Free radical mechanisms in relation to tissue injury.
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Animals↗
Biomedical subjects
T F Slater
Publications and source records attributed to T F Slater.
Production of prostacyclin, 6-keto-PGF1 alpha and thromboxane B2 by human umbilical vessels increases from the placenta towards the fetus.
The aim of this study was to investigate the production of prostacyclin (PGI2) and thromboxane B2 (TXB2) by incubated samples of umbilical arteries and veins taken at different distances (2, 10, 20, 30 cm) from the placenta to provide additional information relevant to the haemodynamics of umbilical blood flow. The production of PGI2, and 6-keto-PGF1 alpha (the stable metabolite of PGI2), was higher in both veins and arteries as the distance from the placenta at which the vessels were sampled was increased. A similar correlation between production by venous rings and distance from the placenta was observed for TXB2, but there was no apparent gradient of TXB2 production by the samples of arterial rings. No statistically significant variations were discernible in the ratio of 6-keto-PGF1 alpha:TXB2 (approximately 50 in the veins and approximately 20 in the arteries) in relation to the sampling distance. The significance of these high ratios is discussed in relation to umbilical blood flow and fetal well-being and development.
6-Ketoprostaglandin F1 alpha↗
Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy.
The purpose of this study was to use a direct method, that of electron spin resonance (ESR) spectroscopy, to demonstrate that reperfusion after a period of ischemia results in a sudden increase in the production of free radicals in the myocardium. The isolated buffer-perfused rat heart was used with N-tert-butyl-alpha-phenylnitrone (PBN) as a spin-trapping agent. Samples of coronary effluent were taken and extracted into toluene for detection of radical adducts by ESR spectroscopy. After 15 minutes of total, global ischemia, aerobic reperfusion resulted in a sudden burst of radical formation that peaked at 4 minutes. When hearts were reperfused with anoxic buffer, no dramatic increase in radical production was observed. Subsequent reintroduction of oxygen, however, resulted in an immediate burst of radical production of a similar magnitude to that seen in the wholly aerobic reperfusion experiments. The ESR signals obtained (aN = 13.60 G, aH = 1.56 G) are consistent with the spin-trapping by PBN of either a carbon-centered species or an alkoxyl radical, both of which could be formed by secondary reactions of initially-formed oxygen radicals with membrane lipid components.
Animals↗
Electron spin resonance studies on isolated hepatocytes treated with ferrous or ferric iron.
Isolated rat hepatocytes incubated with iron salts in the presence of the spin trapping agent alpha-4-pyridyl-l-oxide N-tert-butyl nitrone (4-POBN) generate a clear electron spin resonance signal; this signal is not detectable in the absence of exogenous iron. The hyperfine splitting constants are identical whether ferrous or ferric iron is used. The free radical trapped does not appear to be an active oxygen species but rather a carbon-centred radical, which we here ascribe to a lipodienyl radical on the basis of its hyperfine splitting features. Support to this interpretation is lent by the fact that no such radical could be generated in hepatocytes fully protected against lipid peroxidation by pretreating the donor rats with alpha-tocopherol.
Animals↗
Effects of alpha-tocopherol on carbon tetrachloride metabolism in rat liver microsomes.
alpha-tocopherol is the major lipid-soluble radical-scavenging antioxidant in rat liver. It has long been used as a putative protective agent in CCl4 induced liver injury but with variable results. We have used alpha-tocopherol loaded rat liver microsomes to study the effect of this vitamin on CCl4 metabolism in vitro. As expected, alpha-tocopherol inhibits CCl4-dependent microsomal lipid peroxidation and, at a very high concentration, will inhibit the covalent binding of CCl3 to microsomal protein by up to 50%. No inhibitory effect was observed towards CCl3 production as measured by the electron spin resonance technique of spin-trapping but this apparent discrepancy may represent a limitation of the technique. The high levels required to inhibit covalent binding probably preclude the likelihood of alpha-tocopherol significantly affecting that phenomenon at endogenous concentrations but may be relevant to other experiments employing high doses of alpha-tocopherol as an experimental hepatoprotective agent.
7-Alkoxycoumarin O-Dealkylase↗
Free radical scavenging properties of modulators of eicosanoid metabolism.
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4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
Free radicals and tissue injury: fact and fiction.
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Animals↗
Lipid peroxidation in regenerating rat liver.
Rats entrained to a strictly regulated lighting and feeding schedule have been subjected to partial hepatectomy or a sham operation. In the partially hepatectomised animals the period of liver regeneration is characterised by regular bursts of thymidine kinase activity. Liver microsomes from rats, at times corresponding to maximum thymidine kinase activity, have much reduced rates of lipid peroxidation compared to control preparations: this is due in part to increased levels of lipid-soluble antioxidant at times of maximal DNA synthesis. This temporal relationship between thymidine kinase and lipid peroxidation is consistent with the view that lipid peroxidation is decreased prior to cell division.
Animals↗
Studies on the photolytic breakdown of hydroperoxides and peroxidized fatty acids by using electron spin resonance spectroscopy. Spin trapping of alkoxyl and peroxyl radicals in organic solvents.
Spin trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used to detect and distinguish between the carbon-centred, alkoxyl, and peroxyl radicals produced during the photolytic decomposition of hydroperoxides. Photolysis of tert-butyl and cumene hydroperoxides, and peroxidized fatty acids, in toluene, with low levels of u.v. light, is shown to lead to the initial production of alkoxyl radicals by homolysis of the oxygen-oxygen bond. Subsequent reaction of these radicals with excess hydroperoxide leads, by hydrogen abstraction, to the production of peroxyl radicals that can be detected as their corresponding adducts with the spin trap. Subsequent breakdown of these adducts produces alkoxyl radicals and a further species that is believed to be the oxidized spin-trap radical 5,5-dimethyl-1-pyrrolidone-2-oxyl. No evidence was obtained at low hydroperoxide concentrations, with either the cumene or lipid alkoxyl radicals, for the occurrence of beta-scission reactions; the production of low levels of carbon-centred radicals is believed to be due to the alternative reactions of hydrogen abstraction, ring closure, and/or 1,2 hydrogen shifts. Analogous experiments with 3,3,5,5-tetramethyl-1-pyrroline N-oxide (TMPO) led only to the trapping of alkoxyl radicals with no evidence for peroxyl radical adducts, this is presumably due to a decreased rate of radical addition because of increased steric hindrance.
Benzene Derivatives↗
Effect of spin traps in isolated rat hepatocytes and liver microsomes.
Spin traps are increasingly employed in the detection of free radicals in biological systems, including liver microsomes and isolated hepatocytes. Two spin traps phenyl-t-butyl nitrone (PBN) and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) have been tested for their effects on hepatocyte viability and mixed-function oxidase activity. High concentration of PBN but not of 4-POBN proved to moderately affect liver cell integrity, without interfering with intracellular ATP or cytochrome P-450 content. PBN also decreased hepatocyte GSH content, probably as the result of its metabolism to benzaldehyde. The two spin traps were found to inhibit aminopyrine demethylase and ethoxycoumarin deethylase activity in hepatocytes and microsomes. At low concentrations (1-5 mM) PBN enhanced aniline hydroxylase while high concentrations of the spin trap inhibited this activity. The inhibition of the monooxygenase system was not caused by damage of microsomal enzymes, but rather by competition with other substrates for the binding to the haemoprotein. The effects of spin traps on mixed function oxidase systems should be taken into account when evaluating the results of spin trapping experiments.
Animals↗
Biochemical studies on a 12-lipoxygenase in human uterine cervix.
Human uterine cervix possesses a high 12-lipoxygenase activity; this enzyme has been isolated in a purified form from the squamous epithelial region of human cervix and its major properties have been investigated. Enzyme activity was present in all subcellular fractions obtained by centrifugation; the highest specific activity was associated with the microsome fraction (160,000 X g pellet). Purification of the enzyme was achieved by acetone precipitation, ion exchange chromatography on CM-cellulose and affinity chromatography on linoleyl-aminoethyl-Sepharose. The product from the incubation of sodium [1-14C]arachidonate with crude enzyme extracts co-chromatographed with authentic 12-hydroxyeicosatetraenoic acid, but the purified enzyme gave a product that behaved like the 12-hydroperoxy derivative. The enzyme had optimum activity at pH 6.5, a Km of 15 microM for arachidonic acid and was stimulated by ATP and Ca2+. Enzyme activity was inhibited by esculetin, nordihydroguaiaretic acid, eicosatetraynoic acid, detergents at concentrations greater than 0.1% (w/v) and preincubation of substrate with GSH and GSH peroxidase. The occurrence of a high 12-lipoxygenase activity is discussed in relation to the specific physiological functions of this tissue.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.
A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.
Animals↗
Lipid peroxidation and molecular damage to polyunsaturated fatty acids in rat liver. Recognition of two classes of hydroperoxides formed under conditions in vivo.
The diene conjugates formed during the autoxidation of microsomal lipid extracts, and in endoplasmic reticulum in vivo after exposing rats to CCl4 have been examined by second derivative absorption spectrophotometry. Within a few minutes after administering CCl4 to a rat there is a pronounced signal in microsomal lipid extracts that is ascribed to the cis-trans diene conjugates of microsomal polyunsaturated fatty acids. Somewhat later a second signal becomes evident that is ascribed to trans-trans isomers. The appearance of the trans-trans isomer is very strongly suppressed by prior administration of vitamin E to the rat. It is concluded that the relative contents of cis-trans and trans-trans dienes in lipid extracts of tissue reflect the tissue contents of hydrogen donors as already established for model experiments with polyunsaturated fatty acids in vitro.
Animals↗
Electron spin resonance spin trapping studies on the photolytic generation of halocarbon radicals.
The technique of free radical spin trapping has been used to study the photolytic behaviour of anaerobic solutions of aliphatic halocarbons such as CCl4, CBrCl3 and halothane in either toluene or water at room temperature. Use of the spin traps N-tert-butyl-alpha-phenylnitrone (PBN) and 2-methyl-2-nitrosopropane provides evidence for photolytic cleavage of carbon-halogen bonds yielding radicals such as .CCl3 (from CCl4 and CBrCl3) and .CHClCF3 (from halothane) which are readily trapped; this method of generating biologically important radicals may be of use in studying model reactions of these species. Under aerobic conditions evidence is obtained, by use of the spin trap PBN, for the production of the corresponding halocarbon peroxyl radicals (such as CCl3O2.) by addition of oxygen to the initially produced halocarbon radical. Though the direct detection of the peroxyl radical adducts to the spin trap proved impossible under these experimental conditions, the observed oxidation products of the spin trap (acyl nitroxides) are shown to be indicative of the presence of such species.
Bromotrichloromethane↗
Quantitative cytospectrophotometric studies on protein thiols and reactive protein disulphides in samples of normal human uterine cervix and on samples obtained from patients with dysplasia or carcinoma-in-situ.
Quantitative microspectophotometric studies have been made on sections of human cervix after staining for reactive protein thiol-groups (PSHr), and the sum of protein thiols with so-called reactive protein disulphides (together abbreviated as TRPS). Measurements were made on normal epithelium, apparently normal epithelium adjacent to a pathological lesion, dysplastic epithelium, carcinoma-in-situ, and adjoining stroma. The numbers of cases studied were: normal healthy controls (53); patients with dysplasias (34) and patients with carcinoma-in-situ (29). In the normal control sections the ratio of PSHr in epithelium:stroma was approximately 2.7 and this ratio was strongly decreased in dysplasias (1.6) and carcinoma-in-situ (1.5); the 3 populations of values had sufficient overlap to prevent this measurement being an effective discriminator. No significant variations were observed with TRPS-values except with changes in the stroma adjacent to apparently normal epithelium. However, the ratio of PSHr:TRPS was effectively discriminatory when this double-staining ratio was calculated for epithelial values:stromal values. These results are discussed in relation to the importance of thiol-groups in cell division and cancer, and the biological implications of similar changes observed in neighbouring apparently normal epithelium.
Carcinoma in Situ↗
Effects of 2-mercaptopropionyl glycine on radiation-induced lipid peroxidation in liposomes and in rat liver microsomal suspensions.
gamma-Irradiation of rat liver microsomal suspensions resulted in the accumulation of both malondialdehyde (MDA) and lipid hydroperoxides. The presence of 2-mercaptopropionylglycine (MPG) during the irradiation period decreased the formation of MDA and lipid hydroperoxides in a dose (MPG)-dependent manner. This may be attributed to the ability of MPG to scavenge the free radicals produced by irradiation. Post-irradiation incubation of microsomes further enhanced the production of both MDA and lipid hydroperoxides; when high concentrations of MPG were present during the incubations the production of MDA and lipid hydroperoxides was substantially decreased. This antioxidant role of MPG was demonstrated for both pre-irradiated microsomes and liposomes and is thought to be due to the conversion of the hydroperoxy to hydroxy fatty acids within the lipid bilayer, as well as the scavenging action on initiating free radicals.
Amino Acids, Sulfur↗
Inhibitory effects of ebselen on lipid peroxidation in rat liver microsomes.
The effects of ebselen(2-phenyl-1,2-benzoisoselenazol-3(2H)-one), a synthetic seleno-organic compound with glutathione peroxidase-like activity were investigated on lipid peroxidation in rat liver microsomes. Ebselen inhibited malondialdehyde production coupled to the lipid peroxidation stimulated by either ADP-iron-ascorbate or CCl4. The inhibitory activity of ebselen on each system was strongly increased by a 5-min preincubation with liver microsomes; the IC50 values against ADP-Fe-ascorbate-stimulated and CCl4-stimulated lipid peroxidation were 1.6 microM and 70 microM respectively. Ebselen also inhibited the endogenous lipid peroxidation with a NADPH-generating system, but it slightly stimulated the endogenous activity of ADP-Fe-ascorbate-stimulated lipid peroxidation (without a NADPH-generating system). Furthermore, ebselen inhibited oxygen uptake coupled to the lipid peroxidation by ADP-Fe-ascorbate and NADPH-ADP-iron; the IC50 values were 2.5 microM and 20.3 microM respectively. Ebselen also prolonged the lag-time of onset of ADP-Fe-ascorbate-stimulated lipid peroxidation significantly, but not that observed with NADPH-ADP-Fe-stimulated lipid peroxidation. These findings suggest that ebselen penetrates into the membrane lipid and acts as an effective antioxidant, and that there may be some differences between the modes of inhibitory action on the several types of lipid peroxidation.
Adenosine Diphosphate↗
Biological antioxidants.
The mechanism of lipid peroxidation and the ways in which the rate of this reaction can be reduced by small quantities of certain specific chemicals, called antioxidants, are described. The types and roles of the different antioxidants found in living systems are considered. Vitamin E (alpha-tocopherol) has long been recognized as an important lipid-soluble, chain-breaking antioxidant. It has an unexpectedly high reactivity towards peroxyl radicals, which can be understood only after detailed consideration of its structure. It is the major antioxidant of its class in human blood and its effectiveness in plasma is greatly improved by a synergistic interaction with water-soluble reducing agents such as ascorbic acid. Experiments designed to locate vitamin E within phospholipid bilayers and to discover the origin of the different biopotencies of stereoisomers of alpha-tocopherol are also described.
Antioxidants↗