甘肃黄芩中的新黄酮——甘黄芩甙元的结构

本文报道从唇形科植物甘肃黄芩(Scutellaria rehderiana Diels)的根中分得五种黄酮类成分。其中一个新黄酮类化合物(Ⅰ)命名为甘黄芩甙元(ganhuangenin)其结构测定为5,7,3′,6′-四羟基-8,2′-二甲氧基黄酮,其余四种分别鉴定为黄芩甙、汉黄芩素、黄芩黄素和千层纸甲素。     

Caffeine, aminoimidazolecarboxamide and dicoumarol, inhibitors of NAD(P)H dehydrogenase (quinone) (DT diaphorase), prevent both the cytotoxicity and DNA interstrand crosslinking produced by 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) in Walker cells

A form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) has been isolated from Walker 256 rat carcinoma cells. This enzyme can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Knox et al. Biochem Pharmacol, 37: 4661-4669 and 4671-4677, 1988). 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and AICA [5(4)-aminoimidazole-4(5)-carboxamide] have previously been reported to be antagonists of the anti-tumour effects of CB 1954. We have shown that both these compounds are inhibitors of the above enzyme and that AICA protects against both the cytotoxicity and the formation of DNA interstrand crosslinks, produced by CB 1954 in Walker cells. Similarly, known inhibitors of NAD(P)H dehydrogenase (quinone) such as dicoumarol, also reduced the cytotoxicity and DNA-interstrand crosslinking of CB 1954 in Walker cells. Caffeine was shown to be a novel inhibitor of NAD(P)H dehydrogenase (quinone) and also elicited the above protective effects. All of the above inhibitors were also shown to potentiate the toxic effects of menadione against the Walker cell. This quinone is known to be detoxified by NAD(P)H dehydrogenase (quinone) and thus emphasises the ability of these compounds to inhibit this enzyme within the cell.     

Glucocorticoid regulation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferases, and NAD(P)H:quinone oxidoreductase in cultured fetal rat hepatocytes

The regulation of polycyclic aromatic hydrocarbon-inducible enzymes, cytochrome P450IA1, NAD(P)H:quinone oxidoreductase, and glutathione S-transferases, by glucocorticoids was investigated using primary fetal rat hepatocyte culture. Treatment of cells in culture with 1,2-benzanthracene (100 microM, 72 hr) resulted in 60-, 2-, and 6-fold increases in cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone reductase activities, respectively. The inductive effect of 1,2-benzanthracene on cytochrome P450IA1 and glutathione S-transferase (1-chloro-2,4-dinitrobenzene conjugation) activities was potentiated approximately 3- and 2- to 3-fold, respectively, when dexamethasone (0.01-1 microM) was included in the culture medium. In contrast, 1 microM dexamethasone was found not to potentiate the induction of NAD(P)H:quinone oxidoreductase activity by 1,2-benzanthracene. Treatment of cultured hepatocytes with dexamethasone alone, at concentrations of up to 100 microM, resulted in a 2- to 4-fold increase in glutathione S-transferase and NAD(P)H:quinone oxidoreductase activity. Both the induction of glutathione S-transferase activity by high concentrations of dexamethasone alone and the potentiation of 1,2-benzanthracene induction by lower concentrations of dexamethasone were observed for other steroids of the glucocorticoid class in conjunction with a variety of polycyclic aromatic hydrocarbons. Western immunoblot analyses indicated that low concentrations of dexamethasone (0.1-1 microM) potentiated 1,2-benzanthracene-dependent induction of cytochrome P450IA1, glutathione S-transferase Ya/Yc subunit and NAD(P)H:quinone oxidoreductase content. Additionally, increased glutathione S-transferase activity in response to concentrations of dexamethasone exceeding 1 microM was associated with concomitant increases in Ya/Yc and Yb subunit content. Potentiation of polycyclic aromatic hydrocarbon induction of cytochrome P450IA1, glutathione S-transferase, and NAD(P)H:quinone oxidoreductase protein content by low concentrations of glucocorticoids and induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase by high concentrations of glucocorticoids alone indicates the importance of these endogenous compounds in the regulation of some hepatic enzymes involved in xenobiotic metabolism.     

Menadione (2-methyl-1,4-naphthoquinone)-induced Ca2+ release from rat-liver mitochondria is caused by NAD(P)H oxidation

Incubation of rat-liver mitochondria with menadione in the presence of succinate and rotenone resulted in rapid glutathione and NAD(P)H oxidation followed by Ca2+ release and mitochondrial swelling. Ca2+ release, NAD(P)H oxidation and mitochondrial swelling, were also observed in mitochondria from selenium-deficient rats. Glutathione was only slowly oxidized, suggesting that glutathione oxidation, and subsequent NAD(P)H oxidation via the glutathione peroxidase-glutathione reductase system were not required for Ca2+ release by menadione. Isocitrate prevented and reversed Ca2+ release dose-dependently but dicoumarol had no effect indicating that NADH-ubiquinone oxidoreductase and not DT-diaphorase was responsible for NAD(P)H oxidation. Superoxide anion radical was formed by cyanide-resistant respiration, suggesting that menadione undergoes a one-electron reduction to an autoxidizable semiquinone radical by NADH-ubiquinone oxidoreductase. The inability of menadione to oxidize glutathione in selenium-deficient mitochondria indicates that the metabolism of the superoxide dismutation product, H2O2, by glutathione peroxidase was probably responsible for the glutathione oxidation in selenium-replete mitochondria.     

The enzymology of doxorubicin quinone reduction in tumour tissue.

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.     

Suggested mechanism for the modulation of the activity of NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by menadione: interpretation of the effect of menadione on 5'-[p-(Fluorosulfonyl)benzoyl]adenosine labeling of rat liver NAD(P)H:quinone acceptor oxidoreductase.

5'-[p-(Fluorosulfonyl)benzoyl]adenosine (5'FSBA) was previously shown to be an active site-directed affinity label of rat liver NAD(P)H:quinone acceptor oxidoreductase [Mol. Pharmacol. 35:818-822 (1989)]. Our recent study revealed that menadione, the substrate of this quinone reductase, had a magnifying effect on inactivation of the enzyme by 5'-FSBA. The dissociation constant for the initial reversible enzyme-inhibitor complex was significantly lower and the rate of inactivation was increased when menadione was present during the incubation. However, [14C]5'FSBA labeling was reduced in the presence of menadione. These results are presented and a possible mechanism for the enzyme is discussed.     

Biochemical,cytotoxic, and genotoxic effects of ES936, a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1, in cellular systems

The specific involvement of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the bioactivation of quinone prodrugs has been shown through the use of the inhibitor of NQO1, dicoumarol. Disadvantages of using dicoumarol to inhibit NQO1 include its lack of specificity and its competitive mechanism of inhibition. The concentration of dicoumarol required for inhibition of NQO1 varies according to the substrate under evaluation, which may lead to either false conclusions of the involvement of NQO1 or the alteration of other cellular processes. We have reported previously on the chemical and biochemical properties of ES936, a mechanism-based inhibitor of NQO1 in cell-free systems. In this study, we investigated the effects of ES936 in cellular systems. ES936 (100 nM) inhibits more than 95% of NQO1 activity within 30 min and is stable in complete media at this concentration for a minimum of 2 h. The duration of inhibition is cell line-specific because a new protein must be generated for resumption of activity. ES936 abrogates the toxicity of streptonigrin, with greater effects seen in cell lines expressing higher levels of NQO1. ES936 does not inhibit other cellular reductases, nor does it alter cellular levels of acid-soluble thiols. Some evidence of DNA strand breaks was observed at the concentrations of ES936 required for the inhibition of NQO1 activity. From our studies, we propose the use of ES936 (100 nM) as a mechanism-based inhibitor of NQO1 in cellular systems and for use as a component of the routine activity assay for NQO1.     

DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase] facilitates redox cycling of menadione in channel catfish (Ictalurus punctatus) cytosol.

Characteristics of DT diaphorase (NAD(P)H: (quinone acceptor) oxidoreductase, DTD) activity in Ictalurus punctatus and the effect of DTD activity on menadione (MND)-mediated reduction of acetylated cytochrome c (AcC) were examined. DTD activity in cytosols of four organs followed a distinct gradient in the order stomach greater than gill greater than liver greater than posterior kidney. A similar gradient was observed in organ-specific rates of in vitro AcC reduction in the presence of either NADH or NADPH as reducing equivalent. A greater proportion of the AcC reduction rate was sensitive to inhibition by dicoumarol (DC) in organs with relatively high DTD specific activity (e.g., stomach) than in organs with low DTD activity (e.g., kidney). No such trend was observed in the superoxide dismutase (SOD)-sensitive proportion of AcC reduction rates. DTD was observed to contribute to MND-mediated superoxide production to a greater extent in organs with high DTD activity than in organs with low DTD activity. DC-sensitive (i.e., DTD-mediated) AcC reduction was observed to increase with organ-specific DTD activity, and the majority of the AcC reduction rate was inhibitable by SOD. These findings demonstrate a direct contribution by DTD activity to MND-mediated superoxide production in this in vitro system. The role of I. punctatus DTD as a possible deleterious agent in quinone metabolism and implications regarding the traditional conception of DTD as a detoxifying enzyme are discussed.     

粘毛黄芩黄酮类成分的研究

粘毛黄芩Scutellaria viscidula Bunge为唇形科黄芩属植物,民间以其根或全草入药,谓其性味苦寒、具有清热、解毒、祛湿、止血的作用,最近临床报道该药对菌痢有显著疗效。鉴于粘毛黄芩的化学成分迄今未见报道,为此我们进行了较系统的研究,在生药的乙醚提取部分通过聚酰胺柱层析,获得8个黄酮甙元结晶,在其甲醇提取部分分离获得2个     

韧黄芩中黄酮类成分的研究

黄芩属植物我国有100多种,多为野生,南北均产。药用黄芩为唇形科黄芩属多种植物干燥的根,不是单一品种,主产于北方的有3种,主产于南方的有4、5种。而正品黄芩     

Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells

Dicoumarol, a competitive inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), increases intracellular superoxide and affects cell growth of tumor cells. This work was set to establish a mechanistic link between dicoumarol, superoxide and cell cycle alterations in HL-60 cells. Using ES936, a mechanism-based irreversible inhibitor of NQO1, we demonstrate that NQO1 inhibition is not a major factor involved in superoxide boost. Mitochondrial Complexes II, III and IV were directly inhibited by dicoumarol. Succinate, which inhibits superoxide generation by reversed electron flow in Complex II, significantly decreased superoxide boost in dicoumarol-treated cells and in isolated mitochondria incubated with dicoumarol and decylubiquinol. Superoxide generation in cells was strongly potentiated by blocking the quinone site of Complex II with thenoyltrifluoroacetone, supporting the involvement of cytochrome b560 to drive electrons for increasing superoxide. Simultaneous inhibition of the mitochondrial chain upstream ubiquinone and displacement of succinate from the Complex II active site is proposed as a major mechanism to explain how dicoumarol increases superoxide in HL-60 cells. Dicoumarol-treated cells accumulated in S phase due to the impairment of pyrimidine biosynthesis at dihydroorotate dehydrogenase step because blockade was overcome by addition of exogenous uridine or orotate, but not by dihydroorotate. We demonstrate for the first time that dicoumarol inhibits mitochondrial electron transport, induces superoxide release by reversed electron flow in Complex II, and inhibits pyrimidines biosynthesis. These actions must be taken into account when considering dicoumarol effects on cells.     

Coumarin-based inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity

The enzyme human NAD(P)H quinone oxidoreductase-1 (NQO1), which is overexpressed in several types of tumor cell, is considered a design target for cancer therapeutics. We identify new coumarin-based competitive inhibitors of NQO1, one of which is nanomolar. Using computational docking and molecular dynamics, we obtain insights into the structural basis of inhibition. Selected inhibitors were then assessed for off-target effects associated with dicoumarol and were found to have differing effects on superoxide formation and mitochondrial respiration. A comparison of NQO1 inhibition and off-target effects for dicoumarol and its derivatives suggests that the ability of dicoumarol to kill cancer cells is independent of NQO1 inhibition, that cellular superoxide production by dicoumarol does not seem linked to NQO1 inhibition but may be related to mitochondrial decoupling, and that superoxide does not appear to be a major determinant of cytotoxicity. Implications are discussed for NQO1 inhibition as an anticancer drug design target and superoxide generation as the dicoumarol-mediated mechanism of cytotoxicity.     

A physiological threshold for protection against menadione toxicity by human NAD(P)H:quinone oxidoreductase (NQO1) in Chinese hamster ovary (CHO) cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) has often been suggested to be involved in cancer prevention by means of detoxification of electrophilic quinones. In the present study, a series of Chinese hamster ovary (CHO) cell lines expressing various elevated levels of human NQO1 were generated by stable transfection. The level of NQO1 over-expression ranged from 14 to 29 times the NQO1 activity in the wild-type CHO cells. This panel of cell lines, allowed investigation of the protective role of NQO1 in quinone cytotoxicity. It could be demonstrated that menadione toxicity was significantly reduced in all NQO1-transfected CHO clones compared to the wild-type cells, but the clones did not show differences in their level of protection against menadione. This observation pointed at a critical threshold concentration of NQO1 above which a further increase does not provide further protection against quinone cytotoxicity. Additional studies in which the NQO1 activity was inhibited by dicoumarol showed that only dicoumarol concentrations of about five times the EC(50) for NQO1 inhibition were able to reduce NQO1 levels below the apparent threshold, making the cells more sensitive. The level of this threshold was estimated to be in the range of base line NQO1 activities observed in several tissues and species. Thus, the results of the present study indicate that beneficial effects of NQO1 induction by, for example, cruciferous vegetables might be absent or present depending on the NQO1 activity threshold for optimal protection and the basal level of NQO1 expression in the tissue and species of interest.     

Dicoumarol relieves serum withdrawal-induced G0/1 blockade in HL-60 cells through a superoxide-dependent mechanism

This work was set to study how dicoumarol affects the cell cycle in human myeloid leukemia HL-60 cells. Cells were accumulated in G0/1 after serum deprivation. However, when cells were treated with 5 microM dicoumarol in serum-free medium, a significant increment in the number of cells in S-phase was observed. Inhibition of G0/1 blockade was confirmed by the increase of thymidine incorporation, the phosphorylation of retinoblastoma protein, and the promotion of cell growth in long-term treatments in the absence of serum. Dicoumarol treatment increased superoxide levels, but did not affect peroxide. Increase of cellular superoxide was essential for inhibition of G0/1 blockade, since scavenging this reactive species with a cell-permeable form of SOD and the SOD mimetics 2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine (ambroxol, 100 microM) and copper[II]diisopropyl salicylate (CuDIPS, 10 microM) completely abolished the effect of dicoumarol. However, N-acetyl-cysteine, overexpression of Bcl-2 or a cell-permeable form of catalase were not effective. 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione (ES936), a mechanism-based irreversible inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), did not promote S phase entry, and dicoumarol still inhibited G0/1 blockade in the presence of ES936. We demonstrate that dicoumarol inhibits the normal blockade in G0/1 in HL-60 cells through a mechanism involving superoxide, but this effect is not dependent solely on the inhibition of the NQO1 catalytic activity. Our results send a precautionary message about use of dicoumarol to elucidate cellular processes involving oxidoreductases.     

Pharmacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells

NAD(P)H quinone oxidoreductase (NQO1) has multiple functions in the cell including an ability to act as a detoxifying enzyme and as a protein chaperone. The latter property is particularly important in oncology as one of the client proteins of NQO1 is p53. The inhibitor, dicoumarol, is classically used to probe the biological properties of NQO1, but interpretation of enzyme function is compromised by the multiple “off-target” effects of this agent. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of recombinant human NQO1 have been identified (Nolan et al., J Med Chem 2009;52:7142-56) The purpose of the work reported here is to demonstrate the functional activity of these agents for inhibiting NQO1 in cells. To do this, advantage was taken of the NQO1-mediated toxicity of the chemotherapeutic drug EO9 (Apaziquone). The toxicity of this drug is substantially reduced when the function of NQO1 is inhibited and many of the coumarin-based compounds are more efficient than dicoumarol for inhibiting EO9 toxicity. The ability to do this appears to be related to their capacity to inhibit NQO1 in cell free systems. In conclusion, agents have been identified that may be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.     

Dissecting the role of multiple reductases in bioactivation and cytotoxicity of the antitumor agent 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1)

2,5-Diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is a novel antitumor diaziridinyl benzoquinone derivative designed to be bioactivated by the two-electron reductase NAD(P)H:quinone oxidoreductase (NQO1) and is currently in clinical trials. NQO1 is expressed at high levels in many solid tumors. RH1 cytotoxicity has been shown previously to be NQO1-dependent. The purpose of this study was to investigate whether other reducing enzymes such as cytochrome b(5) reductase (b5R), cytochrome P450 reductase (P450R), dihydronicotinamide riboside:quinone oxidoreductase 2 (NQO2), and xanthine oxidase/xanthine dehydrogenase (XO/XDH) also contribute to the bioactivation and cytotoxicity of RH1 in human tumor cells. For these studies, we established a series of stable MDA468 breast cancer cell lines overexpressing various levels of NQO1, b5R, P450R, and NQO2 and compared RH1-induced growth inhibition [3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium and sulforhodamine B analysis] and interstrand DNA cross-linking (comet analysis) in both parental MDA468 cells and transfected clones. RH1 toxicity correlated with NQO1 and NQO2 but not with either b5R or P450R activity levels in the respective series of transfected MDA468 cell clones. Enzymatic assays showed that RH1 was an in vitro substrate for xanthine oxidase. However, XO/XDH protein and activity could not be detected in a variety of human tumor cell lines. These studies suggest that NQO1 and NQO2 are the principal enzymatic determinants of RH1 bioactivation in MDA468 tumor cells and that b5R, P450R, and XDH/XO are unlikely to play major roles. Our studies also suggest that NQO2 may be particularly relevant as a bioactivation system for RH1 in NQO1-deficient tumors such as leukemias and lymphomas.     

Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP)-glucuronosyltransferase,and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-quinone oxidoreductase in mice

Ruta graveolens (the common rue) has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP), uridine diphosphate (UDP)-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H):quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg) induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(P)H:quinone oxidoreductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (F_mix) increased Cyp2b activities in males. The mixture of rutin and F_mix increased Cyp1a and Cyp2b activities. These results revealed that rutin and F_mix contributed at least in part to the P450 induction by rue.     

Inhibitory effects of baicalein and wogonin on lipopolysaccharide-induced nitric oxide production in macrophages.

In order to elucidate the mechanism of the antiinflammatory action of baicalein and wogonin, flavonoids from the root of Scutellaria baicalensis, the effects of these compounds were investigated on lipopolysaccharide-induced nitric oxide production in a macrophage-derived cell line, RAW 264.7. Baicalein (5-25 microM) and wogonin (5-50 microM) inhibited lipopolysaccharide-induced nitric oxide generation in a concentration-dependent manner. The inhibitory effect of these compounds was observed only when they were added at the start of cell incubation soon after the stimulation with lipopolysaccharide. Baicalein (25 microM) and wogonin (25 microM) also inhibited protein expression of inducible nitric oxide synthase. This inhibitory effect of wogonin was stronger than that of baicalein, which agrees with the result that wogonin showed stronger inhibition of nitric oxide production than baicalein. These results suggest that baicalein and wogonin attenuate lipopolysaccharide-stimulated nitric oxide synthase induction in macrophages and thus may help to explain the antiinflammatory action of these flavonoid compounds.     

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.

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.     

Role of NAD(P)H:quinone oxidoreductase 1 (DT diaphorase) in protection against quinone toxicity

NQO1-/- mice, along with Chinese hamster ovary (CHO) cells, were used to determine the in vivo role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cellular protection against quinone cytotoxicity, membrane damage, DNA damage, and carcinogenicity. CHO cells permanently expressing various levels of cDNA-derived P450 reductase and NQO1 were produced. Treatment of CHO cells overexpressing P450 reductase with menadione, benzo[a]pyrene-3,6-quinone (BPQ), and benzoquinone led to increased cytotoxicity as compared with CHO cells expressing endogenous P450 reductase. In a similar experiment, overexpression of NQO1 significantly protected CHO cells against the cytotoxicity of these quinones. Knockout (NQO1-/-) mice deficient in NQO1 protein and activity had been generated previously in our laboratory and were used in the present studies. Wild-type (NQO1+/+) and knockout (NQO1-/-) mice were given i.p. injections of menadione and BPQ, followed by analysis of membrane damage and DNA damage. Both menadione and BPQ induced lipid peroxidation in hepatic and non-hepatic tissues, indicating increased membrane damage. Exposure to BPQ also resulted in increased hepatic DNA adducts in NQO1-/- mice as compared with NQO1+/+ mice. The skin application of BPQ alone and BPQ + 12-O-tetradecanoylphorbol-13-acetate (TPA) failed to induce papillomas, or other lesions, for up to 50 weeks in either NQO1+/+ or NQO1-/- mice. The various results from CHO cells and NQO1-/- mice indicated that NQO1 protects against quinone-induced cytotoxicity, as well as DNA and membrane damage. The absence of BPQ-induced skin carcinogenicity in NQO1-/- mice may be related to the strain (C57BL/6) of mice used in the present study and/or due to poor BPQ absorption into the skin and/or due to detoxification of BPQ by cytosolic NRH:quinone oxidoreductase 2 (NQO2).