Estradiol regulates leptin sensitivity to control feeding via hypothalamic Cited1.

Until menopause, women have a lower propensity to develop metabolic diseases than men, suggestive of a protective role for sex hormones. Although a functional synergy between central actions of estrogens and leptin has been demonstrated to protect against metabolic disturbances, the underlying cellular and molecular mechanisms mediating this crosstalk have remained elusive. By using a series of embryonic, adult-onset, and tissue/cell-specific loss-of-function mouse models, we document an unprecedented role of hypothalamic Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (Cited1) in mediating estradiol (E2)-dependent leptin actions that control feeding specifically in pro-opiomelanocortin (Pomc) neurons. We reveal that within arcuate Pomc neurons, Cited1 drives leptin's anorectic effects by acting as a co-factor converging E2 and leptin signaling via direct Cited1-ERα-Stat3 interactions. Together, these results provide new insights on how melanocortin neurons integrate endocrine inputs from gonadal and adipose axes via Cited1, thereby contributing to the sexual dimorphism in diet-induced obesity.

Assessment of the association of exposure to polycyclic aromatic hydrocarbons, oxidative stress, and inflammation: A cross-sectional study in Augsburg, Germany.

BACKGROUND: Exposure to polycyclic aromatic hydrocarbons (PAHs) has been linked to acute and chronic health effects through the suggested pathways of oxidative stress and inflammation. However, evidence is still limited. We aimed to investigate jointly the relationship of PAHs, oxidative stress, and inflammation. METHODS: We measured 13 biomarkers of PAH exposure (n = 6: hydroxylated polycyclic aromatic hydrocarbons, [OH-PAHs]), oxidative stress (n = 6: malondialdehyde (MDA); 8-hydroxy-2'-deoxyguanosine (8-OHdG); and 4 representatives of the compound class of F2α-isoprostanes) in urine, and inflammation (n = 1: high-sensitivity C-reactive protein, [hs-CRP]) in serum from 400 participants at the second follow-up (2013/2014) of the German KORA survey S4. Multiple linear regression models were applied to investigate the interplay between biomarkers. RESULTS: Concentrations of biomarkers varied according to sex, age, smoking status, season, and a history of obesity, diabetes, or chronic kidney disease. All OH-PAHs were significantly and positively associated with oxidative stress biomarkers. An interquartile range (IQR) increase in sum OH-PAHs was associated with a 13.3% (95% CI: 9.9%, 16.9%) increase in MDA, a 6.5% (95% CI: 3.5%, 9.6%) increase in 8-OHdG, and an 8.4% (95% CI: 6.6%, 11.3%) increase in sum F2α-isoprostanes. Associations were more pronounced between OH-PAHs and F2α-isoprostanes but also between OH-PAHs and 8-OHdG for participants with potential underlying systemic inflammation (hs-CRP ≥ 3 mg/L). We observed no association between OH-PAHs and hs-CRP levels. While 8-OHdG was significantly positively associated with hs-CRP (13.7% [95% CI: 2.2%, 26.5%] per IQR increase in 8-OHdG), F2α-isoprostanes and MDA indicated only a positive or null association, respectively. CONCLUSION: The results of this cross-sectional study suggest, at a population level, that exposure to PAHs is associated with oxidative stress even in a low exposure setting. Oxidative stress markers, but not PAHs, were associated with inflammation. Individual risk factors were important contributors to these processes and should be considered in future studies. Further longitudinal studies are necessary to investigate the causal chain of the associations.

A comparative study of persistent DNA oxidation and chromosomal instability induced in vitro by oxidizers and reference airborne particles.

Adverse health effects driven by airborne particulate matter (PM) are mainly associated with reactive oxygen species formation, pro-inflammatory effects, and genome instability. Therefore, a better understanding of the underlying mechanisms is needed to evaluate health risks caused by exposure to PM. The aim of this study was to compare the genotoxic effects of two oxidizing agents (menadione and 3-chloro-1,2-propanediol) with three different reference PM (fine dust ERM-CZ100, urban dust SRM1649, and diesel PM SRM2975) on monocytic THP-1 and alveolar epithelial A549 cells. We assessed DNA oxidation by measuring the oxidized derivative 8-hydroxy-2'-deoxyguanosine (8-OHdG) following short and long exposure times to evaluate the persistency of oxidative DNA damage. Cytokinesis-block micronucleus cytome assay was performed to assess chromosomal instability, cytostasis, and cytotoxicity. Particles were characterized by inductively coupled plasma mass spectrometry in terms of selected elemental content, the release of ions in cell medium and the cellular uptake of metals. PM deposition and cellular dose were investigated by a spectrophotometric method on adherent A549 cells. The level of lipid peroxidation was evaluated via malondialdehyde concentration measurement. Despite differences in the tested concentrations, deposition efficiency, and lipid peroxidation levels, all reference PM samples caused oxidative DNA damage to a similar extent as the two oxidizers in terms of magnitude but with different oxidative DNA damage persistence. Diesel SRM2975 were more effective in inducing chromosomal instability with respect to fine and urban dust highlighting the role of polycyclic aromatic hydrocarbons derivatives on chromosomal instability. The persistence of 8-OHdG lesions strongly correlated with different types of chromosomal damage and revealed distinguishing sensitivity of cell types as well as specific features of particles versus oxidizing agent effects. In conclusion, this study revealed that an interplay between DNA oxidation persistence and chromosomal damage is driving particulate matter-induced genome instability.

Adenine derivatization for LC-MS/MS epigenetic DNA modifications studies on monocytic THP-1 cells exposed to reference particulate matter.

The aim of this study was to explore the impact of three different standard reference particulate matter (ERM-CZ100, SRM-1649, and SRM-2975) on epigenetic DNA modifications including cytosine methylation, cytosine hydroxymethylation, and adenine methylation. For the determination of low levels of adenine methylation, we developed and applied a novel DNA nucleobase chemical derivatization and combined it with liquid chromatography tandem mass spectrometry. The developed method was applied for the analysis of epigenetic modifications in monocytic THP-1 cells exposed to the three different reference particulate matter for 24 h and 48 h. The mass fraction of epigenetic active elements As, Cd, and Cr was analyzed by inductively coupled plasma mass spectrometry. The exposure to fine dust ERM-CZ100 and urban dust SRM-1649 decreased cytosine methylation after 24 h exposure, whereas all 3 p.m. increased cytosine hydoxymethylation following 24 h exposure, and the epigenetic effects induced by SRM-1649 and diesel SRM-2975 were persistent up to 48 h exposure. The road tunnel dust ERM-CZ100 significantly increased adenine methylation following the shorter exposure time. Two-dimensional scatters analysis between different epigenetic DNA modifications were used to depict a significantly negative correlation between cytosine methylation and cytosine hydroxymethylation supporting their possible functional relationship. Metals and polycyclic aromatic hydrocarbons differently shapes epigenetic DNA modifications.

Sorption and biodegradation parameters of selected pharmaceuticals in laboratory column experiments.

Pharmaceutically active compounds have increasingly been detected in groundwater worldwide. Despite constituting a risk for human health and ecosystems, their fate in the environment has still not been exhaustively investigated. This study characterizes the transport behavior of five selected pharmaceutically active compounds (antipyrine, atenolol, caffeine, carbamazepine and sulfamethoxazole) in two sediments (coarse quartz sand and sandy loam) using column experiments with long-term injection of spiked groundwater. Transport parameters were estimated using an analytical reactive transport model. When five selected compounds were injected simultaneously, transport behavior of antipyrine, carbamazepine and the antibiotic sulfamethoxazole were similar to the conservative tracer in both sediments and under varying redox conditions. Atenolol and caffeine were retarded significantly stronger in the sandy loam sediment than in the coarse quartz sand. Biodegradation of caffeine was observed in both sediments after an adaption period and depended on dissolved oxygen. The identification of biodegradation processes was supported by monitoring of intracellular adenosine triphosphate (ATPitc) as a measure for microbial activity. ATPitc was present in varying concentrations in all sediments and was highest when biodegradation of pharmaceuticals, especially caffeine, was observed. When only caffeine and sulfamethoxazole were injected simultaneously, sulfamethoxazole was degraded while caffeine degradation was reduced. The latter seemed to be influenced by low concentrations in dissolved oxygen rather than the presence of the antibiotic sulfamethoxazole. Results of these experiments emphasize the impact on pharmaceutical sorption and (bio)degradation of sediment type and redox conditions, as well as available time for microbial adaption and the combination of pharmaceuticals that are released together into groundwater.

Lipidomic Phenotyping Reveals Extensive Lipid Remodeling during Adipogenesis in Human Adipocytes.

Differentiation of preadipocytes into mature adipocytes is a highly complex cellular process. At lipidome level, the adipogenesis remains poorly characterized. To investigate the lipidomic changes during human adipogenesis, we used the LipidyzerTM assay, which quantified 743 lipid species from 11 classes. The undifferentiated human SGBS cell strain showed a heterogeneous lipid class composition with the most abundant classes, phosphatidylethanolamines (PE), phosphatidylcholines (PC), and sphingomyelins (SM). The differentiation process was accompanied by increased ceramide concentrations. After completion of differentiation around day 4, massive lipid remodeling occurred during maturation, characterized by substantial synthesis of diacylglycerols (DAG), lysophosphatidylethanolamines (LPE), PC, PE, SM, and triacylglycerols (TAG). Lipid species composition became more homogeneous during differentiation to highly concentrated saturated and monounsaturated long-chain fatty acids (LCFA), with the four most abundant being C16:0, C16:1, C18:0, and C18:1. Simultaneously, the amount of polyunsaturated and very long-chain fatty acids (VLCFA) markedly decreased. High negative correlation coefficients between PE and PC species containing VLCFA and TAG species as well as between ceramides and SM imply that PE, PC, and ceramides might have served as additional sources for TAG and SM synthesis, respectively. These results highlight the enormous remodeling at the lipid level over several lipid classes during adipogenesis.

Substrate multispecificity among 20ß-hydroxysteroid dehydrogenase type 2 members.

Steroids regulate many physiological processes. Hydroxysteroid dehydrogenases (HSDs) modulate the levels of steroids in pre- and post-receptor metabolism. The subfamily of 20ß-HSD type 2 currently comprises six members from six different species. The zebrafish ortholog converts cortisone to 20ß-dihydrocortisone and is involved in the catabolism of the stress hormone cortisol. Here, we elucidated the substrate preferences of all 20ß-HSD type 2 enzymes towards a selected panel of steroids. For quantification of the substrates and their respective 20ß-reduced products, we first developed and validated a liquid chromatography-mass spectrometry based method. Applying this method to activity assays with recombinantly expressed enzymes, our findings indicate that the 20ß-HSD type 2 enzymes catalyze the 20ß-reduction of a plethora of steroids of the glucocorticoid biosynthesis pathway. The observed multispecificity among the homologous 20ß-HSD type 2 enzymes implies different physiological roles in different species.

Cholesterol metabolism promotes B-cell positioning during immune pathogenesis of chronic obstructive pulmonary disease.

The development of chronic obstructive pulmonary disease (COPD) pathogenesis remains unclear, but emerging evidence supports a crucial role for inducible bronchus-associated lymphoid tissue (iBALT) in disease progression. Mechanisms underlying iBALT generation, particularly during chronic CS exposure, remain to be defined. Oxysterol metabolism of cholesterol is crucial to immune cell localization in secondary lymphoid tissue. Here, we demonstrate that oxysterols also critically regulate iBALT generation and the immune pathogenesis of COPD In both COPD patients and cigarette smoke (CS)-exposed mice, we identified significantly upregulated CH25H and CYP7B1 expression in airway epithelial cells, regulating CS-induced B-cell migration and iBALT formation. Mice deficient in CH25H or the oxysterol receptor EBI2 exhibited decreased iBALT and subsequent CS-induced emphysema. Further, inhibition of the oxysterol pathway using clotrimazole resolved iBALT formation and attenuated CS-induced emphysema in vivo therapeutically. Collectively, our studies are the first to mechanistically interrogate oxysterol-dependent iBALT formation in the pathogenesis of COPD, and identify a novel therapeutic target for the treatment of COPD and potentially other diseases driven by the generation of tertiary lymphoid organs.

Detection of monohydroxylated polycyclic aromatic hydrocarbons in urine and particulate matter using LC separations coupled with integrated SPE and fluorescence detection or coupled with high-resolution time-of-flight mass spectrometry.

A high-performance liquid chromatographic (HPLC) method with integrated solid-phase extraction for the determination of 1-hydroxypyrene and 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene in urine was developed and validated. After enzymatic treatment and centrifugation of 500 µL urine, 100 µL of the sample was directly injected into the HPLC system. Integrated solid-phase extraction was performed on a selective, copper phthalocyanine modified packing material. Subsequent chromatographic separation was achieved on a pentafluorophenyl core-shell column using a methanol gradient. For quantification, time-programmed fluorescence detection was used. Matrix-dependent recoveries were between 94.8 and 102.4%, repeatability and reproducibility ranged from 2.2 to 17.9% and detection limits lay between 2.6 and 13.6 ng/L urine. A set of 16 samples from normally exposed adults was analyzed using this HPLC-fluorescence detection method. Results were comparable with those reported in other studies. The chromatographic separation of the method was transferred to an ultra-high-performance liquid chromatography pentafluorophenyl core-shell column and coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF-MS). The resulting method was used to demonstrate the applicability of LC-HR-TOF-MS for simultaneous target and suspect screening of monohydroxylated polycyclic aromatic hydrocarbons in extracts of urine and particulate matter.

Determination of air pollution-related biomarkers of exposure in urine of travellers between Germany and China using liquid chromatographic and liquid chromatographic-mass spectrometric methods: a pilot study.

OBJECTIVE: The influence of different exposures to PM2.5 (particulate matter with an aerodynamic diameter below 2.5 µm) on the concentrations of biomarkers of exposure and oxidative stress should be investigated. For this purpose, urine samples from individuals travelling from Germany to China were collected and analysed. MATERIALS: Robust LC and LC-MS/MS methods were established for the determination of biomarkers including 8-hydroxy-2'-deoxyguanosine, malondialdehyde, F2α-isoprostanes and hydroxylated polycyclic aromatic hydrocarbons. As a pilot study, nine volunteers travelled from Germany (mean daily concentration of PM2.5: 21 µg/m3) to China (mean daily concentration of PM2.5: 108 µg/m3). Urine samples were collected before and after the trip. RESULTS: In samples collected after return to Germany, the median concentrations of oxidative stress biomarkers were observed to be higher than in samples collected before leaving Germany. Decreasing trends were observed in the sequences of samples collected after return in the following weeks. Correlations were found between exposure and oxidative stress biomarkers. CONCLUSION: Travellers are ideal models for PM pollution-induced acute health effects study. Exposure to PM pollution can cause oxidative stress and damage.

Aerosol emissions of a ship diesel engine operated with diesel fuel or heavy fuel oil.

Gaseous and particulate emissions from a ship diesel research engine were elaborately analysed by a large assembly of measurement techniques. Applied methods comprised of offline and online approaches, yielding averaged chemical and physical data as well as time-resolved trends of combustion by-products. The engine was driven by two different fuels, a commonly used heavy fuel oil (HFO) and a standardised diesel fuel (DF). It was operated in a standardised cycle with a duration of 2 h. Chemical characterisation of organic species and elements revealed higher concentrations as well as a larger number of detected compounds for HFO operation for both gas phase and particulate matter. A noteworthy exception was the concentration of elemental carbon, which was higher in DF exhaust aerosol. This may prove crucial for the assessment and interpretation of biological response and impact via the exposure of human lung cell cultures, which was carried out in parallel to this study. Offline and online data hinted at the fact that most organic species in the aerosol are transferred from the fuel as unburned material. This is especially distinctive at low power operation of HFO, where low volatility structures are converted to the particulate phase. The results of this study give rise to the conclusion that a mere switching to sulphur-free fuel is not sufficient as remediation measure to reduce health and environmental effects of ship emissions.

Particulate matter from both heavy fuel oil and diesel fuel shipping emissions show strong biological effects on human lung cells at realistic and comparable in vitro exposure conditions.

BACKGROUND: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. OBJECTIVES: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. METHODS: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. RESULTS: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. CONCLUSIONS: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.

MRI-based detection of alkaline phosphatase gene reporter activity using a porphyrin solubility switch.

The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with MRI could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water-soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by postmortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets.

Airborne indoor particles from schools are more toxic than outdoor particles.

High concentrations of particulate matter (PM(10)) were measured in classrooms. This study addresses the hazard of indoor particles in comparison to the better-studied outdoor particles. Samples were taken from six schools during teaching hours. Genome-wide gene expression in human BEAS-2B lung epithelial cells was analyzed and verified by quantitative PCR. Polycyclic aromatic hydrocarbons, endotoxin, and cat allergen (Fel d 1) were analyzed by standard methods. Enhancement of allergic reactivity by PM(10) was confirmed in human primary basophils. Acceleration of human blood coagulation was determined with supernatants of PM(10)-exposed human peripheral blood monocytes. Indoor PM(10) induced serine protease inhibitor B2 (involved in blood coagulation) and inflammatory genes (such as CXCL6, CXCL1, IL6, IL8; all P < 0.001). Outdoor PM(10) induced xenobiotic metabolizing enzymes (cytochrome P450 [CYP] 1A1, CYP1B1, TIPARP; all P < 0.001). The induction of inflammatory genes by indoor PM(10) was explained by endotoxin (indoor 128.5 ± 42.2 EU/mg versus outdoor 13.4 ± 21.5 EU/mg; P < 0.001), the induction of CYP by outdoor polycyclic aromatic hydrocarbons (indoor 8.3 ± 4.9 ng/mg versus outdoor 16.7 ± 15.2 ng/mg; P < 0.01). The induction of serine protease inhibitor B2 was confirmed by a more rapid human blood coagulation (P < 0.05). Indoor PM(10) only affected allergic reactivity from human primary basophils from cat-allergic individuals. This was explained by varying Fel d 1 concentrations in indoor PM(10) (P < 0.001). Indoor PM(10), compared with outdoor PM(10), was six times higher and, on an equal weight basis, induced more inflammatory and allergenic reactions, and accelerated blood coagulation. Outdoor PM(10) had significantly lower effects, but induced detoxifying enzymes. Therefore, preliminary interventions for the reduction of classroom PM(10) seem reasonable, perhaps through intensified ventilation.

A liquid chromatography-atmospheric pressure photoionization tandem mass spectrometric method for the determination of azaarenes in atmospheric particulate matter.

The development, optimization and validation of a liquid chromatography-atmospheric pressure photoionization tandem mass spectrometric (LC-APPI/MS/MS) method for the determination of 15 azaarenes (4-azafluorene, benzo[h] and -[f]quinoline, phenanthridine, acridine, 1-azafluoranthene, 4-azapyrene, benz[a]- and -[c]acridine, -10-azabenzo[a]pyrene, 7,9- and 7,10-dimethylbenz[c]acridine, dibenz[a,j]-, -[c,h] and [a,i]acridine) in airborne particulate matter is described. Each compound was detected and quantified operating in multiple reaction monitoring mode. Extraction of azaarenes was achieved using accelerated solvent extraction (ASE) with dichlormethane/methanol (50/50, v/v). After extraction, no additional clean-up procedure like solid phase or liquid/liquid extraction was necessary. Limits of quantification (S/Nx10) ranged from 0.2 pg/microl to 1.4 pg/microl, matrix dependent recoveries were between 57% and 94%, with relative standard deviations from 8% to 17%. Applicability of the method was demonstrated analyzing 10 samples of particulate matter (PM(2.5)) collected in winter 2008. In all samples dimethylbenz[c]acridines as well as dibenzacridines were below the limit of quantification, concentration of the remaining analytes were in the range from 0.002 ng/m(3) to 0.356 ng/m(3).

Differential impact of diesel particle composition on pro-allergic dendritic cell function.

Diesel exhaust particles (DEP) were described as potent adjuvant in the induction and maintenance of allergic diseases, suggesting that they might play a role in the increase of allergic diseases in the industrialized countries. However, the cellular basis by which these particles enhance allergic immune responses is still a matter of debate. Thus, we exposed immature murine bone marrow-derived dendritic cells (BMDC) to different particles or particle-associated organic compounds in the absence or presence of the maturation stimuli lipopolysaccharide (LPS) and analyzed the cellular maturation, viability, and cytokine production. Furthermore, we monitored the functionality of particle-exposed BMDC to suppress B cell isotype switching to immunoglobulin (Ig) E. Only highly polluted DEP (standard reference material 1650a [SRM1650a]) but not particle-associated organic compounds or less polluted DEP from modern diesel engines were able to modulate the dendritic cell phenotype. SRM1650a particles significantly suppressed LPS-induced IL-12p70 production in murine BMDC, whereas cell-surface marker expression was not altered. Furthermore, SRM1650a-exposed immature BMDC lost the ability to suppress IgE isotype switch in B cells. This study revealed that highly polluted DEP not only interfere with dendritic cell maturation but also additionally with dendritic cell function, thus suggesting a role in T(h)2 immune deviation.

Isolation of two Pseudomonas strains producing pseudomonic acid A.

Two novel Pseudomonas strains were isolated from groundwater sediment samples. The strains showed resistance against the antibiotics tetracycline, cephalothin, nisin, vancomycin, nalidixic acid, erythromycin, lincomycin, and penicillin and grew at temperatures between 15 and 37 degrees C and pH values from 4 to 10 with a maximum at pH 7 to 10. The 16S ribosomal RNA gene sequences and the substrate spectrum of the isolates revealed that the two strains belonged to the Pseudomonas fluorescens group. The supernatants of both strains had an antibiotic effect against Gram-positive bacteria and one Gram-negative strain. The effective substance was produced under standard cultivation conditions without special inducer molecules or special medium composition. The antibiotically active compound was identified as pseudomonic acid A by off-line high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The measurement on ultra performance liquid chromatography (UPLC, UV-vis detection) confirmed the determination of pseudomonic acid A which was produced by both strains at 1.7-3.5mg/l. Our findings indicate that the ability to produce the antibiotic pseudomonic acid A (Mupirocin) is more spread among the pseudomonads then anticipated from the only producer known so far.

Determination of aliphatic low-molecular-weight and biogenic amines by capillary zone electrophoresis.

Low-molecular-weight (LMW) aliphatic amines play a key role in the global nitrogen cycle, are involved in nutrient transfer, and act as buffer in the ecosystem. They are widely used as intermediates in chemical synthesis and were shown to cause occupational asthma. Biogenic amines occur in all living organisms and have an effect on the cell growth, although at high concentrations they can be toxic; some are used as cancer markers in health protection or as spoilage markers in foods. Their identification and quantification from different matrices such as human tissues or foods is of high importance. The electrophoretic separation of amines is possible as cations as a result of their high basicity; their detection, however, is more difficult because these amines contain no chromophore group. Indirect ultraviolet (UV) detection is the first presented possibility and widely used for the separation of nonderivatized amines. Otherwise, derivatization of the amines is necessary to directly detect them with laser-induced fluorescence (LIF) detection. Other detection modes such as pulse amperometric, chemiluminescence, or mass spectrometry have been also used for the determination of LMW and biogenic amines, but not on a routine basis. In this chapter, three capillary electrophoretic methods with indirect UV and LIF detection for determination of LMW aliphatic and biogenic amines are described.

Environmental polycyclic aromatic hydrocarbons (PAHs) enhance allergic inflammation by acting on human basophils.

Diesel exhaust particles (DEPs) have been implicated in the worldwide increased incidence of allergic airway diseases over the past century. There is growing evidence that DEP-associated polycyclic aromatic hydrocarbons (PAHs) participate in the development and maintenance of immunoglobulin (Ig) E-mediated allergic diseases. To address this issue we investigated the impact of U.S. Environmental Protection Agency (EPA) priority PAHs as well as of PAH-containing airborne extracts on antigen-induced CD63 upregulation and mediator release from human basophils. Whole blood samples from birch pollen allergic and control subjects were incubated in the presence of organic extracts of urban aerosol (AERex) or EPA-PAH standard with or without rBet v 1. Basophils were analyzed for CD63 expression as a measure of basophil activation by using multiparameter flow cytometry. In addition, purified basophils from birch pollen allergic donors were incubated for 2 h in the presence of 1 muM benzo[a]pyrene (BaP) or phenanthrene (Phe) and then stimulated with rBet v 1 for 45 min. Supernatants were assayed for histamine, interleukin (IL)-4, and IL-8 by means of enzyme-linked immunosorbent assay (ELISA). Basophils exposed in vitro simultaneously to AERex or EPA-PAH standard and rBet v 1 expressed CD63 significantly more than with antigen alone. PAHs synergized with rBet v 1 dose dependently, but did not activate basophils from nonallergic donors. BaP and Phe significantly enhanced cytokine secretion (IL-4, IL-8) and histamine release from purified basophils without antigen added, and secretion was not further enhanced by rBet v 1 stimulation. In conclusion, PAHs from roadside emissions can directly activate sensitized basophils to cytokine secretion and drive proallergic processes through enhanced Fcepsilon RI-coupled mediator release from human basophils.

Determination of oxygenated polycyclic aromatic hydrocarbons in particulate matter using high-performance liquid chromatography-tandem mass spectrometry.

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method with a rapid and simple sample preparation was optimized and validated for the determination of phenanthrene-9,10-dione, chrysene-5,6-dione, benzo[a]pyrene-1,6-dione, benzo[a]pyrene-3,6-dione, benzo[a]pyrene-4,5-dione, benzo[a]pyrene-6,12-dione, benzo[a]pyrene-7,8-dione, benzo[a]pyrene-11,12-dione and 6-oxo-7-oxa-benzo[a]pyrene in particulate matter. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode leading to high sensitivity and selectivity. The limits of quantification (S/N=10) ranged from ca. 0.1 pg/microl to ca. 5.8 pg/microl and matrix dependent recoveries varied between 49 and 92%. The applicability of the LC-MS/MS method was shown by the analysis of particulate matter (PM(2.5)) collected during the course of 2005 in the Munich area, Germany. All oxy-PAHs determined exhibited higher mean and peak concentrations in the winter months compared to the concentration levels in the warmer season.