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.

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.

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells.

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4-7 and 6-9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.

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.

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.

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.

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 microM) 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 microM 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.

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 NH(4)HCO(3) + 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.

Curcumin protected PC12 cells against beta-amyloid-induced toxicity through the inhibition of oxidative damage and tau hyperphosphorylation.

One of the pathological hallmarks of Alzheimer's disease is the progressive accumulation of beta-amyloid (Abeta) in the form of senile plaques, and Abeta insult to neuronal cells has been identified as one of the major causes of the onset of the disease. Curcumin, the major and most active antioxidant of Curcuma longa, protects neuronal cells against Abeta-induced toxicity. Therefore, in this study, we investigated the neuroprotective mechanisms by which curcumin acts against Abeta (25-35)-induced toxicity in PC12 cells. Following the exposure of PC12 cells to 10 microM Abeta (25-35) for 24h, significant increases in the level of antioxidant enzymes, and DNA damage were observed, and these increases were accompanied by a decrease in cell viability, and an increase in intracellular calcium levels and tau hyperphosphorylation. In addition, pretreatment of PC12 cells with 10 microg/ml curcumin for 1h significantly reversed the effect of Abeta, by decreasing the oxidative stress, and DNA damage induced by Abeta, as well as attenuating the elevation of intracellular calcium levels and tau hyperphosphorylation induced by Abeta. Taken together, these data indicate that curucmin protected PC12 cells against Abeta-induced neurotoxicity through the inhibition of oxidative damage, intracellular calcium influx, and tau hyperphosphorylation.

Gene expression profiling in lung tissues from rats exposed to formaldehyde.

Formaldehyde is a ubiquitous toxic organic compound recently classified as a carcinogen by the International Agency for Research on Cancer and one of the major factors causing sick building syndrome. In this study, we have investigated the effects of formaldehyde on mRNA expression in rat lung tissues by applying genomics. Rats were exposed to ambient air and two different concentrations of formaldehyde (0, 5, 10 ppm) for 2 weeks at 6 h/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 the lungs of rats exposed to FA were found to be dose dependently increased. Gene expression was evaluated by using a bio-array hybridization analysis. A total of 21 (2 up- and 19 down-regulated) genes were identified as biomarkers for formaldehyde effects. Several differentiated gene groups were found. Genes involved in apoptosis, immunity, metabolism, signal transduction, transportation, coagulation and oncogenesis were found to be up- and down-regulated. Among these genes, the mRNA expressions of cytochrome P450, hydroxymethylbilane synthase, glutathione reductase, carbonic anhydrase 2, natriuretic peptide receptor 3, lysosomal associated protein transmembrane 5, regulator of G-protein signaling 3, olfactomedin related ER localized protein, and poly (ADP-ribose) polymerase-1 were confirmed by quantitative RT-PCR analysis. In summary, the MDA lipid peroxidation and the carbonyl protein oxidation assays showed that cytotoxic effects increased with increasing formaldehyde levels. Genomic analysis showed that 21 genes were up- or down-regulated. Of these genes, nine were confirmed by quantitative RT-PCR and could be potential biomarkers for human diseases associated with formaldehyde exposure.