Ralph Feltens

Discrimination of different species from the genus Drosophila by intact protein profiling using matrix-assisted laser desorption ionization mass spectrometry

BackgroundThe use of molecular biology-based methods for species identification and establishing phylogenetic relationships has supplanted traditional methods relying on morphological characteristics. While PCR-based methods are now the commonly accepted gold standards for these types of analysis, relatively high costs, time-consuming assay development or the need for a priori information about species-specific sequences constitute major limitations. In the present study, we explored the possibility to differentiate between 13 different species from the genus Drosophila via a molecular proteomic approach.ResultsAfter establishing a simple protein extraction procedure and performing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) with intact proteins and peptides, we could show that most of the species investigated reproducibly yielded mass spectra that were adequate for species classification. Furthermore, a dendrogram generated by cluster analysis of total protein patterns agrees reasonably well with established phylogenetic relationships.ConclusionConsidering the intra- and interspecies similarities and differences between spectra obtained for specimens of closely related Drosophila species, we estimate that species typing of insects and possibly other multicellular organisms by intact protein profiling (IPP) can be established successfully for species that diverged from a common ancestor about 3 million years ago.

Chlorobenzene induces oxidative stress in human lung epithelial cells in vitro

Chlorobenzene is a volatile organic compound (VOC) that is widely used as a solvent, degreasing agent and chemical intermediate in many industrial settings. Occupational studies have shown that acute and chronic exposure to chlorobenzene can cause irritation of the mucosa of the upper respiratory tract and eyes. Using in vitro assays, we have shown in a previous study that human bronchial epithelial cells release inflammatory mediators such as the cytokine monocyte chemoattractant protein-1 (MCP-1) in response to chlorobenzene. This response is mediated through the NF-kappaB signaling pathway. Here, we investigated the effects of monochlorobenzene on human lung cells, with emphasis on potential alterations of the redox equilibrium to clarify whether the chlorobenzene-induced inflammatory response in lung epithelial cells is caused via an oxidative stress-dependent mechanism. We found that expression of cellular markers for oxidative stress, such as heme oxygenase 1 (HO-1), glutathione S-transferase pi1 (GSTP1), superoxide dismutase 1 (SOD1), prostaglandin-endoperoxide synthase 2 (PTGS2) and dual specificity phosphatase 1 (DUSP1), were elevated in the presence of monochlorobenzene. Likewise, intracellular reactive oxygen species (ROS) were increased in response to exposure. However, in the presence of the antioxidants N-(2-mercaptopropionyl)-glycine (MPG) or bucillamine, chlorobenzene-induced upregulation of marker proteins and release of the inflammatory mediator MCP-1 are suppressed. These results complement our previous findings and point to an oxidative stress-mediated inflammatory response following chlorobenzene exposure.

Styrene induces an inflammatory response in human lung epithelial cells via oxidative stress and NF-κB activation

Styrene is a volatile organic compound (VOC) that is widely used as a solvent in many industrial settings. Chronic exposure to styrene can result in irritation of the mucosa of the upper respiratory tract. Contact of styrene with epithelial cells stimulates the expression of a variety of inflammatory mediators, including the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). To characterise the underlying mechanisms of the induction of inflammatory signals by styrene, we investigated the influence of this compound on the induction of oxidative stress and the activation of the nuclear factor-kappa B (NF-kappaB) signalling pathway in human lung epithelial cells (A549). The results demonstrate that styrene-induced MCP-1 expression, as well as the expression of the oxidative stress marker glutathione S-transferase (GST), is associated with a concentration dependent pattern of NF-kappaB activity. An inhibitor of NF-kappaB, IKK-NBD, and the anti-inflammatory antioxidant N-acetylcysteine (NAC) were both effective in suppressing styrene-induced MCP-1 secretion. In addition, NAC was capable of inhibiting the upregulation of GST expression. Our findings suggest that the activation of the NF-kappaB signalling pathway by styrene is mediated via a redox-sensitive mechanism.

Proteome changes in human bronchoalveolar cells following styrene exposure indicate involvement of oxidative stress in the molecular-response mechanism

Styrene is a volatile organic compound that is widely used as an intermediate in many industrial settings. There are known adverse health effects at environmentally significant concentrations, but little is known about the molecular effect of exposure to styrene at sub‐acute toxic concentrations. We exposed human lung epithelial cells, at a wide range of concentrations (1 mg/m3–10 g/m3), to styrene and analyzed the effects on the proteome level by 2‐DE, where 1380 proteins spots were detected and 266 were identified unambiguously by MS. A set of 16 protein spots were found to be significantly altered due to exposure to styrene at environmentally significant concentrations of 1–10 mg/m3 (0.2–2.3 ppm). Among these, superoxide dismutase as well as biliverdin reductase A could be correlated with the molecular pathway of oxidative stress, while eukaryotic translation initiation factor 5A‐1, ezrin, lamin B2 and voltage‐dependent anion channel 2 have been reported to be involved in apoptosis. Treatment with styrene also caused the formation of styrene oxide–protein adducts, specifically for thioredoxin reductase 1. These results underline the relevance of oxidative stress as a primary molecular response mechanism of lung epithelial cells to styrene exposure at indoor‐relevant concentrations.

Maternal phthalate exposure promotes allergic airway inflammation over 2 generations through epigenetic modifications

Background: Prenatal and early postnatal exposures to environmental factors are considered responsible for the increasing prevalence of allergic diseases. Although there is some evidence for allergy‐promoting effects in children because of exposure to plasticizers, such as phthalates, findings of previous studies are inconsistent and lack mechanistic information. Objective: We investigated the effect of maternal phthalate exposure on asthma development in subsequent generations and their underlying mechanisms, including epigenetic alterations. Methods: Phthalate metabolites were measured within the prospective mother‐child cohort Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk (LINA) and correlated with asthma development in the children. A murine transgenerational asthma model was used to identify involved pathways. Results: In LINA maternal urinary concentrations of mono‐n‐butyl phthalate, a metabolite of butyl benzyl phthalate (BBP), were associated with an increased asthma risk in the children. Using a murine transgenerational asthma model, we demonstrate a direct effect of BBP on asthma severity in the offspring with a persistently increased airway inflammation up to the F2 generation. This disease‐promoting effect was mediated by BBP‐induced global DNA hypermethylation in CD4+ T cells of the offspring because treatment with a DNA‐demethylating agent alleviated exacerbation of allergic airway inflammation. Thirteen transcriptionally downregulated genes linked to promoter or enhancer hypermethylation were identified. Among these, the GATA‐3 repressor zinc finger protein 1 (Zfpm1) emerged as a potential mediator of the enhanced susceptibility for TH2‐driven allergic asthma. Conclusion: These data provide strong evidence that maternal BBP exposure increases the risk for allergic airway inflammation in the offspring by modulating the expression of genes involved in TH2 differentiation through epigenetic alterations.

Di-(2-Ethylhexyl)-Phthalate (DEHP) Causes Impaired Adipocyte Function and Alters Serum Metabolites

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.

Species determination of Culicoides biting midges via peptide profiling using matrix-assisted laser desorption ionization mass spectrometry

BackgroundCulicoides biting midges are vectors of bluetongue and Schmallenberg viruses that inflict large-scale disease epidemics in ruminant livestock in Europe. Methods based on morphological characteristics and sequencing of genetic markers are most commonly employed to differentiate Culicoides to species level. Proteomic methods, however, are also increasingly being used as an alternative method of identification. These techniques have the potential to be rapid and may also offer advantages over DNA-based techniques. The aim of this proof-of-principle study was to develop a simple MALDI-MS based method to differentiate Culicoides from different species by peptide patterns with the additional option of identifying discriminating peptides.MethodsProteins extracted from 7 Culicoides species were digested and resulting peptides purified. Peptide mass fingerprint (PMF) spectra were recorded using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peak patterns analysed in R using the MALDIquant R package. Additionally, offline liquid chromatography (LC) MALDI-TOF tandem mass spectrometry (MS/MS) was applied to determine the identity of peptide peaks in one exemplary MALDI spectrum obtained using an unfractionated extract.ResultsWe showed that the majority of Culicoides species yielded reproducible mass spectra with peak patterns that were suitable for classification. The dendrogram obtained by MS showed tentative similarities to a dendrogram generated from cytochrome oxidase I (COX1) sequences. Using offline LC-MALDI-TOF-MS/MS we determined the identity of 28 peptide peaks observed in one MALDI spectrum in a mass range from 1.1 to 3.1 kDa. All identified peptides were identical to other dipteran species and derived from one of five highly abundant proteins due to an absence of available Culicoides data.ConclusionShotgun mass mapping by MALDI-TOF-MS has been shown to be compatible with morphological and genetic identification of specimens. Furthermore, the method performs at least as well as an alternative approach based on MS spectra of intact proteins, thus establishing the procedure as a method in its own right, with the additional option of concurrently using the same samples in other MS-based applications for protein identifications. The future availability of genomic information for different Culicoides species may enable a more stringent peptide detection based on Culicoides-specific sequence information.

Prenatal phthalate exposure associates with low regulatory T-cell numbers and atopic dermatitis in early childhood: Results from the LINA mother-child study

Citation for published version (APA): Herberth, G., Pierzchalski, A., Feltens, R., Bauer, M., Röder, S., Olek, S., ... Lehmann, I. (2017). Prenatal phthalate exposure associates with low regulatory T-cell numbers and atopic dermatitis in early childhood: Results from the LINA mother-child study. Journal of Allergy and Clinical Immunology, 139(4), 1376-1379.e8. https://doi.org/10.1016/j.jaci.2016.09.034

Evaluation of Population and Individual Variances of Urinary PhthalateMetabolites in terms of Epidemiological Studies

In the context of an epidemiological study, urinary concentrations of nine phthalic diester metabolites (monoethyl-, mono-(3-carboxypropyl)-, mono-n-butyl-, monoisobutyl-, monobenzyl-, mono-(2-ethylhexyl)-, mono-(5-hydroxy-2- ethylhexyl)-, mono-(5-oxo-2-ethylhexyl)- and mono-(5-carboxy-2-ethylpentyl)-phthalate) were quantified via LC-MS/ MS. As in the majority of epidemiological studies only single spot samples were available for urine analysis, the implicit assumption in this case is, that exposure data obtained from single spot samples are representative for a longer exposure period. To validate the relevance of single spot analyses we quantified the respective intra-individual variances of urine samples collected from ten volunteers once daily over a period of 30 days. Using the values for the daily variances, approximate values for the underlying population variances in the cohort samples representing the differences between the average individual metabolite levels were calculated. For most of the volunteers, daily metabolites variations were lower, than the variations observed in the epidemiological setup. The results showed that by accounting for the contribution of daily variance, the standard deviations of the log-transformed phthalate values of the cohort samples are reduced (14% to 28%) but still larger (3% to 66%) than daily standard deviation values, with the exception of MCPrP concentrations.