Comparative physiological and proteomic response to phosphate deficiency between two wheat genotypes differing in phosphorus utilization efficiency.

Genetic variation in phosphorus utilization efficiency (PUE) widely exists among wheat genotypes. However, the underlying mechanisms are still unclear. Two contrasting wheat genotypes, Heng4399 (H4399) and Tanmai98 (TM98), were screened out from 17 bread wheat genotypes based on shoot soluble phosphate (Pi) concentrations. The TM98 had a significantly higher PUE than the H4399, especially under Pi deficiency. The induction of genes in the PHR1-centered Pi signaling pathway was significantly higher in TM98 than in H4399. Collectively, through a label-free quantitative proteomic analysis, 2110 high-confidence proteins were identified in shoots of the two wheat genotypes. Among them, 244 and 133 proteins were differentially accumulated under Pi deficiency in H4399 and TM98, respectively. The abundance of proteins related to nitrogen and phosphorus metabolic processes, small molecule metabolic process, and carboxylic acid metabolic process weas significantly affected by Pi deficiency in the shoots of the two genotypes. The abundance of proteins in energy metabolism, especially photosynthesis, was decreased by Pi deficiency in the shoots of H4399. Inversely, the PUE-efficient genotype TM98 could maintain protein abundance in energy metabolism. Moreover, the proteins involved in pyruvate metabolism, glutathione metabolism, and sulfolipid biosynthesis were significantly accumulated in TM98, which probably contributed to its high PUE. SIGNIFICANCE: Improving the PUE of wheat is urgent and crucial for sustainable agriculture. Genetic variation among wheat genotypes provides materials for exploring the underlying mechanisms for high PUE. This study selected two wheat genotypes with contrasting PUE to reveal the differences in the physiological and proteomic responses to phosphate deficiency. The PUE-efficiency genotype TM98 greatly induced the expression of genes in the PHR1-centered Pi signaling pathway. Subsequently, the TM98 could maintain the abundance of proteins related to energy metabolism and enhance the abundance of proteins involved in pyruvate metabolism, glutathione metabolism, and sulfolipid biosynthesis to increase PUE under Pi deficiency. The differentially expressed genes or proteins between the genotypes with contrasting PUE would provide potential and basis for breeding wheat varieties with improved phosphorus use efficiency.

NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation induced skin injury.

Oxidative stress and lipid peroxidation are major causes of skin injury induced by ultraviolet (UV) irradiation. Ferroptosis is a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids and contributes to kinds of tissue injuries. However, it remains unclear whether the accumulation of lipid peroxides in UV irradiation-induced skin injury could lead to ferroptosis. We generated UV irradiation-induced skin injury mice model to examine the accumulation of the lipid peroxides and iron. Lipid peroxides 4-HNE, the oxidative enzyme COX2, the oxidative DNA damage biomarker 8-OHdG, and the iron level were increased in UV irradiation-induced skin. The accumulation of iron and lipid peroxidation was also observed in UVB-irradiated epidermal keratinocytes without actual ongoing ferroptotic cell death. Ferroptosis was triggered in UV-irradiated keratinocytes stimulated with ferric ammonium citrate (FAC) to mimic the iron overload. Although GPX4 protected UVB-injured keratinocytes against ferroptotic cell death resulted from dysregulation of iron metabolism and the subsequent increase of lipid ROS, keratinocytes enduring constant UVB treatment were markedly sensitized to ferroptosis. Nicotinamide mononucleotide (NMN) which is a direct and potent NAD+ precursor supplement, rescued the imbalanced NAD+/NADH ratio, recruited the production of GSH and promoted resistance to lipid peroxidation in a GPX4-dependent manner. Taken together, our data suggest that NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation-induced skin injury and inhibits oxidative skin damage. NMN or ferroptosis inhibitor might become promising therapeutic approaches for treating oxidative stress-induced skin diseases or disorders.

Protective Effects of a Lutein Ester Prodrug, Lutein Diglutaric Acid, against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells.

Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.

Phenolic compounds and hepatoprotective potential of Anastatica hierochuntica ethanolic and aqueous extracts against CCl4-induced hepatotoxicity in rats.

OBJECTIVE: To investigate the effect of Anastatica hierochuntica ethanolic (KEE), aqueous (KAE) extracts, and their combination against CCl4-induced hepatotoxicity in rats. METHODS: The HPLC analysis for KEE and KAE was quantitatively carried out. Biochemical liver markers, antioxidant enzymes, and histopathological alterations were examined then total hepatoprotection potential was calculated. RESULTS: Among 9 identified phenolic compounds (PC) in KEA, sinapic acid was the highest while syringic acid was the highest among 21 identified PC in KAE. Six flavonoids were identified in KEE and two in KAE using HPLC, respectively. Oral administration of KEE, KAE, and KEE + KAE at 250 mg/kg body weight significantly reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels, alkaline phosphatase (ALP), total bilirubin (TBILI), and also attenuated histopathological changes. Additionally, they reduced malondialdehyde (MOD), restored reduced-glutathione (GSH), and enhanced superoxide dismutase (SOD) levels. KEE, KAE, and KEE+KAE protected the liver from CCl4-hepatotoxicity as they mainly attenuating oxidative stress. Total hepatoprotection was about 128.3%, 114.5%, and 103.8% for KEE, KAE, and KEE+KAE, respectively. CONCLUSION: Biochemical observations, supplemented by histopathological examination revealed that AH affords extract-depending protection against CCl4-hepatotoxicity.

Selenium-enriched Saccharomyces cerevisiae improves the meat quality of broiler chickens via activation of the glutathione and thioredoxin systems.

The aim of this study was to investigate the effects of selenium (Se)-enriched Saccharomyces cerevisiae (SSC) on meat quality and to elucidate the underlying mechanisms in broilers. A total of 200 one-day-old Arbor Acres broiler chickens were randomly allocated to one of four treatments with 5 replications of 10 chickens each. Group 1 served as a control and was fed a basal diet without Se supplementation, while groups 2, 3, and 4 were fed the basal diet supplemented with S. cerevisiae (SC), sodium selenite (SS), and SSC, respectively. Breast muscle samples were collected to evaluate meat quality, selenium concentration, oxidative stability, and the mRNA levels of antioxidant enzyme genes on day 42. As compared with groups 1 and 2, SS and SSC supplementation increased Se concentration, glutathione peroxidase (GPx) and thioredoxin reductase (TR) activities, total antioxidant capacity, and the mRNA levels of GPx-1, GPx-4, TR-1, and TR-3 (P < 0.05) and decreased drip loss and malondialdehyde (MDA) content (P < 0.05). As compared with group 3, SSC supplementation increased pH, lightness, yellowness, Se concentration, GPx and superoxide dismutase activities, and the mRNA levels of GPx-1 and GPx-4 (P < 0.05) but decreased drip loss and MDA content (P < 0.05). Thus, SSC improved meat quality and oxidative stability by activating the glutathione and thioredoxin systems, which should be attributed to the combined roles of Se and SC.

Management of Citrus sinensis peels for protection and treatment against gastric ulcer induced by ethanol in rats.

Introduction: Stomach ulcer is one of the most prevalent disorders worldwide. The study was aimed to isolate and characterize the major polymethoxylated flavonoids in Citrus sinensis peels petroleum ether extract and investigate its protective and curative effect on gastric ulcer.Material and methods: Some spectral analyses were used for identification of the isolated compounds from the petroleum ether extract of Citrus sinensis peels. One oral dose (0.5 ml/100 g b.wt.) of absolute ethanol was orally given to rats after starvation for 24 h to induce gastric ulcer. To explore the protective and curative role of the plant extract, it was orally (250 mg/kg b.wt.) given for 1 week either before or post-ulcer induction. A reference drug, ranitidine (100 mg/kg b.wt.), was also evaluated. Stomach acidity, gastric volume, lesion counts, glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), acid phosphatase (AP), interlukin-10 (IL-10) and prostaglandin E2 (PGE2) were estimated. Stomach histopathological features were monitored.Results: Nine polymethoxy flavonoids were identified from the extract. Treatment with C. sinensis peels extract recorded amelioration in all parameters.Conclusion: Citrus sinensis petroleum ether peels extract had protective and curative effects against gastric ulcer. Therefore, the extract recorded anti-secretory, anti-ulcerative, anti-inflammatory, and antioxidant effects. Its healing action exceeded its protective role due to its richness in polymethoxylated flavonoids.

Separation and Characterization of Phenolamines and Flavonoids from Rape Bee Pollen, and Comparison of Their Antioxidant Activities and Protective Effects Against Oxidative Stress.

Phenolamines and flavonoids are two important components in bee pollen. There are many reports on the bioactivity of flavonoids in bee pollen, but few on phenolamines. This study aims to separate and characterize the flavonoids and phenolamines from rape bee pollen, and compare their antioxidant activities and protective effects against oxidative stress. The rape bee pollen was separated to obtain 35% and 50% fractions, which were characterized by HPLC-ESI-QTOF-MS/MS. The results showed that the compounds in 35% fraction were quercetin and kaempferol glycosides, while the compounds in 50% fraction were phenolamines, including di-p-coumaroyl spermidine, p-coumaroyl caffeoyl hydroxyferuloyl spermine, di-p-coumaroyl hydroxyferuloyl spermine, and tri-p-coumaroyl spermidine. The antioxidant activities of phenolamines and flavonoids were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays. It was found that the antioxidant activity of phenolamines was significantly higher than that of flavonoids. Moreover, phenolamines showed better protective effects than flavonoids on HepG2 cells injured by AAPH. Furthermore, phenolamines could significantly reduce the reactive oxygen species (ROS), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and increase the superoxide dismutase (SOD) and glutathione (GSH) levels. This study lays a foundation for the further understanding of phenolamines in rape bee pollen.

Mapping of quantitative trait loci for antioxidant molecules in tomato fruit: Carotenoids, vitamins C and E, glutathione and phenolic acids.

The nutritional value of a crop lies not only in its protein, lipid, and sugar content but also involves compounds such as the antioxidants lycopene, ß-carotene and vitamin C. In the present study, wild tomato Solanum pimpinellifolium LA 1589 was assessed for its potential to improve antioxidant content. This wild species was found to be a good source of alleles for increasing ß-carotene, lycopene, vitamin C and vitamin E contents in cultivated tomato. Characterization of an LA 1589 interspecific inbred backcross line (IBL) mapping population revealed many individuals with transgressive segregation for the antioxidants confirming the usefulness of this wild species for breeding of these traits. Molecular markers were used to identify QTLs for the metabolites in the IBL population. In total, 64 QTLs were identified for the antioxidants and their locations were compared to the map positions of previously identified QTLs for confirmation. Four (57 %) of the carotenoid QTLs, four (36 %) of the vitamin QTLs, and 11 (25 %) of the phenolic acid QTLs were supported by previous studies. Furthermore, several potential candidate genes were identified for vitamins C and E and phenolic acids loci. These candidate genes might be used as markers in breeding programs to increase tomato's antioxidant content.

Antioxidant supplementation partially rescues accelerated ovarian follicle loss, but not oocyte quality, of glutathione-deficient mice†.

The tripeptide thiol antioxidant glutathione (GSH) has multiple physiological functions. Female mice lacking the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations, ovarian oxidative stress, preimplantation embryonic mortality, and accelerated age-related decline in ovarian follicles. We hypothesized that supplementation with thiol antioxidants, N-acetyl cysteine (NAC), or α-lipoic acid (ALA) will rescue this phenotype. Gclm-/- and Gclm+/+ females received 0 or 80 mM NAC in drinking water from postnatal day (PND) 21-30; follicle growth was induced with equine chorionic gonadotropin (eCG) on PND 27, followed by an ovulatory dose of human CG and mating with a wild type male on PND 29 and zygote harvest 20 h after hCG. N-acetyl cysteine supplementation failed to rescue the low rate of second pronucleus formation in zygotes from Gclm-/- versus Gclm+/+ females. In the second study, Gclm-/- and Gclm+/+ females received diet containing 0, 150, or 600 mg/kg ALA beginning at weaning and were mated with wild type males from 8 to 20 weeks of age. α-Lipoic acid failed to rescue the decreased offspring production of Gclm-/- females. However, 150 mg/kg diet ALA partially rescued the accelerated decline in primordial follicles, as well as the increased recruitment of follicles into the growing pool and the increased percentages of follicles with γH2AX positive oocytes or granulosa cells of Gclm-/- females. We conclude that ovarian oxidative stress is the cause of accelerated primordial follicle decline, while GSH deficiency per se may be responsible for preimplantation embryonic mortality in Gclm-/- females.

Effect of Moringa oleifera stem extract on hydrogen peroxide-induced opacity of cultured mouse lens.

BACKGROUND: Moringa oleifera, also known as horseradish tree or drumstick tree, has strong antioxidant properties. In the present study, we investigated the potential effect of Moringa oleifera stem extract (MOSE) on cataract formation induced by oxidative stress in cultured mouse lenses. METHODS: Mouse lenses cultured in vitro were pretreated with MOSE (0.5 and 1 mg/mL) for 24 h. Then, 1 mM hydrogen peroxide was added, and mouse lenses were cultured for a further 24 h. The medium was then changed to normal culture medium. After 48 h, lens opacification, reactive oxygen species (ROS) generation, reduced glutathione (GSH) content, and activities of superoxide dismutase (SOD) and catalase (CAT) were measured in lens tissues. In addition, the protein expression of peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor with potential benefits to improve vision-threatening eye diseases, was assayed. RESULTS: MOSE (1 mg/mL) alleviated lens opacification, reduced ROS generation, increased GSH content, and elevated SOD and CAT activities in cultured lenses. Moreover, MOSE upregulated the expressions of SOD, CAT, and PPARα. CONCLUSIONS: This study showed that MOSE alleviates oxidative stress-induced cataract formation, and the mechanism of the effect is mainly related to its improvement of the endogenous antioxidant system in the lens.

High-Dose Aspirin Reverses Tartrazine-Induced Cell Growth Dysregulation Independent of p53 Signaling and Antioxidant Mechanisms in Rat Brain.

Tartrazine, an azo dye used in food, cosmetics, and pharmaceuticals with the effects on cell cycle, is not well understood. Therefore, we investigated the toxicity of tartrazine in rat brain with high-dose aspirin. Male Wistar rats (n = 24) were divided into (C) control, (T) tartrazine (700 mg/kg body weight [BW] at weeks 1 and 2), (A) aspirin (150 mg/kg [BW] at weeks 1, 2, and 3), and (TA) aspirin + tartrazine (150 mg/kg [BW] aspirin at weeks 1, 2, and 3 and 700 mg/kg [BW] tartrazine at weeks 1 and 2) groups. The expression of p53, B cell lymphoma-2 extra-large (Bcl-xL), cyclin-dependent kinase 2 (CDK2), p27, and Ki67 was evaluated by quantitative reverse-transcription PCR. A histopathological analysis of brain tissue and oxidative stress level was assessed based on reduced glutathione (GSH), ascorbic acid (AA), and malondialdehyde levels. We found that Bcl-xL, Ki67, CDK2, and p27 were upregulated and p53 was downregulated in the tartrazine-treated group as compared to the control group. Aspirin administration reversed these changes except P53 expression. Tartrazine had no effect on lipid peroxidation but altered AA and GSH levels with no reversal by aspirin treatment. Histopathological analysis revealed that aspirin prevented tartrazine-induced damage including increased perivascular space and hemorrhage. These results indicate that aspirin protects the brain from tartrazine-induced toxicity independent of p53 signaling and antioxidant mechanisms.

Pulicaria crispa mitigates gastric ulcer induced by ethanol in rats: role of treatment and auto healing.

Context: Stomach ulcers are the common gastrointestinal disorders worldwide. Objective: This study aimed to investigate the therapeutic impact of Pulicaria crispa aerial parts ethanol extract against gastric ulcer in rats. Materials and methods: Ulcer was induced by one oral dose of ethanol (0.5 ml/100g body weight) on 24 hours empty stomach, then the plant extract (500 mg/kg b.wt.) was orally administered daily for one week. Ranitidine (100 mg/kg b.wt.); as a reference drug was evaluated. Stomach acidity and volume, as well as lesion counts were measured. Levels of malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were estimated. Assay of different marker enzymes; succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), glucose-6-phosphatase (G-6-Pase), acid phosphatase (AP) and 5'-nucleotidase (5'NT) were determined. Interlukin-10 (IL-10), intracellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor alpha (TNF-α) were also determined. Stomach histopathological assessment was detected. Results: Gastric ulcer showed drastic changes in oxidative stress, cell organelles and inflammatory markers. These biomarkers served as good tools to identify the presence of gastric ulcer. Treatment with P. crispa recorded amelioration in most parameters exceeding the auto healing effect. Conclusion: Healing potency of P. crispa is possibly related to its content of glycosides, coumarins, flavonoids, tannins, sterols and triterpenes.

Dietary Supplementation of Probiotic Bacillus subtilis Affects Antioxidant Defenses and Immune Response in Grass Carp Under Aeromonas hydrophila Challenge.

This study investigated whether Bacillus subtilis can provide protection for grass carp against oxidative stress damage induced by Aeromonas hydrophila. A total of 240 healthy grass carp (Ctenopharyngodon idellus) (average weight of 71.42 ± 4.36g) were randomly divided into four groups with three replicates: control group, A. hydrophila group, B. subtilis + A. hydrophila group, and A. hydrophila + B. subtilis group. After challenge with A. hydrophila, the lipid oxidative damage, antioxidant defenses, and the gene expression of inflammatory cytokines of the grass carp were investigated. Our results showed that A. hydrophila caused lipid oxidative damage, led to significant decreases in antioxidant defenses, and induced inflammatory responses of grass carp. However, the grass carp group fed the probiotic B. subtilis diet for 42 days before the challenge and the group fed the probiotic B. subtilis diet immediately after the challenge both showed (i) a reduced level of oxidative stress with a decrease in the level of MDA; (ii) an increase in antioxidant defenses, including an increase in total antioxidant capacity (T-AOC), increased activities of SOD and CAT, increased levels of GSH, and upregulated gene expression of antioxidant enzymes (SOD, CAT, and Gpx); and (iii) an improved immune response with the level of antiinflammatory cytokines IL-10 messenger RNA (mRNA) upregulated and the levels of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-8 mRNA downregulated. Based on this study, B. subtilis can provide effective protection of fish against oxidative stress damage induced by A. hydrophila infection.

Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy.

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease.

Curcumin attenuates heat-stress-induced oxidant damage by simultaneous activation of GSH-related antioxidant enzymes and Nrf2-mediated phase II detoxifying enzyme systems in broiler chickens.

The object of this study was to investigate the effect of curcumin on modulating the glutathione (GSH)-related antioxidant enzymes and antioxidant responses via NF-E2-related factor 2 (Nrf2) signaling pathway in heat-stressed broiler chickens. A total of 400 one-day-old male Arbor Acres broiler chicks was reared in an environmentally controlled room. At 21 d, broiler chicks were divided into 5 treatment groups and were fed one of 4 diets under 2 temperature conditions: 22°C + a basal diet (CON treatment); 34°C for 8 h (0900-1700) + a basal diet supplemented with 0, 50, 100, or 200 mg/kg curcumin (HS, CMN1, CMN2, and CMN3 treatments, respectively). The heat treatment lasted for 20 consecutive days. The results showed that heat stress significantly increased (P < 0.05) the weekly rectal temperature and average head and feet temperature. Compared to the HS treatment, feed conversion was significantly decreased (P < 0.05) in CMN1 and CMN2 treatments. CMN1 administration significantly improved (P < 0.05) the pH24 of muscle. The abnormal changes of serum malonaldehyde and corticosterone concentrations were prevented (P < 0.05) by curcumin. Mitochondrial GSH concentration in the liver was significantly increased (P < 0.05) in CMN1 and CMN2 treatments compared with the HS treatment. The CMN1, CMN2, and CMN3 supplementations significantly increased (P < 0.05) γ-GCL, GSH-Px, and GST activities. Curcumin significantly increased (P < 0.05) the expression of Nrf2, HO-1, and γ-GCLc in the liver as compared to the CON diet. The expression of Cu/ZnSOD and CAT were increased (P < 0.05) by feeding CMN2, respectively, as compared to the HS treatment. It was concluded that curcumin supplementation enhanced the resistance of broilers to heat stress, as evidenced by reversing the FC, increasing the GSH content and GSH-related enzyme activities, and inducing the expression of Nrf2 and Nrf2-mediated phase II detoxifying enzyme genes.

3-Bromo-4,5-dihydroxybenzaldehyde Enhances the Level of Reduced Glutathione via the Nrf2-Mediated Pathway in Human Keratinocytes.

A natural bromophenol found in seaweeds, 3-bromo-4,5-dihydroxybenzaldehyde (BDB), has been shown to possess antioxidant effects. This study aimed to investigate the mechanism by which BDB protects skin cells subjected to oxidative stress. The effect of BDB on the protein and mRNA levels of glutathione-related enzymes and the cell survival of human keratinocytes (HaCaT cells) was investigated. BDB treatment increased the protein and mRNA levels of glutathione synthesizing enzymes and enhanced the production of reduced glutathione in HaCaT cells. Furthermore, BDB activated NF-E2-related factor 2 (Nrf2) and promoted its localization into the nucleus by phosphorylating its up-stream signaling proteins, extracellular signal-regulated kinase and protein kinase B. Thus, BDB increased the production of reduced glutathione and established cellular protection against oxidative stress via an Nrf2-mediated pathway.

Structural insights into the binding of buckwheat glutaredoxin with GSH and regulation of its catalytic activity.

Glutaredoxins (Grxs) are ubiquitous thioltransferases and members of the thioredoxin (Trx) fold superfamily. They have multiple functions in cells including oxidative stress responses and cell signaling. A novel glutaredoxin from buckwheat (rbGrx) with higher catalytic activity was identified, cloned, and purified. The structures of glutathionylated rbGrx and an rbGrx mutant, in which cysteine 39 was mutated to alanine, were solved by x-ray diffraction at a resolution of 2.05Å and 2.29Å, respectively. In rbGrx, GSH (glutathione) is bound at the conserved GSH-binding site, and its structure shows that it has the potential to function as a scaffold protein for the assembly and delivery of GSH. The crystal structure shows that GSH does not bind to the C39A rbGrx mutant, and the C39A mutant had no catalytic activity, indicating that C39 is a key residue that is involved in both the binding of rbGrx to GSH and the regulation of its catalytic activity. The model showing the binding of GSH with rbGrx provides a basis for understanding its molecular function and its potential future applications in medicinal food science.

Effect of dietary n-3 polyunsaturated fatty acids on antioxidant defense and sperm quality in rainbow trout (Oncorhynchus mykiss) under regular stripping conditions.

This study examined the effect of dietary n-3 polyunsaturated fatty acids (PUFA) on malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and reduced glutathione (GSH) levels; semen and liver fatty acid compositions; and spermatological values (semen volume and pH, sperm density, percentage and duration of sperm motility) in rainbow trout (Oncorhynchus mykiss) under regular stripping conditions. For this purpose, one control and two experimental diets were prepared as isonitrogenous and isocaloric. The control diet did not contain n-3 PUFA. However, the D1 and D2 diets were supplemented with n-3 PUFA concentrated anchovy oil at a 1% and 2% level, respectively. The n-3 PUFA content in the semen and liver, semen volume, initial sperm motility, duration of 50% sperm motility, total duration of sperm motility and sperm density values of the control fish fed the n-3 PUFA-deficient diet were decreased and were accompanied by a reduction of the antioxidant defense (SOD, CAT, GSH-Px and GSH) and an elevation of MDA in the blood, gonad, liver and kidney at all of the sampling periods (P<0.01 for each case). However, the effects of the sampling period on the MDA and antioxidant defense values in the blood, gonad, liver and kidney of the control diet fish (with the exception of the GSH and GSH-Px activities) and the D1 and D2 diet fish were not significant (P>0.01). However, supplementation with n-3 PUFA protected the fish from these adverse effects. The modulations were clearly observed in the fish fed the D2 diet because they were under lower oxidative stress, as indicated by MDA. The increased enzyme activity corresponds with the physiological mechanisms combating the elevation of free radicals under oxidative stress. The highest n-3 PUFA levels in the semen and liver and spermatological values were obtained from the fish fed the D2 diet at all of the sampling periods. On the other hand, the effects of the sampling stage on the spermatological values of the fish fed the D1 and D2 diets were not significant (P>0.01). However, the effects of the sampling stage in the fish fed the control diet on these values (with the exception of semen pH) were significant (P<0.01). In conclusion, the addition and balance of n-3 PUFA in the diet of broodstock fish can improve sperm quality and ultimately cause successful reproduction.

Smartamine M and MetaSmart supplementation during the peripartal period alter hepatic expression of gene networks in 1-carbon metabolism, inflammation, oxidative stress, and the growth hormone-insulin-like growth factor 1 axis pathways.

Peripartal cows likely require greater amounts of Met not only at the tissue and cell level for methylation reactions but also for milk protein synthesis after calving. Thirty-nine Holstein cows were fed throughout the peripartal period (-21 d to 30 d in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo Inc., Antony, France; n=12), or CON plus Smartamine M (SM; Adisseo Inc.; n=13). The Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 or 0.07% (dry matter) of feed for MS or SM. Liver tissue was collected on -10, 7, and 21 d for transcriptome profiling of genes associated with Met and glutathione metabolism as well as components of the inflammation, oxidative stress, growth hormone/insulin-like growth factor-1 axis, and DNA methylation pathways. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrasts CON versus SM + MS and SM versus MS. The S-adenosylhomocysteine hydrolase (SAHH) gene was the most abundant among all genes evaluated, with overall greater expression in Met-supplemented cows than CON, and in SM than MS. Expression of Met adenosyltransferase 1A (MAT1A) was greater in Met-supplemented cows than CON by 21 d postpartum. A greater overall expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) occurred in Met-supplemented cows than CON. In contrast, the expression of glutathione synthase (GSS); glutamate-cysteine ligase, catalytic subunit (GCLC); and superoxide dismutase 1, cytosolic (SOD1) was lower in Met-supplemented cows than CON. A greater overall expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1) and greater upregulation of haptoglobin (HP) on d 7 occurred in Met-supplemented cows than CON. Expression of DNA cytosine-5-methyltransferase 3 alpha (DNMT3A) was greater but expression of DNMT1 was lower in Met-supplemented cows than CON. The response observed in SAHH reflects its importance to Met supplementation during the peripartum period. Despite greater HP expression after calving, the lower expression of glutathione (GSS and GCLC) metabolism genes and SOD1 due to Met reflect a lower oxidative stress and mild inflammatory status. The extent to which changes in expression of DNMT3A and DNMT1 result in epigenetic effects partly responsible for the previously observed enhanced performance in Met-supplemented cows remains to be examined. Increasing the supply of Met as SM or MS can affect expression of genes in the Met cycle to various extents and, hence, the supply of methyl donors such as S-adenosylmethionine and antioxidants such as glutathione. These compounds likely are in high demand during the peripartum period.

Metabolic characterization of hepatocellular carcinoma using nontargeted tissue metabolomics.

Hepatocellular carcinoma has a poor prognosis due to its rapid development and early metastasis. In this report, we characterized the metabolic features of hepatocellular carcinoma using a nontargeted metabolic profiling strategy based on liquid chromatography-mass spectrometry. Fifty pairs of liver cancer samples and matched normal tissues were collected from patients having hepatocellular carcinoma, including tumor tissues, adjacent noncancerous tissues, and distal noncancerous tissues, and 105 metabolites were filtered and identified from the tissue metabolome. The principal metabolic alternations in HCC tumors included elevated glycolysis, gluconeogenesis, and ß-oxidation with reduced tricarboxylic acid cycle and Δ-12 desaturase. Furthermore, increased levels of glutathione and other antioxidative molecules, together with decreased levels of inflammatory-related polyunsaturated fatty acids and phospholipase A2, were observed. Differential metabolite levels in tissues were tested in 298 serum specimens from patients with chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Betaine and propionylcarnitine were confirmed to confer good diagnostic potential to distinguish hepatocellular carcinoma from chronic hepatitis and cirrhosis. External validation of cirrhosis and hepatocellular carcinoma serum specimens further showed that this combination biomarker is useful for diagnosis of hepatocellular carcinoma with a supplementary role to α-fetoprotein.