Induction of antioxidant and phase 2 drug-metabolizing enzymes by falcarindiol isolated from Notopterygium incisum extract, which activates the Nrf2/ARE pathway, leads to cytoprotection against oxidative and electrophilic stress

In the present study, we isolated falcarindiol from Notopterygium incisum and investigated the effect of falcarindiol on the expression of antioxidant enzymes (AOEs), such as catalase, and phase 2 drug-metabolizing enzymes (DMEs), such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1, in a cultured cell line from normal rat liver, Clone 9 cells. Exposure of Clone 9 cells to falcarindiol resulted in the significant induction of AOEs and phase 2 DMEs. Western blot analysis and transfection studies using a luciferase reporter construct demonstrated that the induction of AOEs and phase 2 DMEs by falcarindiol was caused through the Nrf2/ARE (nuclear factor-E2-related factor 2/antioxidant response element) pathway. Pretreatment of cells with falcarindiol accelerated the detoxification of a potentially toxic quinone (menadione) and mitigated menadione-induced cytotoxicity. We found that falcarindiol was a novel inducer of AOEs and phase 2 DMEs and falcarindiol might exhibit chemopreventive activity.     

Luteolin inhibits the Nrf2 signaling pathway and tumor growth in vivo

Nuclear factor erythroid 2-related factor 2 (Nrf2) is over-expressed in many types of tumor, promotes tumor growth, and confers resistance to anticancer therapy. Hence, Nrf2 is regarded as a novel therapeutic target in cancer. Previously, we reported that luteolin is a strong inhibitor of Nrf2 in vitro. Here, we showed that luteolin reduced the constitutive expression of NAD(P)H quinone oxidoreductase 1 in mouse liver in a time- and dose-dependent manner. Further, luteolin inhibited the expression of antioxidant enzymes and glutathione transferases, decreasing the reduced glutathione in the liver of wild-type mice under both constitutive and butylated hydroxyanisole-induced conditions. In contrast, such distinct responses were not detected in Nrf2^−/− mice. In addition, oral administration of luteolin, either alone or combined with intraperitoneal injection of the cytotoxic drug cisplatin, greatly inhibited the growth of xenograft tumors from non-small-cell lung cancer (NSCLC) cell line A549 cells grown subcutaneously in athymic nude mice. Cell proliferation, the expression of Nrf2, and antioxidant enzymes were all reduced in tumor xenograft tissues. Furthermore, luteolin enhanced the anti-cancer effect of cisplatin. Together, our findings demonstrated that luteolin inhibits the Nrf2 pathway in vivo and can serve as an adjuvant in the chemotherapy of NSCLC.     

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe²⁺) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n = 13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe²⁺, EE-CAT plus Fe²⁺, EE-GPx plus Fe²⁺ and EE-SOD plus Fe²⁺). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6 h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6 h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2 h after thawing (P < 0.05). Catalase supplementation, however, improved DNA integrity at 4 h (P < 0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6 h, linear motility at 6 h, mitochondrial activity at 6 h, membrane integrity at 2 and 6 h, and DNA integrity at 4 h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2 h after thawing (P < 0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2 h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe²⁺ negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P < 0.05). After thawing, there were, however, no significant differences between the control plus Fe²⁺ and the antioxidative enzymes supplementation plus Fe²⁺ groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.     

Coffee constituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression

Oxidative cellular stress initiates Nrf2 translocation into the nucleus, thus inducing antioxidant response element (ARE)-mediated expression of Phase II enzymes involved in detoxification and antioxidant defence. We investigated whether coffee extracts (CEs) of different proveniences and selected constituents have an impact on the Nrf2/ARE pathway in human colon carcinoma cells (HT29). Assessed as increased nuclear Nrf2 protein, Nrf2 nuclear translocation was modulated by different CEs as observed by Western blot analysis. In addition to the known Nrf2 activator 5-O-caffeoylquinic acid (CGA), pyridinium derivatives like the N-methylpyridinium ion (NMP) were identified as potent activators of Nrf2 nuclear translocation and ARE-dependent gene expression of selected antioxidative Phase II enzymes in HT29. Thereby, the substitution pattern at the pyridinium core structure determined the impact on Nrf2-signalling. In contrast, trigonelline was found to interfere with Nrf2 activation, effectively suppressing the NMP-mediated induction of Nrf2/ARE-dependent gene expression. In conclusion, several coffee constituents, partly already present in the raw material as well as those generated during the roasting process, contribute to the Nrf2-translocating properties of consumer-relevant coffee. A fine tuning in the degradation/formation of activating and deactivating constituents of the Nrf2/ARE pathway during the roasting process appears to be critical for the chemopreventive properties of the final coffee product.     

Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus

Glutathione S-transferases (GSTs; EC 2.5.1.18) are major enzymes that function in Phase II detoxification reactions by catalyzing the conjugation of reduced glutathione through cysteine thiol. In this study, we cloned and sequenced four GST genes from the monogonont rotifer Brachionus koreanus. The domain regions of four Bk-GSTs showed a high similarity to those of other species. In addition, to evaluate the potential of GST genes as an early warning signal for oxidative stress, we exposed sublethal concentrations of copper (Cu) to B. koreanus and measured glutathione (GSH) contents and several antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.8.1.7). The reactive oxygen species (ROS) at 12 h and 24 h after copper exposure increased significantly. GSH contents however did not increase significantly and even it decreased at 0.24 mg/L at 12 h. The activities of several antioxidant enzymes, particularly GPx and GR, showed a dramatic increase in 0.24 mg/L of CuCl₂. Messenger RNAs of each Bk-GST showed different patterns of modulations according to GST types, and particularly, Bk-GST-omega, Bk-GST-sigma, and Bk-GST zeta genes were highly sensitive to Cu. These results indicate that Bk-GSTs, functioning as one of the enzymatic defense mechanisms particularly in the early stage of oxidative stress response, were induced by Cu exposure. This also suggests that these genes and related enzymes have a potential as biomarkers for a more sensitive initial stress response.     

Zeaxanthin induces Nrf2-mediated phase II enzymes in protection of cell death

Zeaxanthin (Zea) is a major carotenoid pigment contained in human retina, and its daily supplementation associated with lower risk of age-related macular degeneration. Despite known property of Zea as an antioxidant, its underlying molecular mechanisms of action remain poorly understood. In this study, we aim to study the regulation mechanism of Zea on phase II detoxification enzymes. In normal human retinal pigment epithelium cells, Zea promoted the nuclear translocation of NF-E2-related factor 2 (Nrf2) and induced mRNA and protein expression of phase II enzymes, the induction was suppressed by specific knockdown of Nrf2. Zea also effectively protected against tert-butyl hydroperoxide-induced mitochondrial dysfunction and apoptosis. Glutathione (GSH) as the most important antioxidant was also induced by Zea through Nrf2 activation in a time- and dose-dependent manner, whereas the protective effects of Zea were decimated by inhibition of GSH synthesis. Finally, Zea activated the PI3K/Akt and MAPK/ERK pathway, whereas only PI3K/Akt activation correlated with phase II enzymes induction and Zea protection. In further in vivo analyses, Zea showed effects of inducing phase II enzymes and increased GSH content, which contributed to the reduced lipid and protein peroxidation in the retina as well as the liver, heart, and serum of the Sprague–Dawley rats. For the first time, Zea is presented as a phase II enzymes inducer instead of being an antioxidant. By activating Nrf2-mediated phase II enzymes, Zea could enhance anti-oxidative capacity and prevent cell death both in vivo and in vitro.     

Treatment of industrial wastewater with two-stage constructed wetlands planted with Typha latifolia and Phragmites australis

Industrial wastewater treatment comprises several processes to fulfill the discharge permits or to enable the reuse of wastewater. For tannery wastewater, constructed wetlands (CWs) may be an interesting treatment option. Two-stage series of horizontal subsurface flow CWs with Phragmites australis (UP series) and Typha latifolia (UT series) provided high removal of organics from tannery wastewater, up to 88% of biochemical oxygen demand (BOD₅) (from an inlet of 420 to 1000 mg L⁻¹) and 92% of chemical oxygen demand (COD) (from an inlet of 808 to 2449 mg L⁻¹), and of other contaminants, such as nitrogen, operating at hydraulic retention times of 2, 5 and 7 days. No significant (P < 0.05) differences in performance were found between both the series. Overall mass removals of up to 1294 kg COD ha⁻¹ d⁻¹ and 529 kg BOD₅ ha⁻¹ d⁻¹ were achieved for a loading ranging from 242 to 1925 kg COD ha⁻¹ d⁻¹ and from 126 to 900 kg BOD₅ ha⁻¹ d⁻¹. Plants were resilient to the conditions imposed, however P. australis exceeded T. latifolia in terms of propagation.     

Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta)

Intertidal alga Gracilaria corticata growing in natural environment experiences various abiotic stresses during the low tides. The aim of this study was to determine whether desiccation exposure would lead to oxidative stress and its effect varies with exposure periods. This study gives an account of various biochemical changes in G. corticata following the exposure to desiccation for a period of 0 (control), 1, 2, 3 and 4 h under controlled conditions. During desiccation, G. corticata thalli showed dramatic loss of water by almost 47% when desiccated for 4 h. The enhanced production of reactive oxygen species (ROS) and increased lipid peroxidation observed during the exposure of 3-4 h were chiefly contributed by higher lipoxygenase (LOX) activity with the induction of two new LOX isoforms (LOX-2, ∼85 kDa; LOX-3, ∼65 kDa). The chlorophyll, carotenoids and phycobiliproteins (phycoerythrin and phycocyanin) were increased during initial 2 h exposure compared to control and thereafter declined in the succeeding exposure. The antioxidative enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX) and the regeneration rate of reduced ascorbate (AsA) and glutathione (GSH) increased during desiccation up to 2-3 h. Further, the isoforms of antioxidant enzymes Mn-SOD (∼150 kDa), APX-4 (∼110 kDa), APX-5 (∼45 kDa), GPX-1 (∼80 kDa) and GPX-2 (∼65 kDa) responded specifically to the desiccation exposure. Compared to control, a relative higher content of both free and bound insoluble putrescine and spermine together with enhanced n-6 PUFAs namely C20:4(n-6) and C20:3(n-6) fatty acids found during 2 h exposure reveals their involvement in defence reactions against the desiccation induced oxidative stress.     

Suppression of Salvinia molesta Mitchell in Texas and Louisiana by Cyrtobagous salviniae Calder and Sands

Release and evaluation studies of the Brazil population of Cyrtobagous salviniae on Salvinia molesta were conducted originally at 18 sites in Texas and Louisiana from 1999 through 2005. However, overall project results could only be continually evaluated at two release and two control sites because the remainder were eventually destroyed or corrupted by floods, droughts, or herbicides. Mean fresh weight biomass of S. molesta ranged from 15.5 kg FW m⁻² during the summer to as low as 2.1 kg FW m⁻² during the winter prior to the release of C. salviniae. Insect populations established within a year of release and initially spread slowly. Damage to S. molesta increased with increasing C. salviniae detections while S. molesta biomass and surface coverage declined at both release sites by more than 99% while remaining unchanged at the control sites. Water in release sites registered higher levels of dissolved oxygen, higher temperatures, and higher pH than water in control sites. This study provides another example of the effectiveness of C. salviniae against S. molesta even in more temperate climates.     

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H₂O₂) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses.     

E-cadherin gene promoter hypermethylation may contribute to the risk of bladder cancer among Asian populations

There are increasing scientific evidences suggesting that E-cadherin gene promoter hypermethylation may contribute to the development and progression of bladder cancer, but existing studies have yielded inconclusive results. This meta-analysis aims to assess the role of E-cadherin promoter hypermethylation in bladder carcinogenesis. We conducted an extensive literature search for relevant studies on PubMed, Embase, Web of Science, Cochrane Library, and CBM databases from their inception through May 1st, 2013. This meta-analysis was performed using the STATA 12.0 software. Crude risk ratio (RR) with 95% confidence interval (CI) was calculated. Ten clinical studies were included in this meta-analysis with a total of 620 bladder cancer samples, 199 normal adjacent samples and 131 normal urothelium tissue. Our meta-analysis revealed that the methylation frequencies in bladder cancer tissues were obviously higher than those in normal control tissues (RR = 2.02, 95%CI: 1.00–4.12, P = 0.050). Subgroup analysis by ethnicity indicated that higher methylation frequencies were observed in bladder cancer tissues among Asian populations (RR = 2.35, 95%CI: 1.11–4.95, P = 0.025), but not among Caucasian populations (RR = 1.62, 95%CI: 0.48–5.53, P = 0.439). Univariate and multivariate meta-regression analyses showed that ethnicity may be the major source of heterogeneity (P < 0.05). No publication bias was detected in this meta-analysis (P = 0.358). The present meta-analysis indicates that E-cadherin gene promoter hypermethylation may contribute to increased risk of bladder cancer among Asian populations.     

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.     

Chemopreventive Properties of Genipin on AGS Cell Line via Induction of JNK/Nrf2/ARE Signaling Pathway

Roles of dietary phytochemicals in cancer chemoprevention via induction of nuclear factor‐erythroid‐2‐related factor 2 (Nrf2)‐mediated antioxidant enzymes have been well established in a number of studies. In this study, FACS analysis was used to reveal that the intracellular reactive oxygen species level decreased at 0–25 μM of genipin treatment. Furthermore, immunofluorescence analysis and Western blotting were used to demonstrate that genipin treatment resulted in the upregulation and nuclear translocation of Nrf2, as well as upregulation of gastrointestinal glutathione peroxidase. Finally, we found that C‐Jun‐NH2‐kinase (JNK) was also dose‐dependently activated, where depleting JNK by using a biochemical inhibitor indicated that JNK was upstream of Nrf2. Interestingly, the antioxidant effects were limited to the treatment in the lower dosage of genipin, where higher dosage of genipin treatment resulted in the increased reactive oxygen species level and cytotoxicity. Thus, this study demonstrates for the first time that lower dosage of genipin results in the induction of JNK/Nrf2/ARE signaling pathway and protection from cell death.