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Biomedical subjects

H de Groot

Publications and source records attributed to H de Groot.

At least 91 records · Page 5Linked to original sources
PubMed1993

Cytotoxicity of nitric oxide in Fu5 rat hepatoma cells: evidence for co-operative action with hydrogen peroxide.

The NO-releasing compounds 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) mediated a rapid loss of viability of Fu5 rat hepatoma cells. SIN-1 in addition to NO also released the superoxide anion radical (O2-.). Its cytotoxicity, however, was not affected by superoxide dismutase. In contrast, the H2O2-converting enzyme catalase significantly, but not completely, diminished cell damage, indicating participation of H2O2 in the tumoricidal activity of SIN-1. Glucose oxidase (5 m-units/ml), producing similar amounts of H2O2 to 5 mM SIN-1, had no effect on cell viability. When 5 m-units/ml glucose oxidase was added to incubations with 5 mM SNP, which alone initiated cell injury of about 40%, cell damage was significantly increased up to 95%. Similar results were observed with 1 mM SNAP and 20 m-units/ml xanthine oxidase, which mediated cytotoxicity of about 90% when both compounds were added together, compared with 35% and 55% cell injury, respectively, induced by the single compounds. The results indicate that a co-operative action with H2O2 enhances the tumoricidal activity of NO in Fu5 cells. No evidence for an interplay of NO with O2-. in cytotoxicity, e.g. via the peroxynitrite anion (ONOO-), was found.

Animals↗
PubMed1993

Loss of alpha-tocopherol upon exposure to nitric oxide or the sydnonimine SIN-1.

SIN-1 which spontaneously decomposes to yield nitric oxide (NO.) and superoxide anion (O2.-) radicals caused a loss of microsomal alpha-tocopherol paralleled by the formation of alpha-tocopheryl quinone. The loss was partially prevented by superoxide dismutase but not by catalase. The SIN-1-induced loss of alpha-tocopherol also occurred when tocopherol was dissolved in ethanol/potassium phosphate buffer (20/80, v/v). Likewise, addition of authentic NO. to alpha-tocopherol dissolved in ethanol resulted in loss of the vitamin and quinone formation. These results suggest that NO. or its products such as peroxynitrite or nitrogen dioxide react with alpha-tocopherol, the quinone derivative being a major oxidation product. Depletion of vitamin E by NO. may contribute to tissue injury, e.g. in neuronal tissues.

Animals↗
PubMed1993

Luminol chemiluminescence in rat macrophages and granulocytes: the role of NO, O2-/H2O2, and HOCl.

Luminol chemiluminescence was increased up to five-fold by L-arginine and markedly inhibited by NG-nitro-L-arginine (L-NNA) in phorbol ester (PMA) or opsonized zymosan-activated rat Kupffer cells, and in PMA-activated rat peritoneal and alveolar macrophages. While in Kupffer cells these effects did occur without pretreatment with lipopolysaccharides (LPS), LPS pretreatment was a requirement in peritoneal and alveolar macrophages. Azide (0.05 mM) had no effect on luminol chemiluminescence in the macrophages. The changes in luminol chemiluminescence were accompanied by parallel changes in nitric oxide (NO) formation. Macrophage superoxide anion radical (O2-) production was not significantly changed by addition of L-arginine and L-NNA nor by pretreatment with LPS. No hypochlorous acid (HOCl) formation was detectable in the macrophages. In contrast, in rat granulocytes activated by a variety of stimuli including PMA, zymosan, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine and the calcium ionophore A23187 with or without pretreatment with LPS, L-arginine and L-NNA had no effect on luminol chemiluminescence. Luminol chemiluminescence, however, was largely inhibited by 0.05 mM azide. The activated granulocytes released significant amounts of HOCl but did not generate NO. These results demonstrate that NO may largely contribute to luminol chemiluminescence in rat macrophages, in which HOCl formation does not occur. On the other hand, HOCl is the reactive oxygen species responsible for luminol chemiluminescence in rat granulocytes, where NO is formed only in minor quantities, if at all.

Animals↗
PubMed1993

Injury to cultured liver endothelial cells during cold preservation: energy-dependent versus energy-deficiency injury.

Previously, we demonstrated an energy-dependent injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution. Here, the effects of Histidine-Tryptophan-Ketoglutarate (HTK) and Euro-Collins (EC) solutions on these cells were studied. In HTK solution, 83% +/- 4% of the cells had lost viability after 9 h of incubation at 4 degrees C. The addition of cyanide (1 mM) to simulate hypoxic conditions protected the cells to the extent that only 9% +/- 1% of the cells lost viability over the same period; the addition of glucose (10 mM) led to increased cell injury. ATP levels were highest in the incubations with the most rapid loss of viability. In Krebs-Henseleit buffer and EC solution, in contrast, cell injury increased upon addition of cyanide; the addition of glucose to Krebs-Henseleit buffer decreased injury. We conclude that the injury to cultured liver endothelial cells during cold incubation in HTK solution is energy-dependent, as it is in UW solution, whereas cells behave differently in EC solution and Krebs-Henseleit buffer.

Adenosine Triphosphate↗
PubMed1993

Has reactive oxygen a role in methylglyoxal toxicity? A study on cultured rat hepatocytes.

The toxicity of methylglyoxal and its ability to generate reactive oxygen species were investigated in cultured rat hepatocytes. Under aerobic and anaerobic conditions methylglyoxal increased lactate dehydrogenase (LDH) release and trypan blue uptake in a concentration dependent manner. Those concentrations of methylglyoxal causing cell injury (1 mM <) also caused the release of reactive oxygen species as indicated by peroxidase-catalyzed luminol chemiluminescence. Release of reactive oxygen was detectable only under aerobic conditions, and only became significant when a large portion of the cells had already lost their viability. It is concluded that methylglyoxal injuries cultured rat hepatocytes and induces the generation of reactive oxygen species. The reactive oxygen species, however, are essentially not involved in methylglyoxal hepatotoxicity but are released by already severely injured cells.

Aerobiosis↗
PubMed1993

Reactive oxygen species associated with cell differentiation in Neurospora crassa.

The conidiation process of Neurospora crassa is characterized by three morphogenetic events: adhesion of hyphae, development of aerial hyphae, and conidia formation. At the onset of all three events a spontaneous, low-level chemiluminescence was detected, indicating the formation of reactive oxygen species. Hyperoxic conditions increased chemiluminescence and accelerated differentiation. Hypoxic conditions abolished both chemiluminescence and differentiation. Chemiluminescence was enhanced by lucigenin and/or luminol. Butylated hydroxytoluene and antioxidants that do not readily enter the cells, like superoxide dismutase and catalase, did not lower the chemiluminescence nor had they an inhibitory effect on the differentiation process. In contrast, N,N'diphenyl-1,4-phenylene diamide, 1,3-dimethyl-2-thiourea, ammonium pyrrolinedimethyl-dithiocarbamate, and N-acetyl-L-cysteine retarded the onset or abolished both the chemiluminescence and the differentiation process. These results further support our hypothesis (Hansberg, W.; Aguirre, J. J. Theor. Biol. 142:201-221; 1990) that a hyperoxidant state triggers cell differentiation events.

Antioxidants↗
PubMed1993

Energy-dependent injury to cultured sinusoidal endothelial cells of the rat liver in UW solution.

The critical injury to liver during cold preservation is believed to occur to the sinusoidal endothelium. In this study the viability of cultured sinusoidal endothelial cells from rat liver was assessed during storage in University of Wisconsin solution at 4 degrees C. The vast majority of cells (83 +/- 12%) died within 24 hr of storage. Addition of KCN (1 mM) to the solution to simulate hypoxia markedly increased survival: only 3 +/- 2% of cells had lost viability after 24 hr in the presence of cyanide. Further experiments showed that other inhibitors of mitochondrial ATP formation (antimycin A 1 microM, rotenone 1 microM, oligomycin 10 microM, carbonyl cyanide m-chlorophenylhydrazone 1 microM) were protective as well, whereas glucose (10 mM) greatly diminished the protective effect of cyanide (loss of viability 38 +/- 7% after 24 hr). ATP measurements confirmed the correlation between the energy state of the cells and cell death: ATP levels after 6 hr of incubation were 19.9 +/- 4.0 nmol/10(6) cells in UW solution, 13.7 +/- 2.9 nmol/10(6) cells in UW + glucose, 6.9 +/- 1.9 nmol/10(6) cells in UW + KCN + glucose and 1.9 +/- 1.5 nmol/10(6) cells in UW + KCN. In contrast to the protective effect observed in UW solution, addition of KCN to Krebs-Henseleit buffer led to increased endothelial cell damage upon cold storage. We therefore conclude that in UW solution damage to the sinusoidal endothelium is energy-dependent.

Adenosine↗
PubMed1993

Improvement of the energy status of hypoxic hepatocytes by calcium channel blockers.

Isolated hepatocytes from rat liver in primary culture rapidly lost viability under hypoxic conditions. Hypoxic injury was significantly decreased by the calcium channel blockers nifedipine (5 microM) and diltiazem (10 microM). The concentrations of the inhibitors which afforded maximum protection also produced the maximum increase in the energy level of the hypoxic hepatocytes, as evidenced by their ATP, ADP, AMP, and total adenine nucleotide content and by their energy charge. The increased hypoxic energy level caused by these calcium channel blocking agents was not due to an increased rate of anaerobic glycolysis; nifedipine did not have any effect on lactate production while diltiazem slightly decreased its rate. During the first 2 h under hypoxic conditions the cytosolic Ca2+ concentration remained constant around 100 nM, subsequently increasing to 400 nM first slowly and later more rapidly. The calcium channel blockers delayed the Ca2+ increase by about 1 h but were without any effect on the rate of this increase. The results suggest that the well-known beneficial effects of calcium channel blockers on hypoxic liver injury are due in large measure to an improved energetic situation of the hepatocytes rather than to the increase in the cytosolic Ca2+ concentration being blocked.

Adenine Nucleotides↗
PubMed1992

Inhibition of superoxide and nitric oxide release and protection from reoxygenation injury by Ebselen in rat Kupffer cells.

Luminol chemiluminescence in phorbolester-activated cultured rat liver Kupffer cells was strongly inhibited by the selenoorganic compound ebselen (IC50 = 2 mumol/L). Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one) also diminished reduction of ferricytochrome c (IC50 = 10 mumol/L), indicating a suppression of superoxide anion formation. Likewise, in lipopolysaccharide-pretreated Kupffer cells, ebselen proved to be a potent inhibitor of the conversion of oxyhemoglobin to methemoglobin (IC50 = 3 mumol/L) as a measure of nitric oxide formation. The sulfur-containing analog (2-phenyl-1,2-benzisothiazol-3[2H]one) and the ebselen derivative, methylselenobenzanilide, were inactive. These results indicate that ebselen is a potent inhibitor of NADPH oxidase in Kupffer cells, as has been reported for other macrophages and granulocytes. In addition, they suggest a novel characteristic of ebselen, namely very effective inhibition of nitric oxide synthase of macrophages. In line with its inhibitory effects on the release of reactive oxygen species by macrophages, complemented by its antioxidant properties, ebselen was potent in the prevention of reoxygenation injury of Kupffer cells (IC50 approximately 5 mumol/L).

Amino Acid Oxidoreductases↗
PubMed1992

Isolated cells in the study of the molecular mechanisms of reperfusion injury.

Isolated cells make it possible to study mechanisms of cell and tissue injury under well-defined conditions, including the interaction of different cells in coculture experiments. Isolated cells, either in suspension or in monolayer cultures, have also been used to study the mechanism of reperfusion injury--in this case better termed as reoxygenation injury in view of the experimental approach taken. In hepatocytes, Kupffer, and endothelial cells, reoxygenation injury resulted in necrosis primarily mediated by reactive oxygen species released by various sources such as mitochondria (hepatocytes) and NADPH oxidase (Kupffer cells). In contrast, contracture was a characteristic feature of reoxygenation injury occurring in cardiomyocytes without loss of cytosolic enzymes. Beside reactive oxygen species, Kupffer cells were activated to release prostanoids and a decrease in endothelial cell-mediated fibrinolysis occurred upon reoxygenation. Reoxygenation injury in endothelial cells was significantly increased when neutrophils were added at the time of reoxygenation, presumably due to additional generation of reactive oxygen species and the release of proteases. As exemplified for the liver, these experiments suggest a mechanism of reperfusion injury in which the various cell types of a given tissue differ significantly in their response to hypoxia-reoxygenation but in which they interact with each other in a complex pathobiochemical network via various mediators such as cytokines, and tissue damaging effector molecules such as reactive oxygen species. Future experiments with isolated cells will allow detailed analysis of the underlying molecular mechanisms.

Cells, Cultured↗
PubMed1992

Role of reactive oxygen species in cell toxicity.

Several types of compound exert their cytotoxicity by generating reactive oxygen species, notably the superoxide anion radical. These include quinoid and nitroaromatic compounds serving as redox cyclers, i.e. producing superoxide at the expense of NADPH and oxygen catalyzed by cellular reductases. In specialized cell-types employed in defense such as granulocytes, eosinophils and macrophages, myeloperoxidase, NADPH oxidase and nitric oxide synthase have been identified as major sources of reactive oxygen species in cell toxicity. These include hypochlorite, singlet oxygen, superoxide, nitric oxide and hydrogen peroxide. The interaction of superoxide and nitric oxide generates further oxidants such as peroxynitrite. Lumino-amplified chemiluminescence generated by Kupffer cells is partially sensitive to inhibitors of NO synthase. Superoxide dismutase has been found to catalyze a novel reaction, the reversible conversion of nitric oxide to the nitroxyl anion, the latter being viewed as another form of EDRF. In the defense against oxidative damage, there are enzymatic and nonenzymatic antioxidants. Regarding compounds used pharmacologically, we have been interested in ebselen, a seleno-organic compound exhibiting GSH peroxidase activity, which protects against reactive oxygen species generated, for example, at reoxygenation following a period of hypoxia. Further, we have studied lipoate and dihydrolipoate as antioxidant redox system and as singlet oxygen quencher, e.g. protecting against damage of deoxyguanosines in plasmid DNA generated by singlet oxygen.

Animals↗
PubMed1992

Production of reactive oxygen by mitochondria from normoxic and hypoxic rat heart tissue.

Reactive oxygen species (ROS), which may be involved in ischemic or reperfusion heart injury, can be produced by mitochondria. Previous work indicated that coupled mitochondria from ischemic heart tissue incubated in calcium-free medium produced less ROS than normal. The effects of calcium, which may be elevated in hypoxic or ischemic tissue, were not examined. The relative production of ROS by mitochondria from normoxic or hypoxic rat heart tissue was estimated by measuring the oxidation of dichlorofluorescin to the fluorescent compound, dichlorofluorescein. ROS were detectable during succinate-stimulated State 4 respiration. In the absence of calcium, mitochondria from hypoxic (60 min) heart tissue produced less ROS than mitochondria from normoxic heart tissue. In the presence of 0.1, 1 or 10 microM calcium, ROS produced by hypoxic mitochondria were increased to normoxic levels. While function was depressed in mitochondria from hypoxic tissue, the presence of 0.1 and 1 microM calcium had no further effect. Respiration was uncoupled in the presence of 10 microM calcium in mitochondria from both normoxic and hypoxic heart tissue. ROS production was increased in mitochondria from hypoxic tissue with both increasing concentrations of calcium and increasing duration of exposure. ROS production in mitochondria from normoxic heart tissue was only stimulated after 200 or more seconds of exposure to 1 or 10 microM calcium. Production of ROS in mitochondria from hypoxic tissue in the presence of 1 microM calcium was inhibited by rotenone (80%), ruthenium red (69%), and a combination of these agents (96%). In contrast, ruthenium red had no effect on ROS production by mitochondria from normoxic heart tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Magic angle spinning NMR studies on the metarhodopsin II intermediate of bovine rhodopsin: evidence for an unprotonated Schiff base.

Magic angle spinning (MAS)13C-NMR spectra of the metarhodopsin II intermediate have been obtained using bovine rhodopsin regenerated with retinal 13C-labeled at the C-13 and C-15 positions to investigate the protonation state of the retinal Schiff base linkage. The 13C-labeled rhodopsin was reconstituted into 1,2-dipalmitoleoylphosphatidylcholine bilayers to increase the amount of meta II trapped at low temperature. Both the 13C-15 (159.2 ppm) and 13C-13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the 13C-15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70-90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules out the possibility that meta II non-covalently binds retinal or is a carbinolamine intermediate and provides convincing evidence that Schiff base deprotonation occurs in the meta I-meta II transition, an event that is likely to be important in triggering the activation of transducin.

Animals↗
PubMed1992

Release of reactive oxygen by hepatocytes on reoxygenation: three phases and role of mitochondria.

Reoxygenation of isolated hepatocytes in primary culture resulted in a three-phase response in the release of reactive oxygen species (ROS) as determined by peroxidase-dependent luminol chemiluminescence. Release of ROS within the first and second phase correlated well with the extent of reoxygenation injury, both being most significant after approximately 4 h of hypoxic incubation. During the third phase, some of the ROS were released by already nonviable cells. Both antimycin A and rotenone significantly increased release of ROS, indicating severe alterations of the mitochondrial respiratory chain caused by hypoxia and suggesting that the altered mitochondrial respiratory chain represents an important source for the release of ROS on reoxygenation. Generation of ROS rose sharply when the O2 content was increased from 0 to 2%, whereas a further increase in the O2 content, of up to 95%, resulted in only small but steady increases in the formation of ROS. The latter suggests that, in addition to enzymatic sources such as the mitochondrial respiratory chain, nonenzymatic reactions may also contribute to the formation of ROS on reoxygenation.

Animals↗
PubMed1992

Morphological changes of cultured rat hepatocytes exposed to methylglyoxal. Calcium-independence of injury.

Methylglyoxal-induced morphological changes were studied in cultured rat hepatocytes. Hepatocytes incubated either in the presence or absence of 2.5 mM Ca2+ were injured by 10 mM methylglyoxal to a similar extent, while 1 mM of the alpha-oxoaldehyde caused a moderate damage only in the absence of Ca2+. In the absence of Ca2+, however, hepatocytes were already injured, even without the presence of methylglyoxal hepatotoxicity proceeds independently of the Ca2+ influx.

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

13C magic-angle spinning NMR studies of bathorhodopsin, the primary photoproduct of rhodopsin.

Magic-angle spinning NMR spectra have been obtained of the bathorhodopsin photointermediate trapped at low temperature (less than 130 K) by using isorhodopsin samples regenerated with retinal specifically 13C-labeled at positions 8, 10, 11, 12, 13, 14, and 15. Comparison of the chemical shifts of the bathorhodopsin resonances with those of an all-trans-retinal protonated Schiff base (PSB) chloride salt show the largest difference (6.2 ppm) at position 13 of the protein-bound retinal. Small differences in chemical shift between bathorhodopsin and the all-trans PSB model compound are also observed at positions 10, 11, and 12. The effects are almost equal in magnitude to those previously observed in rhodopsin and isorhodopsin. Consequently, the energy stored in the primary photoproduct bathorhodopsin does not give rise to any substantial change in the average electron density at the labeled positions. The data indicate that the electronic and structural properties of the protein environment are similar to those in rhodopsin and isorhodopsin. In particular, a previously proposed perturbation near position 13 of the retinal appears not to change its position significantly with respect to the chromophore upon isomerization. The data effectively exclude charge separation between the chromophore and a protein residue as the main mechanism for energy storage in the primary photoproduct and argue that the light energy is stored in the form of distortions of the bathorhodopsin chromophore.

Chlorides↗