Rudolf Reiter

Bio: Rudolf Reiter is an academic researcher from University of Tübingen. The author has contributed to research in topics: Glutathione peroxidase & Glutathione. The author has an hindex of 6, co-authored 6 publications receiving 253 citations.

Selenium and drug metabolism—I: Multiple modulations of mouse liver enzymes

Abstract: Male albino mice were raised on diets containing less than 10 ppb selenium (Se−) or supplemented with 0.5 ppm selenium (Se+) for 6 months. In the (Se−) group total liver selenium was less than 10% of the control, liver selenium-dependent glutathione peroxidase (GSH-Px) less than 2%. The specific activities of catalase and Superoxide dismutase showed essentially no differences between the dietary groups. Several phase I-related specific enzyme activities were measured in liver microsomes. No significant differences between the two animal groups were found for cytochrome P-450 and b 5 content, NADH-cytochrome b 5 reductase, as well as for aniline hydroxylation and aminopyrine dealkylation rates. In (Se−) microsomes, NADPH-cytochrome P-450 reductase activity was about half that found in (Se+) microsomes. An increase in microsomes from (Se−) mice was found for 7-ethoxycoumarine deethylation rate (460%), cytochrome P-450 hydroperoxidase activity (170%), and heme oxygenase (276%). The N -oxidation rate of the flavin-containing monooxygenase decreased by 35%, the N -demethylation rate by 50% in (Se−) animals. Stopped-flow measurements of the reduction rates of microsomal pigments did not support evidence for limitations in microsomal electron supply during selenium deficiency. Among the phase II reactions examined, sulfotransferase activity towards 4-nitrophenol was 47% of the controls in Se-deficient liver cytosols while UDP-glucuronyl transferase activity towards this substrate increased to 215%. Glutathione- S -transferase activity was much higher in (Se−) livers than in (Se+): 310% with 1,2-dichloro-4-nitrobenzene, 255% with 1-chloro-2,4-dinitrobenzene and 120% with ethacrynic acid as substrate. The data indicate that in addition to GSH-Px many other enzyme activities in mouse liver are affected by prolonged dietary selenium deficiency. These effects might be useful in assessing the severity of selenium deficiency. A microsomal selenium-dependent metabolic modulator is discussed as a possible mechanism.

Selenium and drug metabolism—II Independence of glutathione peroxidase and reversibility of hepatic enzyme modulations in deficient mice

Abstract: Male mice were fed a diet containing less than 0.01 ppm selenium (Se−) for 6 months. A control group received the same diet containing 0.5 ppm selenium (Se+). In the livers of the Se− animals a drastic decrease in glutathione peroxidase (GSH-Px) activity was observed. It reached undetectable levels after 17 days of the Se− diet. At that time, GSH-transferase activity began to increase significantly, followed by changes in many other enzyme activities. After the 60th day, these enzyme modulations had reached a plateau with the following percentage changes compared to controls: GSH-transferases: 320% (1,2-dichloro-4-nitrobenzene), 218% (1-chloro-2,4-dinitrobenzene); glutathione reductase: 160%; ethoxycoumarin deethylase: 330%; cytochrome P-450-hydroperoxidase: 230%; heme oxygenase: 240%; UDP-glycuronyltransferase: 200%; GSH-thioltransferase: 64%; sulphotransferase: 62%; NADPH-cytochrome-P-450-reductase: 65%; flavin-containing mono-oxygenase: 57%. No significant changes were observed for GSH-transferase activity assayed with ethacrynic acid or for microsomal H2O2 formation and aniline hydroxylase activity. In single-pulse repletion experiments by injection of 250 μg selenium/kg body wt, different individual time constants for the recovery process of the enzymatic perturbations were observed. The half-times for the recovery ranged from 5.7 hr for the microsomal NADPH-cytochrome-P-450 reductase to over 29 hr for GSH-Px up to 44 hr for part of the GSH-transferase activity. 250 μg selenium/kg bodt wt were needed to restore 50% of GSH-Px activity in the long-term Se− mice compared to Se+ controls. All other enzymatic changes in the Se− mice needed a dose of 7 μg selenium/kg body wt for 50% restorage. The results demonstrate that processes other than those related to GSH-Px take place in a later phase of selenium deficiency in mouse liver with a chronologically common beginning. The different repletion and depletion kinetics as well as the different need of these processes for the trace element are discussed with respect to the existence of two separate selenium pools.

Selenium and drug metabolism—III: Relation of glutathione-peroxidase and other hepatic enzyme modulations to dietary supplements

Abstract: Male mice were fed a torula yeast-based diet containing different amounts of added selenium for a period of 4 months. Liver glutathione peroxidase activity assayed with H2O2 showed a logarithmic dependence on dietary selenium with a saturation plateau above 2 ppm Se and an extrapolated zero of 0.02 ppm Se. In contrast, liver selenium content and GSH-Peroxidase activity showed a linear correlation. Glutathione peroxidase activity became undetectable at a liver Se content of about 90 ng Se/g liver wet wt. Thus, about 10% of liver selenium is not related to GSH-Px activity. Five dietary groups were supplemented, respectively, with 0, 0.05, 0.5, 5.0 and 10 ppm Se in the form of Na2SeO3. Some changes in drug metabolism enzymes were observed with the high Se diets. An increase occurred in Non-Se-GSH activity as well as in ethacrynic acid-assayed GSH transferase, these are interpreted as early signs of Se toxicity. The diet containing 0.01 ppm Se with no supplementary Se produced the multiple hepatic enzyme modulations which were previously reported. The animals raised on this very low Se diet had normal hepatic contents of glutathione, alpha-tocopherol, calcium, magnesium, iron, zinc, copper and manganese compared to controls supplemented with 0.5 ppm Se. However, significant changes in the microsomal fatty acid pattern were observed while the total phospholipid content as well as membrane fluidity showed no differences between the two dietary groups.

Chemically-induced glutathione depletion and lipid peroxidation.

Abstract: Malondialdehyde (MDA) formation in mouse liver homogenates was measured in the presence of various glutathione depletors (5 mmol/l). After a lag phase of 90 min, the MDA formation increased from 1.25 nmol/mg protein to 14.5 nmol/mg in the presence of diethyl maleate (DEM), to 10.5 with diethyl fumarate (DEF) and to 4 with cyclohexenon by 150 min. It remained at 1.25 nmol/mg with phorone and in the control. On the other hand, glutathione (GSH) dropped from 55 nmol/mg to 50 nmol/mg in the control to, < 1 with DEM, to 46 with DEF, to 3 with cyclohexenon and to 7 with phorone. The data show that the potency to deplete GSH is not related to MDA production in this system. DEM stimulated in vitro ethane evolution in a concentration-dependent manner and was strongly inhibited by SKF 525A. From type I binding spectra to microsomal pigments the following spectroscopic binding constants were determined: 2.5 mmol/l for phorone, 1.2 mmol/l for cyclohexenon, 0.5 mmol/l for DEM and 0.3 mmol/l for DEF. In isolated mouse liver microsomes NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activity were unaffected by the presence of DEM, whereas ethoxycoumarin dealkylation was inhibited. Following in vivo pretreatment, hepatic microsomal electron flow as determined in vitro was augmented in the presence of depleting as well as non-depleting agents, accompanied by a shift from O2− to H2O2 production. It is concluded that it is not the absence of GSH which causes lipid peroxidation after chemically-induced GSH depletion but rather the interaction of the chemicals with the microsomal monoxygenase system.

Drug-induced lipid peroxidation in mice—IV: In vitro hydrocarbon evolution, reduction of oxygen and covalent binding of acetaminophen

Abstract: A method is described to measure in vitro lipid peroxidation by the hydrocarbon evolution technique in cell homogenates or subcellular suspensions of mouse liver. The drug acetaminophen (paracetamol) was shown to quench Fe/ADP-induced as well as NADPH-induced microsomal lipid peroxidation. The pH dependence of ethoxycoumarin deethylation, NADPH-cytochrome P-450 or NADH-cytochrome b5 reductase activities, and of NADPH-induced microsomal H2O2 formation in the presence or absence of acetaminophen was investigated. The presence of the drug at 1 mmole/l caused a shift of the NADPH- or the (NADPH + NADH)-supported H2O2 formation to a more alkaline pH. Both processes were inhibited by monooxygenase inhibitors. The dependence of microsomal parameters on the NADP/NADPH ratio was studied and related to the profile of the superoxide dismutase sensitive adrenochrome formation or H2O2 formation in the presence and absence of acetaminophen. With a maximum at an NADP/NADPH ratio of 100 a microsomal H2O2 production of 5 nmoles/mg protein/min without a concomitant adrenochrome formation was observed only in the presence of acetaminophen. At a NADP/NADPH ratio of 0.01, 8 nmoles/mg/min H2O2 and 16 nmoles/mg/min adrenochrome were formed in the absence of acetaminophen. In the presence of 1 mmole/l of the drug, this was quenched to 7 nmoles/mg/min H2O2 and 13.5 nmoles/mg/min adrenochrome, respectively. The steady state of the oxy-cytochrome P-450 complex paralleled H2O2 formation in the absence of acetaminophen, while it did not so in the presence of it. In further experiments the isocitrate dehydrogenase activity was used to modulate the pyridine nucleotide redox potential: H2O2 formation as well as ethane evolution was dissociated into two phases where acetaminophen acted antioxidatively if the pyridine nucleotide couple was reduced. The in vitro covalent binding of the drug paralleled H2O2 formation as well as ethane evolution. These observations seem to be of general importance with respect to oppositely directed effects of drugs in vitro and in vivo. Possible mechanisms relating H2O2 production, ethane evolution and protein binding to a common radical intermediate are discussed.

Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress

Abstract: Increases in the intracellular levels of reactive oxygen species (ROS), frequently referred to as oxidative stress, represents a potentially toxic insult which if not counteracted will lead to membrane dysfunction, DNA damage and inactivation of proteins. Chronic oxidative stress has numerous pathological consequences including cancer, arthritis and neurodegenerative disease. Glutathione-associated metabolism is a major mechanism for cellular protection against agents which generate oxidative stress. It is becoming increasingly apparent that the glutathione tripeptide is central to a complex multifaceted detoxification system, where there is substantial inter-dependence between separate component members. Glutathione participates in detoxification at several different levels, and may scavenge free radicals, reduce peroxides or be conjugated with electrophilic compounds. Thus, glutathione provides the cell with multiple defences not only against ROS but also against their toxic products. This article discusses how glutathione biosynthesis, glutathione peroxidases, glutathione S-transferases and glutathione S-conjugate efflux pumps function in an integrated fashion to allow cellular adaption to oxidative stress. Co-ordination of this response is achieved, at least in part, through the antioxidant responsive element (ARE) which is found in the promoters of many of the genes that are inducible by oxidative and chemical stress. Transcriptional activation through this enhancer appears to be mediated by basic leucine zipper transcription factors such as Nrf and small Maf proteins. The nature of the intracellular sensor(s) for ROS and thiol-active chemicals which induce genes through the ARE is described. Gene activation through the ARE appears to account for the enhanced antioxidant and detoxification capacity of normal cells effected by many cancer chemopreventive agents. In certain instances it may also account for acquired resistance of tumours to cancer chemotherapeutic drugs. It is therefore clear that determining the mechanisms involved in regulation of ARE-driven gene expression has enormous medical implications.

Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy.

Abstract: Glutathione, which is synthesized within cells, is a component of a pathway that uses NADPH to provide cells with their reducing milieu. This is essential for (a) maintenance of the thiols of proteins (and other compounds) and of antioxidants (e.g. ascorbate, α-tocopherol), (b) reduction of ribonucleotides to form the deoxyribonucleic precursors of DNA, and (c) protection against oxidative damage, free radical damage, and other types of toxicity. Glutathione interacts with a wide variety of drugs. Despite its many and varied cellular functions, it is possible to achieve therapeutically useful modulations of glutathione metabolism. This article emphasizes an approach in which the synthesis of glutathione is selectively inhibited in vivo leading to glutathione deficiency. This is achieved through use of transition-state inactivators of γ-glutamylcysteine synthetase, the enzyme that catalyzes the first and rate-limiting step of glutathione synthesis. The effects of marked glutathione deficiency, thus produced in the absence of applied stress, include cellular damage associated with severe mitochondrial degeneration in a number of tissues. Such glutathione deficiency is not prevented or reversed by giving glutathione. The cellular utilization of GSH involves its extracellular degradation, uptake of products, and intracellular synthesis of GSH. This is a normal pathway by which cysteine moieties are taken up by cells. Glutathione deficiency induced by inhibition of its synthesis may be prevented or reversed by administration of glutathione esters which, in contrast to glutathione, are readily transported into cells and hydrolyzed to form glutathione intracellularly. Research derived from this model has led to several potentially useful therapeutic approaches, one of which is currently in clinical trial. Thus, certain tumors, including those that exhibit resistance to several drugs and to radiation, are sensitized to these modalities by selective inhibition of glutathione synthesis. An alternative interpretation is suggested which is based on the concept that some resistant tumors have high capacity for glutathione synthesis and that such increased capacity may be as significant or more significant in promoting the resistance of some tumors than the cellular levels of glutathione. Therapeutic approaches are proposed in which normal cells may be selectively protected against toxic antitumor agents and radiation by cysteine- and glutathione-delivery compounds. Current studies suggest that research on other modulations of glutathione metabolism and transport would be of interest.

The glutathione peroxidases.

Abstract: There are several proteins in mammalian cells that can metabolize hydrogen peroxide and lipid hydroperoxides. These proteins include four selenium-containing glutathione peroxidases that are found in different cell fractions and tissues of the body. This review considers the structure and distribution of the selenoperoxidases and how this relates to their biological function. The functions of the selenoperoxidases were originally studied in systems where their activity was manipulated by changing dietary selenium levels. More recently, molecular techniques have allowed overexpression of selenoperoxidases in cell lines and animals. Additionally, cellular glutathione peroxidase knockout mice have been used to investigate the functions of this protein. From this work it is clear that the selenoperoxidases are involved in cell antioxidant systems. However, they also have more subtle functions in ensuring the regulation and formation of arachadonic acid metabolites that are derived from hydroperoxide intermediates. The range of biological processes, which are potentially dependent on optimal selenoperoxidase activity in mammals, emphasises the importance of achieving adequate selenium intake in the diet.

Antioxidant activity of albumin-bound bilirubin.

Abstract: Bilirubin, when bound to human albumin and at concentrations present in normal human plasma, protects albumin-bound linoleic acid from peroxyl radical-induced oxidation in vitro. Initially, albumin-bound bilirubin (Alb-BR) is oxidized at the same rate as peroxyl radicals are formed and biliverdin is produced stoichiometrically as the oxidation product. On an equimolar basis, Alb-BR successfully competes with uric acid for peroxyl radicals but is less efficient in scavenging these radicals than vitamin C. These results show that 1 mol of Alb-BR can scavenge 2 mol of peroxyl radicals and that small amounts of plasma bilirubin are sufficient to prevent oxidation of albumin-bound fatty acids as well as of the protein itself. The data indicate a role for Alb-BR as a physiological antioxidant in plasma and the extravascular space.

Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase

Abstract: Numerous studies in animal models and more recent studies in humans have demonstrated cancer chemopreventive effects with Se. There is extensive evidence that monomethylated forms of Se are critical metabolites for chemopreventive effects of Se. Induction of apoptosis in transformed cells is an important chemopreventive mechanism. Apoptosis can be triggered by micromolar levels of monomethylated forms of Se independent of DNA damage and in cells having a null p53 phenotype. Cell cycle protein kinase cdk2 and protein kinase C are strongly inhibited by various forms of Se. Inhibitory mechanisms involving modification of cysteine residues in proteins by Se have been proposed that involve formation of Se adducts of the selenotrisulfide (S-Se-S) or selenenylsulfide (S-Se) type or catalysis of disulfide formation. Selenium may facilitate reactions of protein cysteine residues by the transient formation of more reactive S-Se intermediates. A novel chemopreventive mechanism is proposed involving Se catalysis of reversible cysteine/disulfide transformations that occur in a number of redox-regulated proteins, including transcription factors. A time-limited activation mechanism for such proteins, with deactivation facilitated by Se, would allow normalization of critical cellular processes in the early stages of transformation. There is uncertainty at the present time regarding the role of selenoproteins in chemoprevention model systems where supranutritional levels of Se are employed. Mammalian thioredoxin reductase is one selenoprotein that shows increased activity with Se supplementation in the nutritional to supranutritional range. Enhanced thioredoxin reduction could have beneficial effects in oxidative stress, but possible adverse effects are considered. Other functions of thioredoxin reductase may be relevant to cell signaling pathways. The functional status of the thioredoxin/thioredoxin reductase system during in vivo chemoprevention with Se has not been established. Some in vitro studies have shown inhibitory effects of Se on the thioredoxin system correlated with growth inhibition by Se. A potential inactivating mechanism for thioredoxin reductase or other selenoenzymes involving formation of a stable diselenide form resistant to reduction is discussed. New aspects of Se biochemistry and possible functions of new selenoproteins in chemoprevention are described.