Proteomic analysis of the renal effects of simulated occupational jet fuel exposure

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss-Webster mice exposed 1 h/day for five days to aerosolized JP-8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large-scale, high-resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass finger-printing and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression in the kidney and provide novel molecular evidence of JP-8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP-8 exposed humans.     

Toxicity of chemical mixtures: proteomic analysis of persisting liver and kidney protein alterations induced by repeated exposure of rats to JP-8 jet fuel vapor

Male Sprague-Dawley rats were exposed by whole body inhalation to 1000 mg/m3 +/- 10% JP-8 jet fuel vapor or room air control conditions for 6 h/day, 5 days/week for six consecutive weeks. Following a rest period of 82 days rats were sacrificed, and liver and kidney tissues examined by proteomic methods for both total protein abundance and protein charge modification. Kidney and lung samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Through the use of peptide mass fingerprinting, confirmed by sequence tag analysis, three altered proteins were identified and quantified. Numerical, but not significantly different increases were found in total abundance of lamin A (NCBI Accession No. 1346413) in the liver, and of 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH, #1346044) and glutathione-S-transferase (GST; #2393724) in the kidneys of vapor-exposed subjects. Protein charge modification index (CMI) analysis indicated significant alterations (P < 0.001) in expressed lamin A and 10-FTHF DH. These persisting changes in liver and kidney proteins are discussed in terms of possible alterations in the functional capacity of exposed subjects.     

A review of analytical methods for the identification and quantification of hydrocarbons found in jet propellant 8 and related petroleum based fuels

Jet propellant 8 (JP-8) is a complex mixture of compounds that varies from batch to batch. Quantification of various compound classes of JP-8, including BTEX, PAHs and VOCs, has been accomplished. Very few papers have tackled total JP-8 quantification because of its complexity. The components in JP-8 tend to co-elute and present at low concentrations, often nondetectable. JP-8 is the major source of chemical exposure for Department of Defense personnel and a potential hazard for civilians and marine animals. Some components of JP-8 have been identified as possible human carcinogens and have been studied extensively. Development of analytical methods to analyze the components of this fuel are essential to measure the extent of exposure, as well as the short-term and long-term exposure in rodents, humans and marine life. To date, JP-8 has been examined in urine, blood, contaminated water and fish tissue. This paper reviews methods currently utilized in the literature for the analysis of JP-8 and its components. This paper also discusses extraction methods and detectors commonly used in JP-8 and hydrocarbon analysis in general. Finally, the effects of exposure and the future of JP-8 and petroleum analysis with respect to human health are discussed.     

A Study of Degradation of a Jet Fuel

Degradation of a jet fuel (JP-5) derived from petroleum and manufactured by the Chinese Petroleum Corporation in Taiwan was determined using various methods. Fuel degradation was monitored in the presence of the following compounds: phenol, 2,6-dimethylphenol, thiophenol, 2-methylpyridine, copper powder and mixtures of some of these compounds both at room temperature and at 80°C. The extent of light scattering and the amount of copper drawn into JP-5 from added copper powder are correlated with the extent of deposit formation due to degradation of the fuel. Copper promotes deterioration of JP-5 because of Cu(II) complex formation. Electron spin resonance and cyclic voltammetric measurements confirm the presence of Cu(II).     

Studies of the Deposit Formation on Storage of a High Energy Fuel

Storage stabilities of a petroleum-derived jet fuel (JP-5), manufactured by the Chinese Petroleum Corporation in Taiwan, were studied. Fuel degradation was monitored in the presence of added specific heteroatomic (O, N, S) and unsaturated compounds, copper, iron and aluminum powders and mixtures of some of these at room temperature and 80°C. A mechanism is suggested for the sediment formation with the sulfur compounds. The extent of light scattering and of metal drawn into JP-5 from added metal powder measured by ICP-AES are correlated with the extent of deposit formation in degradation of the fuel. Metal promotes deterioration of JP-5, because of metal complex formation.     

Protective Effect of Hydroxytyrosol Against Oxidative Stress Mediated by Arsenic-Induced Neurotoxicity in Rats

The present study reports beneficial effect of hydroxytyrosol (HT) against arsenic (As)-induced oxidative stress in the rat brain. Rats were orally administered with sodium arsenite dissolved in distilled water (25 ppm, by oral gavage) for 8 weeks or HT (10 mg/kg b. wt.) in combination with As. Results showed increase in protein oxidation and lipid peroxidation, while catalase and superoxide dismutase (SOD) activities as well as GSH content were decreased after As exposure in rat brain. Fourier transform infrared analysis showed significant alteration in peak area values that also validated the oxidative damage to lipids and proteins. In addition, As exposure caused increase in protein expression of caspase-3 and Bax, while Bcl-2 expression was downregulated resulting in translocation of cytochrome c from mitochondria to cytosol. Treatment of HT with As reversed protein oxidation, lipid peroxidation, and increased GSH content as well as catalase and SOD activities. Administration of HT also prevented translocation of cytochrome c from mitochondria and increased mitochondria/cytosol ratio of cytochrome c. Hence, treatment of HT with As improved antioxidant system and efficiently lowered the generation of oxidative stress in rat brain.     

GC–MS Determination of Antioxidants in Ground Water Contaminated with JP-8

Accidental spills and leaks of kerosene-based fuel require the differentiation of the exact fuel type between kerosene and JP-8. The detection of the antioxidants, 2,6-Di-tert-butylphenol (DTBP) and 2,4-dimethyl-6-tert-butylphenol (DMTBP) in ground water can be an important clue to distinguish between the two. We have developed a method to determine trace phenolic antioxidants in ground water without derivatization by a gas chromatography–mass spectrometry, and then applied it to distinguish JP-8 from kerosene. 25 ground water samples were collected from 25 monitoring wells in an area contaminated with kerosene-based fuel. The antioxidants from ground water were extracted with methylene chloride. Solid phase extraction (SPE) was compared to liquid extraction (LLE), but LLE was selected due to poorer reproducibility and recovery of SPE. Extraction of the compounds from ground water gave recoveries of about 90% and a detection limit of 0.02 μg L^?1. The method was used to analyze groundwater samples contaminated with fuel. DMTBP was detected in concentrations of 0.05–4.65 μg L^?1 in 12 of the samples. Since DMTBP is the only antioxidant used in JP-8 in Korea, this suggests that the fuel in the contaminated samples is JP-8.     

Comparison and evaluation of analysis procedures for the quantification of (2-methoxyethoxy)acetic acid in urine

Several extraction and derivatization procedures were evaluated for the quantification of (2-methoxyethoxy)acetic acid (MEAA) in urine. MEAA is a metabolite and a biomarker for exposure to 2-(2-methoxyethoxy)ethanol, a glycol ether with widespread use in various industrial applications and the specific use as an anti-icing additive in the military jet fuel formulation JP-8. Quantification of glycol ether biomarkers is an active area of analytical research. Various sample preparation procedures were evaluated: liquid–liquid extraction (LLE) using ethyl acetate yielded the highest recovery, and solid-phase extraction (SPE) gave low recovery of MEAA. Two derivatization procedures were thoroughly investigated and validated, namely, silylation of MEAA with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and esterification of MEAA using ethanol. Quantification was performed by gas chromatography (GC) with a mass spectrometer as detector and using a polydimethylsiloxane (HP-1) capillary column. Deuterated 2-butoxyacetic acid (d-BAA) was used as an internal standard. Recovery studies of spiked human urine demonstrated the accuracy and precision of both procedures. The limit of detection (LOD) and other figures of merit for both derivatization procedures will be discussed in detail. Applications of these analysis procedures are also discussed. Disclaimers Mention of company names and/or products does not constitute endorsement by the Centers for Disease Control and Prevention (CDC). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.     

GC–MS Determination of Antioxidants in Ground Water Contaminated with JP-8

Accidental spills and leaks of kerosene-based fuel require the differentiation of the exact fuel type between kerosene and JP-8. The detection of the antioxidants, 2,6-Di-tert-butylphenol (DTBP) and 2,4-dimethyl-6-tert-butylphenol (DMTBP) in ground water can be an important clue to distinguish between the two. We have developed a method to determine trace phenolic antioxidants in ground water without derivatization by a gas chromatography–mass spectrometry, and then applied it to distinguish JP-8 from kerosene. 25 ground water samples were collected from 25 monitoring wells in an area contaminated with kerosene-based fuel. The antioxidants from ground water were extracted with methylene chloride. Solid phase extraction (SPE) was compared to liquid extraction (LLE), but LLE was selected due to poorer reproducibility and recovery of SPE. Extraction of the compounds from ground water gave recoveries of about 90% and a detection limit of 0.02 μg L⁻¹. The method was used to analyze groundwater samples contaminated with fuel. DMTBP was detected in concentrations of 0.05–4.65 μg L⁻¹ in 12 of the samples. Since DMTBP is the only antioxidant used in JP-8 in Korea, this suggests that the fuel in the contaminated samples is JP-8.

     

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18-week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin beta-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53-related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.     

Shock Tube Study of JP-10 Ignition Delay Time

JP-10 (exo-tetrahydrodicyclopentadiene, C10H16) ignition delay times were measured in a preheated shock tube. The vapor pressures of the JP-10 were measured directly by using a high-precision vacuum gauge, to remedy the difficulty in determining the gaseous concentrations of heavy hydrocarbon fuel arising from the adsorption on the wall in shock tube experiments. The whole variation of pressure and emission of the OH or CH radicals were observed in the ignition process by a pressure transducer and a photomultiplier with a monochromator. The emission of the OH or CH radicals was used to identify the time to ignition. Experiments were performed over the pressure range of 151-556 kPa, temperature range of 1000-2100 K, fuel concentrations of 0.1%-0.55% mole fraction, and stoichiometric ratios of 0.25, 0.5, 1.0 and 2.0. The experimental results show that for the lower and higher temperature ranges, there are different dependency relationships of the ignition time on the temperature and the concentrations of JP-10 and oxygen.     

Protein expression patterns associated with advanced stage ovarian cancer

This is a comparative proteomic study on biopsies from patients with ovarian cancer to identify potential diagnostic/prognostic biomarkers in both healthy and tumor tissue, interstitial fluid (normal interstitial fluid and tumoral interstitial fluid and peritoneal effusion. Protein expression/identification was evaluated by 2-DE and MS analysis: six proteins showed differential expression in tumoral interstitial fluid and tumor tissue compared to normal interstitial fluid and healthy tissue: five were found to be downregulated and identified as galectin 3, glutathione S-transferase A-2, retinol binding protein 1, phosphatidylethanolamine-binding protein and annexin 5, while the calgranulin, was significantly upregulated in all pathological samples, including the ascitic fluid. Validation of S100A8 overexpression in carcinoma tissue was obtained by immunohistochemistry. To our knowledge, this is the first study to report an over-expression of calgranulin by 2-DE associated with MS/MS analysis on surgical biopsy. The reduced expression of galectin 3 and retinol binding protein 1 in cystic fluid and serum of patients with early stage disease is confirmed in this study. The results highlight alterations in proteins that control cell-cycle progression and apoptosis, as well as factors that modulate the activity of signal transduction pathways. Moreover, this study suggests that calgranulin expression may be used as a diagnostic and/or prognostic biomarker.     

A label-free differential proteomic analysis of mouse bronchoalveolar lavage fluid exposed to ultrafine carbon black

Ultrafine carbon black (ufCB) is a potential hazard to the lung. It causes changes in protein expression and it increases alveolar-capillary permeability in the lung. Label-free quantitative proteomic methods allow a sensitive and accurate analytical method for identifying and quantifying proteins in a protein mixture without chemically modifying the proteins. We used a label-free quantitative proteomic approach that combined and aligned LC-MS and LC-MS/MS spectra to analyze mouse bronchoalveolar lavage fluid (BALF) protein changes associated with exposure to ufCB. We developed a simple normalization method for quantification without spiking the internal standard. The intensities of unchanged peptides were used as normalization factors based on a statistical method to avoid the influence of peptides changed because of ufCB. LC-MS/MS spectra and then database searching were used to identify proteins. The relative abundances of the aligned peptides of identified proteins were determined using LC-MS spectra. We identified 132 proteins, of which 77 are reported for the first time. In addition, the expression of 15 inflammatory proteins and surfactant-associated proteins was regulated (i.e., 7 upregulated and 8 downregulated) compared with the controls. Several proteins not previously reported provide complementary information on the proteins present in mouse BALF, and they are potential biomarkers for the understanding of mechanisms involved in ufCB-induced lung disorders hypothesize that using the label-free quantitative proteomic approach introduced here is well suited for more rigorous, large-scale quantitative analysis of biological samples. We hypothesize that this label-free quantitative proteomic approach will be suited for a large-scale quantitative analysis of biological samples.     

Analysis of rat testicular proteome following 30‐day exposure to 900 MHz electromagnetic field radiation

The use of electromagnetic field (EMF) generating apparatuses such as cell phones is increasing, and has caused an interest in the investigations of its effects on human health. We analyzed proteome in preparations from the whole testis in adult male Sprague‐Dawley rats that were exposed to 900 MHz EMF radiation for 1, 2, or 4 h/day for 30 consecutive days, simulating a range of possible human cell phone use. Subjects were sacrificed immediately after the end of the experiment and testes fractions were solubilized and separated via high‐resolution 2D electrophoresis, and gel patterns were scanned, digitized, and processed. Thirteen proteins, which were found only in sham or in exposure groups, were identified by MALDI‐TOF/TOF‐MS. Among them, heat shock proteins, superoxide dismutase, peroxiredoxin‐1, and other proteins related to misfolding of proteins and/or stress were identified. These results demonstrate significant effects of radio frequency modulated EMFs exposure on proteome, particularly in protein species in the rodent testis, and suggest that a 30‐day exposure to EMF radiation induces nonthermal stress in testicular tissue. The functional implication of the identified proteins was discussed.     

Exploration of Potential Tumor Markers for Lung Adenocarcinomas by Two‐Dimensional Gel Electrophoresis Coupled with Nano‐LC/MS/MS

To explore the potential tumor markers for lung adenocarcinoma, two‐dimensional gel electrophoresis (2DE) coupled with nano‐LC/MS/MS was used to analyze the differentially expressed proteins in 10 surgical resected lung adenocarcinoma tissues. 16 proteins were significantly different between the cancer tissue and adjacent normal tissue. Galectin‐1, peroxiredoxin II (Prx II), proapolipoprotein, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), aldolase A, enolase 1, neuropolypeptide h3, Prx V, cyclophilin A, vimentin, protein disulfide isomerase (PDI), tropomyosin 3 (TPM 3), glutathione S‐transferase Pi (GST‐Pi), manganese superoxide dismutase (MnSOD), and cofilin 1 were up‐regulated in the cancer tissue. On the other hand, profilin was down‐regulated in the cancer tissue. Among these proteins, six proteins were validated by Western blot analysis. The identified proteins contributing to the spectrum of cancer progression may be used as potential diagnostic biomarkers for lung adenocarcinomas.     

Cross-matching marsupial proteins with eutherian mammal databases: proteome analysis of cells from UV-induced skin tumours of an opossum (Monodelphis domestica)

The identification and characterisation of Monodelphis proteins has required cross-species analysis. Protein expression was investigated in normal, nonirradiated adult fibroblasts and also in fibroblastic cells from a benign cutaneous tumour after chronic ultraviolet (UVB) exposure and a metastatic cutaneous tumour after intermittent exposure. Proteins were separated and visualised by two-dimensional gel electrophoresis (2-D PAGE) and a peptide mass fingerprint (PMF) was obtained for protein spots using matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDITOF-MS). Cross-species PMF database analysis facilitated the identification of 120 proteins, constituting 46.5% of the proteins analysed. The identification of two proteins was confirmed by internal amino acid sequencing using tandem MS. Differential protein expression was observed between normal fibroblasts and those in tumours chronically or intermittently exposed. A number of tropomyosin and vimentin isoforms were expressed only in cells from the metastatic tumour induced by intermittent exposure to UV radiation. These results highlight the value of cross-species PMF analysis for the rapid characterisation of proteins from a poorly defined species and also show how proteomics can be used to detect changes in protein expression in differentially treated cells.     

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.     

Different protein expression of myocardium from Chinese mini-swine model of myocardial infarct

High-resolution two-dimensional gel electrophoresis (2-DE), followed by computer-assisted image analysis was used to screen protein patterns of normal and infarcted myocardial tissues for quantitative and qualitative differences in protein expression. In the gels of pH 5–8 immobilized pH gradient (IPG) strips, 851 protein spots were detected in normal myocardial tissue and 1 032 protein spots were resolved in infarcted myocardial tissue. Thirteen protein spots only expressed in normal myocardial tissue, and 14 protein spots only expressed in infarcted myocardial tissue. Results also showed that 49 protein spots displayed quantitative changes in expression between normal and infarcted myocardial tissue. Eleven protein spots were subjected to mass spectrometry (MS) analysis and seven proteins were identified by peptide mass fingerprinting (PMF). These proteins may be involved in cardiovascular injury, and could play an important role in the treatment of coronary heart disease. __________ Translated from Chemical Journal of Chinese Universities, 2006, 8(27): 1467–1471 [译自: 高等学校化学学报]     

Ignition and combustion characteristics and agglomerate evolution mechanism of aluminum in nAl/JP-10 nanofluid fuel

Journal of Thermal Analysis and Calorimetry - The ignition and combustion processes of concentrated nAl/JP-10 nanofluid fuel were studied by using a CO2 laser ignition system with online diagnosis,...     

中国小型猪心肌梗死模型的比较蛋白质组学研究

利用二维电泳(2DE)分离中国小型猪心肌梗死模型的正常与梗死心肌组织的蛋白提取液, 采用 PDQuest 软件对比分析了两种心肌组织在pH=5─8范围内的2DE谱图. 正常心肌组织检出851个蛋白点, 梗死组织检出1 032个蛋白点. 发现13个蛋白质点只在小型猪的正常心肌组织中表达, 而有14个蛋白质点只在梗死心肌组织中表达. 另外, 还有49个蛋白点在两种组织中表达量上有显著性变化(P<0.05), 选择进行质谱分析其中11个蛋白点, 成功地鉴定出7种蛋白, 蛋白功能分析结果表明, 这些蛋白的差异表达与心肌梗死过程相关.