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Reactive oxygen species produced in metal-catalyzed oxidation of bis(trifluoromethyl)disulfide and protection by ZE®
Abstract
Bis(trifluoromethyl)disulfide (TFD), used as an industrial fumigant, was found to generate a thiyl free radical as seen by EPR/spin trapping. Oxygen appears to be an absolute requirement for radical production. The results obtained in this investigation implicate the production of thiyl and reactive oxygen species (ROS), superoxide radical anion and hydroxyl radicals, during TFD autoxidation. The rate of production of these free radical intermediates was found to increase in the presence of iron(III) and copper(II). In addition, the metal ion chelator DETAPAC and ROS scavengers ethanol, mannitol, and PEG-SOD/catalase were found to inhibit free radical production. Reactive oxygen species were not formed when a high-potency zinc plus antioxidant, ZE caps, was present. These results provide support for the pro-oxidation of TFD and a protective role for zinc.
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The oxidation of L-cysteine by horseradish peroxidase in the presence of oxygen forms a thiyl free radical as demonstrated with the spin-trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. Cysteine sulfinic acid, a cysteine metabolite, is a poorer substrate for horseradish peroxidase than cysteine and is oxidized to form both sulfur-centered and carbon-centered free radicals.
This book is about selectively toxic agents. That is to say, it is about those substances that affect certain cells without harming others, even when they are close neighbours. Toxicity need not be fatal. It can be made easily reversible, as is the case with general anaesthetics. Selective toxicity covers an immense field: most of the drugs used for treating illness in man and his economic animals, as well as all of the fungicides, insecticides, and weed killers that are used in agriculture. Essentially, this book is a discussion of the physical and chemical means which contribute to selectivity, and this is the basis of molecular pharmacology. _Selective Toxicity began as a course of lectures that Professor F. G. Young encouraged me to give in University College London, in 1948 and again in 1949. The first edition appeared in 1951, as a very small book because little was then known about the factors that provide selectivity. Since those early days, the subject has undergone tremendous development. At first, industry was un receptive to the word 'toxicity', however qualified! Yet the market was being supplied with biologically powerful substances of which several had the potential to cause harm. This aspect was brought to light by two events of the early 1960s. The first of these was the discovery that a sedative, thalidomide, administered to expectant mothers, after what was then considered to be adequate testing, had caused permanent deformities in about 10000 children.
Spin trapping is a technique in which a reactive free radical reacts with a double bond of a diamagnetic compound, the spin trap, to form a less reactive free radical, the spin adduct. Spin trapping is often necessary when the primary free radical cannot be observed by conventional ESR either because of low concentration or very short radical relaxation times which lead to very broad lines (Janzen, 1980).
New organophosphorus compounds containing one or two different S-trifluoromethyl functionalities are synthesized in the reaction between the tri-coordinate phosphorus esters and bis-(trifluoromethane) disulfide. The catalytic effect exerted by the nucleophilic species such as the fluoride anion or tertiary amine on the decomposition of S-trifluoromethyl organophosphorus derivatives is documented. This observation is utilized for the preparation of different types of organophosphorus fluoridates.
A number of free-radical reactions have been examined by recording the e.s.r. spectra of nitroxides which are formed when the reactions are carried out in the presence of 2-methyl-2-nitrosopropane. Examples are presented in which the nitroso-compound scavenges either primary radicals from the decomposition of various initiators, or secondary radicals produced by hydrogen abstraction from a component of the solvent. In the majority of cases the identity of the scavenged radical is at once evident from the e.s.r. spectrum obtained. The general utility of this technique, including its possible application to quantitative studies, is discussed.
Hydroxyl radicals (OH−) can be formed on incubation of an oxygenated solution of ferrous sulphate and cysteine. This has been demonstrated by esr using the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide), catalase, and the radical scavengers ethanol and propan-2-ol. Hydroxyl radicals are not formed when excess zinc sulphate is present. These results provide support for the pro-oxidant action of iron and cysteine and a possible protective role for zinc.
The use of 3,3,5,5-tetramethylpyrroline-N-oxide as a spin trap has been investigated. Spin adducts are found to be more persistent than for 5,5-dimethylpyrroline-N-oxide (DMPO) although the esr spectra are quite similar.
Addition reactions of molecular oxygen to organic sulfur radicals generally appear to be slower than to cor-responding carbon centered radicals. No reaction at all has been found to occur between O 2 and the radical cations RSSR +·, R2S +·, and (R 2S) 2+·. The rate constant for the reaction of oxygen with the thiyl radicals from penicillamine k(PenS· + O 2) = 4.0 × 10 7 M -1 s -1 has been determined from direct pulse radiolysis experiments, and k = 3.4 × 10 8, 7.8 × 10 8, and 2.3 × 10 8 M -1 s -1 have been evaluated by a competition method for the addition of O 2 to C 2H 5S·, t-Bu-S·, and HOCH 2CH 2S·, respectively. Higher rate constants up to 10 9 M -1 s -1 were measured for the addition of O 2 to mesomeric carbon-sulfur radical centers, 〉Ċ-S- ↔ 〉C=Ṡ-. The results are discussed in terms of other O 2 addition reactions to various organic and inorganic radicals.
In the biosynthesis of prostaglandin E1 (PGE1) with an enzyme fraction of sheep vesicular glands glutathione and a moderate amount of antioxidant are required to obtain maximum yield; two oxygen molecules are consumed per mole PGE1 formed. By altering the incubation conditions, besides PGE1 various other products can be obtained.
A scheme for the mechanism of the conversion is proposed in which the formation of PGE1 as well as that of the byproducts is assumed to occur on the enzyme in concerted reactions via peroxy-radicals and a cyclic peroxide.
Abstract—The photodecomposition of sulfanilamide, 4-aminobenzoic acid and other related analogs has been studied with the aid of the spin trap 2-methyl-2-nitrosopropane. UV photolysis of an aqueous solution of sulfanilamide yielded the following radicals C˙6H4SO2NH2, C6H5SO2˙, S˙O2NH2 and SO-#˙ (or SO-2dot;). Under the same conditions 4-aminobenzoic acid gave C˙6H4COOH. In addition both sulfanilamide and 4-aminobenzoic acid, but not 4-dimethylaminobenzoic acid, generated e-aq or hydrogen atoms during UV irradiation. The C6H4SO2NH2 radical was also produced by photolysis of 4-iodobenzenesul-fonamide and 4-nitrobenzenesulfonamide. The C6H4COOH radical was generated by photolysis of 4-nitrobenzoic acid and 4-iodobenzoic acid. Finally the C6H4NO2 radical was formed during the irradiation of 4-nitroaniline, 1,4-dinitrobenzene and 4-iodonitrobenzene. The free radicals generated by sulfanilamide and 4-aminobenzoic acid may play an important role in the phototoxic and photoallergic responses elicited by these drugs in certain individuals.
Evidence is presented supporting the argument that various tissue disorders are the result of iron becoming decompartmentalized. In healthy cells, vital molecules are protected from the action of decompartmentalized iron by the presence of zinc. This protection is particularly important during processes leading to cell division. Should excessive decompartmentalization occur and this protective mechanism become overloaded or should it be weakened by ill health, damaging oxidative free-radical reactions may take place. If these are extensive, death may result. When they are only limited, possibly owing to a low oxygen tension or the presence of copper or a carcinogen, the cell may survive but cancer may result. This 'antioxidant' theory of cancer may provide a unifying mechanism for the action of many carcinogenic agents. Carcinogens are considered to be activated not only by the more usually accepted enzymic pathways but by free-radical reactions, catalysed by iron, in the vicinity of critical sites.
The superoxide radical spin adduct of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide was found to be relatively unstable in aqueous solution. The half-life of the electron spin resonance signal is approximately 80 sec at pH 6 and only about 35 sec at pH 8. These observations as well as the possible reaction products of that may develop in the time course of an experiment, must be considered when planning or interpreting data from a spin trapping experiment.
The protective effects of pretreatment with zinc sulphate aerosols against bronchoconstriction induced by egg albumen or histamine aerosols were assessed in sensitized or non-sensitized guinea-pigs respectively. Pretreatment with an adequate concentration of zinc sulphate aerosol significantly prolonged the time of onset of bronchoconstriction in sensitized guinea-pigs challenged with egg albumen, but did not appreciably alter the onset time of histamine-induced bronchoconstriction in non-sensitized animals. These findings suggest that zinc aerosols may be of prophylactic value against bronchoconstriction of allergic origin.
Sideroblastic anaemia is a group of disorders characterized at the morphological level by the presence of ring sideroblasts in the bone marrow and at a biochemical level by defective haem synthesis in erythroblasts. In the hereditary form, the anaemia may result from a quantitative or qualitative defect of one or other enzyme in haem synthesis, particularly of δ-aminolaevulinic acid (ALA) synthetase or of haem synthetase. In the primary acquired disease or when the disease is associated with leukaemia or other myeloproliferative disorder, it is likely that the enzyme defect arises because of a somatic mutation which affects not only haem synthesis but often also DNA synthesis. The activity of ALA synthetase is almost invariably low in erythroblasts in these disorders. Sideroblastic anaemia due to drugs (e.g. chloramphenicol, lead, isoniazid, cycloserine, pyrazinamide or alcohol) is usually due to antagonism of pyridoxine metabolism or to direct inhibition of haem synthesis or a combination of these. It seems likely that ‘pure’ pyridoxine deficiency may also cause ring sideroblast formation in man, but no primary disturbance of red cell pyridoxine metabolism has been described in primary sideroblastic anaemias, even those which respond to pyridoxine. Iron loading of the erythroblasts has been postulated as further impairing haem synthesis, by inhibition of haem synthetase. Attempts to remove iron by venesection or by iron-chelating drugs given by mouth, by subcutaneous infusion and at the time of blood transfusion intravenously should, therefore, be made in all iron-overloaded patients with sideroblastic anaemia, particularly those needing regular blood transfusions.
The spin trapping technique was used in an attempt to detect the free radical nitric oxide (NO) in solution. Five different spin traps were examined, alpha-phenyl-N-tert butyl nitrone (PBN), alpha-(4-pyridyl-N-oxide) N-tert-butylnitrone (POBN), 5,5-dimethyl-pyrroline-N-oxide (DMPO), 2-methyl-2-nitrosopropane (MNP) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS). Our results suggest that the nitroso spin traps (MNP, DBNBS) are better suited for the identification of NO-related signals, than the nitrones, DMPO, PBN and POBN. In addition, it is shown that spin trapping of NO-related signals with nitroso and nitrone spin traps is subject to many artifacts.
The effects of zinc gluconate have been studied on rat peritoneal mast cells and rat basophilic leukemia cells (RBL 2H3) stimulated by various secretagogues. The IC50's of zinc gluconate on peritoneal cells were (microM): 1.6, 1.9, 5.4 and 18 for ionophore A23187, phorbol 12-myristate 13-acetate, substance P and immunoglobulin E-antigen, respectively. Higher concentrations of zinc gluconate were required to inhibit histamine secretion from RBL 2H3 cells, i.e. 12 microM (ionophore A23187) and 140 microM (immunoglobulin E-antigen). Zinc gluconate (10(-4) to 10(-3) M) also inhibited the IgE-dependent contraction of guinea pig trachea but was unable to affect that induced by exogenous histamine. These results suggest that zinc gluconate acts intracellularly and is selective of "typical" or "connective tissue" mast cells.
Bis(trifluoromethyl) disulfide (TFD) was originally designed for use as an agricultural fumigant. Inhalation of toxic doses of TFD results in varying degrees of pulmonary edema. The purpose of this study was to determine if exhaustive exercise would potentiate the toxic effects of TFD. One group of treadmill-acclimated rats was exercised to exhaustion following a 10-minute whole-body exposure to TFD. A second group was similarly exposed but not exercised. Two other groups of rats were sham exposed; one was exercised while one remained sedentary following the sham exposure. Twenty-four hours after exposure, the animals were sacrificed; the lungs were removed and weighed, and a portion was collected for histopathologic examination. The remaining lung tissue was allowed to dry to constant weight. There was no difference in endurance times between exposed and sham-exposed rats. There was a significant increase in the amount of pulmonary edema and associated pulmonary pathology in rats exercised following exposure to TFD. Eleven of twelve animals exercised following exposure to TFD and three of twelve animals which remained sedentary following exposure died by 24 hours. The degree of pulmonary pathology in all rats exposed to TFD was profound.
The purpose of this review is to consider whether an essential biochemical function of zinc (Zn) is to serve as an antioxidant. Zn has been shown to have an antioxidant role(s) in defined chemical systems. Two mechanisms have been elucidated; the protection of sulfhydryl groups against oxidation and the inhibition of the production of reactive oxygens by transition metals. Supraphysiological concentrations of Zn have antioxidant-like effects in organelle-based systems and isolated cell-based systems in vitro. Administration of pharmacological doses of Zn in vivo has a protective effect against general and liver-specific prooxidants. Dietary Zn deficiency causes increased susceptibility to oxidative damage in membrane fractions from some tissues suggesting that increased oxidative stress may be a small but significant component of the pathology observed in dietary Zn deficiency. However, the biochemical basis for Zn deficiency pathology remains unelucidated; critical antioxidant functions for Zn may still be uncovered.
Exposure of isolated rat hepatocytes to toxic doses of menadione (2-methyl-1,4-naphthoquinone) results in enhanced formation of active oxygen species, depletion of cellular glutathione and protein thiols, and perturbation of intracellular calcium ion homeostasis. An increase in cytosolic Ca2+ concentration, resulting from inhibition of the plasma membrane Ca2+ translocase by menadione metabolism, appears to be critically involved in the development of cytotoxicity.
Thiyl radicals are shown to be readily trapped with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO) giving characteristic spin adducts with hyperfine coupling constants aN 1.52-1.58, aH 1.52-1.80 mT, and g values in the range 2.0065-2.0067 for the DMPO adducts and aN 1.50-1.56, aH 1.70-1.92 mT, g 20049-2.0051 for the TMPO adducts. Kinetic data obtained from pulse radiolysis studies show that, in general, thiyl radicals react rapidly with these spin traps with rate constants of the order of 10(7)-10(8) dm3 mol-1 s-1. The tetramethylated spin trap TMPO though giving slightly less intense electron spin resonance (ESR) spectra, produces longer lived adducts, and is suggested to be of greater utility due to the more characteristic nature of the coupling constants of the observed adducts; reaction of certain thiyl radicals with DMPO produces adducts which are superficially similar to the hydroxyl radical adduct to the same trap.
The study of free radical reactions is not an isolated and esoteric branch of science. A knowledge of free radical chemistry and biochemistry is relevant to an understanding of all diseases and the mode of action of all toxins, if only because diseased or damaged tissues undergo radical reactions more readily than do normal tissues. However it does not follow that because radical reactions can be demonstrated, they are important in any particular instance. We hope that the careful techniques needed to assess the biological role of free radicals will become more widely used.
It has long been known that zinc is essential to life as an integral part of a number of enzymes (1,2). Increasing evidence also suggests that zinc is important in the stability of macromolecules, particularly the components of various biological membranes. This brief review presents a personal and admittedly incomplete view of the latter aspect of the role of zinc. In this review, I shall combine experimental data with speculations in the hopes of stimulating research along new lines.
Stable nitroxide free radicals, when combined with biological materials in aqueous solution, showed a decreased electron spin resonance signal. This phenomenon was due to the reduction of the nitroxide radicals by reducing agents identified to be sulfhydryl groups. This finding suggests a new approach to the study of cell membrane sulfhydryl functions.
Diethyldithiocarbamate (DDC), penicillamine and sodium ethyldiaminetetraacetate (Na-EDTA) in 1 mM concentrations significantly stabilized the membrane of isolated liver lysosomes, as shown by a decreased release of β-glucuronidase upon incubation at 37° and pH 5·0. Thiol compounds which are readily auto-oxidized (cysteine, glutathione and dithiothreitol) did not affect lysosomal fragility when subjected to 1 mM concentration; at a higher concentration (5 mM), these substances increased the release of β-glucuronidase. Stabilization by DDC and penicillamine, both of which contain sulphydryl-like groups, may be related to their nonauto-oxidizability, although their chelation effect cannot be excluded. Various chelating agents relatively specific for Fe2+ or Fe3+, such as l,10-phenanthroline, 5,6-dimethyl-l,10-phenanthroline, 2-2′-dipyridyl, Desferal, and pyrocatechol-3,5-disulfonic acid (Tiron), had no effect on lysosomal stability. Zinc ions (0·05–2·5 mM) significantly stabilized lysosomal membranes; this stabilization was concentration dependent, was not affected by thiol compounds, and was potentiated by equimolar complex with 8-hydroxyquinoline (8-HQ) ; 8-HQ alone (1 mM) labilized lysosomes. Calcium ions had no effect, whereas Cu2+ and Hg2+ labilized the lysosomes. Labilization by Cu2+ was reversed by chelating agents and decreased by thiol compounds. Incubation of lysosomes in nitrogen and dark prevented labilization by copper, and did not affect stabilization by zinc. The effects of certain chelating agents (8-HQ, DDC, penicillamine, EDTA) or metals (Zn2+, Cu2+, Hg2+) on the stability of the lysosomal membranes are discussed in terms of the reactivity of these agents with components of the membrane, or interference with metal-catalyzed lipid peroxidation.
In rats treated for 20 or 34 days with CCl4 (0.2 ml/100 g, ip, twice weekly) a significant increase in the content of malonaldehyde product in microsomal and mitochondrial fractions of the liver occurred. The raise in this indirect index of lipid peroxidation correlated with labilization of lysosomes, as evidenced by the increased portion of free β-glucuronidase present in the 15,000g supernatant of the liver homogenate. At the same time, the amount and the synthesis of collagen was significantly elevated. After zinc acetate administration (5 mg/100 g/day, intragastrically) to rats receiving CCl4, the content of malonaldehyde in the microsomal, as well as the mitochondrial fraction of the liver, was significantly lower. This correlated with a decreased portion of free β-glucuronidase and a decrease in the amount and in the rate of biosynthesis of collagen.Administration of zinc to normal rats had no effect on the amount and synthesis of collagen in the liver. The free and total activity of lysosomal β-glucuronidase in the liver was also unaltered by zinc in normal animals. The level of malonaldehyde was inhibited only in the microsomal fraction. In rats kept on a diet high in zinc (1000 ppm), the content of endogenously-, as well as exogenously-induced malonaldehyde in the liver was lower. Some factors influencing the content of malonaldehyde in intact rat liver are described. We believe that zinc protects the liver against the noxious effect of CCl4 primarily by interference with lipid-peroxidation-related tissue damage. Other possibilities of the protective effect of zinc administration in tissue injury have not been ruled out.
Hematoporphyrin derivative, used in photodynamic therapy of cancer, was found to generate the cysteinyl free radical as seen by spin-trapping. Oxygen appears to be an absolute requirement for thiyl radical production. Singlet oxygen may be the initiating species since azide inhibits oxygen uptake and radical production. In addition, the hydroxyl radical, or a radical with similar reactivity, is also observed and is proposed as the precursor for thiyl free radical production. Hematoporphyrin derivative Cysteine Thiyl free radical Singlet oxygen Hydroxyl radical Spin-trapping.
Zinc, at physiologic concentrations, inhibits in vitro histamine release from human basophils induced by several immunologic (i.e., antigen and anti-immunoglobulin E (IgE) and nonimmunologic [Ca++ ionophore A23187 and formylated tripeptide formyl-methionyl-leucyl-phenylalanine (f-met peptide)] stimuli in a dose-dependent manner. Inhibition begins at about 10(-6) (ionophore A23187, anti-IgE and antigen) or 10(-5) M (f-met peptide) and is maximum at 10(-4) M (80--100% inhibition of histamine release). The activity of zinc is about 25-fold greater with respect to ionophore A23187 (ID50 = 1.1 x 10(-6) M) than to f-met peptide-induced (ID50 = 4 x 10(-5) M) histamine release. Its activity on IgE-mediated histamine release is intermediate between these two extremes (ID50 = 9.7 x 10(-6) M). Zinc does not affect the first stage of histamine release but acts on the calcium-dependent second stage. It is a competitive antagonist of the action of Ca++ in histamine secretion induced by antigen, anti-IgE and f-met peptide (but not by A23187) with a dissociation constant of about 1.2 x 10(-5) M. The addition of colchicine with zinc fails to increase the inhibition caused by the ion alone, suggesting the two compounds work via a common mechanism of action. Deuterium oxide reversed, in a dose-dependent manner, the inhibition of histamine release caused by zinc. These results suggest that the effect of zinc on histamine release from human basophils may be related to its influence on the microtubule system, directly or via its interaction with calcium.
Oxygen radicals have been implicated in the pathogenesis of permeability pulmonary edema. To determine directly if O2 radicals can cause increased alveolar-capillary membrane (ACM) permeability and low-pressure permeability edema, we chemically produced O2 radicals in the sale perfusates of isolated rabbit lungs. The O2 radicals generated by xanthine oxidase caused protein-rich edema and increases in lung perfusion pressures that were inhibitable by catalase (hydrogen peroxide scavenger) or dimethylthiourea (hydroxyl radical scavenger) but not by superoxide dismutase. To determine the effect of O2 radicals on ACM permeability without interference from increased perfusion pressures, we used papaverine to maintain baseline perfusion pressures during O2 radical exposure and then assessed ACM integrity by evaluating the response of isolated lungs to elevated outflow pressures (10 mmHg for 10 min). Under these conditions, increased ACM permeability manifested by weight gains and lavage albumin accumulations occurred in lungs treated with xanthine oxidase but not in control lungs. We conclude that O2 radicals can cause increased ACM permeability and vasoconstriction in isolated lungs.
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