H-quinone Oxidoreductase Research Articles

The effect of thetert-butylquinone metabolite of butylated hydroxyanisole on cytochrome P-450 monooxygenase activity

1. The t-butylquinone metabolite of BHA was shown to redox cycle with NADPH-cytochrome P-450 reductase leading to enhanced NADPH-oxidase activity for both the purified and liver microsome-bound flavoprotein. Likewise, addition of t-butylquinone (20-100 microM) strikingly inhibited electron transfer from the flavoprotein reductase to cytochrome P-450 of liver microsomes from phenobarbital-treated rats. 2. When the effect of t-butylquinone on metabolism of biphenyl was evaluated with liver microsomal fractions or isolated hepatocytes, t-butylquinone was less effective as an inhibitor then BHA alone or vitamin K3 (menadione). Addition of dicoumarol had little or no effect on the inhibitory potency of either t-butylquinone or vitamin K3 in isolated hepatocytes. 3. t-Butylquinone was not an effective reductant for exogenous oxidants, such as cytochrome c, in the presence of purified, cytosolic NAD(P)H-quinone oxidoreductase (DT-diaphorase). This property is most probably due to the lower rate of reoxidation of t-butylquinone by molecular oxygen, relative to vitamin K3 (menadione).

Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O 2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed a 1:1:1 relationship between the amount of NADPH and O 2 consumed and the amount of H 2O 2 formed (measured fluorometrically). The amount of O 2 consumed during the redox cycling of resorufin decreased ~50% in the presence of catalase, whereas the rate of O 2 consumption decreased in the presence of Superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O 2 is converted to H 2O 2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O 2 to H 2O 2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O 2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O 2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O 2 to superoxide anion. The superoxide anion formed is then converted to hydrogen peroxide, either by a second, one-electron oxidation of reduced resorufin or by dismutation. In the accompanying paper, we show that the redox cycling of the 7-alkoxyresorufins by NADPH-cytochrome P450 reductase significantly affects their 0-dealkylation by purified isozymes of rat liver microsomal cytochrome P450.

Butylated hydroxytoluene (BHT)-mediated increases in NAD(P)H-quinone oxidoreductase (QR) in mouse lung: Evidence for the alveolar type II cell as a site of QR activity

Administration of butylated hydroxytoluene (BHT) to inbred A J mice increased lung cytosolic NAD(P)H-quinone oxidoreductase (QR) activity. Increased QR activity was observed using a dose of BHT of 100 mg/kg ip, although maximal increases in activity were observed following BHT 300 mg/kg ip. Increased lung QR activity could be observed as early as 24 hr post-BHT (300 mg/kg ip). Bronchiolar Clara cells and pulmonary macrophages isolated using an elastase digestion procedure from both control and BHT-treated mice exhibited minimal QR activity. The majority of basal and BHT-increased QR activity remained in the undigested lung. As isolation of many cell types to a high degree of purity from mouse lung is not yet feasible, QR activity was measured in established mouse lung cell lines and in cell isolates from rat lung. QR activity was low in a Clara tumor cell line, but higher activity was observed in three alveolar type II cell lines. Type II cell-enriched populations isolated from rat lung exhibited sixfold greater QR activity than the remaining undigested lung. Pulmonary QR activity was significantly greater in rat than in A J or C57 mice. Treatment of cultured Clara or type II cells with BHT in vitro did not increase QR activity.

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.

Mutagenicity of quinones: pathways of metabolic activation and detoxification.

The mutagenicity of various quinones, a class of compounds widely distributed in nature, is demonstrated in the Salmonella TA104 tester strain. The metabolic pathways by which four quinones, menadione, benzo[a]pyrene 3,6-quinone, 9,10-phenanthrenequinone, and danthron, caused mutagenicity in this test system were investigated in detail as were the detoxification pathways. The two-electron reduction of these quinones by NAD(P)H-quinone oxidoreductase (DT-diaphorase) was not mutagenic, whereas the one-electron reduction, catalyzed by NADPH-cytochrome P-450 reductase, was mutagenic, except for danthron, which was only slightly mutagenic. The mutagenicity of the quinones via this pathway was found to be attributable to the generation of oxygen radicals. The cytochrome P-450 monooxygenase also played a significant role in the detoxification and bioactivation of these quinones. For example, phenanthrenequinone was converted to a nonmutagenic metabolite in a cytochrome P-450-dependent reaction, whereas danthron was converted to a highly mutagenic metabolite. These studies show the complexity of metabolic pathways involved in the mutagenicity of quinones.

Vanillic acid metabolism by selected soft-rot, brown-rot, and white-rot fungi

Metabolism of vanillic acid, a product of lignin degradation, has been studied in selected representatives of soft-rot, brown-rot and white-rot fungi. All of the brown-and white-rot species examined decarboxylated vanillate to methoxyhydroquinone oxidatively. Mycelium extracts of all these fungi, except Pleurotus ostreatus contained high levels of an NAD(P)H-dependent vanillate hydroxylase. P. ostreatus also released 14CO2 from 14COOH-vanillate but by a different mechanism possibly involving phenoloxidases. Most of these fungi also contained a dioxygenase which catalysed the intra-diol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to form maleylacetate. No 3-O-demethylase activity was detected, and data indicate that in some of the fungi examined cleavage of the aromatic ring occurs without prior removal of the methoxyl group. None of the soft-rot fungi tested contained vanillate hydroxylase or hydroxyquinol 1,2-dioxygenase, but very low levels of protocatechuate 3,4-dioxygenase were detected in mycelium extracts. Vanillate catabolism among members of this group occurs via a different route which may involve ring demethylation although no 3-O-demethylase activity was detected in this study. The enzyme NAD(P)H-quinone oxidoreductase was demonstrated to exist in all the studied groups of fungi.