Up-regulation of NAD（P）H quinone oxidoreductase 1 during human liver injury

AIM:To investigate the expression and activity ofNAD(P)H quinone oxidoreductase 1(NQO1)in humanliver specimens obtained from patients with liver damagedue to acetaminophen(APAP)overdose or primarybiliary cirrhosis(PBC).METHODS:NQO1 activity was determined in cytosolfrom normal,APAP and PBC liver specimens.Westernblot and immunohistochemical staining were used todetermine patterns of NQO1 expression using a specificantibody against NQO1.RESULTS:NQO1 protein was very low in normal humanlivers.In both APAP and PBC livers,there was stronginduction of NQO1 protein levels on Western blot.Correspondingly,significant up-regulation of enzymeactivity(16-and 22-fold,P<0.05)was also observed inAPAP and PBC livers,respectively.Immunohistochemicalanalysis highlighted injury-specific patterns of NQO1staining in both APAP and PBC livers.CONCLUSION:These data demonstrate that NQO1protein and activity are markedly induced in humanlivers during both APAP overdose and PBC.Up-regulationof this cytoprotective enzyme may represent an adaptivestress response tO limit further disease progression bydetoxifying reactive species.     

Prevalence of the inactivating 609C-->T polymorphism in the NAD(P)H:quinone oxidoreductase (NQO1) gene in patients with primary and therapy-related myeloid leukemia.

NAD(P)H:quinone oxidoreductase (NQO1) converts benzene-derived quinones to less toxic hydroquinones and has been implicated in benzene-associated hematotoxicity. A point mutation in codon 187 (Pro to Ser) results in complete loss of enzyme activity in homozygous subjects, whereas those with 2 wild-type alleles have normal activity. The frequency of homozygosity for the mutant allele among Caucasians and African Americans is 4% to 5% but is higher in Hispanics and Asians. Using an unambiguous polymerase chain reaction (PCR) method, we assayed nonmalignant lymphoblastoid cell lines derived from 104 patients with myeloid leukemias; 56 had therapy-related acute myeloid leukemia (t-AML), 30 had a primary myelodysplastic syndrome (MDS), 9 had AML de novo, and 9 had chronic myelogenous leukemia (CML). All patients had their leukemia cells karyotyped. Eleven percent of the t-AML patients were homozygous and 41% were heterozygous for the NQO1 polymorphism; these proportions were significantly higher than those expected in a population of the same ethnic mix (P =.036). Of the 45 leukemia patients who had clonal abnormalities of chromosomes 5 and/or 7, 7 (16%) were homozygous for the inactivating polymorphism, 17 (38%) were heterozygous, and 21 (47%) had 2 wild-type alleles for NQO1. Thus, NQO1 mutations were significantly increased compared with the expected proportions: 5%, 34%, and 61%, respectively (P =.002). An abnormal chromosome no. 5 or 7 was observed in 7 of 8 (88%) homozygotes, 17 of 45 (38%) heterozygotes, and 21 of 51 (41%) patients with 2 wild-type alleles. Among 33 patients with balanced translocations [14 involving bands 11q23 or 21q22, 10 with inv(16) or t(15;17), and 9 with t(9;22)], there were no homozygotes, 15 (45%) heterozygotes, and 18 (55%) with 2 wild-type alleles. Whereas fewer than 3 homozygotes were expected among the 56 t-AML patients, 6 were observed; 19 heterozygotes were expected, but 23 were observed. The gene frequency for the inactivating polymorphism (0. 31) was increased approximately 1.4-fold among the 56 t-AML patients. This increase was observed within each of the following overlapping cohorts of t-AML patients: the 43 who had received an alkylating agent, the 27 who had received a topoisomerase II inhibitor, and the 37 who had received any radiotherapy. Thus, the frequency of an inactivating polymorphism in NQO1 appears to be increased in this cohort of myeloid leukemias, especially among those with t-AML or an abnormality of chromosomes 5 and/or 7. Homozygotes and heterozygotes (who are at risk for treatment-induced mutation or loss of the remaining wild-type allele in their hematopoietic stem cells) may be particularly vulnerable to leukemogenic changes induced by carcinogens.     

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) and sulfotransferase 1A1 (SULT1A1) polymorphisms and urothelial cancer risk in Taiwan

Purpose To investigate whether the NAD(P)H:quinone oxidoreductase 1 (NQO1) and sulfotransferase 1A1 (SULT1A1) polymorphisms are associated with urothelial cancer (UC) risk in Taiwan. Methods In this study, 600 study subjects (including 300 UC patients and 300 cancer-free controls) were recruited from September 1998 to December 2005. We analyzed the NQO1 and SULT1A1 polymorphisms by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. A comprehensive interview was conducted to collect information, including baseline characteristics and cigarette smoking status. We used an unconditional multivariate logistic regression to calculate the odds ratio (OR) and 95% confidence interval (CI). Results We found a significantly increased UC risk in study subjects with the NQO1 C/T and T/T genotypes (OR = 1.5; 95% CI: 1.03–2.1). A significantly increased UC risk was found in those with the SULT1A1 G/G genotype (OR = 2.0; 95% CI: 1.3–3.2). Subjects who had ever smoked with either the NQO1 C/T and T/T genotypes or the SULT1A1 G/G genotype had significantly increased UC risks, showing ORs of 3.0 and 5.3, respectively. Subjects carrying both the NQO1 C/T and T/T genotypes and the SULT1A1 G/G genotype had a significantly increased UC risk (OR = 3.7; 95% CI, 1.4–9.7). Moreover, those who had ever smoked with both the NQO1 C/T and T/T genotypes and the SULT1A1 G/G genotype had the highest UC risk (OR = 8.6; 95% CI: 2.5–29.7). Conclusions These findings suggest that NQO1 and SULT1A1 polymorphisms are associated with the risk of UC, particularly among those who have ever smoked.     

A potential mechanism underlying the increased susceptibility of individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene toxicity

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase that detoxifies quinones derived from the oxidation of phenolic metabolites of benzene. A polymorphism in NQO1, a C609T substitution, has been identified, and individuals homozygous for this change (T/T) have no detectable NQO1. Exposed workers with a T/T genotype have an increased risk of benzene hematotoxicity. This finding suggests NQO1 is protective against benzene toxicity, which is difficult to reconcile with the lack of detectable NQO1 in human bone marrow. The human promyeloblastic cell line, KG-1a, was used to investigate the ability of the benzene metabolite hydroquinone (HQ) to induce NQO1. A concentration-dependent induction of NQO1 protein and activity was observed in KG-1a cells cultured with HQ. Multiple detoxification systems, including NQO1 and glutathione protect against benzene metabolite-induced toxicity. Indeed, exposure to a noncytotoxic concentration of HQ induced both NQO1 and soluble thiols and protected against HQ-induced apoptosis. NQO1 protein and activity increased in wild-type human bone marrow cells (C/C) exposed to HQ, whereas no NQO1 was induced by HQ in bone marrow cells with the T/T genotype. Intermediate induction of NQO1 by HQ was observed in heterozygous bone marrow cells (C/T). NQO1 also was induced by HQ in wild-type (C/C) human bone marrow CD34(+) progenitor cells. Our data suggest that failure to induce functional NQO1 may contribute to the increased risk of benzene poisoning in individuals homozygous for the NQO1 C609T substitution (T/T).     

Role of NAD(P)H:quinone oxidoreductase 1 on tumor necrosis factor-alpha-induced migration of human vascular smooth muscle cells

OBJECTIVES: In a preliminary study, NAD(P)H:quinone oxidoreductase 1 (NQO1) was found to be highly expressed in cultured human aortic smooth muscle cells (HASMC) and dicumarol, a NQO1 inhibitor and a coumarin-derived natural anticoagulant, suppressed tumor necrosis factor (TNF)-alpha-induced HASMC migration. Therefore, it was hypothesized that NQO1 plays an important role in the regulation of vascular smooth muscle cells (VSMC) migration activated by TNF-alpha. METHODS AND RESULTS: Gelatin zymography, reporter gene, electrophoretic mobility shift and Western blotting assays showed that dicumarol, but not other coumarin-derived anticoagulants, inhibited TNF-alpha-induced HASMC migration and suppressed TNF-alpha-induced matrix metalloproteinase (MMP)-9 expression and secretion in a dose-dependent manner. In addition, down-regulation of NQO1 by transfection of its small interfering RNA similarly inhibited TNF-alpha-induced MMP-9 secretion, indicating that dicumarol-mediated inhibition of MMP-9 expression is due in large part to inhibition of NQO1. Down-regulation of NQO1 inhibits MMP-9 gene expression by suppressing activation of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1), well-known key elements mediating MMP-9 gene expression in its promoter, via the p38 MAPK and JNK pathways. CONCLUSION: The results of the present study demonstrate that down-regulation of NQO1 effectively suppresses TNF-alpha-induced HASMC migration through inhibition of MMP-9 expression, suggesting that NQO1 may be a potential target for the prevention of vascular disorders related to migration of VSMC.     

The frequencies of NAD(P)H quinone oxidoreductase (NQO1) variant allele in Israeli ethnic groups and the relationship of NQO1*2 to adult acute myeloid leukemia in Israeli patients

NAD(P)H:quinone oxidoreductase (NQO1) detoxifies quinones. The NQO1*2 variant enzyme (codon 609 C-->T, encoding a proline to serine substitution) with greatly reduced activity has been reported to predispose to acute myeloid leukemia (AML). Our aim was to examine the relationship between NQO1*2 and AML in Israeli patients. We analyzed for NQO1*2 in 262 adult Israeli patients with de novo AML and 688 controls of the same ethnic groups (Arabs, and Caucasian and Ethiopian Jews). Our analysis showed significant differences in the frequencies of NQO1*2 by ethnic group (p=0.000068). However, NQO1*2 frequencies did not differ between AML patients and controls. Karyotype was not found to be associated with NQO1*2. In Israeli patients, NQO1*2 does not predispose to de novo AML.     

Subunit functional studies of NAD(P)H:quinone oxidoreductase with a heterodimer approach.

NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric enzyme which catalyzes the reduction of quinones, azo dyes, and other electron acceptors by NADPH or NADH. To pursue subunit functional studies, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coli. The wild-type subunit of the heterodimer was tagged with polyhistidine and the other subunit contained a His-194-->Ala mutation (H194A), a change known to dramatically increase the Km for NADPH. This approach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions. The composition of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis. The enzyme kinetics of the purified heterodimer were studied with two two-electron acceptors, 2,6-dichloroindophenol and menadione, and a four-electron acceptor, methyl red, as the substrates. With two-electron acceptors, the Km(NADPH) and Km(NADH) values of the heterodimer H194A/HNQOR were virtually identical to those of the wild-type homodimer, but the kcat-(NADPH) and kcat(NADH) values were only about 50% those of the wild-type homodimer. With the four-electron acceptor, the Km and kcat values of H194A/HNQOR for NADPH and NADH were similar to those of the low-efficiency mutant homodimer. These results suggest that the subunits of NQOR function independently with two-electron acceptors, but dependently with a four-electron acceptor. This heterodimer approach may have general applications for studying the functional and structural relationships of subunits in dimeric or oligomeric proteins.     

Low NAD(P)H:quinone oxidoreductase 1 activity is associated with increased risk of acute leukemia in adults

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that detoxifies quinones and reduces oxidative stress. A cysteine-to-threonine (C --> T) substitution polymorphism at nucleotide 609 of the NQO1 complementary DNA (NQO1 C609T) results in a lowering of NQO1 activity. Individuals homozygous for this mutation have no NQO1 activity, and heterozygotes have low to intermediate activity compared with people with wild type. DNA samples from 493 adult de novo acute leukemia patients and 838 matched controls were genotyped for NQO1 C609T. The majority of cases were diagnosed as acute myeloid leukemia (AML) (n = 420); 67 as acute lymphoblastic leukemia (ALL); and 6 as other forms of acute leukemia. The frequency of cases with low or null NQO1 activity (heterozygote + homozygous mutant) was significantly higher among total acute leukemia case subjects compared with their matched controls (odds ratio [OR] = 1.49; 95% confidence interval [CI], 1.17-1.89). Both ALL (OR = 1.93; 95% CI, 0.96-3.87) and AML case subjects (OR = 1.47; 95% CI, 1.13-1.90) exhibited a higher frequency of low or null NQO1 genotypes than controls. For de novo AML, the most significant effect of low or null NQO1 activity was observed among the 88 cases harboring translocations and inversions (OR = 2.39; 95% CI, 1.34-4.27) and was especially high for those harboring inv(16) (OR = 8.13; 95% CI, 1.43-46.42). These findings were confirmed in a second group of 217 de novo AML cases with known cytogenetics. Thus, inheritance of NQO1 C609T confers an increased risk of de novo acute leukemia in adults, implicating quinones and related compounds that generate oxidative stress in producing acute leukemia.     

P53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1

Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent "hot-spot" p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.     

Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin

NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates the stability of the tumor suppressor WT p53. NQO1 binds and stabilizes WT p53, whereas NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. Here, we show that curcumin, a natural phenolic compound found in the spice turmeric, induced ubiquitin-independent degradation of WT p53 and inhibited p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Like dicoumarol, curcumin inhibited the activity of recombinant NQO1 in vitro, inhibited the activity of endogenous cellular NQO1 in vivo, and dissociated NQO1-WT p53 complexes. Neither dicoumarol nor curcumin dissociated the complexes of NQO1 and the human cancer hot-spot p53 R273H mutant and therefore did not induce degradation of this mutant. NQO1 knockdown by small-interfering RNA induced degradation of both WT p53 and the p53 R273H mutant. The results indicate that curcumin induces p53 degradation and inhibits p53-induced apoptosis by an NQO1-dependent pathway.     

Chloroplast lipid droplet type II NAD(P)H quinone oxidoreductase is essential for prenylquinone metabolism and vitamin K1 accumulation

Lipid droplets are ubiquitous cellular structures in eukaryotes and are required for lipid metabolism. Little is currently known about plant lipid droplets other than oil bodies. Here, we define dual roles for chloroplast lipid droplets (plastoglobules) in energy and prenylquinone metabolism. The prenylquinones--plastoquinone, plastochromanol-8, phylloquinone (vitamin K(1)), and tocopherol (vitamin E)--are partly stored in plastoglobules. This work shows that NAD(P)H dehydrogenase C1 (NDC1) (At5g08740), a type II NAD(P)H quinone oxidoreductase, associates with plastoglobules. NDC1 reduces a plastoquinone analog in vitro and affects the overall redox state of the total plastoquinone pool in vivo by reducing the plastoquinone reservoir of plastoglobules. Finally, NDC1 is required for normal plastochromanol-8 accumulation and is essential for vitamin K(1) production.     

NAD(P)H:醌氧化还原酶基因多态性与阿尔茨海默病遗传易感性的关系

目的 探讨NAD（P）H：醌氧化还原酶（NQ01）基因C609T位点基因多态性与散发性阿尔茨海墨病（SAD）遗传易感性的关系。方法 应用聚合酶链反应－限制性片段长度多态性（PCR－RFLP）方法检测了120例 SAD患者和122名健康老年人的NQ01基因多态性布特征。结果 SAD组C等位基因频率低于对照组,SAD组为45．0％,对照组为57．0％（OR＝0．63,P＜0．05）。SAD组C／C基因频率明显低于对照组,SAD组为12．5％,对照组为29．5％（OR＝0．34,P＜0．01）。结论 NQ01基因多态性与SAD发病明显关联,NQ01基因609序列C／C基因型、C等位基因与SAD发病呈明显负关联。NQ01基因609序列C等位基因对SAD发病可能有保护作用。     

Low NAD(P)H:quinone oxidoreductase activity is associated with increased risk of leukemia with MLL translocations in infants and children

An inactivating polymorphism at position 609 in the NAD(P)H:quinone oxidoreductase 1 gene (NQO1 C609T) is associated with an increased risk of adult leukemia. A small British study suggested that NQO1 C609T was associated with an increased risk of infant leukemias with MLL translocations, especially infant acute lymphoblastic leukemia (ALL) with t(4;11). We explored NQO1 C609T as a genetic risk factor in 39 pediatric de novo and 18 pediatric treatment-related leukemias with MLL translocations in the United States. Children with de novo B-lineage ALL without MLL translocations and a calculation of the expected genotype distribution in an ethnically matched population of disease-free subjects served as the comparison groups. Patients with de novo leukemias with MLL translocations were significantly more likely to be heterozygous at NQO1 C609T (odds ratio [OR] = 2.77, 95% confidence intervals [CI] 1.17-6.57; P =.02), and significantly more likely to have low/null NQO1 activity than patients with de novo B-lineage ALL without MLL translocations (OR = 2.47, 95% CI 1.08-5.68; P =.033). They were also significantly more likely to have low/null NQO1 activity than expected in an ethnically matched population of disease-free subjects (OR = 2.50, P =.02). Infants younger than 12 months old at diagnosis of leukemia with t(4;11) were most likely to have low/null NQO1 activity (OR > 10.0). Conversely, the distribution of NQO1 genotypes among patients with treatment-related leukemias with MLL translocations was not statistically different than in the comparison groups. The inactivating NQO1 polymorphism is associated with an increased risk of de novo leukemia with MLL translocations in infants and children.     

NAD(P)H:quinone oxidoreductase1 (DT diaphorase) specifically prevents the formation of benzo[a]pyrene quinone-DNA adducts generated by cytochrome P4501A1 and P450 reductase.

Monkey kidney COS1 cells transiently transfected with plasmids pMT2-cytochrome P450 1A1 (CYP1A1), pMT2-cytochrome P450 reductase (P450 reductase), and pMT2-NAD(P)H:quinone oxidoreductase1 (NQO1 or DT diaphorase), individually or in combination, expressed significantly elevated levels of the respective enzyme(s). The transfected cells were homogenized to break cell membranes without affecting the nuclei and incubated with benzo[a]pyrene (BP) to determine the role of cDNA-encoded enzymes in metabolic activation and/or detoxification of BP. These studies were performed by measuring the capacity of the transfected cells to form DNA adducts as determined by 32P postlabeling and protein adduct detection. Cotransfection of the COS1 cells with cDNAs encoding CYP1A1 and P450 reductase resulted in eight distinct BP-DNA adducts. Inclusion of cDNA encoding NQO1 along with CYP1A1 and P450 reductase in transfection reduced the number of DNA adducts to six. The two lost DNA adducts were specifically eliminated due to the presence of cDNA-derived NQO1 activity. Subsequent experiments with BP-1,6-quinone, BP-3,6-quinone, and BP-6,12-quinone identified these two adducts as those of BP quinones. In an in vitro system, BP-3,6-quinone produced two adducts with deoxyguanosine (dG) but not with dA, dC, and dT. Furthermore, the positions of BP-3,6-quinone-dG adducts on TLC plate correspond to those that are prevented by cDNA-derived NQO1, thus identifying these adducts as BP quinones of dG. In addition, NQO1 reduced the amount of protein-BP adducts generated by CYP1A1 and P450 reductase into transfected COS1 cells. These results show that semiquinones can directly bind to DNA and demonstrate that NQO1 activity can specifically reduce the binding of quinone metabolites of BP generated by CYP1A1 and P450 reductase to DNA and protein.