Sohee Phark

Anti-oxidative and anti-inflammatory activities of placental extracts in benzo[a] pyrene-exposed rats

OBJECTIVE Placental extracts (PE) have been used for years as a folk remedy in Asian countries. PE mediates alleviation of menopausal symptoms, wound healing, liver regeneration and anti-inflammatory responses. In this study, we evaluated the protective effects of PE on rats exposed to benzo[a]pyrene (BaP). METHODS The composition of amino acids, sugars and fatty acids in PE was analyzed. The effect of PE on DNA damage was determined by Comet assay, and oxidative damage was determined by measuring the activity of superoxide dismutase and the levels of lipid peroxidation. The effect of PE on cytokines and immunoglobulin levels was determined by western blot analysis. RESULTS Exposure of rats to BaP significantly increased the Olive Tailmoments compared to controls, while pre-treatment with PE composed of diverse amino acids, monosaccharides and fatty acids significantly decreased the Olive Tailmoments induced by BaP. In addition, oxidative stress induced by BaP was attenuated by pre-treatment with PE. Furthermore, PE pre-treatment significantly decreased the levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. CONCLUSION Pre-treatment of rats with PE significantly attenuates oxidative damage and immunotoxicity induced by BaP. These findings suggest the further studies regarding the protective effects of PE against environmental toxicants in humans.

Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis.

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also 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 cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.

Evaluation of plasma carcinogenic markers in rat hepatic tumors models induced by rat hepatoma N1-S1 cells and benzo[a]pyrene

Benzo[a]pyrene (BaP) is a known carcinogen. Grilled or smoked meat is the major source of BaP intake for human beings. Previously, we established hepatic tumor animal models by injecting rat hepatoma N1-S1 cells or concomitant injection of N1-S1 cells and BaP into healthy Sprague-Dawley rats. In this study, we performed proteomic analyses of rat plasma collected from a hepatic tumor model and compared them to controls using a 3–10 pI range and large two dimensional gel electrophoreses. Proteomic analyses of rat plasma with hepatic tumors induced by the injection of N1-S1 cells resolved 1295 protein spots. Among them, 10 proteins were identified by ESI-MS-MS; four proteins were up-regulated and six proteins were down-regulated as compared to the controls. In addition, 1295 protein spots were also resolved from rats with hepatic tumors by the injection of N1-S1 cells plus BaP; five proteins were upregulated, and seven proteins were down-regulated. Of these 12 proteins, 10 proteins were identified by ESI-MS-MS. Out of 20 identified proteins, alpha-1-inhibitor 3 and zero beta-1 globin were down-regulated in both rats with hepatic tumors by N1-S1 cell-only and rats with hepatic tumors by the injection of N1-S1 cell plus BaP as compared to the controls. In addition, the identities of four proteins, including dermcidin, serum amyloid P-component (SAP), proteasome subunit alpha type-4 and glutathione peroxidase 3 (GPX-3) were confirmed by western blot analysis. Therefore, the importance of those proteins as candidate biomarkers for the development of hepatic tumors should be further elucidated.

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two-dimensional electrophoresis.

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH4HCO3 + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.

Identification of proteins expressed differently among surgically resected stage I lung adenocarcinomas.

RATIONALE Among patients with surgically resected stage I lung adenocarcinoma, some succumb to early recurrence, while others survive for more than 5 years. Few markers to predict prognoses in these patients have been accepted. Recent advances in proteomic methodologies offer a unique chance to identify new candidate biomarkers. The aim of this study is to find differences in protein expression in resected lung cancer tissue of stage I adenocarcinoma from patients with no recurrence for more than 5 years and from those with early recurrence. METHODS Lung cancer tissues were obtained from 15 patients with pathologically confirmed stage I adenocarcinoma. The patients were divided into two groups, those with recurrence within 36 months (early recurrence group, n=9) and those that were disease-free for over 5 years (disease free group, n=6). Tissue proteins were separated by a two-dimensional electrophoresis long gel system (30 × 40 cm) with set ranges (3-10 NL) and examined by nano-LC-ESI-MS/MS. Western blot assays were performed to validate these proteins. RESULTS Twelve protein spots were up-regulated and 8 were down-regulated in the disease-free group as compared with the recurrence group. Of the 12 up-regulated proteins, haptoglubin, tau-tubulin kinase-2 (TTBK2), thymidine phosphorylase, annexin-1, PIN1, CAPG, and SEC23 were validated by Western blot. Among the 8 down-regulated proteins, serpinB6 and trangelin-2 were validated. CONCLUSIONS A total of 9 differentially expressed proteins were successfully extracted, identified, and confirmed from stage I lung adenocarcinoma tissues. The increased or decreased expression of these proteins according to prognosis may be the basis for further studies of proteomics in developing prognostic biomarkers.

Proteomic analysis of proteins secreted by HepG2 cells treated with butyl benzyl phthalate.

Proteomic changes in proteins secreted by human hepatocellular carcinomas (HepG2) cells exposed to butyl benzyl phthalate (BBP) were evaluated. HepG2 cells were treated with three different concentrations of BBP (0, 10, or 25 μM) for 24 or 48 h. Following incubation, the cells were subjected to proteomic analysis using two different pI ranges (4–7 and 6–9) and large-size two-dimensional gel electrophoresis. Results showed resolution of a total of 2776 protein spots. Of these, 29, including 19 upregulated and 10 downregulated proteins, were identified by electrospray ionization–mass spectrometry–mass spectrometry (ESI-MS/MS). Among these, the identities of cystatin C, Rho guanine nucleotide dissociation inhibitor, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, heptaglobin-related protein, inter-alpha-trypsin inhibitor heavy chain H2, and electron transfer flavoprotein subunit beta were confirmed by Western blot analysis. These proteins were found to be involved in apoptosis, signaling, tumor progression, energy metabolism, and cell structure and motility. Therefore, these proteins have potential to be employed as biomarkers of BBP exposure and may be useful in understanding mechanisms underlying the adverse effects of BBP.

Effects of Benzo(a)pyrene on the Expression of Heat Shock Proteins, Pro-inflammatory Cytokines and Antioxidant Enzymes in Hepatic Tumors Induced by Rat Hepatoma N1-S1 Cells

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is easily introduced to humans via consumption of grilled or smoked meat. BaP causes harmful oxidative effects on cell development, growth and survival through an increase in membrane lipid peroxidation, oxidative DNA damage and mutagenesis. Therefore, the present study was conducted to evaluate the synergistic effects of BaP on oxidative stress in hepatic tumors. In this study, we established a hepatic tumor model by injecting rat hepatoma N1-S1 cells into healthy rats. Changes in the abundance of heat shock proteins (HSPs), antioxidant enzymes and pro-inflammatory cytokines were then investigated by western blot analysis. In addition, we examined changes in oxidative stress levels. Injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells resulted in the formation of hepatic tumors at the injection site. Evaluation of rat plasma reveals that hepatic tumors induced by BaP and N1-S1 cells expresses higher levels of Hsp27, superoxide dismutase (SOD), and tumor necrosis factor-α (TNF-α) when compared to those induced by N1-S1 cells only. The collective results of this study suggest that BaP exerts synergistic effects on the expression of HSP, cytokines and antioxidant enzymes in hepatic tumors induced by rat hepatoma N1-S1 cells.

Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expressions in the plasma of rats and incineration workers.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.