Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals.

Glutathione transferases (GSTs) are enzymes devoted to the protection of cells against many different toxins. In erythrocytes, the isoenzyme (e-GST) mainly present is GSTP1-1, which is overexpressed in humans in case of increased blood toxicity, as it occurs in nephrophatic patients or in healthy subjects living in polluted areas. The present study explores the possibility that e-GST may be used as an innovative and highly sensitive biomarker of blood toxicity also for other mammals. All distinct e-GSTs from humans, Bos taurus (cow), Sus scrofa (pig), Capra hircus (goat), Equus caballus (horse), Equus asinus (donkey) and Ovis aries (sheep), show very similar amino acid sequences, identical kinetics and stability properties. Reference values for e-GST in all these mammals reared in controlled farms span from 3.5±0.2 U/gHb in the pig to 17.0±0.9 U/gHb in goat; such activity levels can easily be determined with high precision using only a few microliters of whole blood and a simple spectrophotometric assay. Possibly disturbing factors have been examined to avoid artifact determinations. This study provides the basis for future screening studies to verify if animals have been exposed to toxicologic insults. Preliminary data on cows reared in polluted areas show increased expression of e-GST, which parallels the results found for humans.

Irreversible inhibition of pig kidney copper-containing amine oxidase by sodium and lithium ions.

Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.

Human glutathione transferase P1-1 and nitric oxide carriers; a new role for an old enzyme.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

Interaction of pig kidney and lentil seedling copper-containing amine oxidases with guanidinium compounds.

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Ki value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.

Metabolism of furazolidone: alternative pathways and modes of toxicity in different cell lines.

1. The metabolism and cytotoxicity of the antimicrobial nitrofuran drug furazolidone have been studied in Caco-2, HEp-2 and V79 cell lines. Free radical production, metabolite pattern, formation of bound residues, inhibition of cellular replication and protection by the antioxidant glutathione were compared for the three cell lines. 2. All three cell lines produced the same nitro-anion radical with similar kinetics. Little further metabolic breakdown was observed in V79 cells, whereas Caco-2 and HEp-2 cells showed extensive degradation of furazolidone, but with different end patterns. 3. Under hypoxic conditions, the colony-forming ability was extensively impaired in HEp-2 cells whereas the other two cell lines were less affected, suggesting that irreversible damage to DNA occurred prevalently in HEp-2 cells. In V79 cells the absence of oxygen caused a 25-fold increase in the formation of protein-bound residues. 4. Brief exposure to furazolidone caused a 50% loss of endogenous glutathione in Caco-2 cells, but no loss could be detected in V79 and HEp-2 cells. Consistently, when glutathione was depleted by buthionine-[S,R]-sulphoximine (BSO) and diethylmaleate (DEM) treatment, the viability of V79 and HEp-2 cells was minimally affected by furazolidone, whereas that of Caco-2 cells was substantially reduced. 5. It is concluded that the cytotoxicity of furazolidone in these cell lines can be exerted by a number of different mechanisms, possibly related to different metabolic pathways. The cytotoxicity of nitrofuran drugs, therefore, cannot be ascribed to a single toxic intermediate, but in Caco-2 cells furazolidone is extensively metabolized and detoxified by GSH, in V79 is only partially activated and then bound to proteins, whereas in HEp-2, once activated, may react with DNA.

Effect of metal substitution in copper amine oxidase from lentil seedlings.

The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k(c) value is different from that of native or Cu-fully-reconstituted enzyme, while K(m) is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.

CuI-semiquinone radical species in plant copper-amine oxidases.

The intermediate CuI-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

Protection of low density lipoprotein oxidation by the antioxidant agent IRFI005, a new synthetic hydrophilic vitamin E analogue.

The oxidative modification of low density lipoprotein (LDL) is thought to be an important factor in the initiation and development of atherosclerosis. Antioxidants have been shown to protect LDL from oxidation and to inhibit atherosclerosis development in animals. Potent synthetic antioxidants are currently being tested, but they are not necessarily safe for human use. We here characterize the antioxidant activity of IRFI005, the active metabolite of Raxofelast (IRFI0016) that is a novel synthetic analog of vitamin E under clinical development, and demonstrate that it prevents oxidative modification of LDL. IFI005 inhibited the oxidative modification of LDL, measured through the generation of MDA, electrophoretic mobility and apo B100 fluorescence. During the oxidation process IRF1005 was consumed with the formation of the benzoquinone oxidation product. The powerful antioxidant activity of IRFI005 is at least in part mediated by a chain breaking mechanism as it is an efficient peroxyl radical scavenger with a rate constant k(IRFI005 + LOO(o)) of 1.8 X 10(6) M(-1)s(-1). 4. IRFI005 substantially preserved LDL-associated antioxidants, alpha-tocopherol and carotenoids, and when co-incubated with physiologic levels of ascorbate provoked a synergistic inhibition of LDL oxidation. Also the co-incubation of IRFI005 with Trolox caused a synergistic effect, and a lag phase in the formation of the trolox-benzoquinone oxidation product. A synergistic inhibition of lipid peroxidation was also demonstrated by co-incubating IRFI005 and alpha-tocopherol incorporated in linoleic acid micelles. These data strongly suggest that IRFI005 can operate by a recycling mechanism similar to the vitamin E/ascorbate sysem.

Inhibitors of plant copper amine oxidases.

In this review, inhibitors of plant copper amine oxidases from Lens esculenta seedlings, Pisum sativum seedlings, and Euphorbia characias latex are described. Reversible competitive inhibitors and non-competitive inhibitors, irreversible active-site directed inhibitors and mechanism-based inactivators are reviewed in regard to their mechanisms of action.

Hypoglycemia, hypoxia, and ischemia in a corticostriatal slice preparation: electrophysiologic changes and ascorbyl radical formation.

Experimental and clinical data suggest that oxygen and/or glucose deprivation alters electrical transmission in the brain and generates free radicals, which may mediate neuronal death. We have analyzed the effects of oxygen and/or glucose deprivation on both excitatory transmission, by measuring field potential amplitude, and free radical production, by using electron spin resonance (ESR) spectroscopy, in a corticostriatal slice preparation. Combined oxygen and/or glucose deprivation (ischemia) lasting 10 to 20 minutes induced a long-term depression of field potential amplitude. The ascorbyl radical could only be detected in brain slices during the reperfusion-phase after 30 minutes of ischemia. It appeared in the early minutes after the washout of ischemic medium and remained stable throughout the reperfusion phase. This radical was never detected in the external medium. Ischemia induced only a slight, but progressive, release of lactate dehydrogenase (LDH) into the external medium during the reperfusion phase. In contrast, exposure of slices to hypoxia or hypoglycemia alone resulted in transient depression of field potential amplitude, and no generation of ascorbyl radicals was observed on reperfusion. We propose that the long-lasting loss of electrical signals is the early sign of neuronal damage during ischemia. On the other hand, ascorbyl radical formation may be considered an indicator of neuronal injury after prolonged energy deprivation.

Characterization of Euphorbia characias latex amine oxidase.

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 x 10(5) M-1 cm-1 and 6000 M-1 cm-1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.

Inhibition of copper amine oxidase by haloamines: a killer product mechanism.

The observation that the alkylamines 2-Br-ethylamine and 2-C1-ethylamine and 1,2-diaminoethane, the shortest diamine, are irreversible inhibitors of several copper amine oxidases led to the investigation of the mechanism by which these compounds react with the highly active amine oxidase from lentil seedlings. 1,2-Diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine were found to be both poor substrates and irreversible inhibitors of lentil amine oxidase; inactivation took place in both the presence and absence of oxygen. All three compounds strongly affected the spectrum of the enzyme, leading to the formation of a stable band at 336 nm both in anaerobiosis and in aerobiosis, consistent with an interaction with the enzyme cofactor 6-hydroxydopa. On the contrary, the corresponding propylamine compounds 1,3-diaminopropane, 3-Br-propylamine, and 3-C1-propylamine were reversible inhibitors of lentil amine oxidase. Inhibition was shown to be due to the aldehyde oxidation products rather than the short chain amines themselves; a reaction mechanism is presented which involves attack of the aldehyde on the 6-hydroxydopa-derived free radical catalytic intermediate. With 1,2-diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine, the complex produced will form a stable 6-membered ring, causing irreversible inhibition of the enzyme.

Tryptamine as substrate and inhibitor of lentil seedling copper amine oxidase.

Copper amine oxidase from lentil seedlings was shown to be able to catalyze the oxidative deamination of the indoleamines tryptamine, 5-hydroxytryptamine, and 5-methoxytryptamine. These compounds showed saturation kinetics with Km values as normal substrates, but their oxidation led to irreversible loss of enzyme activity suggesting a covalent interaction with the enzyme, most probably through its cofactor 6-hydroxydopa (2,4,5-trihydroxyphenylalanine). These indoleamines acted as irreversible inhibitors of the enzyme only in the absence of oxygen but they brought about changes in the electronic spectra of the enzyme both in aerobiosis and in anaerobiosis. This study reports on the mechanism by which these compounds inhibit lentil amine oxidase which involves first the oxidation of indoleamines bound to 6-hydroxydopa followed by the formation of an irreversible covalent derivative. The same inhibitory mechanism could possibly lead to inactivation of mammalian amine oxidases involved in serotonin neurotransmitter metabolism in conditions of ischemia or hypoxia.

Substrate specificity of lentil seedling amine oxidase.

Copper diamine oxidase from lentil (Lens culinaris) seedlings was shown to be able to catalyze the oxidative deamination of a wide range of aliphatic and aromatic monoamine compounds, including some amino acids. Although the catalytic efficiencies were only 1-3% of that measured with the diamine substrate putrescine, they were still comparable to those of specialized monoamine oxidases. In particular, the lentil enzyme oxidized benzylamine and histamine with K(m) and Vmax values similar to those found for the mammalian enzymes benzylamine oxidase and histaminase. Cysteamine was found to be a substrate of the enzyme, whereas hypotaurine and taurine were found to be neither substrates nor inhibitors of the enzyme. Quite unexpectedly the amino acids L-ornithine and L-lysine were oxidized by lentil enzyme, and beta-alanine and gamma-aminobutyric acid were oxidized only at high concentrations of enzyme. These results suggest that enzymes normally classified as diamine oxidases could in fact have a more diversified role in metabolism than recognized so far.

N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone activation by the human intestinal cell line Caco-2 monitored through noninvasive electron spin resonance spectroscopy.

The pathways participating in the metabolism of the nitrofuran antimicrobial drug N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone (furazolidone) in intact cells were investigated in the human intestinal cell line Caco-2. One-electron reduction of furazolidone led to the formation of a free radical intermediate that could be monitored in dense cell suspensions by noninvasive electron spin resonance spectroscopy. The effects of enzyme inhibitors on the kinetics of radical production and decay were used to estimate the relative contribution of different enzymes to the reductive activation of the drug. Although many enzymes are known to reduce nitrofurans in vitro (e.g., xanthine oxidase, aldehyde oxidase, DT-diaphorase, mitochondrial redox chain components), their contributions were insignificant in living Caco-2 cells. The first reducing equivalent required for the formation of the nitroanion derivative of furazolidone appeared to be provided essentially by the microsomal cytochrome P450 reductase. This was confirmed through studies of the NADPH-dependent radical formation by microsomes. Differentiated Caco-2 cells, an established enterocyte model, showed only modestly increased radical formation and the same enzyme-specificity pattern as undifferentiated cells. Consistently, only a small increase in P450 reductase activity was found in differentiated cells, in contrast to the 10-fold increase seen in typical differentiation marker enzymes. With the electron spin resonance method that we describe, it is possible to distinguish between sites of bioactivation of redox active drugs in intact cells.

The reaction mechanism of copper amine oxidase: detection of intermediates by the use of substrates and inhibitors.

Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy. Using highly purified apo- and holoenzyme in combination with a poor substrate and a range of inhibitors, under both aerobic and anaerobic conditions, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate species in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for putrescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequent intramolecular electron transfer to form the characteristic free radical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibitors bind to the 6-hydroxydopa cofactor in the same way as active substrates, but rearrangements lead to formation of stable intermediate adducts at the step preceding release of aldehyde. The semicarbazide-6-hydroxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-hydroxydopa close to the copper site.

Autoxidation of 4-methylcatechol: a model for the study of the biosynthesis of copper amine oxidases quinonoid cofactor.

The autoxidation of 4-methylcatechol under quasi-physiological conditions, leading to 2-hydroxy-5-methyl-1,4-benzoquinone, was investigated. The effects of pH and metal ions were examined. An electrophilic attack of dioxygen to the 4-methylcatechol monoanion to form a transient peroxo species is proposed. It was concluded that such a non-enzymic conversion is likely for this model compound and for its physiological counterpart, a specific tyrosyl residue incorporated in the protein chain at the active site of copper amine oxidases.

Autocatalytic acylation of phospholipase-like myotoxins.

Several snake venoms contain a phospholipase A2 in which position 49 in the active site is occupied by a lysine or a serine instead of the aspartate residue normally found. Although these proteins do not bind Ca2+ and are devoid of catalytic activity, they are still highly specific myotoxins and have recently been shown to induce membrane leakage by a new type of cytolytic mechanism. Three of these toxins, myotoxin II from Bothrops asper, ammodytin L from Vipera ammodytes, and the K49 protein from Agkistrodon piscivorus piscivorus, were examined for their interaction with fatty acids and were found to bind long-chain fatty acids covalently by a rapid, spontaneous, autocatalytic process. The fatty acids could be released by treatment with 1 M NH2OH or NaOH, but not with 1 M NaCl or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron spin resonance studies using spin-labeled fatty acids showed that only the carboxyl headgroup of the fatty acid was linked to protein amino acid, the carbon chain had free mobility and did not bind tightly to the protein surface. Stearic acid methyl esters and short-chain fatty acids did not bind to the toxins. Acylated myotoxins bound to the surface of liposomes and isolated muscle membranes, with the fatty acid moiety inserted into the lipid bilayer and possibly acting as an anchor. The phospholipase-like myotoxins represent the first group of proteins able to undergo acylation by spontaneous reaction with free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

A potent chain-breaking antioxidant activity of the cardiovascular drug dipyridamole.

The antioxidant properties of the antithrombotic drug dipyridamole have been studied using lipid oxidation assays based on the generation of peroxy radicals by azo compounds. Dipyridamole was observed to prevent both peroxidation of arachidonic acid micelles in aqueous solution and peroxidation of methyl linoleate in organic solvents; in contrast to vitamin E, dipyridamole was found to scavenge both hydrophilic and hydrophobic radicals. The rate constant for the reaction of dipyridamole with methyl linoleate peroxyl radicals at 37 degrees C was calculated as 2 x 10(6) M-1s-1, in comparison to 1 x 10(6) M-1s-1 of vitamin E under the same conditions. The antioxidant efficiency of the drug was confirmed in experiments with radiolysis-induced oxidation and through measurements of malondialdehyde production and diene formation. As a result of radical scavenging, a relatively stable dipyridamole radical was formed that could be detected by electron spin resonance spectroscopy. The particular antioxidant properties of dipyridamole may explain the vasodilating and antiplatelet effects of this cardiovascular drug.