The organoselenium compound 1,4-phenylenebis(methylene)selenocyanate inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced tumorgenesis and enhances glutathione-related antioxidant levels in A/J mouse lung

Selenium, in the form of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) but not Se-enriched yeast (Se-yeast), was highly effective at inhibiting lung tumors induced by the tobacco specific nitrosamine (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice and at reducing NNK-induced DNA methylation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the lung. Our goal was to determine if p-XSC but not Se-yeast is effective at inducing levels of glutathione (GSH)-related antioxidants and reducing markers of GSH oxidation in the NNK-induced lung tumor model. In the first bioassay, 6-week-old mice were fed either control or experimental diets (containing 10 ppm as selenium from p-XSC or Se-yeast) and, beginning at 8 weeks of age, received NNK (3 micromol) by gavage once weekly for 8 weeks. After 18 weeks, p-XSC significantly reduced NNK-induced tumor burden by 74% (10.4 +/- 6.0 versus 2.7 +/- 1.5 tumors/mouse, P < 0.001) and tumor incidence from 96% to 68% (P < 0.01), whereas, Se-yeast had no effect. Lung GSH levels were unchanged by either NNK or Se-yeast, but were increased 70% in mice treated with both NNK and p-XSC (P < 0.01) and 41% in mice treated with p-XSC alone. In the second bioassay, the time course of effects of p-XSC was examined. As early as one week after initiation of p-XSC feeding lung and blood selenium levels were increased nearly six- and two-fold, respectively. Increases of 120% for GSH and 65% for Cys were observed in p-XSC groups compared to controls within one week after initiation of p-XSC feeding (P < 0.01). The levels of protein-bound:free GSH ratios and Cys ratios were significantly decreased in p-XSC-treated mice, regardless of NNK status, suggesting a decrease in the levels of oxidative stress. Altogether, these results indicate that p-XSC is a potent inducer of GSH and related thiol antioxidants in the lung leading to decreased levels of oxidative stress and suggest that p-XSC inhibits tumor formation, in part, by protecting against oxidative damage.     

Stimulation of GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: activation of Nrf2 and JNK-p62/SQSTM1 pathways

The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. By activation of Nrf2, cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1) and γ-glutamyl-cysteine ligase (GCL) are induced. GCL-induced glutathione (GSH) production is believed to affect redox signaling, cell proliferation and death. We here report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil, was found to induce phase II enzymes and GSH, thus protect t-BHP-induced mitochondrial dysfunction and apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target gene (GCLc, GCLm, HO-1, NQO-1) messenger RNA (mRNA) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by the PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism behind HT's strengthening of the antiapoptotic actions of the endogenous antioxidant system.     

Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress

Butein and phloretin are chalcones that are members of the flavonoid family of polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory activities. In rat primary hepatocytes, we examined whether butein and phloretin affect tert-butylhydroperoxide (tBHP)-induced oxidative damage and the possible mechanism(s) involved. Treatment with butein and phloretin markedly attenuated tBHP-induced peroxide formation, and this amelioration was reversed by l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced both HO-1 and GCL protein and mRNA expression and increased intracellular glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2 signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear protein-DNA binding activity, and ARE-luciferase reporter activity. The roles of the ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit (GCLC) expression were determined by using RNA interference directed against ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1 and GCLC by butein. In an animal study, phloretin was shown to increase GSH content and HO-1 expression in rat liver and decrease carbon tetrachloride-induced hepatotoxicity. In conclusion, we demonstrate that butein and phloretin up-regulate HO-1 and GCL expression through the ERK2/Nrf2 pathway and protect hepatocytes against oxidative stress.     

Major differences among chemopreventive organoselenocompounds in the sustained elevation of cytoprotective genes

Cytoprotective enzyme elevation through the nuclear erythroid 2 p45‐related factor 2 (Nrf2)–Kelch‐like ECH‐associated protein 1/antioxidant response element pathway has been promulgated for cancer prevention. This study compares the redox insult and sustained cytoprotective enzyme elevation by organoselenocompounds and sulforaphane (SF) in lung cells. SF elicited a rise in reactive oxygen species (ROS) and drop in glutathione (GSH) at 2 h; nuclear accumulation of Nrf2 at 4 h; and a GSH rebound and elevation in NAD(P)H quinone oxidoreductase (NQO1), thioredoxin reductase (TR1), and glutamate–cysteine ligase (GCL) at 24 h. Selenocystine (SECY) elicited a similar 24 h response, despite lesser earlier time‐point changes. 2‐Cyclohexylselenazolidine‐4‐carboxylic acid effects were similar to SECY's but with a larger Nrf2 change and the largest 24 h increase in GSH, GCL, TR1, and NQO1 of any compound investigated. Selenomethionine elicited a similar acute rise in ROS, but lesser depletion of GSH, no 4 h increase in nuclear Nrf2, only minor 24 h elevations in TR1 and NQO1, and a GCL elevation insufficient to elevate GSH. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:344–353, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21417     

Comparative excretion and tissue distribution of selenium in mice and rats following treatment with the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate

In a previous preliminary investigation, we reported on the excretion, tissue disposition and metabolism of the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) in the rat, but similar studies in the mouse have not been explored. Following the oral administration of p-XSC (50 micromol/kg body weight), selenium excretion in feces was comparable to that in urine in mice, but in rats, feces was the major route of excretion. Tetraselenocyclophane (TSC) was the major metabolite detected in mouse and rat feces. In both species, levels of selenium in exhaled air were negligible. At termination, in the mouse, the stomach had the highest selenium content followed by liver and blood, but lung and kidney contained negligible levels of selenium; in the rat, the selenium level in liver was the highest followed by kidney, stomach, blood and lung. The identification of TSC as a fecal metabolite in both species let us to postulate the following metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->a selenol (p-XSeH)-->TSC. Since the glutathione conjugate appears to be the proximal precursor for the selenol metabolite that may be an important intermediate in cancer chemoprevention, we report for the first time the synthesis of p-XSeSG and its other potential metabolites, namely the cysteine- and N-acetylcysteine-conjugates of p-XSC. HPLC analysis of the urine and bile showed a few metabolites of p-XSC; none of which eluted with the synthetic standards described above. When we examined the conversion of p-XSC and p-XSeSG in vitro using rat cecal microflora, TSC was formed from p-XSeSG but not from p-XSC. The formation of TSC from p-XSC in vivo but not in vitro suggests that p-XSC needs to be metabolized to p-XSeSG or an intermediate derived from its further metabolism. Thus, p-XSeSG was given orally to rats and the results showed that the pattern of selenium excretion after p-XSeSG treatment was similar to that of p-XSC; TSC was also identified as a fecal metabolite of p-XSeSG. It may be that the conversion of p-XSeSG to TSC is too facile, or the mere conjugation of p-XSC with glutathione does not occur in rats and mice.     

Enhanced Nrf2-dependent induction of glutathione in mouse embryonic fibroblasts by isoselenocyanate analog of sulforaphane

Epidemiological and laboratory studies have highlighted the potent chemopreventive effectiveness of both dietary selenium and cruciferous vegetables, particularly broccoli. Sulforaphane (SFN), an isothiocyanate, was identified as the major metabolite of broccoli responsible for its anti-cancer properties. An important mechanism for SFN chemoprevention is through the enhancement of glutathione (GSH), the most abundant antioxidant in animals and an important target in chemoprevention. Enhancement of GSH biosynthetic enzymes including the rate-limiting glutamate cysteine ligase (GCL), as well as other Phase II detoxification enzymes results from SFN-mediated induction of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signaling pathway. While isothiocyanate compounds such as SFN are among the most potent Nrf2 inducers known, we hypothesized that substitution of sulfur with selenium in the isothiocyanate functional group of SFN would result in an isoselenocyanate compound (SFN-isoSe) with enhanced Nrf2 induction capability. Here we report that SFN-isoSe activated an ARE-luciferase reporter in HepG2 cells more potently than SFN. It was also found that SFN-isoSe induced GCL and GSH in MEF cells in an Nrf2-dependent manner. Finally, we provide evidence that SFN-isoSe was more effective in killing HepG2 cancer cells, yet was less toxic to non-cancer MEF cells, than SFN.These data support our hypothesis, and suggest that SFN-isoSe and potentially other isoselenocyanates may be highly effective chemoprotective agents in vivo due to their ability to induce Nrf2 with low toxicity in normal cells and high efficiency at killing cancer cells.     

Induction of adaptive response and enhancement of PC12 cell tolerance by 7-hydroxycholesterol and 15-deoxy-delta(12,14)-prostaglandin J2 through up-regulation of cellular glutathione via different mechanisms

Increasing evidence suggests an adaptive response induced by reactive oxygen species and other physiologically existing oxidative stimuli. We have recently reported that a variety of lipid peroxidation products at sublethal concentrations could induce adaptive response and enhance PC12 cell tolerance, although the detailed underlying molecular mechanisms have not been clearly clarified. In the present study, we found that both 7-hydroxycholesterol (7-OHCh) and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) at sublethal concentrations significantly increased the cellular GSH as well as the enzyme activity of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. Depletion of cellular GSH by buthionine sulfoximine completely abolished the adaptive response. Interestingly, treatment with 15d-PGJ2 significantly increased the gene expression of both subunits of GCL in an NF-E2-related factor 2 (Nrf2)-dependent manner, whereas neither 7-OHCh induced any considerable changes on the GCL gene expression nor did the Nrf2-small interfering RNA treatment exert any appreciable effects on the GSH elevation and subsequent adaptive response induced by 7-OHCh. These results demonstrate that the adaptive response induced by both 7-OHCh and 15d-PGJ2 is mediated similarly through the up-regulation of GSH but via different mechanisms.     

Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.     

Mechanism and Significance of Changes in Glutamate-Cysteine Ligase Expression during Hepatic Fibrogenesis

GSH is synthesized sequentially by glutamate-cysteine ligase (GCL) and GSH synthase and defends against oxidative stress, which promotes hepatic stellate cell (HSC) activation. Changes in GSH synthesis during HSC activation are poorly characterized. Here, we examined the expression of GSH synthetic enzymes in rat HSC activation and reversion to quiescence. Expression of the GCL catalytic subunit (GCLC) fell during HSC activation and increased when activated HSCs revert back to quiescence. Blocking the increase in GCLC expression kept HSCs in an activated state. Activated HSCs have higher nuclear levels and binding activity of MafG to the antioxidant response element (ARE) of GCLC but lower Nrf2/MafG heterodimer binding to the ARE. Quiescent HSCs have a lower nuclear MafG level but higher Nrf2/MafG heterodimer binding to ARE. This occurred because of enhanced sumoylation of Nrf2 and MafG by SUMO-1, which promoted Nrf2 binding to ARE and heterodimerization with MafG. In vivo, knockdown of GCLC exacerbated bile duct ligation-induced liver injury and fibrosis. Ursodeoxycholic acid and S-adenosylmethionine are anti-fibrotic in bile duct ligation, but this effect was nearly lost if GCLC induction was blocked. In conclusion, sumoylation of Nrf2 and MafG enhances heterodimerization and increases GCLC expression, which keeps HSCs in a quiescent state. Antifibrotic agents require activation of GCLC to fully exert their protective effect.     

The Catalytic Subunit of Drosophila Glutamate-Cysteine Ligase Is a Nucleocytoplasmic Shuttling Protein

GSH concentration is considerably lower in the nucleus than in the cytoplasm; however, it is significantly elevated during active cell proliferation. The main purpose of this study was to understand the mechanism underlying these variations in nuclear/cytoplasmic distribution of GSH. The rate-limiting step in the de novo GSH biosynthesis pathway is catalyzed by glutamate cysteine ligase (GCL), a heterodimer, composed of a catalytic subunit (GCLc) and a modulatory subunit (GCLm). In Drosophila, GCLc, but not GCLm, contains a nuclear localization signal (NLS). Drosophila S2 cells, constitutively expressing regular GCLc protein or expressing GCLc protein with a mutated NLS motif, were generated by transfection. In quiescent S2 cells, GCLc is aggregated in the perinuclear cytosol and the nucleus, whereas GLCm resides solely in the cytosol. In actively proliferating S2 cells, expressing the normal NLS motif, GCLc migrates from the perinuclear cytoplasm into the nucleus, and the nuclear GSH level becomes elevated; in contrast, in proliferating cells, expressing the mutated NLS motif, neither does the GCLc migrate into the nucleus nor does the nuclear GSH amount rise. In S2 cells expressing wild type GCLc, perturbation of cellular redox state by exposure to cadmium resulted in the migration of GCLc into the nucleus but not in cells expressing GCLc with the mutated NLS motif. Overall, results indicated that GSH biosynthesis in the nucleus is associated with migration of only the GCLc subunit from the cytoplasm into the nucleus, and this migration requires the presence of an intact NLS.The tripeptide, γ-glutamylcysteinylglycine or GSH, is the most abundant intracellular nonprotein thiol. It serves multiple physiological functions, including maintenance of redox homeostasis, providing reducing equivalents for the elimination of reactive oxygen species, protection against electrophilic xenobiotics, maintenance of protein structure, and storage/transport of l-cysteine. GSH is synthesized de novo by two ATP-dependent consecutive reactions: ligation of l-glutamate to l-cysteine by the activity of glutamate-cysteine ligase (GCL²; EC 6.3.2.2), a rate-limiting step in the pathway, followed by the coupling of glycine to γ-glutamylcysteine by glutathione synthase (EC 6.3.2.3). The GCL holoenzyme is heterodimeric, consisting of a catalytic (GCLc) and a modifier (GCLm) subunit, each encoded by a unique gene. The intracellular ratios of GLCc and GCLm are, however, not necessarily equimolar and may be altered under conditions of oxidative stress. Although GCLc by itself is fully competent to catalyze the biosynthesis of GSH (1), dimerization with GCLm lowers the K_m for glutamate and also decreases sensitivity to feedback inhibition by GSH (2).Overexpression of GCL subunits in cultured cells has been reported to increase GSH production and confer enhanced protection against oxidative stress (3), apoptosis (4), and oxidant-induced DNA lesions (5). We have shown that overexpressions of GCLc or GCLm boost GSH biosynthesis and extend life span in Drosophila melanogaster (6). In contrast, inhibition of GCL activity results in decreased GSH levels, enhanced susceptibility to oxidative or nitrosative stress, increased DNA damage, and cell cycle arrest (7, 8). The homozygous knock-out for the catalytic GCLc subunit is embryonic lethal (9).GSH is predominantly synthesized in the cytoplasm, but its levels greatly vary among the different intracellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cytosol (10–12). Currently available evidence suggests the existence of two alternate mechanisms for the differential distribution of GSH in subcellular compartments: (i) GSH may first be synthesized in the cytoplasm and then transported into the organelles either actively or via passive diffusion; (ii) GSH may be synthesized in those organelles that display activities of GCL and glutathione synthase (13, 14).It has been demonstrated that GSH levels in the nucleus of proliferating cells are much higher than those in the confluent cells (15); however, the mechanism underlying this variation is presently unclear. In the present study, we provide evidence that GSH is synthesized in the nucleus and that this synthesis is dependent upon the shuttling of the GCLc, but not the GCLm, subunit from the cytoplasm to the nucleus. The ability of GCLc to migrate into the nucleus is due to the presence of a nuclear localization signal (NLS).     

Comparative action of 1,4-phenylenebis(methylene)selenocyanate and its metabolites against 7,12-dimethylbenz[a]anthracene-DNA adduct formation in the rat and cell proliferation in rat mammary tumor cells

1,4-phenylenebis(methylene)selenocyanate (p-XSC) inhibits 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis and DMBA-DNA binding in the rat mammary gland. Tetraselenocyclophane (TSC) was identified in rat feces as a metabolite of p-XSC. This led us to postulate the metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->aromatic selenol (p-XSeH)-->TSC. Whether p-XSC or one of its metabolites is responsible for cancer prevention is the focus of this study. We utilized the DMBA-DNA binding assay with p-XSC as a positive control to evaluate the chemopreventive potential of p-XSC metabolites at dietary selenium levels of 10 ppm. Rats were fed AIN-76A diet supplemented with various selenium compounds for 1 week prior to the oral administration of a single dose of [3H]DMBA (5 mg per rat, specific activity 51.3 mCi/mmol). The rats were sacrificed 24 h later and DNA was isolated from the mammary fat pads. Relative levels of total binding were: [pmol/mg DNA, mean +/- S.D., n=6]; DMBA [7.2 +/- 1.6]; DMBA+p-XSC [3.5 +/- 2.7]; DMBA+p-XSeSG [2.2 +/- 1.1]; DMBA+TSC [5.6 +/- 2.9]. All selenium compounds, except TSC, significantly inhibited DMBA-DNA adduct formation; however, the difference between p-XSC and p-XSeSG was not statistically significant. The inhibition of total binding was attributed to a reduction in the formation of the three major adducts derived from bay-region diol epoxides of DMBA. On the basis of their chromatographic characteristics, these were identified as anti-diol-epoxide:deoxyguanosine, syn-diol-epoxide:deoxyadenosine, and anti-diol-epoxide:deoxyadenosine. Our results suggest that p-XSeSG, but not TSC, is the likely inhibitor of mammary cancer. Selenium levels measured by atomic absorption spectroscopy in the target organ (mammary fat pads) and in plasma following the dietary administration of selenium compounds were in the order of p-XSeSG congruent with p-XSC>TSC. These results appear to be consistent with their order of inhibitory effects on total DMBA-DNA binding. Further in vitro studies of the effect of selenium compounds on cell proliferation suggest that, depending on the dose and time point selected, p-XSC is comparable to or better than p-XSeSG; but both are more effective than TSC. Collectively, our in vivo and in vitro results indicate that p-XSC and its conjugate are better candidates than TSC for future studies on mammary cancer chemoprevention.     

Age-associated perturbations in glutathione synthesis in mouse liver

The nature of the mechanisms underlying the age-related decline in glutathione (GSH) synthetic capacity is at present unclear. Steady-state kinetic parameters of mouse liver GCL (glutamate-cysteine ligase), the rate-limiting enzyme in GSH synthesis, and levels of hepatic GSH synthesis precursors from the trans-sulfuration pathway, such as homocysteine, cystathionine and cysteine, were compared between young and old C57BL/6 mice (6- and 24-month-old respectively). There were no agerelated differences in GCL V(max), but the apparent K(m) for its substrates, cysteine and glutamate, was higher in the old mice compared with the young mice (approximately 800 compared with approximately 300 microM, and approximately 710 compared with 450 microM, P<0.05 for cysteine and glutamate in young and old mice respectively). Amounts of cysteine, cystathionine and Cys-Gly increased with age by 91, 24 and 28% respectively. Glutathione (GSH) levels remained unchanged with age, whereas GSSG content showed an 84% increase, suggesting a significant pro-oxidizing shift in the 2GSH/GSSG ratio. The amount of the toxic trans-sulfuration/glutathione biosynthetic pathway intermediate, homocysteine, was 154% higher (P<0.005) in the liver of old mice compared with young mice. The conversion of homocysteine into cystathionine, a rate-limiting step in trans-sulfuration catalysed by cystathionine beta-synthase, was comparatively less efficient in the old mice, as indicated by cystathionine/homocysteine ratios. Incubation of tissue homogenates with physiological concentrations of homocysteine caused an up to 4.4-fold increase in the apparent K(m) of GCL for its glutamate substrate, but had no effect on V(max). The results suggest that perturbation of the catalytic efficiency of GCL and accumulation of homocysteine from the trans-sulfuration pathway may adversely affect de novo GSH synthesis during aging.