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Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes, can be irreversibly degraded by the membrane-bound S1P lyase (S1PL) yielding (2E)-hexadecenal and phosphoethanolamine. It is discussed that (2E)-hexadecenal is further oxidized to (2E)-hexadecenoic acid by the long-chain fatty aldehyde dehydrogenase ALDH3A2 (also known as FALDH) prior to activation via coupling to coenzyme A (CoA). Inhibition or defects in these enzymes, S1PL or FALDH, result in severe immunological disorders or the Sjogren-Larsson syndrome, respectively. Hence, it is of enormous importance to simultaneously determine the S1P breakdown product (2E)-hexadecenal and its fatty acid metabolites in biological samples. However, no method is available so far. Here, we present a sensitive and selective isotope-dilution high performance liquid chromatographyelectrospray ionizationquadrupole/time-of-flight mass spectrometry method for simultaneous quantification of (2E)-hexadecenal and its fatty acid metabolites following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Optimized conditions for sample derivatization, chromatographic separation, and MS/MS detection are presented as well as an extensive method validation. Finally, our method was successfully applied to biological samples. We found that (2E)-hexadecenal is almost quantitatively oxidized to (2E)-hexadecenoic acid, that is further activated as verified by cotreatment of HepG2 cell lysates with (2E)-hexadecenal and the acyl-CoA synthetase inhibitor triacsin C. Moreover, incubations of cell lysates with deuterated (2E)-hexadecenal revealed that no hexadecanoic acid is formed from the aldehyde. Thus, our method provides new insights into the sphingolipid metabolism and will be useful to investigate diseases known for abnormalities in long-chain fatty acid metabolism, e.g., the Sjogren-Larsson syndrome, in more detail.
Pak Choi Fed to Mice: Formation of DNA Adducts and Influence on Xenobiotic-Metabolizing Enzymes
(2015)
Dopamine (DA) and serotonin (SRT) are monoamine neurotransmitters that play a key role in regulating the central and peripheral nervous system. Their impaired metabolism has been implicated in several neurological disorders, such as Parkinson's disease and depression. Consequently, it is imperative to monitor changes in levels of these low-abundant neurotransmitters and their role in mediating disease. For the first time, a rapid, specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DA and SRT in the nematode Caenorhabditis elegans (C. elegans). This model organism offers a unique approach for studying the effect of various drugs and environmental conditions on neurotransmitter levels, given by the conserved DA and SRT biology, including synaptic release, trafficking and formation. We introduce a novel sample preparation protocol incorporating the usage of sodium thiosulfate in perchloric acid as extraction medium that assures high recovery of the relatively unstable neurotransmitters monitored. Moreover, the use of both deuterated internal standards and the multiple reaction monitoring (MRM) technique allows for unequivocal quantification. Thereby, to the best of our knowledge, we achieve a detection sensitivity that clearly exceeds those of published DA and SRT quantification methods in various matrices. We are the first to show that exposure of C elegans to the monoamine oxidase B (MAOB) inhibitor selegiline or the catechol-O-methyltransferase (COMT) inhibitor tolcapone, in order to block DA and SRT degradation, resulted in accumulation of the respective neurotransmitter. Assessment of a behavioral output of the dopaminergic system (basal slowing response) corroborated the analytical LC-MS/MS data. Thus, utilization of the C elegans model system in conjunction with our analytical method is well-suited to investigate drug-mediated modulation of the DA and SRT system in order to identify compounds with neuroprotective or regenerative properties. (C) 2015 Elsevier B.V. All rights reserved.
Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C-elegans
(2015)
Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C. elegans
(2015)
Overexposure to the essential metal manganese (Mn) can result in an irreversible condition known as manganism that shares similar pathophysiology with Parkinson's disease (PD), including dopaminergic (DAergic) cell loss that leads to motor and cognitive impairments. However, the mechanisms behind this neurotoxicity and its relationship with PD remain unclear. Many genes confer risk for autosomal recessive, early-onset PD, including the parkin/PARK2 gene that encodes for the E3 ubiquitin ligase Parkin. Using Caenorhabditis elegans (C. elegans) as an invertebrate model that conserves the DAergic system, we previously reported significantly increased Mn accumulation in pdr-1/parkin mutants compared to wildtype (WT) animals. For the current study, we hypothesize that this enhanced accumulation is due to alterations in Mn transport in the pdr-1 mutants. While no change in mRNA expression of the major Mn importer proteins (smf-1-3) was found in pdr-1 mutants, significant downregulation in mRNA levels of the putative Mn exporter ferroportin (fpn-1.1) was observed. Using a strain overexpressing fpn-1.1 in worms lacking pdr-1, we show evidence for attenuation of several endpoints of Mn-induced toxicity, including survival, metal accumulation, mitochondrial copy number and DAergic integrity, compared to pdr-1 mutants alone. These changes suggest a novel role of pdr-1 in modulating Mn export through altered transporter expression, and provides further support of metal dyshomeostasis as a component of Parkinsonism pathophysiology.
Understanding penetration not only in intact, but also in lesional skin with impaired skin barrier function is important, in order to explore the surplus value of nanoparticle-based drug delivery for anti-inflammatory dermatotherapy. Herein, short-termex vivo cultures of (i) intact human skin, (ii) skin pretreated with tape-strippings and (iii) skin pre-exposed to sodium lauryl sulfate (SLS) were used to assess the penetration of dexamethasone (Dex). Intradermal microdialysis was utilized for up to 24 h after drug application as commercial cream, nanocrystals or ethyl cellulose nanocarriers applied at the therapeutic concentration of 0.05%, respectively. In addition, Dex was assessed in culture media and extracts from stratum corneum, epidermis and dermis after 24 h, and the results were compared to those in heat-separated split skin from studies in Franz diffusion cells. Providing fast drug release, nanocrystals significantly accelerated the penetration of Dex. In contrast to the application of cream and ethyl cellulose nanocarriers, Dex was already detectable in eluates after 6 h when applying nanocrystals on intact skin. Disruption of the skin barrier further accelerated and enhanced the penetration. Encapsulation in ethyl cellulose nanocarriers delayed Dex penetration. Interestingly, for all formulations highly increased concentrations in the dialysate were observed in tape-stripped skin, whereas the extent of enhancement was less in SLS-exposed skin. The results were confirmed in tissue extracts and were in line with the predictions made by in vitro release studies and ex vivo Franz diffusion cell experiments. The use of 45 kDa probes further enabled the collection of inflammatory cytokines. However, the estimation of glucocorticoid efficacy by Interleukin (IL)-6 and IL-8 analysis was limited due to the trauma induced by the probe insertion. Ex vivo intradermal microdialysis combined with culture media analysis provides an effective, skin-sparing method for preclinical assessment of novel drug delivery systems at therapeutic doses in models of diseased skin. (C) 2016 Elsevier B.V. All rights reserved.
Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.
Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver of mice
(2017)
The average intake of the essential trace element selenium (Se) is below the recommendation in most European countries, possibly causing sub-optimal expression of selenoproteins. It is still unclear how a suboptimal Se status may affect health. To mimic this situation, mice were fed one of three physiologically relevant amounts of Se. We focused on the liver, the organ most sensitive to changes in the Se supply indicated by hepatic glutathione peroxidase activity. In addition, liver is the main organ for synthesis of methyl groups and glutathione via one-carbon metabolism. Accordingly, the impact of Se on global DNA methylation, methylation capacity, and gene expression was assessed. We observed higher global DNA methylation indicated by LINE1 methylation, and an increase of the methylation potential as indicated by higher S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio and by elevated mRNA expression of serine hydroxymethyltransferase in both or either of the Se groups. Furthermore, increasing the Se supply resulted in higher plasma concentrations of triglycerides. Hepatic expression of glycolytic and lipogenic genes revealed consistent Se dependent up-regulation of glucokinase. The sterol regulatory element-binding transcription factor 1 (Srebf1) was also up-regulated by Se. Both effects were confirmed in primary hepatocytes. In contrast to the overall Se-dependent increase of methylation capacity, the up-regulation of Srebf1 expression was paralleled by reduced local methylation of a specific CpG site within the Srebf1 gene. Thus, we provided evidence that Se-dependent effects on lipogenesis involve epigenetic mechanisms. (C) 2017 The Authors. Published by Elsevier Inc.
Preclinical assessment of penetration not only in intact, but also in barrier‐disrupted skin is important to explore the surplus value of novel drug delivery systems, which can be specifically designed for diseased skin. Here, we characterized physical and chemical barrier disruption protocols for short‐term ex vivo skin cultures with regard to structural integrity, physiological and biological parameters. Further, we compared the penetration of dexamethasone (Dex) in different nanoparticle‐based formulations in stratum corneum, epidermis and dermis extracts of intact vs. barrier‐disrupted skin as well as by dermal microdialysis at 6, 12 and 24 hours after topical application. Dex was quantified by liquid‐chromatography ‐ tandem‐mass spectrometry (LC‐MS/MS). Simultaneously, we investigated the Dex efficacy by interleukin (IL) analysis. Tape‐stripping (TS) and 4 hours sodium lauryl sulfate 5 % (SLS) exposure were identified as highly effective barrier disruption methods assessed by reproducible transepidermal water loss (TEWL) changes and IL‐6/8 increase which was more pronounced in SLS‐treated skin. The barrier state has also a significant impact on the Dex penetration kinetics: for all formulations, TS highly increased dermal Dex concentration despite the fact that nanocrystals quickly and effectively penetrated both, intact and barrier‐disrupted skin reaching significantly higher dermal Dex concentration after 6 hours compared to Dex cream. The surplus value of encapsulation in ethyl cellulose nanocarriers could mostly be observed when applied on intact skin, in general showing a delayed Dex penetration. Estimation of cytokines was limited due to the trauma caused by probe insertion. In summary, ex vivo human skin is a highly interesting short‐term preclinical model for the analysis of penetration and efficacy of novel drug delivery systems.