Activation of the glutathione peroxidase 2 (GPx2) promoter by beta-catenin

GPx2, formerly named gastrointestinal glutathione peroxidase, is highly expressed in the proliferative area of the intestinal crypt-to-villus axis and in Paneth cells. Additionally, GPx2 is transiently up-regulated during development of gastrointestinal adenocarcinomas. Because both normal proliferation and differentiation of intestinal epithelial cells as well as carcinogenesis are regulated by the Wnt pathway, it was tested whether GPx2 may be a target of the beta-catenin/TCF complex which transfers Wnt signals. The GPx2 promoter contains five putative beta-catenin/TCF binding sites. Accordingly, the promoter was active in two cell lines with a constitutively active Wnt pathway, HepG2 and SW480, but not in BHK-21 cells in which the pathway is silent. Overexpression of beta-catenin/TCF activated the GPx2 promoter in all three cell lines. Overexpression of wild-type adenomatous polyposis coli (APC) in SW480 cells which harbor a mutated APC gene decreased basal GPx2 promoter activity. Truncation of the promoter identified one beta-catenin/TCF binding site that was sufficient for activation. Mutation of this site reduced the response to beta-catenin/TCF by more than 50%. These findings suggest a function of GPx2 in the maintenance of normal renewal of the intestinal epithelium. Whether up-regulation of GPx2 during carcinogenesis supports tumor growth or can rather be considered as a counteracting effect remains to be investigated.     

Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx,GPx4) in mammalian cells

Reactive oxygen species (ROS) are known mediators of intracellular signal cascades. Excessive production of ROS may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Lipid hydroperoxides are one type of ROS whose biological function has not yet been clarified. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide in mammalian cells. This contrasts with most antioxidant enzymes, which cannot reduce intracellular phospholipid hydroperoxides directly. In this review, we focus on the structure and biological functions of PHGPx in mammalian cells. Recently, molecular techniques have allowed overexpression of PHGPx in mammalian cell lines, from which it has become clear that lipid hydroperoxides also have an important function as activators of lipoxygenase and cyclooxygenase, participate in inflammation, and act as signal molecules for apoptotic cell death and receptor-mediated signal transduction at the cellular level.     

Growth condition optimization for docosahexaenoic acid (DHA) production by Moritella marina MP-1

The marine organism Moritella marina MP-1 produces the polyunsaturated fatty acid docosahexaenoic acid (DHA). While the basic metabolic pathway for DHA production in this organism has been identified, the impact of growth conditions on DHA production is largely unknown. This study examines the effect of supplemental carbon, nitrogen and salts, growth temperature and media composition and pH on DHA and biomass production and the fatty acid profile. The addition of supplemental nitrogen significantly increased the overall DHA titer via an increase in biomass production. Supplemental glucose or glycerol increased biomass production, but decreased the amount of DHA per biomass, resulting in no net change in the DHA titer. Acidification of the baseline media pH to 6.0 increased DHA per biomass. Changes in growth temperature or provision of supplemental sodium or magnesium chloride did not increase DHA titer. This organism was also shown to grow on defined minimal media. For both media types, glycerol enabled more DHA production per biomass than glucose. Combination of these growth findings into marine broth supplemented with glycerol, yeast extract, and tryptone at pH?6.0 resulted in a final titer of 82?±?5 mg/L, a nearly eightfold increase relative to the titer of 11?±?1 mg/L seen in the unsupplemented marine broth. The relative distribution of other fatty acids was relatively robust to growth condition, but the presence of glycerol resulted in a significant increase in myristic acid (C14:0) and decrease in palmitic acid (C16:0). In summary, DHA production by M. marina MP-1 can be increased more than fivefold by changing the growth media. Metabolic engineering of this organism to increase the amount of DHA produced per biomass could result in additional increases in titer.     

Effect of docosahexaenoic acid (DHA) on arachidonic acid metabolism and platelet function

Studies from our laboratory have suggested a role for ferrous iron in the metabolism of arachidonic acid and demonstrated that inhibitors of prostaglandin synthesis exert their effect by complexing with the heme group of cyclooxygenase. Docosahexaenoic acid (DHA) is a potent competitive inhibitor of arachidonic acid metabolism by sheep vesicular gland prostaglandin synthetase. In this study we have evaluated the effect of exogenously added DHA on platelet function and arachidonic acid metabolism. DHA at 150 microM concentration inhibited aggregation of platelets to 450 microM arachidonic acid. At this concentration DHA also inhibited the second wave of the platelet response to the action of agonists such as epinephrine, adenosine diphosphate and thrombin. Inhibition induced by this fatty acid could be overcome by the agonists at higher concentrations. DHA inhibited the conversion of labeled arachidonic acid to thromboxane by intact, washed platelet suspensions. However, platelets in plasma incubated first with DHA then washed and stirred with labeled arachidonate generated as much thromboxane as control platelets. These results suggest that the polyenoic acids, if released in sufficient quantities in the vicinity of cyclooxygenase, could effectively compete for the heme site and inhibit the conversion of arachidonic acid.     

Application of lipase to concentrate the docosahexaenoic acid (DHA) fraction of fish oil

A commercial lipase preparation from Rhizopus niveus was used to concentrate the omega-3 fatty acid, docosahexaenoic acid (DHA) component in fish oil. The DHA content of cod-liver oil was 9.64% (w/w) of total fatty acids. Enzymatic digestion conditions were established which produced a DHA content in the monoglycerides fraction of 29.17% (w/w) of total fatty acid, triglyceride, and diglyceride components were 5.72, 9.95, and 15316%, respectively.     

Efficient production of triacylglycerols rich in docosahexaenoic acid (DHA) by osmo-heterotrophic marine protists

Thraustochytrids have recently emerged as a promising source for docosahexaenoic acid (DHA) production due to their high growth rate and oil content. In this study, two thraustochytrid isolates, Aurantiochytrium sp. PKU#SW7 and Thraustochytriidae sp. PKU#Mn16 were used for DHA production. Following growth parameters were optimized to maximize DHA production: temperature, pH, salinity, and glucose concentration. Both isolates achieved the highest DHA yield at the cultivation temperature of 28 °C, pH 6, 100 % seawater, and 2 % glucose. A DHA yield of 1.395 g/l and 1.426 g/l was achieved under the optimized culture conditions. Further investigation revealed that both isolates possess simple fatty acids profiles with palmitic acid and DHA as their dominant constituents, accounting for ～79 % of total fatty acids. To date, very few studies have focused on the DHA distribution in various lipid fractions which is an important factor for identifying strains with a potential for industrial DHA production. In the present study, the lipids profiles of each strain both revealed that the majority of DHA was distributed in neutral lipids (NLs), and the DHA distribution in NLs of PKU#SW7 was exclusively in the form of triacylglycerols (TAGs) which suggest that PKU#SW7 could be utilized as an alternative source of DHA for dietary supplements. The fermentation process established for both strains also indicating that Aurantiochytrium sp. PKU#SW7 was more suitable for cultivation in fermenter. In addition, the high percentage of saturated fatty acids produced by the two thraustochytrids indicates their potential application in biodiesel production. Overall, our findings suggest that two thraustochytrid isolates are suitable candidates for biotechnological applications.     

Phenotype and genotype relationship of glutathione peroxidase1 (GPx1) and rs 1800668 variant: the homozygote effect on kinetic parameters

GPx1 is one of the most important enzymes involved in oxidative balance so that, we studied the phenotype and genotype relationship of GPx1 activity and rs 1800668 (C/T) site and also evaluated the changes of GPx1 kinetic parameters in the rs 1800668 homozygotes. One hundred fifty eight subjects were recruited after clinical exams. The rs 1800668 (C/T) genotype distribution was identified using RFLP-PCR method. The hemolysate GPx1 activity was spectrophotometrically measured in a reaction coupled with glutathione reductase (GR). The GPx1 enzyme was purified using gel filtration chromatography with Sephacryl S-300 column and, Km(app) was studied in the rs 1800668 TT and CC homozygotes. The results showed that the GPx1 activity is significantly associated to the rs 1800668 (C/T) genotype distribution (P<0.05) so that, the GPx1 activity was high among the CC homozygotes (P<0.03). In addition, Km(app) for TBHP substrate in the TT homozygote (8.48 μM) was higher than the CC homozygote (5.74 μM). We concluded that the C allele within rs 1800668 position is related to the GPx1 activity and may be a potential factor involved in development of inflammatory events.     

Identification of cytotoxic mediators and their putative role in the signaling pathways during docosahexaenoic acid (DHA)-induced apoptosis of cancer cells

Docosahexaenoic acid (DHA), an important w-3 fatty acid exhibits differential behavior in cancer cells of neural origin when compared to that in normal healthy astrocytes. Treatment of C6 glioma and SH-SY5Y cell lines and primary astrocytes, representing the neoplastic cells and normal healthy cells respectively, with 100 µM DHA for 24 h showed significant loss of cell viability in the both the cancer cells as determined by MTT assay, whereas the primary astrocytes cultures were unaffected. Such loss of cell viability was due to apoptosis as confirmed by TUNEL staining and caspase-3 activation in cancer cells. Proteomic approach, employing 2-dimensional gel electrophoresis (2DE), difference gel electrophoresis (DIGE), and MALDI-TOF-TOF analysis identified six proteins which unlike in the astrocytes, were differently altered in the cancer cells upon exposure to DHA, suggesting their putative contribution in causing apoptosis in these cells. Of these, annexin A2, calumenin, pyruvate kinase M2 isoform, 14-3-3ζ were downregulated while aldo keto reductase-1B8 (AKR1B8) and glutathione–S-transferase P1 subunit (GSTP1) showed upregulation by DHA in the cancer cells. siRNA-mediated knockdown of AKR1B8 and GSTP1 inhibit DHA-induced apoptosis confirming their role in apoptotic process. Furthermore, western blot analysis identified upregulation of PPARα and the MAP kinases, JNK and p38 as well as increased ROS production selectively in the cell lines. Results suggest that DHA selectively induces apoptosis in the neural cell lines by regulating the expression of the above proteins to activate multiple apoptotic pathways which in association with excess ROS and activated MAPKs promote cell death.     

Elucidation of the mechanism of selenoprotein glutathione peroxidase (GPx)-catalyzed hydrogen peroxide reduction by two glutathione molecules: a density functional study

The mechanism of the hydrogen peroxide reduction by two molecules of glutathione catalyzed by the selenoprotein glutatione peroxidase (GPx) has been computationally studied. It has been shown that the first elementary reaction of this process, (E-SeH) + H(2)O(2) --> (E-SeOH) + H(2)O (1), proceeds via a stepwise pathway with the overall barrier of 17.1 kcal/mol, which is in good agreement with the experimental barrier of 14.9 kcal/mol. During reaction 1, the Gln83 residue has been found to play a key role as a proton acceptor, which is consistent with experiments. The second elementary reaction, (E-SeOH) + GSH --> (E-Se-SG) + HOH (2), proceeds with the barrier of 17.9 kcal/mol. The last elementary reaction, (E-Se-SG) + GSH --> (E-SeH) + GS-SG (3), is initiated with the coordination of the second glutathione molecule. The calculations clearly suggest that the amide backbone of the Gly50 residue directly participates in this reaction and the presence of two water molecules is absolutely vital for the reaction to occur. This reaction proceeds with the barrier of 21.5 kcal/mol and is suggested to be a rate-determining step of the entire GPx-catalyzed reaction H(2)O(2) + 2GSH --> GS-SG + 2H(2)O. The results discussed in the present study provide intricate details of every step of the catalytic mechanism of the GPx enzyme and are in good general agreement with experimental findings and suggestions.     

Vasodilator action of docosahexaenoic acid (DHA) in human coronary arteries in vitro

Coronary arterial tissues obtained from mammalian hearts are known to develop spontaneous phasic contractions. The aim of the present study was to investigate the vasodilatory effects of docosahexaenoic acid (DHA) on the rhythmic contractions of isolated human coronary arterial (HCA) preparations obtained from the recipient hearts of patients undergoing cardiac transplantation. Results from 8 hearts show that: (i) most HCA tissues displayed spontaneous rhythmic phasic contractions with a cycle length around 10 min in the absence or presence of PGF2alpha or elevated [K+]0 (20 mM); (ii) the rhythmic activity could be suppressed by a free fatty acid DHA (30 microM); (iii) high [K+]0 (20 and 80 mM) could induce sustained tonic contraction in addition to phasic contractions in HCA tissues, the tonic contraction could be antagonized by L-type Ca(2+) channel blockers or by DHA (depending on [K+]0); (iv) a digitalis substance ouabain also could induce tonic contraction and suppress phasic contraction; (v) in isolated HCA vascular smooth muscle cells, DHA increased the magnitude of outward voltage-gated K+ (IKV) currents and the inwardly rectifying IK1 currents. Enhancement of K+ currents could be related to vasorelaxation induced by DHA in HCA preparations. Further studies on the effects of DHA on various ionic currents and intracellular Ca(2+) transient are needed to clarify the Ca(2+)-dependent and the Ca(2+)-independent actions of DHA in HCA.     

Translational studies on regulation of brain docosahexaenoic acid (DHA) metabolism in vivo

One goal in the field of brain polyunsaturated fatty acid (PUFA) metabolism is to translate the many studies that have been conducted in vitro and in animal models to the clinical setting. Doing so should elucidate the role of PUFAs in the human brain, and effects of diet, drugs, disease and genetics on this role. This review discusses new in vivo radiotracer kinetic and neuroimaging techniques that allow us to do this, with a focus on docosahexaenoic acid (DHA). We illustrate how brain PUFA metabolism is influenced by graded reductions in dietary n-3 PUFA content in unanesthetized rats. We also show how kinetic tracer techniques in rodents have helped to identify mechanisms of action of mood stabilizers used in bipolar disorder, how DHA participates in neurotransmission, and how brain DHA metabolism is regulated by calcium-independent iPLA₂β. In humans, regional rates of brain DHA metabolism can be quantitatively imaged with positron emission tomography following intravenous injection of [1-¹¹C]DHA.     

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS*+ radical cation: 65% and 89% inhibition for compound [3] at 10(-4) M and 5 x 10(-5) M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.     

Mitochondria-specific transgenic overexpression of phospholipid hydroperoxide glutathione peroxidase (GPx4) attenuates ischemia/reperfusion-associated cardiac dysfunction

Ischemia/reperfusion (I/R) injury elicits damage to mitochondria. Antioxidants provide protection from I/R-induced mitochondrial damage. The goal of this study was to determine the impact of mitochondria-specific overexpression of GPx4 (PHGPx) on cardiac function following I/R. Transgenic mice were created in which PHGPx was overexpressed solely in the mitochondrion (mPHGPx). MPHGPx and littermate control hearts were subjected to global no-flow ischemia (20 min) followed by reflow reperfusion (30, 60, and 90 min). Following I/R, mPHGPx hearts possessed significantly better rates of contraction, developed pressures, and peak-systolic pressures as compared to controls (P<0.05). No differences were observed in rates of relaxation or end-diastolic pressures. Lipid peroxidation was significantly lower in mitochondria from mPHGPx hearts as compared to controls, following I/R (P<0.05). Electron transport chain (ETC) complex I, III, and IV activities were significantly higher in mPHGPx hearts as compared to controls, following I/R (P<0.05). MPHGPx overexpression enhanced ETC complex I, III, and IV activities in subsarcolemmal mitochondria (SSM; P<0.05), and ETC complex I and III activities in interfibrillar mitochondria (IFM; P<0.05) following I/R. These results indicate that mitochondria-specific GPx4 overexpression protects cardiac contractile function and preserves ETC complex activities following I/R. These results provide further rationale for the use of mPHGPx as a therapeutic protectant.     

Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen

The purpose of the current study was to further investigate the role of the antioxidant selenium-dependent enzyme glutathione peroxidase (GPx) in reproductive organs and semen from bulls. To this end a fast and convenient combined method for immune detection and substrate localization was adapted, which allows the assessment of both molecular weight and peroxidase activity of proteins on one and the same SDS-PAGE gel plate. After routine semen analysis of ejaculates, a spectrophotometrical assay of GPx activity in bovine semen was performed. For the immunological analyses performed, a rabbit polyclonal monospecific antibody against GPx was raised. Substrate detection and immunolocalization of GPx in lysates from bovine testis, epididymis, spermatozoa, and seminal plasma was performed. In order to determine the localization of GPx in spermatozoa, immunofluorescence analysis was performed. A positive correlation was established between GPx activity in semen and the number of motile spermatozoa. A negative correlation was observed between GPx activity and the number of immotile spermatozoa. The combined method for immunodetection and substrate localization was tested and proved reliable. Both tetramer and monomer forms of GPx were detected in lysates from testis, epididymis, and spermatozoa. We found no GPx activity in seminal plasma. Immunofluorescence shows the presence of GPx chiefly in the mitochondrial and in the acrosome regions of spermatozoa. GPx activity remained stable under the extreme experimental conditions.     

Two weeks of docosahexaenoic acid (DHA) supplementation increases synthesis-secretion kinetics of n-3 polyunsaturated fatty acids compared to 8 weeks of DHA supplementation

Docosahexaenoic acid (DHA, 22:6n-3) must be consumed in the diet or synthesized from n-3 polyunsaturated fatty acid (PUFA) precursors. However, the effect of dietary DHA on the metabolic pathway is not fully understood. Presently, 21-day-old Long Evans rats were weaned onto one of four dietary protocols: 1) 8 weeks of 2% ALA (ALA), 2) 6 weeks ALA followed by 2 weeks of 2% ALA + 2% DHA (DHA), 3) 4 weeks ALA followed by 4 weeks DHA and 4) 8 weeks of DHA. After the feeding period, ²H₅-ALA and ¹³C₂₀-eicosapentaenoic acid (EPA, 20:5n-3) were co-infused and blood was collected over 3 h for determination of whole-body synthesis-secretion kinetics. The synthesis-secretion coefficient (ml/min, means ± SEM) for EPA (0.238±0.104 vs. 0.021±0.001) and DPAn-3 (0.194±0.060 vs. 0.020±0.008) synthesis from plasma unesterified ALA, and DPAn-3 from plasma unesterified EPA (2.04±0.89 vs. 0.163±0.025) were higher (P<.05) after 2 weeks compared to 8 weeks of DHA feeding. The daily synthesis-secretion rate (nmol/d) of DHA from EPA was highest after 4 weeks of DHA feeding (843±409) compared to no DHA (70±22). Liver gene expression of ELOVL2 and FADS2 were lower (P<.05) after 4 vs. 8 weeks of DHA. Higher synthesis-secretion kinetics after 2 and 4 weeks of DHA feeding suggests an increased throughput of the PUFA metabolic pathway. Furthermore, these findings may lead to novel dietary strategies to maximize DHA levels while minimizing dietary requirements.     

Docosahexaenoic acid (DHA) sensitizes brain tumor cells to etoposide-induced apoptosis

In this study, we investigated whether DHA, a nutritionally important n-3 unsaturated fatty acid, modulated the sensitivity of brain tumor cells to the anticancer drug, etoposide (VP16). Medulloblastoma (MB) cell lines, Daoy and D283, and glioblastoma (GBM) cell lines, U138 and U87, were exposed to DHA or VP16 alone or in combination. The effects on cell proliferation and the induction of apoptosis were determined by using MTS and Hoechest 33342/PI double staining. U87 and U138 cells were found to be insensitive to the addition of DHA and VP16, whereas the two MB cell lines showed high sensitivity. DHA or VP16 alone showed little effect on cell proliferation or death in either the MB or GBM cell lines, but pretreatment with DHA enhanced the responsiveness to VP16 in the MB cell lines. To understand the mechanisms of combined DHA and VP16 on MB cells, pathway specific oligo array analyses were performed to dissect possible signaling pathways involved. The addition of DHA and VP16, in comparison to VP16 added alone, resulted in marked suppression in the expression of several genes involved in DNA damage repair, cell proliferation, survival, invasion, and angiogenesis, including PRKDC, Survivin, PIK3R1, MAPK14, NFκB1, NFκBIA, BCL2, CD44, and MAT1. These results suggest (1) that the effects of DHA and VP16 in brain tumor cells are mediated in part by the down regulation of events involved in DNA repair and the PI3K/MAPK signaling pathways and (2) that brain tumors genotypically mimicked by MB cells may benefit from therapies combining DHA with VP16.