Two new isoquinoline alkaloids from Cryptocarya wrayi and their biological activities.

Two new isoquinoline alkaloids, cryptowrayines A (1) and B (2), along with one known pavine alkaloid (-)-12-hydroxyeschscholtzidine (3), were isolated from the twigs of Cryptocarya wrayi. The structures of new compounds were elucidated by extensive spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Both compounds 1 and 2 exhibited moderate quinone reductase inducing activity in Hepa 1c1c7 cells.

Protective effects of hyperbaric oxygen preconditioning against LPS-induced acute lung injury in rats.

INTRODUCTION: Acute lung injury (ALI) is generally caused by oxidative damages and pulmonary overinflammations. Hyperbaric oxygen preconditioning (HBO2-PC) has been proven protective against oxidative-stress-related injuries. In this study, we investigated the effect of HBO2-PC on lipopolysaccharide (LPS)-induced ALI in rats. METHODS: Thirty-two Sprague-Dawley rats randomly assigned into Sham, HBO2-PC, ALI and HBO2-PC÷ALI groups (eight in each group) were sacrificed at 12 hours after the injection of LPS. The severity of ALI in rats was assessed in terms of histopathological changes in addition to wet/dry weight ratios. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß in serum and lung homogenates were measured by enzyme-linked immunosorbent and qRT-PCR assays. Activities by hydrogen peroxide (H2O2), malondialdehyde (MDA), myeloperoxidase (MPO) as well as superoxide dismutase (SOD) in rat lungs were tested for neutrophil infiltration. Meanwhile the oxidative stress molecular markers nuclear factor-kappa B(NF-κB) p65 and nuclear factor erythroid 2-related factor 2 (Nrf2), together with its downstream heme-oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) were also quantified. RESULTS: HBO2-PC significantly alleviated LPS-induced ALI, lowered the lung injury scores, reduced lung water content, and reduced H2O2, MDA levels as well as MPO activity, while simultaneously improving the arterial partial oxygen pressure (PaO2) and SOD activity. Furthermore, HBO2-PC inhibited the nuclear translocation of NF-κB p65 while enhancing the nuclear translocation of Nrf2, thus upregulating HO-1 and NQO1. CONCLUSIONS: Our results suggest that HBO2-PC was potentially protective for LPS-induced ALI lungs in rats, with a presumed mechanism that suppressed NF-κB while activating Nrf2. We propose that HBO2-PC should be considered a potential therapeutic strategy against ALI in rats.

DNA damage signalling barrier, oxidative stress and treatment-relevant DNA repair factor alterations during progression of human prostate cancer.

The DNA damage checkpoints provide an anti-cancer barrier in diverse tumour types, however this concept has remained unexplored in prostate cancer (CaP). Furthermore, targeting DNA repair defects by PARP1 inhibitors (PARPi) as a cancer treatment strategy is emerging yet requires suitable predictive biomarkers. To address these issues, we performed immunohistochemical analysis of multiple markers of DNA damage signalling, oxidative stress, DNA repair and cell cycle control pathways during progression of human prostate disease from benign hyperplasia, through intraepithelial neoplasia to CaP, complemented by genetic analyses of TMPRSS2-ERG rearrangement and NQO1, an anti-oxidant factor and p53 protector. The DNA damage checkpoint barrier (γH2AX, pATM, p53) mechanism was activated during CaP tumorigenesis, albeit less and with delayed culmination compared to other cancers, possibly reflecting lower replication stress (slow proliferation despite cases of Rb loss and cyclin D1 overexpression) and progressive loss of ATM activator NKX3.1. Oxidative stress (8-oxoguanine lesions) and NQO1 increased during disease progression. NQO1 genotypes of 390 men did not indicate predisposition to CaP, yet loss of NQO1 in CaP suggested potential progression-opposing tumour suppressor role. TMPRSS2-ERG rearrangement and PTEN loss, events sensitizing to PARPi, occurred frequently along with heterogeneous loss of DNA repair factors 53BP1, JMJD1C and Rev7 (all studied here for the first time in CaP) whose defects may cause resistance to PARPi. Overall, our results reveal an unorthodox DNA damage checkpoint barrier scenario in CaP tumorigenesis, and provide novel insights into oxidative stress and DNA repair, with implications for biomarker guidance of future targeted therapy of CaP.

Direct and indirect antioxidant activity of polyphenol- and isothiocyanate-enriched fractions from Moringa oleifera.

Moringa oleifera Lam. is a fast-growing, tropical tree with various edible parts used as nutritious food and traditional medicine. This study describes an efficient preparatory strategy to extract and fractionate moringa leaves by fast centrifugal partition chromatography (FCPC) to produce polyphenol and isothiocyanate (ITC) rich fractions. Characterization and further purification of these fractions showed that moringa polyphenols were potent direct antioxidants assayed by oxygen radical absorbance capacity (ORAC), whereas moringa ITCs were effective indirect antioxidants assayed by induction of NAD(P)H quinone oxidoreductase 1 (NQO1) activity in Hepa1c1c7 cells. In addition, purified 4-[(α-l-rhamnosyloxy)benzyl]isothiocyanate and 4-[(4'-O-acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate were further evaluated for their ORAC and NQO1 inducer potency in comparison with sulforaphane (SF). Both ITCs were as potent as SF in inducing NQO1 activity. These findings suggest that moringa leaves contain a potent mixture of direct and indirect antioxidants that can explain its various health-promoting effects.

Butterbur (Petasites japonicus Max.) extract improves lipid profiles and antioxidant activities in monosodium L-glutamate-challenged mice.

We investigated the effect of the butanol fraction from the methanol extract of butterbur (Petasites japonicus Max.) (BMP) on the plasma lipid profiles and oxidative damage of liver in mice challenged with monosodium l-glutamate (MSG). ICR mice (6-8 weeks old, male) were fed BMP (0.1% or 0.3%) for 1 week, and on day 7, MSG (4 mg/g) was administered intraperitoneally to the mice. Administration of MSG resulted in a significant decrease of antioxidant biomarkers such as total glutathione level and antioxidant enzyme activities such as glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferase (GST), and quinone reductase (NQO1), whereas the thiobarbituric acid-reactive substances (TBARS) value was increased in liver tissue. However, BMP supplementation markedly enhanced the hepatic antioxidant enzyme activities, including GPx, GR, GST, and NQO1, whereas it lowered TBARS, compared to the MSG-treated group. Moreover, BMP supplementation decreased total cholesterol, atherogenic index, and low-density lipoprotein-cholesterol, compared to the MSG-treated group. Based on these results, it was proposed that BMP improve the plasma lipid profiles and decrease the oxidative stress by up-regulating the hepatic antioxidant enzymes in mice challenged with MSG.

Candidate dietary phytochemicals modulate expression of phase II enzymes GSTP1 and NQO1 in human lung cells.

Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. We applied human lung cells in vitro to investigate the effects of several candidate phytopreventive agents, including green tea extracts (GTE), broccoli sprout extracts (BSE), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC), on inducing phase II enzymes glutathione S-transferase P1 (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) at mRNA and protein levels. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and lung adenocarcinoma cells (A549) were exposed to diet-achievable levels of GTE and BSE (0.5, 1.0, 2.0 mg/L), or individual index components EGCG, SFN, PEITC, BITC (0.5, 1.0, 2.0 micromol/L) for 24 h, 48 h, and 6 d, respectively. mRNA assays employed RNA-specific quantitative RT-PCR and protein assays employed Western blotting. We found that in NHBE cells, while GSTP1 mRNA levels were slightly but significantly increased after exposure to GTE or BSE, NQO1 mRNA increased to 2- to 4-fold that of control when exposed to GTE, BSE, or SFN. Effects on NQO1 mRNA expression in HBEC cells were similar. NQO1 protein expression increased up to 11.8-fold in SFN-treated NHBE cells. Both GSTP1 and NQO1 protein expression in A549 cells were constitutively high but not induced under any condition. Our results suggest that NQO1 is more responsive to the studied chemopreventive agents than GSTP1 in human lung cells and there is discordance between single agent and complex mixture effects. We conclude that modulation of lung cell phase II metabolism by chemopreventive agents requires cell- and agent-specific discovery and testing.

The chemopreventive effect of taxifolin is exerted through ARE-dependent gene regulation.

Phase II detoxification enzymes are responsible for the detoxification and elimination of activated carcinogens, and thus act as important biomarkers for chemoprevention. In this study, we tested the chemopreventive activity of taxifolin, a flavanon compound purified from a mongolian medicinal plant, by measuring quinone reductase (QR) activity in HCT 116 cells. Taxifolin induced significant QR activity, but displayed relatively low cytotoxicity in cells (chemoprevention index=5.75). To identify the target genes regulated by taxifolin, DNA microarray was performed with a 3K human cancer chip containing 3096 human genes associated with carcinogenesis. Significant analysis of microarray (SAM) revealed 428 differentially expressed (DE) genes as statistically significant, with a false discovery rate (FDR) of 57.2% (delta=0.3366). Sixty-five genes, including a few detoxification enzymes (NQO1, GSTM1) and an antioxidant enzyme (TXNRD1), were up-regulated and 363 genes were down-regulated in the presence of 60 microM taxifolin. In view of the finding that selected genes of interest contained antioxidant response element (ARE), we hypothesize that taxifolin modulates chemopreventive genes through activation of the ARE. Transient transfection experiments using the ARE QR-CAT construct demonstrate that taxifolin significantly activates ARE, but not xenobiotic response element (XRE). In conclusion, taxifolin acts as a potential chemopreventive agent by regulating genes via an ARE-dependent mechanism.

Polyphenolic compounds, antioxidant capacity, and quinone reductase activity of an aqueous extract of Ardisia compressa in comparison to mate (Ilex paraguariensis) and green (Camellia sinensis) teas.

Aqueous extracts of the leaves of Ardisia compressa (AC) have been used in folk medicine to treat various liver disorders including liver cancer. The objective of this study was to partially characterize and determine the total polyphenol content, antioxidant capacity, and quinone reductase activity of A. compressa tea in comparison to mate (Ilex paraguariensis, MT) and green (Camellia sinensis,GT) teas. Total polyphenol content, antioxidant capacity, and phase II enzyme induction capacity were measured by the modified Folin-Ciocalteu, ORAC, and quinone reductase (QR) assays, respectively. The major polyphenols in AC were not catechins. HPLC retention times and standard spikes of AC indicated the presence of gallic acid, epicatechin gallate, ardisin and kaempferol. Using catechin as standard, the total polyphenol value of AC (36.8 +/- 1.1 mg/mg DL) was significantly lower than GT (137.2 +/- 5.8 mg equivalent of (+)-catechin/mg dried leaves, DL) and MT (82.1 +/- 3.8 mg/mg DL) (P < 0.001). Antioxidant capacity (AC, 333; GT, 1346; MT, 1239 mmol Trolox equivalents/g DL) correlated with total polyphenol values (r(2) = 0.86, P < 0.01). AC (4.5-12.5 microg/mL) induced QR enzyme, in Hepa1c1c7 cells, up to 15%. MT and GT showed no induction at the concentrations tested (0.5-10.5 and 0.5-12.5 mg/mL, respectively). These results suggest that AC has a different mechanism of protection against cytotoxicity that is not related to its antioxidant capacity. Further studies are needed to determine such mechanisms and to explore its potential as a chemopreventive or therapeutic agent.

Vitamin E and selenium deficiency induces expression of the ubiquinone-dependent antioxidant system at the plasma membrane.

We have used a model of dietary deficiency that leads to a chronic oxidative stress to evaluate responses that are adaptations invoked to boost cellular defense systems. Long-Evans hooded rats were fed with a diet lacking vitamin E (E) and selenium (Se) for 7 wk from weaning leading to animals deficient in both nutrients (-E -Se). In the absence of an electron donor, liver plasma membranes from these rats were more sensitive to lipid peroxidation, although they contained 40% greater amounts of ubiquinone than the plasma membranes from rats consuming diets with sufficient vitamin E and Se (+E +Se). The incubation of plasma membranes with NAD(P)H resulted in protection against peroxidation, and this effect was more pronounced in -E -Se membranes. Deficiency was accompanied by a twofold increase in redox activities associated with trans plasma membrane electron transport such as ubiquinone reductase and ascorbate free radical reductase. Staining with a polyclonal antibody against pig liver cytochrome b5 reductase, which acts as one ubiquinone reductase in the plasma membrane, showed an increased expression of the enzyme in membranes from -E -Se rats. Little DT-diaphorase activity was measured in +E +Se plasma membranes, but this activity was dramatically increased in -E -Se plasma membranes. No such increase was found in liver cytosols, which contained elevated activity of calcium-independent phospholipase A2. Thus, ubiquinone-dependent antioxidant protection in +E +Se plasma membranes is based primarily on NADH-cytochrome b5 reductase, whereas additional protection needed in -E -Se plasma membranes is supported by the increase of ubiquinone levels, increased expression of the cytochrome b5 reductase, and translocation of soluble DT-diaphorase to the plasma membrane. Our results indicate that, in the absence of vitamin E and Se, enhancement of ubiquinone-dependent reductase systems can fulfill the membrane antioxidant protection.

Immunological quantitation of DT-diaphorase in carcinoma cell lines and clinical colon cancers: advanced tumors express greater levels of DT-diaphorase.

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) plays a major role in activating mitomycin C (MMC) in human colon and gastric carcinoma cell lines. Thus, measurement of DTD in clinical tumor samples could be beneficial in designing adjuvant chemotherapy. We explored immunological quantitation of DTD protein using a monoclonal antibody against DTD, demonstrating a close correlation between protein expression and enzyme activity of DTD in colon and gastric carcinoma cell lines and in colorectal tumor samples. This indicates that such immunoblot analysis is a simple alternative method for quantitating DTD in clinically excised samples. In most colorectal tumor samples, the tumors expressed larger amounts of DTD than did the peripheral normal tissues, suggesting a selective toxicity of MMC toward tumor cells. Also tumors with nodal metastases showed significantly higher DTD levels than did tumors without metastasis. These results raise the possibility that DTD expression is related to tumorigenesis and malignant progression of colorectal tumors. Measurement of DTD by the immunological method described here could be beneficial in designing a rational adjuvant chemotherapy with MMC.

Morphology of neurons in the white matter of the adult human neocortex.

Neurons in the human cerebral cortical white matter below motor, visual, auditory and prefrontal orbital areas have been studied with the Golgi method, immunohistochemistry and diaphorase histochemistry. The majority of white matter neurons are pyramidal cells displaying the typical polarized, spiny dendritic system. The morphological variety includes stellate forms as well as bipolar pyramidal cells, and the expression of a certain morphological phenotype seems to depend on the position of the neuron. Spineless nonpyramidal neurons with multipolar to bitufted dendritic fields constitute less than 10% of the neurons stained for microtubule associated protein (MAP-2). Only 3% of the MAP-2 immunoreactive neurons display nicotine adenine dinucleotide-diaphorase activity. The white matter pyramidal neurons are arranged in radial rows continuous with the columns of layer VI neurons. Neuron density is highest below layer VI, and decreases with increasing distance from the gray matter. White matter neurons are especially abundant below the primary motor cortex, and are least frequent below the visual cortex area 17. In contrast to other mammalian species, the white matter neurons in man are not only present during development, but persist throughout life.