Cytosolic NADPH-UQ reductase, the enzyme responsible for cellular ubiquinone redox cycle as an endogenous antioxidant in the rat liver.

Cellular ubiquinone (UQ) is expected to act as an endogenous antioxidant against oxidative stress. To confirm this, UQ-reductases which are necessary to regenerate ubiquinol (UQH2) were investigated in rat tissue, and a novel NADPH-dependent UQ (NADPH-UQ) reductase was found in cytosol. The cytosolic NADPH-UQ reductase activity accounted for more than 80% of UQ-10 reduction by the rat liver homogenate in the presence of NADPH. Furthermore, the NADPH-UQ reductase activities in various tissues were correlated to the redox states of UQ in the corresponding tissues. Rat liver cytosol with NADPH protected lecithin liposomes containing UQ-10, as well as UQH2-10 from AMVN (2,2'-azobis(2,4-dimethylvaleronitrile))-induced lipid peroxidation. The enzyme purified from rat liver cytosol, reduced UQ-10 in lecithin liposomes at approximately the same rate as did cytosol. These results supported that cytosolic NADPH-UQ reductase is the enzyme responsible for nonmitochondrial UQ reduction acting as an endogenous antioxidant against oxidative stress. The antioxidant role of the UQ redox cycle and NADPH-UQ reductase was discussed in relation to other cellular NADPH-dependent antioxidant enzymes.     

Reactivity of ubiquinone and ubiquinol with superoxide and the hydroperoxyl radical: implications for in vivo antioxidant activity

Endogenous ubiquinones (UQ) such as coenzyme Q(10) are essential electron carriers in the mitochondrial respiratory chain, and the reduced ubiquinol form (UQH(2)) is a chain-breaking antioxidant, decreasing oxidative damage caused by lipid peroxidation within mitochondria. Consequently, exogenous UQ are used as therapies to decrease mitochondrial oxidative damage. The proximal radical produced during mitochondrial oxidative stress is superoxide (O(2)(.-)) and the reaction between UQ and O(2)(.-) to form the ubisemiquinone radical anion (UQ(.-)) may also be important for the scavenging of O(2)(.-) by exogenous UQ. The situation in vivo is that many UQ are predominantly located in the hydrophobic membrane core, from which O(2)(.-) will be excluded but its conjugate acid, HOO(.), can enter. The reactivity of UQ or UQH(2) with HOO(.) has not been reported previously. Here a pulse radiolysis study on the reactions between UQ/UQH(2) and O(2)(.-)/HOO(.) in water and in solvent systems mimicking the surface and core of biological membranes has been undertaken. O(2)(.-) reacts very rapidly with UQ, suggesting that this may contribute to the scavenging of O(2)(.-) in vivo. In contrast, UQH(2) reacts relatively slowly with HOO(.), but rapidly with other oxygen- and carbon-centered radicals, indicating that the antioxidant role of UQH(2) is mainly in preventing lipid peroxidation.     

Electrochemical modeling of electron and proton transfer to ubiquinone-10 in a self-assembled phospholipid monolayer.

Ubiquinone-10 (UQ) was incorporated at concentrations ranging from 0.5 to 2 mol% in a self-assembled monolayer of dioleoylphosphatidylcholine (DOPC) deposited on a mercury drop electrode, and its electroreduction to ubiquinol (UQH2) was investigated in phosphate and borate buffers over the pH range from 7 to 9.5 by a computerized chronocoulometric technique. The dependence of the applied potential for a constant value of the faradaic charge due to UQ reduction upon the electrolysis time t at constant pH and upon pH at constant t was examined on the basis of a general kinetion approach. This permitted us to conclude that the reduction of UQ to UQH2 in DOPC monolayers takes place via the reversible uptake of one electron with the formation of the semiubiquinone radical anion UQ.-, followed by the rate-determining protonation of this anion with UQH. formation; this neutral radical is more easily reduced than UQ, yielding the ubiquinol UQH2. In spite of the very low concentration of hydrogen ions as compared with that of the acidic component of the buffer, the only effective proton donor is the proton itself; this strongly suggests that the protonation step takes place inside the polar head region of the DOPC monolayer, which is only accessible to protons.     

Effect of Dicumarol,a NAD(P)H: Quinone Acceptor Oxidoreductase 1 (DT-diaphorase) Inhibitor on Ubiquinone Redox Cycling in Cultured Rat Hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 &#119 M dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro, was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 &#119 M dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Effect of dicumarol,a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 μM dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro , was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 μM dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Ubiquinone redox cycle as a cellular antioxidant defense system

Ubiquinone (UQ) reductase responsible for reduction of non-mitochondrial UQ was investigated in rats toward demonstrating an antioxidant role of UQ. In the liver, most of cellular UQ-10 reductase activity was attributable to a novel NADPH-UQ reductase in cytosol. The enzyme was not inhibited by dicumarol and rotenone, and had a Km of 19 microM for NADPH and 307 microM for NADH at the optimum pH 7.4. The enzyme was purified 300-fold to apparent homogeneity from the liver cytosol by an affinity chromatographic method. The purified enzyme reduced UQ-10 in lecithin liposomes, and protected the liposomes from lipid peroxidation. Furthermore, supplementation of rats with UQ-10 was observed to increase the enzyme level in their livers without affecting levels of other antioxidant factors. The observations suggested that cytosolic NADPH-UQ reductase is responsible for cellular UQ redox cycle as an endogenous antioxidant.     

Subcellular distribution of OM cytochrome b-mediated NADH-semidehydroascorbate reductase activity in rat liver

Tissue, cellular, and subcellular distributions of OM cytochrome b-mediated NADH-semidehydroascorbate (SDA) reductase activity were investigated in rat. NADH-SDA reductase activity was found in the post-nuclear particulate fractions of liver, kidney, adrenal gland, heart, brain, lung, and spleen of rat. Liver, kidney, and adrenal gland had higher NADH-SDA reductase activity than other tissues, and OM cytochrome b-dependent activity was 60-70% of the total activity. On the other hand, almost all of the reductase activity of heart and brain cells was mediated by OM cytochrome b. The ratio of the OM cytochrome b-mediated activities of NADH-SDA reductase to rotenone-insensitive NADH-cytochrome c reductase varied among these tissues. OM cytochrome b-mediated NADH-SDA reductase and rotenone-insensitive NADH-cytochrome c reductase activities were mainly present in the parenchymal cells of rat liver. The localization of the cytochrome-mediated reductase activities in the outer mitochondrial membrane was confirmed by subfractionation of liver mitochondria. Among the submicrosomal fractions, OM cytochrome b-mediated NADH-SDA reductase activity was highest in the cis-Golgi membrane fraction, in which monoamine oxidase activity was also highest. On the other hand, OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity showed a slightly different distribution pattern from the NADH-SDA reductase activity. Thenoyltrifluoroacetone (TTFA), a metal chelator, effectively inhibited the NADH-SDA reductase activity, though other metal chelators did not affect the activity. TTFA failed to inhibit rotenone-insensitive NADH-cytochrome c reductase activity at the concentration which gave complete inhibition of NADH-SDA reductase activity.     

Rat strain variations in liver cytosolic DT-diaphorase activity and possible significance of the trait in carcinogenesis by azo dyes

1. Strain variations among female rats in terms of cytosolic DT-diaphorase activity were studied in liver, heart and glandular stomach tissues with or without administration of 3-tert-butyl-4-hydroxyanisole (BHA). 2. BHA induced liver DT-diaphorase activity in all strains examined, and both the basal and induced activities varied according to strain. Among the five strains tested, Brown Norway (BN) and Sprague-Dawley (SD) rats showed relatively high levels of enzyme activity in the liver, whereas Fischer (F344) rats showed a relatively low level of activity. Results of examination of Fischer-BN-F1 rats indicated that a lower level of liver DT-diaphorase activity was inherited essentially as a dominant trait. 3. Liver DT-diaphorase activity in male rats was significantly lower than in female rats. Small strain variations of the activity, if any, were observed in the heart and stomach cytosolic fractions with or without induction by BHA. The magnitude of induction by BHA was also small, if any, in heart and stomach cytosolic fractions. 4. From these and other observations, we discussed the differences between rats and mice in these strain and tissue variations of DT-diaphorase activity, and also the possible significance of liver DT-diaphorase activity in carcinogenesis by azo dyes.     

Mouse strain variations in the magnitude of induction of liver DT-diaphorase and hereditary transmission of the trait

1. Strain variations among mice in terms of cytosolic DT-diaphorase activity were studied in liver, kidney, stomach and heart tissues with or without the administration of 3-tert-butyl-4-hydroxyanisole (BHA). 2. BHA induced DT-diaphorase activity in all strains examined, and the magnitude of induction varied depending on the strain and tissue. Among the 10 inbred strains tested, BALB/c and C57BL mice showed relatively large magnitudes of induction for liver DT-diaphorase, whereas C3H and CBA mice showed relatively small magnitudes. 3. Results of examinations of BALB/c-C3H-F1, -F2 and C57BL-CBA-F1 mice revealed that smaller magnitudes of induction of liver DT-diaphorase were inherited essentially as a dominant trait. The hereditary trait could be adequately explained by postulating two gene loci that regulate the magnitude of induction. 4. The possible significance of DT-diaphorase activity in chemical carcinogenesis was discussed.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Molecular characterization of NAD(P)H:quinone oxidoreductase of tobacco leaves

Summary The NAD(P)H:quinone oxidoreductase activity of tobacco leaves is catalyzed by a soluble flavoprotein [NAD(P)H-QR] and membrane-bound forms of the same enzyme. In particular, the activity associated with the plasma membrane cannot be released by hypoosmotic and salt washing of the vesicles, suggesting a specific binding. The products of the plasma-membrane-bound quinone reductase activity are fully reduced hydroquinones rather than semi-quinone radicals. This peculiar kinetic property is common with soluble NAD(P)H-QR, plasma-membrane-bound NAD(P)H:quinone reductase purified from onion roots, and animal DT-diaphorase. These and previous results demonstrate that soluble and plasma-membrane-bound NAD(P)H:quinone reductases are strictly related flavo-dehydrogenases which seem to replace DT-diaphorase in plant tissues. Following purification to homogeneity, the soluble NAD(P)H-QR from tobacco leaves was digested. Nine peptides were sequenced, accounting for about 50% of NAD(P)H-QR amino acid sequence. Although one peptide was found homologous to animal DT-diaphorase and another one to plant monodehydroascorbate reductase, native NAD(P)H-QR does not seem to be structurally similar to any known flavoprotein.Abbreviations MDAR monodehydroascorbate reductase - PM plasma membrane - NAD(P)H-QR NAD(P)H:quinone oxidoreductase - DPI diphenylene iodonium - DQ duroquinone - CoQ₂ coenzyme Q₂     

Antioxidant enzyme activities in embryologic and early larval stages of turbot

The antioxidant enzymes superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), selenium-dependent glutathione peroxidase (SeGPX; EC 1.11.1.9), glutathione reductase (EC 1.6.4.2) and DT-diaphorase (EC 1.6.99.2), plus total GPX activity (sum of SeGPX and Se-independent GPX activities), were studied in 13 500 g supernatants of embryos and 3-day and 11-day post-hatch larvae of turbot Scophthalmus maximus L. SOD activity decreased progressively during development from embryos to 11-day-old larvae, indicative of a decreased need to detoxify superoxide anion radical (O₂⁻). In contrast, catalase, SeGPX and glutathione reductase activities increased progressively from embryos to 11-day-old larvae, indicative of an increased need to metabolize hydrogen peroxide (H₂O₂) and organic peroxides. Consistent with the latter changes, levels of lipid peroxides (i.e. thiobarbituric acid reactive substances) increased 13-fold from embryos to 3-day-old larvae, whilst total peroxidizable lipid was indicated to decrease. Increases were seen for NADPH-dependent DT-diaphorase (after hatching) and total GPX (between 3 and 11 days post-hatch) activities, whilst no change was found in NADH-dependent DT-diaphorase activity. Overall, the results demonstrate a capacity for early life-stages of S. maximus to detoxify reactive oxygen species (O₂⁻ and H₂O₂) and other pro-oxidant compounds (organic peroxides, redox cycling chemicals). Furthermore, qualitative and quantitative antioxidant changes occur during hatching and development, possibly linked to such events as altered respiration rates (SOD changes) and tissue reorganization and development (catalase, SeGPX, lipid peroxidation).     

DT-diaphorase as a quinone reductase: a cellular control device against semiquinone and superoxide radical formation

Rat liver microsomes incubated in the presence of NADPH catalyze the oxidation of menadione (2-methyl-1,4-naphthoquinone) by two pathways: NADPH-cytochrome P-450 reductase and DT-diaphorase. The former pathway gives rise to labile semiquinones which are readily autooxidized as revealed by a nonstoichiometric NADPH oxidation and a concomitant O₂ consumption. The reduction of menadione catalyzed by DT-diaphorase on the other hand results in a relatively stable hydroquinone accompanied by a stoichiometric oxidation of NADPH and no O₂ consumption. The total amount of NADPH oxidized by a given amount of menadione reflects the relative contributions of the two pathways which can be demonstrated by the addition of selective inhibitors of the two enzymes or by treatment of the rats with phenobarbital or 3-methylcholanthrene which preferentially induces NADPH-cytochrome P-450 reductase and DT-diaphorase, respectively. Addition of cytosol, which contains the bulk of cellular DT-diaphorase, minimizes the formation of semiquinones and the concomitant O₂ consumption. Data relating to other quinones are also presented. The results support the earlier proposal that DT-diaphorase serves as a cellular control device against quinone toxicity.     

Characterization of cellular uptake and distribution of coenzyme Q10 and vitamin E in PC12 cells

Coenzyme Q (CoQ) is a well-known electron transporter in the mitochondrial respiratory chain. Furthermore, ubiquinol (UQH(2))--a reduced form of ubiquinone (UQ)--has been shown to act as a radical-scavenging antioxidant. Some studies have reported the beneficial effect of CoQ addition to cultured cells; however, the cellular uptake and distribution of CoQ have not been elucidated. In the present study, we used rat pheochromocytoma PC12 cells to investigate and compare the cellular uptake and distribution of CoQ(10) and alpha-tocopherol (alphaT). UQ(10) or UQ(10)H(2) treatment resulted in an increase in the cellular content of both CoQ(10) in a time- and concentration-dependent manner. A subcellular fractionation study revealed that the added UQ(10) as well as UQ(10)H(2) mainly localized in the mitochondrial fraction, which is similar to the localization of endogenous CoQ but different from that of alphaT. The cellular distribution of alphaT directly corresponded to the lipid distribution, while the CoQ distribution did not show any relationship with the lipid distribution, particularly in the mitochondrial and microsomal fractions. These results indicate that the cellular distribution of CoQ is completely different from that of alphaT; moreover, a certain system which accumulates CoQ preferentially in mitochondria may be suggested.     

Diurnal changes in the fraction of 3-hydroxy-3-methylglutaryl-CoA reductase in the active form in rat liver microsomal fractions.

'Initial' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in cold-clamped samples of liver from rats at 2h intervals throughout the 24h light/dark cycle. Initial activities were obtained in microsomes (microsomal fractions) isolated and assayed in the presence of 100mM-KF, whereas 'total' activities were measured in microsomes prepared from the same homogenates but washed free of KF and incubated with exogenous partially purified rat liver protein phosphatase. The initial/total-activity ratio for HMG-CoA reductase underwent a diurnal cycle, which had a nadir 4h into the light phase (when initial activity was 28% of total activity) and a peak 12h later, i.e. 4h into the dark phase (when initial activity was 80% of total activity). These low and high points of the cycle were separated by gradual steady changes in the ratio. The characteristics of this diurnal cycle were different from those of the cycle observed for total activity, which had a plateau of high activity between 2 and 10h into the dark cycle preceded and succeeded by a very rapid increase and decrease, respectively, in the total activity of HMG-CoA reductase. The combination of the two cycles resulted in the dampening of the resultant cycle for the initial or effective activity of HMG-CoA reductase, such that the changes in initial activity around the beginning and and end of the dark phase were more gradual than would otherwise have been the case if the initial/total-activity ratio for HMG-CoA reductase were constant throughout the diurnal cycle. The physiological implications of the observed diurnal variation in the fraction of hepatic HMG-CoA reductase in the active form are discussed.     

Vitamin K antagonism of coumarin anticoagulation. A dehydrogenase pathway in rat liver is responsible for the antagonistic effect.

In the liver, it appears that there are two different pathways for vitamin K reduction. One pathway is irreversibly inhibited by coumarin anticoagulant drugs. The other pathway has been shown in the present study to be composed of enzymes that are not effected by physiological 'in vivo' concentrations of these drugs. This pathway appears to be responsible for the antidotal effect of vitamin K in overcoming coumarin poisoning. In rat liver the pathway has been shown to be composed of DT-diaphorase (EC.1.6.99.2) and a microsomal dehydrogenase(s). The activity of the microsomal dehydrogenase(s) was 3.6-fold higher with NADH than with NADPH present in the test system. It appears that this enzyme is the physiologically important enzyme in the pathway. In contrast with DT-diaphorase, this enzyme(s) is shown to be tightly associated with the mirosomal membrane. The enzyme(s) is not identical with either of the quinone-reducing enzymes cytochrome P-450 reductase or cytochrome-b5 reductase. Our data thus postulate the existence of an as-yet-unidentified microsomal dehydrogenase that appears to have an important function in the pathway.     

Effects of diabetes on the expressed and total activities of 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver in vivo. Reversal by insulin treatment.

The expressed and total activities of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase (EC 1.1.1.34) were measured in microsomal fractions prepared from cold-clamped liver samples [Easom & Zammit (1984) Biochem. J. 220, 733-738] from control or insulin-treated diabetic animals. Streptozotocin-induced diabetes resulted in a marked decrease in total activity of HMG-CoA reductase and in the fraction of the enzyme in the active form, but appreciable effects were only observed in the liver of animals in which the blood glucose was above 20 mM. Intravenous infusion of insulin into diabetic rats resulted in a rapid (less than 20 min) and total dephosphorylation of the enzyme in vivo without any change in total activity. Longer-term (4 h) treatment with insulin (injected intraperitoneally) produced a rapid increase in expressed/total HMG-CoA reductase activity ratio to about 90%, followed, after a lag of 2-3 h, by a 5-6-fold increase in total activity. These observations are discussed with respect to the possible role of insulin in generating and maintaining the respective diurnal rhythms in total and in expressed/total HMG-CoA reductase activity ratio observed for normal animals in vivo [Easom & Zammit (1984) Biochem. J. 220, 739-745].     

Modulatory effect of Urtica dioica L. (Urticaceae) leaf extract on biotransformation enzyme systems, antioxidant enzymes, lactate dehydrogenase and lipid peroxidation in mice.

The effects of two doses (50 and 100 mg/kg body wt given orally for 14 days) of an ethanol-water (80%-20%) extract of Urtica dioica L. and butylated hydroxyanisole (BHA) were investigated, for phase I and phase II enzymes, antioxidant enzymes, lactate dehydrogenase, lipid peroxidation and sulfhydryl groups in the liver of Swiss albino mice (8-9 weeks old). A modulatory effect of two doses and BHA was also observed for the activities of glutathione S-transferase, DT-diaphorase, superoxide dismutase and catalase in the kidney, lung and forestomach, as compared with the control group. The activities of cytochrome b5 (cyt b5), NADH-cytochrome b5 reductase (cyt b5 R), glutathione S-transferase (GST), DT-diaphorase (DTD), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) showed a significant increase in the liver at both dose levels of extract. Both extract-treated showed significantly lower activity of cytochrome P450 (cyt P450), lactate dehydrogenase (LDH), NADPH-cytochrome P450 reductase (cyt P450 R), total sulfhydryl groups (T-SH), nonprotein sulfhydryl groups (NP-SH) and protein-bound sulfhydryl groups (PB-SH). BHA-treated Swiss albino mice showed a notable increase in levels of cyt b5, DTD, T-SH, PB-SH, GPx, GR, and SOD in the liver while, LDH, cyt P450, cyt P450 R, Cyt b5 R, GST, NP-SH, and CAT levels were reduced significantly as compared to control values. The extract was effective in inducing GST, DTD, SOD and CAT activity in the forestomach and SOD and CAT activity in the lung at both dose levels. BHA-treated Swiss albino mice induced DTD, GST and all antioxidative parameters in the kidney, lung and forestomach.     

Reduction of Brain Antioxidant Defense Upon Treatment with Butylated Hydroxyanisole (BHA) and Sudan III in Syrian Golden Hamster

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1. 4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.Deceased 4th November 1998     

NAD(P)H: quinone oxidoreductase 1 expression in kidney podocytes.

NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase; DTD) is a cytosolic two-electron reductase, and compounds of the quinone family such as mitomycin C are efficiently bioactivated by this enzyme. The observation that DT-diaphorase is highly expressed in many cancerous tissues compared to normal tissues has provided us with a potentially selective target that can be exploited in the design of novel anticancer agents. Because of the relative lack of information about the cell-specific expression of DT-diaphorase, the purpose of this study was to map the distribution of this enzyme in normal human tissues. Fifteen tissue samples from normal human kidney were analyzed for expression of DT-diaphorase by immunohistochemistry (two-step indirect method). We found a specific high expression of DT-diaphorase in glomerular visceral epithelial cells (podocytes). These results suggest that a high expression of DT-diaphorase in podocytes could play a major role in the pathogenesis of renal toxicity and mitomycin C-induced hemolytic uremic syndrome, in which injury to the glomerular filtration mechanism is the primary damage, leading to a cascade of deleterious events including microangiopathic hemolytic anemia and thrombocytopenia. This observation has potential therapeutic implications because the DT-diaphorase metabolic pathway is influenced by many agents, including drugs, diet, and environmental cell factors such as pH and oxygen tension.