GSH Redox Cycle Research Articles

Interactive impacts of CO2-induced seawater acidification and cadmium exposure on antioxidant defenses of juvenile tongue sole Cynoglossus semilaevis

Antioxidant responses of juvenile sole exposed to seawater acidification (SA) and Cd were investigated. SA increased lipid peroxidation (LPO) in the fish, independent of Cd concentrations. Cd at medium and high levels inflated LPO under no or moderate SA conditions. This effect was absent under high SA levels, due to SA effect exceeding and obscuring Cd effect. SA and Cd collaborated to provoke LPO, with SOD and CAT being stimulated to defend against oxidative stress, while those related to GSH redox cycle were inhibited under SA exposure. Responses of GSH-related antioxidants to Cd impact varied contingent on their interactions with SA. This defensive strategy was insufficient to protect fish from increased LPO. Antioxidants responded more sensitively to SA than Cd exposure. GSH, GR, SOD and CAT are sensitive biomarkers for SA conditions. The findings offer insights into assessing fish's antioxidant defense strategy under Cd and SA circumstances in natural habitats.

A water-soluble two-photon fluorescent probe for rapid and reversible monitoring of redox state

Redox homeostasis is the guarantee for the normal operation of living organisms. Given its importance, monitoring dynamic changes of redox state will facilitate the study of relevant physiological and pathological processes. Herein, a two-photon fluorescent probe NA-Se with selenomorpholine as the response site has been developed for reversible detection of redox state. NA-Se exhibits outstanding analytical performance, including a rapid response to ClO− within 8 s, a detection limit as low as 13.3 nM, and at least 10 redox reversible cycles. Furthermore, NA-Se has improved water solubility and extremely low cytotoxicity, making it ideal for bioimaging. Finally, NA-Se has been successfully applied to monitor the ClO−/GSH redox cycle in living cells and zebrafish. This work may provide an effective tool for studying redox state and related diseases in vivo.

The safe Laccase@ZIF-8-prodrug system with GSH redox cycle for effective targeted cancer therapy with low off-target toxicity

Nanotechnology has been widely used in cancer treatment but only a small fraction (0.7 %) of the administered nanoparticle was delivered into a solid tumor, while the remaining fraction causes off-target toxicity in healthy tissues. The activation of prodrugs by exogenous enzymes can be used as an effective anticancer strategy to reduce systemic toxicity. In this study, the laccase@zeolitic imidazolate framework-8 (LA@ZIF-8) enzyme-activated prodrug system was created based on the high catalytic activity of LA in an acidic environment and the pH response (pH∼5.5) of ZIF-8. Quercetin (QU) was selected as the prodrug, which is non-toxic and even beneficial without being activated by LA. LA could be precisely released in the acidic tumor microenvironment for activating nontoxic QU successfully to produce toxic oxidized quercetin (OQU), and the LA was steady loaded on the LA@ZIF-8 under physiological conditions (pH∼7.4). Especially, the high concentrations of glutathione (GSH) in tumors could accelerate the oxidation of QU by LA. Meanwhile, GSH could be consumed continuously by the reduction of OQU for regenerating QU, thus a LA-QU-GSH redox was formed ingeniously. Therefore, the synergistic effect of OQU toxicity and GSH depletion induced reactive oxygen species (ROS) accumulation and tumor cell apoptosis. Overall, the LA@ZIF-8-QU prodrug system provides ideas for safe and effective cancer treatment with low off-target toxicity.

Pharmacological Effects of Gami-Yukmijihwang-Tang on the Lipopolysaccharide-Induced Hippocampus Oxidation and Inflammation via Regulation of Sirt6.

Yukmijihwang-Tang is widely used in traditional Korean medicine to treat age-related disorders. In the present study, we re-prescribed Gami-Yukmijihwang-Tang (YJT), which is slightly modified from Yukmijihwang-Tang by adding more medicinal plants to evaluate its pharmacological effects on underlying mechanisms against repeated lipopolysaccharide (LPS)-injection-induced neuroinflammation in the hippocampus regions. C57BL/6J male mice (16–24 weeks old) were divided into six groups: (1) the control group (DW with 0.9% saline injection), (2) LPS group (DW with LPS injection), YJT groups ((3) 100, (4) 200, or (5) 400 mg/kg of YJT with LPS injection), and (6) glutathione (GSH) group (100 mg/kg of GSH with LPS injection), respectively. Mice were orally administrated with various doses of YJT or glutathione (GSH) for the first five days. Neuroinflammation in the hippocampus region was induced by repeated injection of LPS during the last three days. As predicted, LPS not only increased oxidative stress–related markers including malondialdehyde, 4-hydroxynonenal, nitrotryptophan, and hydrogen peroxide, but also drastically enhanced inflammatory reactions including nitric oxide, inducible nitric oxide synthase, p65, and toll-like receptor 4, respectively. YJT administration, on the other hand, notably decreased the above pathological alterations by enhancement of antioxidant capacities such as superoxide dismutase and catalase activities. To explain the underlying pharmacological actions of YJT, we focused on a representative epigenetic regulator, a nicotinamide adenine dinucleotide + (NAD+)–dependent chromatin enzyme, Sirtuin 6 (Sirt6). Neuroinflammation in hippocampus regions depleted Sirt6 at the protein level and this alteration directly affected the nuclear factor erythroid 2–related factor (Nrf2)/hemeoxygenase (HO)-1 signaling pathway in the LPS group; however, YJT significantly recovered the Sirt6 protein levels, and it could recover the abnormal status of Nrf2/HO-1 signaling pathways in the hippocampus regions. Additionally, Sirt6 led to the up-regulation of GSH sub-enzymes of mRNA expression and protein levels of total GSH content. These findings suggest that YJT can protect against LPS-induced neuroinflammation and oxidative stress by regulating the Sirt6-related pathways and normalizing the GSH redox cycle.

Effects of Selenium on Serotonin Synthesis and the Glutathione Redox Cycle in Plum Leaves

Selenium (Se), an essential micronutrient for humans, is also a beneficial nutrient for growth and development of many plants. The aim of the present study was to determine whether low-dose Se application could increase plum growth and serotonin synthesis, as well as upregulate the glutathione redox cycle. Two-year-old plum trees were treated with Se6+ (Na2SeO4) and Se2− (SeMet). Growth parameters, levels of serotonin synthesis precursors, compounds and enzymes of the glutathione (GSH) redox cycle, and the antioxidant capacity were analyzed. Results showed that plant height and stem diameter were increased by Se treatment. The leaf Se content was significantly increased; tryptophan was not affected by Se treatment, while the 5-hydroxytryptophan content was significantly higher than control. Although serotonin levels were also increased, the effect was not significant, and no tryptamine was detected in our study. On the 30th day, the MDA content in Se2− was highest followed by the control and Se6+ treatment, and the activities of T-AOT and CAT were increased, while GST was reduced by Se treatment. The two forms of Se treatments increased the activity of GPX and GR, and the total content of GSH + glutathione disulfide (GSSG) at the end of treatment. Furthermore, the level of GSH and GSH/GSSG was instable changed. GSSG content was similar during the treatment time except on the 20 days. Conclusively, the pathway of plum serotonin was clear, both Se6+ and Se2− could influence the serotonin synthesis. Additionally, the GSH redox cycle was sensitive to the Se application. Moreover, Se6+ performed better than Se2− on leaf Se content, plant growth parameters, and antioxidant capacity.

Synergistic effects of tanshinone IIA and andrographolide on the apoptosis of cancer cells via crosstalk between p53 and reactive oxygen species pathways.

Tanshinone IIA (Tan IIA) and andrographolide (Andro) are natural compounds that are reported to exhibit anticancer activities against various types of cancers. The aim of this study is to evaluate the synergistic anticancer effects of the combination of Tan IIA and Andro, and to investigate the mechanisms of pharmacological effect and their potential applications as an anticancer therapy in clinics. The anticancer effects of the combination of Tan IIA and Andro on MCF7, SMMC7721, and BGC823 cells were explored. The apoptosis of the cancer cells was determined by MTT and AV-PI dual stain assays. The intracellular GSH level was measured by DTNB assay, and the intracellular levels of reactive oxygen species (ROS) were examined by flow cytometry. The expression of the proteins in the apoptosis pathway was determined by immunobloting. The combination of Tan IIA and Andro exhibited significant synergistic anticancer effects against cancer cells, especially at low concentrations. Andro reacted with the thiol group of intracellular GSH, thus disrupting the GSH redox cycle and eventually increasing the level of intracellular ROS. Tan IIA triggered p53 responses and apoptosis by binding to the DNA of cancer cells. The crosstalk between ROS and p53 exhibited a synergistic effect on the apoptosis of cancer cells. The combination of Tan IIA and Andro showed significant synergistic effects on cancer cell apoptosis by promoting crosstalk between ROS and p53, providing a novel and effective combination that has the potential to be applied in clinical anticancer therapy.

Survival of glioblastoma cells in response to endogenous and exogenous oxidative challenges: possible implication of NMDA receptor-mediated regulation of redox homeostasis.

Cancer cells are highly metabolically active and produce high levels of reactive oxygen species (ROS). Drug resistance in cancer cells is closely related to their redox status. The role of ROS and its impact on cancer cell survival seems far from elucidation. The mechanisms through which glioblastoma cells overcome aberrant ROS and oxidative stress in a milieu of hypermetabolic state is still elusive. We hypothesize that the formidable growth potential of glioma cells is through manipulation of tumor microenvironment for its survival and growth, which can be attributed to an astute redox regulation through a nexus between activation of N-methyl-d-aspartate receptor (NMDAR) and glutathione (GSH)-based antioxidant prowess. Hence, we examined the NMDAR activation on intracellular ROS level, and cell viability on exposure to hydrogen peroxide (H2 O2 ), and antioxidants in glutamate-rich microenvironment of glioblastoma. The activation of NMDAR attenuated the intracellular ROS production in LN18 and U251MG glioma cells. MK-801 significantly reversed this effect. On evaluation of GSH redox cycle in these cells, the level of reduced GSH and glutathione reductase (GR) activity were significantly increased. NMDAR significantly enhanced the cell viability in LN18 and U251MG glioblastoma cells, by attenuating exogenous H2 O2 -induced oxidative stress, and significantly increased catalase activity, the key antioxidant that detoxifies H2 O2 . We hereby report an unexplored role of NMDAR activation induced protection of the rapidly multiplying glioblastoma cells against both endogenous ROS as well as exogenous oxidative challenges. We propose potentiation of reduced GSH, GR, and catalase in glioblastoma cells through NMDAR as a novel rationale of chemoresistance in glioblastoma.

143 - Selenoprotein loss resulting from Alkbh8 deficiency engages senescence and metabolic programming and predicts poor clear cell renal cancer cell survival

Over 14,000 Americans die each year from kidney cancer with clear cell Renal Cell Carcinoma (ccRCC) being the most prevalent and malignant type. Disruptions in glutathione (GSH) metabolism have emerged as predictors of poor survival in ccRCC. Critically important to the maintenance of the GSH redox cycle are the activities of many selenocysteine-containing GSH metabolizing enzymes whose translation is controlled by the tRNA methyltransferase alkylation repair homolog 8 (Alkbh8). Epitransciptomic marks, in the form of tRNA modifications, can regulate gene expression at the level of translation. Our studies indicate that defective epitranscriptomic regulation and impairment in selenocysteine (SEC) incorporation, resulting from a deficiency in tRNA methytransferase Alkbh8 (Alkbh8-/-), alters glutathione (GSH) metabolism (4) and triggers a gene signature that is highly predictive (Hazard Ratio 2.65, p

Cystine and Theanine Improve Survival after Gut Ischemia-Reperfusion

Background and Aim: Oral administration of cystine and theanine (CT) may modulate antioxidant glutathione (GSH) metabolism, thereby improving outcomes after gut ischemia reperfusion. Methods: Experiment 1: Institute of Cancer Research mice (n = 35) were assigned to a Vehicle (n = 11), a CT140 (n = 14), or a CT280 (n = 10) group. The CT140 and 280 groups were given CT at respective dosages of 140 and 280 mg/kg (cystine: theanine = 5: 2) once daily via gavage for 5 days. All mice underwent 75-min occlusion of the superior mesenteric artery (SMA). Survival after reperfusion was observed. Experiment 2: Mice (n = 67) were pretreated for 5 days (Vehicle: n = 24, CT280: n = 20, vehicle/sham: n = 23). The Vehicle and CT280 groups underwent 60-min SMA occlusion. Levels of GSH, the oxidized form of GSH, Glutathione-S-S-Glutathione (GSSG), and GSH-related amino acids (cysteine and glutamic acid) in the small intestine, and plasma cytokine (IL-6, IL-1β, TNFα) levels, were evaluated before (0 h), 3, 6, or 9 h after reperfusion. Results: Experiment 1: The CT280 group showed significantly better survival than the Vehicle group. Experiment 2: Gut GSSG, cysteine, and glutamic acid levels were higher in the CT280 than in the Vehicle group after reperfusion. Plasma IL-6 and TNFα levels rose more rapidly in the CT280 than in the Vehicle group. Conclusion: Oral administration of CT improves survival after gut I/R, possibly through the modulation of the GSH-redox cycle and cytokine responses.

Glutathione reductase mediates drug resistance in glioblastoma cells by regulating redox homeostasis

Glutathione (GSH) and GSH-related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)-resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ-resistant cells than in sensitive cells. Furthermore, silencing GSR in drug-resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over-expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro-oxidant L-buthionine -sulfoximine. The modulation of redox state by GSH or L-buthionine -sulfoximine regulated GSR-mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress-free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment.

126 - Loss of Epitranscriptomic Control of Seleno-cysteine Utilization Engages Senescence and Metabolic Reprogramming through mTOR Activation and Predicts Poor Survival in Clear Cell Renal Cell Cancer Patients

Over 14,000 Americans die each year from kidney cancer with clear-cell Renal Cell Carcinoma (ccRCC) being the most prevalent and malignant type of kidney cancer. Recent work has implicated disruptions in glutathione (GSH) metabolism as predictors of poor survival in ccRCC. Maintenance of the GSH redox cycle is reliant on the activities of selenocysteine-containing GSH metabolizing enzymes whose translation is controlled by the alkylation repair homolog 8 (Alkbh8) tRNA methyltransferase. In cells from Alkbh8-/- mice the absence of the tRNA methyltransferase Alkbh8 decreases the levels of many GSH metabolizing selenoproteins, promotes increased ROS and DNA damage levels, and confers enhanced sensitivity to oxidizing agents. Surprisingly the Alkbh8-/- mice reproduce, thrive normally but display phenotypic characteristics of premature ageing such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine) when compared to their aged-matched WT control. Alkbh8-deficiency also triggers a senescence and metabolic gene signature that is predictive (Hazard Ratio 2.94, p

283 - Epitranscriptomic Control of Selenoprotein Synthesis, Senescence, Mitochondrial Bioenergetics and Clear Cell Renal Cell Carcinoma

Maintenance of the GSH redox cycle is reliant on the activities of selenocysteine-containing GSH metabolizing enzymes. Selenocysteine (SEC) is the 21 st amino acid and does not contain a dedicated codon. SEC incorporation during translation requires UGA-stop-codon recoding, which uses specifically modified tRNA for accurate decoding. We have shown that the stress-induced translation of many SEC containing ROS detoxifying enzymes is dependent on the Alkbh8 tRNA methyltransferase. Alkbh8 enzymatically methylates the uridine wobble base on tRNA SEC to promote UGA-stop codon decoding. In cells from Alkbh8 -/- mice, the absence of the tRNA methyltransferase Alkbh8 decreases the levels off many GSH metabolizing selenoproteins, promotes increased ROS and DNA damage levels, and confers enhanced sensitivity to oxidizing agents. Surprising to us was the ability of the Alkbh8 -/- MEFs and mice to thrive with the functional absence many key SEC containing proteins. Thus, we performed focused gene arrays and biochemical analysis that demonstrated dramatic alterations in both senescence and mitochondrial function. Senescence engagement and bioenergetic reprogramming are commonly observed during carcinogenesis, oncogenic transformation and metastatic disease. Thus, we next evaluated whether the adaptive gene responses that accompany Alkbh8 deficiency may be of prognostic significance. Analysis for the Alkbh8 adaptive genes of illumineseq gene expression data from 37 cancer types in The Cancer Genome Atlas revealed a significant decrease in survival percentage of only patients with the kidney cancer, clear cell renal cell carcinoma and increase of the adapt gene signature with tumor grade. Our observation that global selenoprotein suppression can accelerate the senescence phenotype and bioenergetics function, suggests that SEC-dependent regulation of these processes may be critical for limiting insults that drives tumor development or age related tissue degeneration.

Effect of quercetin, genistein and kaempferol on glutathione and glutathione-redox cycle enzymes in 3T3-L1 preadipocytes

Context and objective: Many studies have shown that cellular redox potential is largely determined by glutathione (GSH), which accounts for more than 90% of cellular nonprotein thiols. The aim of this study was to delineate the effect of three flavonoids – namely, quercetin, kaempferol and genistein – and exogenous GSH on oxidative damage by the Fenton’s pathway through the GSH and GSH-redox cycle enzymes in 3T3-L1 cells. Materials and methods: 3T3-L1 preadipocytes were exposed to each flavonoid and GSH at concentrations of 0, 5, 10, 15, 20 and 25 µM and then GSH levels and activities of glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rx) and superoxide dismutase (SOD) were measured. Results: Exogenous GSH did not have significant effect on intracellular GSH although slight decrease was observed at 15–25 µM doses. However, each of the three flavonoids sustained intracellular GSH levels in the cells as compared to the respective controls. Quercetin had the most profound effect, followed by kaempferol and genistein in that order. GSH-Px, GSH-Rx and SOD activities increased for all the doses tested compared to their respective controls. Again, quercetin had the maximum increase in enzyme activities followed by kaempferol and genistein for the enzymes tested. Discussion and conclusion: These findings suggest that the flavonoids play an important role in diminishing oxidation-induced biochemical damages. The enhancement of these enzymes may increase the resistance of the organism against oxidative damage by the Fenton’s pathway.

Medium-chain TAG attenuate hepatic oxidative damage in intra-uterine growth-retarded weanling piglets by improving the metabolic efficiency of the glutathione redox cycle.

The present study investigated the effects of medium-chain TAG (MCT) on hepatic oxidative damage in weanling piglets with intra-uterine growth retardation (IUGR). At weaning (mean 21 (SD 1·06) d of age), twenty-four IUGR piglets and twenty-four normal-birth weight (NBW) piglets were selected according to their birth weight (BW; IUGR: mean 0·95 (SD 0·04) kg; NBW: mean 1·58 (SD 0·04) kg) and weight at the time of weaning (IUGR: mean 5·26 (SD 0·15) kg; NBW: mean 6·98 (SD 0·19) kg) and fed either a soyabean oil (SO) diet (containing 5% SO) or a MCT diet (containing 1% SO and 4% MCT) for 28 d. IUGR piglets exhibited poor (P<0·05) growth performance, lower (P<0·05) metabolic efficiency of hepatic glutathione (GSH) redox cycle, and increased (P<0·05) levels of reactive oxygen species, apoptosis and necrosis in hepatocytes compared with NBW piglets. The MCT diet increased (P<0·05) the average daily gain and feed efficiency of piglets during the first 4 weeks after weaning. Furthermore, MCT diet-fed piglets had a higher (P<0·05) GSH:oxidised glutathione ratio and increased (P<0·05) activities of glucose-6-phosphate dehydrogenase (G6PD) and GSH reductase. The expression of G6PD was up-regulated (P<0·05) by the MCT diet irrespective of BW. Moreover, malondialdehyde concentrations in the liver and apoptosis and necrosis levels in hepatocytes were decreased (P<0·05) by the MCT diet irrespective of BW. These results indicate that MCT might have auxiliary therapeutic potential to attenuate hepatic oxidative damage in IUGR offspring during early life, thus leading to an improvement in the metabolic efficiency of the hepatic GSH redox cycle.

Increased glutaredoxin-1 and decreased proteinS-glutathionylation in sputum of asthmatics

To the Editor: Total reduced glutathione (GSH), the main pulmonary antioxidant, is increased in asthma patients and, in some studies, increased amounts of oxidised glutathione (GSSG) were also found. In addition, enzymes that regulate the GSH redox cycle are altered in asthmatics (reviewed in [1]). GSH, with its redox cycle partners, serves to maintain the reduced state of protein thiols, which can be achieved by scavenging oxidants or by the covalent reversible binding of GSH to protein thiols. The latter occurs under physiological conditions, is induced upon mild oxidative stress and is known as S -glutathionylation (PSSG) [2]. PSSG protects targeted thiols from irreversible oxidations and can modulate protein function. Of significant relevance in asthma, SERCA (sarco/endoplasmic reticulum calcium ATPase) is activated by PSSG, increasing smooth muscle relaxation, and PSSG of the RyR (ryanodine receptor) calcium channel was associated with impaired coupling. With respect to inflammation, nuclear factor-κB and activator protein-1 are negatively affected by PSSG (reviewed in [2]). GSH can be removed from proteins by glutaredoxins (Grx), which restores the function of proteins targeted by PSSG [2]. The Grx1 isoform localises to the cytosol, and Grx2 to mitochondria and the nucleus. Grx1 can also reduce low molecular weight disulfides and, thus, proteins with functionally important disulfide bonds could also be affected by Grx1 alterations. In contrast to damaging oxidations and measurements of GSH, PSSG and Grx have rarely been studied in lung diseases, and never in conjunction. In chronic obstructive pulmonary disease (COPD), Grx1-positive alveolar macrophages were negatively correlated with forced expiratory volume in 1 s (FEV1) and sputum Grx1 levels were higher during exacerbations [3]. In a murine model of allergic airway disease, we found increased Grx1 expression and activity [4]. Given the importance of …

Purification and characterization of glutathione reductase (E.C. 1.8.1.7) from bovine filarial worms Setaria cervi

Antioxidant enzymes are the parasite's premier resource to defend themselves against reactive oxygen species generated by macrophages, neutrophils and eosinophils of the host. These enzymes may be particularly important for parasites involved in chronic infections, such as parasitic helminths. Glutathione (GSH) and glutathione reductase (GR) are parts of the GSH redox cycle, which protects cells against damage by oxidants. Both GSH and GR are present in significant amounts in Setaria cervi female worms. GR has a central role in glutathione metabolism and as such is a potential target for chemotherapy. The aim of the work was to purify and characterize GR from S. cervi and to compare the properties of the helminth enzyme with its mammalian counterpart. GR was purified from filarial parasites S. cervi and preliminary steady state kinetics was performed. The purified protein was observed to be a dimer of 55kDa subunit as evident from SDS-PAGE analysis. Kinetic studies revealed significant differences in the properties of S. cervi GR from its mammalian counterpart which may be exploited in chemotherapy of filariasis. Filarial GR is thus proposed as a potential drug target.

Glutathione redox cycle in small intestinal mucosa and peripheral blood of pediatric celiac disease patients

The celiac disease is an autoimmune gastrointestinal disorder caused by gluten from wheat, rye or barley. In genetically predisposed persons, gluten induces the immune-mediated inflammation of small intestinal mucosa. Histological lesions include intraepithelial lymphocytosis, crypt hypertrophy and villous atrophy, resulting in malabsorption of micro- and macronutrients. The only treatment for celiac patients is a permanent gluten-free diet (GFD). Reactive oxygen species (ROS) and oxidative stress are strongly associated with the celiac disease. Glutathione (GSH) is a main detoxifier of endogenous and exogenous ROS in the intestine. In order to explain the role of glutathione redox cycle in celiac patients, we examined the activities of GSH-related antioxidant (AO) enzymes glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the concentration of GSH in small intestinal biopsies and peripheral blood of children affected by the celiac disease. The concentration of lipid hydroperoxides (LOOH) as markers of oxidative damage was measured in the same samples. The results clearly demonstrate a significant malfunction of GSH redox cycle with a concomitant decrease in the capacity to regenerate GSH and detoxify LOOH in celiac patients, even after several years of GFD. The oral administration of GSH and a diet rich in natural antioxidants, as well as appropriate dietary supplements, could be of great benefit to the patients.

NADPH-dependent Reductases Involved in the Detoxification of Reactive Carbonyls in Plants

Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.

Complete glutathione system in probiotic Lactobacillus fermentum ME-3

There is much information about glutathione (GSH) in eukaryotic cells, but relatively little is known about GSH in prokaryotes. Without GSH and glutathione redox cycle lactic acid bacteria (LAB) cannot protect themselves against reactive oxygen species. Previously we have shown the presence of GSH in Lactobacillus fermentum ME-3 (DSM14241). Results of this study show that probiotic L. fermentum ME-3 contains both glutathione peroxidase and glutathione reductase. We also present that L. fermentum ME-3 can transport GSH from environment and synthesize GSH. This means that it is characterized by a complete glutathione system: synthesis, uptake and redox turnover ability that makes L. fermentum ME-3 a perfect protector against oxidative stress. To our best knowledge studies on existence of the complete glutathione system in probiotic LAB strains are still absent and glutathione synthesis in them has not been demonstrated.

Altered lipid profile, oxidative status and hepatitis B virus interactions in human hepatocellular carcinoma

Altered membrane integrity in hepatocellular carcinoma (HCC) tissue was indicated by an elevation in cholesterol and significant decrease in phosphatidylcholine (PC). The resultant decreased phosphatidylcholine/phosphatidylethanolamine (PC/PE) and increased cholesterol/phospholipid ratios are associated with decreased fluidity in the carcinoma tissue. The lower PC was associated with a decrease in the quantitative levels of the saturated (C16:0, C18:0), ω6 (C18:2, C20:4) and ω3 (C22:5, C22:6) fatty acids (FAs), resulting in reduced long-chain polyunsaturated fatty acids (LCPUFAs), total PUFA and an increase in ω6/ω3 FA ratio. In PE, the saturated and ω3 (C22:5, C22:6) FAs were reduced while the total ω6 FA level was not affected, leading to an increased ω6/ω3 FA ratio. Increased levels of C18:1ω9, C20:2ω6 and reduction of 22:6ω3 in PC and PE suggest a dysfunctional delta-6 desaturase. The reduced PC/PE ratio resulted in a decreased C20:4ω6 (PC/PE) ratio, implying a shift towards synthesis of the 2-series eicosanoids. Lipid peroxidation was reduced in both hepatitis B negative (HBV −) and positive (HBV +) HCC tissues. Glutathione (GSH) was decreased in HCC while HBV had no effect, suggesting an impairment of the GSH redox cycle. In contrast HBV infection enhanced GSH in the surrounding tissue possibly to counter oxidative stress as indicated by the increased level of conjugated dienes. Apart from the reduced LCPUFA, the low level of lipid peroxidation in the carcinoma tissue was associated with increased superoxide dismutase and glutathione peroxidase activity. The disruption of the redox balance, resulting in increased cellular antioxidant capacity, could create an environment for resistance to oxidative stress in the carcinoma tissue. Alterations in membrane cholesterol, phospholipids, FA parameters, C20:4ω6 membrane distribution and low lipid peroxidation are likely to be important determinants underlying the selective growth advantage of HCC cells.