Biomedical subjects

S Puntarulo

Publications and source records attributed to S Puntarulo.

41 records · Page 3Linked to original sources

PubMed1988

Oxidation of pyrazole to 4-hydroxypyrazole by intact rat hepatocytes.

4-Hydroxypyrazole has been identified as a major metabolite found in the urine of rats and mice after in vivo administration of pyrazole, a potent inhibitor of alcohol dehydrogenase and of ethanol metabolism. The locus and the enzyme systems responsible for the oxidation of pyrazole have not been identified. In the current report, isolated hepatocytes from fed rats were shown to oxidize pyrazole to 4-hydroxypyrazole. An HPLC procedure employing UV and electrochemical detection was utilized to separate and quantify the 4-hydroxypyrazole. The apparent Km for pyrazole by intact hepatocytes was about 2 mM, whereas the apparent Vmax was about 0.06 nmol 4-hydroxypyrazole per min per mg liver cell protein. The production of 4-hydroxypyrazole was inhibited by carbon monoxide and metyrapone, as well as by competitive drug substrates such as aniline or aminopyrine. These results implicate a role for cytochrome P-450 in the oxidation of pyrazole by the hepatocytes. Ethanol was an effective inhibitor of pyrazole oxidation. Hepatocytes were also isolated from rats treated with acetone and 4-methylpyrazole, to attempt to evaluate whether pyrazole oxidation is induced. The rate of 4-hydroxypyrazole production by hepatocytes after acetone and 4-methylpyrazole treatment was actually lower than that of controls. Kinetic assays suggested the presence of an endogenous inhibitor (perhaps the inducer itself) in the induced hepatocytes. In contrast, hepatocytes isolated from rats fasted for 48 hr showed a 2-fold increase in the oxidation of pyrazole to 4-hydroxypyrazole. The Km for pyrazole was the same in hepatocytes from fasted and fed rats, whereas Vmax was increased after fasting. The locus and enzyme system responsible for the oxidation of pyrazole to 4-hydroxypyrazole, and the site of sensitivity to ethanol, appears to be the cytochrome P-450 system of the hepatocyte.

Animals↗

PubMed1988

Increased microsomal interaction with iron and oxygen radical generation after chronic acetone treatment.

In vivo administration of acetone influences a variety of reactions catalyzed by rat liver microsomes. The effect of chronic treatment with acetone (1% acetone in the water for 10-12 days) on interaction with iron and subsequent oxygen radical generation by liver microsomes was evaluated. Microsomes from the acetone-treated rats displayed elevated rates of H2O2 generation, an increase in iron-dependent lipid peroxidation, and enhanced chemiluminescence upon the addition of t-butylhydroperoxide. The ferric EDTA-catalyzed production of formaldehyde from DMSO or of ethylene from 2-keto-4-thiomethylbutyrate was increased 2-fold after acetone treatment. This increase in hydroxyl radical generation was accompanied by a corresponding increase in NADPH utilization and was sensitive to inhibition by catalase and a competitive scavenger, ethanol, but not to superoxide dismutase. In vitro addition of acetone to microsomes had no effect on oxygen radical generation. Associated with the chronic acetone treatment was a 2-fold increase in the microsomal content of cytochrome P-450 and in the activity of NADPH-cytochrome-P-450 reductase. It appears that increased oxygen radical generation by microsomes after chronic acetone treatment reflects the increase in the major enzyme components which comprise the mixed-function oxidase system.

Acetone↗

PubMed1988

Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination.

Hydrogen peroxide steady state levels of 5 micromolar were determined in soybean (Glycine max) embryonic axes incubated for 2 hours and in axes pretreated with aminotriazole or cyanide, where these levels were 50 and 1 micromolar, respectively. The activities of catalase (105 picomoles H(2)O(2) per minute per axis), peroxidase (10-44 picomoles H(2)O(2) per minute per axis), glutathione peroxidase (3 picomoles H(2)O(2) per minute per axis) and superoxide dismutase (3.5 units per axis), were also determined. Catalase seems to be the most important H(2)O(2) consuming enzyme at the physiological concentration of H(2)O(2). A short treatment with aminotriazole, while substantially increasing H(2)O(2) level, did not affect the growth of the axes. The production of superoxide anion by the mitochondria isolated from soybean axes was measured from the superoxide dismutase-sensitive rate of adrenochrome formation in the presence of NADH or succinate as substrate and amounted to 1.3 and 0.8 nanomole O(2) (-) per minute per milligram protein, respectively. According to the stoichiometry of O(2) (-) and H(2)O(2) dismutation reactions, it is apparent that about 0.9 to 1.5% of the total oxygen uptake proceeds through the formation of the free intermediates of the partial reduction of oxygen.

Journal Article↗

PubMed1987

Chemiluminescence studies on the generation of oxygen radicals from the interaction of NADPH-cytochrome P-450 reductase with iron.

The ability of NADPH-cytochrome P-450 reductase to interact with iron and generate oxygen radicals was evaluated by assaying for low level chemiluminescence. The basic reaction system which contained the reductase, an NADPH-generating system, ferric-EDTA as an electron acceptor, and t-butyl hydroperoxide as the oxidant acceptor, resulted in the production of chemiluminescence. Omission of any of these components resulted in a complete loss of chemiluminescence. The light emission was completely sensitive to inhibition by glutathione and butylated hydroxytoluene, partially sensitive (about 60% decrease) to catalase and hydroxyl radical scavengers, and relatively insensitive (about 20% decrease) to superoxide dismutase. The ability of other ferric chelates to replace ferric-EDTA in catalyzing the reductase-dependent chemiluminescence was evaluated. Ferric-citrate, -ADP, -ATP, and ferric-ammonium sulfate were ineffective in promoting chemiluminescence, whereas ferric-diethylenetriaminepentaacetic acid was even more effective than ferric-EDTA. Thus, the ferric chelates, which catalyze reductase-dependent chemiluminescence, are those which are efficient electron acceptors from the reductase and were previously shown to be those capable of catalyzing hydroxyl radical production by microsomes and the reductase. It is suggested that chemiluminescence results from (a) the direct interaction of the reduced iron chelate with the hydroperoxide (Fenton-type of reaction) to generate alkoxyl and peroxyl radicals, and (b) the generation of hydroxyl radicals, which subsequently react with the hydroperoxide to generate secondary radicals. The latter, but not the former, would be sensitive to inhibition by catalase and competitive hydroxyl radical scavengers, whereas both would be sensitive to antioxidants such as butylated hydroxytoluene. Chemiluminescence appears to be a versatile tool for studying the reductase-dependent generation of oxygen radicals and for the interaction of reductase with iron.

Animals↗

PubMed1987

Production of 4-hydroxypyrazole from the interaction of the alcohol dehydrogenase inhibitor pyrazole with hydroxyl radical.

Pyrazole, an effective inhibitor of alcohol dehydrogenase, was previously shown to be a scavenger of the hydroxyl radical. 4-Hydroxypyrazole is a major metabolite in the urine of animals administered pyrazole in vivo. Experiments were conducted to show that 4-hydroxypyrazole was a product of the interaction of pyrazole with hydroxyl radical generated from three different systems. The systems utilized were the iron-catalyzed oxidation of ascorbate, the coupled oxidation of hypoxanthine by xanthine oxidase, and NADPH-dependent microsomal electron transfer. Ferric-EDTA was added to all the systems to catalyze the production of hydroxyl radicals. A HPLC procedure employing either uv detection or electrochemical detection was utilized to assay for the production of 4-hydroxypyrazole. The three systems all supported the oxidation of pyrazole to 4-hydroxypyrazole by a reaction which was sensitive to inhibition by competitive hydroxyl radical scavengers such as ethanol, mannitol, or dimethyl sulfoxide and to catalase. The sensitivity to catalase implicates H2O2 as the precursor of the hydroxyl radical by all three systems. Superoxide dismutase inhibited production of 4-hydroxypyrazole only in the xanthine oxidase reaction system. In the absence of ferric-EDTA (and azide), microsomes catalyzed the oxidation of pyrazole to 4-hydroxypyrazole by a cytochrome P-450-dependent reaction which was independent of hydroxyl radicals. This latter pathway may be primarily responsible for the in vivo metabolism of pyrazole to 4-hydroxypyrazole. The production of 4-hydroxypyrazole from the interaction of pyrazole with hydroxyl radicals may be a sensitive, rapid technique for the detection of these radicals in certain tissues or under certain conditions, e.g., increasing oxidative stress.

Alcohol Dehydrogenase↗