Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.8.1.4 (diaphorase)
 2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme (NADPH-dependent diaphorase) present in rat brain microsomes has been solubilised and shown to utilise both nitrobluetetrazolium and cytochrome c as electron acceptors, when reduced by NADPH. The kinetics of the enzyme have been determined using cytochrome c (Km = 1.3 microM), NADPH (Km = 1.4 microM) and the Vmax (4.7 nmol/min/mg solubilised microsome protein). The subunit Mr is approximately 73,000 D and that of the native enzyme is 170,000-180,000 D, indicating that the enzyme is probably a dimer. Evidence is also provided to show that the enzyme is a flavoprotein, and that it has equimolar amounts of FAD and FMN with respect to the subunit concentration. It seems a possibility that the rat brain diaphorase enzyme may be cytochrome P450 reductase, EC 1.6.2.4.
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PMID:Rat brain NADPH-dependent diaphorase. A possible relationship to cytochrome P450 reductase. 313 10

The reduction and the potential autoxidation of quinoid compounds may be viewed as taking place in three cell compartments. In microsomal fractions (endoplasmic reticulum) one-electron reduction by NAPDH-cytochrome P450 reductase leads to the formation of semiquinones which rapidly react with oxygen to form the parent quinone and superoxide anions. The formation of superoxide through this futile cycle leads ultimately to other damaging species (H2O2 and .OH). A similar futile cycle in mitochondria involves NADH dehydrogenase. In this instance, mitochondria initiation of such a cycle with quinones results not only in the formation of toxic radical species but also in the diversion of electrons from phosphorylating pathways. The consequent diminution of cellular ATP may have as important a consequence with respect to the toxicity of quinones as the generation of radicals. Finally, cytosolic DT diaphorase, which carries out a two-electron reduction of quinones to more stable hydroquinones, may compete with the one-electron systems and participate in the detoxification of quinones by supplying hydroquinones for conjugation reactions. The extent of quinone-induced damage may thus vary from cell to cell depending on the integration of these pathways.
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PMID:Futile redox cycling: implications for oxygen radical toxicity. 631 61

The metabolism of the o-quinone derivative of estrone, 3,4-estrone quinone (3,4-EQ), has been investigated in human breast cancer cells. Unlike the p-quinone, diethylstilbestrol 4',4"-quinone, 3,4-EQ was not a substrate for the two-electron reduction catalyzed by the putative detoxifying enzyme, NAD(P)H:quinone reductase (DT diaphorase; DT D). Accordingly, the DNA damage induced by 3,4-EQ in human MCF-7 cells was not affected by an inhibitor of DT D. Although 3,4-EQ was not an apparent substrate for the two-electron reduction catalyzed by DT D, this o-quinone was a substrate for the one-electron reduction catalyzed by cytochrome P450 reductase. The one-electron reduction of 3,4-EQ catalyzed by cytochrome P450 reductase occurred in the face of a significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH. The impact of purified superoxide dismutase (SOD) upon the production of hydrogen peroxide produced as a consequence of 3,4-EQ metabolism was evaluated; surprisingly, SOD inhibited the hydrogen peroxide produced by this o-quinone. Possible reasons for the SOD-mediated inhibition of redox cycling of 3,4-EQ are discussed. In summary, important differences in the metabolism of 3,4-EQ vis-a-vis o- and p- quinones have been observed, and the implications of these differences are discussed.
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PMID:Cellular biochemical determinants modulating the metabolism of estrone 3,4-quinone. 784 38

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.
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PMID: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. 807 96

This study was undertaken to elucidate the mechanism(s) of differential sensitivity of human bladder cancer cell lines J82 and SCaBER to mitomycin C (MMC) and its analogue, BMY 25067. The IC50 values for MMC and BMY 25067 in the SCaBER cell line were respectively 5- and 4-fold higher than in J82. BMY 25282 and BMY 25067 were significantly more cytotoxic, on a molar basis, than MMC in both the cell lines. NADPH cytochrome P450 reductase and DT diaphorase activities were significantly higher in the J82 cell line than in SCaBER, suggesting that relatively lower sensitivity of the SCaBER cell line to MMC and BMY 25067 may be due to deficient drug activation. This conclusion was supported by the observation that IC50 values for BMY 25282, which has lower quinone reduction potential than MMC and BMY 25067, did not differ significantly in these cell lines. A correlation between drug sensitivity, oxyradical formation and levels of antioxidative enzymes was not observed. These results suggest that the relatively lower sensitivity of SCaBER cells to MMC or BMY 25067 may be independent of differential oxyradical formation. MMC-induced DNA interstrand cross-link (ISC) formation was markedly lower in the SCaBER cell line than in J82. However, it remains to be seen if the reduced ISC frequency in the SCaBER cell line is a consequence of deficient drug activation or results from increased repair of the damaged DNA.
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PMID:Mechanism of differential sensitivity of human bladder cancer cells to mitomycin C and its analogue. 829 21

Female F344 rats received an i.p. injection of iron-dextran (600 mg Fe/kg) and then after 1 week were fed a diet containing 0.02% hexachlorobenzene (HCB) for up to 65 weeks. All rats (8/8) which received HCB after iron overload developed multiple hepatic nodules whereas only 3/8 rats administered HCB alone had nodules (average of one per positive liver). These hyperplastic regions were depleted of iron and were often positive for gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase P (GST-P). Telangiectasis and peliosis were prominent features in the lesions. Short-term experiments (5-15 weeks of iron/HCB treatments) showed that GGT and GST-P were induced early in the neoplastic process but not in discrete focal areas. Iron alone also caused some induction of these enzymes. Some cells with induced GST-P in either short or long term experiments also stained positively for this enzyme in the nucleus. Studies of cytochrome P450 mediated activities showed that at 5 and 15 weeks HCB had induced EROD (an estimate of CYP1A1), PROD (CYP2B1 activity) and BROD activities (CYP2B1 but also other isoenzymes). Under the influence of iron overload EROD was significantly depressed from HCB alone, but not the others or cytochrome P450 reductase. Cytosolic glutathione S-transferase activities were also induced by HCB, but, unlike microsomal EROD, preloading with iron enhanced the effects. In contrast, although cytosolic diaphorase activity was induced by HCB, this response was depressed in combination with iron. Glutathione peroxidase (with H2O2 as substrate) was depressed by both iron and HCB. Clearly, iron overload potentiates the neoplastic process induced by HCB in rats, with both enhancing and depressing effects on various enzyme activities induced by this chemical.
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PMID:Enhancement by iron of hepatic neoplasia in rats caused by hexachlorobenzene. 833 Mar 54

MPP+ is redox active in the presence of cytochrome P450 reductase and induces the formation of O2.- and HO(.). In this study, we report the redox cycling capability of MPP+ with additional enzymes and with UV photolysis detected through ESR techniques. The treatment of MPP+ with UV light resulted in the production of HO. trapped as a spin adduct. Two of the enzymes examined in this study, xanthine oxidase and aldehyde dehydrogenase, produced O2.- in the presence of substrate. However, when MPP+ was added to the incubations, the radical trapped by DMPO was HO(.). This indicates that MPP+ redox cycles in the presence of these two enzymes or UV light, which produces HO.. Our data also suggest that MPP+ is reduced by lipoamide dehydrogenase. MPP+ stimulated the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by the enzyme at concentrations between 2 mM and 8 mM of MPP+. Higher concentrations of MPP+ inhibited lipoamide dehydrogenase. MPP+ appears to be redox active with a number of redox enzymes. The mechanism involved may be hydride transfer from the enzymes to MPP+, rather than a direct single-electron reduction.
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PMID:Redox cycling of MPP+: evidence for a new mechanism involving hydride transfer with xanthine oxidase, aldehyde dehydrogenase, and lipoamide dehydrogenase. 839 42

Mitomycin C (MMC), an alkylating anti-tumor agent, was activated by non-enzymatic and enzymatic mechanisms leading to DNA binding and adduct formation. However, it was enzymatically, not non-enzymatically, activated MMC which induced inter-strand DNA cross-linking, a major determinant of cell death. The enzymatic activation of MMC was catalyzed by microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic enzyme activities. Human P450 reductase, transiently expressed from its cDNA in the COSI cells, metabolically activated MMC to generate 9 specific MMC-DNA adducts and induced inter-strand DNA cross-linking. Co-chromatography of the MMC-DNA adducts generated by P450 reductase and sodium borohydride in separate experiments indicated that MMC was metabolized by P450 reductase to produce 2,7-diaminomitosenes that exhibited binding to deoxyguanosine. Several experiments indicated that cytosolic enzymes which catalyzed reductive activation of MMC and DNA cross-linking included NAD(P)H:quinone oxidoreductaseI (NQOI or DT diaphorase) when present in extremely high concentrations and a unique cytosolic activity. The unique cytosolic activity was present in several mammalian cells and mouse colon and liver but absent in mouse kidney. The unique activity had properties of a diaphorase but was distinct from NQOI because of a lack of correlation between NQOI (2,6-dichlorophenolindophenol reduction) activity and the amount of MMC-reductive activation leading to DNA cross-linking. This activity was also distinct from xanthine oxidoreductase and NADH-cytochrome b5 reductase, 2 other enzymes that catalyze metabolic activation of MMC, because the unique activity was not inhibited by allopurinol (an inhibitor of xanthine oxidoreductase) and its activity was the same with NADH and NADPH (cytochrome b5 reductase is specific to NADH).
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PMID:Non-enzymatic and enzymatic activation of mitomycin C: identification of a unique cytosolic activity. 856 27

o-Quinones are easily formed by oxidation of physiologically relevant catechols. These reactions mainly occur in two specialized cells, catecholaminergic neurons and melanocytes. Both types of cells are related ontogenetically, as they arise from the neural crest during the developmental differentiation. o-Quinones are used to form melanin, a protective pigment formed by different mechanisms in melanocytes and catecholaminergic neurons. However, the reactivity of these quinones makes their presence in the cytosol dangerous for the cell survival and these compounds have been proposed as degenerative and apoptotic agents. Thus, melanin-producing cells show several potential mechanisms to protect themselves against the noxious effects of o-quinones. In melanocytes, the most effective autoprotecting mechanisms are the existence of malanosomes as a confined site for melano-synthesis and the action of tyrosinase-related protein 2 (TRP2) to drive L-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid minimizing the formation of 5,6-dihydroxyindole. In catecholaminergic neurons, recent data suggest that glutathione transferase (GST M2-2 isoenzyme) and macrophage migration inhibitory factor (MIF) are very effective in preventing long-lived formation of dopaminechrome and noradrenochrome, although the detoxification reactions are different (conjugation to GSH or isomerization respectively). These mechanisms are less efficient for adrenochrome, although MIF and GST M1-1 could also catalyze similar reactions using this compound as substrate. In addition, the formation of adrenochrome is still under discussion, and adrenolutin formation could contribute to deactivate its harmful effects. The contribution of D-dopachrome tautomerase to these mechanisms is yet unknown, although in contrast to MIF, that enzyme does not recognize catecholaminechromes as substrates. Diaphorase could also be protective against quinones, since this enzyme catalyzes their bielectronic reduction back to catechols, thus preventing the formation of chrome species. This activity has been described in melanocytes and neurons, so that its contribution should be further investigated. In contrast to diaphorase, cytochrome P450 reductase should not be considered a protective enzyme, since its monoelectronic reduction of quinones leads to formation of semiquinones, that is, even more noxious than the quinones.
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PMID:Neurotoxicity due to o-quinones: neuromelanin formation and possible mechanisms for o-quinone detoxification. 1283 99

Enzymatic reduction of physiological Fe(III) complexes of the "labile iron pool" has not been studied so far. By use of spectrophotometric assays based on the oxidation of NAD(P)H and formation of [Fe(II) (1,10-phenanthroline)3]2+ as well as by utilizing electron paramagnetic resonance spectrometry, it was demonstrated that the NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase (diaphorase, EC 1.8.1.4) effectively catalyzes the one-electron reduction of Fe(III) complexes of citrate, ATP, and ADP at the expense of the co-enzymes NAD(P)H. Deactivated or inhibited lipoyl dehydrogenase did not reduce the Fe(III) complexes. Likewise, in the absence of NAD(P)H or in the presence of NAD(P)+, Fe(III) reduction could not be detected. The fact that reduction also occurred in the absence of molecular oxygen as well as in the presence of superoxide dismutase proved that the Fe(III) reduction was directly linked to the enzymatic activity of lipoyl dehydrogenase and not mediated by O2. Kinetic studies revealed different affinities of lipoyl dehydrogenase for the reduction of the low molecular weight Fe(III) complexes in the relative order Fe(III)-citrate > Fe(III)-ATP > Fe(III)-ADP (half-maximal velocities at 346-485 microm). These Fe(III) complexes were enzymatically reduced also by other flavoenzymes, namely glutathione reductase (EC 1.6.4.2), cytochrome c reductase (EC 1.6.99.3), and cytochrome P450 reductase (EC 1.6.2.4) with somewhat lower efficacy. The present data suggest a (patho)physiological role for lipoyl dehydrogenase and other flavoenzymes in intracellular iron metabolism.
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PMID:Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool. 1296 36


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