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)

3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233; WIN 59075) is a highly selective hypoxic cell cytotoxin soon to enter phase I clinical trial. The compound is thought to exert its action through a toxic one-electron reduced free radical intermediate. Preliminary data have suggested that SR 4233 may be metabolized by DT-diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2)] to both two- and four-electron reduced products and that this route of biotransformation may represent a bioprotection pathway. In this study, a highly purified enzyme preparation was employed in order to investigate further the metabolism of SR 4233 by DT-diaphorase and to examine the mechanism of reduction in more detail. Spectrophotometric analysis showed that SR 4233 underwent reduction by DT-diaphorase with an apparent Km of 1.23 +/- 0.27 mM and Vmax of 8.55 +/- 1.67 nmol/min/microgram protein. This reaction was inhibited completely by dicoumarol (100 microM) and partially by an antiserum raised against the purified enzyme. Characterization of the products of SR 4233 reduction by reverse-phase HPLC confirmed that both two- (SR 4317) and four- (SR 4330) electron reduction products were generated, the latter being the predominant metabolite, particularly in prolonged incubations. Further experiments showed that the four-electron reduction product, but not the two-electron reduction product, was also a substrate for DT-diaphorase with an apparent Km of 1.14 mM and a Vmax of 57.12 nmol/min/micrograms protein. The results presented confirm that SR 4233 is indeed a substrate for DT-diaphorase and that a mixture of two-, four- and six-electron reduced products may be formed. The possible toxicological and pharmacodynamic significance of this metabolism is discussed.
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PMID:Enzymology of the reduction of the potent benzotriazine-di-N-oxide hypoxic cell cytotoxin SR 4233 (WIN 59075) by NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2) purified from Walker 256 rat tumour cells. 173 5

SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel benzotriazine di-N-oxide which shows unusually high selective toxicity towards hypoxic cells, probably as a result of reductive bioactivation. Using an HPLC assay for the parent drug and its 2- and 4-electron reduction products (SR 4317 and SR 4330, respectively), we have examined the enzymology of SR 4233 reductive metabolism in vitro using a variety of different enzyme preparations. SR 4233 was converted extremely rapidly to SR 4317 under N2 by mouse liver microsomes, and showed a marked preference for NADPH over NADH as a reduced cofactor. The reaction was inhibited completely in air and boiled preparations. It was also inhibited by 78-86% in carbon monoxide (CO), implicating cytochrome P-450 as the major microsomal SR 4233 reductase. The kinetics of reductive metabolism of SR 4233 to SR 4317 by mouse liver microsomes conformed to Michaelis-Menten kinetics, with a Km of 1.4 mM and a Vmax of 950 nmol/min/mg protein. SR 4233 reduction was also catalysed by mouse liver cytosol under N2. However, rates were markedly slower than for microsomes and showed an equal dependency on NADH and NADPH. The cytosolic enzymes aldehyde oxidase and xanthine oxidase both catalysed SR 4233 reduction to SR 4317 under N2. Purified buttermilk xanthine oxidase also catalysed this reaction. In contrast to other enzyme preparations, DT-diaphorase from Walker 256 tumour cells reduced SR 4233 predominantly to SR 4330, and this reaction occurred under aerobic conditions. These data illustrate that SR 4233 is reduced rapidly by a wide variety of reductases. We propose that the therapeutic selectivity of SR 4233 will be controlled by the relative expression of reductases in tumour versus normal tissues, and in particular by the differential participation of putative activating versus detoxifying enzymes.
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PMID:Enzymology of the reductive bioactivation of SR 4233. A novel benzotriazine di-N-oxide hypoxic cell cytotoxin. 234 70

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

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is an anti-tumour agent that has a highly selective toxicity to hypoxic cells. In this study we delineate the role of several different bioreductive enzymes in the metabolism of SR 4233 by two tumour cell lines HT 1080 (human fibrosarcoma) and SCCVII (mouse carcinoma). Enzyme kinetics demonstrates similar KM of HT 1080 and SCCVII cell sonicates and differing Vmax. Among all cofactors tested, NADPH was the most important one in reducing SR 4233 by both tumour cell sonicates. NADH was the second most important cofactor while hypoxanthine and N-methylnicotinamide were less involved in the reduction of SR 4233. Carbon monoxide inhibited the reduction by about 60% suggesting that cytochrome P-450 may play a major role in the reduction of SR 4233 under hypoxia in both SCCVII and HT 1080 cells. DT diaphorase is also involved, particularly in HT 1080 cells, in this drug reduction. The level of functional cytochrome P-450, cytochrome P-450 reductase activity and DT diaphorase activity in both cell lines were assayed. These enzyme levels were all higher in SCCVII cells than in HT 1080 cells. This result correlated the higher Vmax of SR 4233 reduction in SCCVII cells than in HT 1080 cells.
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PMID:Metabolism of the bioreductive cytotoxin SR 4233 by tumour cells: enzymatic studies. 843 60

Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide, SR 4233, WIN 59075) is a bioreductive antitumor agent with a high selective toxicity for hypoxic cells. The selective hypoxic toxicity of TPZ results from the rapid reoxidation of the one-electron reduction product, the TPZ radical, in the presence of molecular oxygen with the concomitant production of superoxide radical. Under hypoxia the TPZ radical kills cells by causing DNA double-strand breaks and chromosome aberrations. However, the mechanism of aerobic cytotoxicity is still a matter of debate. In this study, we investigated the mechanism of aerobic cytotoxicity by adapting human lung adenocarcinoma A549 cells to aerobic TPZ exposure and characterizing the changes associated with drug resistance. The adapted cells were resistant to aerobic TPZ exposures (with dose-modifying factors of up to 9.2), although hypoxic sensitivity was largely unchanged. Relative to the parental A549 cell line, adaptation to continuous aerobic TPZ exposure resulted in increased levels of manganese superoxide dismutase (up to 9.4-fold), moderate increases in glutathione reductase (up to 2.1-fold), and loss of both quinone oxidoreductase (DT-diaphorase) activity and NADPH cytochrome P450 reductase activity. There was essentially no change in the activity of the cytoplasmic form of superoxide dismutase (CuZnSOD), catalase, or glutathione peroxidase. The increased activity of antioxidant enzymes in the resistant cell lines (in particular MnSOD) strongly suggests that reactive oxygen species are, in large part, responsible for the toxicity of TPZ under aerobic conditions, and is consistent with aerobic and hypoxic drug cytotoxicity resulting from different mechanisms.
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PMID:Adaptation of human tumor cells to tirapazamine under aerobic conditions: implications of increased antioxidant enzyme activity to mechanism of aerobic cytotoxicity. 927 29