Toxicity of aromatic disulphides. III. In vivo haemolytic activity of aromatic disulphides

Diphenyl disulphide, 4,4'-diaminodiphenyl disulphide, 2,2'-diaminodiphenyl disulphide, 4,4'-dimethyldiphenyl disulphide and 4,4'-dinitrodiphenyl disulphide, when administered orally to rats, induced haematological and pathological changes indicative of erythrocyte destruction in vivo. No evidence of haemolysis was detected, however, in animals receiving diphenyl disulphide-2,2'-dicarboxylic acid or dibenzyl disulphide. The order of activity of the various aromatic disulphides in provoking in vivo haemolysis was similar to that previously recorded for 'active oxygen' generation and erythrocyte damage in vitro. The results of this investigation suggest that in vivo haemolysis may be anticipated from any disulphide or thiol which undergoes appreciable autoxidation at neutral pH. While aromatic or alpha beta-unsaturated thiols and disulphides would be expected to be the most active haemolytic agents, other thiols or disulphides may precipitate the destruction of erythrocytes whose defences against oxidative attack are deficient.     

Steric effects on the haemolytic activity of aromatic disulphides in rats

Certain derivatives of diphenyl disulphide are known to cause haemolytic anaemia in rats, by a mechanism possibly involving intra-erythrocytic redox cycling with concomitant generation of 'active oxygen' species. In ring-substituted diphenyl disulphide derivatives, electronic effects of substituents have been shown markedly to affect the rate of 'active oxygen' production in vitro and toxicity in vivo. In the present study, the influence of steric effects of substituents on these parameters has been investigated. The severity of the haemolysis induced in groups of seven rats by oral dosing with 4,4'-dimethoxydiphenyl disulphide and 4,4'-dimethyldiphenyl disulphide at doses of 500 mumol/kg/day for 6 days was greater than that of the 2,2' isomers and the haemolytic activity of a series of 2,2'-dialkyl derivatives decreased with increasing size of the alkyl group. In vitro, the haematin-catalysed oxidation rates and the rates of redox cycling of the corresponding thiols in the presence of glutathione were similarly influenced by steric hindrance. The structure-activity relationships identified in the present investigation, together with knowledge of the electronic effects of substituents, should permit accurate prediction of the toxicity of new or untested aromatic thiols and disulphides.     

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.     

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. 2--Evidence for intracellular generation of superoxide radical from sporidesmin

Formation of hydrogen peroxide, the dismutation product of superoxide radical, has been demonstrated in erythrocytes incubated with the mycotoxin sporidesmin. Erythrocytic thiols, both non-protein and protein-bound, were depleted in the presence of sporidesmin, whilst haemoglobin was oxidized to methaemoglobin. Irreversible haemoglobin oxidation also occurred in these cells, shown by the formation of Heinz bodies; purified haemoglobin likewise suffered oxidative damage when incubated with sporidesmin in the presence of glutathione. Sporidesmin has previously been shown to generate superoxide radical in vitro; the erythrocytic changes induced by the mycotoxin, which are characteristically produced by compounds which generate 'active oxygen' species, suggest that it is also capable of generating this radical intracellularly.     

Dialuric acid autoxidation. Effects of transition metals on the reaction rate and on the generation of "active oxygen" species

The autoxidation of dialuric acid, a process which is believed to be of crucial importance in the diabetogenic action of alloxan, was found to be strongly catalysed by copper, iron and manganese. Superoxide radical and hydrogen peroxide were generated in both the uncatalysed and the metal-catalysed reactions. In contrast, hydroxyl radical was formed during dialuric acid autoxidation only in the presence of added iron salts. Production of the latter radical was strongly inhibited by catalase but only weakly by superoxide dismutase, implying that the metal-catalysed Haber-Weiss reaction is of comparatively little importance in hydroxyl radical generation from dialuric acid.     

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.     

Toxicity of aromatic thiols in the human red blood cell

Thiophenol and 4-aminothiophenol were used to study levels of toxicity in human red blood cells. Thiophenols caused conversion of oxyhemoglobin to methemoglobin. Reduction of corresponding disulfides by intracellular glutathione caused cyclic reduction/oxidation reactions, resulting in increased oxidative flux. Three levels of oxidative stress were observed in these experiments: the lowest level resulted from incubation with 0.25 mM thiophenol; the intermediate level with 0.50 mM thiophenol or 0.25 mM 4-aminothiophenol; the highest levels with 0.50 mM 4-aminothiophenol. Methemoglobin formation increased with increasing level of oxidative stress. Glycolysis and the hexose monophosphate shunt were inhibited at the intermediate and highest levels of stress, respectively. Above the highest level of stress non-intact hemoglobin was formed and cell lysis occurred. These metabolic responses were reflected in cellular levels of NADH, NADPH and reduced glutathione. At the lowest level of oxidative stress, both glycolysis and hexose monophosphate shunt were increased such that near-normal levels of NADH, NADPH and reduced glutathione were maintained and methemoglobin formation was kept to a minimum. The response of red cells to 0.25 mM thiophenol appears to represent a level of oxidative stress to which the cell is capable of adaptive metabolic response. Glycolysis contributes approximately one-quarter of the total reducing equivalents from glucose metabolism in response to the oxidative challenge by thiophenol. The results suggest that the metabolic response to autoxidation of endogenous thiols is thiol exchange with glutathione and reduction of resulting glutathione disulfide by the hexose monophosphate shunt.     

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.     

Oxidation of thiol drugs and biochemicals by the lactoperoxidase/hydrogen peroxide system

The thiol moiety is prone to oxidative free radical formation, which may be important in mediating the toxicity of some thiol-containing compounds. The oxidation of the compounds cysteine, cysteamine, N-acetylcysteine, glutathione, penicillamine, and captopril were studied using ESR and oxygen uptake techniques. Lactoperoxidase, with hydrogen peroxide to provide oxidizing equivalents, was used to initiate the oxidation. The reaction appears to be strongly peroxide dependent, with either exogenous H2O2 or thiol-derived peroxide driving the reaction.     

Thiol-dependent DNA damage produced by anthracycline-iron complexes. The structure-activity relationships and molecular mechanisms

Doxorubicin (Adriamycin) and daunomycin analogs have been examined for their ability to chelate iron and catalyze the oxidative cleavage of DNA. The results show that the C-11-hydroxyl group is essential for iron binding and DNA damage. Thus, the iron complexes of doxorubicin, daunomycin, carminomycin, and 4-demethoxydaunomycin are potent redox catalysts capable of reducing molecular oxygen in the presence of physiologic concentrations of glutathione. They are also effective catalysts of hydroxyl radical formation from hydrogen peroxide. With the exception of daunomycin, generation of hydroxyl radical from hydrogen peroxide is stimulated by greater than 200% by DNA addition. Analogs that lack the C-11-hydroxyl group are relatively inefficient at oxygen reduction, hydroxyl radical formation, and DNA cleavage. The potencies of the anthracycline analogs tested in the H2O2-dependent DNA cleavage reaction correlated well with their relative cardiac toxicities.     

Generation of DNA-Damaging Reactive Oxygen Species via the Autoxidation of Hydrogen Sulfide under Physiologically Relevant Conditions: Chemistry Relevant to Both the Genotoxic and Cell Signaling Properties of H(2)S

Hydrogen sulfide (H(2)S) has long been known for its toxic properties; however, in recent years, evidence has emerged that this small, gaseous molecule may serve as an endogenous cell-signaling agent. Though perhaps surprising in light of its potential role as an endogenous signaling agent, a number of studies have provided evidence that H(2)S is a DNA-damaging mutagen. In the work reported here, the chemical mechanisms of DNA damage by H(2)S were examined. Using a plasmid-based DNA strand cleavage assay, we found that micromolar concentrations of H(2)S generated single-strand DNA cleavage. Mechanistic studies indicate that this process involved autoxidation of H(2)S to generate superoxide, hydrogen peroxide, and, ultimately, the well-known DNA-damaging agent hydroxyl radical via a trace metal-mediated Fenton-type reaction. Strand cleavage by H(2)S proceeded in the presence of physiological thiol concentrations, and the known byproducts of H(2)S oxidation such as thiosulfate, sulfite, and sulfate do not contribute to the strand cleavage process. However, initially generated oxidation products such as persulfide (S(2)(2-)) likely undergo rapid autoxidation reactions that contribute to the generation of superoxide. The potential relevance of autoxidation processes to the genotoxic and cell signaling properties of H(2)S is discussed.     

Hydroxyl radical mediated demethylenation of (methylenedioxy)phenyl compounds.

The oxidative demethylenation reactions of (methylendioxy)phenyl compounds (MDPs), (methylenedioxy)benzene (MDB), (methylenedioxy)amphetamine (MDA), and (methylenedioxy)methamphetamine (MDMA), were evaluated by using two hydroxyl radical generating systems, the autoxidation of ascorbate in the presence of iron-EDTA and the iron-catalyzed Haber-Weiss reaction conducted by xanthine/xanthine oxidase with iron-EDTA. Reaction products generated when MDB, MDA, and MDMA were incubated with the ascorbate or xanthine oxidase system were catechol, dihydroxyamphetamine (DHA), and dihydroxymethamphetamine (DHMA), respectively. The reaction required the presence of either ascorbic acid or xanthine oxidase. Levels of each catechol increased in proportion to ferric ion concentration and were suppressed by desferrioxamine B methanesulfonate (desferal). Catalase (CAT) inhibited the oxidation by the ascorbate system whereas superoxide dismutase (SOD) had little effect. The addition of hydrogen peroxide to the reaction mixture stimulated the oxidation, but the reaction was not initiated by hydrogen peroxide alone, suggesting that hydrogen peroxide acts as a precursor of hydroxyl radical. SOD and CAT suppressed the demethylenation reactions in the xanthine oxidase system. Hydroxyl radical scavenging agents such as ethanol, benzoate, DMSO, and thiourea effectively inhibited the oxidation by both systems. Urea, which has little effect on hydroxyl radical, was without any effect. These results indicated that hydroxyl radical can effect the cleavage of methylenedioxy group on MDPs.     

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.     

Reaction of chromium (VI) with hydrogen peroxide in the presence of glutathione: reactive intermediates and resulting DNA damage

The reaction of chromium(VI) with hydrogen peroxide was studied in the presence of glutathione. In vitro, reaction of chromium(VI) with hydrogen peroxide alone led to production of hydroxyl radical as the significant reactive intermediate, while reaction of chromium(VI) with glutathione led to formation of two chromium(V)-glutathione complexes and the glutathione thiyl radical. Incubation of chromium(VI) with glutathione prior to addition of hydrogen peroxide led to formation of peroxochromium(V) species and a dramatic increase in hydroxyl radical production over that detected in the reaction of chromium(VI) with hydrogen peroxide alone. In contrast, addition of chromium(VI) to a preincubated mixture of glutathione and hydrogen peroxide led to a decrease in hydroxyl radical production over that obtained in the reaction of chromium(VI) with hydrogen peroxide. When pBR322 DNA was added to the above reactions, the extent of chromium(VI)-induced DNA strand breakage correlated with the relative amount of hydroxyl radical formed. Reaction of chromium(VI) with calf thymus DNA in the presence of a preincubated mixture of glutathione and hydrogen peroxide led to detection of the 8-hydroxydeoxyguanosine adduct, whose formation correlated with that of hydroxyl radical production. No significant chromium-DNA adduct formation was detected. The results suggest that, in the cellular metabolism of chromium(VI), preformed chromium(V)-glutathione complexes may react with hydrogen peroxide in a Fenton-type manner to produce hydroxyl radical as the DNA-damaging agent. However, if glutathione reacts with hydrogen peroxide prior to exposure to chromium(VI), the amount of hydroxyl radical generated may not be sufficient to cause significant DNA damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiol-group reactivity, hydrophilicity and stability of alloxan, its reduction products and its N-methyl derivatives and a comparison with ninhydrin

The diabetogenic agent, alloxan, is a hydrophilic and chemically unstable compound. The logarithm of the octanol/water partition coefficient of alloxan was found to be -1.86; its half-life at pH 7.4 and 37 degrees in phosphate buffer was 1.5 min. The partition coefficients and half-lives of the alloxan reduction products, alloxantin and dialuric acid, were very similar to those of the parent compound; N-methylalloxan and N,N'-dimethylalloxan were less hydrophilic but more unstable. Ninhydrin was found also to be hydrophilic although this compound, in contrast to alloxan and its derivatives, was quite stable in aqueous solution. Alloxan and its N-methyl derivatives were reduced by thiols and in the presence of glutathione and cysteine, rapid redox cycling occurred, with formation of 'active oxygen' species; no such reaction was observed, however, with ninhydrin. Comparatively slow redox cycling was recorded with alloxan derivatives and dithiothreitol although rapid cycling occurred with ninhydrin and this dithiol. Such differences may explain why ninhydrin does not share with alloxan a selective toxic effect upon the pancreatic B-cell.     

Mechanism of activation of rat liver microsomal glutathione transferase by noradrenaline and xanthine oxidase.

Activation of glutathione transferase activity in rat liver microsomes under a variety of conditions producing oxidative stress was investigated. Neither hydrogen peroxide (10 mM) (added or produced endogenously by glucose + glucose oxidase) nor duroquinone together with an NADPH-regenerating system (which generates the superoxide anion radical) had any significant effect on the glutathione transferase activity towards 1-chloro-2,4-dinitrobenzene. On the other hand, incubation of microsomes with 1 mM noradrenaline (which autooxidizes and generates superoxide anion radical) gave a 160% activation, as shown earlier (Aniya and Anders, J Biol Chem 264: 1998-2002, 1989). This was taken as an indication that microsomal glutathione transferase could be activated by oxidative stress. Here, we demonstrate that activation by this compound is due to covalent binding (presumably of the quinone formed during autooxidation). The xanthine/xanthine oxidase system, which generates the superoxide anion radical and hydrogen peroxide, increases microsomal glutathione transferase activity, but this activation was not dependent on the presence of xanthine. Western blots of microsomes treated with xanthine oxidase revealed that activation was due to proteolysis (presumably by contaminating proteases in the xanthine oxidase). In conclusion, there is no firm evidence that rat liver microsomal glutathione transferase is activated directly by reduced oxygen species in the microsomal system. The possibility remains that oxidative stress triggers secondary mechanisms such as generation of reactive intermediates and/or activation of proteolysis, which can in turn increase enzyme activity.     

汉防己甲素对大鼠肝保护和抗氧化作用研究

目的:使用汉防己甲素评价对四氯化碳诱导的大鼠肝损伤的保护作用以及抗氧化作用。方法:每日给予四氯化碳损伤大鼠不同的浓度汉防己甲素剂量分别为20,60,100mg/kg,随后检测血清和组织中谷胱甘肽(GSH),谷胱甘肽过氧化物酶,过氧化氢酶(CAT),超氧化和物歧化酶(SOD),谷胱甘肽S-转移酶(GST),脂质过氧化效应等指标,体外抗氧化使用使用超氧化物和过氧化物清除实验评价。结果:与对照组和四氯化碳模型组比较后发现,汉防己甲素在所用剂量下均显示出较为理想的肝保护和抗氧化效应。进一步的体外过氧化自由基清除和超氧化自由基清除实验表明,汉防己甲素较维生素C有更为优越的清除活性。结论:汉防己甲素能有效保护四氯化碳对大鼠造成的肝损伤,其保护机制可能与抗氧化和自由基清除密切相关。     

Generation of volatile hydrocarbons from amino acids and proteins by an iron/ascorbate/GSH system

Incubation of free, but not of peptide-bound methionine in an iron/ascorbate system resulted in ethylene generation, which was inhibited by glutathione. Leucine and isoleucine, however, when incubated in an iron/ascorbate/GSH system, released small amounts of propane and ethane, respectively. Peptide-bound leucine additionally yielded butane, as did bovine serum albumin or casein. Hydrocarbon generation from amino acids was inhibited by hydroxyl radical scavengers, but catalase and superoxide dismutase were more efficient. Additionally, ethane and propane generation in this system was optimal at pH 6.2 suggesting the involvement of protonated superoxide besides OH-radicals which attack the side chains of Leu and Ile and very probably produce carbon-centered radicals, which should abstract a hydrogen atom from the thiol group of GSH resulting in the formation of saturated hydrocarbons.     

Autoxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine

The indolic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) has been widely speculated to express its neurodegenerative effects as a result of intraneuronol autoxidation. Until recently, it was believed that autoxidation led to reactive electrophilic quinone imine species which alkylated neuronal membrane proteins and that byproducts of the autoxidation reaction were cytotoxic reduced-oxygen species. This study reveals that at physiological pH carbanions of 5,7-DHT act as the primary electron-donor species to yield C(4)- and C(6)-centered free radical superoxide complexes in a 1:2 ratio. The C(4)-centered complex reacts to yield, ultimately, 5-hydroxytryptamine-4,7-dione which has been shown to be a significantly more powerful neurotoxin than 5,7-DHT. The C(6)-centered radical superoxide complexes react to give 6,6'-bis(5-hydroxytryptamine-4,7-dione). It is likely that the latter reaction yields O2.- as a cytotoxic byproduct.