NADPH-dependent Process Research Articles

Proteome analysis reveals differential expression of proteins involved in triacylglycerol accumulation by Rhodococcus jostii RHA1 after addition of methyl viologen.

Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and β-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.

Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new anti-schistosomal drugs.

Investigations regarding the chemistry and mechanism of action of 2-methyl-1,4-naphthoquinone (or menadione) derivatives revealed 3-phenoxymethyl menadiones as a novel anti-schistosomal chemical series. These newly synthesized compounds (1-7) and their difluoromethylmenadione counterparts (8, 9) were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR), which has been identified as a potential target for anti-schistosomal drugs. The compounds were also tested in enzymic assays using both human flavoenzymes, i.e. glutathione reductase (hGR) and selenium-dependent human thioredoxin reductase (hTrxR), to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chemical studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively compared to hGR and hTrxR enzymes, in particular those bearing an α-fluorophenol methyl ether moiety, which improves anti-schistosomal action. Furthermore, the (substituted phenoxy)methyl menadione derivative (7) displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox cycling of SmTGR, and potent anti-schistosomal action in worms cultured ex vivo. In contrast, the difluoromethylmenadione analog 9, which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in worms cultured ex vivo. Despite ex vivo activity, none of the compounds tested was active in vivo, suggesting that the limited bioavailability may compromise compound activity. Therefore, future studies will be directed toward improving pharmacokinetic properties and bioavailability.

Rat liver microsomal and nuclear activation of methanol to hydroxymethyl free radicals

Recent studies from other laboratories reported that during methanol intoxication lipid peroxidation and protein oxidation in liver occurred. Further, they detected free radicals-PBN adducts in bile and urine of methanol poisoned rats. In this work, we report the presence in liver microsomes and nuclei of NADPH dependent processes of hydroxymethyl (HMet) radical formation. The detection of HMet radicals was performed by GC/MS of the trimethylsilyl derivatives of the PBN (N- tert-butyl-a-phenylnitrone)-radical adducts. The formation of HMet radicals was observed only under nitrogen, in these in vitro conditions. Formation of formaldehyde from methanol was observed in aerobic incubation mixtures containing either microsomes or nuclei but also under nitrogen using microsomes. The latter process was not inhibited by diphenyleneiodonium while the anaerobic microsomal one producing HMet was strongly inhibited by it. This shows that they are independent processes. Results suggest that both, liver nuclei and microsomes are able to generate free radicals during NADPH-mediated methanol biotransformation.

Cytochrome P450 reductase-mediated anaerobic biotransformation of ethanol to 1-hydroxyethyl-free radicals and acetaldehyde

The ability of cytochrome P450 reductase to metabolize ethanol (EtOH) to acetaldehyde (AC) and 1-hydroxyethyl free radicals (1HEt) in anaerobic media was studied. Determination of AC was made by GC-FID analysis of the head space of incubation mixtures. The formation of 1HEt was established by GC-MS analysis of the adduct formed between the radical and the spin trap PBN. Results showed that pure human P450 reductase is able to biotransform EtOH to AC and 1HEt in a NADPH-dependent process under an oxygen-free nitrogen atmosphere. Pure FAD in the presence of NADPH was also able to generate AC and 1HEt from the alcohol. Anaerobic incubation mixtures containing either rat liver microsomes or pure nuclei were also able to biotransform EtOH to AC and 1HEt in the presence of NADPH. These processes were inhibited by antibody against rat liver microsomal P450 reductase. Results suggest that semiquinone forms of the flavin in P450 reductase may biotransform EtOH. These reactions might be of some significance in tissues where the P450 reductase is present in the absence of specific forms of cytochrome P450 known to be involved in EtOH metabolism (e.g. CYP2E1). However the toxicological significance of this enzymatic process remains to be established.

Aryl acetylenes as mechanism-based inhibitors of cytochrome P450-dependent monooxygenase enzymes.

Aryl acetylenes have been investigated as inhibitors of cytochrome P450 (P450)-dependent alkoxyresorufin dealkylation dealkylation activities in liver microsomes prepared from rats exposed to beta-naphthoflavone, isosafrole, or phenobarbital. Many of the acetylenes investigated produce pseudo-first-order time-dependent and NADPH-dependent losses of the dealkylation activities characteristic of mechanism-based irreversible inactivation (suicide inhibition). Replacing the terminal hydrogen of aryl acetylenes with a methyl group to convert ethynes into propynes enhances the inhibition of P450 1A enzymes; in some instances, this modification converts a reversible inhibitor of P450s into a suicide inhibitor. In contrast, ethynes are more effective suicide inhibitors of P450 2B-dependent dealkylations than the corresponding propynes. Aryl acetylenes with an ethynyl group on the 2 position of naphthalene or on the 9 position of phenanthrene and arylalkyl acetylenes with alkyl chains containing 2, 3, or 4 methylene groups are selective inhibitors of P450 2B1/2B2 in liver microsomes from rats. Aryl acetylenes also act as suicide inhibitors of P450 1A2 in human liver microsomes, of purified P450 1A2 from rabbit or rat liver in reconstituted systems, and of purified recombinant human P450 1A2 and 1A1 in reconstituted systems. 4-(1-Propynyl)biphenyl (4PBi) inactivated P450 1A2-dependent ethoxyresourfin deethylation (EROD) activity in human liver microsomes in an NADPH-dependent process (k(inactivation), 0.23 min-1; KI, 2.3 microM). 4PBi also inactivated purified recombinant human P450 1A2 (k(inactivation), 0.24 min-1; KI, 4.3 microM). In agreement with previous reports [Yun, C.-H., Hammons, G. J., Jones, G., Martin, M. V., Hopkins, N. E., Alworth, W. L., and Guengerich, F. P. (1992) Biochemistry 31, 10556-10563], 2-ethynylnaphthalene (2EN) was not a suicide inhibitor of the P450 1A2 activity in human liver microsomes but did inactivate purified human P450 1A2. Neither 4PBi nor 2EN affected diagnostic activities of human microsomal P450 2E1, 2C9/10, 3A4, or 2C19. In the systems examined, the losses of P450-dependent activity produced by these aryl acetylenes were not accompanied by corresponding decreases in the measured P450 absorption spectra. Thus P450 inactivation by these aryl acetylenes does not involve labeling and destruction of the heme. Incubation of 4PBi with microsomal P450 1A1 or 1A2 from rat liver under conditions that lead to P450-dependent, enzyme inactivations generates a 2-biphenylylpropionic acid product. This suggests that the suicide inhibition of P450s by propynylaryl acetylenes proceeds via a methylaryl ketene formed by a 1,2-methyl rearrangement, analogous to the mechanism of suicide inhibition by ethynyl acetylenes that proceed via ketene intermediates formed by 1,2-hydrogen shifts [Ortiz de Montellano, P. R., and Kunze, K. L. (1981) Arch. Biochem. Biophys. 209, 710-712].

Evidence for presence of a reduced form of digoxin-like immunoreactive factor (dihydro-DLIF) in mammalian tissues

Digoxin-like immunoreactive factor (DLIF) from adrenal glands is an endogenous ligand structurally related to the plant-derived cardiac glycoside digoxin. Cardiac glycosides regulate the activity of the sodium pump and thus play key roles in disease processes involving regulation of ion transport. We now report the discovery of an endogenous dihydro-DLIF analogous to dihydrodigoxin. We used HPLC, ultraviolet spectrophotometry, and cross-reactivity with two antibodies, one specific for digoxin and one for dihydrodigoxin, to support the hypothesis that dihydro-DLIF contains a chemically reduced lactone ring. The spectral absorbance maximum for dihydro-DLIF is at 196 nm, identical to dihydrodigoxin. DLIF and dihydro-DLIF are 975- and 2588-fold less immunoreactive than digoxin and dihydrodigoxin for their respective antibodies. The molar ratio of dihydro-DLIF to DLIF is approximately 5.3 in bovine adrenocortical tissue and approximately 0.38 in human serum. Dihydrodigoxin (reduced lactone ring) added to microsomes isolated from bovine adrenal cortex produced a 4.5-fold increase in digoxin-like immunoreactivity (oxidized lactone ring) after 3 h of incubation. The biotransformation is likely mediated by a cytochrome P-450 NADPH-dependent process. Our findings demonstrate the presence of a dihydro-DLIF in mammals and suggest a metabolic route for synthesis of endogenous DLIF in mammalian tissue.

Fate of a terminal olefin with Drosophila microsomes and its inhibitory effects on some P-450 dependent activities.

In vitro bioassays were used to analyze the metabolism of the 11-dodecenoic acid (11-DDNA) by microsomes prepared from Drosophila melanogaster RalDDTR strain. 11-DDNA is metabolized to 11,12-epoxylauric acid (epoxyLA) in a NADPH-dependent way. The microsomal production of epoxyLA reaches a plateau very quickly, suggesting the occurrence of an enzyme inactivation process. After incubation of microsomes with (1-14C)11-DDNA, three proteins of Mr approximately 50 kDa were labeled. 11-DDNA inhibits the microsomal metabolism of lauric acid and 7-ethoxycoumarin in a time and NADPH-dependent process. An inhibition of metabolites generated from DDT and testosterone was also obtained but at higher concentrations. These results are discussed according to the fact that RalDDTR is an insecticide resistant strain characterized as a high metabolizer of the insecticide DDT and also of lauric acid, testosterone, and ethoxycoumarin.

In vitro studies on the interaction of the pyridoindole antioxidant stobadine with rat liver microsomal P450.

1. Stobadine, a pyridoindole antioxidant agent, elicited medium affinity, low capacity interaction with type I binding sites of the hepatic microsomal cytochromes P450 derived from control and acetone-pretreated rats. Reverse type I interaction of low affinity and low capacity was observed in microsomes from phenobarbital-treated rats. 2. Stobadine led to an increase of H2O2 production when added to liver microsomes derived from differently pretreated rats in an NADPH-dependent process with concomitantly increased oxygen consumption. 3. Stobadine, at concentrations stimulating H2O2 formation, was found to prevent NADPH-induced microsomal lipid peroxidation, assessed as thiobarbituric acid-reactive product accumulation. 4. Only a weak inhibitory effect of stobadine on either NADPH- or cumene hydroperoxide-dependent aminopyrine N-demethylation and aniline hydroxylation was observed in microsomes from control and phenobarbital-pretreated rats. An approximately 10 times higher inhibitory potency towards aminopyrine N-demethylase activity was observed in acetone-pretreated rats. 5. In spite of the direct interaction of stobadine with microsomal P450, the compound only marginally affected aminopyrine and aniline metabolism both by monooxygenase and peroxidase modes of action of the P450 enzyme system. The potent antioxidant activity of stobadine was not diminished by the ability of the drug to stimulate the oxidase function of P450.

Sulfamethoxazole is metabolized to the hydroxylamine in humans.

The oxidation of sulfamethoxazole to its hydroxylamine metabolite was investigated in vitro with human liver microsomes and in vivo by detection in the urine. Sulfamethoxazole was oxidized to the hydroxylamine in an NADPH-dependent process by liver microsomes prepared from two human livers. Three healthy volunteers ingested 1000 mg sulfamethoxazole, and urine was collected for 24 hours. Sulfamethoxazole hydroxylamine constituted 3.1% +/- 0.7% of the drug excreted in the urine in 24 hours. Fifty-four percent of the ingested dose was excreted during this same time period. We conclude that sulfamethoxazole hydroxylamine is an authentic in vivo metabolite in humans, probably formed predominantly by cytochrome P450 in the liver. It could be responsible for mediation of sulfonamide adverse reactions, particularly hypersensitivity reactions.

An in vitro investigation of predisposition to sulphonamide idiosyncratic toxicity in dogs

Sulphonamide idiosyncratic toxicosis has been reported in 28 dogs. Non-septic polyarthritis and fever occurring after 8 to 21 days therapy was the most common manifestation. Of 22 dogs with this syndrome, 7 were Doberman Pinschers. In humans, inherited decreased ability to detoxify sulphonamide hydroxylamine metabolites (as reflected in an in vitro mononuclear leukocyte (MNL) toxicity assay) has been associated with susceptibility to sulphonamide idiosyncratic toxicity. We have demonstrated that microsomes obtained from the liver of a dog were capable of metabolizing sulphamethoxazole to sulphamethoxazole hydroxylamine (SMX-HA). Production of SMX-HA was an NADPH dependent process and the yield was increased by the presence of 1 mmol/L ascorbic acid. SMX-HA was toxic to isolated MNL from mixed breed dogs (MBD) and Doberman Pinschers. The toxicity of SMX-HA to MNL from Dobermans was significantly different from that to MNL from MDB. MNL from 7 out of 15 Dobermans (including a dog with a history of an idiosyncratic reaction to a sulphonamide) had an LD-50 (concentration of SMX-HA required to produce 50% cytotoxicity in MNL) less than 100 mumols/L, while MNL from 0 out of 10 MBD had an LD-50 less than 100 mumols/L. These results suggest that the basis for the observed predisposition of Dobermans to sulphonamide idiosyncratic toxicity may be a limited capacity to detoxify the hydroxylamine metabolites of sulphonamides.

The mechanisms of reduction of protein mixed disulfides (dethiolation) in cardiac tissue

Dethiolation of proteins (reduction of protein mixed disulfides) by NADPH-dependent and glutathione (GSH)-dependent enzymes, and by nonenzymatic reaction with GSH, was studied by electrofocusing methodology with glycogen phosphorylase b and creatine kinase as substrates. Phosphorylase b was not rapidly dethiolated by reduced glutathione alone, but a cardiac extract catalyzed rapid dethiolation by both an NADPH-dependent and a GSH-dependent process. In contrast, creatine kinase was actively dethiolated by GSH. This GSH-dependent dethiolation was not enhanced by a soluble extract of bovine heart. Creatine kinase was also not dethiolated by an NADPH-dependent process. Partial purification of the phosphorylase dethiolases showed that the NADPH-dependent dethiolase had both a high-molecular-weight and a low-molecular-weight component. The properties of these components were similar to those of thioredoxin and thioredoxin reductase. These two components were sensitive to inhibition by phenylarsine oxide and inhibition was reversed by addition of a dithiol. In contrast, GSH-dependent dethiolation required a single component of low molecular weight. This process was less sensitive to phenylarsine oxide inhibition. These studies show that two cytosolic proteins, phosphorylase b and creatine kinase, were dethiolated by different mechanisms. Phosphorylase b was dethiolated by both NADPH-dependent and GSH-dependent enzymes found in a soluble extract of bovine heart. In contrast, creatine kinase was rapidly dethiolated nonenzymatically by GSH alone.

Derailment product in NADPH-dependent synthesis of a dihydroisocoumarin 6-hydroxymellein by elicitor-treated carrot cell extracts.

Synthetic activity of 6-hydroxymellein, the immediate precursor of carrot phytoalexin 6-methoxymellein, from acetyl-CoA and malonyl-CoA was induced in carrot cell extracts when the root disks were treated with CuCl2 or oligogalacturonide elicitor. These elicitors showed specific inducing activity of phytoalexin production and did not affect fatty acid synthesis in carrot tissues which may share some common properties with 6-hydroxymellein biosynthesis. 6-Hydroxymellein production was an NADPH-dependent process and, in the absence of the reagent, triacetic acid lactone was produced as a derailment product of the reaction process. This finding suggested that the reduction of the double bond at the 3,4-position of the phytoalexin takes place during the elongation of the poly(oxomethylene) chain. This NADPH-dependent reduction seems to occur at the triacetate stage before the condensation of the third malonyl-CoA as the conversion of carbonyl to hydroxyl group.

715 PARENTERAL FAT AS NITROGEN SPARER IN LOW BIRTHWEIGHT(LBT) INFANTS

The nitrogen sparing effect of calories has been documented, however the influence of the quality of these calories has not been studied in LBW infants. The aim of this study was to compare the metabolic response of glucose and fat in parenterally fed LBW infants. In a paired design, 6 LBW infants (mean ± SD, birthweight: 2055±586; gestational age: 34±2wk; age: 19±20d) received 2 isoproteinic (Travasol C: 2.8±0.1g/kg.d) and isocaloric (67±13kcal/kg.d) regimens over 12 days. During a high glucose phase (CHO) 77±3% of nonproteinic energy was infused as glucose, versus 56±10% as fat during a high lipid phase (LIP). Nitrogen retention (N2 Ret)> triglycerides (TG), plasma phenylalanine (PHE) and tyrosine (TYR), insulin (INS), pCO2 (n=4, free of RDS) were compared: No significant plasma amino acid imbalance was noted. Lipogenesis, a NADPH dependent process, was suggested by the higher PHE/TYR ratio found during CHO. Free fatty acids provided by a high fat (3g/kg.d) fuel mixture, produced a better N2 Ret without compromising the respiratory function. The results emphasize the relation between the composition of infused energy and the quality of growth in parenterally fed infants as opposed to adults.

Metabolism of chloroethanes by rat liver nuclear cytochrome P-450.

1,2-Dichloroethane, 1,1,1-trichloroethane and 1,1,2,2-tetrachloroethane appear to be metabolized by hepatic nuclear cytochrome P-450. All of these compounds are converted to chlorinated metabolites after incubation with hepatic nuclei and an NADPH-generating system plus EDTA, with the omission of any component eliminating metabolite production. In addition, CO, an inhibitor of cytochrome P-450, diminished the production of the chlorinated metabolites by hepatic nuclear preparations. The major metabolites of the chlorinated ethanes from hepatic microsomal cytochrome P-450, viz. chloroacetaldehyde from 1,2-dichloroethane, 2,2,2-trichloroethanol from 1,1,1-trichloroethane, and dichloroacetic acid from 1,1,2,2-tetrachloroethane, were also produced from the three chloroalkanes by hepatic nuclear cytochrome P-450. The levels of the metabolites produced were 65, 0.09 and 4.4 nmol/nmol cytochrome P-450/60 min. It is proposed that the pathways for the formation of these metabolites by hepatic nuclear cytochrome P-450 are as for their production by hepatic microsomal cytochrome P-450. Chloral hydrate was produced from 1,1,1-trichloroethane by hepatic nuclei plus NADPH, but not by hepatic microsomes. The presence of reactive species or transient enzyme bound intermediates in the pathways for the cytochrome P-450 dependent metabolism of the chloroethanes in hepatic nuclei is suggested by the observation that nuclear cytochrome P-450 is degraded in the presence of the chloroethanes in a NADPH dependent process which is inhibited by CO. It is proposed that, although the cytochrome P-450 dependent metabolism of the chloroethanes in microsomes can greatly exceed that in nuclei, the metabolism of 1,2-dichloroethane and 1,1,2,2-tetrachloroethane by nuclear cytochrome P-450 may in part mediate the mutagenicity and carcinogenicity of parent compounds.

Lysis of vitamin E-deficient rat erythrocytes by rat liver microsomes in an NADPH-dependent process in the presence of inhibitors of lipid peroxidation.

Haemolytic substances are generated by rat liver microsomes undergoing NADPH-dependent lipid peroxidation. In the present experiments, vitamin E-deficient red cells were found to be haemolysed in an NADPH-dependent process by rat liver microsomes in the presence of EDTA, an inhibitor of microsomal lipid peroxidation. This is in contrast to the behaviour of normal, vitamin E-adequate red cells which are resistant to lysis under similar conditions. Aminopyrine (AP), a substrate for the microsomal mixed function oxidase system, was found to prevent lysis of vitamin E-deficient red cells in NADPH-microsome-EDTA mixtures. Experiments designed to detect only the more stable haemolysins formed in such mixtures involved sampling and assaying for haemolytic activity in the presence of AP. The result support the conclusion that haemolysis was due to a short-lived species. If EDTA is omitted from the NADPH-microsome mixture other more stable haemolysins are produced.

On the significance of the cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism.

1. Reconstituted membrane vesicles containing purified preparations of cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase effectively destroyed 2-deoxy-D-ribose in an NADPH-dependent process. 2. The destruction was mediated by hydroxyl radicals formed in an iron-catalysed Haber-Weiss reaction between superoxide anions and hydrogen peroxide liberated from the haemoprotein. 3. Administration of ethanol or benzene to rabbits, compounds known to be oxygenated by the hydroxyl radical-dependent mechanism, resulted in induction of a species of cytochrome P-450 effective in the radical-dependent metabolism of both chemicals. 4. Benzene treatment of rabbits also resulted in an enhanced hydroxyl radical-dependent metabolism of ethanol and benzene in liver microsomes. 5. It is suggested that, for certain substrates, hydroxyl radical-mediated cytochrome P-450-dependent oxygenation reactions are of importance for the microsomal metabolism of these compounds. 6. It is speculated that radical-producing species of cytochrome P-450 may contribute to hydroxyl radical-mediated cell damage.

Amaranth suppresses the mutagenicity of 2-acetylaminofluorene by lowering the concentration of nadph in top agar

Addition of 1 mg amaranth (FD&C Red No.2) to the top agar of Salmonella/S9 assay plates decreased the yield of revertants induced by 20 μg 2-acetylaminofluorene (AAF) by over 50% and additional amaranth completely eliminated the mutagenic response. Similar suppression of AAF mutagenicity was seen with sulfonazo III, another azo dye. The suppressive effect of amaranth was greatest at low S9 concentrations and decreased as the amount of S9 was increased. When N-hydroxyacetylaminofluorene (N-OH-AAF) was used as mutagen, amaranth had little or no effect on either the number of revertants obtained or the S9 optimum. Similarly, 1-naphthylamine-4-sulfonic acid (a reduction product of amaranth) did not significantly affect the mutagenicity of AAF. The rate of metabolism of [ 14C]AAF by the S9 preparations was shown to be markedly decreased by amaranth, as were the levels of both the phenolic metabolites and of N-OH-AAF. Thus, it appeared that amaranth acts by blocking the conversion of AAF to N-OH-AAF and that this effect is caused by the amaranth itself and not by its constituent amines. Further experiments indicated that amaranth greatly decreased the levels of NADPH formed in reaction mixtures comparable to S9 mix in top agar and that such reaction mixtures also metabolized amaranth to colourless compounds. It appears likely that in top agar, NADPH reacts with amaranth at a fast enough rate to limit severely the level of the reduced co-factor (which must be formed from NADPH + by the action of endogenous glucose-6-phosphate dehydrogenase) and thus decreases the rate of activation of mutagens by other NADPH-dependent processes.