Christina Lind

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

Abstract 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.

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase.

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.

Metabolism of benzo(a)pyrene-3,6-quinone and 3-hydroxybenzo(a)pyrene in liver microsomes from 3-methylcholanthrene-treated rats: A possible role of DT-diaphorase in the formation of glucuronyl conjugates

Abstract Benzo( a )pyrene (BP) quinones and 3-OH-BP are the metabolites preferentially converted to glucuronyl conjugates when BP is metabolized by microsomes from 3-methyl-cholanthrene (MC)-treated rats in the presence of NADPH, O 2 , and UDP-glucuronic acid (UDPGA). No glucuronyl conjugates of [ 14 C]BP-3,6-quinone are formed in the absence of NAD(P)H, indicating that BP quinones must be reduced prior to glucuronylation. The observations that NADH can replace NADPH in BP-3,6-quinone glucuronylation and that these reactions are equally sensitive to dicoumarol, a potent inhibitor of DT-diaphorase, suggest that the reduction of BP-3,6-quinone preceding glucuronylation is catalyzed by DT-diaphorase. Furthermore, trypsin-treated microsomes, which have unchanged DT-diaphorase activity but less than 5% of original NADPH-cytochrome c reductase activity, exhibit unaltered capacity for the conversion of BP-3,6-quinone to glucuronyl conjugates. Analysis of ethyl acetate extracts from incubations with [ 14 C]BP-3,6-quinone by high pressure liquid chromatography reveals that BP-3,6-quinone can be further metabolized by MC-induced microsomes to more polar but still water-insoluble products. These metabolites are not formed in the absence of NADPH or in trypsin-treated microsomes, which have no detectable aryl hydrocarbon monooxygenase (AHM) activity, indicating that the further metabolism of BP-3,6-quinone proceeds through the AHM system. The rate of this reaction and of glucuronylation of BP-3,6-quinone is very similar. The glucuronylation of 3-OH-BP by MC-induced microsomes is also inhibited by dicoumarol, whereas that of p -nitrophenol, methylumbelliferone or phenolphtalein is not. Trypsin treatment of microsomes strongly enhances 3-OH-BP glucuronylation. Evidence is presented suggesting that the dicoumarol effect is probably not due to an inhibition of UDP-glucuronosyltransferase, and the possibility is considered that a DT-diaphorase dependent rearrangement of 3-OH-BP prior to glucuronylation may be responsible for the observed dicoumarol sensitivity.

Effect of superoxide dismutase on the autoxidation of various hydroquinones. A possible role of superoxide dismutase as a superoxide: semiquinone oxidoreductase

The autoxidation of DT-diaphorase-reduced 1,4-naphthoquinone, 2-OH-1,4-naphthoquinone, and 2-OH-p-benzoquinone is efficiently prevented by superoxide dismutase. This effect was assessed in terms of an inhibition of NADPH oxidation (over the amount required to reduce the available quinone), O2 consumption, and H2O2 formation. Superoxide dismutase also affects the distribution of molecular products -hydroquinone/quinone-involved in autoxidation, by favoring the accumulation of the reduced form of the above quinones. In contrast, the rate of autoxidation of DT-diaphorase-reduced 1,2-naphthoquinone is enhanced by superoxide dismutase, as shown by increased rates of NADPH oxidation, O2 consumption, and H2O2 formation and by an enhanced accumulation of the oxidized product, 1,2-naphthoquinone. These findings suggest that superoxide dismutase can either prevent or enhance hydroquinone autoxidation. The former process would imply a possible new activity displayed by superoxide dismutase involving the reduction of a semiquinone by O2-.. This activity is probably restricted to the redox properties of the semiquinones under study, as indicated by the failure of superoxide dismutase to prevent autoxidation of 1,2-naphthohydroquinone.

Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol: I. Purification of cytosolic DT-diaphorase from control and 3-methylcholanthrene-treated rats

Abstract Liver cytosolic DT-diaphorase has been purified from both control and 3-methylcholanthrene (MC)-treated rats, employing a new efficient affinity chromatography gel azodicoumarol coupled to divinyl sulfone cross-linked Sepharose 6B. A subsequent gel filtration on Sephacryl S-200 results in a homogeneous enzyme preparation with a yield of 50–55%. The enhanced DT-diaphorase activity observed in MC-treated rats is most probably due to an increase in enzyme concentration. This conclusion is based on the following results: (a) cycloheximide, an inhibitor of protein synthesis, prevents the increase in DT-diaphorase activity normally caused by MC; (b) purifications of DT-diaphorase from control and MC-treated rats result in enzyme preparations exhibiting closely similar specific activities; 15 times higher amounts of enzyme are obtained from MC-treated rats as compared to controls; (c) immunological identity between DT-diaphorase isolated from control and MC-treated rats are found with the Ouchterlony immunodiffusion method employing antisera raised against either type of enzyme preparation; (d) rocket immunoelectrophoresis reveals a severalfold higher specific content of DT-diaphorase in cytosol from MC-induced rats as compared to controls. The investigated physicochemical properties of DT-diaphorase are not altered after MC treatment of rats. The minimum molecular weight based on the flavin content of the enzyme is close to that obtained with SDS-slab gel electrophoresis, i.e., approximately 28,000, indicating that the dimer of DT-diaphorase contains two molecules of FAD. The molecular activities of DT-diaphorase with various electron acceptors have been investigated; no significant differences between DT-diaphorase isolated from control and MC-treated rats are found. However, the molecular activity of the enzyme with 2,6-dichlorophenolindophenol and menadione varies considerably from one preparation to another, irrespective of source. Employing fused rocket immunoelectrophoresis, it has been possible to detect at least three antigenic forms of DT-diaphorase with different reactivities toward electron acceptors such as 2,6-dichlorophenolindophenol and menadione. The possible existence of several forms of DT-diaphorase is discussed.

Benzo(α)pyrene quinones can be generated by lipid peroxidation and are conjugated with glutathione by glutathione S-transferase B from rat liver

Abstract We have investigated the effect of adding purified glutathione S -transferases A, B, and C, to rat liver microsomes metabolizing benzo(α)pyrene. Glutathione S -transferase B was found to be effective in metabolizing benzo(α)pyrene 1,6 and 3,6 quinones to a water soluble metabolite presumably a glutathione conjugate. Also we show that non-enzymic lipid peroxidation in microsomes by ascorbate and ADP-Fe2+ in the presence of benzo(α)pyrene gives rise chiefly to quinones derived from this compound.

DT-diaphorase-catalyzed two electron reduction of quinone epoxides

DT-diaphorase catalyzes the two-electron reduction of the unsubstituted quinone epoxide, 2,3-epoxy-p-benzoquinone, at expense of NAD(P)H with formation of 2-OH-p-benzohydroquinone as the reaction product. The further conversion reactions of 2-OH-p-benzohydroquinone are influenced by the presence of O2 in the medium. Under aerobic conditions, 2-OH-p-benzohydroquinone undergoes autoxidation--probably with formation of 2-OH-semiquinone intermediates--to 2-OH-p-benzoquinone. The latter product is rapidly reduced by DT-diaphorase and, thus, its accumulation can be only observed upon exhaustion of NADPH. Under anaerobic conditions, 2-OH-p-benzohydroquinone does not undergo autoxidation and its accumulation is stoichiometrically (1:1) related to the amount of NADPH oxidized and epoxide substrate reduced. DT-diaphorase also catalyzes the reduction of the disubstituted quinone epoxide, 2,3-dimethyl-2,3-epoxy-1,4-naphthoquinone. Neither the aliphatic epoxide, trans-stilbene oxide, nor the aromatic epoxide, 4,5-epoxy-benzo[a]pyrene are substrates for DT-diaphorase. The reduction of 2,3-epoxy-p-benzoquinone is also catalyzed by the one-electron transfer enzyme, NADPH-cytochrome P450 reductase at a rate similar to that found with DT-diaphorase. However, this reaction differs from that catalyzed by DT-diaphorase in the distribution of molecular products as well as in the relative contribution of nonenzymatic reactions, i.e. semiquinone disproportionation and autoxidation.

Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol: II. Purification of mitochondrial and microsomal DT-diaphorase from 3-methylcholanthrene-treated rats

Abstract Liver mitochondrial and microsomal DT-diaphorase have been purified from 3-methylcholanthrene-treated rats. A 1150-fold and 3500-fold purification of mitochondrial and microsomal DT-diaphorase, respectively, is achieved after solubilization of the membranes with deoxycholate followed by affinity chromatography on azodicoumarol Sepharose 6B and subsequent gel filtration on Sephadex G-100. From this purification procedure, 65–70% of mitochondrial DT-diaphorase is recovered and the purified enzyme has a specific activity comparable to that of cytosolic DT-diaphorase; i.e., 50.4 kat/kg protein. Microsomal DT-diaphorase is obtained with a yield of 45% and a specific activity of 15.5 kat/kg protein. Purified mitochondrial DT-diaphorase exhibits an absorption spectrum characteristic of a flavoprotein and very similar to that of the cytosolic enzyme. Purification of both mitochondrial and microsomal DT-diaphorase results in fractions enriched in a polypeptide with a molecular weight of 28,000 which comigrates with purified cytosolic DT-diaphorase on SDS-polyacrylamide gel electrophoresis. Employing antiserum raised against cytosolic DT-diaphorase, immunological identity between DT-diaphorase isolated from the three cell fractions is observed with both the Ouchterlony immunodiffusion technique and fused rocket immunoelectrophoresis. The latter method also reveals that DT-diaphorase isolated from mitochondria and microsomes contains several antigenic forms identical to those observed in purified cytosolic DT-diaphorase. Furthermore, this antiserum inhibits DT-diaphorase to about the same extent whether the enzyme is isolated from mitochondria, microsomes, or cytosol. In addition, this antiserum efficiently inhibits membrane-bound microsomal DT-diaphorase.

DT-diaphorase-catalyzed two-electron reduction of various p-benzoquinone- and 1,4-naphthoquinone epoxides

The oxidation of various quinones by H2O2 results in quinone epoxide formation. The yield of epoxidation is inversely related to the degree of methyl substitution of the quinone and seems not to be dependent on the redox potential of the quinones studied. The following order of H2O2-mediated epoxidation of quinones was found: p-benzoquinone greater than or equal to 1,4-naphthoquinone greater than 2-methyl-p-benzoquinone greater than 2,6-dimethyl-p-benzoquinone greater than or equal to 2-methyl-1,4-naphthoquinone greater than 2,3-dimethyl-1,4-naphthoquinone. DT-Diaphorase reduces several quinone epoxides at different rates. The rate of quinone epoxide reduction cannot be related to either the redox potential of the quinone epoxide (as reflected by the half-wave potential calculated from the corresponding hydrodynamic voltamograms) or the degree of substitution of the quinone epoxide. It appears, however, that a quinone epoxide redox potential more negative than -0.5 to -0.6 volts settles a threshold for the electron transfer reaction. This does not exclude that specificity requirements, i.e. the formation of the quinone epoxide substrate-enzyme complex may chiefly determine the rate of reduction of quinone epoxides by DT-diaphorase. DT-diaphorase-catalyzed two-electron transfer to quinone epoxides--resulting in epoxide ring opening--yields 2-OH-p-benzohydroquinone or 2-OH-1,4-naphthohydroquinone products. These hydroxy-derivatives show a higher rate of autoxidation than do the parent hydroquinones lacking the OH substituent.

Formation of benzo[a]pyrene-3,6-quinol mono- and diglucuronides in rat liver microsomes

The formation of benzo[a]pyrene (BP)-3,6 quinol glucuronides in liver microsomes in the presence of UDP-glucuronic acid and NAD(P)H appears to occur by a sequence of three reactions: BP-3,6-quinone----BP-3,6 hydroquinone----BP-3,6-quinol monoglucuronide----BP-3,6-quinol diglucuronide. This conclusion is based on the following results. Incubations with [14C]BP-3,6-quinone or UDP-[14C]glucuronic acid and analysis of the samples by TLC established the existence and identity of the two BP-3,6-quinol glucuronides which exhibit different fluorescence spectra. The nature of the monoglucuronide, i.e., a quinol and not a semiquinone glucuronide, was suggested by the finding that the rate of diglucuronide formation was the same with or without NAD(P)H provided that a sufficient amount of monoglucuronide had been formed prior to oxidation of the nucleotides. Furthermore, BP-3,6-quinol monoglucuronides can serve as substrates in the formation of diglucuronides. The ratio between the decrease in monoglucuronides and the formation of diglucuronides was found to be close to 1, suggesting that the conversion of the monoglucuronide of BP-3,6-quinol to the diglucuronide is also catalyzed by UDP-glucuronosyltransferase. However, great differences in the pattern of induction of mono- and diglucuronide formation indicate that two different UDP-glucuronosyltransferases are involved. The yield of BP-3,6-quinol glucuronides with NADH relative to NADPH and the increase in glucuronide formation observed in the presence of cytosolic DT-diaphorase (NAD(P)H-quinone oxidoreductase) are discussed with regards as to whether DT-diaphorase plays an important role as a BP-3,6-quinone reductase in the formation of BP-3,6-quinol glucuronides compared to other NAD(P)H-oxidizing flavoproteins.