Activation of Nrf2 by electrophiles is largely independent of the selenium status of HepG2 cells

Selenoenzymes, whose activity depends on adequate selenium (Se) supply, and phase II enzymes, encoded by target genes of nuclear factor erythroid 2-related factor 2 (Nrf2), take part in governing cellular redox homeostasis. Their interplay is still not entirely understood. Here, we exposed HepG2 hepatoma cells cultured under Se-deficient, Se-adequate, or Se-supranutritional conditions to the Nrf2 activators sulforaphane, cardamonin, or diethyl maleate. Nrf2 protein levels and intracellular localization were determined by immunoblotting, and mRNA levels of Nrf2 target genes and selenoproteins were assessed by qRT-PCR. Exposure to electrophiles resulted in rapid induction of Nrf2 and its enrichment in the nucleus, independent of the cellular Se status. All three electrophilic compounds caused an enhanced expression of Nrf2 target genes, although with differences regarding extent and time course of their induction. Whereas Se status did not significantly affect mRNA levels of the Nrf2 target genes, gene expression of selenoproteins with a low position in the cellular “selenoprotein hierarchy”, such as glutathione peroxidase 1 (GPX1) or selenoprotein W (SELENOW), was elevated under Se-supplemented conditions, as compared to cells held in Se-deficient media. In conclusion, no major effect of Se status on Nrf2 signalling was observed in HepG2 cells

Analysis of Marine Biotoxins using LC-MS/MS

Different clinical types of algae-related poisoning have attracted scientific and commercial attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, mainly due to the low sensitivity and the absence of specialized variations. Therefore, many efforts have been made to determine algal toxins using different chemical methods. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins. This chapter describes state-of-the-art LC-MS/MS methods for the detection and quantitation of different classes of phycotoxins in shellfish matrices. These classes include the highly hydrophilic paralytic shellfish poisoning (PSP) toxins. , which have been traditionally separated by ion pair chromatography followed by derivatization and fluorescence detection. However, the ion pair reagents added to the mobile phases are not compatible with mass spectrometry, so a new chromatographic system had to be developed. Hydrophilic interaction liquid chromatography (HILIC) has been shown to be useful in the separation of PSP toxins and is described in detail within this chapter. Another important class of phycotoxins are diarrhetic shellfish poising (DSP) toxins. This group traditionally comprises okadaic acid and dinophysistoxins (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). The latter two groups are often associated with diarrhoea causing DTXs, but recently turned out not to have diarrhetic effects by themselves, which means in the strict sense they are no DSP toxins. For this reason the term “lipophilic” toxins rather than DSP is increasingly applied to for this group of toxins. The most recently described shellfish poisoning syndrome, azaspiracid shellfish poising (AZP) is caused by azaspiracids, which in turn are diarrhetic, but usually are treated separately as AZP. The last group of regulated shellfish toxins is the amnesic shellfish poisoning (ASP) toxin domoic acid, produced by species of the genus Pseudo-nitzschia

Selective activation of cellular stress response pathways by fumaric acid esters

The cellular response to oxidants or xenobiotics comprises two key pathways, resulting in modulation of NRF2 and FOXO transcription factors, respectively. Both mount a cytoprotective response, and their activation relies on crucial protein thiol moieties. Using fumaric acid esters (FAEs), known thiol‐reactive compounds, we tested for activation of NRF2 and FOXO pathways in cultured human hepatoma cells by dimethyl/diethyl as well as monomethyl/monoethyl fumarate. Whereas only the diesters caused acute glutathione depletion and activation of the stress kinase p38MAPK, all four FAEs stimulated NRF2 stabilization and upregulation of NRF2 target genes. However, no significant FAE‐induced activation of FOXO‐dependent target gene expression was observed. Therefore, while both NRF2 and FOXO pathways are responsive to oxidants and xenobiotics, FAEs selectively activate NRF2 signaling

Non-linear impact of glutathione depletion on C. elegans life span and stress resistance

The redox environment in cells and organisms is set by low-molecular mass and protein-bound thiols, with glutathione (GSH) representing a major intracellular redox buffer. Subtle thiol oxidation elicits signal transduction processes and adaptive responses to cope with stressors, whereas highly oxidizing conditions may provoke cell death. We here tested how thiol depletion affects life span, stress resistance and stress signaling in the model organism Caenorhabditis elegans. Diethyl maleate (DEM), an α,β-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1 mM. In contrast, low and moderate doses of DEM (10–100 µM) increased mean and maximum life span and improved resistance against oxidative stress. DEM-induced life span extension was not detectable in worms deficient in either the FoxO orthologue, DAF-16, or the Nrf2 orthologue, SKN-1, pointing to a collaborative role of the two transcription factors in life span extension induced by thiol depletion. Cytoprotective target genes of DAF-16 and SKN-1 were upregulated after at least 3 days of exposure to 100 µM DEM, but not 1 mM DEM, whereas only 1 mM DEM caused upregulation of egl-1, a gene controlled by a p53-orthologue, CEP-1. In order to test whether depletion of GSH may elicit effects similar to DEM, we suppressed GSH biosynthesis in worms by attenuating γ-glutamylcysteine synthetase (gcs-1) expression through RNAi. The decline in GSH levels elicited by gcs-1 knockdown starting at young adult stage did not impair viability, but increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown commencing right after hatching impaired nematode stress resistance and rendered young adult worms prone to vulval ruptures during egg-laying. Thus, modest decrease in GSH levels in young adult worms may promote stress resistance and life span, whereas depletion of GSH is detrimental to freshly hatched and developing worms

Study protocol: evaluation of specialized outpatient palliative care in the German state of Hesse (ELSAH study) – work package I: assessing the quality of care

Abstract Background In Germany, patients suffering from life-limiting conditions are eligible for specialized outpatient palliative care (SOPC). Evaluation of the quality of this service lacks currently integration of patient-relevant outcomes. There is also no scientific consensus how to prove quality of care in the special context of SOPC adequately. Existing quality reports are primarily based on descriptive structural data which do not allow for estimation of process quality or result quality. The ELSAH study (‘Evaluation of Specialized Outpatient Palliative Care in the German state of Hesse’) aims to choose - or, if necessary, to adopt - to evaluate and to implement a suit of measures to assess, evaluate and monitor the quality of specialized, home-based palliative care. Methods All 22 SOPC teams providing their services in the state of Hesse, Germany, participate in the ELSAH study. The study is divided in two phases: a preparation phase and a main study phase. Based on the findings of the preparation phase we have chosen a preliminary set of instruments including the Integrated Palliative Outcome Scale, Views on Care, Zarit Burden Interview, Phase of Illness, Goal Attainment Scaling, Eastern Cooperative Oncology Group Performance Status, Consumer Quality Indices Palliative Care and Sense of Security in Care. During the main study phase, we will use a mixed-methods approach to evaluate the instruments’ psychometric properties (reliability, validity, feasibility and practicability), to identify barriers, facilitators and limitations of their routine use and to explore how their use affects the care within the SOPC setting. Discussion At the end of this study, an outcome- and patient-centered, validated measurement approach should be provided, adapted for standardized evaluations in SOPC across patient groups, palliative care services and regions nationwide. The standardized application of instruments should allow for making valid statements and comparisons of health care quality in SOPC based on process- and outcome-evaluation rather than relying on structural data only. Moreover, the instruments might directly influence the care of patients in palliative situations. Trial registration German Clinical Trials Register (DRKS-ID: DRKS00012421)

Study protocol : evaluation of specialized outpatient palliative care (SOPC) in the German state of Hesse (ELSAH study) - work package II ; palliative care for pediatric patients

Background: In 2007, the European Association of Palliative Care (EAPC) provided a comprehensive set of recommendations and standards for the provision of adequate pediatric palliative care. A number of studies have shown deficits in pediatric palliative care compared to EAPC standards. In Germany, pediatric palliative care patients can be referred to specialized outpatient palliative care (SOPC) services, which are known to enhance quality of life, e.g. by avoiding hospitalization. However, current regulations for the provision of SOPC in Germany do not account for the different circumstances and needs of children and their families compared to adult palliative care patients. The "Evaluation of specialized outpatient palliative care (SOPC) in the German state of Hesse (ELSAH)" study aims to perform a needs assessment for pediatric patients (children, adolescents and young adults) receiving SOPC. This paper presents the study protocol for this assessment (work package II). Methods/Design: The study uses a sequential mixed-methods study design with a focus on qualitative research. Data collection from professional and family caregivers and, as far as possible, pediatric patients, will involve both a written questionnaire based on European recommendations for pediatric palliative care, and semi-structured interviews. Additionally, professional caregivers will take part in focus group discussions and participatory observations. Interviews and focus groups will be tape- or video-recorded, transcribed verbatim and analyzed in accordance with the principles of grounded theory (interviews) and content analysis (focus groups). A structured field note template will be used to record notes taken during the participatory observations. Statistical Package for Social Sciences (SPSS, version 22 or higher) will be used for descriptive statistical analyses. The qualitative data analyses will be software-assisted by MAXQDA (version 12 or higher). Discussion: This study will provide important information on what matters most to family caregivers and pediatric patients receiving SOPC. The results will add valuable knowledge to the criteria that distinguish SOPC for pediatric from SOPC for adult patients, and will provide an indication of how the German SOPC rule of procedure can be optimized to satisfy the special needs of pediatric patients. Trial registration: Internet Portal of the German Clinical Trials Register (www.germanctr.de, DRKS-ID: DRKS00012431)

Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

Diethyl maleate (DEM), a thiol-reactive α,β-unsaturated carbonyl compound, depletes glutathione (GSH) in exposed cells and was previously shown by us to elicit a stress response in Caenorhabditis elegans that, at lower concentrations, results in enhanced stress resistance and longer lifespan. This hormetic response was mediated through both the Nrf2 ortholog, SKN-1, and the forkhead box O (FOXO) family transcription factor DAF-16. As FOXO signaling is evolutionarily conserved, we analyzed here the effects of DEM exposure on FOXO in cultured human cells (HepG2, HEK293). DEM elicited nuclear accumulation of GFP-coupled wild-type human FOXO1, as well as of a cysteine-deficient FOXO1 mutant. Despite the nuclear accumulation of FOXO1, neither FOXO1 DNA binding nor FOXO target gene expression were stimulated, suggesting that DEM causes nuclear accumulation but not activation of FOXO1. FOXO1 nuclear exclusion elicited by insulin or xenobiotics such as arsenite or copper ions was attenuated by DEM, suggesting that DEM interfered with nuclear export. In addition, insulin-induced FOXO1 phosphorylation at Thr-24, which is associated with FOXO1 nuclear exclusion, was attenuated upon exposure to DEM. Different from FOXO-dependent expression of genes, Nrf2 target gene mRNAs were elevated upon exposure to DEM. These data suggest that, different from C. elegans, DEM elicits opposing effects on the two stress-responsive transcription factors, Nrf2 and FOXO1, in cultured human cells. Keywords: FOXO, DAF-16, Glutathione, Stress response, Nrf-2, Thiols, Nuclear expor

Variations of PSP toxin profiles during different growth phases in Gymnodinium catenatum (Dinophyceae) strains isolated from three locations in the Gulf of California,

In vitro experiments were performed with Gymnodinium catenatum Graham strains isolated from three locations in the Gulf of California to determine the variability in toxicity and toxin profiles. Strains were cultivated in GSe at 20° C ± 1° C, 150 μmol photons·m-2·s-1 (12:12 light:dark cycle), and harvested during different growth phases. Growth rates were higher than in previous studies, varying between 0.70 and 0.82 day-1. The highest cell yields were reached at 16 and 19 days, with maximum densities between 1090 and 3393 cells· mL-1. Bahía de La Paz (BAPAZ) and Bahía de Mazatlán (BAMAZ) were the most toxic (101 pg STXeq-cell 1), whereas strains from Bahía Concepción (BACO) were the least toxic (13 pg STXeq·cell-1). A strain isolated from cyst germination was one of the least toxic strains. No significant changes in toxin content with culture age were observed (0.2 and 0.6pmol paralytic shellfish poisoning·cell-1). All strains contained neosaxitoxin (NEOSTX), decarbamoyl-saxitoxin (dcSTX), decarbamoyl-gonyautoxin-2,-3, (dcGTX2-3), N-sulfocarbamoylsaxitoxin (Bl), N-sulfo-carbamoylneosaxitoxin (B2), and N-sulfo-carbamoylgonyautoxin-2,-3 (C1-2). Bahía Concepción strains had the highest content of Cl; BAPAZ and BAMAZ strains had a higher percentage of NEOSTX. Differences in toxin composition with culture age were observed only in BAMAZ and BAPAZ strains. Cultures with a higher percentage of long chains had more NEOSTX, while those with a higher proportion of short chains had a lower content of NEOSTX. Gulf of California strains are characterized by a high proportion of NEOSTX, and seem to have evolved particular physiological responses to their environment that are reflected in the toxin profile, suggesting different populations