmed by Western blot assay, and apo E was further analyzed by using 2-DE immunoblot assays to determine isoform patterns. Two cytokine including IL4 and INF-γ were measured in plasma with respect to fibrinogen γ changes. In summary, cytotoxicity, and genotoxicity assays, namely MDA lipid peroxidation assay, the carbonyl protein oxidation assay, and Comet genotoxic assay showed that these effects increased on increasing FA levels. Proteomic analysis with three different pI ranges and long size 2-DE gel electrophoresis showed that 32 protein spots were up-or down-regulated. Of these 32 proteins, 7 proteins were confirmed by western blot assay. They could be potential biomarkers for human diseases associated with FA exposure. Keywords: formaldehyde • sick building syndrome • proteomics • rat plasma • two-dimensional polyacrylamide gel electrophoresis • matrix-assisted laser desorption /ionization-time-of-flight mass spectrometry • apolipoprotein A-1 • apolipoprotein E

1. Introduction Formaldehyde (FA) is a low molecular weight organic compound and one of major components that causes sick * To whom correspondence should be addressed. These authors contributed equally to this work. Tel.: + 82-2-922-6174. Fax: +82-2-927-7220. E-mail address: [email protected]. † School of Public Health, Korea University. ‡ Environmental Toxico-Genomic and Proteomic Center, College of Medicine, Korea University. § Department of Preventive Medicine, School of Medicine and Institute for Environmental Health, Medical Science Research Center, Korea University. | School of Biological Sciences and Technology, Chonnam University. ⊥ MyGene Bioscience Institute. # Department of Pathology, College of Medicine, Korea University. ∧ Department of Environmental Health, College of Health Science, Korea University. O Graduate School of Medicine, Korea University.

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Published on Web 04/29/2006

building syndrome (SBS).1-3 FA is widely used in occupational and household indoor environments and is commonly used in the construction, textile, paper product, resin, wood composite, insulating material, paint, plastic, fabric, adhesive, and cosmetic industries.1,4,5 It is suspected that exposure to low levels of FA induces or aggravates airway inflammation, which is mediated by immunological and neurological reactions.6 FA is also classified as a probable human carcinogen based on animal studies with neoplastic lesions at the point of contact and in the nasal cavity, but with only limited evidence of human respiratory tract carcinogenicity.7-9 In addition, FA is a well-known cross-linking agent and can react with many different macromolecules, such as proteins and nucleic acids or with low molecular weight substance as amino acids.9-11 10.1021/pr050437b CCC: $33.50

 2006 American Chemical Society

Toxicological Monitoring Markers of Formaldehyde

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Figure 1. Schematic diagram of the experimental design. FA was generated using a permeater and mixed with air in a mixing chamber. FA concentration, temperature, and humidity in the main exposure chamber were monitored by HPLC, and by using a thermometer, and a hygrometer. These conditions were adjusted by altering humidified airflow.

Thus, levels of FA-induced DNA adducts in nasal cells should serve as a biomarker of FA exposure and its effects.12 Geno-toxicological studies have shown that environmental low concentrations of FA induce DNA damage, growth inhibition and delay DNA repair after UV irradiation in various human cell types, in primary cultures of rat tracheal epithelial cells, and in mouse cells.13-17 In previous studies, toxicological effects, such as, DNA single strand breakage and the formation of DNA adducts were found to change gene regulation and protein expression in organisms.18-21 Recently, proteome analysis has been introduced as a means of analyzing differential gene expression at the protein level, of identifying biomarkers, which is achieved by comparing the 2-DE patterns of proteomes of biosystems exposed to different conditions particularly, after exposure or not to compounds of toxicological relevance. Moreover, a large range of immobilized pH gradients (IPG) strips and more advanced forms of 2-DE analysis, have made it possible to identify proteins whose levels are significantly increased or decreased in cells and animals after exposure to toxic compounds.15 In addition, plasma or serum proteins are useful target molecules to search clinical and toxicological biomarkers.22 However, 2-DE analysis has limitations in terms of plasma protein analysis, because plasma proteins are composed of many high molecular weight molecules. Therefore, in present study, we utilized a large size 2-DE system and three different pI strips to increase the resolution of proteomic analysis. We chose three different concentrations of FA, and cytotoxic and genotoxic assays were carried out on the plasma, and the blood or liver cells of rats treated with FA by inhalation. Profiles of plasma protein changes determined using 2-DE analysis also were evaluated with the aim of identifying toxicological monitoring markers in rats exposed to FA. In addition, associations between the results of cytotoxic and genotoxic assays with respect to plasma protein changes are discussed.

2. Materials and Methods Chemicals. FA was purchased from Sigma Chemical (St Louis, MO). Urea, thiourea, 3- [(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), dithiothreitol (DTT), acrylamide, NN′-methylene-bisacrylamide, iodoacetamide, acetonitrile, sodium thiosulfate, trifluoroactic acid, 2-thiobarbituric acid(TBA),butylatedhydroxytoluene(BHT),1,1,3,3-tetramethoxypropane (TMP) were purchased from Sigma Chemical (St Louis,

MO). 2,4-dintrophenylhydrazine (DNPH) was purchased from Fluka (Buchs, Switzerland) and protease inhibitor cocktail from Roche (Mannheim, Germany). Animals. Specific pathogen free male, Sprague-Dawley rats were obtained from the Samtaco Animal Breeding Company (Osan, Korea), and housed under standard laboratory conditions (Tm; 24 ( 2 °C, humidity; 50 ( 10% and 12-hour day/ night cycles). Animals were allowed to acclimatize to the facility for 1-2 weeks, and were then observed for abnormal behavior. They were given free access to Samtaco Animal chow diet (PMI Nutritional Int. LLC, MO) and drinking water ad libitum, and were 6-8 weeks old on the first day of exposure. Experimental Design. Two formaldehyde exposure groups i.e., at 5 and 10 ppm and an unexposed control group were used in this study. Animals were exposed for 2 weeks at 6 hours/day and 5 days/week in an inhalation exposure chamber. To determine the formaldehyde concentrations, air samples were collected and FA concentrations were determined using an HPLC. Ten rats were exposed in each experiment (Total: 30 rats). Rats were finally sacrificed (09:00 to 12:00) and blood and tissue samples were collected for proteomic analysis and to determine the extents of DNA damage, protein oxidation, and lipid peroxidation. Formaldehyde Exposure. FA exposures were conducted for 2 weeks in 1-m3 stainless steel-and-glass inhalation chambers, at target FA concentrations of 5 and 10 ppm. Figure 1 shows a schematic of the exposure process. FA concentrations were generated using a Permeater PD-1B (Gastec, Japan) and were adjusted by altering the humidified airflow in a second mixing chamber. Exposure chamber air was conditioned to 22 ( 2 °C and 50 ( 5% humidity. FA concentrations in inhalation were monitored using a HPLC. Blood Cell ans Tissue Sample Preparation. Blood samples (3-5 mL of heparinized whole blood) were collected by cardiac puncture from each rat, and comet assays were carried out within 3 h. Lymphocytes were prepared by removing red blood cells from whole blood, via centrifugation with Ficoll-Paque solution. Lymphocytes were washed three times with 10 mL of phosphate buffered saline and centrifuged at 300 × g. Collected cells were immediately subjected to Comet assay. Livers were chosen for examination of the MDA, protein carbonyl formation and Comet assay, because livers were the main organ tissue to change the plasma protein profiles. Liver tissue samples were prepared for Comet assay according to the Journal of Proteome Research • Vol. 5, No. 6, 2006 1355

research articles methods described by Farris, with minor modifications.23 Livers were minced with a scalpel blade and then mashed with a syringe plunger. The cell suspension so obtained was filtered through a 25-µm metal mesh and the filtered suspension was placed in 15 mL conical tubes and allowed to settle for five minutes to form a 2-3 mL of upper single cell suspension. These collected liver cells were washed with PBS buffer in preparation for the Comet assay. Comet Assay. The comet assay was performed as described by Singh, with minor modifications.24 In brief, normal melting point agarose (Ameresco, NMA) and low melting point agarose (Ameresco, LMA) were dissolved in PBS (Gibco, BRL) using microwave heating. Then, 100 µL of 1% NMA was added to a fully frosted slides precoated with 50 µL of 1% NMA for a firm attachment, and the slides were allowed to solidify with cover slips in the refrigerator for 5 min. After solidification of the gel, the cover slips were removed and 50 µL lymphocytes mixed with 50 µL of 1% LMA were added. The cover slips were added to the layer and the slides were again allowed to solidify in the refrigerator for 5 min. After removing the cover slips, 100 µL of 0.5% LMA was added to the third layer, and the slides were placed with cover slips in the refrigerator again for 5 min. The slides were submersed in the lysing solution (2.5 M NaCl, 100 mM EDTA-2Na, 10 mM Tris-HCl, pH 10; 1% Triton X-100 and 10% DMSO, pH 10 were added fresh) for 1 h. The slides were then placed in unwinding buffer (1 mM EDTA and 300 mM NaOH, pH 13) for 20 min and electrophoresis was carried out using the same solution for 20 min at 25 V and 300 mA (0.8 V/cm). After electrophoresis, the slides were neutralized via three washings with neutralization buffer (400 mM Tris-HCl, pH 7.4) 5 min each and were stained with 50 µL of 10 µg/mL ethidium bromide. The slides were examined using a Komet 4.0 image analysis system (Kinetic Imaging, Liverpool, UK) fitted with an Olympus BX50 fluorescence microscope equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm. For each treatment group, two slides were prepared and each 50-100 randomly chosen cells (total 100-200 cells) were scored manually. The parameter, Olive tail moment ()(Tail.mean-Head.mean)*Tail%DNA /100), was calculated automatically using the Komet 4.0 image analysis system, which was used for global comet description. Determination of Malondialdehyde (MDA). The high performance liquid chromatographic (HPLC) method described by Agarwal and Chase25 for MDA was used, with slight modification for the analysis of plasma and liver samples as follows: MDA in plasma or liver was measured using a HPLC (Gilson, France) equipped with a Synergi Polar-RP C18 5 µm column (4.6 mm in inner diameter and 300 mm in length). Fifty microliters of plasma or 20% liver homogenate were mixed with 50 µL of 0.05% BHT, 400 µL of 0.44 M phosphoric acid (H3PO4) solution, and 100 µL of TBA solution. The samples were then incubated in 100 °C water bath for 1 h. After incubation, the TBA-MDA adduct was extracted with n-butanol. Aliquots of 100 µL were then removed from the n-butanol layer and injected into the HPLC system. Standards and quality control samples were prepared using 97% TEP (0, 0.125, 0.5, 1.0, and 2.0 µmol/L). Protein Oxidation. Protein carbonyls in the livers and plasma were determined using a spectrometric DNPH assay according to Fagan with minor modifications.26 Brifely, liver tissues were homogenized by sonication in lysis buffer containing PBS (pH 7.2), 1% Triton ×100, 1mM EDTA, and 1 X protease inhibitor cocktail and the insoluble cellular debris were re1356

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moved by centrifugation. Aliquots of protein samples were precipitated with 10 volumes of HCl-acetone (3:100) and then washed with 5 mL of 10% of TCA solution. Pellets were resuspended in 500 ul buffer solution and then reacted with 500 µL of 10 mM DNPH (in 2 M HCl) by vortexing for 15 min. In case of plasma, 100 µL of plasma was mixed with 400 µL of buffer solution. Protein blanks were prepared with 500 µL of 2 M of HCl solution. After mixing, 500 µL of 30% TCA was added to each tube, vortexed and placed on ice for 10 min. To remove unreacted DNPH, the centrifuged pellets were washed with 5 mL of 20% TCA and 5 mL of ethanol: ethyl acetate mixture (1:1) (v/v). The final precipitate was resolved in 1 mL of 6 M guanidine HCl, and the absorbance 380 nm was determined for the sample treated with DNPH and HCl, which was subtracted as background. The carbonyl content was calculated from the absorbance measurement at 380 nm and an absorption coefficient of 22 000 mol/cm. 2-DE PAGE. Sample Preparation. Plasma samples were prepared for 2-DE as described previously.21 Lipids and salts were removed from samples using a molecular weight cut off column (3 kDa; Amicon, Millipore, Bradford, MA). For this step, plasma and sample buffer containing of 7 M urea, 2 M thiourea, 40 mM Tris (0.5 M, pH 8.5), 4% CHAPS, 65 mM DTT, 1% IPG buffer (pH 4-7 L) and 1% protease inhibitors were mixed in equal volume. After centrifugation of a molecular weight cut off column 4 times at 3500 rpm for 1 h at 12 °C, each the supernatant was aliquoted and stored at -70 °C. Protein concentration was measured by modified Bradford assay method.27 Isoelectric Focusing (IEF) and SDS-PAGE. In the first dimension of the 2-DE, the proteins were separated according to their isoelectric points. The protein sample solution (150 µg) was mixed with a rehydration buffer containing 8 M urea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTT a trace of bromophenol blue (BPB) to a total volume a 450 µL per sample. The IEF was carried out with commercially available immobilized pH gradients (pH 3-11 nonlinear, 3-5.6 nonlinear, 5.3-6.5, 6-9, 24 cm), using the IPGphor (Amersham Biotech, Amersham, UK) apparatus. The gel was rehydrated in the presence of the sample for 12 h and focused for 60, 85, and 130 kVh. After IEF, the IPG gel strips were equilibrated twice for 15 min, under gentle shaking at room temperature, first in the solutions (equilibration buffer: 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 1% w/v SDS) containing 1% DTT, and in an equilibration buffer containing 2.5% iodoacetamide. In the second dimension, SDS-PAGE, the proteins were resolved solely on the basis of their molecular masses in 11.5% gradient polyacrylamide gels (size 35 × 45 cm) using an Owl separation system runner (Owl Separation System Co, Portamouth, NH). The IPG strips were embedded in 0.5% w/v melted agarose prior to running on the SDS-PAGE slabs. The agarose contained 0.001% w/v BPB as a tracking dye. The running conditions were 1 w/gel for 30 min and 20 w/gel for 14-16 h until the BPB reached the end of the gel. Visualization and Image Analysis. After separation in the SDS-PAGE gels, the proteins were visualized using a silver staining kit (Amersham Biotech, Amersham, UK), according to the manufacture’s instruction. The silver-stained gels were scanned using an 3600 × 4900 dpi instrument (Epson Expression 10000XL, Epson, Japan) and the image files were transformed into TIF format with linear gray scale values. The computer analysis of the 2D-image was carried out using Progenesis Discovery, 2-DE image analysis software (Nonlinear


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Figure 2. DNA damage in lymphocytes (A) and in the livers (B) of rats exposed to varying concentrations of FA; 0 (control), 5, and 10 ppm. After 2 weeks, genotoxicity was assessed by DNA single strand breakage via the Comet assay. Data represent the means ( SD (n ) 10). *: Significantly different with formaldehyde concentration 0 ppm (control) by Duncan test and ANOVA (p < 0.05) #: Significantly different with formaldehyde concentration 5 ppm by Duncan test and ANOVA (p < 0.05).

Figure 3. Lipid peroxidation in plasma (A) and in the livers (B) of rats exposed to varying concentrations of FA; 0 (control), 5, and 10 ppm. After 2 weeks, MDA levels were determined by HPLC. Data represent the means ( SD (n ) 10). *: Significantly different with formaldehyde concentration 0 ppm (control) by Duncan test and ANOVA (p < 0.05) #: Significantly different with formaldehyde concentration 5 ppm by Duncan test and ANOVA (p < 0.05).

Dynamics, Newcastle upon Tyne, UK) according to the Manufacture’s protocol. Intensity levels were normalized between gels as a proportion of the total protein intensity detected for the entire gel. MALDI-TOF-MS Analysis and Protein Identification. Protein spots were obtained from gels stained using 0.12% Coomassie Brilliant Blue G (Amersham Biotech, Amersham, UK) and were digested previously described but some modifications.28,29 Briefly, gel spots were excised with a scalpel, crushed, and destained by washing with 25 mM ammonium bicarbonate, 50% acetonitrile. Gels were then dehydrated by adding acetonitrile, rehydrated in ice by adding 10-20 µL of 25 mM ammonium bicarbonate with 10 µg/mL of sequencing grade trypsin (Promega), and incubated at 37 °C for 12-15 h. Peptides were extracted three times by adding 25 µL of a solution containing 50% acetonitrile, 0.1% trifluoroacetic acid and completed by adding 20 µL of acetonitrile. The extracted solutions were pooled and evaporated to dryness in a Speed Vac centrifuge. Samples were reconstituted in 10 µL of 0.1% trifluoroacetic acid and treated with ZipTips containing C18 resin (Millipore Co. Bedford, MA) according to the manufacturer’s instructions. The washed peptides were eluted using saturated matrix solution (R-cyano-4-hydroxycinnamic acid in 60% acetonitrile, 0.1% trifluoroacetic acid). Monoisotopic masses (M+1) of tryptic fragments were determined using a Perspective Biosystem MALDI-TOF-MS voyager DE-STR Mass

Spectrometer (Framingham, MA). The spectra obtained were internally calibrated versus trypsin peaks. The interpreted tandem mass spectra of peptides were searched against the NCBInr (NCBInr.1.6.2005) using the MS-Fit (http://prospector.ucsf.edu) program. Known keratin masses and trypsin autodigestion products were excluded from the searches. The parameters were set as one of missed cleavage and acrylamide modification. Protein identities were assigned if at least five peptides masses were matched within a maximum error of 50 ppm and the candidate agreed with the estimated pI and Mw estimates from 2-DE gel. Western Blotting. Rat plasma proteins were separated by 12% SDS-PAGE. Proteins were electroblotted onto PVDF membranes and membranes were blocked overnight at 4 °C in TPBS (0.05% Tween 20 in PBS) with 5% dried skimmed milk, and then incubated in a 1:500 dilution of primary antibody (fibrinogen γ, apo A, apo E, kinesin, SNAP 23, clustein, interleukin 4, INF-γ and tubulin, supplied by Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Membranes were then washed with TPBS and incubated in a 1:2000 dilution of anti-rabbit, mouse, goat IgG conjugated to peroxidase (supplied by by Santa Cruz Biotechnology, Inc) for 45 min at room temperature. Bands containing rat plasma proteins were visualized by chemiluminescence (Amersham ECL kit) and analyzed by scanning with a flat-bed scanner and digitalizing using Scion image analysis software (Scion Co, Frederick, MD). For 2-DE Journal of Proteome Research • Vol. 5, No. 6, 2006 1357

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Figure 4. Protein oxidation in plasma (A) and in the livers (B) of rats exposed to varying concentrations of FA; 0 (control), 5, and 10 ppm. After 2 weeks, carbonyl contents were determined using spectrometric DNPH assay. Data represent the means ( SD (n ) 10). *: Significantly different with formaldehyde concentration 0 ppm (control) by Duncan test and ANOVA (p < 0.05).

immunoblot analysis of Apo E, rat plasma proteins were separated by 2-DE using pH 4-7 NL strips and by 11.5% SDSPAGE and then electroblotted onto PVDF membranes (20 × 14 cm). Statistical Analysis. Statistic analyses were performed using SAS version 8.2. We used the analysis of variance (ANOVA) method with Duncan and Tukey to determine the differences between exposure and control groups. The level of statistical significance employed in all cases was p < 0.05.

3. Results Rat Inhalation Exposure to FA. FA exposures to rats were carried out after FA concentration had been equilibrated in the mixing chamber. FA concentrations were monitored using HPLC. The actual mean FA concentrations achieved over the two weeks study were 5.10 ( 0.01 and 10.08 ( 0.01 ppm. DNA Damage in Lymphocytes. Comet assay results of rat peripheral lymphocytes and livers are shown in Figure 2. The mean value of the Olive tail moment of the control lymphocytes was 1.24 ( 0.04. After 2 weeks of FA exposure, the mean values of the Olive tail moments of lymphocytes from rats exposed to 5 or 10 ppm FA were 1.72 ( 0.11 (p ) 0.0019) and 2.16 ( 0.14 (p ) 0.0001), respectively indicating significant level of DNA damage. The mean values of the Olive tail moments of the lymphocytes of rats exposed to FA increased with FA concentration. In livers, a similar pattern of DNA damage was found 1358


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Figure 5. 2-DE analysis of plasma proteins using three different ranges of pI strips (3.0-5.6, 5.3-6.5, and 6-9). Large size gels (24 × 36 cm) were used to analyze protein profiles and protein spots were visualized by silver staining.

and this too increased in an FA dose dependent manner. The mean Olive tail moment value of control livers was 1.19 ( 0.08, and after 2 weeks of FA exposure to 5 or 10 ppm FA, the mean Olive tail moment values of rat livers were 1.73 ( 0.10 (p ) 0.0001) and 2.49 ( 0.20 (p ) 0.0001), respectively. Determination of Lipid Peroxidation. Rat plasma and livers after 2 weeks of exposure to 0, 5, or 10 ppm FA were subjected to MDA assay (Figure 3). The levels of MDA in rat plasma and livers of rats treated with 5 ppm FA were no different from those of the controls, but MDA levels were significantly higher when animals were exposed to FA at 10 ppm. The mean MDA values of the control plasma and livers were 0.48 ( 0.07 µmol/L and 0.29 ( 0.02 nmol/mg protein and after 2 weeks of exposure to FA at 5 or 10 ppm, these mean MDA values in plasma and livers of rats increased to 0.56 ( 0.09 and 0.96 ( 0.05 µmol/L in plasma (p ) 0.0001) and 0.31 ( 0.03 and 0.35 ( 0.05 nmol/mg protein in liver (p ) 0.0165), respectively. Protein Oxidation. Protein oxidation was determined by contents of carbonyl groups on amino acids, which are derivatized with 2,4-dinitrophenyl hydrazine (Figure 4). The mean carbonyl content of the control livers was 0.69 ( 0.23 nmol/ mg protein. After 2 weeks of FA exposure, this mean value increased to 1.18 ( 0.18 and 1.91 ( 0.35 nmol/mg protein in rats exposed to 5 or 10 ppm FA (p ) 0.0011), respectively. Protein oxidation in the livers of rats exposed to FA thus increased with increasing FA concentration. In plasma, a similar pattern of protein oxidation was found and carbonyl contents increased in a FA dose dependent manner. The mean carbonyl content of control plasma was 3.31 ( 0.02 nmol/mg protein,


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Figure 6. A. 2-DE pattern of plasma proteins obtained using a 3-5.6 pI strip. Gels were visualized by silver staining. The images of protein spots were analyzed using the Image Master 2-DE Progenesis Discovery Software program (Nonlinear Dynamics, Newcastle upon Tyne, UK). The 2-DE image demonstrates the plasma proteome pattern of untreated control rats. The images of each changed spot were compared at increased FA concentration. B. Spot volumes were calculated by normalizing versus total spot volumes. The quantity presented by each spot is expressed as a relative intensity. Data represent the means ( SD (n ) 10).

and after 2 weeks of FA exposure, this mean value increased to 3.37 ( 0.06 and 3.37 ( 0.08 nmol/mg protein, respectively, in rats exposed to 5 or 10 ppm FA (p ) 0.000 03). 2-DE Analysis of FA-Induced Changes in the Plasma Protein Profiles. Proteomic analysis was carried out using three different pI ranges (i.e., 3.0-5.6, 5.3-6.5, 6-9) and a large size 2-DE system (Figures 5-8). Rats were exposed to FA at 0, 5, or 10 ppm. Figure 5 shows the 2DE-patterns of plasma proteins of rats exposed to FA using the three different ranges of pI strips (at 3.0-5.6, 5.3-6.5, 6-9), 1496, 1362, and 633 protein spots were observed in gels, respectively. Thus, a total of 3491 protein spots were resolved (Figure 5).

A total of 32 proteins were found to be up- and downregulated in exposed cells, which is summarized in Tables 1 and 2 (Figures 6-8). In plasma from exposed rats, 19 spots were up-regulated in a dose dependent manner using strips of pI 3.0-5.6, 5.3-6.5, and 6-9 strips (Figures 6 and 8). Eight spots, 4 spots, and 1 spots were down-regulated in dose dependently in the pI 3.0-5.6, 5.3-6.5, and 6-9 strips, respectively. The identification of these differentially expressed proteins was carried out by MALDI-TOF-MS which allowed the identification of 32 proteins, which included proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and cell motility (Tables 1 and 2). Journal of Proteome Research • Vol. 5, No. 6, 2006 1359

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Figure 7. A. 2-DE gel pattern of plasma proteins obtained using a 5.3-6.5 pI strip. Gels were visualized by silver staining. The image of protein spots were analyzed using the Image Master 2-DE Progenesis Discovery Software program (Nonlinear Dynamics, Newcastle upon Tyne, UK). The 2-DE image demonstrates the plasma proteome pattern of untreated control rats. The images of each changed spot were compared at increased FA concentration. B. Spot volumes were calculated by normalizing versus total spot volumes. The quantity presented by each spot is expressed as a relative intensity. Data represent the means ( SD (n ) 10).

Confirmation of The Identities of Proteins by Western Blotting. Among a total of 3491 resolved protein spots, 32 were found to be dose-dependently up- or down-regulated in rat plasma using different pI strips. Western blot assays were conducted to confirm the protein identities (Figure 9). Commercially SNAP 23, apolipoprotein A-1 (Apo A-1) and E, clusterin, kinesin, and fibrinogen γ monoclonal antibodies were purchased and used to confirm their identities as useful candidates of toxicological biomarkers of FA. The expressions of five proteins, i.e., SNAP-23, Apo A-1, clusterin, fibrinogen γ, and kinesin were significantly down-regulated on increasing concentration of FA, but Apo E was up-regulated at 5 ppm FA and down-regulated at 10 ppm FA (Figure 9). Expression of Cytokines in Plasma of Rats Exposed to FA. To investigate the expressions of inflammatory cytokines in plasma from rats exposed to FA, Western blot assays were 1360


performed (Figure 10). Th2 type cytokine, IL-4 was upregulated, but Th1 cytokine, IFN-gamma was down-regulated in the plasma of FA exposed rats in a dose dependent manner. These data suggest that FA has an inflammatory effect. Determination of Apo E Expression using 2-DE Immunoblot Assay. The 2-DE immunoblot assay was carried out to determine the expression pattern of Apo E isoforms (Figure 11). Two isoforms of Apo E (D1 and D2) were significantly downregulated and all nine isoforms of Apo E (UI-U9) were significantly up-regulated (p < 0.05, n ) 10). Further study is needed to determine the characteristics of the Apo E isoforms.

4. Discussion The IARC has classified FA in Group2A, due to the existence of sufficient evidence in animals and limited evidence in humans for carcinogenicity on the basis of epidemiological


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Figure 8. A. 2-DE gel pattern of plasma proteins obtained using a 6-9 pI strip. Gels were visualized by silver staining. The image of protein spots were analyzed using an Image Master 2-DE Progenesis Discovery Software program (Nonlinear Dynamics, Newcastle upon Tyne, UK). The 2-DE image demonstrates the plasma proteome pattern of untreated control rats. The images of each changed spot were compared at increased FA concentration. B. Spot volumes were calculated by normalizing versus total spot volumes. The quantity presented by each spot is expressed as a relative intensity. Data represent the means ( SD (n ) 10).

studies.8 In present study, we evaluated the general toxic and genotoxic effects of FA on lymphocytes, liver cells and in plasma before investigating the differential expressions of plasma proteins. MDA assays were carried out to determine lipid peroxidation levels and Comet assays were used for genotoxicity evaluations. In addition, spectro-immunoassays were carried out to determine for the presence of DNPconjugated proteins. In a previous study, the general toxicity and the genetic damage caused by of FA was investigated in germ cells at different stage. A significant increase in the micronuclei ratio was observed in early spermatogenic cells and in the sister chromatid exchange ratio in the medium and high dose groups.30 On the other hand, FA has been found to increase the frequency of chromatid/chromosome aberrations, sister chromatid exchange, and gene mutations in a variety of rodent and human cell types. Exposure to FA was found to increase

DNA damage in human fibroblasts and in rat tracheal epithelial cells, and to increase unscheduled DNA synthesis in rat nasoturbinate and maxilloturbinate.9 Other genotoxic studies show that FA has an inhibitory effect on different DNA repair pathways,13,14,19 and the presence of DNA adducts in nasal cells, which could serve as a biomarker of FA exposure and of its effects.12 FA is also a well-known cross-linking agent that can react with many different macromolecules, including proteins and nucleic acids, and with low molecular weight substance as amino acids.10 In addition, FA induced DNA protein crosslinks can block either DNA polymerase or the entire replication complex.20 FA was also found to cause persistent DNA damage in bronchial cells, which were initially damaged by exposure to N-methyl-N-nitrosourea, and to inhibit the repair of single strand DNA breakages caused by UV. The present study also produced a similar finding as DNA damage was significantly Journal of Proteome Research • Vol. 5, No. 6, 2006 1361

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Table 1. Up-Regulated Protein Spots in the Rat Plasma Exposed to Formaldehydea spot no.

up-regulated proteins

mass (kDa)/pI theoretic

coverage (%)

NCBI accession no.

958 1058 1166 1307 1348 1461 1705 2572 2656 2704 2811 3251 3253 3426 3446 3453 3539 3632 3718

Similar to Crk-like protein 12/15-lipoxygenase Type 2X myosin heavy chain Unidentified Glutathione S-transferase Yb-2 NG,NG-dimethylarginine dimethylaminohydrolase 1 Apolipoprotein E Inositol (myo)-1(or 4)-monophosphatase 1 Calcium-binding protein p22 Sulfotransferase family 4A, member 1 Proliferin Fructose-1,6-bisphosphatase Unidentified Unidentified Unidentified Phosphatidylinositol transfer protein alpha isoform Similar to CGI-112 protein 27 kDa Golgi SNARE protein Proteasome activator complex subunit 1

33865/6.3 26863/6.3 21347/8.0 29066/8.3 25703/6.9 31426/5.8 35754/5.2 30512/5.2 22432/5.0 33054/5.3 27822/4.8 39610/5.5 46012/5.5 15055/4.6 32088/5.5 31908/6.0 22942/5.8 24650/6.8 28577/5.8

32 29 33

55249763 M 765332 M 5802178

34 33 27 15 21 27 28 26

28933457 M 11560131 M 55824759 M 14091736 M 13162318 M 13928882 M 16758092 M 51036635

20 25 21 22

8393962 M 55562756 M 12643479 M 8394088 M

a Protein spots were determined using a Perspective Biosystem MALDI-TOF-MS voyager DE-STR Mass Spectrometer (Framingham, MA). The spectra obtained were internally calibrated versus trypsin peaks. The interpreted tandem mass spectra of peptides were searched against the NCBInr (NCBInr.1.6.2005) using the MS-Fit (http://prospector.ucsf.edu) program. The spots 1307, 3253, 3426, and 3446 were unidentified, because of inability to obtain MS data.

Table 2. Down-Regulated Protein Spots in the Rat Plasma Exposed to Formaldehydea spot no.

down-regulated proteins

mass (kDa)/pI theoretic

coverage (%)

NCBI accession no.

185 476 2243 2496 2497 2719 2738 2964 2987 3069 3125 3423 3731

Growth factor receptor bound protein 14 Fibrinogen gamma chain precursor Phosducin-like protein Kinesin-like protein KIF4 SH3P13S Tyrosine 3/tryptophan 5 -monooxygenase activation protein Clusterin Synaptosomal-associated protein, 23kD Apolipoprotein A-I Guanine nucleotide-binding protein G(q), alpha subunit Unidentified Heme oxygenase (decycling) 2 Similar to RIKEN cDNA 9430023L20

60593/8.8 50633/5.4 34274/4.7 29384/4.9 31554/4.8 28212/4.8 24931/4.9 23235/4.8 29831/5.5 41470/5.6 49252/6.0 35763/5.3 25016/5.8

27 34 17 24 28 36 29 22 49 29

13928858 M 1346007 11560050 M 5070670 6497032 6981710 M 11127974 12083641 M 2145143 9296968

22 38

13242295 M 50925751 M

a Protein spots were determined using a Perspective Biosystem MALDI-TOF-MS voyager DE-STR Mass Spectrometer (Framingham, MA). The spectra obtained were internally calibrated versus trypsin peaks. The interpreted tandem mass spectra of peptides were searched against the NCBInr (NCBInr.1.6.2005) using the MS-Fit (http://prospector.ucsf.edu) program. The spot 3125 was unidentified because of inability to obtain MS data.

induced in lymphocytes and livers of rats by increasing FA concentration. Lipid peroxidation is commonly regarded as a deleterious process that leads to the structural modifications of complex lipid protein assemblies, such as, biomembranes and lipoproteins, and is usually associated with cellular malfunction.31 The levels of MDA in the plasma and livers of rats exposed to FA were found to be increased in a dose dependent manner in present study. It has also been reported that MDA levels in testicle germ cells at different stage are significantly increased in mice exposed to high levels of FA.30 In general, lipid peroxidation processes may occur nonenzymatically due to the actions of reactive oxygen species or may be catalyzed by enzymes such as alpha-dioxygenases, lipoxygenases, or peroxidases.32 In our proteomic analysis, lipoxygenase expression was found to be up-regulated in exposed rat plasma in a dose dependent manner. Lipoxygenases are a family of enzymes that dioxygenate unsaturated fatty acids, and thus initiate the lipid peroxidation of membranes and the synthesis of signaling molecules. Consequently, they induce structural and metabolic changes in cells in a number of pathophysiological conditions. Recently, lipoxygenase and hydroperoxides were reported to 1362


have a pro-apoptotic effects in different cells and tissues, leading to cell death.33 Although the mode of action of FA is not well understood, based on data derived from laboratory studies, the regenerative proliferation associated with its cytotoxicity appears to be an obligatory intermediate step in putative cancer induction by FA.9 In a 14C-FA study, hydroxymethyl arginines were found to be endogenous FA carrier molecules, in that they preferentially transfer bound FA to tumorous cells, and significantly inhibit cell proliferation and induce apoptosis.34 In the present study, FA exposure induced Crk expression in rat plasma. Smith et al. reported that the adaptor protein Crk is required for apoptotic signaling in egg extracts, including regulation of cell growth and cell motility.35,36 Regarding the effects of FA on detoxifying species, sulfotransferases and glutathione S-transferases (GSTs) were significantly up-regulated by FA. Sulfotransferases catalyze sulfonation and the cytosolic sulfotransferases are involved in detoxification, hormone regulation, and drug metabolism.37 GSTs are multifunctional and multigene products. The GST enzymes are traditionally considered to be detoxifiers of electrophiles by glutathione conjugation. In one study, FA


research articles

Figure 9. A. Western blots of (1) SNAP-23, (2) Apo A-1, (3) Apo E, (4) clusterin, (5) fibrinogen γ, (6) kinesin, and (7) tubulin. Plasma proteins (20 g) from 0 ppm (control), 5 and 10 ppm FA exposed rats were loaded in each lane. B. Quantities represented by gel bands are expressed as intensities relative to tubulin. (1) SNAP-23, (2) Apo A-1, (3) Apo E, (4) clusterin, (5) fibrinogen γ, (6) kinesin. Data represent the means ( SD (n ) 10).

inhalation (3.6 ppm) for 3 days failed to affect the activities of GSTs in rat nasal epithelium.38 However, in the present study FA exposure (5 ppm) for 10 days was found to increase their activities in plasma. On the other hand, other antioxidant molecule, such as, heme oxygenases (HO) were significantly down-regulated in plasma. HO is a stress-response enzyme and

is involved in the catabolism of haem; it appears to be a novel protective factor with potent antiinflammatory, anti-oxidant, and anti-proliferative effects39,40 In terms of cell homeostasis and transportation, some apoproteins, including apo A, E, and J, were found to be downor up-regulated. Clusterin/Apolipoprotein J (Apo J) is a hetJournal of Proteome Research • Vol. 5, No. 6, 2006 1363

research articles

Im et al.

Figure 10. A. Expression of IL-4 and INF-γ by Western blotting. Plasma proteins (20 g) from 0 ppm (control), 5 and 10 ppm FA exposed rats were loaded in each lane. (1) IL-4, (2) INF-γ, and (3) tubulin. B. Quantities represented by gel bands are expressed as intensities relative to tubulin. (1) IL-4, (2) INF-γ. Data represent the means ( SD (n ) 10).

erodimeric multifunctional glycoprotein and is expressed in a wide variety of tissues and is found in all human fluids.41 Its demonstrated and proposed functions include the transport of lipoproteins, the inhibition of complement-mediated cell lysis, and the modulation of cell-cell interactions. On the basis of the findings of previous studies, clusterin is a cell survival gene, and exerts a protective function on the surviving bystander cells.42 In cells undergoing apoptosis clusterin expression is not enhanced, but rather down-regulated.42 In our study, clusterin expression in plasma was down-regulated dosedependently, and it may be a useful biomarker of FA exposure. Apo A was significantly down-regulated in the highly acidic range (pI 3.0-5.6). The formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that accompanies lipid peroxidation.43 Apolipoprotein E (Apo E) is a 34-kDa lipidassociated protein that is present in plasma and in the central nervous system. Previous studies have demonstrated that Apo E has multiple functions, which include the ability to transport lipids, regulate cell homeostasis, and inhibit lipid oxidation. Moreover, the receptor binding domain of Apo E is responsible for its antioxidant activity.44 In present study, Western blotting showed that Apo E expression was significantly up-regulated in plasma on increasing FA concentration which suggests that it plays a role in cell homeostasis and antioxidant activity, because plasma levels of DNA damage, lipid peroxidation, and protein oxidation were increased in exposed rats. In addition, 2-DE immunoblot patterns showed that the expressions of the several isoforms of Apo E were revealed down- or up-regulated, and thus they could be specific biomarkers of FA in plasma. 1364


Further study is needed to determine the characteristics of the Apo E isoforms. Proteins functionally associated with energy metabolism, structure, or transportation were either down- or up-regulated. Fructose 1, 6-biphosphatase, inositol-1-monophosphatase, myosin heavy chain, calcium binding protein p22 were significantly up-regulated, whereas kinesin and guanine nucleotide binding protein were significantly down-regulated. The kinesins are a superfamily of mechanochemical enzymes and motor proteins that transport membranous organelles and macromolecules required for cellular functions along microtubules.45 Moreover, recent studies have revealed that kinesins play an integral role in the mechanics of mitotic spindle assembly, chromosome segregation, and the shaping of connections in the brain.46 The fibrinogens are a major group of plasma coagulation proteins; they bind using their gamma chains to cell surface receptors, growth factors, and coagulation factors and fulfill key roles in fibrin clot formation, platelet aggregation, and wound healing.47 It has been suggested that fibrinogen could be a useful biomarker of vascular disease and inflammations,48-51 and it is suspected that exposure to low levels of FA induces or aggravates airway inflammation mediated by immunological and neurological reactions.6 In addition, certain cytokines play pivotal roles as inducer and regulators of fibrinogen biosynthesis, for example, interleukins 4, 10, and 13 down-regulate the biosynthesis of fibrinogen.52-54 In the present study, similar results were obtained. IL-4 was found to be up-regulated, whereas fibrinogen and interferon gamma were down-regulated on increasing FA concentration.


Figure 11. 2-DE immunoblot pattern of Apo E. Plasma proteins (500 g) from A. 0 ppm (control), B. 5 ppm and C. 10 ppm FA exposed rats were separated by 2-DE using pH 4-7 NL (24 cm) strips and 11.5% SDS-PAGE. Proteins were electroblotted onto PVDF membranes (20 × 14 cm). Two isoforms were found to be down-regulated (D) and nine to be up-regulated (U) by FA exposure.

In summary, cytotoxicity and genotoxicity assays, namely MDA lipid peroxidation assay, the carbonyl protein oxidation assay, and Comet genotoxic assay showed that these effects increased on increasing FA levels. Th2 type cytokine, IL-4 was up-regulated, but Th1 cytokine, IFN-gamma was downregulated in the plasma of FA exposed rats in a dose dependent manner. These data suggest that FA has an inflammatory effect. Proteomic analysis using three different pI ranges and large size 2-DE resolved 3491 protein spots and showed that 19 proteins were up-regulated and 13 proteins were downregulated after exposure to FA. Of these 32 proteins, 27 proteins were identified by MALDI-TOF-MS. The identities of six proteins, including kinesin, fibrinogen, clusterin, Apo E, Apo A-1, and SNAP 23 were confirmed by Western blot assays, and may be useful biomarkers for human diseases associated with FA exposure. However, these plasma proteins might be similarly altered by other oxidative reagents. Apo E was further analyzed to determine the expression patterns of their isoforms. Apo E revealed a distinctive expression pattern whereby its isoforms were down- or up-regulated, thus indicating that it might be of use as a specific biomarker for FA exposure.

Acknowledgment. This work was supported by the Medical Research Center for Environmental Toxico-Genomics & Proteomics of Korea University and by the Ministry of Environment as “The Eco-Technopia 21 project”. References (1) Kim, W. J.; Terada, N.; Nomura, T.; Takahashi, R.; lee, S. D.; Park, J. H.; Kinno A. Clin. Exp. Allergy 2002, 32, 287-295.

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