Studies on the mechanism of toxicity of the mycotoxin sporidesmin. 3--Inhibition by metals of the generation of superoxide radical by sporidesmin

The mycotoxin sporidesmin has previously been shown to generate superoxide radical. This reaction involves autoxidation of the reduced form of the mycotoxin, a dithiol. In the present study, a number of mercaptide-forming metals have been shown to inhibit superoxide formation from sporidesmin in vitro. Furthermore, these metals decreased the rate of sporidesmin-induced hydrogen peroxide formation in erythrocytes and ameliorated the subsequent oxidative damage to these cells. These effects were found to be specific to sporidesmin; mercaptide-forming metals did not inhibit the changes induced by compounds which are not dependent upon thiol groups for active oxygen generation. Zinc was one of the most potent inhibitors of superoxide generation from sporidesmin in these test systems; only mercury and cadmium were significantly more active. Salts of zinc are known to provide effective protection against the harmful effects of sporidesmin in vivo. The results of these studies provide a possible explanation for this effect.     

Studies on the mechanism of toxicity of the mycotoxin, sporidesmin. V. Generation of hydroxyl radical by sporidesmin

Sporidesmin, the mycotoxin responsible for "facial eczema' in ruminants, has previously been shown to generate superoxide radical and hydrogen peroxide. In the present study, the formation of the third "active oxygen' species, hydroxyl radical, has been demonstrated. This species is produced both during the autoxidation of the reduced (dithiol) form of the mycotoxin and in the cyclic reduction/autoxidation reaction between sporidesmin and glutathione. In view of the exceptional reactivity of the hydroxyl radical, this substance may be the proximate agent responsible for the toxic effects of sporidesmin.     

Studies on the mechanism of toxicity of the mycotoxin sporidesmin. IV. Inhibition by copper-chelating agents of the generation of superoxide radical by sporidesmin

Sporidesmin, the mycotoxin responsible for "facial eczema" in ruminants, has previously been shown to generate superoxide free radical, the latter being formed during autoxidation of the reduced form of the mycotoxin, a dithiol. The autoxidation of reduced sporidesmin is catalysed by iron and by copper, although cobalt, nickel, manganese, cerium, vanadium and molybdenum were found to be without effect. The catalytic activity of copper was some 900-fold that of iron. Cyanide and certain organic complexing agents inhibit superoxide production from reduced sporidesmin by virtue of their ability to chelate copper. Some proteins behave likewise, again through binding of the metal catalyst, although amino acid-bound copper was found to be as effective as ionic copper in catalysing the autoxidation reaction. From the results of the present experiments, it is suggested that any superoxide production from sporidesmin in vivo would be mediated by the intracellular transport pool of copper. Furthermore, the ability of zinc to inhibit intestinal absorption of copper may be involved in the mechanism whereby salts of this metal afford protection against the harmful effects of sporidesmin in the living animal.     

The involvement of the disulphide group of sporidesmin in the action of the toxin on swelling and respiration of liver mitochondria

The disulphide group of the mycotoxin sporidesmin appears to be involved in the action of the toxin on the swelling and increased respiration of mitochondria. The requirements of the swelling produced by sporidesmin and gliotoxin, a toxic antibiotic which also contains the epidithiapiperazinedione moiety, are similar. The swelling produced by oxidized glutathione is quite different as there is no requirement for substrate or phosphate and much higher concentrations of the disulphide are required. Dithiothreitol but not a number of mono- or other dithiols inhibits the action of 70 μM sporidesmin, 50 per cent reduction in the rate of swelling being observed at dithiothreitol concentrations of 42 μM. Dithiothreitol but not the other mono- and dithiols reacts with sporidesmin to form a number of products. It is suggested that effects of sporidesmin on mitochondria in vitro may involve a reaction between the disulphide group of the toxin and reactive thiols in the membrane and that sporidesmin may exert its primary effect in vivo by reacting with specific thiol groups of certain hepatic membranes.     

Effects of the mycotoxin sporidesmin on swelling and respiration of liver mitochondria

In the presence of both oxidizable substrate and phosphate, low concentrations of sporidesmin produced rapid swelling of mitochondria isolated from guinea-pig liver. There is no requirement for alkali metal ions. Uncoupling agents or respiratory inhibitors inhibited the swelling. Mitochondria that had been swollen in the presence of sporidesmin could be contracted by addition of ATP, magnesium ions and bovine serum albumin. The rate of swelling was dependent on the concentration of both sporidesmin and mitochondrial protein and half-maximum effect was observed at 140 nmoles of sporidesmin per mg of protein. A lag period occurred before the maximal effect of sporidesmin and this period was also dependent on the concentration of both sporidesmin and mitochondrial protein. Sporidesmin decreased the respiratory control index of mitochondria by increasing the rate of state 4 respiration and decreasing that of state 3. As sporidesmin had no effect on the respiration of mitochondrial preparations that had either been treated with Triton X-100 or been subject to sonication, the toxin does not directly inhibit the respiratory chain. It is suggested that sporidesmin produces the effects described above by altering the permeability of the mitochondrial membrane.     

Studies with primaquine in vitro: superoxide radical formation and oxidation of haemoglobin.

1. The production of superoxide radicals from primaquine diphosphate in aqueous solution has been demonstrated, using as indicator the reduction of cytochrome C with inhibition of the reaction by superoxide dismutase. 2. Primaquine-mediated oxidation of haemoglobin to methaemoglobin was reduced by the addition of catalase and increased by superoxide dismutase. Mannitol, a hydroxyl radical scavenger, abolished the increase in methaemoglobin observed in the presence of superoxide dismutase. EDTA reduced the oxidation of haemoglobin with and without superoxide dismutase. 3. Although the oxidation of haemoglobin in the presence of primaquine includes the effects of hydrogen peroxide, superoxide and hydroxyl radicals and metal ions, the results indicate that hydrogen peroxide, rather than the superoxide radical, is the main oxidizing species. The increase in haemoglobin oxidation occurring with superoxide dismutase may result from the augmented rate of hydrogen peroxide formation from superoxide radicals.     

Toxicity of aromatic disulphides. I. Generation of superoxide radical and hydrogen peroxide by aromatic disulphides in vitro

The aromatic thiol, thiophenol, is readily autoxidized at neutral pH in a reaction which generates superoxide radical and hydrogen peroxide. The oxidation product, diphenyl disulphide, may be reduced back to thiophenol by glutathione and in the presence of an excess of the latter thiol a reduction/autoxidation cycle for generation of 'active oxygen' species is established. The autoxidation reaction is strongly catalysed by haematin; haemoglobin is also an effective mediator of 'active oxygen' generation from the diphenyl disulphide/glutathione couple, being oxidized to methaemoglobin in the process. Certain derivatives of diphenyl disulphide also generate superoxide radical and hydrogen peroxide in the presence of glutathione, although the rate of the reaction is strongly influenced by the nature of the substituent groups. Among the ring-substituted derivatives of diphenyl disulphide investigated, the rate of 'active oxygen' production decreased in the order 4-amino greater than 2-amino greater than 4-methyl greater than unsubstituted greater than 4-nitro greater than 2-carboxyl; little reaction was detected with the homologous compound, dibenzyl disulphide.     

Primary Impacts of the Fungal Toxin Sporidesmin on HepG2 Cells: Altered Cell Adhesion without Oxidative Stress or Cell Death

The fungal metabolite sporidesmin is responsible for severe necrotizing inflammation of biliary tract and liver of livestock grazing on pasture containing spores of Pithomyces chartarum that synthesizes the toxin. The toxin is secreted into bile causing the erosion of the biliary epithelium accompanied by inflammation and damage to surrounding tissues. Toxicity has been suggested to be due to cycles of reduction and oxidation of sporidesmin leading to oxidative damage from the formation of reactive oxygen species. The current work is the first test of the oxidative stress hypothesis using cultured cells. Oxidative stress could not be detected in HepG2 cells incubated with sporidesmin using a dichlorodihydrofluorescein diacetate assay or by use of two-dimensional electrophoresis to search for oxidized peroxiredoxins. There was also no evidence for necrosis or apoptosis, although there was a loss of cell adhesion that was accompanied by the disruption of intracellular actin microfilaments that have known roles in cell adhesion. The results are consistent with a model in which altered contact between cells in situ leads to altered permeability and subsequent inflammation and necrosis, potentially from the leakage of toxic bile into surrounding tissues. There is now a need for the further characterization of the damage processes in vivo, including the investigation of altered permeability and mechanisms of cell death in the biliary tract and other affected organs.     

Production of superoxide during the metabolism of nitrazepam

Nitrazepam is metabolized in both humans and rats to 7-amino-nitrazepam OFFicating that this drug is reduced to a number of metabolic intermediates including several free radical species. When rat-hepatic microsomes are incubated with NADPH in the presence of nitrazepam, its nitro anion free radical was observed under anaerobic conditions. In the presence of oxygen, this free radical reduced oxygen giving nitrazepam and superoxide. 7-Nitroxyl-nitrazepam was produced by the chemical oxidation of 7-amino-nitrazepam using m-chloroperbenzoic acid. Reaction of this reactive free radical with hepatic microsomes led to the covalent spin labelling of microsomal protein. This phenomenon was also observed by the enzymic oxidation of 7-amino-nitrazepam with hepatic microsomes, obtained from a phenobarbital-induced rat, in the presence of a NADPH-generating system. With the generation of superoxide and hydrogen peroxide (arising from the dismutation of superoxide), it is not surprising that nitrazepam-enhanced lipid peroxidation was demonstrated by monitoring the production of lipid peroxyl radicals using spin-trapping techniques.     

Studies on the antioxidant activity of Lippia citriodora infusion: scavenging effect on superoxide radical,hydroxyl radical and hypochlorous acid

Lippia citriodora is an herbal species which contains several flavonoids and phenolic acids. In view of the pharmacological interest in natural phenolic compounds as antioxidants, this study examined the superoxide radical, hydroxyl radical and hypochlorous acid scavenging activities of L. citriodora infusion. Superoxide radical was generated either in an enzymatic or in a chemical system, and scavenging ability was assessed by the inhibition of nitroblue tetrazolium reduction. Hydroxyl radical was generated by the reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, and was assayed by evaluating deoxyribose degradation. Hypochlorous acid scavenging activity was tested by measuring the inhibition of 5-thio-2-nitrobenzoic acid oxidation. The results demonstrate that this infusion has a potent superoxide radical scavenging activity and a moderate scavenging activity of hydroxyl radical and hypochlorous acid. The chemical composition of the lyophilized infusion was also determined in an attempt to establish its relationship with the antioxidant activity found in the present study.     

Metabolic activation of oxygen by nitrofurantoin in the young chick

Anaerobic incubations containing nitrofurantoin, and NADPH-generating system, and chick hepatic microsomes produced an electron spin resonance spectrum identified as the nitro anion free radical. Aerobically, nitrofurantoin markedly stimulated oxygen consumption, superoxide formation, and NADPH oxidation in hepatic microsomal preparations from control and selenium-deficient chicks. The nitrofurantoin-stimulated oxidation of NADPH was inhibited by superoxide dismutase (SOD). The superoxide-dependent oxidation of NADPH did not appear to be mediated by an NADP^? radical, as has been shown for lactate dehydrogenase. Further, the aerobic metabolism of the nitro drug was also affected by SOD, suggesting the existence of a previously unreported metabolic pathway for nitrofurantoin. These studies support the growing body of evidence which suggests that nitrofurantoin toxicity is mediated, at least in part, by the metabolic activation of oxygen by the nitro aromatic anion radical. Further, these data suggest that superoxide may be involved in the oxidative metabolism of the aromatic nitro compounds.     

Studies on the mechanisms of oxidation in the erythrocyte by metabolites of primaquine

The interaction of certain metabolites of the 8-aminoquinoline antimalarial primaquine with both normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes and with haemoglobin preparations was studied in an attempt to elucidate the mechanisms of methaemoglobin formation and haemolytic anaemia associated with the use of primaquine. Studies using erythrocytes revealed that oxidation of haemoglobin and reduced glutathione (GSH) was due to the metabolites rather than the parent drug. Incubation of free haemoglobin with 5-hydroxylated metabolites of primaquine also led to oxidation of oxyhaemoglobin and GSH. Oxidation of GSH also occurred in the absence of oxyhaemoglobin. The results suggest a dual mechanism for these oxidative effects, involving autoxidation of the 5-hydroxy-8-aminoquinolines and their coupled oxidation with oxyhaemoglobin. The initial products of these processes would be drug metabolite free radicals, superoxide radical anions, hydrogen peroxide and methaemoglobin. Further free radical reactions would lead to oxidation of GSH, more haemoglobin and probably other cellular constituents. NADPH had no effect on the oxidative effects of the primaquine metabolites in these experiments. In the G6PD-deficient erythrocyte, the oxidation of haemoglobin and GSH leads to Heinz body formation and eventually to haemolysis, the mechanisms of which are as yet unclear. The possible role of oxygen free radicals in the mode of action of 8-aminoquinolines against the malaria parasite is also briefly discussed.     

Nitric oxide trapping of the tyrosyl radical-chemistry and biochemistry

The quenching of the Y(D) tyrosyl radical in photosystem II by nitric oxide was reported to result from the formation of a weak tyrosyl radical-nitric oxide complex. This radical/radical reaction is expected to generate an electron spin resonance (ESR)-silent nitrosocyclohexadienone species that can reversibly regenerate the tyrosyl radical and nitric oxide or undergo rearrangement to form 3-nitrosotyrosine. It has been proposed that 3-nitrosotyrosine can be oxidized by one electron to form the tyrosine iminoxyl radical (>C=N-O.). This proposal was put forth as a result of ESR detection of the iminoxyl radical intermediate when photosystem II was exposed to nitric oxide. Although the detection of the iminoxyl radical in photosystem II strongly suggested a mechanism involving 3-nitrosotyrosine, the iminoxyl radical ESR spectrum was not unequivocally identified as originating from tyrosine. Subsequently, non-protein L-tyrosine iminoxyl radical was generated by two methods: (1) peroxidase oxidation of synthetic 3-nitroso-N-acetyl-L-tyrosine; and (2) peroxidase oxidation of free L-tyrosine in the presence of nitric oxide. The determination of protein nitrotyrosine content has become a frequently used technique for the detection of nitrosative tissue damage. Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e.g. peroxynitrite) formed in reactions between nitric oxide (NO.) and oxygen-derived species such as superoxide. The enzyme prostaglandin H synthase-2 also forms a tyrosyl radical during its enzymatic catalysis of prostaglandin formation. In the presence of the NO.-generator diethylamine nonoate, the tyrosyl radical of prostaglandin H synthase-2 also changes to that of an iminoxyl radical. Western blot analysis of prostaglandin H synthase-2 after exposure to the NO.-generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.     

Oxidative damage of DNA induced by methylglyoxal in vitro

Methylglyoxal is an endogenous metabolic by-product of glycolysis and has genotoxic effects. Previous studies suggested that the reaction of methylglyoxal with amino acid leads to the production of free radicals. In this study, oxidative damage of DNA by the reaction of methylglyoxal with amino acid was investigated. When plasmid DNA was incubated with methylglyoxal and lysine, DNA strand was cleaved. Cu(2+) enhanced DNA strand breakage induced by the reaction of methylglyoxal with lysine. The formation of superoxide anion was detected during the glycation reaction of methylglyoxal with lysine. Radical scavengers, catalase, and copper chelators inhibited the DNA breakage. The deoxyribose assay showed that hydroxyl radicals were generated during the reaction of methylglyoxal with lysine in the presence of Cu(2+). The generation of hydroxyl radicals was inhibited by radical scavenger, catalase, and copper chelator. These results suggest that superoxide anion and H2O2 may generate from the glycation reaction of methylglyoxal with lysine and then Cu(2+) likely participates in a Fenton's type reaction to produce hydroxyl radicals, which may cause DNA cleavage. This mechanism may be linked to several diverse biological processes including mutagenesis, aging, carcinogenesis, and diabetic complications.     

Determination of metal-based hydroxyl radical generating capacity of ambient and diesel exhaust particles

Numerous studies have suggested the association of reactive oxygen species (ROS) with adverse health effects derived from exposure to airborne particulate matter (PM) and diesel exhaust particles (DEP). This redox activity has been attributed to both inorganic and organic species present in these particles, but a clear distinction has not been established between the contribution of each. This article describes an application of an analytical procedure, based on the reaction of salicylic acid with hydroxyl radical to form dihydroxybenzoate (DHBA) isomers, to measure transition metal-based redox activity associated with ambient and diesel exhaust particles. In the procedure, ascorbic acid (AA) is used as electron source for reduction of metal ions and oxygen to generate superoxide, which is further reduced to hydroxyl radical in the presence of transition metal ions. Hydroxyl radical reacts with salicylate to generate DHBA isomers, which are measured by high-performance liquid chromatography (HPLC) with electrochemical detector. Both copper (Cu) and iron (Fe) ions generated DHBA isomers in a concentration-dependent manner but at different rates. The procedure was applied to DEP and ambient particles and the results showed Cu ion to be the major contributor to DHBA formation. The procedure provides a quantitative measure of transition metal-based redox activity associated with ambient samples with different physicochemical properties.     

Free radical production and DNA cleavage by copper chelating peptide-anthraquinones

Two pseudopeptides incorporating a peptide metal-chelating moiety (Gly-His-Lys) and a polyhydroxy anthraquinone ring related to the nuclei of anti-tumor drugs such as mitoxantrone and ametantrone, have been synthesized. The goal was to conjugate the redox effects of a quinone ring with the iron-chelating properties of the peptide in order to generate free radical species capable of damaging DNA. Indeed quinone-containing drugs undergo, in vivo, one-electron reduction to the corresponding semiquinone radicals which, in the presence of molecular oxygen, produce a superoxide anion radical, hydrogen peroxide and ultimately, in the presence of metal, hydroxyl radical (Fenton reaction). Hydroxyl radicals (OH.) are short-lived and extremely reactive with their bioenvironment. The interaction of both drugs with DNA has been studied by fluorescence quenching and DNA melting experiments. Spectroscopic and e.s.r. studies demonstrated that several types of Cu-complex are formed depending on the copper-drug ratio. The production of free radicals, as evidenced by spin-trapping, is optimum with a Cu/drug ratio of 0.1; in this case the metal ion is chelated by the peptide moiety. This latter complex is able to induce DNA breakage at a high level. Thus, it appears that the proposed concept works but that care must be taken in the choice of the relative concentration of copper.     

Mechanisms of oxygen activation by nitrofurantoin and relevance to its toxicity

Purified ferredoxin-(cytochrome c)-NADP+ oxidoreductase and xanthine oxidase were found to catalyse the reduction of nitrofurantoin to the free radical. Under aerobic conditions, the nitrofurantoin radical underwent autoxidation to regenerate the parent compound with the concomitant production of superoxide and eventually hydrogen peroxide. The nitrofurantoin radical was also shown to react with hydrogen peroxide to generate a highly reactive species which was capable of oxidising methionine to ethylene. This active oxygen radical appeared to be identical with the crypto-OH . radical, previously proposed as being formed from the analogous reaction of the methyl viologen radical with hydrogen peroxide [R.J. Youngman and E.F. Elstner, FEBS Lett. 129, 265 (1981)]. Catalase inhibited nitrofurantoin-dependent ethylene formation in both enzyme systems, whereas superoxide dismutase was only inhibitory in the xanthine oxidase mediated reaction. Although the primary function of the respective enzyme systems is to generate the nitrofurantoin radical, the xanthine oxidase reaction is markedly more complex than that of ferredoxin-(cytochrome c)-NADP+ oxidoreductase. The differences between the two enzyme reactions appear to be due to the endogenous autoxidation of xanthine oxidase. The aerobic activation of nitrofurantoin by xanthine oxidase involved the superoxide anion as an intermediate, whereas the nitrofuran was directly reduced by ferredoxin-(cytochrome c)-NADP+ oxidoreductase without a requirement for active oxygen species.     

Toxicity of aromatic disulphides. II. Intraerythrocytic hydrogen peroxide formation and oxidative damage by aromatic disulphides

Diphenyl disulphide has been shown to generate hydrogen peroxide in erythrocytes in vitro. It also induces oxidative damage (reversible and irreversible haemoglobin oxidation, depletion of non-protein and protein-bound thiols) in these cells. Such changes were also recorded in erythrocytes exposed to 4,4'-diaminodiphenyl disulphide, 2,2'-diaminodiphenyl disulphide, 4,4'-dimethyldiphenyl disulphide, 4,4'-dinitrodiphenyl disulphide, diphenyl disulphide-2,2'-dicarboxylic acid and dibenzyl disulphide. The relative potency of these compounds in causing erythrocyte damage is correlated with their ability to generate 'active oxygen' species in vitro.