Oxidative changes in lipids, proteins, and antioxidants in yogurt during the shelf life.

Oxidation processes in milk and yogurt during the shelf life can result in an alteration of protein and lipid constituents. Therefore, the antioxidant properties of yogurt in standard conditions of preservation were evaluated. Total phenols, free radical scavenger activity, degree of lipid peroxidation, and protein oxidation were determined in plain and skim yogurts with or without fruit puree. After production, plain, skim, plain berries, and skim berries yogurts were compared during the shelf life up to 9 weeks. All types of yogurts revealed a basal antioxidant activity that was higher when a fruit puree was present but gradually decreased during the shelf life. However, after 5-8 weeks, antioxidant activity increased again. Both in plain and berries yogurts lipid peroxidation increased until the seventh week of shelf life and after decreased, whereas protein oxidation of all yogurts was similar either in the absence or presence of berries and increased during shelf life. During the shelf life, a different behavior between lipid and protein oxidation takes place and the presence of berries determines a protection only against lipid peroxidation.

Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State.

The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release.

Inhibition of thioredoxin reductase by lanthanum chloride.

Lanthanum chloride (LaCl(3)) is a good inhibitor of both the cytosolic and mitochondrial thioredoxin reductase with IC(50) values of 1.75 and 7.46 µM, respectively. On the contrary, the related enzyme glutathione reductase is not inhibited by lanthanum ions even at relatively high concentrations. In the presence of LaCl(3), steady-state kinetics shows a non-competitive type of inhibition with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as a substrate suggesting no interaction of this ion with the thiol/selenol active site of thioredoxin reductase. Comparison of the electrostatic surface potential of thioredoxin reductase shows that the presence of a trivalent cation such as La(3+) decreases the negatively charged area of the enzyme surface particularly in the region closed to the NADPH binding site. Human ovarian carcinoma cells (A2780 cells) incubated with lanthanum ions show a noticeable inhibition of thioredoxin reductase activity indicating the ability of this ion to reach the active site of the enzyme even in a cellular setting. In addition, A2780 cells treated with LaCl(3) show an increase in reactive oxygen species production in part dependent on thioredoxin reductase inhibition.

Contrasting effects of selenite and tellurite on lipoamide dehydrogenase activity suggest a different biological behaviour of the two chalcogens.

The effects of selenite and tellurite on the mammalian enzyme lipoamide dehydrogenase were compared. Selenite acts as a substrate of lipoamide dehydrogenase in a process requiring the presence of lipoamide. In contrast, tellurite is a potent inhibitor, effective in the low micromolar range. The inhibitory effect of tellurite on lipoamide dehydrogenase is partially reverted by dithiothreitol indicating the participation of the thiol groups of the enzyme. Tellurite, but not selenite, stimulates the diaphorase activity of lipoamide dehydrogenase. In a mitochondrial matrix protein preparation, which contains lipoamide dehydrogenase, an inhibitory action similar to that observed on the purified enzyme was also elicited by tellurite. Human embryonic kidney cells (HEK 293 T) treated with tellurite show a partial inhibition of lipoamide dehydrogenase. In addition to the toxicological implications of tellurium compounds, the reported results suggest that tellurite and its derivatives can be used as potential tools for studying biochemical reactions.

Interaction of selenite and tellurite with thiol-dependent redox enzymes: Kinetics and mitochondrial implications.

The interactions of selenite and tellurite with cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and glutathione reductases (GR) from yeast and mammalian sources were explored. Both TrxR1 and TrxR2 act as selenite and tellurite reductases. Kinetic treatment shows that selenite has a greater affinity than tellurite with both TrxR1 and TrxR2. Considering both k(cat) and K(m), selenite shows a better catalytic efficiency than tellurite with TrxR1, whereas with TrxR2, the catalytic efficiency is similar for both chalcogens. Tellurite is a good substrate for GR, whereas selenite is almost completely ineffective. Selenite or tellurite determine a large mitochondrial permeability transition associated with thiol group oxidation. However, with increasing concentrations of both chalcogens, only about 25% of total thiols are oxidized. In isolated mitochondria, selenite or tellurite per se does not stimulate H2O2 production, which, however, is increased by the presence of auranofin. They also determine a large oxidation of mitochondrial pyridine nucleotides. In ovarian cancer cells both chalcogens decrease the mitochondrial membrane potential. These results indicate that selenite and tellurite, interacting with the thiol-dependent enzymes, alter the balance connecting pyridine nucleotides and thiol redox state, consequently leading to mitochondrial and cellular alterations essentially referable to a disulfide stress.

Cytotoxicity in human cancer cells and mitochondrial dysfunction induced by a series of new copper(I) complexes containing tris(2-cyanoethyl)phosphines.

Over the last few years a lot of research has been done to develop novel metal-based anti-cancer drugs, with the aim of improving clinical effectiveness, reducing general toxicity, and broadening the spectrum of activity. The search for novel metal-based antitumour drugs other than Pt agents includes the investigation of the cytotoxic activity of copper(I/II) compounds. Among these copper agents, particular attention has been recently devoted to hydrophilic copper(I) species bearing phosphines because of their noteworthy stability in aqueous media together with their remarkable in vitro cytotoxic activity. In this study we report on the synthesis, characterization and cytotoxic assays of a series of Cu(I) complexes with tris(2-cyanoethyl)phosphine (PCN) and bis(2-cyanoethyl)phenylphosphine (PCNPh). They were prepared by reaction of [Cu(CH(3)CN)(4)](+) or CuX(2) precursors with the pertinent phosphine in acetone or acetonitrile solutions producing compounds of the following formulation: [Cu(PCN)(2)](+) 2, [Cu(CH(3)CN)(PCN)](+) 3, [Cu(X)(PCN)] (X = Cl, 4; Br, 5), and [Cu(PCNPh)(2)](+) 6. The new copper(I) complexes were tested for their cytotoxic properties against a panel of several human tumour cell lines. Cellular copper uptake rate was correlated with cell growth inhibition in 2008 human ovarian cancer cells. Moreover, copper(I)-PCN complexes were evaluated for their ability to alter the most relevant mitochondrial pathophysiological parameters such as respiration, coupling, ATP-synthetase activity and membrane potential in isolated mitochondria. These data were correlated with changes in mitochondrial membrane potential and production of reactive oxygen species (ROS) in drug-treated 2008 cells.

Hypericins and thioredoxin reductase: Biochemical and docking studies disclose the molecular basis for effective inhibition by naphthodianthrones.

Cytosolic (TrxR1) and mitochondrial (TrxR2) thioredoxin reductases experience pronounced concentration- and time-dependent inhibition when incubated with the two naphthodianthrones hypericin and pseudohypericin. Pseudohypericin turned out to be a quite strong inhibitor of TrxR1 (IC(50)=4.40µM) being far more effective than hypericin (IC(50)=157.08µM). In turn, the IC(50) values measured toward TrxR2 were 7.45µM for pseudohypericin and 43.12µM for hypericin. When compared to pseudohypericin, the inhibition caused by hypericin usually required significantly longer times, in particular on TrxR1. These important differences in the inhibitory potencies and profiles were analysed through a molecular modeling approach. Notably, both compounds were found to accommodate in the NADPH-binding pocket of the enzyme. The binding of the two naphthodianthrones to thioredoxin reductase seems to be particularly strong as the inhibitory effects were fully retained after gel filtration. Also, we found that TrxR inhibition by hypericin and pseudohypericin does not involve the active site selenol/thiol motif as confirmed by biochemical and modeling studies. The resulting inhibition pattern is very similar to that produced by the two naphthodianthrones on glutathione reductase. As the thioredoxin system is highly overexpressed in cancer cells, its inhibition by hypericin and pseudohypericin, natural compounds showing appreciable anticancer properties, might offer new clues on their mechanism of action and open interesting perspectives for future tumor therapies.

Gold(I) complexes determine apoptosis with limited oxidative stress in Jurkat T cells.

In Jurkat T cells, S-triethylphosphinegold(I)-2,3,4,6-tetra-O-acetyl-1-thio-beta-d-glucopyranoside (auranofin) and triethylphosphine gold(I) chloride (TepAu) induced apoptosis, as estimated by DNA fragmentation and visualised by fluorescence microscopy. Apoptosis was characterised by mitochondrial cytochrome c release which was not prevented by cyclosporin A. Apoptosis appeared to be triggered by inhibition exerted by gold(I) compounds on the cytosolic and mitochondrial isoforms of thioredoxin reductase, which determined a definite increase in hydrogen peroxide, whereas glutathione and its redox state were not modified. Total thiols showed a slight decrease, particularly in the presence of auranofin. However, no significant lipid peroxidation or nitric oxide formation were observed after incubation with gold(I) complexes, indicating that the cells had not been subjected to extensive oxidative stress. Interestingly, the gold(I) compound aurothiomalate was poorly effective, both in inhibiting thioredoxin reductase and in inducing apoptosis. These results demonstrate that the increased production of hydrogen peroxide determines an oxidative shift responsible for the occurrence of apoptosis and not involving lipid peroxidation.

Inhibitory effect by new monocyclic 4-alkyliden-beta-lactam compounds on human platelet activation.

In the present study some new beta-lactam compounds were screened for their ability to inhibit human platelet activation. In particular four compounds differing in the group on the nitrogen atom of the azetidinone ring were investigated. A beta-lactam having an ethyl 2-carboxyethanoate N-bound group was demonstrated to inhibit, in the micromolar range, both the Ca(2+) release from endoplasmic reticulum, induced either by thrombin or by the ATPase inhibitor thapsigargin, and the Ca(2+) entry in platelets driven by emptying the endoplasmic reticulum. The compound also inhibited the platelet aggregation induced by a variety of physiological agonists including ADP, collagen, thrombin and thrombin mimetic peptide TRAP. The beta-lactam reduced the phosphorylation of pleckstrin (apparent MW 47 kDa), elicited by thrombin but not by the protein kinase C activator phorbol ester. Accordingly it did not significantly affect the aggregation evoked by phorbol ester or Ca(2+) ionophore. It was concluded that the beta-lactam likely exerts its anti-platelet-activating action by hampering the agonist induced cellular Ca(2+) movements. The beta-lactam concentration, which significantly inhibited platelet activation, only negligibly affected the cellular viability. Even if it is still premature to draw definitive conclusions, the present results suggest that this new compound might constitute a tool of potential clinical interest and the starting-point for the synthesis of new more beneficial anti-thrombotic compounds.

Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells.

Cisplatin is an effective antitumor agent for the treatment of several carcinomas. However, the development of resistance to cisplatin represents a serious clinical problem. The effects of auranofin, a gold(I) compound clinically used as an antirheumatic agent, on cisplatin-sensitive (2008) and-resistant (C13*) cancer cells were studied. Auranofin is more effective than cisplatin in decreasing cell viability and its action is particularly marked in C13* cells, indicating that no cross-resistance occurs. Furthermore, auranofin is able to permeate C13* cells more efficiently than 2008 cells. Treatment with auranofin determines a consistent release of cytochrome c in both cell lines, while cisplatin is effective only in sensitive cells. Both auranofin and cisplatin induce apoptosis in 2008 cells, while in C13* cells only auranofin is effective. Apoptosis is accompanied by an increased production of hydrogen peroxide that, however, is inhibited by N-acetyl-L-cysteine. In resistant cells, H(2)O(2) production is counteracted by a large overexpression of thioredoxin reductase that constitutes the preferred target of the inhibitory action of auranofin. This specific effect of auranofin might rationalize its ability in overcoming cisplatin resistance in human ovarian cancer cells.

Differential effect of calcium ions on the cytosolic and mitochondrial thioredoxin reductase.

The effect of calcium ions has been studied on three different isoforms of thioredoxin reductase. The cytosolic (TrxR1), mitochondrial (TrxR2), and the Escherichia coli enzymes were examined and compared. In our condition, TrxR1 appears extremely sensitive to Ca2+ showing an IC50 of about 160 nM, while Ca2+ exerts only a weak inhibitory effect on the mitochondrial isoform. The thioredoxin reductase purified from E. coli is almost completely insensitive to calcium ions. Circular dichroism analysis of highly purified mitochondrial and cytosolic thioredoxin reductases reveals that Ca2+ induces conformational alterations that are particularly relevant only in the cytosolic isoform. These observations are discussed with reference to the physiological role and, in particular, to the regulatory functions of the thioredoxin system.

An in vitro study of the interaction of sea-nine with rat liver mitochondria.

The interactions of the antifouling compound Sea-Nine with rat liver mitochondria have been studied. The results indicate that low doses of this compound inhibit adenosine 5'-triphosphate (ATP) synthesis. Further investigations indicate that ATP synthesis inhibition should be due to an interaction of Sea-Nine with the succinic dehydrogenase in the mitochondrial respiratory chain.

The modulation of thiol redox state affects the production and metabolism of hydrogen peroxide by heart mitochondria.

In rat heart mitochondria, auranofin, arsenite, diamide, and BCNU increase H2O2 formation, further stimulated by antimycin. However, in submitochondrial particles, H2O2 formation and oxygen uptake are not affected, indicating that these substances do not alter respiration. Mitochondria are also able to rapidly metabolize added H2O2 in a process partially prevented by BCNU or auranofin. Calcium does not modify the production of H2O2 and the mitochondrial thioredoxin system is not affected by calcium ions. Auranofin, arsenite, and diamide determine a large mitochondrial permeability transition, while BCNU and acetoacetate are ineffective. Thiols and glutathione are modified only by BCNU and diamide. However, all the compounds tested cause the release of cytochrome c that occurs also in the absence of mitochondrial swelling. In conclusion, the compounds utilized share the common feature of shifting the mitochondrial thiol-linked redox balance towards a more oxidized condition that is responsible of the observed effects.

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase.

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.

Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions.

The effects of gold(I) complexes (auranofin, triethylphosphine gold and aurothiomalate), gold(III) complexes ([Au(2,2'-diethylendiamine)Cl]Cl(2), [(Au(2-(1,1-dimethylbenzyl)-pyridine) (CH(3)COO)(2)], [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine)(OH)](PF(6)), [Au(bipy(dmb)-H)(2,6-xylidine)](PF(6))), metal ions (zinc and cadmium acetate) and metal complexes (cisplatin, zinc pyrithione and tributyltin) on mitochondrial thioredoxin reductase and mitochondrial functions have been examined. Both gold(I) and gold(III) complexes are extremely efficient inhibitors of thioredoxin reductase showing IC(50) ranging from 0.020 to 1.42 microM while metal ions and complexes not containing gold are less effective, exhibiting IC(50) going from 11.8 to 76.0 microM. At variance with thioredoxin reductase, auranofin is completely ineffective in inhibiting glutathione peroxidase and glutathione reductase, while gold(III) compounds show some effect on glutathione peroxidase. The mitochondrial respiratory chain is scarcely affected by gold compounds while the other metal complexes and metal ions, in particular zinc ion and zinc pyrithione, show a more marked inhibitory effect that is reflected on a rapid induction of membrane potential decrease that precedes swelling. Therefore, differently from gold compounds, the various metal ions and metal complexes exert their effect on different targets indicating a lower specificity. It is concluded that gold compounds are highly specific inhibitors of mitochondrial thioredoxin reductase and this action influences other functions such as membrane permeability properties. Metal ions and metal complexes markedly inhibit the activity of thioredoxin reductase although to an extent lower than that of gold compounds. They also inhibit mitochondrial respiration, decrease membrane potential and, finally, induce swelling.

A possible transport mechanism for aluminum in biological membranes.

The transport mechanism of aluminum in lysosomes extracted from rat liver has been investigated in this paper. The experimental evidence supports the hypothesis that aluminum is transported inside lysosomes in the form of an Al(OH)(3) electroneutral compound, the driving force being the internal acidic pH. This mechanism could help to explain the presence of aluminum in cells in many illnesses.

Evaluation of the antioxidant properties of propofol and its nitrosoderivative. comparison with homologue substituted phenols.

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole > propofol > 2,6-dimethylphenol > 2,6-di-tertbutylphenol > butylated hydroxytoluene. In cumene hydroperoxide-dependent microsomal lipid peroxidation, propofol acts as the most effective antioxidant, while butylated hydroxyanisole, 2,6-di-tertbutylphenol and butylated hydroxytoluene exhibit a rather similar effect, although lower than propofol. In the iron/ascorbate-dependent lipid peroxidation propofol, at concentrations higher than 10 microM, exhibits antioxidant properties comparable to those of butylated hydroxytoluene and butylated hydroxyanisole, 2,6-Dimethylphenol is scarcely effective in both lipoperoxidative systems. The antioxidant properties of the various molecules depend on their hydrophobic characteristics and on the steric and electronic effects of their substituents. However, the introduction of the nitroso group in the 4-position almost completely removes the antioxidant properties of the examined compounds. The nitrosation of the aromatic ring of antioxidant molecules and the consequent loss of antioxidant capacity can be considered a condition potentially occurring in vivo since nitric oxide and its derivatives are continuously formed in biological systems.

Mitochondrial thioredoxin reductase inhibition by gold(I) compounds and concurrent stimulation of permeability transition and release of cytochrome c.

The effects of auranofin, chloro(triethylphosphine)gold(I) (TEPAu), and aurothiomalate on mitochondrial respiration, pyridine nucleotide redox state, membrane permeability properties, and redox enzymes activities were compared. The three gold(I) derivatives, in the submicromolar range, were extremely potent inhibitors of thioredoxin reductase and stimulators of the mitochondrial membrane permeability transition (MPT). Auranofin appeared as the most effective one. In the micromolar range, it inhibited respiratory chain and glutathione peroxidase activity only slightly if not at all. TEPAu and aurothiomalate exhibited effects similar to auranofin, although TEPAu showed a moderate inhibition on respiration. Aurothiomalate inhibited glutathione peroxidase at concentrations where auranofin and TEPAu were without effect. Under nonswelling conditions, the presence of auranofin and aurothiomalate did not alter the redox properties of the mitochondrial pyridine nucleotides indicating that membrane permeability transition occurred independently of the preliminary oxidation of pyridine nucleotides. Under the same experimental conditions, TEPAu showed a moderate stimulation of pyridine nucleotides oxidation. Mitochondrial total thiol groups, in the presence of the gold(I) derivatives, slightly decreased, indicating the occurrence of an oxidative trend. Concomitantly with MPT, gold(I) compounds determined the release of cytochrome c that, however, occurred also in the presence of cyclosporin A and, partially, of EGTA, indicating its independence of MPT. It is concluded that the specific inhibition of thioredoxin reductase by gold(I) compounds may be the determinant of MPT and the release of cytochrome c.

Correlation between fluidising effects on phospholipid membranes and mitochondrial respiration of propofol and p-nitrosophenol homologues.

Nitrosopropofol (2-6-diisopropyl-4-nitrosophenol) has dramatic consequences for respiration, ATP synthesis and the transmembrane potential of isolated rat liver mitochondria at concentrations at which propofol (2-6-diisopropylphenol) does not cause any apparent effects. These results correlate well with the observation that nitrosopropofol is also a stronger perturbing agent of phospholipid membranes. In this paper we verify the possible biological activity of different phenols and nitrosophenols on mitochondrial respiration. We then discuss their interactions with phospholipid liposomes, studied with differential scanning calorimetry, spin labelling techniques and UV-Vis spectrophotometry, in order to obtain information on drug distribution and the modifications they impose on lipid bilayer. The results of the experiments performed on mitochondria and model membranes prove an interesting correlation between the effects of the molecules on both systems.

Effects of nitrosopropofol on mitochondrial energy-converting system.

Nitrosopropofol (NOPR) is a relatively stable compound obtained from the reaction between the general anesthetic 2,6 diisopropylphenol (propofol) and nitrosoglutathione (GSNO) and bearing a more acidic phenol group than propofol. It interfered with mitochondrial energetic metabolism in a concentration-dependent manner. Concentrations as high as 100 or 200 microM disrupted both oxidative phosphorylation and electron transport. Low concentrations of NOPR (50 microM) markedly slowed down the electron transport rate which was insensitive both to ADP and uncoupler stimulation and spontaneously gradually stopped. Consequently, both the transmembrane potential production and the ATP synthesis system were affected. In the presence of 10 or 20 microM NOPR, mitochondria respired but showed a worsening of the respiratory control and produced a transmembrane potential useful to respond to a phosphorylation pulse, but were not able to restore it. These results were consistent with ATP synthesis and swelling experiments. NOPR was effective at concentrations lower than those required by the combination of propofol and GSNO, suggesting that mitochondria might be able to catalyze the reaction between GSNO and propofol and that the resulting metabolite was more active on mitochondrial membrane structure than the parent compounds. Although the details of the process are yet unknown, the mechanism presented may be of potential relevance to rationalize the pathophysiological effects of propofol.