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Biomedical subjects

H Sies

Publications and source records attributed to H Sies.

At least 217 records · Page 12Linked to original sources
PubMed1991

Nitric oxide production by cultured aortic endothelial cells in response to thiol depletion and replenishment.

The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells. NO production was diminished when cells were pretreated with thiol-depleting agents (IC50: N-ethylmaleimide, 30 microM; 1-chloro-2,4-dinitrobenzene, 200 microM; diamide, 1.5 mM; diethyl maleate, 20 mM). The depletion of glutathione (45-99% loss at the various IC50 values) and protein thiols (3-25% loss at IC50) showed no consistent relationship to decreased NO production. The effects of the agents on NO production were not linked to altered sensitivity to the stimulant (calcium ionophore A23187; maximal effect at 10 microM), but roughly paralleled the appearance of cell damage (17-44% lactate dehydrogenase release at IC50). The decrease in NO production due to 1-chloro-2,4-dinitrobenzene was partially reversed by cysteine, dithioerythritol, and dihydrolipoate, whereas cystine partially reversed the decrease due to diamide or diethyl maleate. On the other hand, several thiols diminished NO production in control cells. Overall, alterations of NO production did not parallel the depletion or replenishment of either glutathione, protein thiol, or soluble thiol pools, and so the results argue against hypotheses that cellular thiols are either substrates or necessary cofactors in the pathway of NO synthesis in endothelial cells.

Animals↗
PubMed1991

Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2.

Acridines↗
PubMed1991

Oxidative stress: from basic research to clinical application.

The occurrence of reactive oxygen species, known as pro-oxidants, is an attribute of normal aerobic life. The steady-state formation of pro-oxidants is balanced by a similar rate of their consumption by antioxidants that are enzymatic and/or nonenzymatic. "Oxidative stress" results from imbalance in this pro-oxidant-antioxidant equilibrium in favor of the pro-oxidants. A number of diseases are associated with oxidative stress, being the basis of antioxidant therapy. Current evidence in clinical research does not show unequivocal distinction between causal or associative relationships of pro-oxidants to the disease process.

Antioxidants↗
PubMed1991

ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver.

Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.

Adenosine Triphosphate↗
PubMed1991

Formation of 8-hydroxy(deoxy)guanosine and generation of strand breaks at guanine residues in DNA by singlet oxygen.

Singlet molecular oxygen (1O2) was generated in aqueous solution (H2O or D2O) at 37 degrees C by the thermal dissociation of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). Guanosine and deoxyguanosine quench 1O2 with overall quenching rate constants of 6.2 X 10(6) M-1 s-1 and 5.2 X 10(6) M-1 s-1, respectively. Reaction with 1O2 results in the formation of 8-hydroxyguanosine (8-OH-Guo) and 8-hydroxydeoxyguanosine (8-OH-dGuo), respectively, with a yield of 1.5% at 1 mM substrate with an NDPO2 concentration of 40 mM; a corresponding 8-hydroxy derivative is not formed from deoxyadenosine. In D2O the yield of 8-OH-Guo is 1.5-fold that in H2O. Sodium azide suppresses 8-OH-Guo and 8-OH-dGuo production. In contrast, the hydroxyl radical scavengers, tert-butanol, 2-propanol, or sodium formate, do not decrease the production of the 8-OH derivatives. The formation of 8-OH derivatives is significantly increased (2-5-fold) by thiols such as dithiothreitol, glutathione, cysteine, and cysteamine. With use of a plasmid containing a fragment of the mouse metallothionein I promoter (pMTP3') and a novel end-labeling technique, the position of 1O2-induced single-strand breaks in DNA was examined. Strand breaks occur selectively at dGuo; no major differences (hot spots) were observed between individual guanines.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1991

Singlet oxygen induced single-strand breaks in plasmid pBR322 DNA: the enhancing effect of thiols.

The biologically occurring thiols, glutathione, cysteamine and cysteine, significantly enhance the single-strand breaks in plasmid pBR322 DNA induced by singlet molecular oxygen (1O2) generated by the thermodissociation of the endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. The enhancing effect was also observed with chemically related sulfhydryl compounds but not by disulfides. In contrast, dihydrolipoate and its disulfide lipoate protected the plasmid DNA. Metal chelators as well as superoxide dismutase or catalase had no effect, whereas mannitol or sodium azide, decreased the thiol-1O2-induced strand breaks. It is concluded that the observed effects are mediated by reactive oxidation products arising from the 1O2-oxidation of thiols.

Chelating Agents↗
PubMed1991

Identification of an abundant S-thiolated rat liver protein as carbonic anhydrase III; characterization of S-thiolation and dethiolation reactions.

An S-thiolated 30-kDa protein has been purified from rat liver by two steps of ion-exchange chromatography. This monomeric protein has two "reactive" sulfhydryls that can be S-thiolated by glutathione (form a mixed disulfide with glutathione) in intact liver. The protein has been identified as carbonic anhydrase III by sequence analysis of tryptic peptides from the pure protein. The two "reactive" sulfhydryls on this protein can produce three different S-thiolated forms of the protein that can be separated by isoelectric focusing. Using this technique it was possible to study the S-thiolation and dethiolation reactions of the pure protein. The reduced form of this protein was S-thiolated both by thiol-disulfide exchange with glutathione disulfide and by oxyradical-initiated S-thiolation with reduced glutathione. The S-thiolation rate of this 30-kDa protein was somewhat slower than that of glycogen phosphorylase b by both S-thiolation mechanisms. The S-thiolated form of this protein was poorly dethiolated (i.e., reduced) by glutathione, cysteine, cysteamine, or coenzyme A alone. Enzymatic catalysis by two different enzymes (glutaredoxin and thioredoxin-like) greatly enhanced the dethiolation rate. These experiments suggest that carbonic anhydrase III is a major participant in the liver response to oxidative stress, and that the protein may be S-thiolated by two different non-enzymatic mechanisms and dethiolated by enzymatic reactions in intact cells. Thus, the S-thiolation/dethiolation of carbonic anhydrase III resembles glycogen phosphorylase and not creatine kinase.

Amino Acid Sequence↗
PubMed1991

Activity of thiols as singlet molecular oxygen quenchers.

Singlet molecular oxygen O2(1 delta g) arising from the thermodissociation of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) was used to assess the quenching ability of various thiols and related compounds in sodium phosphate buffer in D2O at 37 degrees C. The overall quenching ability decreases in the sequence ergothioneine, methionine, cysteine, beta,beta-dimethyl cysteine (penicillamine), mercaptopropionylglycine, mesna, glutathione (GSH), dithiothreitol, N-acetyl cysteine and captopril. Cystine, glutathione disulphide, dimesna, methionine sulphone and methionine sulphoxide have no quenching effect. Comparison of the rate constants for physical (kq) with chemical (kr) quenching by thiols indicates that chemical reactivity accounts fully for their ability to quench O2(1 delta g), and pD dependence indicates that the thiolate anion reacts with O2(1 delta g). Loss of thiol groups, as exemplified by GSH, is not affected by the free radical scavengers superoxide dismutase and mannitol. However, sodium azide, a scavenger of O2(1 delta g), completely prevents NDPO2-induced thiol depletion. Depletion of GSH by NDPO2 is accompanied by the formation of its disulphide, sulphinate, sulphonate, sulphoxide and other products.

Glutathione↗
PubMed1991

Cholestasis and changes of portal pressure caused by chlorpromazine in the perfused rat liver.

Chlorpromazine (10 mumol/L) causes a marked increase in portal pressure in perfused rat liver. Simultaneously, oxygen consumption, hepatic clearance of taurocholate and bile flow are diminished. These effects are prevented by the cyclooxygenase inhibitors indomethacin (15 mumol/L), acetylsalicylate (3 mmol/L) or ibuprofen (200 mumol/L). On addition of chlorpromazine the liver releases increased amounts of prostaglandin D2; this increase does not occur in the presence of indomethacin. At higher concentrations of chlorpromazine (100 mumol/L) the inhibition of taurocholate clearance and bile flow is accompanied by only a moderate increase of portal pressure, and indomethacin is without effect. At this high concentration, substantial cell damage, as indicated by the release of lactate dehydrogenase, is present. We conclude that arachidonic acid-derived metabolites, notably prostanoids, are involved in the inhibition of bile flow and of taurocholate clearance observed at low concentrations of chlorpromazine. The data suggest that changes in the microcirculation are responsible for the impairment of the liver functions. At higher concentrations of chlorpromazine the cell toxicity of the drug becomes prominent.

Animals↗
PubMed1991

Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols.

Reactive oxygen species occur in tissues and can damage DNA, proteins, carbohydrates, and lipids. These potentially deleterious reactions are controlled by a system of enzymatic and nonenzymatic antioxidants which eliminate prooxidants and scavenge free radicals. The ability of the lipid-soluble carotenoids to quench singlet molecular oxygen may explain some anticancer properties of the carotenoids, independent of their provitamin A activity. Tocopherols are the most abundant and efficient scavengers of hydroperoxyl radicals in biological membranes. Water-soluble antioxidants include ascorbate and cellular thiols. Glutathione is an important substrate for enzymatic antioxidant functions and is capable of nonenzymatic radical scavenging. Thiols associated with membrane proteins may also be important to the antioxidant systems. Interactions between the thiols, tocopherols, and other compounds enhance the effectiveness of cellular antioxidant defense.

Animals↗
PubMed1990

Oxidative damage to mitochondria and protection by ebselen and other antioxidants.

Iron/ascorbate induced lipid peroxidation in liver mitochondria isolated from normal and glutathione-depleted rats was monitored by low-level chemiluminescence and by accumulation of thiobarbituric acid-reactive substances (TBARS). Antioxidant capacity was assessed by the duration of the lag phase preceding the onset of active peroxidation. The lag phases in state 4 and in the presence of uncouplers were similar, but shorter in the presence of ADP (state 3). In glutathione-depleted rats the lag periods were less than those in normal mitochondria. A biphasic pattern of loss of membrane alpha-tocopherol was typical in state 4 with about 55% remaining after 40 min, while in presence of ADP there was a steady and rapid loss to about 30% of the initial level. Synthetic antioxidants such as ebselen or its glutathione adduct protected mitochondrial membranes against peroxidative reactions. There was a 5-fold increase in the lag phase with 1 microM ebselen in state 4 (lag doubling concentration, 0.4 microM) and a significantly lower rate of loss of alpha-tocopherol with about 90% of the initial level still remaining after 40 min. Likewise, the lag doubling concentrations were 0.04 microM for diethyldithiocarbamate, 0.3 microM for 5-hydroxyindole, 10 microM for dihydroxyphenylalanine and serotonin, and about 40 microM for epinephrine and norepinephrine.

Animals↗
PubMed1990

Inverse relationship of epidermal growth factor receptor and HER2/neu gene expression in human renal cell carcinoma.

Expression of the oncogenes, epidermal growth factor (EGF) receptor, HER2/neu, c-myc, and c-fos, in renal cell carcinoma and corresponding nonneoplastic kidney tissue of 30 patients has been analyzed by Northern blot analysis. In renal cell carcinoma an inverse relationship of EGF receptor and HER2/neu gene expression was detected, with high expression of the EGF receptor gene in 22 of 30 (73%) cases and low expression of the HER2/neu gene in 28 of 30 (93%) cases. Furthermore, altered expression of the oncogenes c-myc and c-fos was detected in renal cell carcinoma, which appears to be related to the tumor grade of malignancy. Additional Southern blot analysis of six renal cell carcinomas gave no indication of chromosomal rearrangement events or gene amplification.

Biomarkers, Tumor↗
PubMed1990

Physical and chemical scavenging of singlet molecular oxygen by tocopherols.

Singlet molecular oxygen (1O2) arising from the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate was used to assess the effectiveness of alpha-, beta-, gamma-, and delta-tocopherol in the physical quenching as well as the chemical reaction of 1O2. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of alpha greater than or equal to beta greater than gamma greater than delta-tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with 1O2 was low, accounting for 0.1-1.5% of physical quenching with beta-tocopherol showing particularly low reactivity, resulting in the sequence alpha greater than gamma greater than delta greater than beta-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from gamma-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with 1O2. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of 1O2, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that beta-tocopherol is as effective as alpha-tocopherol in physical quenching of 1O2 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense.

Kinetics↗
PubMed1990

Phosphorylase and creatine kinase modification by thiol-disulfide exchange and by xanthine oxidase-initiated S-thiolation.

The reaction of glycogen phosphorylase b and creatine kinase with glutathione disulfide, cystine, and cystamine was compared by direct analysis on electrofocusing gels. This method was useful for individual proteins or for mixtures of the proteins. Millimolar concentrations of glutathione disulfide were required for both proteins and the rate of modification of each protein was similar. The reaction of glutathione disulfide with creatine kinase was inhibited by reduced glutathione (GSH), but the effect on the reaction with phosphorylase was minimal. Cystine and cystamine were required in micromolar amounts to effectively form the disulfide adducts. Both proteins were modified by cystine but cystamine reacted only with phosphorylase. Cystamine (10 microM) was an effective inhibitor of the reaction of phosphorylase b with 2 mM glutathione disulfide. S-thiolation of creatine kinase inactivated the enzyme and a direct assay of the enzyme activity could be used to quantitate S-thiolation of this protein by each of the disulfides. The effect of each disulfide on enzyme activity confirmed the results obtained by gel electrofocusing. Glutathione disulfide and cystine both inactivated the enzyme while cystamine had no effect on the activity. S-thiolation of phosphorylase had no observable effect on any activity parameter, but it effectively prevented binding of phosphorylase to high-molecular-weight glycogen, probably at the glycogen storage site of phosphorylase. The rate of S-thiolation of a mixture of phosphorylase and creatine kinase by thiol-disulfide exchange with glutathione disulfide was compared to the rate of S-thiolation of these proteins by a xanthine oxidase-initiated process (presumably due to protein sulfhydryl activation by reactive oxygen species). The xanthine oxidase-initiated mechanism was somewhat faster than thiol-disulfide exchange with both proteins. It was shown that GSH inhibited S-thiolation of creatine kinase by this mechanism as well as by thiol-disulfide exchange. It is suggested that both mechanisms may play a role in protein S-thiolation in vivo. For proteins that are typified by creatine kinase, the concentration of GSH in the cells may determine whether the S-thiolated form of the protein accumulates. For proteins typified by phosphorylase b, the accumulation of S-thiolated forms may be more independent of GSH.

Animals↗
PubMed1990

Reactivity of ebselen and related selenoorganic compounds with 1,2-dichloroethane radical cations and halogenated peroxyl radicals.

The reactivity of ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, and structurally related analogues was studied by pulse radiolysis. The rate constant for the reaction of ebselen with trichloromethylperoxyl radicals was determined to be 2.9 X 10(8) M-1 s-1, while its sulfur analogue, 2-phenyl-1,2-benzisothiazol-3(2H)one, was oxidized at much lower rates, k less than or equal to 10(7) M-1 s-1. Among several derivatives studied, the only other compound that exhibited a high rate constant was 2-(methylseleno)-benzoic acid-N-phenylamide. Oxidation of ebselen by other halogenated peroxyl radicals was also carried out and revealed a direct relationship between rate constant and the degree of halogenation of the oxidant. The transient radicals generated during oxidation of ebselen and the analogues were characterized by optical absorption and conductivity measurements and were attributed to one-electron-oxidized radical cations. The oxidation potentials were determined by cyclic voltammetry. Comparative evaluation of the in vitro behavior during microsomal lipid peroxidation revealed ebselen to be the most potent antioxidant of the compounds investigated, 2-(Methylseleno)-benzoic acid-N-phenylamide, despite its high rate constant for oxidation by halogenated peroxyl radicals, was found to be a poor antioxidant. The rate constant of oxidation of ebselen by trichloromethylperoxyl radicals is comparable to that of alpha-tocopherol under similar conditions, underscoring the potential pharmacological interest of ebselen as an antioxidant.

Antioxidants↗