Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K₁ epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase. E.C. I.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes. but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K₁ epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K, 10^?5 as rapidly as it reduced dichlorophenylindophenol(DCPIP). Reduction of 10 μM vitamin K, by200 μM NADH was not inhibited by 10μM dicoumarol. whereas DCPIP reduction was fully inhibited. In contrast to vitamin K, (menadione). vitamin K₁ (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions. suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K₁ epoxide and quinone. and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin. lapachol. and sulphaquinoxaline     

A note on the inhibition of DT-diaphorase by dicoumarol.

The participation of DT-diaphorase or NAD(P)H:(quinone acceptor) oxidoreductase (E.C. 1.6.99.2) in metabolism or in events leading to toxicity is often implied on the basis of the inhibitory effects of dicoumarol. DT-diaphorase functions via a ping pong bi-bi kinetic mechanism involving oxidized and reduced flavin forms of the free enzyme. Dicoumarol, a potent (Ki = 10 nM) inhibitor, binds to the oxidized form of the enzyme, competitively versus reduced pyridine nucleotide. Inhibition is effectively complete at 1 microM dicoumarol in typical studies using DCPIP, one of the best known substrates for the enzyme, as electron acceptor. The antitumor quinone Diaziquone (AZQ) is a poor substrate for DT-diaphorase relative to DCPIP, but effective inhibition of its reduction requires ten-fold higher concentrations of dicoumarol than for inhibition of DCPIP reduction under otherwise similar conditions. The variable inhibition of DT-diaphorase by dicoumarol dependent on the efficiency of the electron acceptor can be explained on the basis of the complete rate equation describing its ping pong type kinetic mechanism. Thus, the concentration of dicoumarol used to inhibit DT-diaphorase must be chosen carefully and consideration should be given to the efficiency of the electron acceptor. The absence of an inhibitory effect using low doses of dicoumarol cannot rule out a reaction mediated by DT-diaphorase. Although higher doses of dicoumarol may be required to inhibit DT-diaphorase mediated metabolism of less efficient electron acceptors, the use of such doses in cells may also affect biochemical processes other than DT-diaphorase and should be approached with caution.     

Epidermal Growth Factor Stimulates Nuclear Factor-κB Activation and Heme Oxygenase-1 Expression via c-Src,NADPH Oxidase,PI3K,and Akt in Human Colon Cancer Cells

Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47^phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation.     

Determinants of MTT reduction in rat hepatocytes

The determinants of reduction of the dye MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in rat hepatocytes have been investigated. NADH, NADPH, and succinate were substrates for MTT reduction in rat liver homogenate, activity being greatest with NADH and least with succinate. Similar results were obtained with submitochondrial particles isolated from rat liver. NAD(P)Hdependent reduction of MTT was also detected in rat liver microsomes and cytosol. Rotenone, at a concentration that inhibited NAD(P)H-dependent MTT reduction in submitochondrial particles, did not inhibit MTT reduction in rat hepatocytes. Malonate, at a concentration that inhibited succinate-dependent MTT reduction in liver homogenate, did not inhibit MTT reduction in rat hepatocytes. Incubation of rat hepatocytes with ethanol or lactate (increase NADH levels), dicoumarol (inhibitor of DT-diaphorase), aminopyrine or hexobarbitone (substrates for the NADPH-requiring cytochrome P450-dependent microsomal monooxygenase) led to significant increases in the level of cellular MTT reduction. From these data, it is concluded that extramitochondrial NAD(P)H is the principal reductant for MTT reduction in rat hepatocytes, with mitochondrial dehydrogenase activity being only a minor contributor. It is also possible that cellular generation of superoxide (as might be expected on redox cycling of endogenous quinones following inhibition of DT diaphorase by dicoumarol) may be another source of MTT reduction. Caution should be exercised in ascribing an alteration in the level of cellular MTT reduction to a change in mitochondrial performance in the absence of corroborating evidence.     

In vitro inhibition of the metabolism and mutagenicity of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by naphthazarin and other naphthol derivatives

Among naphthol derivatives tested in the Ames assay, 5,8-dihydroxy-1,4-naphthoquinone or naphthazarin was found to be the most effective inhibitor of benzo(a)pyrene mutagenicity. The inhibitory activity is due in part to the redox cycling of naphthazarin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen, thus diverting electrons from cytochrome P-450 enzymes. Metabolite separations showed a decrease in microsomal metabolism of benzo(a)pyrene and of benzo(a)pyrene-7,8-dihydrodoil upon addition of naphthazarin. Since both NADP and dicoumarol inhibited the naphthazarin-stimulated non-stoichiometric consumption of NADPH and oxygen then naphthazarin redox cycling probably involves both DT-diaphorase and NADPH cytochrome P-450 reductase.     

Pre-CFU-f: young-type stromal stem cells in murine bone marrow following administration of DNA inhibitors

Occurrence of young-type stromal stem cells (defined here as "pre-CFU-f") in murine bone marrow is reported in this study. Two consecutive intraperitoneal (i.p.) cytosine arabinoside (ara-C) injections were administered to C57B1 mice (2 X 200 mg/kg at 6-h intervals). Two days later the bone marrow was collected and assayed for colony-forming units-fibroblastoid (defined here as "CFU-f"). In additional experiments, ara-C-treated marrow was exposed in vitro to hydroxyurea (HU; "hydroxyurea killing test"), prior to plating, to establish the cycling state of stromal stem cells. In separate cultures of ara-C-treated marrow, replating of adherent cells was carried out up to quaternary sub-cultures. The results indicate ara-C-treated marrow produces approximately 20% "huge" fibroblastoid colonies (approximately 5 mm diameter versus 0.5-2 mm normal size); most stromal stem cells producing huge colonies are cycling cells; and adherent cells from primary ara-C-treated marrow cultures replated to secondary cultures produce adherent layers with double the number of cells than in the control secondary cultures. We conclude that the ara-C-treated murine bone marrow contains certain young-type cycling stromal stem cells which we refer to as pre-CFU-f. These stem cells produce huge fibroblastoid colonies in culture, indicating that they probably go through more cell cycles than CFU-f during the culture period. Alternatively, pre-CFU-f may have a higher self-replicative capacity than CFU-f.     

Dicoumarol-sensitive glucuronidation of benzo(a)pyrene metabolites in rat liver microsomes

The effect of dicoumarol on glucuronidation of 3-OH-benzo(a)pyrene (BP) appears to be due to inhibition of UDPglucuronosyltransferase (UDPGT) and not to an inhibited DT-diaphorase (NAD(P)H:quinone oxidoreductase); to date the only enzyme known to be inhibited by dicoumarol. This dicoumarol-sensitive form of UDPGT does not seem to be identical to the major form catalyzing the glucuronidation of p-nitrophenol or methylumbelliferone, nor to the isozyme involved in the formation of phenolphthalein glucuronides. These conclusions are based on the following observations: In solubilized microsomes, devoid of DT-diaphorase, a 3-OH-BP glucuronidation activity is found which is very similar to that observed in microsomes before passing through an azodicoumarol Sepharose 6B column that binds more than 98% of DT-diaphorase; in the eluate from this column the inhibition by dicoumarol of 3-OH-BP glucuronidation is the same as in microsomes containing DT-diaphorase; other coumarin derivatives, which are either modified or substituted in the methylene bridge between the two coumarin entities in dicoumarol, are potent inhibitors of DT-diaphorase but not of UDPGT; a concentration of 10(-6) M dicoumarol is sufficient to inhibit 3-OH-BP glucuronidation 50%. In contrast, to inhibit glucuronidation of p-nitrophenol or methylumbelliferone the concentration of dicoumarol must be raised to the substrate level: i.e., 10(-4) M. Phenolphthalein glucuronidation is almost unaffected even by this high concentration of dicoumarol. The present investigation also reveals that DT-diaphorase and NADPH-cytochrome P-450 reductase can both catalyze the reduction of BP-3,6-quinone for the formation of BP-3,6-quinol glucuronides. In the eluate from the azodicoumarol Sepharose 6B column, no NADH-supported glucuronidation of BP-3,6-quinone can be detected unless DT-diaphorase is added. However, NADPH-supported formation of BP-3,6-quinol glucuronides can still be observed. The rate of the latter reaction is sufficient enough to allow studies on the effect of dicoumarol on BP-3,6-quinone glucuronidation. These results show that glucuronidation of BP-3,6-quinols is also catalyzed by a dicoumarol-sensitive UDPGT. However, not only is the formation of BP-3,6-quinol monoglucuronides inhibited by dicoumarol, but the conversion of monoglucuronides to diglucuronides is inhibited as well. The former reaction is inhibited 50% by 3.5 X 10(-6) M dicoumarol (close to the I50 for 3-OH-BP glucuronidation), whereas 10 times less dicoumarol (2 X 10(-7) M) is sufficient for 50% inhibition of the latter reaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6(-/-) mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.     

Quinone-induced apoptosis in human colon adenocarcinoma cells via DT-diaphorase mediated bioactivation

DT-diaphorase (DTD) activity has been related to bioactivation and cytotoxicity of antitumor quinones. A pair of human colon adenocarcinoma cell lines, HT29 and BE, were used in this study to examine the role of DTD in antitumor quinone induced apoptosis. HT29 cells have elevated levels of DTD whereas BE cells lack functional DTD due to a point mutation which results in a complete lack of DTD activity. MeDZQ, a quinone that is efficiently bioactivated by DTD, induced apoptosis both in HT29 and BE cells, but with a much higher incidence in HT29, as assessed by morphological criteria and the formation of oligonucleosomal fragments of DNA. Two other quinone compounds which are also substrates for DTD, i.e. streptonigrin and mitomycin C, also preferentially induced apoptosis in HT29 cells, which could be inhibited by dicoumarol. Our data suggest that bioreductive activation of antitumor quinones by DTD results in induction of apoptosis in human colon carcinoma cells.     

TNF inhibits production of stromal cell-derived factor 1 by bone stromal cells and increases osteoclast precursor mobilization from bone marrow to peripheral blood

Introduction

The objective of the present study was to investigate the role of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis in TNF-induced mobilization of osteoclast precursors (OCPs) from bone marrow.

Methods

OCPs were generated from bone marrow cells of TNF-transgenic mice or wild-type mice treated with TNF or PBS. The percentage of CD11b⁺/Gr-1^-/lo OCPs was assessed by fluorescence-activated cell sorting. OCP migration to the SDF-1 gradient and the osteoclast forming potency were assessed in chemotaxis/osteoclastogenic assays. SDF-1 expression was assessed by real-time RT-PCR, ELISA and immunostaining in primary bone marrow stromal cells, in the ST2 bone marrow stromal cell line, and in bones from TNF-injected mice.

Results

OCPs generated in vitro from wild-type mice migrated to SDF-1 gradients and subsequently gave rise to osteoclasts in response to RANKL and macrophage colony-stimulating factor. TNF reduced SDF-1 expression by ST2 cells. Bone marrow stromal cells from TNF-transgenic mice produced low levels of SDF-1. TNF treatment of wild-type mice decreased the SDF-1 concentration in bone marrow extracts and decreased the SDF-1 immunostaining of bone marrow stromal cells, and it also increased the circulating OCP numbers. The percentage of bone marrow CXCR4⁺ OCPs was similar in TNF-transgenic mice and wild-type littermates and in TNF-treated and PBS-treated wild-type mice.

Conclusion

Systemically elevated TNF levels inhibit bone marrow stromal cell production of SDF-1 and increase the release of bone marrow OCPs to the peripheral blood. Disruption of the SDF-1/CXCR4 axis by TNF may play an important role in mediating OCP mobilization from the bone marrow cavity in chronic inflammatory arthritis.     

HFCL细胞对U937细胞的诱导分化作用

为了观察正常人骨髓成纤维样细胞系HFCL对急性单核细胞白血病U937细胞促分化作用,及其对经典诱导分化剂TPA诱导分化作用的影响,先建立U937细胞和HFCL细胞共培养体系,以细胞形态学改变、硝基四氦唑蓝(NBT)、流式细胞仪检测细胞周期和CD11b、CD13、CD14、CD33细胞表面抗原作为诱导分化指标;Western印迹检测P38蛋白的表达变化。结果发现,与HFCL细胞共培养后,U937细胞出现分化成熟的形态学改变,且与HFCL细胞直接接触组的诱导分化作用大于用transwell组。同时发现U937细胞与HFCL细胞共培养后,G1期细胞增高,S期细胞减少;CD11b、CD13、CD14和CD33表达增高;且NBT阳性细胞增高至46、3％。Western印迹检测结果显示,直接接触组总P38蛋白表达增加。而且HFCL细胞还能增强TPA对U937的诱导分化作用。     

In vitro genotoxicity of danthron and its potential mechanism

To ascertain the in vitro genotoxicity of danthron and its potential mechanism of action, we performed an Ames test, a cytokinesis-block micronucleus assay and a comet assay in Balb/c 3T3 cells. The Ames test revealed that danthron was mutagenic only toward Salmonella typhimurium strain TA102 in the presence of an exogenous metabolic activation system (S9 mix). Danthron (25, 50 and 100μg/ml) increased the frequencies of micronuclear cells with or without S9 mix, and the comet length, tail length and Olive tail moment in comet assays without S9 mix in a dose-dependent manner. These results demonstrated the in vitro genotoxicity of danthron and that 3T3 cells are capable of activating danthron. When NADP was replaced by NAD in the S9 mix, danthron remained mutagenic toward strain TA102. The addition of dicoumarol, a DT-diaphorase inhibitor, decreased the number of danthron-induced histidine revertants by 35-39%, indicating that DT-diaphorase is involved in the metabolic activation of danthron in the presence of NADH as an electron donor. In 3T3 cells, increases in reactive oxygen species (ROS) formation and 8-hydroxydeoxyguanosine levels as well as a reduction in GSH levels were induced by danthron in a dose-dependent manner, indicating that oxidative stress may be a major contributing pathway in the genotoxicity of danthron.     

Sialoadhesin expression by bone marrow macrophages derived from Ehrlich-tumor-bearing mice

Sialoadhesin (sheep erythrocyte receptor, SER) is a macrophage-restricted adhesion molecule that binds certain sialylated ligands. It is borne by bone marrow stromal macrophages, promoting the interaction with developing myeloid cells, and by a subset of tissue macrophages involved in antigen presentation and activation of tumor-reactive T cells. The expression of sialoadhesin on SER⁺ macrophages is not constitutive but requires the continuous supply of a sialoadhesin-inducing serum factor. Tumor growth is often associated with marked alterations of myelopoiesis and impairment of T cell activation; yet the expression of sialoadhesin in macrophages derived from tumor bearers has not been addressed. The aim of this study was to assess whether Ehrlich tumor (ET) – a murine mammary carcinoma – growth may modify the sialoadhesin expression by bone marrow macrophages and/or sialoadhesin-inducing activity in ET-bearing sera. Moreover, putative functional sialoadhesin inhibitors produced by ET cells were tested. The results indicate that bone marrow cells from ET bearers show a seven- to eight-fold decrease in SER⁺ cells as detected by flow cytometry. This is accompanied by an overall decrease in sheep erythrocyte binding to tumor-bearer-derived bone marrow cells, but also by lower numbers of plastic-adherent cells. Functional sialoadhesin expression is preserved at the single-cell level and no inhibitors are found in ET-bearing sera or ET cell culture supernatants. Tumor progression does not impair the sialoadhesin-inducing activity of ET-bearing sera, or the ability of SER⁻ macrophages (e.g. peritoneal macrophages) to respond to such an induction. In conclusion, while SER⁺ macrophages are greatly decreased in bone marrow from ET bearers, this is not due to a down-regulation of sialoadhesin expression, nor to an impairment of sialoadhesin-inducing factor or to the presence of sialoadhesin-binding moieties of tumor origin, but, more likely, to a decrease of fully mature macrophages. Received: 18 March 1999 / Accepted: 22 July 1999     

The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form

The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells. Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells. The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones. It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts. The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow). In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.     

Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells

This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.     

Study of DT-diaphorase in pigment-producing cells.

DT-diaphorase is an FAD-containing enzyme capable of a two-electron reduction of ortho- and paraquinones. Nicotinamide coenzymes (NADH + H+ and NADPH + H+) serve as hydrogen sources in these reactions. The role of DT-diaphorase has been thoroughly investigated in situations when the enzyme is able to reduce exogenous and endogenous quinones, hence protecting the cells against these reactive intermediates. The enzyme has also been studied in connection with its ability to activate some quinoid cytostatics. It is surprising that DT-diaphorase has never been investigated in pigment-producing cells that are known to generate considerable amounts of ortho-quinones. Using a spectrophotometric method we could readily measure the activity of DT-diaphorase in epidermis and various cultured pigment cells. The melanocytes isolated from dark skin showed generally higher DT-diaphorase activity than those from fair skin samples. Also, darkly pigmented congenital naevus cells exhibited higher activity of this enzyme. The most striking was the high DT-diaphorase activity in melanoma cell cultures. In these cells DT-diaphorase activity could be induced by incubation of the cells with 4-hydroxyanisole. A similar effect was seen when a catechol-O-methyltransferase (COMT) inhibitor (3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione (OR-462) was utilised. The induction was inhibited by cyclohexidine.     

The nature of DT-diaphorase (EC 1.6.99.2) activity in plasma membrane of astrocytes in primary cultures

This is the confirmation of an earlier indication (Mersel, M., Malviya, A.N., Hindelang, C. and Mandel, P. (1984) Biochim. Biophys. Acta 778, 144-154) that the plasma membrane of astrocytes in primary cultures is endowed with DT-diaphorase (EC 1.6.99.2) activity. It is observed that the NADPH-2,6-dichloroindophenol diaphorase activity found in the isolated plasma membrane is not inhibited by dicoumarol. DT-diaphorase-type activity is also observed on the cell surface employing dichloroindophenol as external electron acceptor and it is found to be a dicoumarol-sensitive NADH dehydrogenase.     

The reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells: Free radical formation and NAD(P)H: Quinone-acceptor oxidoreductase (DT-diaphorase) activity

The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ.