Sulphydryl oxidase: Properties and applications

Sulphydryl oxidase has been isolated and purified from bovine milk and partially characterized. The enzyme is obtained from the whey fraction produced by chymosin (rennin) treatment of skim milk. Several procedures have been developed for its isolation, all of which use to advantage its concentration-dependent aggregate size. This enzyme exhibits a much broader substrate specificity than that shown by other ‘aerobic oxidases’ presently characterized which catalyse the formation of disulphides from thiols. Using molecular oxygen as an electron acceptor in a twoelectron reduction to hydrogen peroxide, the enzyme catalyses oxidation of cysteine and its analogues, some volatile thiols, peptides, and also thiols in proteins.Vis-a-vis protein-disulphide oxidoreductase and protein-disulphide isomerase, this enzyme does not catalyse thiol-disulphide interchange and thus functions only in de novo formation of disulphides. Enzyme-bound iron and most likely a free sulphydryl group are essential for catalytic activity; however, the carbohydrate moiety of this glycoprotein is probably not required. The broad substrate specificity suggests several potential industrial uses for the enzyme, including flavour modification in the food industry or synthesis of disulphides in pharmaceuticals. Consequently, immobilized forms were developed and characterized. Reactors containing sulphydryl oxidase covalently immobilized on porous glass and silica have been examined for their efficacy in eliminating the cooked flavour in ultra-high temperature sterilized milk. Both the degree of oxidation and cooked flavour were correlated with a normalized residence time in the reactor.     

Taurine protects the antioxidant defense system in the erythrocytes of cadmium treated mice

The present study was undertaken to investigate the protective role of taurine (2-aminoethanesulfonic acid) against cadmium (Cd) induced oxidative stress in murine erythrocytes. Cadmium chloride (CdCl(2)) was chosen as the source of Cd. Experimental animals were treated with either CdCl(2) alone or taurine, followed by Cd exposure. Cd intoxication reduced hemoglobin content and the intracellular Ferric Reducing/Antioxidant Power of erythrocytes, along with the activities of antioxidant enzymes, glutathione content, and total thiols. Conversely, intracellular Cd content, lipid peroxidation, protein carbonylation, and glutathione disulphides were significantly enhanced in these cells. Treatment with taurine before Cd intoxication prevented the toxin-induced oxidative impairments in the erythrocytes of the experimental animals. Overall, the results suggest that Cd could cause oxidative damage in murine erythrocytes and that taurine may play a protective role in reducing the toxic effects of this particular metal.     

The genotoxicity of selenium

Selenium at nutritional levels has been shown to have numerous anticarcinogenic or preventative effects against carcinogen-induced breast, colon, liver and skin cancer in animals. Many of these anticarcinogenic effects have been summarized. In addition, numerous mutagenic and antimutagenic effects of selenium compounds have been reported. Some of the selenium compounds frequently tested for mutagenicity are listed in Table 1. Because of the numerous reported anticarcinogenic and preventative effects of selenium, many individuals are supplementing their diets with amounts of selenium that are greater than the recommended daily requirement. Selenium is also used widely in industrial products such as selenium rectifiers, photoelectric batteries, alloys and paints. Because selenium at higher levels is known to be toxic, there should be a greater understanding about its genotoxic as well as its beneficial effect. The object of this review is to summarize experimental evidence both for the antimutagenic and the mutagenic effect of selenium.     

The histochemistry of thiols and disulphides. III. Staining patterns in rat tissues

Synopsis With the aid of new staining methods, thiol groups produced by the reduction of disulphide bonds were positively distinguished from pre-existing groups in paraffin sections of several organs of the rat. Good preservation of structures in which the natural thiol-disulphide balance had been maintained was sought by fixing the tissues in neutral formalin containing an organomercurial. After dissociation of the resulting mercaptide bonds that protected the native thiols, these were shown in one colour and then disulphide sites in another within the same sections. Intracellular granules and extracellular membranes rich in disulphides thereby stood out in red against the predominantly blue labelling of the cellular ground plasm. Intimate mixtures of the two forms in some places and the presumed transformation of thiols to disulphides in others, notably the keratinizing epithelium of the tongue, were readily seen. Supplemented by separate visualization of thiols and disulphides along with suitable controls for specificity of staining, the results obtained diverged in some major respects from those of previous investigations.     

The histochemistry of thiols and disulphides. IV. Protective fixation by organomercurial-formalin mixtures

Summary Formation of mercaptides as the result of adding organomercuric salts to neutral formalin used for fixation was found to protect protein thiols from autoxidation, provided the tissues were washed in distilled and not tap water. Such bloking, in contrast to that given by HgCl₂, could be reversed quantitatively by mercaptoethanol made strongly acid to keep it from reducing disulphides. However, some cleavage of disulphides by the mercurials themselves caused slight arbfactual thiol staining in a limited number of sites. Three of the nine compounds tested are sufficiently soluble to penetrate tissues with reasonable speed, stable enough to preclude more than incidental mercurial deposits and currently available commercially. Of them, the diuretic mercurial Mersalyl is at present the protecting, agent of choice since methyl- and ethylmercuric chlorides are too toxic to recommend for routine use.     

Oxidative stress in cell culture: an under-appreciated problem?

Cell culture studies have given much valuable information about mechanisms of metabolism and signal transduction and of regulation of gene expression, proliferation, senescence, and death. However, cells in culture may behave differently from cells in vivo in many ways. One of these is that cell culture imposes a state of oxidative stress on cells. I argue that cells that survive and grow in culture might use ROS-dependent signal transduction pathways that rarely or never operate in vivo. A further problem is that cell culture media can catalyse the oxidation of compounds added to them, resulting in apparent cellular effects that are in fact due to oxidation products such as ROS. Such artefacts may have affected many studies on the effects of ascorbate, thiols, flavonoids and other polyphenolic compounds on cells in culture.     

Amino acids and their derivatives as radioprotective agents

Summary Numerous amino acids and their analogs are capable of protecting biological systems from the toxic effects of ionizing radiation. These radioprotective agents can be classified into two broad groups, depending upon the presence or absence of a free or potentially free sulfhydryl group. The sulfhydryl-containing compounds have been studied extensively and are thought to exert their radioprotective effects by several mechanisms, including free radical scavenging and hydrogen atom donation. Several non-sulfhydryl-containing amino acids are also being investigated for their radioprotective effects. These agents are less well known than the familiar sulfhydryl compounds, but possess very interesting protective qualities. In short, the study of amino acids and their derivatives as radioprotective agents continues to contribute to an understanding of processes involved in radiation toxicity and to offer new compounds with potential application to situations of human exposure.     

Abnormal radiosensitizing and cytotoxic properties of ortho-substituted nitroimidazoles

Various 5-substituted 4-nitroimidazoles have been shown to be much more efficient radiosensitizers and much more toxic than would have been predicted from their electron affinities, as measured by values of one-electron reduction potential, E17. Using Chinese hamster V79 cells in vitro, a comparison has been made with some isomeric 4-substituted 5-nitroimidazoles. These compounds have E17 values some 64mV greater than the 4-nitroimidazoles, yet show much lower sensitizing efficiency and also lower toxicity. Neither series of compounds shows the greater toxicity towards hypoxic cells usually associated with nitroaromatic and nitroheterocyclic compounds. The second-order rate constants, k2, for reaction of these isomeric nitroimidazoles with glutathione and dithiothreitol were determined. Within each series the value of k2 increased with increasing electron affinity, however, the 4-nitroimidazoles were always more reactive than their corresponding 5-nitro isomers. The sensitizing and toxic properties of these compounds may involve depletion of intracellular thiols; this possibility is discussed.     

Submicrodeterminations of thiols, disulphides and thiol esters in serum by using o-hydroxymercuribenzoic acid and dithiofluorescein

1. Methods are described for selective estimation of thiols, disulphides and thiol esters in standard solutions and in serum. The methods are based on the reaction with the excess of o-hydroxymercuribenzoic acid (HMB) in alkaline solution with subsequent addition of dithiofluorescein in excess and determination of the extinction at 588mmu. The sensitivity of the methods amounts to 1.5x10(-9)g.equiv. in 5ml. of final solution. Of results obtained on standard solutions 80% have the errors within the range +/-4%. 2. It has been found that serum contains an unidentified substance (substance X) producing green complexes with dithiofluorescein which undergo decomposition on addition of formaldehyde. The correction for substance X must be estimated in a separate sample and taken into account. The concentration of substance X can be calculated from extinctions measured at 588mmu and 635mmu in the presence of dithiofluorescein in excess. 3. The selective determination of thiols and disulphides is based on different reaction rates with formaldehyde. The complexes between HMB and cysteine can be selectively decomposed by formaldehyde, and free glutathione can be selectively removed by formaldehyde in the presence of protein thiols. 4. Thiols are determined in the presence of triethylamine, thiols plus disulphides in the presence of triethylamine and sulphite, and thiols plus thiol esters in the presence of dimethylamine, with subsequent addition of ammonium sulphate.     

Antioxidant and pro-oxidant effect of the thiolic compounds N-acetyl-L-cysteine and glutathione against free radical-induced lipid peroxidation

The antioxidant ability of thiol compounds has been the subject of much of the current research about oxidative stress. The direct scavenging of hydroxyl radicals by thiols has been suggested as their protection mechanisms. Nevertheless, the interaction of thiols with reactive radicals can generate thiyl radicals, which, in turn, may impart a pro-oxidant function. The purpose of this study has been to establish the effect of the thiol compounds N -acetyl- l -cysteine (NAC) and glutathione (GSH) against the peroxidative processes involving membrane lipids. The results obtained support the ability of NAC and GSH to suppress the 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH)-dependent or to enhance the Fe 2+ /H 2 O 2 -dependent oxidative actions. The evaluation of thiobarbituric acid reactive substances (TBARS) production, the study of the influence of oxidants on membrane fluidity and the measurements of the changes in the fluorescence of bilayer probes, such as 3-( p -(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA), have shown the antioxidant and pro-oxidant effects of both NAC and GSH. Also their dependence on the nature of the radicals generated by the oxidative systems used has been shown. The use of ESR spectroscopy has allowed us to establish the ability of these compounds to scavenge the AAPH-derived radicals, to determine the formation of thiyl radicals in the iron-mediated oxidation and to evaluate the enhanced production of hydroxyl radicals by NAC and GSH.     

Studies on the fungitoxicity of captan

The cellular distribution of ³⁵S after incubating labelled captan with Neurospora crassa conidia has been determined. Nearly all the ³⁵S in the spores is bound to the water-soluble and protein fractions. Thin-layer chromatography of the hot-water extract of spores has shown that ³⁵S occurs largely in oxidized glutathione (GSSG) and in a product tentatively identified as a thiazolidine derivative of glutathione. It is suggested that this derivative, which only forms above pH 6·5, is produced by reaction between glutathione (GSH) and the thiocarbonyl chloride liberated on the decomposition of captan. Captan toxicity could not be completely reversed by pretreatment with thiols and disulphides capable of penetrating the cell membrane, confirming the previous hypothesis that fungitoxicity is due to irreversible changes following the oxidation of the protein thiols to disulphides.     

Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis: proteomic identification of target proteins

Protein thiols are targets of oxidative stress. Their modification was analysed in gill extracts of the mussel Mytilus edulis, exposed to menadione. Diagonal gel electrophoresis revealed two clusters of carbonylated proteins involved in interchain disulphide linkages. Immunoblotting identified these as being associated with protein disulphide isomerase (PDI) and actin and this was confirmed by immunoprecipitation. Protein free thiols (-SH) were identified in 2-DE separations by labelling with 5-iodoacetamidofluorescein (IAF). Cysteines involved in disulphide bridges were identified by blocking free -SH with N-ethylmaleimide, reducing disulphides with DTT and IAF labelling. Several protein spots containing free thiols disappeared on exposure to menadione. Conversely, new protein spots containing disulphides appeared in response to menadione which may be protective against oxidative stress. In-gel tryptic digestion followed by LC/MS-MS and database searching identified some of the free thiol targets: PDI; hsp gp96; calreticulin; heavy metal binding protein. Tubulin, PDI, enolase and gelsolin contained new disulphide bridges in response to menadione. Our findings indicate a protein level response to oxidative stress principally involving PDI, chaperone-like and cytoskeletal proteins. Since many environmental pollutants cause oxidative stress, studies on PDI and structural proteins may be particularly relevant to understanding toxicity in this popular sentinel species.     

Mini-review highlighting toxic effects of triflumuron in insects and mammalian systems

Pesticides have been used to kill pests such as insects, fungi, rodents, and unwanted plants. As these compounds are potentially toxic to the target organisms, they could also be harmful to human health and the environment. Several chronic adverse effects have been identified even after months or years of exposure. The adverse effects of pesticides on the agricultural ecosystem have been a matter of concern in recent decades. In this review, we present an overview of the studies, including our previous studies, monitoring currently used pesticides in the Tunisian agricultural soils that belong to the class of insect growth regulators (IGRs). Triflumuron (TFM) is a benzoyl phenyl urea insecticide belonging to the class of IGRs. TFM is widely used around the world to increase crop yield by protecting them from damage caused by insects. TFM works by inhibiting the synthesis of chitin, an essential part of the insect cuticle, making it susceptible to pathogens and deformities. Consequently, insects become more susceptible to pathogens and malformations. However, studies revealing its toxicity and its mode of action in mammalian systems remain very limited. The aim of this review is to better inform the community about the impact of TFM on crops, the environment, and human beings by summarizing its toxic effects.     

Kinetics of inhibition of aldehyde dehydrogenase isoenzymes of rat liver by fluorine-containing disulfides

New disulphides synthesized on the basis of dithiocarboxylic acid derivatives and heterocyclic thiols containing the fluorine atoms were studied as applied to inhibit aldehyde dehydrogenase (ALDH) isozymes of the rat liver mitochondria. The most effective rat liver inhibitors of ALDH isozymes were revealed. Inhibition of the rat liver isozymes by disulphides I, II, IV, VI-VIII and fluorinated pyridine disulphide was found to be irreversible. The values of isozyme inactivation rate constants are reported. The ALDH inhibition by disulphides I, IV, VI-VIII was competitive both for the cofactor and for the substrate of the reaction. The protective effect of the NAD+ against ALDH I and II inactivation by disulfiram and disulphides I, IV, VI-VIII and X is shown. NADP+ protects isozyme II against inactivation by disulfiram and also disulphides I, VI-VIII.     

The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium

Glutathione (GSH) plays an important neuroprotective role, and its synthesis depends on the amount of available cysteine (CSH) in the cells. Various kinds of evidence suggest that astrocytes can provide CSH or GSH to neurons, but the delivery mechanism of the thiol-compounds has not been elucidated. In this study, the dynamics of CSH, GSH and their disulphides in astrocyte culture medium were investigated by following the time-course of concentration changes and by computer simulation and curve fitting to experimental data using a mathematical model. The model consists of seven reactions and three transports, which are grouped into four categories: autoxidation of thiols into disulphides, thiol-disulphide exchange and reactions of thiols with medium components, as well as the cellular influx and efflux of thiols and disulphides. The obtained results are interpreted that cystine (CSSC) after entering astrocyte is reduced to CSH, most of which is released to medium and autoxidized to CSSC. The efflux of GSH was estimated to be considerably slower than that of CSH, and most of the excreted GSH is converted to cysteine-glutathione disulphide principally through the thiol-disulphide exchange. The results seem to indicate that astrocytes provide neurons mainly with CSH, rather than GSH, as the antioxidant material for neuroprotection.     

Toxicity of secondary compounds to the seed-eating larvae of the bruchid beetle Callosobruchus maculatus

By incorporating various secondary compounds in the normal diet of larval Callosobruchus maculatus bruchids, we show that the effects of any particular compound are dosage-dependent. Alkaloids are generally the most toxic of the compounds tested. Non-protein amino acids are more toxic than protein amino acids but the latter can be toxic at 1 and 5% incorporation in the diet. The non-protein amino acid homoarginine has a salutary effect on larval survival at low concentrations. A variety of other secondary compounds found in seeds are toxic at various levels representative of those levels found in seeds in nature, and for all secondary compounds tested a 0.1–5% incorporation in the diet often has a detrimental effect on production of adult beetles. We conclude that many of the secondary compounds found in seeds are likely to be toxic to at least some animal, and thus are likely to be responsible at least in part for the extreme host-specifity shown by seed-eating insects.     

Toxicity and mutagenicity of selenium compounds in Saccharomyces cerevisiae

Selenium (Se) is an essential trace element for humans, animals and some bacteria which is important for many cellular processes. Se's bio-activity is mainly influenced by its chemical form and dose. The use of Se supplements in the human diet emphasizes the need to establish both the beneficial and detrimental doses of each Se compound. We have evaluated three different Se compounds, sodium selenite (SeL), selenomethionine (SeM) and Se-methylselenocysteine (SeMC), with respect to their potential DNA damaging effects. The budding yeast Saccharomyces cerevisiae was used as a model system to test the toxic and mutagenic effects as well as the DNA double-strand breakage potency of these Se compounds in both exponentially growing and stationary yeast cells. Only SeL manifested any significant toxic effects in the yeast which were more pronounced in the exponentially growing cells than in those cells in the stationary phase of growth. The toxic effects of SeL were however accompanied with the pro-mutagenic effects in the stationary cell phase of growth. The toxic and mutagenic effects of SeL are likely associated with the ability of this compound to generate DNA double-strand breaks (DSB). We also show that SeL significantly increased frame-shift mutations, especially 1-4 bp deletions, in the CAN1 mutational spectrum of the yeast genome when compared to untreated control. We propose that SeL is acting as an oxidizing agent in S. cerevisiae producing superoxide and oxidative damage to DNA accounting for the observed DSB and cell death.     

Capillary electrophoresis in the evaluation of aminothiols in body fluids

Thiols play a fundamental role in cell biology, biochemistry and pharmacology. Altered thiol levels in body fluids are linked to specific pathological conditions. Glutathione is the most abundant intracellular low-molecular-mass thiol, playing an essential role in protecting cells from toxic species; other relevant thiol-containing compounds are homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly). Plasma aminothiols can be bound to proteins but they also occur free in the disulfide (symmetrical and mixed) and in the reduced forms. The simultaneous determination of these aminothiols, their precursor and metabolites is a useful tool in studying oxidative stress, metabolic and redox regulation. Many capillary electrophoresis methods have been proposed for this purpose, the aim of the present review is to support researchers in the choice of suitable methods for the determination of thiols in body fluids evaluating the different approaches and technologies proposed from the literature.     

Attempts to relate enzyme inactivation to degradation in vivo

Inactivation of liver cytosol proteins has been measured in vitro in the presence of various membranes and disulphides. Inactivation rates correlate with the known degradation rate constants of the enzymes in the intact liver. More extensive studies were carried out with glucose-6-phosphate dehydrogenase (G6PD) and phosphoenolpyruvate carboxykinase (PEPCK) using either cytosol as a source of these enzymes or alternatively highly purified preparations of each enzyme. All membranes purified from liver had a considerable capacity to inactivate the enzymes with higher activity found in the hepatocyte plasma membrane. Various lipid preparations or plasma membranes from other tissues were virtually ineffective. Inactivation was dependent on disulphides in the membranes as shown by the inhibition of activity if membranes were pretreated with thiols. Preliminary experiments of the fate of inactivated G6PD or PEPCK show binding to membranes and subsequent proteolysis. A model is proposed for the degradation of labile enzymes.     

Aluminum Toxicity: A Case Study on Tobacco (<i>Nicotiana tabacum</i> L.)

Aluminum is an abundant metal in the earth’s crust that turns out to be toxic in acidic environments. Many plants are affected by the presence of aluminum at the whole plant level, at the organ level, and at the cellular level. Tobacco as a cash crop (Nicotiana tabacum L.) is a widely cultivated plant worldwide and is also a good model organism for research. Although there are many articles on Al-phytotoxicity in the literature, reviews on a single species that are economically and scientifically important are limited. In this article, we not only provide the biology associated with tobacco Al-toxicity, but also some essential information regarding the effects of this metal on other plant species (even animals). This review provides information on aluminum localization and uptake process by different staining techniques, as well as the effects of its toxicity at different compartment levels and the physiological consequences derived from them. In addition, molecular studies in recent years have reported specific responses to Al toxicity, such as overexpression of various protective proteins. Besides, this review discusses data on various organelle-based responses, cell death, and other mechanisms, data on tobacco plants and other kingdoms relevant to these studies.