NQO1 Activity Research Articles

Abstract 1613: β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs)

Abstract Atypical teratoid rhabdoid tumors (ATRTs) are aggressive malignant neoplasms that occur in the CNS, most commonly in children <3 years of age. Despite intensive chemo-radiotherapy and surgery, the outcomes for patients with this disease remain dismal, with ∼75% of patients dying from their disease at a median of 10 months after diagnosis. Thus, it is critical to identify and validate novel ATRT targets for new therapies. We found that the pathognomonic genetic lesion of ATRTs, loss of the SWI/SNF chromatin remodeling complex member INI1/hSNF5/SMARCB1, leads to high expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a two-electron oxidoreductase. Thus, we hypothesized that ATRTs will be vulnerable to drugs, such as ß-lapachone (ß-lap, ARQ761 in clinical form), that selectively kill NQO1-expressing cells. A panel of ATRT cell lines exhibited overall high NQO1 expression and low micromolar sensitivity to ß-lap (IC50 1.6-4 uM), with the exception of cell lines that were homozygous for the C609T NQO1 (*2) polymorphism, which exhibited no NQO1 activity and were resistant to ß-lap treatment (IC50 >15 uM). A two-hour pulse with ß-lap caused a burst of ROS-mediated DNA damage and PARP1 hyperactivation, resulting in NAD+ and ATP depletion and caspase-independent, mu-calpain-mediated apoptosis. Co-treatment with dicoumarol, a specific inhibitor of NQO1, prevented these effects by blocking the NQO1-dependent bioactivation of ß-lap. Low expression of NQO1 in normal brain and other pediatric tissues suggests a wide therapeutic window for ß-lap. These findings encourage the continued study of ß-lap and other NQO1 bioactivated therapeutics for the treatment of this recalcitrant disease. This work was supported by NIH/NCI R01 CA102972 to DAB and a REACH award from the Alex's Lemonade Stand Foundation to JFA and TWL. Citation Format: Zachary R. Moore, Sarai Stuart, Agnieszka Cholka, Ashwin Venkataraman, Jingying Xu, Anat Erdreich-Epstein, Dinesh Rakheja, David A. Boothman, James F. Amatruda, Theodore W. Laetsch. β-lapachone: a novel targeted therapy for atypical teratoid rhabdoid tumors (ATRTs). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1613. doi:10.1158/1538-7445.AM2015-1613

Abstract 550: Evaluation of NQO1 as a potential diagnostic marker for bladder cancer

Abstract The present method for detection of bladder cancer involves ultrasonography followed by cystoscopy. This process is time intensive, requires skilled personnel and is unpleasant for the patient as a result of its invasive nature. This results in patient drop-out and patients that have recovered from bladder cancer are lost to follow-up due to the necessity for hospital visits for monitoring. We have used a novel test in order to monitor NAD(P)H quinone oxidoreductase 1 (NQO1) activity in cells recovered from routine urine samples since expression of NQO1 has previously been reported to be increased in several tumour types including transitional cell carcinoma of the bladder. Informed consent and urine samples were obtained from 73 bladder cancer patients and 42 age-matched bladder cancer-negative outpatients at the University Hospital of Wales, Cardiff. Live exfoliated cells were collected from the urine and assessed for their ability to metabolise an NQO1-specific substrate into a highly fluorescent product. Fluorescence intensity was then measured in a simple, inexpensive instrument. Results were checked against clinical correlates, bladder cancer stage, age, medication, and smoking status. Initial tests in cell lines showed excellent correlation with NQO1 levels detected by other methods. We found that in patient samples, while cell numbers in urine and expression of NQO1 per cell were not in themselves significant, NQO1 per ml of urine was highly significantly increased in bladder cancer patients (p = 0.009). Further, NQO1 was higher in patients that had high grade tumours as described in the WHO 2004 guidelines (p = 0.003). The sensitive fluorescence-based method to detect NQO1 activity in exfoliated urothelial cells shows exceptional promise in the development of rapid, non-invasive bladder cancer diagnostics. The method will be used on larger numbers of samples in order to be able to verify these initial findings. Note: This abstract was not presented at the meeting. Citation Format: Simone Cuff, Ruth Lewis, Mohammed Jaffar, Richard Knox, Ian Weeks. Evaluation of NQO1 as a potential diagnostic marker for bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 550. doi:10.1158/1538-7445.AM2015-550

Modulation of aryl hydrocarbon receptor-regulated enzymes by trimethylarsine oxide in C57BL/6 mice: In vivo and in vitro studies

Arsenic is a worldwide environmental pollutant that is associated with skin and several types of internal cancers. Recent reports revealed that arsenic biomethylation could activate the toxic and carcinogenic potential of arsenic. Therefore, we investigated the effect of trimethylarsine oxide (TMAO) on the activation of AhR-regulated genes in vivo and in vitro. In vivo, C57BL/6 mice received TMAO (13mg/kg i.p.) with or without the prototypical AhR ligand, TCDD (15μg/kg), then the livers were harvested at 6 and 24h post-treatment. In vitro, isolated hepatocytes from C57BL/6 mice were treated with TMAO (5μM) in the absence and presence of TCDD (1nM) for 6 and 24h. Our in vivo results demonstrated that, TMAO alone increased Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Gsta1, and Ho-1 at mRNA level. Upon co-exposure to TMAO and TCDD, TMAO potentiated the TCDD-mediated induction of Cyp1a1, Cyp1b1, and Nqo1 mRNA levels. Western blotting revealed that, TMAO alone increased Cyp1a1, Cyp1a2, Nqo1, Gsta1/2, and Ho-1 protein levels, and potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 protein level. In addition, TMAO alone significantly increased Cyp1a1, Cyp1a2, Nqo1, Gst, and Ho-1 activities and significantly potentiated the TCDD-mediated induction of Cyp1a1 activity. At the in vitro level, TMAO induced Cyp1a1 and potentiated the TCDD-mediated induction of Cyp1a1 at mRNA, protein and activity levels. In addition, TMAO increased the nuclear localization of AhR and AhR-dependent XRE-driven luciferase activity. Our results demonstrate that the TMAO, modulates AhR-regulated genes which could potentially participate, at least in part, in arsenic induced toxicity and carcinogenicity.

Antioxidant activity of Moringa oleifera isothiocyanates

Moringa oleifera Lam. (Brassicales) is an edible tropical tree with medicinal properties. Here, we describe a fractionation process of moringa leaves by fast centrifugal partition chromatography (FCPC) to generate polyphenol and isothiocyanate (ITC) rich fractions (Fig. 1). The polyphenol-rich fraction showed direct antioxidant activity assayed by oxygen radical absorbance capacity (ORAC), while the ITC-rich fraction demonstrated indirect antioxidant properties assayed by NQO1 activity in Hepa1c1c7 cells. Also, purified 4-[(α-l-rhamnosyloxy) benzyl] isothiocyanate (ITC-1), and 4-[(4'-O-acetyl-α-l-rhamnosyloxy)benzyl] isothio-cyanate (ITC-4) were evaluated for their ORAC and NQO1 inducer potency in comparison with sulforaphane (SF) at 5µM (Fig. 2). Both ITCs were as potent as SF in inducing NQO1 activity. These findings suggest that edible M. oleifera leaves contain a potent mixture of antioxidants that could explain its health promoting effects.

Modulation of aryl hydrocarbon receptor regulated genes by acute administration of trimethylarsine oxide in the lung, kidney and heart of C57BL/6 mice

1. Arsenite alters the expression of aryl hydrocarbon receptor (AhR)-regulated genes in extrahepatic tissues; yet, the effect of organic arsenicals still unknown. Therefore, C57BL/6 mice received trimethylarsine oxide (TMAO; 13 mg/kg i.p.) with or without 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 15 μg/kg), and euthanized at 6 or 24 h.2. Our results demonstrated that TMAO increased Cyp1a1 and Cyp1b1 mRNA, protein and activity in the lung. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1a2 mRNA, protein and activity in the lung. In the kidney, TMAO increased Cyp1b1 mRNA and protein. TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA, protein and activity. In the heart, TMAO potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 mRNA.3. Moreover, TMAO induced Nqo1 mRNA in the lung, kidney and heart, with subsequent increase in Nqo1 protein and activity in the lung. TMAO increased Gsta mRNA in the heart; and increased Gsta protein and activity in the lung and kidney. TMAO increased Nqo1 mRNA as compared to TCDD in the kidney and heart, and potentiated the TCDD-mediated induction of Gsta protein and activity in the kidney.4. In conclusion, TMAO modulates AhR-regulated genes in a tissue- and enzyme-specific manner.

The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance

The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance

An electrochemical method to assay the activity of NAD(P)H: Quinone oxidoreductase 1

A simple and rapid electrochemical method is reported in this paper to assay the activity of NAD(P)H: quinone oxidoreductase 1 (NQO1), a potential target for anticancer therapy. Firstly, a quinone derivative, the substrate of NQO1, is designed to be covalently immobilized on the surface of gold electrode. In the presence of the enzyme, the quinone derivative transforms to the corresponding hydroquinone derivative, followed by the expected cyclizative cleavage reaction via the gem-dialkyl effect. So, the hydroquinone derivative species are released from the electrode surface into the bulk solution. As the electrochemical probe, quinone derivative can generate a pair of redox waves in aqueous buffer and its peak current is correlated with the activity of NQO1, so an electrochemical method to determine the enzyme activity can be proposed. Under the optimized conditions, NQO1 activity can be assayed in the range from 0 to 6U/mL with a detection limit of 0.22U/mL. Moreover, resveratrol, an antitumor compound, is evaluated with the maximum inhibition rate of 95% and IC50 value of 23.70μM. Therefore, with wide detection range, high sensitivity, acceptable reliability and rapid response, the established method can be used for not only the investigation of NQO1 activity but also the screening of the enzyme inhibitor.

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.

Drug-metabolizing and antioxidant enzymes in monosodium L-glutamate obese mice.

The prevalence of obesity is rapidly increasing across the world. Physiologic alterations associated with obesity are known to alter enzyme expression and/or activities. As drug-metabolizing and antioxidant enzymes serve as defense system against potentially toxic compounds, their modulation might have serious consequences. In this work, we studied selected antioxidant and drug-metabolizing enzymes (DME) in monosodium glutamate-mouse model of obesity. Specific activities, protein, and mRNA expressions of these enzymes in liver as well as in small intestine were compared in obese male mice and in their lean counterparts. Furthermore, expression of the NF-E2-related factor 2 (Nrf2) and its relation to obesity were tested. Obtained results showed that obesity affects expression and/or activities of some DME and antioxidant enzymes. In obese mice, upregulation of UDP-glucuronosyltransferases 1A (UGT1A), NAD(P)H:quinone oxidoreductase 1 (NQO1), nuclear transcription factor Nrf2, and downregulation of some isoforms of glutathione S-transferases (GST) were observed. Most of these changes were tissue and/or isoform specific. NQO1 seems to be regulated transcriptionally via Nrf2, but other enzymes might be regulated post-transcriptionally and/or post-translationally. Enhanced expression of Nrf2 in livers of obese mice is expected to play a role in protective adaptation. In contrast, elevated activities of NQO1 and UGT1A may cause alterations in drug pharmacokinetics in obese individuals. Moreover, decreased capacity of GST in obese animals indicates potentially reduced antioxidant defense and weaker chemoprotection.

Role of Nrf2 dysfunction in uremia-associated intestinal inflammation and epithelial barrier disruption.

Gut inflammation is prevalent in chronic kidney disease (CKD) and likely contributes to systemic inflammation via disruption of the epithelial tight junction with subsequent endotoxin and bacterial translocation. To study the expression profile of inflammatory and tight junction proteins in the colon from CKD rats compared to healthy controls, and demonstrate the role of Nrf2 (transcription factor nuclear factor erythroid 2-related factor 2) using a potent Nrf2 activator. CKD was induced via 5/6 nephrectomy in Sprague-Dawley rats, and dh404 (2 mg/kg/day) was used to study the effects of systemic Nrf2 activation. The experimental groups included sham, CKD and CKD+ dh404 rats. Blood and colon tissues were analyzed after a 10-week study period. Colon from CKD rats showed histological evidence of colitis, depletion of epithelial tight junction proteins, significant reduction of Nrf2 and its measured target gene products (NQO1, catalase, and CuZn SOD), activation of NFkB, and upregulation of pro-inflammatory molecules (COX-2, MCP-1, iNOS, and gp91(phox)). Treatment with dh404 attenuated colonic inflammation, restored Nrf2 activity and levels of NQO1, catalase and CuZn SOD, decreased NFkB and lowered expression of COX-2, MCP-1, iNOS, and gp91(phox). This was associated with restoration of colonic epithelial tight junction proteins (occludin and claudin-1). CKD rats exhibited colitis, disruption of colonic epithelial tight junction, activation of inflammatory mediators, and impairment of Nrf2 pathway. Treatment with an Nrf2 activator restored Nrf2 activity, attenuated colonic inflammation, and restored epithelial tight junction proteins.

Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ~80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells.

Comparative Study of 17-AAG and NVP-AUY922 in Pancreatic and Colorectal Cancer Cells: Are There Common Determinants of Sensitivity?

The use of heat shock protein 90 (Hsp90) inhibitors is an attractive antineoplastic therapy. We wanted to compare the effects of the benzoquinone 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and the novel isoxazole resorcinol–based Hsp90 inhibitor NVP-AUY922 in a panel of pancreatic and colorectal carcinoma cell lines and in colorectal primary cultures derived from tumors excised to patients. PANC-1, CFPAC-1, and Caco-2 cells were intrinsically resistant to 17-AAG but sensitive to NVP-AUY922. Other cellular models were sensitive to both inhibitors. Human epidermal growth factor receptor receptors and their downstream signaling pathways were downregulated in susceptible cellular models, and concurrently, Hsp70 was induced. Intrinsic resistance to 17-AAG did not correlate with expression of ATP-binding cassette transporters involved in multidrug resistance. Some 17-AAG-resistant, NVP-AUY922–sensitive cell lines lacked NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme and activity. However, colorectal LoVo cells still responded to both drugs in spite of having undetectable levels and activity of NQO1. Pharmacological and biologic inhibition of NQO1 did not confer resistance to 17-AAG in sensitive cell lines. Therefore, even though 17-AAG sensitivity is related to NQO1 protein levels and enzymatic activity, the absence of NQO1 does not necessarily convey resistance to 17-AAG in these cellular models. Moreover, NVP-AUY922 does not require NQO1 for its action and is a more potent inhibitor than 17-AAG in these cells. More importantly, we show in this report that NVP-AUY922 potentiates the inhibitory effects of chemotherapeutic agents, such as gemcitabine or oxaliplatin, and other drugs that are currently being evaluated in clinical trials as antitumor agents.

Induction of Anticarcinogenic Enzymes by Dichloromethane-soluble Fraction of Physalis alkekengi var. francheti Hort. in Mouse Hepatoma Cells

Physalis alkekengi var. francheti Hort. is known as an insecticide and traditional remedy for liver related diseases. Therefore, this study investigated the chemopreventive effects of extracts and several solvent fractions (n-hexane, dichloromethane, n-butanol, water) of Physalis alkekengi var. francheti Hort. First, their cytotoxicity and NQO1 activity were measured using an MTT assay, plus a quinone reductase [NAD(P)H dehydrogenase (quinone); NAD(P)H: (quinone acceptor) oxidoreductase, EC 1.6.99.2]-inducing activity assay was performed using cultured murine hepatoma cells (Hepa1c1c7) and its mutant cells(BpRc1). The reduction of electrophilic quinones by NQO1 is an important detoxification pathway and major mechanism of chemoprevention. When compared with the other solvent soluble fractions with different polarities, the dichloromethane fraction of Physalis alkekengi var. francheti Hort. showed a higher NQO1-inducing activity that was also dose-dependent. Moreover, the dichloromethane fraction of Physalis alkekengi var. francheti Hort. induced ARE-luciferase activities in HepG2-C8 cells that were generated by transfecting the ARE-luciferase gene construct, suggesting the Nrf2-ARE-mediated induction of anti-oxidative enzymes. In conclusion, the dichloromethane-soluble fraction of Physalis alkekengi var. francheti Hort. showed a relatively strong induction of detoxifying enzymes, thereby meriting further study to identify the active components and evaluate their potential as cancer preventive agents.

Correlation between CYP1A1 transcript, protein level, enzyme activity and DNA adduct formation in normal human mammary epithelial cell strains exposed to benzo[a]pyrene.

quinone oxidoreductase 1 (NQO1), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58-836 for CYP1A1, 336-5587 for CYP1B1 and 5943-40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251-13234 for CYP1A1, 4133-57078 for CYP1B1 and 4456-55887 for NQO1. There were 3.5 (mean, range 0.2-15.8) BPdG adducts/10(8) nucleotides in the NHMECs (n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG (P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO1 protein, or NQO1 enzyme activity.

Protective effect of Artemisia annua L. extract against galactose-induced oxidative stress in mice.

Artemisia annua L. (also called qinghao) has been well known as a source of antimalarial drug artemisinins. In addition, the herb was reported to have in vitro antioxidative activity. The present study investigated the protective effect of aqueous ethanol extract of Qinghao (AA extract) against D-galactose-induced oxidative stress in C57BL/6J mice. Feeding AA extract-containing diet lowered serum levels of malondialdehyde and 8-OH-dG that are biomarkers for lipid peroxidation and DNA damage, respectively. Furthermore, AA extract feeding enhanced the activity of NQO1, a typical antioxidant marker enzyme, in tissues such as kidney, stomach, small intestine, and large intestine. In conclusion, AA extract was found to have antioxidative activity in mouse model.

Hydrogen peroxide responsive miR153 targets Nrf2/ARE cytoprotection in paraquat induced dopaminergic neurotoxicity

Epidemiological and animal studies suggest that environmental toxins including paraquat (PQ) increase the risk of developing Parkinson's disease (PD) by damaging nigrostriatal dopaminergic neurons. We previously showed that overexpression of a group of microRNAs (miRs) affects the antioxidant promoting factor, Nrf2 and related glutathione-redox homeostasis in SH-SY5Y dopaminergic neurons. Although, dysregulation of redox balance by PQ is well documented, the role for miRs and their impact have not been elucidated. In the current study we investigated whether PQ impairs Nrf2 and its related cytoprotective machinery by misexpression of specific fine tune miRs in SH-SY5Y neurons. Real time PCR analysis revealed that PQ significantly (p<0.05) increased the expression of brain enriched miR153 with an associated decrease in Nrf2 and its function as revealed by decrease in 4× ARE activity and expression of GCLC and NQO1. Also, PQ and H2O2-induced decrease in Nrf2 3′ UTR activity was restored on miR153 site mutation suggesting a 3′ UTR interacting role. Overexpression of either anti-miR153 or Nrf2 cDNA devoid of 3′ UTR prevented PQ and H2O2-induced loss in Nrf2 activity confirming that PQ could cause miR153 to bind to and target Nrf2 3′ UTR thereby weakening the cellular antioxidant defense. Adenovirus mediated overexpression of cytoplasmic catalase (Ad cCAT) confirmed that PQ induced miR153 is hydrogen peroxide (H2O2) dependent. In addition, Ad cCAT significantly (p<0.05) negated the PQ induced dysregulation of Nrf2 and function along with minimizing ROS, caspase 3/7 activation and neuronal death. Altogether, these results suggest a critical role for oxidant mediated miR153-Nrf2/ARE pathway interaction in paraquat neurotoxicity. This novel finding facilitates the understanding of molecular mechanisms and to develop appropriate management alternatives to counteract PQ-induced neuronal pathogenesis.

Curcumin alleviated liver oxidative stress injury of rat induced by paraquat

To investigate the effect of curcumin on liver injury in rats induced by paraquat-mediated oxidative stress and the mechanism underlying its effect. Sixty rats were randomly divided into 4 groups: control group, curcumin control group (curcumin 50 mg/kg), paraquat group (2% paraquat solution 100 mg/kg), and curcumin intervention group (curcumin 50 mg/kg at 15 min, 24 h, or 48 h after paraquat exposure). On days 1, 3, or 7 after paraquat administration, and liver tissue was collected thereafter. The content of malonaldehyde (MDA) and the activities of superoxide dismutase activity (SOD) and catalase (CAT) in the liver tissue were determined by chemical colorimetry. The activities of heme oxygenase 1 (HO-1) and quinone oxidoreductase 1 (NQO-1) in the liver tissue were determined by ELISA. The mRNA and protein levels of NF-E2-related factor 2 (Nrf2) were determined by RT-PCR and Western blot, respectively. The pathological changes of liver tissue were examined by optical microscopy. No significant change was observed between the control group and the curcumin control group in any examination of this study (P > 0.05). Both paraquat group and curcumin intervention group showed increase in MDA content, decreases in SOD and CAT activities, increases in HO-1 and NQO-1 activities, and increases in the protein and mRNA levels of Nrf2, in comparison with the control group (P < 0.05 for all except HO-1 activity in paraquat group on day 7). In comparison with the parquet group on the same day, the curcumin intervention group showed decrease in MDA content, increases in the activities of SOD, CAT, HO-1, and NQO-1, and increases in the mRNA and protein levels of Nrf2 on days 1, 3, and 7 (P < 0.05). The pathological examination revealed that the damage of liver tissue in the paraquat group was the most serious on the 3rd day after paraquat exposure, and the damage was consistently alleviated by curcumin intervention on days 1, 3, and 7, as compared with the paraquat group. Oxidative stress plays an important role in paraquat-induced acute liver damage in rats, and curcumin can exert a hepatoprotective effect against oxidative stress by increasing the expression of Nrf2 and the activities of HO-1, NQO-1, SOD, and CAT and reducing the content of MDA.

C9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway

Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis.

The effect of aristolochic acid I on expression of NAD(P)H:quinone oxidoreductase in mice and rats—A comparative study

Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H:quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined.We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver.NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Synergy of bioactives from camelina seed meal for health promotion (647.16)

Camelina sativa L. Crantz is under development as a novel oil seed crop yet there are few studies about the bioefficacy of camelina. Camelina seed meal contains several potentially bioactive compounds: two major aliphatic GSLs, glucoarabin (9‐(methylsulfinyl)nonylglucosinolate; GSL9), glucocamelinin (10‐(methylsulfinyl)decylglucosinolate; GSL10), with trace amount of a third, 11(methylsulfinyl)undecylglucosinolate (GSL11) and several flavonoids, mostly as quercetin glycosides. We tested hydrolyzed glucosinolates (hGSL) and flavonoids quercetin and rutin for their ability to upregulate detoxification enzymes, as biomarkers of anti‐cancer activity. None of these compounds either caused induction of CYP1A1 activity, or inhibition of β‐naphthoflavone‐induced CYP1A1 activity. In contrast, hGSL9 and hGSL10, quercetin and rutin alone and in combination all caused induction of quinone reductase (NQO1) activity. We found synergistic induction of NQO1 activity when Hepa1c1c7 cells were treated with a combination of all the four major bioactives in the proportions present in camelina seed extract. Although potency of hGSL9 was somewhat less than that of sulforaphane, we found that GSL9 and quercetin appear responsible for this synergy. As Camelina seed extract possesses NQO1 inducing activity, it should be evaluated for anticancer activity, similar to other Brassicaceae family members, such as Broccoli.Grant Funding Source: Supported by USDA/NIFA #AG 2010‐65200