scholarly journals Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats

1986 ◽  
Vol 233 (3) ◽  
pp. 755-761 ◽  
Author(s):  
G Ekström ◽  
T Cronholm ◽  
M Ingelman-Sundberg
Keyword(s):  
Amino Acid ◽  
Hydroxyl Radical ◽  
Ethanol Oxidation ◽  
Liver Microsomes ◽  
Radical Scavenger ◽  
Ethanol Treatment ◽  
Radical Production ◽  
Apparent Homogeneity ◽  
Nadph Oxidation ◽  
Cytochrome P 450

In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an ‘uncoupling’ of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.

scholarly journals Ethanol Oxidation by a Component of Liver Microsomes Rich in Cytochrome P-450

1973 ◽  
Vol 248 (4) ◽  
pp. 1183-1187
Author(s):  
Esteban Mezey ◽  
James J. Potter ◽  
W. Douglas Reed
Keyword(s):  
Ethanol Oxidation ◽  
Liver Microsomes ◽  
Cytochrome P 450

scholarly journals Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted

1991 ◽  
Vol 276 (3) ◽  
pp. 753-757 ◽  
Author(s):  
M Ingelman-Sundberg ◽  
H Kaur ◽  
Y Terelius ◽  
J O Persson ◽  
B Halliwell
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Liver Microsomes ◽  
Iron Chelator ◽  
Radical Formation ◽  
Rabbit Liver ◽  
Nadph Cytochrome ◽  
Substrate Hydroxylation ◽  
Cytochrome P 450

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation.

scholarly journals Fe-EDTA-Bisamide and Fe-ADR-925, The Iron-Bound Hydrolysis Product of the Cardioprotective Agent Dexrazoxane, Cleave DNA Via the Hydroxyl Radical

1997 ◽  
Vol 4 (4) ◽  
pp. 199-205 ◽  
Author(s):  
Thomas J. Magliery ◽  
Lizabeth K. Vitellaro ◽  
Ndeye Khady Diop ◽  
Rosemary A. Marusak
Keyword(s):  
Side Effects ◽  
Hydroxyl Radical ◽  
Plasmid Dna ◽  
Dna Cleavage ◽  
Antitumor Agents ◽  
Hydrolysis Product ◽  
Solution Chemistry ◽  
Radical Scavenger ◽  
Radical Production ◽  
Radical Generation

Use of the antitumor drug doxorubicin is limited by cardiomyopathic side-effects which are believed to be due to iron-mediated hydroxyl radical generation. Dexrazoxane reduces this cardiotoxicity, possibly by removal of iron from doxorubicin by the EDTA-like hydrolysis product of dexrazoxane, ADR-925. However, EDTA-diimides like dexrazoxane, previously used as antitumor agents, are themselves carcinogenic, and recent studies have found that Fe-ADR-925 can also promote hydroxyl radical production. This study demonstrates that, like Fe-EDTA, Fe-ADR-925 and a related desmethyl complex can cleave plasmid DNA under Fenton conditions, and suggests by radical scavenger study that this cleavage is probably via the hydroxyl radical. Differences in DNA cleavage dependence upon concentrations of Fe-EDTA, Fe-ADR-925 and Fe-EDTA-bisamide can be explained by differences in the solution chemistry of the complexes.

scholarly journals Purification and characterization of a vitamin D3 25-hydroxylase from pig liver microsomes

1992 ◽  
Vol 287 (3) ◽  
pp. 725-731 ◽  
Author(s):  
E Axén ◽  
T Bergman ◽  
K Wikvall
Keyword(s):  
Liver Enzyme ◽  
Rat Liver ◽  
Vitamin D3 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Pig Liver ◽  
Specific Content ◽  
Apparent Homogeneity ◽  
Structural And Functional Properties ◽  
Cytochrome P 450

A cytochrome P-450 which catalyses 25-hydroxylation of vitamin D3 has been purified to apparent homogeneity from pig liver microsomes. The specific content of cytochrome P-450 was 12 nmol.mg of protein-1, and the preparation showed a single band with an apparent M(r) of 50,500 upon SDS/PAGE. A monoclonal antibody raised against the vitamin D3 25-hydroxylase reacted strongly with the purified 25-hydroxylating cytochrome P-450 from pig kidney microsomes [Bergman & Postlind (1990) Biochem. J. 270, 345-350]. The liver enzyme showed structural and functional properties very similar to those of the kidney enzyme. The two enzymes differed with respect to only one of the first 16 N-terminal amino acids. The vitamin D3 25-hydroxylase in pig liver microsomes exhibited a turnover and an apparent Km for 25-hydroxylation of vitamin D3 which were of the same order of magnitude as those of a well-characterized male-specific 25-hydroxylating cytochrome P-450 in rat liver microsomes. The two enzymes differed structurally. The pig liver enzyme was, in contrast to the rat liver enzyme, not sex-specific, and did not catalyse 16 alpha-hydroxylation of testosterone. These properties of the 25-hydroxylase in rat liver microsomes have led to questions on the role of microsomal 25-hydroxylation of vitamin D3. It is concluded that studies on microsomal 25-hydroxylation with the rat may be misleading. The results of the present study show that the pig appears to be a representative species for evaluation of vitamin D3 hydroxylases in other mammals, including man.

scholarly journals On the amino acid sequence of cytochrome P-450 isozyme 4 from rabbit liver microsomes.

1984 ◽  
Vol 81 (14) ◽  
pp. 4260-4264 ◽  
Author(s):  
V. S. Fujita ◽  
S. D. Black ◽  
G. E. Tarr ◽  
D. R. Koop ◽  
M. J. Coon
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Liver Microsomes ◽  
Rabbit Liver ◽  
Cytochrome P 450

scholarly journals Effect of oxygen concentration on microsomal oxidation of ethanol and generation of oxygen radicals

1988 ◽  
Vol 251 (3) ◽  
pp. 787-794 ◽  
Author(s):  
S Puntarulo ◽  
A I Cederbaum
Keyword(s):  
Lipid Peroxidation ◽  
Hydroxyl Radical ◽  
Oxygen Radicals ◽  
Liver Microsomes ◽  
Reaction System ◽  
Rat Liver Microsomes ◽  
Biphasic Response ◽  
Microsomal Lipid Peroxidation ◽  
Cytochrome P 450 ◽  
Oxidation Of Ethanol

The iron-catalysed production of hydroxyl radicals, by rat liver microsomes (microsomal fractions), assessed by the oxidation of substrate scavengers and ethanol, displayed a biphasic response to the concentration of O2 (varied from 3 to 70%), reaching a maximal value with 20% O2. The decreased rates of hydroxyl-radical generation at lower O2 concentrations correlates with lower rates of production of H2O2, the precursor of hydroxyl radical, whereas the decreased rates at elevated O2 concentrations correlate with lower rates (relative to 20% O2) of activity of NADPH-cytochrome P-450 reductase, which reduces iron and is responsible for redox cycling of iron by the microsomes. The oxidation of aniline or aminopyrine and the cytochrome P-450/oxygen-radical-independent oxidation of ethanol also displayed a biphasic response to the concentration of O2, reaching a maximum at 20% O2, which correlates with the dithionite-reducible CO-binding spectra of cytochrome P-450. Microsomal lipid peroxidation increased as the concentration of O2 was raised from 3 to 7 to 20% O2, and then began to level off. This different pattern of malondialdehyde generation compared with hydroxyl-radical production probably reflects the lack of a role for hydroxyl radical in microsomal lipid peroxidation. These results point to the complex role for O2 in microsomal generation of oxygen radicals, which is due in part to the critical necessity for maintaining the redox state of autoxidizable components of the reaction system.

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