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.

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.

Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate.

Diethyl maleate (DEM), a thiol-reactive α,ß-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.

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,ß-unsaturated carbonyl compound that conjugates to GSH and other thiols, decreased C. elegans life span at a concentration of 1mM. In contrast, low and moderate doses of DEM (10-100µM) 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 100µM DEM, but not 1mM DEM, whereas only 1mM 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.

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. 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. 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 is diarrhetic shellfish poisoning (DSP) toxins. This group traditionally comprises okadaic acid and dinophysistoxins (DTXs), pectenotoxins (PTXs), and yessotoxins (YTXs). The most recently described shellfish poisoning syndrome, azaspiracid shellfish poisoning (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.

Effect of E-beam treatment on the chemistry and on the antioxidant activity of lycopene from dry tomato peel and tomato powder.

Tomato powder (TP) and dry tomato peel (DTP) have been previously used in our laboratory as a source of lycopene to manufacture meat products ready-to-eat (RTE) submitted to E-beam irradiation with good technological and sensory results. Present work describes the studies performed in order to investigate the effect of radiation on chemical changes and antioxidant properties of lycopene. DTP and TP were irradiated (4 kGy). Changes on lycopene were analyzed by HPLC; inhibition of reactive oxygen species (ROS), possible modulation of mitogen-activated protein kinases (MAPK) cascade, nuclear factor κ-light-chain-enhancer of activated B cells (NP-κB) activation and expression of proteins involved in oxidation stress were analyzed in RAT-1 fibroblasts cell culture. Radiation reduced the content of all-E-lycopene and increased (Z)-lycopene, lycopene isomerization, and degradation being higher in DTP than in TP. E-Beam treatment increased the antioxidant ability of both DTP and TP in inhibiting spontaneous and H2O2-induced oxidative stress in cultured fibroblasts. Antioxidant activity was higher in DTP than in TP samples.

Characteristic profiles of Ciguatera toxins in different strains of Gambierdiscus spp.

Ciguatera fish poisoning characterizes the intoxication caused by consumption of fish from tropical and subtropical areas, which have accumulated ciguatoxins (CTXs). The observed pattern of ciguatoxins in fish highly depends on the marine region and the causative organisms. It is evident that differences exist between ciguatoxins produced by certain strains of the dinoflagellate Gambierdiscus toxicus and other Gambierdiscus spp. In this context cultured strains purchased from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) and strains from Vietnam were analyzed. Besides, lyophilized samples of several Gambierdiscus spp. from the National Oceanic and Atmospheric Administration (NOAA), USA and lyophilized samples of G. toxicus from Vietnam were analyzed. The latter has been cultured at different salinities. We observed differences between the toxin ratios of the analogues in the strain from Vietnam depending on the salinity. The CTX profiles of the Vietnamese samples were compared with cultures of Gambierdiscus spp. from CCMP and the National Oceanic and Atmospheric Administration (NOAA) resulting in an overview of toxins in cultures from different regions. Hence, it was obvious that the strain from Vietnam forms a characteristic CTX profile which is not directly comparable to CTX pattern observed in other tropical marine regions.

Saxitoxins accumulation by freshwater tilapia (Oreochromis niloticus) for human consumption.

The accumulation of saxitoxins (STXs) in fish from freshwater aquaculture was investigated for the first time in the present study. Cyanotoxins have been monitored in liver and muscle samples of Oreochromis niloticus by chromatographic methods, both before and after the depuration process. The results show that tilapia can accumulate STXs. Our findings suggest that depuration with clean water is an alternative process to eliminate STXs from fish and, therefore, improve the safety of tilapia for consumers.

Paralytic shellfish poisoning toxin profile of mussels Perna perna from southern Atlantic coasts of Morocco.

During the monitoring programme of harmful algal blooms established along the south Atlantic coast of Morocco, a bimonthly determination of harmful algae and phycotoxins analysis in Perna perna was carried out from May 2003 to December 2004. Results of mouse bioassay (in organs and whole flesh) showed a seasonal evolution of paralytic shellfish poisoning (PSP) toxin. The mussel's contamination was associated with the occurrence in water of Alexandrium minutum. The PSP toxin profile obtained with high-performance liquid chromatography (HPLC/FD) revealed the dominance of gonyautoxins GTX2 and GTX3 and a minority of GTX1, GTX4 and saxitoxin (STX). This profile explains that the toxicity was mainly associated with A. minutum.

Application of a new zwitterionic hydrophilic interaction chromatography column for determination of paralytic shellfish poisoning toxins.

A novel method for paralytic shellfish poisoning (PSP) toxins which is based on the chromatographic separation of the toxins using a zwitterionic (ZIC) hydrophilic interaction chromatography (HILIC) column is presented. Efficient retention of the polar PSP toxins on the ZIC-HILIC column allowed their selective and sensitive determination by the application of mass spectrometric (MS/MS) detection or as derivatives after oxidation prior to fluorescence detection (FD). Low buffer concentrations and the omission of ion-pair reagents decreased the limits of detection (LODs) by MS/MS analysis and showed a good linearity for both methods of detection. This method can be applied for the qualitative and quantitative determination of PSP toxins in various types of phytoplankton, and for the routine analysis of seafood.

Detection of harmful cyanobacteria and their toxins by both PCR amplification and LC-MS during a bloom event.

We briefly report here the occurrence of toxic blooms in the eutrophic reservoir Billings, São Paulo city, Brazil. Water samples were collected in May 2004, during a cyanobacterial bloom. The presence of toxic species was confirmed by using PCR amplifications of a fragment region of genes encoding microcystin synthetase-mcyB. The determination of toxins was performed by liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analyses of the toxins from the bloom revealed variants of microcystins (MC), such as MC-LR, MC-RR and MC-YR. HPLC-FLD was used to determine the paralytic shellfish poisoning (PSP) saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 (GTX2) and 3 (GTX3). GTX2, GTX3 and NEO were detected for the first time in a natural sample from Billings reservoir. These results are a contribution to the knowledge of the biogeography of toxic cyanobacteria and their toxins, specifically in São Paulo.