NADPH Quinone Oxidoreductase Research Articles

QS459. Heme Oxygenase-1 and NADPH Quinone Oxidoreductase-1 Expression is Enhanced by Flaxseed in a Mouse Model of Lung Ischemia Reperfusion Injury

QS459. Heme Oxygenase-1 and NADPH Quinone Oxidoreductase-1 Expression is Enhanced by Flaxseed in a Mouse Model of Lung Ischemia Reperfusion Injury

QS458. Reperfusion Injury Induces HEME Oxygenase-1 and NADPH Quinone Oxidoreductase-1 Enzymes in an in Vivo Model of Lung Ischemia Reperfusion Injury

QS458. Reperfusion Injury Induces HEME Oxygenase-1 and NADPH Quinone Oxidoreductase-1 Enzymes in an in Vivo Model of Lung Ischemia Reperfusion Injury

Oral sulforaphane increases Phase II antioxidant enzymes in the human upper airway

Background Cellular oxidative stress is an important factor in asthma and is thought to be the principle mechanism by which oxidant pollutants such as ozone and particulates mediate their pro-inflammatory effects. Endogenous Phase II enzymes abrogate oxidative stress through the scavenging of reactive oxygen species and metabolism of reactive chemicals. Objective We conducted a placebo-controlled dose escalation trial to investigate the in vivo effects of sulforaphane, a naturally occurring potent inducer of Phase II enzymes, on the expression of glutathione-s-transferase M1 (GSTM1), glutathione-s-transferase P1 (GSTP1), NADPH quinone oxidoreductase (NQO1), and hemoxygenase-1 (HO-1) in the upper airway of human subjects. Methods Study subjects consumed oral sulforaphane doses contained in a standardized broccoli sprout homogenate (BSH). RNA expression for selected Phase II enzymes was measured in nasal lavage cells by RT-PCR before and after sulforaphane dosing. Results All subjects tolerated oral sulforaphane dosing without significant adverse events. Increased Phase II enzyme expression in nasal lavage cells occurred in a dose-dependent manner with maximal enzyme induction observed at the highest dose of 200 g broccoli sprouts prepared as BSH. Significant increases were seen in all sentinel Phase II enzymes RNA expression compared to baseline. Phase II enzyme induction was not seen with ingestion of non-sulforaphane containing alfalfa sprouts. Conclusion Oral sulforaphane safely and effectively induces mucosal Phase II enzyme expression in the upper airway of human subjects. This study demonstrates the potential of antioxidant Phase II enzymes induction in the human airway as a strategy to reduce the inflammatory effects of oxidative stress. Clinical implications This study demonstrates the potential of enhancement of Phase II enzyme expression as a novel therapeutic strategy for oxidant induced airway disease. Capsule summary A placebo-controlled dose escalation trial demonstrated that naturally occurring sulforaphane from broccoli sprouts can induce a potent increase in antioxidant Phase II enzymes in airway cells.

Functional Polymorphisms in Oxidant Metabolism and DNA Repair Pathways and Risk of Leukemia and Transient Myeloproliferative Disorder in Children with Down Syndrome

Children with Down syndrome can have a wide range of developmental abnormalities related to the presence of additional genetic material from a small region of chromosome 21. Additional copies of the Cu/Zn superoxide dismutase gene and related genes at this locus are believed to contribute to high oxidative stress, and increased endogenous DNA damage and deficient repair observed in cells from patients with Down syndrome. Children with Down syndrome also have a markedly increased risk for the development of serious hematological disorders, including an approximately 20-fold increased risk for the development of acute myeloid leukemia (AML). In addition, in early infancy, as many as 10% may develop a transient myeloproliferative disorder (TMD). The basis for this overall increased risk is unknown, as are the risk factors influencing which specific patients with Down syndrome develop AML/TMD. We sought to test the hypothesis that functional polymorphisms in genes involved in oxidant metabolism and DNA repair contribute to the increased risk of AML and TMD in children with Down syndrome. We studied functional polymorphisms in the oxidant metabolizing enzyme NADPH quinone oxidoreductase 1 (NQO1), and in the DNA repair proteins X-ray repair cross complementing 1 protein (XRCC1) and Fanconi anemia complementation group A protein (FANCA). Genotyping was conducted in 170 patients with Down syndrome AML or TMD, and 202 healthy blood donor controls. All genotype frequencies in the control populations were consistent with those expected from Hardy-Weinberg equilibrium. We found that the variant NQO1 Pro187Ser allele, associated with reduced quinone reductase activity, occurred with a greater frequency in the Down syndrome AML/TMD patient population compared to healthy controls (OR, 1.61; 95% CI, 1.067–2.426; p=0.0231). The FANCA-1501 variant allele frequency was observed more frequently in Down syndrome AML/TMD patients compared to healthy controls (OR, 1.77; 95%CI, 1.220–2.568; p=0.0025). The variant XRCC-1 Arg399Gln allele was observed less frequently in the TMD/DS AML population as compared to controls (OR, 0.536; CI, 0.354–0.811; p=0.0031). These latter findings parallel the observation of a protective effect against AML in non-Down syndrome individuals with at least one copy of the XRCC-1 Arg399Gln allele (Seedhouse et al. Blood 2002, 100:3761). Conclusion: These preliminary studies, representing the only reported data in Down Syndrome TMD/AML patients, suggest that functional polymorphic variants of the NQO1, FANCA, and XRCC1 genes involved in oxidant metabolism and DNA repair may influence the risk for developing leukemia. Studies involving future cohorts will be needed to confirm these observations.

The Natural Compound Ascorbigen Modulates NADPH-Quinone Oxidoreductase (NQO1) mRNA and Enzyme Activity Levels in Cultured Liver Cells and in Laboratory Rats

Ascorbigen (ABG) is a natural compound that represents a breakdown product of the glucosinolates that are present in Brassica vegetables. It is postulated that ABG may have anticarcinogenic activity; however, the underlying molecular and cellular mechanisms are largely unknown. In the present study we investigated the effect of ABG on the mRNA and enzyme activity levels of NADPH-quinone oxidoreductase (NQO1), which is centrally involved in the detoxification of xenobiotics, in cultured liver cells and in rats. The mRNA levels of NQO1 showed an increase of up to 100% in cultured liver cells (HepG2) following incubation with different concentrations of ABG (3–100 µmol/l) compared to control cells. Furthermore, NQO1 activity was elevated (up to 20%) by ABG treatment. The in vitro results were confirmed in rats who received either 5 mg/day ABG or vehicle for 7 days. Significantly higher mRNA (a 90% increase) and enzyme activity levels (a 40% increase) of NQO1 were detected in the liver of ABG-treated rats as compared to control animals. Current data indicate that ABG is a moderate inducer of the phase II enzyme NQO1, both in cultured hepatocytes and in vivo.

Resveratrol protects primary rat hepatocytes against oxidative stress damage:: Activation of the Nrf2 transcription factor and augmented activities of antioxidant enzymes

Oxidative stress is recognized as an important factor in the development of liver pathologies. The reactive oxygen species endogenously generated or as a consequence of xenobiotic metabolism are eliminated by enzymatic and nonenzymatic cellular systems. Besides endogen defences, the antioxidant consumption in the diet has an important role in the protection against the development of diseases product of oxidative damage. Resveratrol is a naturally occurring compound which is part of the human diet. This molecule has been shown to have many biological properties, including antioxidant activity. We decided to test if resveratrol could protect primary hepatocytes in culture from oxidative stress damage and if so, to determine if this compound affects the cellular detoxifying systems and their regulation through the Nrf2 transcription factor that regulates the expression of antioxidant and phase II detoxifying enzymes. Cell death by necrosis was detected by measuring the activity of lactate dehydrogenase liberated to the medium. The activities of antioxidant and phase II enzymes were measured using previously described methods. Activation of the Nrf2 transcription factor was studied by confocal microscopy and the Nrf2 and its coding mRNA levels were determined by western blot and quantitative PCR respectively. Resveratrol pre-treatment effectively protected hepatocytes in culture exposed to oxidative stress, increasing the activities of catalase, superoxide dismutase, glutathione peroxidase, NADPH quinone oxidoreductase and glutathione- S-transferase. Resveratrol increases the level of Nrf2 and induces its translocation to the nucleus. Also, it increases the concentration of the coding mRNA for Nrf2. In this work we show that resveratrol could be a useful drug for the protection of liver cells from oxidative stress induced damage.

Arsenic Methylation Capability, Heme Oxygenase-1 and NADPH Quinone Oxidoreductase-1 Genetic Polymorphisms and the Stage and Grade of Urothelial Carcinomas

Background: Arsenic exposure is associated with an increased risk of urothelial carcinoma (UC). To explore the distribution of the arsenic methylation capability in patients with different stages and grades of UCs, 100 UC cases were recruited between September 2002 and May 2004 for this study. Methods: Urinary arsenic species, including inorganic arsenic (As^III + As^V), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA), were determined with a high-performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. Determining the percentages of various arsenic species among the total urinary arsenic amount assessed the arsenic methylation capability. The primary methylation index (PMI) was defined as the ratio between MMA and inorganic arsenic. The secondary methylation index (SMI) was determined as the ratio between DMA and MMA. Results: Differential effects of the arsenic methylation capability were found among patients with different stages of UCs; however, none was found among different grades. Conclusion: A significantly different distribution of the HO-1 genotype was found in subjects with different-stage UCs; however, it was not related to the NAD(P)H:quinone oxidoreductase 1 genotype.

Selenium Deficiency, but Not Vitamin E Deficiency, Activates the Mouse Liver Nrf2‐ARE Pathway

Selenium (Se) and vitamin E are essential micronutrients with antioxidant properties. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2‐antioxidant response element (ARE) pathway was investigated using ARE‐reporter mice and Nrf2−/− mice. Weanling male mice were fed Se‐deficient (0 Se), vitamin E‐deficient (0 E), or control diet for 16 or 22 weeks. The ARE‐reporter was elevated 450 fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2‐ARE—glutathione S‐transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase‐1 (HO‐1)—were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO‐1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2‐ARE as well as other stress‐response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. It follows that the oxidant defense mechanisms in which these antioxidant nutrients function are autonomous. Supported by NIH grant ES02497.

Association analysis among polymorphisms of the various genes and chronic alcoholic pancreatitis

Chronic and excessive consumption of alcohol is an important factor responsible for the onset of pancreatitis. However, the incidence of chronic pancreatitis in heavy drinkers differs in individuals, suggesting that these individual differences may involve various genetic and environmental factors. In the present study, we investigated an association of alcoholic pancreatitis with polymorphisms of the various genes related to metabolism of the oxidative compounds. We analyzed polymorphisms of NADPH-quinone oxidoreductase 2 (NQO2), multidrug resistance 1 (MDR1), alcohol dehydrogenase 1B (ADH1B) and lipoprotein lipase (LPL). The subjects consisted of 53 patients with chronic alcoholic pancreatitis (AlCP), 54 alcoholic patients without pancreatic dysfunction (Alc), and 42 healthy individuals. DNA samples were prepared from the peripheral blood of all subjects, and the genetic mutations were analyzed by polymerase chain reaction and restriction fragment length polymorphism methods. The ADH1B gene frequencies were significantly different between healthy controls and Alc patients (P < 0.001), and also between AlCP and Alc patients (P < 0.05). However, no significant difference was found between healthy controls and AlCP patients. The gene frequencies of MDR1 (3435C > T) and MDR1 (2677G > A/T) of patients with AlCP or Alc were different when compared with healthy controls, although the difference was not significant. The NQO2 and LPL genes showed no relation with Alc and AlCP patients. The ADH1B*1 gene frequency in AlCP was significantly lower compared with Alc. We speculate that the ADH1B*1 gene may function by reducing vulnerability to the onset of alcoholic pancreatitis. Other genes analyzed in the present study lacked association with AlCP.

The significance of Nrf2 pathway in (photo)‐oxidative stress response in melanocytes and keratinocytes of the human epidermis

The expression of genes encoding antioxidant and/or phase 2 detoxifying enzymes can be enhanced in response to various environmental stresses. The main transcription factor involved in this response is nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 activity is negatively regulated by the protein Kelch-like-Ech-associated-protein 1 (Keap1). While the roles of Nrf2 and phase 2 genes in chemoprevention of carcinogenesis have been well described; only few studies have dealt with their role in skin cancer. Normal human keratinocytes (NHK) and melanocytes (NHM) were treated by chemical inducers of the Nrf2 pathway or by small interfering RNAs (siRNA) used to knock down Keap1 mRNA. The above treatments resulted in significant stimulation of NQO-1 (NADPH-Quinone-Oxidoreductase 1) gene expression. GCL (gamma-Glutamyl-cysteinyl-ligase) gene was also induced but interestingly increased mRNA encoding the catalytic, heavy subunit GCLC was mainly stimulated in NHK, whereas the mRNA encoding the modifier, light subunit GCLM was mostly induced in NHM. HO-1 (Heme Oxygenase 1) gene induction was relatively strong in NHM, but generally absent in NHK, except when the cells were subjected to cytotoxic doses of the above chemicals. Exposure to solar UV (UVB + UVA, 300-400 nm) or to UVA alone (320-400 nm) confirmed this trend, but interestingly, at doses where cell growth reduction was comparable, UVA was generally more efficient than solar UV in inducing phase 2 genes. When siRNAs directed against Nrf2 were used, a strong down-regulation of NQO-1 expression was observed in both, NHM and NHK, whereas reduction of HO-1 expression was mainly detected in NHM. To our knowledge, this is the first study comparing phase 2 gene modulation in NHK and NHM. The results hereby presented should contribute to a better understanding of the molecular mechanisms involved in skin adaptation to environmental stress.

Selenium deficiency activates mouse liver Nrf2–ARE but vitamin E deficiency does not

Selenium (Se) and vitamin E are antioxidant micronutrients. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2–antioxidant response element (ARE) pathway was investigated using ARE-reporter mice and Nrf2−/− mice. Weanling males were fed Se-deficient (0 Se), vitamin E-deficient (0 E), or control diet for 16 or 22 weeks. The ARE reporter was elevated 450-fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2–ARE (glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase-1 (HO-1)) were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO-1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2–ARE and other stress-response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. This suggests that the oxidant defense mechanisms in which these antioxidant nutrients function are independent of one another.

The NADPH quinone reductase MdaB confers oxidative stress resistance to Helicobacter hepaticus

An mdaB mutant strain in a quinone reductase (MdaB) of Helicobacter hepaticus type strain ATCC51449 was constructed by insertional mutagenesis, and the MdaB protein was purified and compared to the Helicobacter pylori enzyme. While wild type H. hepaticus cells could tolerate 6% O 2 for growth, the mdaB strain was clearly inhibited at this oxygen level. Disruption of the gene downstream of mdaB (HH1473) did not affect the oxidative stress phenotype of the strain. The mdaB mutant was also more sensitive to oxidative stress reagents such as H 2O 2, cumene hydroperoxide, t-butyl hydroperoxide, and paraquat. All H. hepaticus mdaB strains isolated constitutively up-expressed another oxidative stress-combating enzyme, superoxide dismutase; this is in contrast to H. pylori mdaB strains. H. hepaticus MdaB is a flavoprotein catalyzing quinone reduction using a two-electron transfer mechanism from NAD(P)H to quinone. The H. hepaticus enzyme specific activity was far less than for the H. pylori enzyme purified in the same manner.

Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2

The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1–4 (Mrp1–4). It was hypothesized that induction of hepatic Mrp1–4 expression following APAP is Nrf2 dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 h after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1–4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.

Flavodoxin:Quinone Reductase (FqrB): a Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in Helicobacter pylori and Campylobacter jejuni

Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k(cat) of 122 s(-1), and an apparent K(m) of 14 muM for menadione and 26 muM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO(2), and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO(2) fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO(2)-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO(2) fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.

Carbonyl Reductases: The Complex Relationships of Mammalian Carbonyl- and Quinone-Reducing Enzymes and Their Role in Physiology

Carbonyl groups are frequently found in endogenous or xenobiotic compounds. Reactive carbonyls, formed during lipid peroxidation or food processing, and xenobiotic quinones are able to covalently modify DNA or amino acids. They can also promote oxidative stress, the products of which are thought to be an important initiating factor in degenerative diseases or cancer. Carbonyl groups are reduced by an array of distinct NADPH-dependent enzymes, belonging to several oxidoreductase families. These reductases often show broad and overlapping substrate specificities and some well-characterized members, e.g., carbonyl reductase (CBR1) or NADPH-quinone reductase (NQO1) have protective roles toward xenobiotic carbonyls and quinones because metabolic reduction leads to less toxic products, which can be further metabolized and excreted. This review summarizes the current knowledge on structure and function relationships of the major human and mammalian carbonyl reductases identified.

BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress

NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.

Polycyclic aromatic hydrocarbon (PAH) metabolizing enzyme activities in human lung, and their inducibility by exposure to naphthalene, phenanthrene, pyrene, chrysene, and benzo(a)pyrene as shown in the rat lung and liver

In order to survey changes and activities in the polycyclic aromatic hydrocarbon (PAH)-metabolizing enzymes implicated in lung cancer susceptibility studies, we investigated enzyme induction by 2-5-ring-sized 'biomarker' PAHs in rat liver and lung, and the activities in five human lung specimens. Naphthalene, phenanthrene, pyrene, chrysene, and benzo[a]pyrene (BaP) were administered to rats for 3 days (25-128 mg/kg/day) and the responses compared with those of model inducers. PAH treatment increased the CYP1A-catalyzed activity of pyrene 1-hydroxylation and 7-ethoxyresorufin O-deethylation in rat liver by up to 28- and 279-fold, and in rat lung by up to 22- and 51-fold, respectively. 1-Naphthol (hUGT1A6), 1-hydroxypyrene (hUGT1A6/1A9), and entacapone (hUGT1A9) are markers of PAH-glucuronidating human uridine diphosphate-glucuronosyltransferases (UGT). These activities increased up to 6.4-fold in rat liver and up to 1.9-fold in rat lung. NADPH:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase activities increased up to 5.3- and 1.6-fold (liver), and up to 4.4- and 1.4-fold (lung), respectively. CYP1A showed the best liver-to-lung relationship (R (2 )=( )0.90). The inducing efficiency by PAHs differed extensively: control <or= naphthalene < phenanthrene, pyrene << chrysene < BaP. In human lung (non-smokers), the marker activities of CYP1A1, UGT1A6/1A9, and NQO1 were lower than those in rat lung. Epoxide hydrolase activity was 1,000-fold higher than the pulmonary CYP1A1 activities. Human UGT and NQO1 displayed large variations (>60-fold), many times greater than the experimental (inducible/constitutive) variation in the rat. Kinetics of 1-hydroxypyrene glucuronidation showed two low-K (m) forms both in rat and human lung. Since the 2-4-ring PAHs (major constituents) were poor enzyme inducers, it appears that the PAH-metabolizing pathways are mainly induced by BaP-type minor constituents. Gene-environmental interactions which magnify polymorphic variability in pulmonary bioactivation/detoxification capacity probably play a key role in individual susceptibility to (or protection against) chemically induced lung cancer. Hence, human exposure to PAH mixtures with high content of BaP-type hydrocarbons confers a potentially higher health risk than PAH mixtures with low content of procarcinogens.

The diverse antioxidant systems of Helicobacter pylori

The gastric pathogen Helicobacter pylori induces a strong inflammatory host response, yet the bacterium maintains long-term persistence in the host. H. pylori combats oxidative stress via a battery of diverse activities, some of which are unique or newly described. In addition to using the well-studied bacterial oxidative stress resistance enzymes superoxide dismutase and catalase, H. pylori depends on a family of peroxiredoxins (alkylhydroperoxide reductase, bacterioferritin co-migratory protein and a thiol-peroxidase) that function to detoxify organic peroxides. Newly described antioxidant proteins include a soluble NADPH quinone reductase (MdaB) and an iron sequestering protein (NapA) that has dual roles - host inflammation stimulation and minimizing reactive oxygen species production within H. pylori. An H. pylori arginase attenuates host inflammation, a thioredoxin required as a reductant for many oxidative stress enzymes is also a chaperon, and some novel properties of KatA and AhpC were discovered. To repair oxidative DNA damage, H. pylori uses an endonuclease (Nth), DNA recombination pathways and a newly described type of bacterial MutS2 that specifically recognizes 8-oxoguanine. A methionine sulphoxide reductase (Msr) plays a role in reducing the overall oxidized protein content of the cell, although it specifically targets oxidized Met residues. H. pylori possess few stress regulator proteins, but the key roles of a ferric uptake regulator (Fur) and a post-transcriptional regulator CsrA in antioxidant protein expression are described. The roles of all of these antioxidant systems have been addressed by a targeted mutant analysis approach and almost all are shown to be important in host colonization. The described antioxidant systems in H. pylori are expected to be relevant to many bacterial-associated diseases, as genes for most of the enzymes carrying out the newly described roles are present in a number of pathogenic bacteria.

Induction of Rat UDP-Glucuronosyltransferases in Liver and Duodenum by Microsomal Enzyme Inducers That Activate Various Transcriptional Pathways

Microsomal enzyme inducers (MEIs) up-regulate phase I biotransformation enzymes, most notably cytochromes P450. Transcriptional up-regulation by MEIs occurs through at least three nuclear receptor mechanisms: constitutive androstane receptor (CAR; CYP2B inducers), pregnane X receptor (PXR; CYP3A inducers), and peroxisome proliferator-activated receptor alpha (PPARalpha; CYP4A inducers). Other mechanisms include transcription factors aryl hydrocarbon receptor (AhR; CYP1A inducers), and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2; NADPH-quinone oxidoreductase inducers). UDP-glucuronosyltransferases (UGTs) are phase II biotransformation enzymes that are predominantly expressed in liver and intestine. MEIs increase UGT activity; however, transcriptional regulation of individual UGT isoforms is not completely understood. The purpose of this study was to examine inducibility of individual UGT isoforms and potential mechanisms of transcriptional regulation in rat liver and duodenum. UGT mRNA levels were assessed in liver and duodenum of rats treated with MEIs that activate various transcriptional pathways. All four CAR activators induced UGT2B1 in liver, but not duodenum. UGT1A1, 1A5, 1A6, and 2B12 were induced by at least two CAR activators in liver only. Two PXR ligands induced UGT1A2, but only in duodenum. Two PPARalpha ligands induced UGT1A1 and 1A3 in liver only. AhR ligands induced UGT1A6 and 1A7 in liver, but not duodenum. Nrf2 activators increased UGT2B3 and 2B12 in both liver and duodenum, and UGT1A6, 1A7, and 2B1 in liver only. In summary, only UGT1A2 and 1A8 were not inducible in liver by MEIs. MEIs differentially regulate hepatic expression of individual UGT isoforms, although no one transcriptional pathway dominated. In duodenum, MEIs had minimal effects on UGT expression.

Cytochrome P450 2A5 expression in a G6PD‐deficient mouse model of oxidative stress

Murine hepatic cytochrome P450 2A5 (CYP2A5), unlike most CYP enzymes, is upregulated during hepatitis and hepatotoxicity. To determine if oxidative stress is responsible for this induction, glucose-6-phosphate dehydrogenase (G6PD)-deficient mice, that are more susceptible to oxidative stress, were treated with the prototypical CYP2A5 inducer pyrazole. Compared to normal mice, G6PD-deficient mice had 3, 1.6, and 5 times greater induction of CYP2A5 mRNA, protein, and activity, respectively. Involvement of oxidative stress was determined using markers of oxidative damage and expression of oxidant-responsive genes. Although no significant elevation in protein carbonyl groups or glutathione peroxidase and superoxide dismutase transcripts were observed, NADPH quinone oxidoreductase 1, which protects against oxidative stress and redox cycling, was increased by pyrazole. Glutathione-S-transferase (GST) alpha, mu, and pi mRNA levels were also elevated by pyrazole, with GST pi showing 1.9 times greater induction in G6PD-deficient mice. Glucose-regulated protein 78, an endoplasmic reticulum stress protein, showed a 1.7 fold greater response to pyrazole in deficient mice. These results suggest that conditions leading to upregulation of CYP2A5 are complex and factors other than oxidative stress may be involved in regulation of CYP2A5. Supported by the Natural Sciences and Engineering Research Council of Canada.