The properties of hydrogen peroxide production under hyperoxic and hypoxic conditions of perfused rat liver.

The properties of H2O2 production in the "haemoglobin-free", "non-circulatory" perfused liver of rats were examined. The H2O2 production with 1 mM-lactate and 0.15 mM-pyruvate was 82nmol/min per g of liver or 333nmol/min per 100g body wt. in the liver of fed rats at 30 degrees C. This rate decreased to almost half in the livers of starved and phenobarbital-pretreated rats. When H2O2 production was stimulated by urate infusion, almost all of the H2O2 produced by the uricase reaction was decomposed by the catalase reaction. During the demethylation reaction of aminopyrine, no change in H2O2 production was detected by the present method; thus microsomal H2O2 production observed in isolated subcellular fractions appeared not to contribute significantly to the H2O2 production in the whole organ. Whereas the rate of the glycolate-dependent H2O2 production was halved at an intracellular O2 concentration that caused a 10 percent increase in the reduction state of cytochrome c, the half-maximal rate of H2O2 production with lactate and pyruvate was observed at an O2 concentration that caused a 40 percent increase in the reduction state of cytochrome c in the liver. No further increase in the rates of H2O2 production was obtained by increasing O2 pressure up to 5 times 10(5) Pa. The rate of ethanol oxidation through the catalase "peroxidatic" reaction varied, depending on the substrate availability. The maximal capability of this pathway in ethanol oxidation reached approx. 1.5 mumol/min per g of liver, when a mixture of urate, glycollate and octanoate was infused to enhance H2O2 production.     

Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.     

Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats

Energy restriction (ER), without malnutrition, is the only environmental intervention that consistently increases maximum life span in laboratory rodents. One theory proposes that a reduction in energy expenditure and reactive oxygen species production is the mechanism responsible for this action of ER. To further test this theory, proton leak, H2O2 production, lipid peroxidation, and protein carbonyls were measured in mitochondria from FBNF1 rats fed either a control or 40% ER diet (onset at 6 mo of age). Liver mitochondria were isolated at 7 and 12 mo of age. Liver weight decreased 25 and 36% at 1 and 6 mo of ER, respectively (P < 0.05). ER resulted in an increase (P < 0.05) in percent total polyunsaturates, n-6 polyunsaturates, and total unsaturates (6 mo only) in mitochondrial lipids. These changes, however, were not associated with significant alterations in mitochondrial function. State 4 respiration and membrane potential were not different (P > 0.05) between groups at either assessment period. Similarly, proton leak kinetics were not different between control and ER animals. Top-down metabolic control analysis and its extension, elasticity analysis, were used at the 6-mo assessment and revealed no difference in control of the oxidative phosphorylation system between control and ER rats. H2O2 production with either succinate or pyruvate/malate substrates was also not different (P > 0.05) between groups at either time point. In conclusion, ER did not alter proton leak or H2O2 production at this age or stage of restriction in liver.     

Metabolism of 5-hydroxytryptophan in isolated perfused liver from normal and x-irradiated rats

Summary Metabolism of¹⁴C-5-OH-tryptophan was studied in isolated perfused liver from normal and whole body irradiated rats (7 to 8 days after 1000 R). Metabolism of 5-OH-tryptophan is slow compared to that of 5-OH-tryptamine, and the metabolites isolated were mainly conjugates of 5-OH-tryptamine and 5-OH-indole acetic acid. Irradiation does not diminish significantly the conversion of 5-OH-tryptophan to 5-OH-tryptamine, rather an increased formation of 5-OH-indole acetic acid and of conjugates could be observed in irradiated liver.

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Zusammenfassung Der Stoffwechsel von¹⁴-C-5-OH-Tryptophan wurde in isoliert perfundierter Leber normaler und bestrahlter Ratten untersucht (7 bis 8 Tage nach 1000 R). Der Abbau von 5-OH-Tryptophan ist gering im Vergleich zu dem von 5-OH-Tryptamin. Die beobachteten Metaboliten waren im wesentlichen die Konjugate von 5-OH-Tryptamin und 5-OH-Indolacetat. Bestrahlung vermindert nicht die Umwandlung von 5-OH-Tryptophan zu 5-OH-Tryptamin und führt sogar zu einer vermehrten Bildung von 5-OH-Indolessigsäure und ihrer Konjugate.

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This is publication No. 706 of the Euratom Biology Division Contract Euratom-C.E.N./ S.C.K. No. 078-69-1 BIAC.     

13C and 31P NMR study of gluconeogenesis: utilization of 13C-labeled substrates by perfused liver from streptozotocin-diabetic and untreated rats

The metabolism of 13C-labeled substrates was followed by 13C and 31P NMR in perfused liver from the streptozotocin-treated rat model of insulin-dependent diabetes. Comparison was made with perfused liver from untreated littermates, fasted either 24 or 12 h. The major routes of pyruvate metabolism were followed by a 13C NMR approach that provided for the determination of the metabolic fate of several substances simultaneously. The rate of gluconeogenesis was 2-4-fold greater and beta-hydroxybutyrate production was 50% greater in liver from the chronically diabetic rats as compared with the control groups. Large differences in the distribution of 13C label in hepatic alanine were measured between diabetic and control groups. The biosyntheses of 13C-labeled glutathione and N-carbamoylaspartate were monitored in time-resolved 13C NMR spectra of perfused liver. Assignments for the resonances of glutathione and N-carbamoylaspartate were made with the aid of 13C NMR studies of perchloric acid extracts of the freeze-clamped livers. 13C NMR spectroscopy of the perfusates provided a convenient, rapid assay of the rate of oxidation of [2-13C]ethanol, the hepatic output of [2-13C]acetaldehyde, and the accumulation of [2-13C]acetate in the perfusate. By 31P NMR spectroscopy, carbamoyl phosphate was measured in all diabetic livers and an unusual P,P'-diesterified pyrophosphate was observed in one-fourth of the diabetic livers examined. Neither of these phosphorylated metabolites was detected in control liver. Both 13C and 31P NMR were useful in defining changes in hepatic metabolism in experimental diabetes.     

Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi.

The glutathione peroxidase-glutathione reductase system, an alternative pathway for metabolic utilization of H2O2 [Chance, Sies & Boveris (1979) Physiol. Rev. 59, 527-605], was investigated in Trypanosoma cruzi, an organism lacking catalase and deficient in peroxidase [Boveris & Stoppani (1977) Experientia 33, 1306-1308]. The presence of glutathione (4.9 +/- 0.7 nmol of reduced glutathione/10(8) cells) and NADPH-dependent glutathione reductase (5.3 +/- 0.4 munit/10(8) cells) was demonstrated in the cytosolic fraction of the parasite, but with H2O2 as substrate glutathione peroxidase activity could not be demonstrated in the same extracts. With t-butyl hydroperoxide or cumene hydroperoxide as substrate, a very low NADPH-dependent glutathione peroxidase activity was detected (equivalent to 0.3-0.5 munit of peroxidase/10(8) cells, or about 10% of glutathione reductase activity). Blank reactions of the glutathione peroxidase assay (non-enzymic oxidation of glutathione by hydroperoxides and enzymic oxidation of NADPH) hampered accurate measurement of peroxidase activity. The presence of superoxide dismutase and ascorbate peroxidase activity in, as well as the absence of catalase from, epimastigote extracts was confirmed. Ascorbate peroxidase activity was cyanide-sensitive and heat-labile, but no activity could be demonstrated with diaminobenzidine, pyrogallol or guaiacol as electron donor. The summarized results support the view that T. cruzi epimastigotes lack an adequate enzyme defence against H2O2 and H2O2-related free radicals.     

Mechanism of the stimulatory effect of fructose on ethanol oxidation in perfused rat liver.

The stimulatory effect of fructose on ethanol oxidation was studied in livers from fasted rats perfused with Krebs-Henseleit-bicarbonate buffer in a non-recirculating system. Two series of experiments were performed: (A) ethanol was infused with stepwise increasing concentrations (0.1-20 mM) in the presence of 4 mM fructose; (B) fructose was infused with stepwise increasing concentrations (0.5-10 mM) in the presence of 2 mM ethanol. From measured metabolic rates the following parameters were calculated: energy-rich phosphates consumed for fructose metabolism which were provided from oxidative phosphorylation (delta approximately P); reducing equivalents derived from stimulated ethanol utilization which were disposed by mitochondrial oxidation (delta2H). Under the various conditions studied a linear relationship between these parameters was observed. The ratio delta approximately P/delta2H was about 2.0. It is suggested that fructose stimulates ethanol oxidation indirectly by increasing the energy consumption of the liver due to the production of glucose from fructose. Consequetnly, the rate of oxidative phosphorylation is increased and, therefore, the capacity of the respiratory chain for oxidizing reducing equivalents derived from ethanol is enhanced. The data support the more general hypothesis that the rate of ethanol oxidation depend upon the rate of hepatic energy consumption in a given metabolic state.     

Lack of melatonin effect on hydrogen peroxide induced bronchoconstriction in isolated and perfused rat lung

The effect of melatonin on hydrogen peroxide- induced broncho-and vasoconstriction was examined in vivo in the model of the isolated, perfused and ventilated lung. The administration of hydrogen peroxide (500 microM) to the perfusate caused a marked decrease in lung compliance, conductance and flow rate. The administration of melatonin (500 microM) to the perfusate 20 min before and during the hydroperoxide exposure did not cause any change in lung function. Exposure of lung microsomes to hydrogen peroxide (1-100 microM) did not induce any significant increase in malonaldehyde (MDA), an index of lipid peroxidation, and it was not affected by treatment with melatonin (500 microM). On the other hand, brain microsomes exposed to hydrogen peroxide (1-100 microM) give rise to increased levels of MDA, which were decreased by pre-treatment with melatonin (500 microM). The results suggest that melatonin may exert an antioxidant effect in conditions were lipid peroxidation is occurring. Its use may not be relevant in conditions where the mechanisms of the reactive oxygen species damage appears to be lipid peroxidation independent, such as the case of hydrogen peroxide induced broncho- and vasoconstriction.     

Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: role for protein-dimer formation.

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 mM hydrogen peroxide at 37 degrees C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.     

Orotate decreases the inhibitory effect of ethanol on galactose elimination in the perfused rat liver.

1. The galactose-elimination rate in perfused livers from starved rats was decreased in the presence of ethanol (2-28mM) to one-third of the control values. Orotate injections partly reversed the effect of ethanol, so that the galactose-elimination rate was about two-thirds of the control values. Orotate alone had no effect on the galactose-elimination rate. 2. Ethanol increased [galactose 1-phosphate] and [UDP-galactose], and decreased (UDP-glucose] and [UTP], both with and without orotate. Orotate increased [UTP], [UDP-galactose], both with and without ethanol. The increase of [galactose 1-phosphate] in the presence of ethanol was inhibited by orotate. Orotate alone had no appreciable effect on [galactose 1-phosphate]. 3. Both the effect of ethanol and that of orotate on the galactose-elimination rate can be accounted for by assuming inhibition of galactokinase by galactose 1-phosphate with Ki about 0.2mM, the inhibition being either non-competitive or uncompetitive. 4. The primary effect of ethanol seems to be inhibition of UDP-glucose epimerase (EC 5.1.3.2), followed by accumulation of UDP-galactose, trapping of UDP-glucose and increase of [galactose 1-phosphate]. Orotate decreased the effect of ethanol, probably by increasing [UDP-glucose].     

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.     

The effect of hydrogen peroxide on spores of Clostridium bifermentans.

The effect of hydrogen peroxide on the germination, colony formation and structure of spores of Clostridium bifermentans was examined. Treatment with 0.35 M-hydrogen peroxide increased the germination rate at 25 degrees C but increasing the temperature or concentration of hydrogen peroxide decreased both the germination rate and colony formation. The presence of Cu2+ increased the lethal effect of hydrogen peroxide on colony formation as much as 3000-fold. Pre-incubation of spores with Cu2+ before treatment with hydrogen peroxide produced a similar increase, but this could be eliminated by washing the spores with dilute spores--apparently from the coat--and treatment with dithiothreitol, which also removes spore-coat protein, increased the lethal effect of hydrogen peroxide 500-fold, suggesting that spore-coat protein has a protective effect against hydrogen peroxide.     

Activation of bovine liver glycerol kinase by ethanol.

The crystallization of bovine glycerol kinase (ATP:glycerol 3-phosphotransferase EC 2.7.1.30) is reported along with a study on the unusual activation of this enzyme by ethanol. The enzyme was extracted from calf lever andusual activation of this enzyme by ethanol. The enzyme was extracted from calf liver and purified 5900-fold giving crystals with a 5% yield. The kinetics of the enzyme with regard to glycerol and ATP were studied by varying the concentration of one substrate while keeping the other at saturating levels, and the effect of ethanol was observed by adding it at levels of 5% (v/v). Ethanol increased the V in both cases almost 2-fold. The apparent Km of ATP was 3.5 - 10(-6) and was increased to 7.6 - 10(-6) in the presence of ethanol. The apparent Km for glycerol was 3 - 10(-5) and was increased to 12 - 10(-5) when ethanol was added. A number of other alcohols had a similar activating effect except for 1,2-diols which only inhibited the enzyme. These findings are consistent with the explanation that alcohols compete with glycerol (hence also with the glycerophosphate product) for a hydroxy binding site on the enzyme. This leads to more rapid dissociation of the glycerophosphate (i.e. an increase in the steady-state constant, "k+2" resulting in an increased V).     

Use of sulfite and hydrogen peroxide to control bacterial contamination in ethanol fermentation.

Lactic acid bacteria isolated from an industrial-scale ethanol fermentation process were used to evaluate sulfite as a bacterial-contamination control agent in a cell-recycled continuous ethanol fermentation process. The viabilities of bacteria were decreased by sulfite at concentrations of 100 to 400 mg liter-1, while sulfite at the same concentrations did not change the viability of the Saccharomyces cerevisiae strain used in this process. Sulfite was effective only in the presence of oxygen. Bacteria showed differences in their susceptibilities to sulfite. Facultatively heterofermentative Lactobacillus casei 4-3 was more susceptible than was obligatory heterofermentative Lactobacillus fermentum 7-1. The former showed higher enzyme activities involved in the production and consumption of hydrogen peroxide than did the latter. The viability of L. fermentum 7-1 could be selectively controlled by hydrogen peroxide at concentrations of 1 to 10 mM. Based on these findings, it is hypothesized that the sulfur trioxide radical anions formed by peroxidase in the presence of hydrogen peroxide are responsible for the control of contaminating bacteria. Sulfite did not kill the yeast strain, which has catalase to degrade hydrogen peroxide. A cell-recycled continuous ethanol fermentation process was run successfully with sulfite treatments.     

Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide.

A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.     

The effect of ethanol on the bile-secreting function of the liver in rats

The effect of long-term application of ethanol on the biochemical composition of rat bile has been studied. These results have been compared with the changes of activity of hepatic microsomal enzymes and ultrastructure of hepatocytes. The changes of biliary lipid synthesis and secretion have been shown to reflect the change of microsomal metabolic functions and to be accompanied by liver destruction processes.