Regulatory Phenomena in the Glutathione Peroxidase Superfamily.

Significance: The selenium-containing Glutathione peroxidases (GPxs)1-4 protect against oxidative challenge, inhibit inflammation and oxidant-induced regulated cell death. Recent Advances: GPx1 and GPx4 dampen phosphorylation cascades predominantly via prevention of inactivation of phosphatases by H2O2 or lipid hydroperoxides. GPx2 regulates the balance between regeneration and apoptotic cell shedding in the intestine. It inhibits inflammation-induced carcinogenesis in the gut but promotes growth of established cancers. GPx3 deficiency facilitates platelet aggregation likely via disinhibition of thromboxane biosynthesis. It is also considered a tumor suppressor. GPx4 is expressed in three different forms. The cytosolic form proved to inhibit interleukin-1-driven nuclear factor κB activation and leukotriene biosynthesis. Moreover, it is a key regulator of ferroptosis, because it reduces hydroperoxy groups of complex lipids and silences lipoxygenases. By alternate substrate use, the nuclear form contributes to chromatin compaction. Mitochondrial GPx4 forms the mitochondrial sheath of spermatozoa and, thus, guarantees male fertility. Out of the less characterized GPxs, the cysteine-containing GPx7 and GPx8 are unique in contributing to oxidative protein folding in the endoplasmic reticulum by reacting with protein isomerase as an alternate substrate. A yeast 2-Cysteine glutathione peroxidase equipped with CP and CR was reported to sense H2O2 for inducing an adaptive response. Critical Issues: Most of the findings compiled are derived from tissue culture and/or animal studies only. Their impact on human physiology is sometimes questionable. Future Directions: The expression of individual GPxs and GPx-dependent regulatory phenomena are to be further investigated, in particular in respect to human health.

Selenium and redox signaling.

Selenium compounds that contain selenol functions or can be metabolized to selenols are toxic via superoxide and H2O2 generation, when ingested at dosages beyond requirement. At supra-nutritional dosages various forms of programmed cell death are observed. At physiological intakes, selenium exerts its function as constituent of selenoproteins, which overwhelmingly are oxidoreductases. Out of those, the glutathione peroxidases counteract hydroperoxide-stimulated signaling cascades comprising inflammation triggered by cytokines or lipid mediators, insulin signaling and different forms of programmed cell death. Similar events are exerted by peroxiredoxins, which functionally depend on the selenoproteins of the thioredoxin reductase family. The thiol peroxidases of both families can, however, also act as sensors for hydroperoxides, thereby initiating signaling cascades. Although the interaction of selenoproteins with signaling events has been established by genetic techniques, the in vivo relevance of these findings is still hard to delineate for several reasons: The biosynthesis of individual selenoproteins responds differently to variations of selenium intakes; selenium is preferentially delivered to privileged tissues via inter-organ trafficking and receptor-mediated uptake, and only half of the selenoproteins known by sequence have been functionally characterized. The fragmentary insights do not allow any uncritical use of selenium for optimizing human health.

Characteristic single glucosinolates from Moringa oleifera: Induction of detoxifying enzymes and lack of genotoxic activity in various model systems.

Leaves of Moringa oleifera are used by tribes as biological cancer medicine. Scientific investigations with M. oleifera conducted so far have almost exclusively used total plant extracts. Studies on the activity of single compounds are missing. Therefore, the biological effects of the two main aromatic multi-glycosylated glucosinolates of M. oleifera were investigated in the present study. The cytotoxic effects of M. oleifera glucosinolates were identified for HepG2 cells (NRU assay), for V79-MZ cells (HPRT assay, SCE assay), and for two Salmonella typhimurium strains (Ames test). Genotoxic effects of these glucosinolates were not observed (Ames test, HPRT assay, and SCE assay). Reporter gene assays revealed a significant increase in the ARE-dependent promoter activity of NQO1 and GPx2 indicating an activation of the Nrf2 pathway by M. oleifera glucosinolates. Since both enzymes can also be induced via activation of the AhR, plasmids containing promoters of both enzymes mutated in the respective binding sites (pGL3enh-hNQO1-ARE, pGL3enh-hNQO1-XRE, pGL3bas-hGPX2-mutARE, pGL3bas-hGPX2-mutXRE) were transfected. Analyses revealed that the majority of the stimulating effects was mediated by the ARE motif, whereas the XRE motif played only a minor role. The stimulating effects of M. oleifera glucosinolates could be demonstrated both at the transcriptional (reporter gene assay, real time-PCR) and translational levels (enzyme activity) making them interesting compounds for further investigation.

Mixed results with mixed disulfides.

A period of research with Helmut Sies in the 1980s is recalled. Our experiments aimed at an in-depth understanding of metabolic changes due to oxidative challenges under near-physiological conditions, i.e. perfused organs. A major focus were alterations of the glutathione and the NADPH/NADP(+) system by different kinds of oxidants, in particular formation of glutathione mixed disulfides with proteins. To analyze mixed disulfides, a test was adapted which is widely used until today. The observations in perfused rat livers let us believe that glutathione-6-phosphate dehydrogenase (G6PDH), i.a. might be activated by glutathionylation. Although we did not succeed to verify this hypothesis for the special case of G6PDH, the regulation of enzyme/protein activities by glutathionylation today is an accepted posttranslational mechanism in redox biology in general. Our early experimental approaches are discussed in the context of present knowledge.

GPx2 Induction Is Mediated Through STAT Transcription Factors During Acute Colitis.

BACKGROUND: The selenoprotein glutathione peroxidase 2 (GPx2) is highly expressed in the gastrointestinal epithelium. During inflammatory bowel disease and colorectal cancer, GPx2 expression is enhanced. METHODS: We analyzed GPx2 expression and transcriptional regulation during the different phases of dextran sulfate sodium (DSS)-induced colitis in mice and in cytokine-treated colorectal cancer cells. RESULTS: In the colon of DSS-treated mice, GPx2 was upregulated during the acute and recovery phase. In the latter, it was specifically localized in regenerating ki67-positive crypts next to ulcerations. In cultured cells, endogenous GPx2 expression and GPx2 promoter activity were enhanced by the anti-inflammatory mediators 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) and interleukin-22 (IL-22), while it was unaffected by classical proinflammatory cytokines like IL-1ß. Induction of GPx2 expression by 15d-PGJ2 was mediated through Nrf2. In contrast, in DSS-treated Nrf2-KO mice GPx2 expression remained upregulated during recovery, which appeared to be independent of Nrf2. IL-22 activates transcription factors of the signal transducers and activators of transcription (STAT) family. Therefore, we analyzed the GPx2 promoter for putative STAT-responsive elements and identified 4 of them. Point mutation of the binding element next to the transcription start completely abolished promoter activation after IL-22 treatment and after cotransfection of STAT expression plasmids. To show in vivo relevance of the obtained results, we performed immunohistochemistry for phospho-STAT3 and GPx2. Especially during acute colitis, GPx2 and nuclear STAT3 colocalized in inflamed areas. CONCLUSIONS: GPx2 is a novel target of STAT transcription factors. The upregulation of GPx2 by IL-22 indicates that GPx2 might be important for the resolution of inflammation.

GPx2 suppression of H2O2 stress links the formation of differentiated tumor mass to metastatic capacity in colorectal cancer.

Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived "colonosphere" cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacity.

Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently.

High consumption of Brassica vegetables is considered to prevent especially colon carcinogenesis. The content and pattern of glucosinolates (GSLs) can highly vary among different Brassica vegetables and may, thus, affect the outcome of Brassica intervention studies. Therefore, we aimed to feed mice with diets containing plant materials of the Brassica vegetables broccoli and pak choi. Further enrichment of the diets by adding GSL extracts allowed us to analyze the impact of different amounts (GSL-poor versus GSL-rich) and different patterns (broccoli versus pak choi) of GSLs on inflammation and tumor development in a model of inflammation-triggered colon carcinogenesis (AOM/DSS model). Serum albumin adducts were analyzed to confirm the up-take and bioactivation of GSLs after feeding the Brassica diets for four weeks. In agreement with their high glucoraphanin content, broccoli diets induced the formation of sulforaphane-lysine adducts. Levels of 1-methoxyindolyl-3-methyl-histidine adducts derived from neoglucobrassicin were the highest in the GSL-rich pak choi group. In the colon, the GSL-rich broccoli and the GSL-rich pak choi diet up-regulated the expression of different sets of typical Nrf2 target genes like Nqo1, Gstm1, Srxn1, and GPx2. GSL-rich pak choi induced the AhR target gene Cyp1a1 but did not affect Ugt1a1 expression. Both colitis and tumor number were drastically reduced after feeding the GSL-rich pak choi diet while the other three diets had no effect. GSLs can act anti-inflammatory and anti-carcinogenic but both effects depend on the specific amount and pattern of GSLs within a vegetable. Thus, a high Brassica consumption cannot be generally considered to be cancer-preventive.

Deletion of glutathione peroxidase-2 inhibits azoxymethane-induced colon cancer development.

The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (-Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the -Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to -Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation.

Selenium in the redox regulation of the Nrf2 and the Wnt pathway.

Selenium deficiency is known to increase cancer risk by so far unclear mechanisms. Selenium exerts its biological effects via selenocysteine as an integral part of selenoproteins. Certain selenoproteins have redox properties, thereby providing a tool to regulate hydroperoxide-mediated signaling. Selenium deficiency does not only reduce synthesis of selenoproteins but also affects the expression of other proteins and even pathways. A moderate Se deficiency activates the Nrf2 and the Wnt pathways. The link between both pathways appears to be GSK3ß which in the active state prepares Nrf2 as well as ß-catenin, the key player in Wnt signaling, for ubiquitination and proteasomal degradation, thus silencing their transcriptional activity. Upon stimulation by Wnt signals, GSK3ß becomes inactivated and transcription factors are stabilized. Many intermediate steps in both pathways can be modulated by hydroperoxides, making them predestined to be regulated by selenoproteins. Oxidation sensors are (i) Keap1 which keeps Nrf2 in the cytosol unless it is modified by hydroperoxides/electrophiles and (ii) nucleoredoxin (Nrx) which is associated with disheveled (Dvl). NOX1-derived H2O2 oxidizes Nrx leading to the liberation of Dvl and the activation of Wnt signaling. Selenium deficiency can support oxidation of both sensors and activate both pathways. The consequences are dual: while the Keap1/Nrf2 system is generally believed to protect against oxidative stress, diverse xenobiotics, inflammation, and carcinogenesis, the Wnt response is considered rather a risky one in these respects. However, not only healthy cells but also malignant ones benefit from intact Keap1/Nrf2 signaling, making a dysregulated hydroperoxide signaling a plausible explanation for the increased cancer risk in selenium deficiency.

Selenoprotein W as biomarker for the efficacy of selenium compounds to act as source for selenoprotein biosynthesis.

Selenium is an essential trace element and, like all elements, present in many different compounds with unequivocal functions. This fact is only sporadically mentioned when recommended intake or supplementation is indicated just as "selenium." In mammals, selenium is an integral part of selenoproteins as selenocysteine. Selenocysteine is formed from serine at the respective tRNA((ser)sec), a reaction that requires selenophosphate formed from selenide and ATP. Thus, only compounds that can be metabolized into selenide can serve as sources for selenoprotein biosynthesis. We therefore tested the ability of selenium compounds such as sodium selenite, methylseleninic acid (MeSeA), Se-methyl selenocysteine, and selenomethionine to increase the activity, protein, or mRNA levels of commonly used biomarkers of the selenium status, glutathione peroxidase-1 (GPx1) and thioredoxin reductase, and of putatively new biomarkers, selenoprotein W1 (SepW1), selenoprotein H, and selenoprotein 15 in three different cell lines. Selenite and MeSeA were most efficient in increasing all markers tested, whereas the other compounds had only marginal effects. Effects were higher in the noncancerous young adult mouse colon cells than in the cancer cell lines HepG2 and HT-29. At the protein level, SepW1 responded as well as GPx1 and at the mRNA level, even better. Thus, the outcome of selenium treatment strongly depends on the chemical form, the cell type, and the biomarker used for testing efficacy.

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples.

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-L-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.

Glutathione peroxidases.

BACKGROUND: With increasing evidence that hydroperoxides are not only toxic but rather exert essential physiological functions, also hydroperoxide removing enzymes have to be re-viewed. In mammals, the peroxidases inter alia comprise the 8 glutathione peroxidases (GPx1-GPx8) so far identified. SCOPE OF THE REVIEW: Since GPxs have recently been reviewed under various aspects, we here focus on novel findings considering their diverse physiological roles exceeding an antioxidant activity. MAJOR CONCLUSIONS: GPxs are involved in balancing the H2O2 homeostasis in signalling cascades, e.g. in the insulin signalling pathway by GPx1; GPx2 plays a dual role in carcinogenesis depending on the mode of initiation and cancer stage; GPx3 is membrane associated possibly explaining a peroxidatic function despite low plasma concentrations of GSH; GPx4 has novel roles in the regulation of apoptosis and, together with GPx5, in male fertility. Functions of GPx6 are still unknown, and the proposed involvement of GPx7 and GPx8 in protein folding awaits elucidation. GENERAL SIGNIFICANCE: Collectively, selenium-containing GPxs (GPx1-4 and 6) as well as their non-selenium congeners (GPx5, 7 and 8) became key players in important biological contexts far beyond the detoxification of hydroperoxides. This article is part of a Special Issue entitled Cellular functions of glutathione.

TrxR1 and GPx2 are potently induced by isothiocyanates and selenium, and mutually cooperate to protect Caco-2 cells against free radical-mediated cell death.

Currently, there is significant interest in the field of diet-gene interactions and the mechanisms by which food compounds regulate gene expression to modify cancer susceptibility. From a nutrition perspective, two key components potentially exert cancer chemopreventive effects: isothiocyanates (ITCs), present in cruciferous vegetables, and selenium (Se) which, as selenocysteine, is an integral part of selenoproteins. However, the role of these compounds in the expression of key selenoenzymes once the cancer process has been initiated still needs elucidation. Therefore, this investigation examined the effect of two forms of selenium, selenium-methylselenocysteine and sodium selenite, both individually and in combination with two ITCs, sulforaphane or iberin, on the expression of the two selenoenzymes, thioredoxin reductase 1 (TrxR1) and gastrointestinal glutathione peroxidase (GPx2), which are targets of ITCs, in Caco-2 cells. Co-treatment with both ITCs and Se induced expression of TrxR1 and GPx2 more than either compound alone. Moreover, pre-treatment of cells with ITC+Se enhanced cytoprotection against H(2)O(2)-induced cell death through a ROS-dependent mechanism. Furthermore, a single and double knockdown of TrxR1 and/or GPx2 suggested that both selenoproteins were responsible for protecting against H(2)O(2)-induced cell death. Together, these data shed new light on the mechanism of interactions between ITC and Se in which translational expression of the enhanced transcripts by the former is dependent on an adequate Se supply, resulting in a cooperative antioxidant protective effect against cell death.

Physiological functions of GPx2 and its role in inflammation-triggered carcinogenesis.

Mammalian glutathione peroxidases (GPxs) are reviewed with emphasis on the role of the gastrointestinal GPx2 in tumorigenesis. GPx2 ranks high in the hierarchy of selenoproteins, corroborating its importance. Colocalization of GPx2 with the Wnt pathway in crypt bases of the intestine and its induction by Wnt signals point to a role in mucosal homeostasis, but GPx2 might also support tumor growth when increased by a dysregulated Wnt pathway. In contrast, the induction of GPx2 by Nrf2 activators and the upregulation of COX2 in cells with a GPx2 knockdown reveal inhibition of inflammation and suggest prevention of inflammation-mediated carcinogenesis. The Janus-faced role of GPx2 has been confirmed in a mouse model of inflammation-associated colon carcinogenesis (AOM/DSS), where GPx2 deletion increased inflammation and consequently tumor development, but decreased tumor size. The model further revealed a GPx2-independent decrease in tumor development by selenium (Se) and detrimental effects of the Nrf2-activator sulforaphane in moderate Se deficiency.

The selenoproteins GPx2, TrxR2 and TrxR3 are regulated by Wnt signalling in the intestinal epithelium.

BACKGROUND: The glutathione peroxidase 2 (GPx2) is expressed at crypt bases of the intestinal epithelium and in tumour tissue. The GPx2 promoter is activated by the Wnt pathway, which might be the reason for the specific expression pattern of GPx2. Together with additional selenoproteins, thioredoxin reductases TrxR2 and TrxR3, which are putative Wnt targets based on microarray analysis, Wnt-dependent GPx2 expression was analysed. METHODS: Two cell culture models for either an activated (3T3 cells with Wnt3a overexpression) or an inhibited Wnt pathway (HT-29 APC cells) were analysed. To provide physiological relevance, crypt base epithelial cells of the jejunum and colon of mice were compared to cells of the villus or crypt table, respectively. In addition, ß-catenin was deleted in crypt base cells ex vivo. RESULTS: In cancer cell lines, the endogenous expression of all three selenoproteins was consistently dependent on Wnt pathway activity. Expression was higher in the proliferative crypt compartment, where also the Wnt pathway is active. An inducible knockout of ß-catenin in isolated colonic crypt base cells reduced basal GPx2 expression. We, thus, demonstrated the regulation of GPx2 expression by the Wnt pathway in vitro and in vivo. Furthermore, the selenoproteins TrxR2 and TrxR3 have been identified as novel Wnt targets. This may imply a role of GPx2, TrxR2 and TrxR3 in proliferation, apoptosis and, therefore, also during cancer development. GENERAL SIGNIFICANCE: Selenium which is essential for the biosynthesis of Wnt-dependent selenoproteins might be important for the renewal of the intestinal epithelium and during carcinogenesis.

The yin and yang of nrf2-regulated selenoproteins in carcinogenesis.

The NF-E2-related factor-2 (Nrf2) is a transcription factor which regulates the major cellular defense systems and thereby contributes to the prevention of many diseases including cancer. Selenium deficiency is associated with a higher cancer risk making also this essential trace element a promising candidate for cancer prevention. Two selenoproteins, thioredoxin reductase-1 (TrxR1) and glutathione peroxidase-2 (GPx2), are targets for Nrf2. Selenium deficiency activates Nrf2 as does a TrxR1 knockout making a synergism between both systems plausible. Although this might hold true for healthy cells, the interplay may turn into the opposite in cancer cells. The induction of the detoxifying and antioxidant enzymes by Nrf2 will make cancer cells chemoresistant and will protect them against oxidative damage. The essential role of TrxR1 in maintaining proliferation makes its upregulation in cancer cells detrimental. The anti-inflammatory potential of GPx2 will help to inhibit cancer initiation and inflammation-triggered promotion, but its growth supporting potential will also support tumor growth. This paper considers beneficial and adverse consequences of the activation of Nrf2 and the selenoproteins which appear to depend on the cancer stage.

Glutathione peroxidase-2 and selenium decreased inflammation and tumors in a mouse model of inflammation-associated carcinogenesis whereas sulforaphane effects differed with selenium supply.

Chronic inflammation and selenium deficiency are considered as risk factors for colon cancer. The protective effect of selenium might be mediated by specific selenoproteins, such as glutathione peroxidases (GPx). GPx-1 and -2 double knockout, but not single knockout mice, spontaneously develop ileocolitis and intestinal cancer. Since GPx2 is induced by the chemopreventive sulforaphane (SFN) via the nuclear factor E2-related factor 2 (Nrf2)/Keap1 system, the susceptibility of GPx2-KO and wild-type (WT) mice to azoxymethane and dextran sulfate sodium (AOM/DSS)-induced colon carcinogenesis was tested under different selenium states and SFN applications. WT and GPx2-KO mice were grown on a selenium-poor, -adequate or -supranutritional diet. SFN application started either 1 week before (SFN4) or along with (SFN3) a single AOM application followed by DSS treatment for 1 week. Mice were assessed 3 weeks after AOM for colitis and Nrf2 target gene expression and after 12 weeks for tumorigenesis. NAD(P)H:quinone oxidoreductases, thioredoxin reductases and glutathione-S-transferases were upregulated in the ileum and/or colon by SFN, as was GPx2 in WT mice. Inflammation scores were more severe in GPx2-KO mice and highest in selenium-poor groups. Inflammation was enhanced by SFN4 in both genotypes under selenium restriction but decreased in selenium adequacy. Total tumor numbers were higher in GPx2-KO mice but diminished by increasing selenium in both genotypes. SFN3 reduced inflammation and tumor multiplicity in both Se-adequate genotypes. Tumor size was smaller in Se-poor GPx2-KO mice. It is concluded that GPx2, although supporting tumor growth, inhibits inflammation-mediated tumorigenesis, but the protective effect of selenium does not strictly depend on GPx2 expression. Similarly, SFN requires selenium but not GPx2 for being protective.

Basic principles and emerging concepts in the redox control of transcription factors.

Convincing concepts of redox control of gene transcription have been worked out for prokaryotes and lower eukaryotes, whereas the knowledge on complex mammalian systems still resembles a patchwork of poorly connected findings. The article, therefore, reviews principles of redox regulation with special emphasis on chemical feasibility, kinetic requirements, specificity, and physiological context, taking well investigated mammalian transcription factor systems, nuclear transcription factor of bone marrow-derived lymphocytes (NF-κB), and kelch-like ECH-associated protein-1 (Keap1)/Nrf2, as paradigms. Major conclusions are that (i) direct signaling by free radicals is restricted to O(2)•- and •NO and can be excluded for fast reacting radicals such as •OH, •OR, or Cl•; (ii) oxidant signals are H(2)O(2), enzymatically generated lipid hydroperoxides, and peroxynitrite; (iii) free radical damage is sensed via generation of Michael acceptors; (iv) protein thiol oxidation/alkylation is the prominent mechanism to modulate function; (v) redox sensors must be thiol peroxidases by themselves or proteins with similarly reactive cysteine or selenocysteine (Sec) residues to kinetically compete with glutathione peroxidase (GPx)- and peroxiredoxin (Prx)-type peroxidases or glutathione-S-transferases, respectively, a postulate that still has to be verified for putative mammalian sensors. S-transferases and Prxs are considered for system complementation. The impact of NF-κB and Nrf2 on hormesis, management of inflammatory diseases, and cancer prevention is critically discussed.

Breakdown products of neoglucobrassicin inhibit activation of Nrf2 target genes mediated by myrosinase-derived glucoraphanin hydrolysis products.

Glucosinolates (GLSs) present in Brassica vegetables serve as precursors for biologically active metabolites, which are released by myrosinase and induce phase 2 enzymes via the activation of Nrf2. Thus, GLSs are generally considered beneficial. The pattern of GLSs in plants is various, and contents of individual GLSs change with growth phase and culture conditions. Whereas some GLSs, for example, glucoraphanin (GRA), the precursor of sulforaphane (SFN), are intensively studied, functions of others such as the indole GLS neoglucobrassicin (nGBS) are rather unknown as are functions of combinations thereof. We therefore investigated myrosinase-treated GRA, nGBS and synthetic SFN for their ability to induce NAD(P)H:quinone oxidoreductase 1 (NQO1) as typical phase 2 enzyme, and glutathione peroxidase 2 (GPx2) as novel Nrf2 target in HepG2 cells. Breakdown products of nGBS potently inhibit both GRA-mediated stimulation of NQO1 enzyme and Gpx2 promoter activity. Inhibition of promoter activity depends on the presence of an intact xenobiotic responsive element (XRE) and is also observed with benzo[a]pyrene, a typical ligand of the aryl hydrocarbon receptor (AhR), suggesting that suppressive effects of nGBS are mediated via AhR/XRE pathway. Thus, the AhR/XRE pathway can negatively interfere with the Nrf2/ARE pathway which has consequences for dietary recommendations and, therefore, needs further investigation.

alpha-Tocopherol enhances degranulation in RBL-2H3 mast cells.

Based on the observation that 3 months alpha-tocopherol supplementation caused an up-regulation of the mRNA of vesicular transport proteins in livers of mice, the functional relevance was investigated in RBL-2H3 cells, a model for mast cell degranulation. In total, 24 h incubation with 100 muM alpha-tocopherol enhanced the basal and phorbol-12-myristyl-13-acetate/ionomycin-stimulated release of beta-hexosaminidase and cathepsin D as measured by enzymatic analysis as well as Western blotting and immunocytochemistry, respectively. beta-Tocopherol exerted the same effect, whereas alpha-tocopheryl phosphate and trolox were inactive, indicating that both the side chain and the 6-OH group at the chroman ring are essential for activation of degranulation. alpha-Tocopherol did not induce mRNA expression of soluble NSF-attachment protein receptor (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins, such as N-ethylmaleimide sensitive fusion protein, complexin-2, SNAP23 or syntaxin-3, in the RBL-2H3 cell model. In view of the well known alpha-tocopherol-mediated activation of protein phosphatases, which regulate soluble NSF-attachment protein receptor activities by dephosphorylation, underlying mechanisms are discussed in terms of preventing oxidative inactivation of protein phosphatases and so far unknown functions in certain membrane domains.