Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytotoxicity of menadione (2-methyl-1,4-naphthoquinone) had been investigated using primary cultures of rat hepatocytes. Menadione was found to induce DNA strand breaks which were actively repaired by the cells. Dicoumarol, an inhibitor of DT diaphorase, did not potentiate menadione-induced DNA strand breaks. Neither had metyrapone, an inhibitor of cytochrome P-450 dependent monooxygenases, any effect on the extent of DNA damage. Covalent binding of menadione metabolite(s) to DNA was detected in the cultured hepatocytes and, in addition, hepatic microsomes were also found to metabolize menadione to DNA-binding products. The extent of binding of menadione to DNA in vitro, was markedly decreased by inclusion of the hepatic cytosol fraction, or reduced glutathione, in the incubations. In the presence of dicoumarol, menadione was also found to induce cell membrane damage. It also caused a rapid loss in cellular glutathione which was augmented by the presence of dicoumarol. The results suggest that both the cell membrane damage and DNA damage induced by menadione are mediated by one-electron reduction of the quinone to free radical intermediate(s). DT diaphorase appears to protect the cell from membrane damage, whereas reduced glutathione may have an important role in the prevention of DNA damage.
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PMID:Induction of DNA damage by menadione (2-methyl-1,4-naphthoquinone) in primary cultures of rat hepatocytes. 620 38

The mitomycin C (MC) analog BMS-181174 (previously designated as BMY25067) has been shown to be active against a variety of solid tumors in mice. The activity of this compound against tumor cell lines resistant to MC and the different toxicity profiles of BMS-181174 and MC suggested that there may be significant differences in the metabolism and the mechanisms of action of these two compounds. Our studies with a mouse mammary tumor cell line (EMT6), a wild-type Chinese hamster cell line (AA8), and three repair-deficient Chinese hamster cell lines (UV4, UV5, and EM9) supported this concept. BMS-181174 was more toxic to all five cell lines in air than in hypoxia; in contrast, MC is more toxic in hypoxia. Dicoumarol (which increases the cytotoxicity of MC in hypoxia and reduces the cytotoxicity of this drug in air) did not alter the cytotoxicity of BMS-181174. This finding suggests that neither DT-diaphorase nor cytochrome b5 reductase is involved in the activation of BMS-181174. Studies with the repair-deficient cell lines suggest that DNA strand breaks are not important to the cytotoxicity of BMS-181174, and that cross-links and adducts may be the critical lesions; these studies also suggest that the lethal lesions produced by BMS-181174 are the same under aerobic and hypoxic conditions.
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PMID:Cytotoxicity of BMS-181174. Effects of hypoxia, dicoumarol, and repair deficits. 748 40

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK alpha of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.
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PMID:Active site studies of DT-diaphorase employing artificial flavins. 753 91

Purified DT-diaphorase [NAD(P)H (quinone acceptor) oxidoreductase (EC.1.6.99.2)] from Walker cells was used to investigate the reductive metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) under aerobic and anaerobic conditions. In the presence of NADPH, under aerobic conditions, HPLC analysis showed the four-electron reduction product 3-amino-1,2,4-benzotriazine (SR 4330) was the major reaction product. In contrast, anaerobically, the 2-electron reduction product 3-amino-1,2,4-benzotriazine-1-oxide (SR 4317) was the predominant metabolite. Anaerobic reduction of SR 4233 to the known metabolites SR 4317 and SR 4330, catalyzed by DT-diaphorase, was 3-fold higher than reduction under aerobic conditions. Anaerobically, approximately half of the substrate utilized could not be accounted for by the formation of known products. Aerobically, the majority of the SR 4233 lost could be accounted for by its conversion to SR 4317 and SR 4330. In Walker cells incubated with SR 4233 anaerobically, SR 4317 was the major metabolite formed. Dicoumarol (100 microM) had little effect on the rate of formation of this metabolite in this cell line or in a rat liver epithelial derived (JBJ) cell line. Dicoumarol did however partially reduce the induction of unscheduled DNA synthesis caused by SR 4233 in Walker cells but not in JB1 cells, suggesting the action of dicoumarol may be specific to Walker cells. It is concluded that DT-diaphorase plays only a minor role in the overall reduction of SR 4233 in the two cell lines studied.
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PMID:Metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) by purified DT-diaphorase under aerobic and anaerobic conditions. 767 76

The metabolism, cytotoxicity, and genotoxicity of streptonigrin (SN) w ere determined in two human colon carcinoma cell lines: HT-29 with high NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, DTD) activity and BE with undetectable DTD activity. Dicumarol-sensitive oxidation of NADH was observed with HT-29 cytosol, but not with BE cytosol. Oxygen consumption was also observed using HT-29 cytosol, but was absent with BE cytosol. Dicumarol inhibited oxygen consumption with HT-29 cytosol, but deferoxamine had no effect, suggesting that divalent metal cations were not necessary for efficient auto-oxidation of SN hydroquinone. In cytotoxicity studies, SN was much more toxic to the DTD-rich HT-29 cells than to the DTD-deficient BE cells. Deferoxamine decreased toxicity in both cell lines, implicating hydroxyl radicals produced during Fenton-type reactions as the toxic species. In the genotoxicity assay, SN induced a much higher incidence of DNA strand breaks in HT-29 cells than in BE cells, and deferoxamine protected against DNA strand breaks in both cell lines. Some evidence of DNA repair was also observed in the two cell lines. These results support an important role for DTD in the cytotoxicity of SN in the high DTD HT-29 colon carcinoma cell line.
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PMID:Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin. 861 1

The reaction mechanism of a 1,4-benzoquinone reductase from the wood-rotting basidiomycete Phanerochaete chrysosporium was investigated. The native, oxidized, FMN-containing enzyme was reduced quantitatively by NADH and the resulting reduced enzyme was reoxidized in the presence of one equivalent of 2,6-di-methoxy-1,4-benzoquinone (DMBQ). The stoichiometry of NADH oxidation versus DMBQ reduction is 1:1. The enzyme catalyzes the reduction of quinones to hydroquinones by a ping-pong steady-state mechanism. However, inhibition is observed at low NADH concentrations. Quinone products derived from the autooxidation of the unstable compounds 1,2,4-trihydroxybenzene and 5-chloro-2,3,4-trihydroxybenzene also appear to be substrates for the quinone reductase. The enzyme reduces the one-electron acceptors ferricyanide and ferricytochrome c (Cc3+) with rates of 58.4 and 0.08%, respectively, compared to DMBQ. The stoichiometry of NADH oxidation versus ferricyanide reduction is 1:2. In the presence of quinones the rates of Cc3+ and ferricyanide reduction are increased, owing to the nonenzymatic reduction of these acceptors by enzyme-generated hydroquinone products. Dicumarol and Cibacron blue are competitive inhibitors with respect to NADH, with Ki values of 2.1 and 0.30 microM, respectively. Reconstitution of the apoprotein with FMN yields a fully active enzyme at an FMN-to-protein ratio of 2:1, suggesting that the flavin content of the enzyme is two molecules of FMN per dimer.
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PMID:1,4-Benzoquinone reductase from basidiomycete Phanerochaete chrysosporium: spectral and kinetic analysis. 866 Jun 80

Incubation of cultured Chinese hamster V79 cells with menadione (2-methyl-1,4-naphthoquinone), a generator of superoxide anion radicals, caused a rapid increase in the level of glutathione disulfide (GSSG) and a decrease in the level of glutathione (GSH), which followed a 1.5- to 2-fold increase in the level of GSH during post-treatment incubation. Menadione also caused a concentration- and time-dependent increase in the activity of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme in the synthesis of GSH. These results suggested that the increase in level of GSH after treatment with menadione was due to the increase in the activity of gamma-GCS. Dicoumarol, an inhibitor of DT-diaphorase, did not influence the increase in the activity of gamma-GCS caused by menadione but it did enhance the cytotoxicity and the increase in the level GSSG caused by menadione. This result suggested that neither the DT-diaphorase-mediated metabolism of menadione nor the increase in level of GSSG caused by menadione was associated with the increase in the activity of gamma-GCS. Chelators of divalent iron and copper (I), and cycloheximide did not influence the increase in the activity of gamma-GCS caused by menadione. Thus, it appeared that reactive oxygen radicals, generated from hydrogen peroxide by an iron- or copper-catalyzed Fenton reaction, were not responsible for the increase in the activity of gamma-GCS and that the increase was not an inducible phenomenon.
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PMID:Menadione causes increases in the level of glutathione and in the activity of gamma-glutamylcysteine synthetase in cultured Chinese hamster V79 cells. 879 48

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), by exposure to O6-benzylguanine leads to a dramatic enhancement in the cytotoxic response of cells to chemotherapeutic alkylnitrosoureas. Benzylated pyrimidines identified as more potent inactivators than O6-benzylguanine in vitro include 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (5-nitroso-BP) and 2,4-diamino-6-benzyloxy-5-nitropyrimidine (5-nitro-BP). In efforts to determine the clinical usefulness of these benzylated pyrimidines, we examined the metabolism and pharmacokinetics of 5-nitroso-BP in Sprague-Dawley rats, together with its potency as an AGT inactivator in mice. The mean plasma half-life, clearance, and volume of distribution of 5-nitroso-BP in rats were, respectively, 3.8 min, 22 liters/hr/kg, and 2.1 liters/kg. Two metabolites were identified in rat plasma (i.e. 5-nitro-BP and 2,4,5-triamino-6-benzyloxypyrimidine) after intravenous administration of 5-nitroso-BP in rat. Reduction of 5-nitroso-BP (100 microM) occurred primarily in cytosol and was inhibited (> 95%) by 1 mM menadione. Dicumarol (100 microM), a DT-diaphorase inhibitor, did not significantly inhibit this reaction. This indicated a possible role of a dicumarol-resistant quinone reductase. At higher substrate and protein concentration, NADPH-dependent oxidation of 5-nitroso-BP to 5-nitro-BP primarily occurred in microsomes and was completely inhibited by 1-aminobenzotriazole (1 mM), a P450 inhibitor. Unfortunately, neither 5-nitroso-BP nor 5-nitro-BP was as effective as O6-benzylguanine at depleting AGT activity in mouse liver or spleen. At 1 hr after injection of 15 mg/kg O6-benzylguanine, 5-nitroso-BP, or 5-nitro-BP, AGT levels in liver fell to 1%, 66%, and 71% basal activity, respectively. Rapid cytosolic reduction of 5-nitroso-BP may explain the lack of potency of the pyrimidines in vivo.
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PMID:Pharmacokinetics and metabolism in rats of 2,4-diamino-6-benzyloxy-5- nitrosopyrimidine, an inactivator of O6-alkylguanine-DNA alkyltransferase. 893 54

A resistant subline (AH130/5A) selected from rat hepatoma AH130 cells after exposure to adriamycin (ADM) showed remarkable resistance to multiple antitumor drugs, including mitomycin C (MMC) and porfiromycin (PFM). PFM, vinblastine (VLB), and ADM accumulated in AH130/5A far less than in the parent AH130 (AH130/P) cells. AH130/5A cells showed overexpression of P-glycoprotein (PGP), an increase in glutathione S-transferase activity, and a decrease in DT-diaphorase and glutathione peroxidase activity. The resistance to MMC and VLB of AH130/5A cells was partly reversed by H-87, an inhibitor of PGP. Buthionine sulfoximine, an inhibitor of glutathione synthase, did not affect the action of MMC. tert-Butylhydroquinone induced DT-diaphorase activity, increased PFM uptake, and enhanced the growth-inhibitory action of MMC in AH130/5A cells. Dicumarol, an inhibitor of DT-diaphorase, decreased PFM uptake and reduced the growth-inhibitory action of MMC in AH130/P cells. These results indicated that the adriamycin treatment of hepatoma cells caused multifactorial multidrug resistance involving a decrease in DT-diaphorase activity.
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PMID:Establishment by adriamycin exposure of multidrug-resistant rat ascites hepatoma AH130 cells showing low DT-diaphorase activity and high cross resistance to mitomycins. 904 1

We investigated the existence of an NADH-dependent paraquat (PQ) reduction system in rat liver mitochondria (Mt) in respect to the cytotoxic mechanisms of PQ. The outer membrane fractions, free from the contamination of inner membranes but with a few microsomes, catalyzed rotenone-insensitive NADH, but not NADPH, oxidation by menadione or PQ. Anti-NADH-cytochrome b5 reductase antibody and its inhibitor p-hydroxymercuribenzonate did not inhibit the NADH-PQ reduction activity. Therefore, the respiratory systems of the inner membranes and microsomal cytochrome P450 systems could not have been responsible for the reaction. Dicoumarol, an inhibitor of NAD(P)H-quinone oxidoreductase (NQO), dose dependently suppressed the NADH oxidation in the outer membrane via PQ as well as menadione, with I50 values of 190 (for menadione) and 150 microM (for PQ). Because of a lower sensitivity to NADPH and the higher doses of dicoumarol required for its inhibition, the activity in the outer membrane may be an "NADH-quinone oxidoreductase" which partly differs from the NQO previously reported. This outer membrane enzyme produced superoxide anions in the presence of both NADH and PQ and was too tightly membrane-bound to be extracted by Triton X-100 and deoxycholate. From these results, we concluded that the free radical-producing mitochondrial NADH-quinone oxidoreductase is a novel oxidation-reduction system participating in PQ toxicity. This is in good agreement with our previous results showing that PQ selectively damaged Mt in vivo and in vitro, resulting in cell death (K.-I. Hirai et al., 1992, Toxicology 72, 1-16).
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PMID:Mitochondrial NADH-quinone oxidoreductase of the outer membrane is responsible for paraquat cytotoxicity in rat livers. 950 Aug 51


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